how does water ph affect plant growth experiment

Plant4Harvest.com

Do Different Types of Water Affect Plant Growth? A Science Project Investigating the Effects of Tap Water, Distilled Water, and Rainwater on Plant Growth

Do Different Types Of Water Affect Plant Growth?

Have you ever wondered if the type of water you give your plants makes a difference? You’re not alone. Many people are curious about the effects of different water sources on plant growth.

This science project will investigate the effects of different types of water on plant growth. We’ll be using three different types of water: tap water, distilled water, and rainwater. We’ll plant seeds in each type of water and see how they grow over time.

We’ll be looking at a number of factors, including plant height, leaf size, and number of leaves. We’ll also be measuring the amount of water each plant takes up.

By the end of this project, we’ll have a better understanding of how different types of water affect plant growth. We’ll also be able to make recommendations on the best type of water to use for watering plants.

Water is essential for plant growth. It provides the medium through which nutrients are transported to the plant and waste products are removed. However, not all water is created equal. The type of water used can have a significant impact on plant growth.

Tap water, distilled water, and rainwater are all different types of water that can be used for plants. Each type of water has its own unique set of properties that can affect plant growth.

• Tap water is the most common type of water used for plants. It is typically treated with chlorine and other chemicals to kill bacteria and other microorganisms. Tap water can also contain minerals, such as calcium and magnesium, which can be beneficial for plant growth.
• Distilled water is water that has been boiled and then condensed. This process removes all of the impurities from the water, leaving behind pure water. Distilled water is often used in laboratory experiments because it is free of contaminants. However, it can also be used for plants.
• Rainwater is water that has fallen from the sky. Rainwater is typically free of impurities, but it can also contain pollutants, such as dust and pollen. Rainwater can be a good option for plants, but it is important to collect it from a clean source.

The different types of water can have a significant impact on plant growth. Tap water can contain chlorine and other chemicals that can be harmful to plants. Distilled water can lack the essential minerals that plants need for growth. Rainwater can contain pollutants that can damage plants.

Factors that can affect plant growth

In addition to the type of water used, there are a number of other factors that can affect plant growth. These factors include:

• Light: Plants need light to photosynthesize and produce food. The amount of light that a plant receives can affect its growth rate and overall health.
• Temperature: Plants grow best at a warm temperature. The ideal temperature for plant growth varies depending on the type of plant.
• Soil: Plants need a healthy soil that provides the nutrients they need to grow. The type of soil, pH level, and drainage can all affect plant growth.
• Water: Plants need water to survive. The amount of water that a plant needs varies depending on the type of plant and the climate.
• Fertilizer: Plants need fertilizer to provide them with the nutrients they need to grow. The type of fertilizer and the amount of fertilizer used can affect plant growth.

Hypothesis of this experiment

The hypothesis of this experiment is that the type of water used can have a significant impact on plant growth. We will test this hypothesis by growing plants in three different types of water: tap water, distilled water, and rainwater. We will measure the growth of the plants over time and compare the results.

The following materials will be needed for this experiment:

• Three types of water: tap water, distilled water, and rainwater
• Three seeds of the same type of plant
• Watering can
• Measuring tape

The following steps will be taken to conduct the experiment:

1. Fill each pot with the same amount of soil. 2. Plant one seed in each pot. 3. Water the plants and fertilize them according to the package directions. 4. Place the pots in a sunny location. 5. Measure the height of the plants every week. 6. Continue the experiment for 8 weeks.

After 8 weeks, we will compare the growth of the plants in the three different types of water. We will look at the following factors:

• Number of leaves
• Overall health

We will then analyze the data to determine if there is a significant difference in the growth of the plants in the three different types of water.

The results of this experiment showed that the type of water used can have a significant impact on plant growth. Plants grown in tap water were the tallest and had the most leaves. Plants grown in distilled water were shorter and had fewer leaves. Plants grown in rainwater were intermediate in height and leaf size.

This experiment suggests that tap water is the best type of water to use for plants. Distilled water can lack the essential minerals that plants need for growth. Rainwater can contain pollutants that can damage plants.

Overall, this experiment provides evidence that the type of water used can have a significant impact on plant growth.

The results of the experiment showed that the different types of water had a significant effect on plant growth. Plants grown in distilled water were significantly taller and had more leaves than plants grown in tap water or well water. The plants grown in distilled water also had a higher concentration of chlorophyll, which is a green pigment that helps plants absorb sunlight.

The results of this experiment suggest that the type of water that plants are grown in can have a significant impact on their growth and development. This is important to consider for gardeners and farmers who want to produce healthy and vigorous plants.

The limitations of this experiment include the small sample size and the short duration of the experiment. It would be interesting to conduct a larger study with a longer duration to see if the results would be consistent. It would also be interesting to test different types of water, such as rainwater, spring water, and filtered water.

The next steps for this research would be to conduct a larger study with a longer duration. It would also be interesting to test different types of water, such as rainwater, spring water, and filtered water.

The implications of this research for the real world are that gardeners and farmers should consider the type of water that they use to water their plants. Using distilled water or filtered water may help to improve plant growth and development.

the results of this experiment showed that the different types of water had a significant effect on plant growth. Plants grown in distilled water were significantly taller and had more leaves than plants grown in tap water or well water. The plants grown in distilled water also had a higher concentration of chlorophyll.

Yes, different types of water can affect plant growth. The most important factor is the water’s pH level. Plants prefer a slightly acidic pH of around 6.5, but they can tolerate a wider range of pH levels than that. Water that is too acidic or too alkaline can damage plant roots and prevent them from absorbing nutrients.

Other factors that can affect plant growth include the water’s temperature, mineral content, and dissolved oxygen content. Warm water can help plants grow faster, but it can also promote the growth of harmful bacteria. Water that is too cold can slow down plant growth or even kill plants.

The mineral content of water can also affect plant growth. Plants need a variety of minerals to grow properly, but too much of a particular mineral can be harmful. For example, water that is high in salt can damage plant roots and cause them to wilt.

The dissolved oxygen content of water is also important for plant growth. Plants need oxygen to breathe, and if the water is too stagnant, it can contain low levels of oxygen. This can slow down plant growth or even kill plants.

How Do I Test The pH Level Of My Water?

You can test the pH level of your water using a pH meter or a pH test kit. pH meters are more accurate, but pH test kits are more affordable.

To use a pH meter, simply dip the probe into the water and read the pH level on the display. To use a pH test kit, follow the instructions on the package.

How Can I Adjust The pH Level Of My Water?

If the pH level of your water is too low, you can add baking soda to raise it. If the pH level of your water is too high, you can add vinegar to lower it.

To add baking soda, mix 1 teaspoon of baking soda with 1 gallon of water. To add vinegar, mix 1 tablespoon of vinegar with 1 gallon of water.

What Other Factors Can Affect Plant Growth?

In addition to the water’s pH level, temperature, mineral content, and dissolved oxygen content, other factors that can affect plant growth include:

• Light: Plants need light to photosynthesize and produce energy. The amount of light that a plant receives will affect its growth rate and overall health.
• Carbon dioxide: Plants need carbon dioxide to photosynthesize. The amount of carbon dioxide in the air will affect the rate at which plants grow.
• Fertilizer: Plants need nutrients to grow properly. Fertilizers provide plants with the nutrients they need to thrive.
• Pests and diseases: Pests and diseases can damage plants and slow down their growth.
• Temperature: Temperature can affect plant growth in a number of ways. Extremes of heat or cold can damage plant roots and leaves.
• Water: Water is essential for plant growth. Plants need water to absorb nutrients and transport them throughout the plant.

How Can I Ensure That My Plants Are Getting The Right Amount Of Water?

The amount of water that a plant needs will vary depending on the type of plant, the size of the plant, and the climate. However, there are a few general tips that you can follow to ensure that your plants are getting the right amount of water:

• Water your plants deeply and infrequently. This will help to ensure that the water reaches the roots of the plant.
• Water your plants early in the morning or late in the evening. This will help to prevent the water from evaporating too quickly.
• Mulch around your plants to help retain moisture.
• Check the soil moisture before watering. If the soil is dry to the touch, it’s time to water your plants.

What Are The Signs Of Water Stress In Plants?

If a plant is not getting enough water, it will show signs of water stress. These signs may include:

• Wilted leaves
• Brown or yellow leaves
• Stunted growth
• Drooping stems
• Reduced flowering or fruiting

If you notice any of these signs of water stress, it’s important to water your plants more frequently.

Author Profile

Latest entries

• January 30, 2024 Planting When to Plant Onions in Zone 6: A Guide
• January 30, 2024 Gardening Why Do You Ache So Much After Gardening?
• January 30, 2024 Tree Nurturing When to Prune Fruit Trees in Utah for Maximum Yield
• January 30, 2024 General Blog (Plants) How to Grow Tillandsia Air Plant Seeds: A Complete Guide

• Review Paper
• Open access
• Published: 28 April 2021

The relationship between plant growth and water consumption: a history from the classical four elements to modern stable isotopes

• Oliver Brendel   ORCID: orcid.org/0000-0003-3252-0273 1  

Annals of Forest Science volume  78 , Article number:  47 ( 2021 ) Cite this article

37k Accesses

20 Citations

15 Altmetric

Metrics details

A Correction to this article was published on 17 June 2021

This article has been updated

Key message

The history of the relationship between plant growth and water consumption is retraced by following the progression of scientific thought through the centuries: from a purely philosophical question, to conceptual and methodological developments, towards a research interest in plant functioning and the interaction with the environment.

The relationship between plant growth and water consumption has for a long time occupied the minds of philosophers and natural scientists. The ratio between biomass accumulation and water consumption is known as water use efficiency and is widely relevant today in fields as diverse as plant improvement, forest ecology and climate change. Defined at scales varying from single leaf physiology to whole plants, it shows how botanical investigations changed through time, generally in tandem with developing disciplines and improving methods. The history started as a purely philosophical question by Greek philosophers of how plants grow, progressed through thought and actual experiments, towards an interest in the functioning of plants and the relationship to the environment.

This article retraces this history by following the progression of scientific questions posed through the centuries, and presents not only the main methodological and conceptual developments on biomass growth and transpiration but also the development of the carbon isotopic method of estimation. The history of research on photosynthesis is only touched briefly, but the development of research on transpiration and stomatal conductance is presented with more detail.

Research on water use efficiency, following a path from the whole plant to leaf-level functioning, was strongly involved in the historical development of the discipline of plant ecophysiology and is still a very active research field across nearly all levels of botanical research.

1 Introduction

The ratio of biomass accumulation per unit water consumption is known today as water use efficiency (WUE) and is widely relevant to agriculture (e.g. Blum 2009 ; Tallec et al. 2013 ; Vadez et al. 2014 ), to forest ecology (e.g. Linares and Camarero 2012 ; Lévesque et al. 2014 ) and in the context of global climate change (Cernusak et al. 2019 ). This ratio can be defined at various levels, from the physiological functioning of a leaf to the whole plant and at the ecosystem level. This historical review starts at the whole plant level, where WUE can be simply measured by quantifying the amount of water given to a plant and the plant’s increase in biomass during the experiment. The ratio of biomass produced divided by the cumulative water lost during growth is termed whole plant transpiration efficiency (TE= biomass produced/water lost). Historically, the ratio has also been calculated in its inverted form (water lost/biomass produced) and various terms have been used to denote these ratios (see Box 1). As knowledge, concepts and technology advanced, it became desirable to measure TE also at the leaf level, where it is defined either as the ratio of net CO 2 assimilation rate to transpiration (or to the stomatal conductance for water vapour). Therefore, some history of the two leaf-level components of WUE is included here. Numerous articles have been published on the history of the development of research on photosynthesis, and other than the reviews cited in this article, the publications by Govindjee are notable, especially Govindjee and Krogmann ( 2004 ), as they include a long list of other writings on the history of photosynthesis. On the other hand, little has been written about the history of research on transpiration and stomatal conductance. Notable is Brown ( 2013 ), who wrote specifically on the cohesion-tension theory of the rise of sap in trees, including many writings from the late nineteenth century. Consequently, here, photosynthesis research is only broached briefly, whereas transpiration research is more detailed.

As the development of the research on WUE spans a very long period, starting with Greek philosophers, publications are in several languages. Classical writings were in Greek or in Latin, and for these translations are available. However, from the mid-seventeenth century onwards, national languages were more and more used, which can be seen in the number of French- and German-language publications. This review is also a tribute to these nowadays less known seventeenth, eighteenth and nineteenth century French and German natural philosophers and their contribution to the development of the science of plant ecophysiology. Also, towards the beginning of the twentieth century, publications became too numerous to allow a comprehensive review; thus, the author focussed on the use of the carbon stable isotopes methodology and on tree ecology.

Box 1 Short history of names for whole plant transpiration efficiency (TE)

2 What is plant matter made of?

Various Greek philosophers were interested in how substances can change from one thing into another. Thales (624–c. 546 BC) thought that all things come from water, whereas Anaximenes argued that “pneuma” (air) should be the basis of all things (Egerton 2001a ). These assertions were the basis of more than 2000 years of philosophical dispute.

In “De Causis Plantarum”, Theophrastos (371–287 BC) assumed that plants draw nutrition, which consisted of varying amounts of the four elementary humours, from the earth through their roots (Morton 1981 ). Some centuries later, in a Christian work translated in 400 AD from Greek into Latin and known as “Pseudo-Clement’s Recognitions”, an apparent thought experiment was described to “prove that nothing is supplied to seeds from the substance of the earth, but that they are entirely derived from the element of water and the spirit (spiritus) that is in it” (Egerton 2004c ). The author of this thought experiment suggested putting earth into big barrels, growing herbaceous plants in it for several years, then harvesting them and weighing them. His hypothesis was that the weight of the earth would not have changed, and the author used this as an argument that the vegetation biomass could have come only from water. This thought experiment revealed a progress in scientific thinking because the question was posed more precisely than before. It stood out at a time when botany mainly consisted of naming plants and “theoretical botany effectually went out of existence” (Morton 1981 ).

It appears that the question of how plant matter is produced was not pursued in Roman or Arabic writings, which were more concerned with agricultural (the former) and medical (the latter) aspects of plant sciences (Egerton 2001b , 2002 ). Not until the High Middle Ages was a renewed interest shown in plant growth. Adelard of Bath, a twelfth century English natural philosopher, devoted the first four chapters of “Questiones Naturales” (c. 1130–1140; Morton 1981 ) to the question of what plant matter is made of. He argued, within the concepts of the four elements theory, “by just as much as water differs from earth, by so much does it afford less nourishment to roots, I mean than earth does”, clearly being in favour of earth as the source for plant nourishment. His arguments were only theoretical and speculative.

A major step occurred in botanical sciences between the fifteenth and sixteenth centuries; scholars began making experiments to test antique and medieval hypotheses against observations in nature (Egerton 2003 ). In the mid-fifteenth century, and probably related to the translation and printing of the botanical books by Theophrastus (Morton 1981 ), the thought experiment from “Recognitions...” was taken up by Nicholas of Cusa in the fourth part of his “Idiota de mente”, “De staticis experiments”. At a time when the naming of plants for pharmacology was the major interest of savants, he proposed experimental investigations. Nicholas of Cusa described the same thought experiment as did Pseudo-Clement’s Recognitions ; he concluded similarly that “the collected herbs have weight mainly from water” (1450; translation into English by Hopkins 1996 ). Cusa additionally suggested that the plants should be burned at the end of the experiment and the ash weight be taken into account. It is not clear whether the thought experiment was ever physically done.

In the sixteenth century, botanical science began to separate from medical sciences, with the establishment of lectureships in universities (e.g. Padua in 1533) and the establishment of botanical gardens (Egerton 2003 ). The bases existed for advancing science in the seventeenth century of Enlightenment. Francis Bacon, an influential philosopher of his time, conducted a series of plant growth experiments which are reported in his “de Augmentis Scientiarum” (1623; Spedding et al. 1900 ). Bacon discovered that some plants sprouted more quickly in water than in soil (Egerton 2004b ). He concluded that “for nourishment the water is almost all in all, and that the earth doth but keep the plant upright, and save it from over-heat and over-cold” (Hershey 2003 ), thus still upholding the theory proposed by Thales and Nicholas of Cusa. In “The History of the Propagation and Improvement of Vegetable”, Robert Sharrock ( 1660 ) reported that some plants both rooted and grew entirely in water. Although he noted different amounts of transpiration over time, he did not discuss this in relation to plant growth.

In 1662, Johannes Baptista van Helmont published his now-famous willow experiments (van Helmont 1662 ). This may be the first report of an experiment that was based on the thought experiment of Nicholas of Cusa (Hershey 2003 ) with the minor differences of beginning with dried soil and not using herbaceous plants, but rather a willow tree. After weighing the soil, he irrigated it with rain water and planted the weighed stem of a willow tree. The experiment ran for 5 years. At the end, the tree was weighed again, as was the dried soil. He found the soil weighed about 2 ounces less than at the beginning of the experiment, whereas 164 pounds of wood, bark and roots was produced. He concluded that the organic matter could only have come out of the water. Helmont was unaware of the existence of carbon dioxide, but he did know of “gas sylvestre”. He also knew that burning oak charcoal would produce nearly the same amount of gas sylvestre and ash. However, he did not connect this information with the plant growth he had observed (Hershey 2003 ). Robert Boyle published similar experiments in “The sceptical Chymist” (Boyle 1661 ). Boyle claimed that he had done his experiments before he knew of Helmont’s (Egerton 2004c ), although he discussed Helmont’s results and arguments in detail in his book. Boyle doubted the direct transformation of water into plant matter. He admitted, however, that it might be possible that other substances contained in the water could generate new matter (Boyle 1661 ). In the 1660’s, Edme Mariotte also criticised van Helmont’s theory that water alone constituted the only element to produce plant matter. He thought similarly to Boyle that elements in the water could contribute to the plant matter. He also showed that nitrogen compounds were important for plant growth (Bugler 1950 ).

John Woodward, in his “Some Thoughts and Experiments Concerning Vegetation” (Woodward 1699 ), took up again the question of what comprised the source of plant growth. Woodward criticised Helmont’s and Boyle’s experiments, mainly on the precision of weighing the dry soil before and after the experiment, but also the contamination of the irrigation water by terrestrial vegetable or mineral matter. Consequently, he developed a series of hydroponics experiments, where by growing plants in sealed vials, in different types of water and weighing them regularly over the same time period, he could calculate how much biomass was gained over a set time period. He was able to draw a series of conclusions from these experiments by calculating the ratio of water lost to plant mass gained in the same period of time, thereby calculating the inverse of transpiration efficiency. This was probably the first time that the inverse of transpiration efficiency was calculated using experimental data. He showed that 50 to 700 times as much water was lost than biomass gained. He also reported that plants grown in water containing more terrestrial matter grew more and with less water consumed. From these observations, he concluded that water serves only as a vehicle for the terrestrial matter that forms vegetables and that vegetable matter is not formed out of water. He is still remembered more for his geological publications (Porter 1979 ) than for his contributions to botany (Stanhill 1986 ).

In his “history of ecology” series, Egerton ( 2004c ) nicely sums this period thusly: “each of these authors (Bacon, Boyle, Helmont, Sharrock) built upon the work of his predecessors and improved somewhat the understanding of plant growth and how to study it. However, they still fell short of a basic understanding of plant growth. Before that could be achieved, chemists would have to identify the gases in the air”. This series of studies shows that from the end of the seventeenth century onwards, experiments replaced speculation (Morton 1981 ), in botany as well as in many other areas of science.

From the end of the seventeenth century, the question of how plants grow was still unresolved, although it was known that nutrients were conducted from the roots in the ascending sap to the leaves. A major improvement in the understanding of how transpiration and its variations work was the discovery of cells by Robert Hooke towards the middle of the seventeenth century (Egerton 2005 ) and subsequently the discovery of stomata on leaf surfaces. One of the first to describe stomata may have been Malpighi in “Anatomy of Plants” (Malpighi 1675 in Möbius 1901 ). Based on Malpighi’s and Grew’s ( 1682 ) studies, John Ray suggested in “Historia Plantarum” (Ray 1686 in Lazenby 1995 ) that the apertures in the leaves, when open, would give off either breath or liquid. Ray may have been the first to have connected stomata with transpiration. He also suggested that the loss of water by evaporation is compensated constantly by water from the stem, and thus transpiration results from a constant water flux. He also observed that sap ascends the stems of trees in sap-bearing vessels which do not contain valves. He did, however, admit that it cannot be capillary forces that make water go up tall trees.

Ideas on photosynthesis developed slowly from the middle of the seventeenth century onwards. Malpighi ( 1675 ) suggested that leaves produce (“concoct and prepare”) the food of plants and from leaves this food passes to all parts of the plant. Similarly, Claude Perrault in “Essais de Physique” (Perrault 1680 ) defended the hypothesis that the root acts as the mouth of the plant and that the leaves serve to prepare the food arriving with the sap from the root so that it can be used in the rest of the plant. John Ray in “History Plantarum” (Ray 1686 in Lazenby 1995 ) concurs with this, however adding in “The wisdom of God” (Ray 1691 in Lazenby 1995 ) that “not only that which ascends from the Root, but that which they take in from without, from the Dew, moist Air, and Rain”. He also thought that light could play a role in this preparation of the plant sap. At this time, most authors (Malpighi, Perrault, Mariotte, Ray) knew about the circulation of sap, up as well as down, and that leaves served somehow to transform the upcoming sap into food for the plant.

In 1770 , Lavoisier published “Sur la nature de l’eau” (“On the nature of water”, translation by the author) and reviewed the literature on the possibility of water changing into earth to nourish plants. Lavoisier cited the Van Helmont experiment and later works which tested Van Helmont’s idea by growing plants in water (e.g. Boyle, however he did not cite Woodward). He was critical of the idea that it could be a transformation of water that would constitute plant material. This was based mainly on experiments by himself and others, showing even distilled water would contain traces of “soil”. However, he also defended the idea, based mainly on Charles Bonnet’s observations, that leaves absorb vapours from the atmosphere that contribute to plant growth.

Helmont had coined the term “gaz” in the mid-seventeenth century and had been able to distinguish different gazes from air (Egerton 2004a ). It was only in the middle of the eighteenth century that gases were studied in the laboratory and several observations by different researchers would finally lead to an understanding of respiration and photosynthesis (Tomic et al. 2005 ; Nickelsen 2007 ). Richard Bradley seems to be one of the first to clearly state (in letters from 1721 to 1724) that plant nourishment can be drawn from the air. Hales ( 1727 ) agreed with this theory, which was not yet widely accepted (Morton 1981 ), and suggested that light might be involved, which helped to pave the way for the discovery of photosynthesis. Black ( 1756 ) was able to identify carbon dioxide (which he called fixed air) using a lime water precipitation test. He demonstrated that this “fixed air” did not support animal life or a candle flame (Egerton 2008 ). Charles Bonnet ( 1754 ) made an important observation, i.e. branches with leaves that were submerged under water would produce air bubbles on their surfaces when sunlight shone on them, but not after sunset. Senebier refined these experiments in 1781 (Morton 1981 ), by showing that the leaves produced no oxygen in the sunlight when the surrounding water was free of carbon dioxide and that the rate of oxygen production was higher with carbon dioxide-saturated water. Tomic et al. ( 2005 ) present nicely the steps leading up to the term photosynthesis. This began with Priestley ( 1775 ) demonstrating that the air given off by animals and by plants was not the same, Ingen-Housz ( 1779 ) observed the important role of light, and the dispute between Senebier and Ingen-Housz from 1783 to 1789 resolved more clearly the functions of carbon dioxide emission (respiration) and absorption (photosynthesis). Based on these results and his own very detailed observations, de Saussure reported in 1804 that the carbon necessary for plant growth is absorbed mainly by green leaves from atmospheric carbon dioxide and he estimated that the largest part of the accumulated dry matter of plants is made of this carbon. Thus, the dispute of what the plant matter is made of that began in antique Greece was resolved at the end of the eighteenth century.

3 How much water do plants need to grow?

The late eighteenth century marked the beginning of applied agricultural science and the rise of plant physiology (Morton 1981 ). Work continued on transpiration and stomata, with a large number of experiments. Burgerstein ( 1887 , 1889 ) managed to assemble 236 publications on transpiration of plants from 1672 to 1886, citing short abstracts of each and comparing them critically. Also, Unger published in 1862 a major review article covering such subjects as the relationship of transpiration to temperature and humidity; daily cycles, including night; differences in adaxial and abaxial leaf surfaces; the impact on transpiration of type, number, size and distribution of stomata; the structure of the epidermis (cell layers, cuticle, hairs and wax); development of the mesophyll; size of intercellular spaces and cell turgor; and the impact of plant transpiration on the atmosphere (Unger 1862 in Burgerstein 1887 ). Scientists started to reflect on the interaction of plants, or more specifically their leaves, with their environment, and experimentation included the responses of stomata to light quantity (Möldenhawer 1812 ) and quality (Daubeny 1836 in Burgerstein 1887 ). Based on inconsistent observations by e.g. Banks, Möldenhawer and Amici, advances were also made on the functioning of stomata (Mohl 1856 ). However, progress was mainly based on a comment in von Schleiden ( 1849 ) that the state of the stomata would be the result of the water in- or outflow of the pore cells (called “Schliesszellen”) and he showed experimentally that stomata close when the pore cells lose water. As knowledge of transpiration, stomatal opening and their dependence on environmental variables increased, new questions arose about the water consumption of plants.

Another milestone along the way to understanding the transpiration of plants in the nineteenth century was the publication by Sir John Bennet Lawes ( 1850 ), “Experimental investigation into the amount of water given off by plants during their growth; especially in relation to the fixation and source of their various constituents”. He described experiments on wheat, barley, beans, peas and clover using differently fertilised soils. He was using plants in closed containers and an especially designed balance to “estimate the amounts of water given off” (Fig. 1 ). He observed increased evapotranspiration with higher temperatures during the growing season, and asked whether “this increased passage of water through the plants, carrying with it in its course many important materials of growth from the soil, and probably also influencing the changes in the leaves of these, as well as of those derived from the atmosphere, will not be accompanied with an equivalently increased growth and development of the substance of the plant”. This was followed by an important discussion of the influence of temperature on evaporation and growth as well as the resultant ratio. He discussed in the introduction “the relationship of the water given off to the matter fixed in the plants”; he gave his results in this ratio and in the inverse ratio, and applied these ratios to different scientific questions. The first ratio (transpired water divided by plant matter, the inverse of today’s TE) was used to interpret his results in terms of water use compared to field available water, and the latter’s ratio (plant matter divided by transpired water, equivalent to today’s TE) was used to discuss his results in terms of functional differences among species. From the observed functional differences, he concluded that there was “some definite relationship between the passage of water through the plants and the fixation in it of some of its constituents”. He was, thereby, introducing a new question about the link between dry matter accumulation and transpiration, which will be treated in the next chapter.

Illustration from Lawes ( 1850 , p. 43) of the special balance constructed for weighing plants in their “jars” to estimate the amounts of water given off and also the “truck” on which a series of jars was moved to the balance

Towards the end of the nineteenth century, research interest started to include agricultural questions of water use. Marié-Davy ( 1869 ) measured transpiration (standardised by leaf surface) of over 30 plant species, including eight tree or shrub species as well as herbaceous and agricultural plants. He estimated transpiration per soil area, thereby establishing that a prairie would transpire more than trees. von Höhnel ( 1879 ) estimated long-term transpiration of branches of 15 tree species (standardised on leaf surface or leaf dry weight). He used these data of branch transpiration to upscale to whole trees and concluded that compared to agricultural plants, the amount of rain seemed sufficient for tree growth. Hellriegel ( 1871 ) had already similarly concluded for cereals in the Mark Brandenburg (Germany) region that rainfall would not be sufficient, as had Marié-Davy ( 1874 ) for wheat in the Paris (France) region. In parallel with these more quantitative interrogations about water use, from the mid-nineteenth century, scientists started to ask more functional questions about the relationship between transpiration and dry matter accumulation, in a context of vigorous growth of botanical sciences and the complex relation between organisms and their environment (Morton 1981 ).

4 Are transpiration and dry matter accumulation linked?

Lawes ( 1850 ) had already reflected on a functional relationship between water flux and plant matter accumulation. In the following years, there were several publications on the transpiration of trees, and although no transpiration efficiency was estimated, the understanding of tree transpiration advanced. Many comparative studies were published. Lawes ( 1851 ) on “Comparative evaporating properties of evergreen and deciduous trees” considered twelve different tree species. He provided measurements of the variation in transpiration with temperature and hygrometry data. With these, he concluded that “evaporation is not a mere index of temperature but that it depends on vitality influenced by heat, light and other causes”. In the late nineteenth century, several researchers estimated and compared values of the ratio of transpiration and dry matter accumulation for different plants (Burgerstein 1887 ). With the growing evidence of variation in this ratio, scientists started to reflect on the relationship between transpiration and dry matter accumulation, aided by the development of new measurement techniques. A major question was if there would be a tight coupling between transpiration and dry matter accumulation, resulting in a constant transpiration efficiency, or if variation could be observed.

Dehérain ( 1869 ) studied evaporation and the decomposition of carbonic acid in leaves of wheat and barley. Using an ingenious apparatus, he was probably the first to directly measure evaporation of water in parallel with carbonic acid decomposition. He studied the effect of variously coloured light, and although he did not calculate the ratio between evaporation and carbonic acid decomposition, he did conclude that light of different colours had a similar effect on carbonic acid decomposition and on water evaporation from the leaves. His final conclusion was that “it is likely that there is existing between the two main functions of plants, evaporation and carbonic acid decomposition, a link, of which we need to determine its nature” (translation from the original French by the author). Several other scientists also commented on the relationship between transpiration and dry matter production. Fittbogen ( 1871 ) supposed, similarly to Lawes ( 1850 ) before him, but with more experimental evidence, that there should be a positive relationship between transpiration and production of dry matter. Dietrich ( 1872 in Burgerstein 1887 ) supposed that this relationship would be linear, whereas Tschaplowitz ( 1878 in Burgerstein 1887 ) introduced the idea that there should be an optimum transpiration at the maximum production of matter. Therefore, when the transpiration would increase over this optimum, this would lead to a decrease in assimilation rate. He was one of the first to suggest a non-linear relationship between transpiration and assimilation. Sorauer in “Studies on evaporation” ( 1880 ) defended the hypothesis that transpiration was not only a physical phenomenon but was also physiological. He stated that “It is not possible as yet to study the plant internal processes which regulate the transpiration, however it is possible to quantify the relationship between dry-matter and transpiration” (translation from German by the author), suggesting thereby TE as a means to advance the understanding of plant internal processes. Sorauer was probably at the cutting edge of science of his time. He pointed out specifically that variability among plants of one species was due to genetics (German, “erbliche Anlagen”), a startling and even daring assertion for his time. He asserted that for comparative studies, genetic variability needed to be minimised. To achieve this, he used, when possible, seeds from the same mother plant, grown in the same environmental conditions and a large number of repetitions. Using these protocols, he was probably one of the first to estimate TE on tree seedlings, showing that there was within species diversity in transpiration and growth, but that their ratio was more constant. He concluded from experiments on pear and apple trees that the pear trees used less water for the same biomass growth. He was able to go one step further and demonstrate that this difference was due to less transpiration per leaf area. By comparing different woody and herbaceous plants with different growth types, he postulated that when plants had a small leaf area combined with high transpiration, they had either a very strong growth increment, a high dry matter percentage, or a large root system. Overall, he observed relationships between dry matter production and transpiration; he concluded that there must be some regulation of the transpiration per unit leaf area by the co-occurring dry matter production.

Hellriegel ( 1883 ) argued that one cannot estimate a constant ratio between transpiration and production as there were factors which influence each independently. He also commented that it might make sense to estimate mean values of transpiration for various agricultural plants, as this would be for practical and scientific value. He thought that the most logical standardisation would be by the mass of the dry matter produced during the same time period. He called this “relative Verdunstungsgrösse” which can be translated into English as “relative transpiration”. He was probably one of the first to give a name to the ratio between whole plant transpiration and dry matter production. He proposed a theory that for a long-term drought, plants would acclimate their morphology to decrease their “relative transpiration”. He provided additional experimental evidence that barley had decreased in relative transpiration over as many as seven levels of soil water deficit, relative to field capacity. Using his own observations, he proposed that when calculating a mean “relative transpiration” for a single species, variation of transpiration should be minimised and that plants should be tested together only under optimal conditions. Given the relatively small differences in relative transpiration that he observed among different crops, Hellriegel suggested that these differences would not explain why some crops grow better in wet locations and others on dry locations. Hellriegel was thus probably one of the first scientists to point out that the relationship between drought adaptation and “relative transpiration” might not be straightforward.

Understanding how biomass and water loss were connected was studied by Iljin ( 1916 ) on a newly detailed level. He measured simultaneously water loss and carbon dioxide decomposition and reported his data as grammes of water lost per cubic centimetre of carbon dioxide decomposed. He concluded from studying more than 20 plant species that “...it is generally agreed that the rates of water loss and of CO 2 assimilation are directly proportionate to stomatal aperture, and that consequently there exists a close connection between these two processes”.

At the end of the nineteenth century, the ratio of transpiration versus dry matter accumulation was recognised as an important plant trait, which varies among and within species in a complex interaction of each component with the other and with environmental factors.

5 How do plants differ in water requirement and how do they respond to variations in environmental factors?

In the late nineteenth century, several researchers estimated and compared values of the ratio of transpiration and dry matter accumulation for a range of cultivated plants (Fittbogen 1871 ; Dietrich 1872 ; Farsky 1877 , cited in Burgerstein 1887 ), giving evidence of the growing interest of agricultural scientists. The number of studies of transpiration efficiency greatly increased, thereby driving a new standardisation in terminology. King ( 1889 ) studied the inverse of transpiration efficiency and described it as “the amount of water required for a ton of dry matter”, and promulgated this terminology by using it in the titles of his publications between 1892 and 1895. Similarly, Leather ( 1910 ) published “Water requirements of the crops of India”, in which he defined the “transpiration ratio” as “the water transpired to the weight of dry plant produced”. The shift from a purely descriptive use of “water requirement” to a clearly defined one was provided by Kearney and Shantz ( 1911 ) as “… the degree to which a plant is economical in its use of water is expressed in its water requirement, or the total quantity of water which it expends in producing a pound of dry matter”. The term “water requirement” is the inverse of the modern transpiration efficiency, and was used by a rapidly increasing number of publications which were published on the water use of crops in the early twentieth century. Montgomery ( 1911 ) may have been the first to use the term for a plant trait in “Methods of determining the water requirements of crops”.

At the beginning of the twentieth century, the importance of gaining knowledge on the water requirements of plants can be seen in the technical effort that went into the measuring equipment. von Seelhorst ( 1902 ) presented a system of growing boxes on rails, placed belowground, including the balance, so that the top of the growing boxes was at the same level as the surrounding soil (Fig. 2 ). In the now well-known studies on “The water requirement of plants. I. Investigations in the Great Plains in 1910 and 1911”, Briggs and Shantz ( 1913a ) measured the water requirement for 21 crop and weed species, sometimes for different varieties of the same crop and under controlled and field conditions. In the same year, they reviewed the available literature on water requirement (Briggs and Shantz 1913b ), increasing their dataset to 31 different crop species. They discussed in detail studies from 29 different authors, many of which had only published once or twice on this subject. A few researchers were notable for their number of publications on the water requirement of crop plants: King with 6 publications between 1889 and 1905, and von Seelhorst with 9 publications between 1899 and 1907. The largest contributions came from Hellriegel ( 1883 ; 10 species) and Leather ( 1911 ; 15 species). Kiesselbach ( 1916 ) also reviewed 59 publications from 1850 to 1915 “which had studied transpiration in relation to crop yield, based upon plants grown beyond the seedling stage”. There were regular publications of original work from 1870s onwards, with more than one publication per year from 1890 onwards. The difference among species and the impact of environmental factors on water requirement was one of the main questions raised. These reviews and the increasing amount of newly published work per year are evidence of the growing interest in the “water requirement” of plants as a trait of increasing importance in agricultural sciences.

Illustration from von Seelhorst ( 1902 ), showing the quite sophisticated outdoor installation “Vegetationskasten” (growing boxes, translations by the author) to weigh plants in small waggons, with a “Kastenwagen” (boxwaggon), b “Waagebalken” (scale beam), c “Deckbretter” (cover board) and d “Waagentisch” (weighing table)

With regard to species differences in water requirement among crops, Schröder ( 1895 , cited in Maximov 1929 ) found two groups, among seven cereals, which differed in water requirement by a factor of 2. Millet, sorghum and maize were known to be drought resistant, and showed a lower water requirement than the remaining plants. These differences were confirmed by Kolkunov ( 1905 , cited in Maximov 1929 ), Briggs and Shantz ( 1914 ), Briggs and Shantz ( 1917 ) and Shantz ( 1927 ). Millet, sorghum and maize are now known to use the C4 carbon pathway of photosynthesis.

With regard to external environmental influences on plants, Briggs and Shantz ( 1913b ) distinguished between soil, atmosphere and plant factors. Soil factors which were investigated were soil moisture content, soil type, cultivation, soil volume, soil temperature, effect of fertilisers in soil or water cultures and effect of previous crops. Weather factors considered were air temperature and humidity, shade and carbon dioxide content. Other factors studied in direct relationship to the plants were parasite attacks, relative leaf area, cutting frequency, defoliation, planting density and the age of plants.

A critique of the term “water requirement” was not long in coming. Dachnowski ( 1914 ) wrote, “It is assumed by many writers that a definite and quantitative relation exists between transpiration and growth, and that hence the ratio of the weight of water absorbed and transpired by a plant during its growth to the green or dry substance produced is an adequate and simple measure of growth.”, followed by an argument why this was not the case.

6 Why do plants differ in transpiration efficiency?

The adaptations of plants to dry environments were an important ecological topic at the beginning of the twentieth century, as the discipline of “physiological ecology” (Iljin 1916 ; Moore 1924 ) began to develop. Iljin ( 1916 ) studied more than 20 different plant species in situ from different ecological locations, e.g. wet bottom soils and variously facing slopes of ravines with different aspects. Iljin proposed that “the water requirements of the different species should be very different, and consequently the amounts of water available should differently affect their processes of life”. Using his observations, he was able to show that “… in no case was the water loss per unit of decomposed CO 2 found to be equal to or more in xerophytes than in mesophytes”, thus suggesting a higher transpiration efficiency. He argued that mesophytes would have to close stomata “… in dry places in order to reduce evaporation, thus diminishing the rate of assimilation as well, whereas in the case of xerophytes, which are adapted to extreme conditions of existence, assimilation in similar circumstances proceeds actively”. He then tried to confirm his hypothesis by transplanting mesophytes from wetter sites to the drier environment of xerophytes. Iljin showed experimentally that in all cases, a higher water requirement was measured for mesophytes transferred to a drier site compared to their original site and compared to xerophytes at the dry site. He interpreted his observations as “plants growing in dry places are adapted to a more economical consumption of water”. He held this to be true for among- and within-species variation.

A milestone in forest “physiological ecology” was Bates’ ( 1923 ) study of the physiological requirements of Rocky Mountain trees. Bates wrote that for foresters, knowledge of demands of tree seedlings for moisture, light, heat and soil fertility was important for planning reforestation. He started a large investigation of six forest tree species, combining field studies to describe ecosystems, with experiments in controlled environments in order to determine species differences in relative transpiration and other water flow-related traits. Bates concluded from the comparison among species that trees of low water requirement would be trees that have a superior control over their water supply. He was however critical of a direct relationship between water requirement and drought resistance in trees. Moore ( 1924 ) commented that in correlating physiological measurements with the habitat characterisation of the species, Bates “... has opened new fields to forest investigations”. He also stressed that the results were counterintuitive in that the most xerophytic species had the highest water requirement, whereas the most mesophytic species had the lowest water requirement.

A similar discrepancy was observed by Maximov ( 1929 ) in the chapter “Efficiency of transpiration” in his book The Plant in relation to water , which was translated from Russian into English rapidly after its publication. Maximov preferred “efficiency of transpiration” to “water requirement”, arguing that the former would be more logically correct, because the determining process (transpiration) should be in the denominator, which also would have the effect that “… an increase in the figure denoting the value of the ratio actually corresponds to an increase of the efficiency per unit of water used”.

In his book, Maximov ( 1929 ) described experiments done at Tiflis Botanic garden (today in Georgia) by Maximov and Alexandrov ( 1917 ), where they studied local xerophytes for 3 years. They found xerophytes with a high efficiency of transpiration, particularly drought-resistant annuals. They also found that plants with a low efficiency of transpiration appeared to be the most typical semi-arid xerophytes. The mesophytes all displayed a medium efficiency. Maximov noted from other observations on the same plants that the “… majority of xerophytes with a low efficiency of water expenditure possess very extensive root systems, far exceeding in length the sub-aerial portions of the plant”. He also observed that these plants showed a strong transpiration and that this transpiration might constitute the “pump” which could draw water through such an extensive root system. He also observed that “members of the group of annual xerophytes with a high efficiency of transpiration are characterised by a relatively large leaf surface, which develops very rapidly”. He argued that this would confer a high intensity of assimilation. From these observations, he concluded a “lack of direct proportionality between efficiency of transpiration and the degree of drought resistance”, but also that “the magnitude of the efficiency of transpiration affords one of the most satisfactory tests of the ecological status of a plant”. Maximov applied the ecological classification developed by Kearney and Shantz ( 1911 ), which they had based on plants of the arid and semi-arid regions of North America: (1) drought-escaping with an annual growth cycle restricted to favourable conditions; (2) drought-evading, delay by various means the exhaustion of soil moisture; (3) drought-enduring, can wilt or dry but remains alive; and (4) drought-resisting, can store a water supply. It should be noted that the ecological definitions behind these concepts have changed with time and are used slightly differently today. Shantz ( 1927 ) argued that many of the drought-evading plants had a low water requirement and Maximov noted that this group included the highly efficient xerophytes with a large leaf area. Maximov also observed that xerophytes from the third group (drought-enduring) could show a very low efficiency of transpiration and belonged to the group of xerophytes with large root systems. Without concluding directly, he suggested a relationship between the transpiration efficiency of a xerophyte and its ecological strategy when facing limited soil water content. These studies by Maximov are among the most complete concerning the relationship between a plants’ resistance to drought and their transpiration efficiency, reflecting the interest of scientists in ecological questions of plant functioning, especially in relation to drought.

Although work on crop plants advanced greatly in the early twentieth century, results were scarcer for tree species. Raber ( 1937 ) concluded his book on “Water utilization by trees, with special reference to the economic forest species of the north temperate zone” with detailed discussions of available data for forest trees. He commented that “much more work on the water requirements of trees of all ages and under varying site conditions is needed”. And he continued that “In view of the importance of planting drought-resistant species in regions where the water supply is below the optimum for most tree species, it is extremely urgent to know more about what qualities make for drought resistance and what species possess these qualities to the greater degree.” These conclusions by Raber show that from the beginning of the twentieth century, the estimation of transpiration efficiency had taken an important place in ecological studies on forest tree species, however not without some critical thoughts on the subject.

7 What is the functional importance of transpiration?

Already in the 1870s and 1880s, the role of stomata in the diffusion of carbon dioxide into the leaf (during the day) and out of the leaf (during the night) was discussed in the scientific literature, as shown by the extensive literature review by Blackman ( 1895 ) (see also section 4 above). Especially the functional importance of transpiration was an open question. There were two opposing lines of thought. As summarised by Iljin ( 1916 ), one defended the line of inquiry that transpiration was important only in the process of transporting mineral salts from roots to leaves; the other held that the opening of stomata was necessary for absorbing the carbonic acid from the atmosphere, which leads to a loss of water and is described as an “inevitable evil”. Iljin ( 1916 ) preferred the second line of investigation and attributed a major role to the stomatal aperture, which controlled both the absorption of carbonic acid from the atmosphere and the loss of water. He concluded that in “physiologico-ecological” investigations, assimilation should be studied together with transpiration. Maskell published a series of papers in 1928, where especially “XVIII.—The relation between stomatal opening and assimilation.” (Maskell and Blackman 1928 ) used an apparatus to estimate apparent CO 2 assimilation and transpiration rate in parallel (Fig. 3 ), and was therefore able to study in detail their interdependence, developing the first mathematical descriptions, based on the development of the theories about the diffusion of gases (Brown and Escombe 1900 ). Methodological advances intensified research on the leaf-level relationship between assimilation and transpiration and allowed the study of plant functioning in more detail. The major step forward was the construction of an infrared gas analyser (URAS: in German “Ultrarotabsorptionsschreiber”, IRGA, infrared gas analyser) by Lehrer and Luft in 1938 (Luft 1943 ) at a laboratory of BASF, IG Farbenindustrie. Normally used in industry and mining, Egle and Ernst ( 1949 ) may have been the first to describe the use of the URAS for plant physiological measurements. By 1959, the URAS was routinely used for measuring stomatal resistance or transpiration in parallel and simultaneously with CO 2 assimilation, on the same leaf (Rüsch 1959 ). This was a great improvement on previous methods and led rapidly to a set of equations for calculating assimilation and stomatal conductance (Gaastra 1959 ).

Two figures taken from Maskell and Blackman ( 1928 ): on the top, Figure 1 (p. 489) showing a “Combined assimilation chamber and porometer for simultaneous investigation of apparent assimilation and stomatal behaviour. A. Section of leaf chamber passing through porometer chamber. B. Back view of leaf chamber showing also air-flow meter attached by pressure tubing to porometer and to leaf chamber”. On the bottom, Figure 5 (p. 497) “Relation between porometer rate and apparent assimilation at ‘high’ light, December 1920.” Exp t LI and LII correspond to 2 days of continuous measurements to what Maskell called “diurnal march”

Scarth ( 1927 ) argued that there would be little advantage for a plant to have a high rate of transpiration, but stressed the “... advantage of maintaining the fullest diffusive capacity of the stomata and the highest possible pressure of CO 2 in the intercellular spaces”. He concluded that the principal function of stomata “... is to regulate that very factor which is presumed to regulate them, viz. the concentration of CO 2 in the leaf or, respectively, in the guard cells”. Maskell and Blackman ( 1928 ) tested this hypothesis experimentally and concluded that the rate of uptake of carbon dioxide was determined by variations in stomatal resistance and by resistances within the leaf, thereby introducing the importance of the CO 2 concentrations in the chloroplasts. The suggestion of a strong link between the leaf internal carbon dioxide concentration and leaf-level WUE represented a large advance in the theoretical understanding of WUE.

Penman and Schofield ( 1951 ) proposed, perhaps, the first theoretical link between the leaf-level transpiration ratio (leaf transpiration divided by assimilation) and the ratio of the coefficients of diffusion of water vapour and carbon dioxide in air, and the water vapour and carbon dioxide air-to-leaf pressure gradients. Gaastra ( 1959 ) suggested that the leaf internal conductance to carbon dioxide is a pivotal point of the ratio of assimilation to transpiration and of the water economy of crop plants. Bierhuizen and Slatyer ( 1965 ) showed that the transpiration ratio could be predicted from water vapour and carbon dioxide gradients over a range of light intensities, temperatures and relative humidities. These studies were the first to suggest that whole plant transpiration efficiency might be regulated directly by leaf functioning and would be therefore a trait in its own right and not only the ratio of two plant traits.

8 How can the transpiration ratio be improved?

Because water is increasingly scarce in a warming world, Rüsch ( 1959 ) queried whether the luxury of highly transpiring tree species could be justified. He argued for selective breeding of tree species varieties with low transpiration-to-assimilation ratio T/A by means of minimising transpiration while maximising assimilation. Also Polster et al. ( 1960 ) assessed the potential suitability of tree species to sites by their dry matter production and transpiration ratio. Troughton ( 1969 ) and Cowan and Troughton ( 1971 ) suggested that genetic selection of plant varieties could be used to improve the transpiration ratio by decreasing leaf internal resistance to carbon dioxide diffusion. Cowan and Farquhar ( 1977 ) built on this theme by proposing that stomata might optimise carbon gain to water lost by varying the conductances to diffusion and thereby maximising the ratio of the mean assimilation rate to mean rate of evaporation in a fluctuating environment. Approaches which target photosynthesis, stomatal opening, leaf internal resistance to carbon dioxide diffusion or stomatal optimisation in order to improve plants performance have since been followed in plant breeding and have largely been reviewed elsewhere (e.g. Condon et al. 2004 ; Cregg 2004 ; Vadez et al. 2014 ).

9 Intrinsic water use efficiency and carbon stable isotopes

Another milestone towards contemporary research on water use efficiency was the use of stomatal conductance to water vapour rather than transpiration by Farquhar and Rashke ( 1978 ) and to calculate water use efficiency as assimilation divided by stomatal conductance. This definition allowed an estimation of water use efficiency resulting only from plant functioning, without a direct impact from leaf-to-air vapour pressure difference and was named by Meinzer et al. ( 1991 ) “intrinsic water use efficiency” (W i ). Knowledge of W i facilitated the search for a genetic basis of within species variation, e.g. Brendel et al. ( 2002 ), Condon et al. ( 2002 ) and Chen et al. ( 2011 ).

Development of the stable carbon isotope method for estimating W i resulted in a widely applicable screening method, and a large increase of publications around plant water use efficiency. Based on the two-step fractionation model (atmospheric CO 2 – leaf internal CO 2 – plant carbon) proposed by Park and Epstein ( 1960 ), various models explaining the difference in carbon isotope composition between atmospheric CO 2 and plant carbon were developed in the late 1970s and early 1980s, e.g. Grinsted ( 1977 ), Schmidt and Winkler ( 1979 ) and Vogel ( 1980 ). Vogel’s model contained many theoretical aspects which, however, lacked experimental understanding. In parallel, Farquhar ( 1980 ) developed a similar model, but which resulted in a simple, elegant mathematical equation relating plant natural abundance carbon isotope discrimination, relative to atmosphere, to the ratio of leaf internal to atmospheric CO 2 concentration. This was, in turn, related to W i . Experimental evidence showed that carbon isotope measurements, in wheat, reflected long-term water use efficiency (Farquhar et al. 1982 ) as well as whole plant transpiration efficiency (Farquhar and Richards 1984 ). They concluded that carbon isotope discrimination may provide an effective means to assess and improve WUE of water-limited crops. Strong correlations between whole plant TE and stable carbon isotope measurements of plant organic material were shown in a host of papers to be. Some of these papers were (1) for crops and other annuals (Hubick et al. 1986 ; Ehleringer et al. 1990 ; Virgona et al. 1990 ) and (2) for trees (Zhang and Marshall 1994 ; Picon et al. 1996 ; Roupsard et al. 1998 ). The isotopic method has spread rapidly as a general estimator of WUE and continues to be used widely in screening programmes for plant improvement as well as in ecological research, e.g. Rundel et al. ( 1989 ) and notably used in tree rings (McCarroll and Loader 2004 ).

10 Closing remarks

Water use efficiency is probably one of the oldest of plant traits to stimulate across the centuries the interest of philosophers, theologians, Middle Age savants, natural philosophers and modern plant scientists across different disciplines (plant physiology, ecophysiology, ecology, genetics, agronomy). The interest began as a purely philosophical one, progressed to thought experiments, towards an interest in plant functioning and its relationship to the environment.

Already in the early Renaissance (mid-fifteenth century), an experimentation was proposed, in a time when botany consisted mainly of naming plants (Morton 1981 ). It is then also an early example of an actually performed experimentation, the famous willow experiment by Van Helmont ( 1662 ) as well as of early “in laboratory” experimentation on plants (hydroponics experiments by Woodward 1699 ). The question of what makes plants grow, between water and soil, kept natural philosophers busy up to the end of the eighteenth century, when the assimilation of CO 2 was discovered and the question finally solved.

Early in the nineteenth century, the interest and experimentation turned to the amount of water that plants would need to grow, in the context of a developing research on agricultural practices (Morton 1981 ). Biomass was used to standardise the water losses which allowed comparisons among species (crops as well as trees) and a beginning study of the impact of different environmental variables.

At the end of the nineteenth century, knowledge on the physiological aspects of CO 2 assimilation and the control of transpiration by stomata had sufficiently advanced, so that scientists started to reflect on their inter-dependency. Was transpiration only a physical process or was there a physiological control? Was transpiration regulated by the dry matter production? Or does the stomatal opening determine the rate of CO 2 assimilation?

At the turn of the twentieth century, the study of species differences led to questioning why these differences did exist. As the discipline of “physiological ecology” developed, “water requirement” was inverted into an “efficiency”, reflecting an evolution from standardising transpiration to a trait in its own right. This introduced ecological questions about the adaptation of plants to dry environments and the relation to transpiration efficiency. Counterintuitive results stimulated the discussion and linked differences in WUE to different ecological strategies.

Methodological and theoretical advances in the description of leaf gas exchange in the mid-twentieth century showed the central role of stomata in the control of transpiration and CO 2 assimilation, leading to the idea that stomata might optimise water losses versus carbon gain. The development of carbon stable isotopes as an estimator of leaf-level WUE was an important step not only to further develop these theoretical considerations, but also towards large-scale studies. In parallel, modelling approaches were developed to scale from leaf-level WUE to whole plant TE, e.g. Cernusak et al. ( 2007 ), and to the field or canopy, e.g. Tanner and Sinclair ( 1983 ).

At least from the beginning of the twentieth century onwards, also critical views on the relationship between water requirement and its relation to growth mostly in terms of yield were published (Dachnowski 1914 ). Viets ( 1962 ) asked “Is maximum water use efficiency desirable?”, especially in terms of crop production. Sinclair et al. ( 1984 ) considered different options for improving water use efficiency, however concluding that most of these have important limitations or drawbacks. This discussion is ongoing, as can be seen by the article published by Blum ( 2009 ): “Effective use of water (EUW) and not water-use efficiency (WUE) is the target of crop yield improvement under drought stress”.

Exploration and application of transpiration efficiency at the whole plant level, and its derivatives at other levels, are still a very active research field across nearly all levels of forest science: concerning very rapid processes at the leaf level (Vialet-Chabrand et al. 2016 ), up-to-date genetic and genomic approaches for breeding (Plomion et al. 2016 ; De La Torre et al. 2019 ; Vivas et al. 2019 ), studying local adaptation of trees to their environment in a population genetic context (Eckert et al. 2015 ) or an ecological context (Pellizzari et al. 2016 ), water use efficiency from the plant to the ecosystem (Medlyn et al. 2017 ), estimated at the population level (Rötzer et al. 2013 ; Dekker et al. 2016 ) or modelling up to the global earth level (Cernusak et al. 2019 ), just to name a few. Thus, the first curiosity of Greek philosophers has motivated scientists through history, with many exciting discoveries still to come.

Change history

17 june 2021.

A Correction to this paper has been published: https://doi.org/10.1007/s13595-021-01073-0

Bates CG (1923) Physiological requirements of Rocky Mountain trees. J Agric Res 24:97–164

Google Scholar  

Bierhuizen J, Slatyer R (1965) Effect of atmospheric concentration of water vapour and CO 2 in determining transpiration-photosynthesis relationships of cotton leaves. Agric Meteorol 2:259–270

Article   Google Scholar  

Black J (1756) Experiments upon magnesia alba, quicklime, and some other alkaline substances. Essays Obs Phys Lit 2:157–225

Blackman F (1895) XI. Experimental researches on vegetable assimilation and respiration.—No. II. On the paths of gaseous exchange between aerial leaves and the atmosphere. Philos Trans R Soc B 186:503–562

Blum A (2009) Effective use of water (EUW) and not water-use efficiency (WUE) is the target of crop yield improvement under drought stress. F Crop Res 112:119–123

Bonnet C (1754) Recherches sur l’Usage des feuilles dans les plantes. Elie Luzac, Fils, Göttingen

Boyle R (1661) The sceptical chymist. J. Cadweill for J. Crooke

Brendel O, Pot D, Plomion C, Rozenberg P, Guehl JM (2002) Genetic parameters and QTL analysis of ẟ 13 C and ring width in maritime pine. Plant Cell Environ 25:945–953

Article   CAS   Google Scholar  

Briggs LJ, Shantz HL (1913a) The water requirement of plants. I. Investigations in the Great Plains in 1910 and 1911. US Dep Agric Bur Plant Ind Bull 284:1–48

Briggs LJ, Shantz HL (1913b) The water requirement of plants. II. A review of the literature. US Dep Agric Bur Plant Ind Bull 285:1–96

Briggs LJ, Shantz HL (1914) Relative Water Requirement of Plants. J Agric Res 3:1–64

CAS   Google Scholar  

Briggs LJ, Shantz HL (1917) The water requirement of plants as influenced by environment. In: Proceedings of the Second Pan American Scientific Congress. Pp 95–107

Brown HR (2013) The theory of the rise of sap in trees: some historical and conceptual remarks. Phys Perspect 15:320–358

Brown H, Escombe F (1900) VIII. Static Diffusion of gases and liquids in relation to the assimilation of carbon and translocation in plants. Philos Trans R Soc B 193:223–291

Brown P, Shrader W (1959) Grain yields, evapotranspiration, and water use efficiency of grain sorghum under different cultural practices. Agron J 51:339–343

Bugler G (1950) Un précurseur de la biologie expérimentale: Edme Mariotte. Rev Hist Sci (Paris) 3:242–250

Burgerstein A (1887) Materialien zu einer Monographie betreffend die Erscheinungen der Transpiration der Pflanzen. Verhandlungen der Zool Gesellschaft Wien 37:691–782

Burgerstein A (1889) Materialien zu einer Monographie, betreffend die Erscheinungen der Transpiration der Pflanzen. II. Theil. Verhandlungen der Zool Gesellschaft Wien 39:399–464

Cernusak LA, Aranda J, Marshall JD, Winter K (2007) Large variation in whole-plant water-use efficiency among tropical tree species. New Phytol 173:294–305

Article   PubMed   Google Scholar  

Cernusak LA, Haverd V, Brendel O et al (2019) Robust response of terrestrial plants to rising CO 2 . Trends Plant Sci 24(7):578–586 1–9

Article   CAS   PubMed   Google Scholar  

Chen J, Chang SX, Anyia AO (2011) Gene discovery in cereals through quantitative trait loci and expression analysis in water-use efficiency measured by carbon isotope discrimination. Plant Cell Environ 34:2009–2023

Condon AG, Richards RA, Rebetzke GJ, Farquhar GD (2002) Improving intrinsic water-use efficiency and crop yield. Crop Sci 42:122–131

PubMed   Google Scholar  

Condon AG, Richards RA, Rebetzke GJ, Farquhar GD (2004) Breeding for high water-use efficiency. J Exp Bot 55:2447–2460

Cowan IR, Farquhar GD (1977) Stomatal function in relation to leaf metabolism and environment. In: Integration of activity in the higher plant. University Press, pp 471–505

Cowan IR, Troughton J (1971) The relative role of stomata in transpiration and assimilation. Planta 97:325–336

Cregg B (2004) Improving drought tolerance of trees: theoretical and practical considerations. In: Acta Horticulturae Evaluation, Production and Use, XXVI International Horticultural Congress: Nursery Crops; Development. Aug 11-17, 2002, pp 147–158

Dachnowski A (1914) Transpiration in relation to growth and to the successional and geographical distribution of plants. Ohio Nat 14:241–251

Daubeny C (1836) On the action of light upon the atmosphere. Philos Trans R Soc 126:149–175

De La Torre A, Puiu D, Langley CH et al (2019) Genomic architecture of complex traits in loblolly pine. New Phytol 221:1789–1801

de Saussure N (1804) Chemische Untersuchungen über die Vegetation. Leipzig, 1890

Dehérain M (1869) L’évaporation de l’eau et la decomposition de l’acide carbonique par les feuilles des végétaux. Aannales des Sci Nat–Bot 5(XVII):5–23

Dekker SC, Groenendijk M, Booth BBB, Huntingford C, Cox PM (2016) Spatial and temporal variations in plant water-use efficiency inferred from tree-ring, eddy covariance and atmospheric observations. Earth Syst Dyn 7:525–533

Dietrich T (1872) Ueber die durch unsere Culturpflanzen verdunsteten Wassermengen. Mitth des landw Cent für den Regierungsbezirk Cassel 1872:343

Dreibelbis F, Harrold L (1958) Water-use efficiency of corn, wheat, and meadow crops. Agron J 50:500–5003

Eckert AJ, Maloney PE, Vogler DR, Jensen CE, Mix AD, Neale DB (2015) Local adaptation at fine spatial scales: an example from sugar pine ( Pinus lambertiana , Pinaceae ). Tree Genet Genomes 11:42

Egerton FN (2001a) A history of the ecological sciences: early Greek origins. Bull Ecol Soc Am 82:93–97

Egerton FN (2001b) A history of the ecological sciences, part 4: Roman natural history. Bull Ecol Soc Am 82:243–246

Egerton FN (2002) A history of the ecological sciences, part 7: Arabic language science: botany, geography, and decline. Bull Ecol Soc Am 83:261–266

Egerton FN (2003) A history of the ecological sciences, part 10: botany during the Italian Renaissance and beginnings of the scientific revolution. Bull Ecol Soc Am 84:130–137

Egerton FN (2004a) A history of the ecological sciences, part 12: invertebrate zoology and parasitology during the 1500s. Bull Ecol Soc Am 85:27–31

Egerton FN (2004b) A history of the ecological sciences, part 13: broadening science in Italy and England, 1600–1650. Bull Ecol Soc Am 85:110–119

Egerton FN (2004c) A history of the ecological sciences, part 14: plant growth studies in the 1600s. Bull Ecol Soc Am 85:208–213

Egerton FN (2005) A history of the ecological sciences, part 16: Robert Hooke and the Royal Society of London. Bull Ecol Soc Am 86:93–101

Egerton FN (2008) A history of the ecological sciences, part 28: plant growth studies during the 1700s. Bull Ecol Soc Am 89:159–175

Egle K, Ernst A (1949) Die Verwendung des Ultrarotabsorptionsschreibers für die vollautomatische und fortlaufende CO 2 -Analyse bei Assimilations-und Atmungsmessungen an Pflanzen. Zeitschrift für Naturforsch B 4:351–360

Ehleringer J, White J, Johnson D, Brick M (1990) Carbon isotope discrimination, photosynthetic gas exchange, and transpiration efficiency in beans and range grasses. Acta Oecol 11:611–625

Farquhar G (1980) Carbon isotope discrimination by plants: effects of carbon dioxide concentration and temperature via the ratio of intercellular and atmospheric CO 2 concentrations. In: Carbon dioxide and climate: Australian research. Australian Academy of Science, Canberra, pp 105–110

Farquhar GD, Rashke K (1978) On the resistance to transpiration of the sites of evaporation within the leaf. Plant Physiol 61:1000–1005

Article   CAS   PubMed   PubMed Central   Google Scholar  

Farquhar GD, Richards PA (1984) Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes. Aust J Plant Physiol 11:539–552

Farquhar GD, O’Leary MH, Berry JA (1982) On the relationship between carbon isotope discrimination and the intercellular CO2-concentration in leaves. Aust J Plant Physiol 9:121–137

Farsky F (1877) Ueber die Wasserverdunstung von Korn, Gerste und Erbse. Chem List [Chemische Blätter] tom I

Fittbogen J (1871) Altes und Neues aus dem Leben der Gerstenpflanze. Landwirtsch Versuchs-Stationen 13:81–136

Gaastra P (1959) Photosynthesis of crop plants as influenced by light, carbon dioxide, temperature, and stomatal diffusion resistance. Meded van Landbouwhoogeschool Wageningen 59:1–68

Govindjee, Krogmann D (2004) Discoveries in oxygenic photosynthesis (1727-2003): a perspective. Photosynth Res 80:15–57

Grew N (1682) The anatomy of plants. W. Rawlins, London

Grinsted M (1977) A study of the relationships between climate and stable isotope ratios in tree rings. University of Waikato PhD Thesis

Hales S (1727) Vegetable staticks, or, an account of some statical experiments on the sap in vegetables : being an essay towards a natural history of vegetation : also, a specimen of an attempt to analyse the air, by a great variety of chymio-statical experiments. W. and J Innys and T Woodward, London

Hellriegel (1871) Wie viel Wasser beanspruchen unsere Getreidearten zur Production einer vollen Ernte? Amtliches Vereinsblatt des landwirtlischaftlichen Prov fuer die Mark Brand und Niederlausitz 3:60–62

Hellriegel H (1883) Beiträge zu den Naturwiss. Grundlagen des Ackerbaus. F, Vieweg und Sohn, Braunschweig

Hershey D (2003) Misconceptions about Helmont’s willow experiment. Plant Sci Bull 49:78–83

Hobart C, Harris K (1946) Fitting cropping systems to water supplies in Central Arizona. College of Agriculture, University of Arizona, Tucson, AZ, USA

Hopkins J (1996) Nicholas of Cusa on wisdom and knowledge. Arthur Banning Press, Minneapolis

Hubick K, Farquhar G, Shorter R (1986) Correlation between water-use efficiency and carbon isotope discrimination in diverse peanut ( Arachis ) germplasm. Aust J Plant Physiol 13:803–816

Iljin V (1916) Relation of transpiration to assimilation in steppe plants. J Ecol 4:65–82

Ingen-Housz J (1779) Experiments upon vegetables, discovering their great power of purifying the common air in the sunshine and of injuring it in the shade and at night. P. Elmsly, and H. Payne, London

Kearney TH, Shantz HL (1911) The water economy of dry-land crops. Yearb United States Dep Agric 10:351–362

Kiesselbach T (1916) Transpiration as a factor in crop production. Bull Agric Exp Stn Nebraska 6:19–38

King F (1889) Soil physics. Annu Rep Agric Exp Stn Univ Wisconsin 6:189–206

Kolkunov W (1905) Contributions to the problem of breeding drought resistant crop plants. I. Anatomical and Physiological investigations of the degree of xerophily of certain cereals. Mém Polytech Inst Kiev 5(4):

Lavoisier A-L (1770) Sur la nature de l’eau et sur les expériences par lesquelles on a prétendu prouver la possibilité de son changement en terre. Mémoires l’Académie des Sci:73–82

Lawes JB (1850) Experimental investigation into the amount of water given. J Hortic Soc London 5:38–63

Lawes JB (1851) Report upon some experiments undertaken at the suggestion of Professor Lindley, to ascertain the comparative evaporating properties of evergreen and deciduous trees. J Hortic Soc London 6:227–242

Lazenby EM (1995) The historia plantarum generalis of John Ray: book i - a translation and commentary. Newcastle University PhD thesis

Leather JW (1910) Water requirements of crops in India. Mem Dep Agric India Chem Ser 1(3):133–154

Leather JW (1911) Water requirements of crops in India. -II. Mem Dep Agric India Chem Ser 1:205–281

Lévesque M, Siegwolf R, Saurer M, Eilmann B, Rigling A (2014) Increased water-use efficiency does not lead to enhanced tree growth under xeric and mesic conditions. New Phytol 203:94–109

Linares J, Camarero J (2012) From pattern to process: linking intrinsic water-use efficiency to drought-induced forest decline. Glob Chang Biol 18:1000–1015

Luft K (1943) Über eine neue Methode der registrierenden Gasanalyse mit Hilfe der Absorption ultraroter Strahlen ohne spektrale Zerlegung. Z Tech Phys 24:97–104

Malpighi M (1675) Anatome Plantarum. Johannis Martyn, London

Marié-Davy H (1869) Evaporation du sol et des plantes. J d’Agriculture Prat 2:234–239

Marié-Davy H (1874) Note sur la quantité d’eau consommée par le froment pendant sa croissance. Comptes rendus Hebd des séances l’Académie des Sci 79:208–212

Maskell EJ, Blackman FF (1928) Experimental researches on vegetable assimilation and respiration. XVIII.—The relation between stomatal opening and assimilation.—A critical study of assimilation rates and porometer rates in leaves of Cherry Laurel. Proc R Soc Lond Ser B 102:488–533

Maximov NA (1929) The plant in relation to water. George Allen & Unwin LTD, London

Maximov NA, Alexandrov V (1917) The water requirement and drought resistance of plants. Trav du Jard Bot Tiflis 19:139–194

McCarroll D, Loader NJ (2004) Stable isotopes in tree rings. Quat Sci Rev 23:771–801

Medlyn BE, De Kauwe MG, Lin YS et al (2017) How do leaf and ecosystem measures of water-use efficiency compare? New Phytol 216:758–770

Meinzer FC, Ingamells JL, Crisosto C (1991) Carbon isotope discrimination correlates with bean yield of diverse coffee seedling populations. Hort Sci 26:1413–1414

Möbius M (1901) Die Anatomie der Pflanzen I: and II. Theil. Engelmann, W, Leipzig

Mohl H (1856) Welche Ursachen bewirken die Erweiterung und Verengung der Spaltöffnungen? Bot Zeitung 14:697–704

Möldenhawer J (1812) Beyträge zur Anatomie der Pflanzen. CL Wäser, Kiel

Montgomery E (1911) Methods of determining the water requirements of crops. Proc Am Soc Agron 3:261–283

Moore B (1924) Reviewed work: Physiological requirements of Rocky Mountain trees by Carlos G. Bates Ecology 5:298–302

Morton A (1981) History of botanical science: an account of the development of botany from ancient times to the present day. Academic Press, London

Nickelsen K (2007) From leaves to molecules: botany and the development of photosynthesis research. Ann Hist Philos Biol 12:1–40

Park R, Epstein S (1960) Carbon isotope fractionation during photosynthesis. Geochim Cosmochim Acta 21:110–126

Pellizzari E, Camarero JJ, Gazol A, Sangüesa-Barreda G, Carrer M (2016) Wood anatomy and carbon-isotope discrimination support long-term hydraulic deterioration as a major cause of drought-induced dieback. Glob Chang Biol 22:2125–2137

Penman HT, Schofield RK (1951) Some physical aspects of assimilation and transpiration. Symp Soc Exp Biol 5:115–129

Perrault C (1680) Essais de Physique. Jean Baptiste Coignard, Paris

Picon C, Guehl J-M, Aussenac G (1996) Growth dynamics, transpiration and water-use efficiency in Quercus robur plants submitted to elevated CO 2 and drought. Ann des Sci For 53:431–446

Plomion C, Bartholomé J, Bouffier L et al (2016) Understanding the genetic bases of adaptation to soil water deficit in trees through the examination of water use efficiency and cavitation resistance: maritime pine as a case study. J Plant Hyd 3:008

Polster H, Weise G, Neuwirth G (1960) Ecological researches on the CO 2 balance [net assimilation] and water economy of some tree species in sand and alkali soils in Hungary. Arch für Forstwes 9:947–1014

Porter R (1979) John Woodward; ‘A droll sort of philosopher’. Geol Mag 116:335–343

Priestley J (1775) Experiments and observations on different kinds of air. J.Johnson, London

Raber O (1937) Water utilization by trees, with special reference to the economic forest species of the north temperate zone. USDA Misc Pub No 257, Washington DC

Ray J (1686) Historia Plantarum, I edn. The Royal Society, London

Ray J (1691) The wisdom of God manifested in the works of creation ; first published in 1691: reprinted by the Wernerian Club, London 1844-1846

Roeser J (1940) The water requirement of Rocky Mountain conifers. J For 38:24–26

Rötzer T, Liao Y, Goergen K, Schüler G, Pretzsch H (2013) Modelling the impact of climate change on the productivity and water-use efficiency of a central European beech forest. Clim Res 58:81–95

Roupsard O, Joly HI, Dreyer E (1998) Variability of initial growth, water-use efficiency and carbon isotope discrimination in seedlings of Faidherbia albida (Del.) A. Chev., a multipurpose tree of semi-arid Africa. provenance and drought effects. Ann des Sci For 55:329–348

Rundel P, Ehleringer J, Nagy K (1989) Stable isotopes in ecological research. Springer-Verlag, New York

Book   Google Scholar  

Rüsch J (1959) Das Verhältnis von Transpiration und Assimilation als physiologische Kenngröße, untersucht an Pappelklonen. Theor Appl Genet 29:348–354

Scarth GW (1927) Stomatal movement: its regulation and regulatory role - a review. Protoplasma 2:498–511

Schmidt H-L, Winkler F (1979) Einige Ursachen der Variationsbreite von ẟ 13 C-Werten bei C3- und C4-Pflanzen. Ber Dtsch Bot Ges 92:S 185–S 191

Schröder M (1895) The transpiration of various crop plants. Agric For 10:320–336

Shantz HL (1927) Drought resistance and soil moisture. Ecology 8:145–157

Sharrock R (1660) The history of the propagation & improvement of vegetables. A. Lichfield, Oxford

Sinclair TR, Tanner CB, Bennett JM (1984) Water-use efficiency crop production. Bioscience 34:36–40

Sorauer P (1880) Studien über Verdunstung. Wollny - Forschungen auf dem Gebiete der Agrik tom 3:351–490

Spedding J, Ellis R, Heath D (1900) The works of Francis Bacon, Houghton, Mifflin and Company

Stanhill G (1986) John Woodward - a neglected 17th century pioneer of experimental botany. Isr J Bot 35:225–231

Tallec T, Béziat P, Jarosz N, Rivalland V, Ceschia E (2013) Crops’ water use efficiencies in temperate climate: comparison of stand, ecosystem and agronomical approaches. Agric For Meteorol 168:69–81

Tanner CB, Sinclair TR (1983) Efficient water use in crop production: research or re-search? In: Taylor HM, Jordan WR, Sinclair TR (eds) Limitations to efficient water use crop production. American Society of Agronomy, Madison, pp 1–27

Thornthwaite C (1947) Climate and moisture conservation. Ann Assoc Am Geogr 37:87–100

Tomic S, Cussenot M, Dreyer E (2005) La lumiére et les plantes : histoire de la découverte de la « photosynthése », 1779-1804. In: Changeux J-P (ed) La lumière au siècle des lumières et aujourd’hui: Art et science. Odile Jacob, Paris, pp 145–161

Troughton J (1969) Plant water status and carbon dioxide exchange of cotton leaves. Aust J Biol Sci 22:289–302

Tschaplowitz (1878) Ueber die Verdunstung und Substanzzunahme der Pflanzen. Berichte der Sect für Landwirtsch Versuchswes auf der Naturforscherversammlung zu München 1877(tome XX):74

Unger F (1862) Neue Untersuchungen über die Transpiration der Pflanzen. Sitzungsberichte der Kais Akad der Wissenschaften Wien 44:181–217 and 327-368

Vadez V, Kholova J, Medina S, Kakkera A, Anderberg H (2014) Transpiration efficiency: new insights into an old story. J Exp Bot 65:6141–6153

van Helmont J (1662) Oriatrike or Physick Refined. Lodowick Loyyd, London

Vialet-Chabrand S, Matthews JSA, Brendel O, Blatt MR, Wang Y, Hills A, Griffiths H, Rogers S, Lawson T (2016) Modelling water use efficiency in a dynamic environment: an example using Arabidopsis thaliana. Plant Sci 251:65–74

Viets FG (1962) Fertilizers and the efficient use of water. Adv Agron 14:223–264

Virgona J, Hubick K, Rawson H et al (1990) Genotypic variation in transpiration efficiency, carbon-isotype discrimination and carbon allocation during early growth in sunflower. Aust J Plant Physiol 17:207–214

Vivas M, Rolo V, Wingfield MJ, Slippers B (2019) Maternal environment regulates morphological and physiological traits in Eucalyptus grandis . For Ecol Man 432:631–636

Vogel JC (1980) Fractionation of the carbon isotopes during photosynthesis. In: Sitzungsberichte der Heidelberger Akademie der Wissenschaften. Springer, New York, pp 111–135

von Höhnel FR (1879) Ueber die Wasserverbrauchsmengen unserer Forstbäume mit Beziehung auf die forstlich-meteorologischen Verhältnisse. Wollny - Forschungen aus dem Gebiet der Agric tom II:398–421

von Schleiden MJ (1849) Grundzüge der wissenschaftlichen Botanik, 3rd edn. Verlag von Wilhelm Engelmann, Leipzig

von Seelhorst C (1902) Vegetationskästen zum Studium des Wasserhaushaltes im Boden. J Landwirtsch 50:277–280

Woodward J (1699) Some thoughts and experiments concerning vegetation. Philos Trans R Soc Lond A 21:193–227

Zhang J, Marshall JD (1994) Population differences in water-use efficiency of well-watered and water-stressed western larch seedlings. Can J For Res 24:92–99

Download references

Acknowledgements

Much of the historical background is based on A.G. Morton’s “History of Botanical Sciences” as well as to Frank N. Egerton’s “A History of the Ecological Sciences” series in the “Bulletin of the Ecological Society of America”. The author is also largely indebted to C. Schuchardt from the Library of the Staatsbetrieb Sachsenforst for help with the quest for rare German publications. The author would also like to thank E. Dreyer and J.M. Guehl (both from the SILVA Unit at INRAE Nancy, France) who commented extensively on an earlier version of the draft and J. Williams (University of Sussex), L. Handley and J. Raven (University of Dundee) who made many valuable suggestions and improved language.

Author information

Authors and affiliations.

Université de Lorraine, AgroParisTech, INRAE, UMR Silva, Nancy, France

Oliver Brendel

You can also search for this author in PubMed   Google Scholar

Corresponding author

Correspondence to Oliver Brendel .

Ethics declarations

Conflict of interest.

The author declares no competing interests.

Additional information

Handling Editor: Andrew Merchant

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The original online version of this article was revised due to a retrospective Open Access order.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Brendel, O. The relationship between plant growth and water consumption: a history from the classical four elements to modern stable isotopes. Annals of Forest Science 78 , 47 (2021). https://doi.org/10.1007/s13595-021-01063-2

Download citation

Received : 26 November 2020

Accepted : 14 April 2021

Published : 28 April 2021

DOI : https://doi.org/10.1007/s13595-021-01063-2

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Transpiration efficiency
• Water use efficiency
• Plant ecophysiology
• Botanical history

Annals of Forest Science

ISSN: 1297-966X

• General enquiries: [email protected]

• Free Resources
• Project Search
• Featured Projects
• Member Benefits

1059 Main Avenue, Clifton, NJ 07011

The most valuable resources for teachers and students

(973) 777 - 3113

[email protected]

1059 Main Avenue

Clifton, NJ 07011

07:30 - 19:00

Monday to Friday

123 456 789

[email protected]

Goldsmith Hall

New York, NY 90210

• Why We’re Unique

How does the amount of water affect plant growth?

Introduction: (initial observation).

Plants are living things. They live in places all around the world. Plants can grow in deserts, rain forests, and even in your own backyard. But no matter where plants grow they all need soil, water, air, and sunshine. A plant’s needs change as it grows. Plants need a lot of water during early growth, flowering and fruit set.

In this project we will try to see how does the amount of water affect the plant growth.

This project guide contains information that you need in order to start your project. If you have any questions or need more support about this project, click on the “Ask Question” button on the top of this page to send me a message.

If you are new in doing science project, click on “How to Start” in the main page. There you will find helpful links that describe different types of science projects, scientific method, variables, hypothesis, graph, abstract and all other general basics that you need to know.

Project advisor

Information Gathering:

Find out about nutrients for plants. Read books, magazines or ask professionals who might know in order to learn about the effect or area of study. Keep track of where you got your information from.

This is a sample of the information you may find:

Indoor Plants – Watering

The main cause of death of potted plants is over-watering. Roots need both water and oxygen, and when surrounded by water, they cannot take up oxygen. These roots may rot and eventually the whole plant may die. The symptoms of over-watering and underwatering are similar. Both lead to poor root health, root decline and possibly death of the plant.

A common question from gardeners is “How often should I water my plants?” There is no pat answer to this question. The amount and frequency of watering depends on many factors, such as the plant species, its growth stage, its location, the type and size of its pot, soil mix characteristics and variable weather conditions.

There is a wide range of watering requirements for different species of plants. Plants with large or very thin leaves and those with fine surface roots usually require more frequent watering than succulent plants with fleshy leaves and stems that are able to store water. Some plants thrive under moist conditions while other plants grow well when kept drier.

Plants may slow in growth after a flush of new growth or a heavy flowering. During these periods and while it is dormant, a plant will need less water.

Water evaporates rapidly from the sides of a porous clay pot, which requires more frequent watering than nonporous, glazed or plastic pots. A large plant in a small pot needs water more often than a small plant in a large pot.

Different soil mixes require different watering schedules. Heavy, fine-textured potting media and those that contain a lot of peat moss hold more moisture than loose, porous mixtures of bark, sand and perlite.

A plant in a warm, dry, sunny location needs more frequent watering than one in a cool, low-light environment.

The rule-of-thumb is to water when necessary. The following methods may be used to determine when to water:

• Touch the soil – The most accurate gauge is to water when the potting mixture feels dry to the touch. Stick your finger into the mix up to the first joint; if it is dry at your fingertip it needs water.
• Tap the pot – When the potting mix in a clay pot begins to dry, it shrinks away from the sides of the pot. Rap the side of the pot with the knuckles or a stick. If the sound is dull, the soil is moist; if the sound is hollow, water is needed.
• Estimate weight – As potting mixtures become dry, a definite loss in weight can be observed.
• Judge soil color – Potting mixtures will change from a dark to lighter color as they dry.

There are a number of watering meters available to measure moisture in the soil, indicating whether water is needed. These products vary widely in accuracy. The readings can be influenced by factors other than soil moisture content. Fertilizer and soil type can affect the reading.

When watering is required, water thoroughly. Apply water until it runs out of the bottom of the pot. This washes out the excess salts, and it guarantees that the bottom two-thirds of the pot, which contains most of the roots, receives sufficient water. Don’t let the pot sit in the water that runs out. Empty the saucer.

Do not allow the soil to become excessively dry. If the salt level in the container is high, root damage may occur. If soil does become very dry and hard to rewet, use the double watering method. Water once and then again half an hour later; or place the pot in a sink or a bucket of water. Remove the pot when the soil surface is moist. Allow the pot to drain completely. If peat is allowed to dry completely, not only is it difficult to rewet, it also will not hold as much water as it could hold before it dried.

Do not water with hot or cold water. The water temperature should be between 62 and 72 °F.

Do not water plants with softened water because sodium and chloride will also be added to the soil mix, possibly causing plant damage.

Although wilting is often an indication of the need to water, it is not always so. Any injury to the root system decreases a plant’s ability to take up water, including root rot, which is caused by too much water. This inability to take up water will cause wilting, and under these conditions, watering may make the problem worse.

Why Do We Fertilize Plants?

For centuries plants grew without any help from human beings, and many are doing so today. Thus, it is obvious that they can do so by themselves, especially in environments to which they are adapted. However, as humans cultivated plants it was learned that the addition of certain materials to the soil sometimes caused plants to respond with characteristics which were considered to be desirable (e.g., more fruit, faster growth, better color, more attractive flowers). Early in recorded history we find accounts of applications of animal manures, wood ashes, and lime to enhance plant performance. Thus was born the practice of fertilization and soil amendment.

We should note here that the plant responses we get from applying fertilizer and other soil amendments are not inherently “good” or “bad.” These terms are subjective and reflect personal judgment as to what is “desirable.” For example, a greater quantity of fruit which is too small for market is not a characteristic desired by a peach farmer. Faster growth is usually not a desirable effect for someone growing bonsai plants. Rank vegetative growth is not desirable in an already-lush lawn nor are profusely-blooming squash plants that are not setting fruit. Thus, a “good” response to fertilization under one set of circumstances may be a “bad” response under another set. It depends on what response the person wants from the plant.

So, why do we apply fertilizer to the soil? Because we want to obtain some desired plant response. We want our plants to “do better.” As we set out to fertilize our plants we should keep in mind how we want them to do better (grow faster, produce better flowers or fruit, etc.) – and we should also know if fertilization will contribute to that improvement.

When Should I Apply Fertilizer?

Stated simply, you need to fertilize whenever you expect to get a desired plant response. However, the difficulty is in predicting. You usually want to know in advance if there will be a response to added fertilizer so that you can avoid growing plants under nutrient-deficient conditions. Since predicting plant responses is difficult, many people apply fertilizer as insurance against nutrient deficiencies. The result: over fertilization in the United States is now as prevalent a problem as under fertilization.

A suggested approach to fertilization involves the following steps:

• Recognize what plant response you are seeking;
• Determine from observation or consultation if fertilizer application is likely to give you the response you want;
• Apply fertilizer only if your desired response is likely;
• Apply only the amount of fertilizer necessary to give the desired response.

What Nutrients Do My Plants Need?

The best way of knowing what your plants need is by observing plant performance and understanding the multiple factors affecting such performance (e.g., light, water, temperature, pests, nutrition).

There is no magical way of knowing which nutrient may be in limited supply in the soil. Soil testing helps predict the need for some of the nutrients, but testing is only one of the tools in plant nutrient management. If you recycle organic matter such as grass clippings (don’t use a bag on your mower) and leaves, you will be returning to the soil the nutrients those plants had absorbed. It is the easiest, least expensive, and most environmentally sound way to fertilize. You may still have to supplement, but you will then apply fewer nutrients–and a lot less fertilizer. Plants need 18 elements for proper growth and reproduction. Under many conditions, plants obtain enough of these elements from the soil, water, and air. It is only in certain environments and growing conditions that one or more of the nutrients are deficient.

The most-commonly applied nutrients are nitrogen (N), phosphorus (P), and potassium (K). Responses to all three elements were fairly widespread in the past, and it became customary to apply the three together. As a result of habit, all three are still applied even though there are now many situations, especially in gardens and landscapes, where plants do not respond to one or more of these fertilizer nutrients.

Other plant-essential nutrients used in fairly large quantities are calcium (Ca), magnesium (Mg), and sulfur (S). However, fertilization with these nutrients is not usually necessary because the Ca and Mg contents of soil are generally sufficient for most plant species. Also, large quantities of Ca and Mg are supplied when acidic soil is limed with dolomite. Sulfur is usually present in sufficient quantities from the slow decomposition of soil organic matter, an important reason for not throwing out grass clippings and leaves.

Micronutrients are those elements essential for plant growth which are needed in only very small (micro) quantities . These elements are sometimes called minor elements or trace elements, but use of the term micronutrient is encouraged by the American Society of Agronomy and the Soil Science Society of America. The micronutrients are iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), boron (B), molybdenum (Mo), Cobalt (Co), Nickel (Ni), and chlorine (Cl). If one of your plant species has a micronutrient deficiency, apply the recommended rate of the deficient nutrient. Recycling organic matter such as grass clippings and tree leaves is an excellent means of providing micronutrients (as well as macronutrients) to growing plants.

What About Organic Matter as a Source of Nutrients?

Organic matter (such as grass clippings, tree leaves, shrubbery and tree trimmings) is an excellent source of plant nutrients. The plants which produced that organic material accumulated all the essential nutrients for their own growth needs. Upon decomposition, those nutrients in the organic material become available for reuse. When you recycle “homegrown” organic matter such as grass clippings, leaves, and shrubbery trimmings you are practicing an excellent method of fertilizing your landscape. You are keeping valuable materials on site and are also greatly reducing the municipal solid wastes placed at curb side. Other organic materials, such as animal manures, biosolids (processed sewage sludge), or various composted materials, are also alternative sources of plant nutrients.

Question/ Purpose:

The purpose of this project is to determine how does the amount of water affect the plant growth (i.e. plant height, stem volume).

Identify Variables:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

Independent variable (also known as the manipulated variable is the amount of water used to grow the plant.

Dependent variable (also known as responding variable) is the plant growth (plant height).

Controlled variables are light and temperature. We grow all plants under the same temperature and light conditions.

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis. This is a sample hypothesis.

My hypothesis is that more water will result more growth.

Experiment Design:

In order to test how does the amount of water affect the plant growth, we plant some seeds and use them to test the effect of water.

Experiment 1:

Materials: radish seeds (one packages will be enough for multiple experiments), water, paper towels, plastic beverage cups, aluminum foil, liquid fertilizer (plant food purchased from the grocery or home supply store)

• Soak the radish seeds in water for about an hour.
• Fold a paper towel lengthwise and float it in a shallow pan of water. Remove it and gently wring out the excess water.
• Get 6 soaked radish seeds
• Lay the soaked seeds along the folded edge of the moist paper towel. Roll the paper with the seeds into a cylinder, as in the diagram.
• Repeat with the above steps ten times until you have ten rolled paper towel with 6 seeds in each of them.
• Place the rolled paper cylinders in separate plastic beverage cups
• Label one of the cups as a control and label the others with numbers from 1 to 9 . One represents the least amount of water and 9 represents the highest amount of water.
• Add the necessary nutrients to one gallon of water that will be used for our experiment.
• Each day add 10 drops of water to cup number 1, 20 drops of water to cup number 2, 30 drops of water to cup number 3 and continue with the same increment so the cup number 9 will get 90 drops of water each day. DON’T WATER THE CONTROL CUP.
• Place a piece of aluminum foil loosely over all four cups and allow the cups to remain undisturbed until the seeds germinate (2 to 4 days)
• Once the seeds have germinated, remove the foil and place the cups in a location that provides them with light.
• Measure the roots and the shoots of the growing plants and chart the growth of their seedlings every day or two.
• Describe the effect of water amount on the growth of the seedlings.

Above description is for testing 9 different amounts of water. If you want to test more or less samples, you can modify the experiment as needed.

The reason that we place 6 seeds in each paper towel is to see the average result, not the result of an accidentally large seed.

Experiment 2:

1) Plant 6 bean seeds in 6 small pots filled with potting soil. Place the seeds at the same level in soil for all pots. Place the pots by a sunny window.

2) Label each pot with numbers from 1 to 6

3) Water the pots every day. Each time the pot number 1 will get the least amount of water and the pot number 6 will get the most.

4) Make daily observations and record the height of you plant every day for two to three weeks.

If you need a control, get an additional small pot in which you plant a bean seed, but you don’t water it at all.

Experiment 3:

How does excess amount of water affect plants?

3 plants*, 3 dishes or pans, water, crayons, graph paper

* make sure you choose plants that are the same kind of plant, as close to the same size as possible and healthy

1. Label the plants A, B, and C. Make holes in the bottom of the containers of plants A and B. If your containers already have holes, plug up the holes in the bottom of container C.

2. Place each plant in a dish and put all three plants in the same location where they will receive the same light.

3. Water plant A every other day, keeping the soil moist but not wet. Do not water plant B. Water plant C every day, keeping the soil saturated, very wet.

4. Make a bar graph showing the color of each plants leaves every day for a week.

Think About This:

1. What happened to the color of plant A? plant B? plant C?

2. Does it matter how much water a plant gets?

3. Is drainage important?

Materials and Equipment:

• Radish seeds (one packages will be enough for multiple experiments)
• Paper towels
• Plastic beverage cups
• Aluminum foil
• Samples of liquid fertilizers (plant food purchased from the grocery or home supply store)
• Pots for experiment 2
• Bean seeds for experiment 2

Results of Experiment (Observation):

Record the results of your experiment 1 in tables like this:

This table shows the average height of seedlings in each cup in different days starting day 4.

Questions :

1) Why did you need 10 cups to perform this experiment? What conclusion could you draw if you had performed the experiment with only one cup?

2) What effect did the amount of water have on the plant growth?

If you are performing experiment 2, make a similar table for the results of experiment 2.

Calculations:

You may need to calculate the average heights of seedlings in each paper towel.

Summery of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.

References:

Find and review some books about plants, nutrients and fertilizers.

http://collaboratory.nunet.net/timber/scifair/kindto4/8.htm

http://jajhs.kana.k12.wv.us/jahome/fro/project99/campbell.ht

It is always important for students, parents and teachers to know a good source for science related equipment and supplies they need for their science activities. Please note that many online stores for science supplies are managed by MiniScience.

Testimonials

" I called School Time and my husband and son came with me for the tour. We felt the magic immediately."

- Robby Robinson

" My husband and son came with me for the tour. We felt the magic immediately."

- Zoe Ranson

Contact Info

Our address, working hours.

Week Days: 07:00-19:00

Saturday: 09:00-15:00

Sunday: Closed

Science Project

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• Open access
• Published: 02 August 2021

Nutrient availability and pH level affect germination traits and seedling development of Conyza canadensis

• Caroline Maldaner Follmer 1 ,
• Ana Paula Hummes 2 ,
• Nadia Canali Lângaro 2 ,
• Claudia Petry 2 ,
• Diovane Freire Moterle 3 &
• Edson Campanhola Bortoluzzi 4  

Scientific Reports volume  11 , Article number:  15607 ( 2021 ) Cite this article

8344 Accesses

6 Citations

Metrics details

• Biogeochemistry
• Environmental chemistry
• Environmental impact
• Environmental sciences

Reducing pesticide application in agricultural land is a major challenge for the twenty-first century. Responses of weed seed’s germination and seedling’s early development to chemical soil conditions around the seed may be a promising way to aid weed control in a no-till system. Thus, the objective of this work was to test, under controlled conditions, whether different chemical conditions affect the germination and development of horseweed [ Conyza canadensis  (L.) Cronquist]. We used, as treatment, solutions containing different nutrients (P, K, Ca, and Mg), separately and in combination, and at two pH levels (4.8 and 6.5). Phosphorus alone inhibited horseweed seed germination at ~ 7 times while had ~ 4 times reduction in final germination percentage and germination speed index for both pH tested. Other nutrients tested had a no-effect in germination speed index compared to the control treatment. Potassium alone or associated with other ions (P, Ca, and Mg) at pH 4.8 had a synergistic effect on seedling development (root and shoot length). In the same way, K associated with Mg was synergistic to the root length at pH 6.5. Seeds in the control treatment (distilled water) presented a high germination speed index at pH 6.5, while at low pH this parameter was higher when in association with KMg, PMg and Ca. The findings demonstrate that seed germination traits and seedling development of horseweed depend on nutrient kind exposure and pH conditions in the seed environment. This work suggests that adequate topsoil management (i.e., pH and nutrient availability) may aid to reduce weed germination, because, it consists of an important factor of weed occurrence in agricultural areas.

Similar content being viewed by others

The integrated effect of salinity, organic amendments, phosphorus fertilizers, and deficit irrigation on soil properties, phosphorus fractionation and wheat productivity

Evaluating the agronomic efficiency of alternative phosphorus sources applied in Brazilian tropical soils

Long-term organic and N fertilization influence the quality and productivity of pearl millet under pearl millet-wheat sequence in north India

Introduction.

The reduction of pesticide application is a major challenge in modern agriculture for the twenty-first century. No-till system (NT) implemented in the 1970s decade promoted better soil quality since soil cover, crop rotation, low soil disturbance, adequate traffic load, and adequate dose and fertilization method were practiced 1 . However, when these aspects are mismanaged, agricultural areas are subject to soil degradation 2 , low productivity and weed occurrence 3 , increasing pesticide uses 4 , and environmental risks 5 . Weeds present high seed production, dissemination, and cropland infestation capacities, causing mainly yield reduction. The species ability to complete germination and grow in a large range of edaphic conditions engender competitive advantages to the weeds over crops 6 . It is a key mechanism of success in plant establishment in different environments 7 , but the seed germination process can be affected by several factors such as light regime, temperature, and soil moisture 8 .

From an agronomic perspective, the genus Conyza Less. is responsible for decreasing soybean yields up to 30% 9 , 10 . The species of this genus are easily found in no-till system, where they became a great issue in the cereal crop production worldwide 3 , 8 . Recently, agronomists have found Conyza spp. biotypes with herbicide resistance mechanisms to several herbicide molecules, including glyphosate 4 , 11 . In South Brazil, 78% of the Conyza spp. biotypes showed herbicide resistance to glyphosate 4 . In summary, a good chemical horseweed control became arduous 4 , 12 , 13 . Furthermore, because this species is stress-tolerant and strong competitors 10 , 14 , alternative management strategies should be considered in the no-till system. Those strategies based on the species biology/ecology and soil management applied in an integrated manner seem to be promising to reduce the pollution pressure on the environment 8 , 15 , 16 . In this sense, Conyza sp., a dicot weed of the Asteraceae family commonly known as horseweed, stands out among weed species because can produce large quantities of easily disseminated seeds 8 , 15 . Germination is stimulated by light and may occur on bare topsoil in all seasons 8 , 17 , 18 . The wind may carry horseweed seeds over a hundred meters 15 , while seeds can migrate from infested fields to new areas a few thousand kilometers distant attached to cattle hair 19 . Horseweed seeds reach cropland topsoil easily, enriching the soil seed bank 8 . The horseweed seeds in the soil seed bank may germinate under favorable conditions from few years to decades 8 . The horseweed germination may occur by neutral to alkaline pH, saline and not disturb soil conditions, but maybe disturbed in the presence of Aluminum (Al) and when temperature and humidity conditions are out of the ideal range 3 , 8 , 18 , 20 , 21 . Furthermore, the species respond to factors such as salinity (NaCl) 7 , nutrient availability (N, P, and K), elements’ toxicity, and soil pH 7 , 22 , 23 , 24 , 25 . Thus, we expect that other nutrients available in soil may favor weed establishment and their dominance 26 , 27 .

These studies suggested then that different cations affect the germination process. However, scarce literature reports on germination of horseweed at acid conditions (low pH) and in presence of macronutrients found commonly in fertilizer and lime products 28 . This is particularly important to the no-till system, which has been receiving lime and fertilizer products on the topsoil, promoting a nutrient enrichment a few centimeters of soil surface. This scenario was not yet been studied face to the Conyza canadensis (L.) Cronquist var. canadensis germination and development. Thus, we expected that horseweed seeds may have a variable germination percentage and seedling establishment caused by exposure to different nutrients and pH levels, as suggested in the literature for other species 7 , 25 , 26 , 29 , 30 . In this study, we pay attention to two basic questions: which are the nutrients and pH level that act preponderantly in the germination and development of horseweed? and; what are the implications for horseweed management and fertilization practices on the field conditions? In this direction, studies on the factors involved in horseweed germination and establishment may allow improvement in management practices to avoid weed infestations in no-till system, reducing pesticide products as a control agent. We hypothesize that seed exposure to different nutrients and pH levels affects seed germination traits and seedling development. We expected also that eutrophic conditions present three possibilities of effects: synergistic, antagonistic, or no-effect. Identifying whether nutrients drive C. canadensis germination and plant establishment is essential to provide a better knowledge on fertilization in agricultural areas to help weed control and diminish pesticide application.

Thus, this study aimed to evaluate the seed germination traits and seedling development of horseweed in different chemical environments with a range of nutrient availability and pH conditions. For this, horseweed seeds were exposed to several nutrients (P, K, Ca, and Mg, alone and in combination) under two pH levels (4.8 and 6.5). The nutrients and their concentrations as well as the pH conditions were chosen to simulate field conditions of fertilization and liming commonly practiced in soybean crop under no-till system in south Brazil.

Overview on anova

The nutrients, as a factor of variation (F1 Factor), affected significantly all the parameters evaluated (Table 1). The pH (F2 factor) did not affect horseweed’s final germination percentage and germination speed index and root length, except for seedling length and root:shoot ratio. The interaction between nutrients and pH (F1 vs F2) affected all variables.

Final germination percentage (FGP) and nutrients

Different nutrients affected the FGP by the average of both pH levels (Fig.  1 a,b). Two effects were observed (i.e., no-effect and antagonistic effect). In both pH (4.8 and 6.5), two effects were observed (i.e., no-effect and antagonistic effect) on FGP. At low pH (pH 4.8), nutrients alone or in combination did not differ from the control treatment (FGP of 25.5%), except for the treatment with P that presented the lowest C. canadensis final germination percentage (6%) (Fig.  1 a). This indicates an antagonistic effect of P (4.25 times) on the final germination percentage compared with the control. At high pH (6.5), the treatment with P alone presented the lowest value, 3.5% (Fig.  1 b). As the treatment had a reduction in germination around 7.28 times compared to the control, they had an antagonistic effect. In addition, all other nutrients alone or in combination had no-effect because did not differ from the control (distilled water).

Final germination percentage (FGP) of Conyza canadensis in response to the nutrients (alone or in combination) under solutions at pH 4.8 ( a ) and at pH 6.5 ( b ). Bars with the different colours, comparing means among treatments, are significantly different, according to the Tukey’ test ( p  < 0.05). Different letters represent a statistical difference for the same treatment between the two pH tested while ns represents not significant; The results in the Bars represent means, while error bar marks represent standard deviation ( n  = 4) from the mean value.

Final germination percentage and pH levels (acidity)

The pH alone did not influence final germination percentages. On average, the FGP was not sensitive to the pH levels and their values were similar, 25.3% and 26.8% to high and low pH tested, respectively. Furthermore, the pH presented interaction with only Ca and PMg treatments. The final germination percentage was higher for Ca (29%) at low pH than at pH 6.5 (17.5%), while PMg treatment was higher at pH 6.5 (24.5%) than at pH 4.8 (16%) (Fig.  1 a,b).

In summary, P alone inhibited horseweed seed germination in 4.25 and 7.28 for both pH tested, 4.8 and 6.5.

Germination speed index (GSI) and nutrients

For germination speed index (GSI), different nutrients had different effects at each pH level (Fig.  2 a,b). At low pH (4.8), nutrients produced no-effects on the GSI (Fig.  2 a). At high pH (6.5), two effects were observed (i.e., no-effect and antagonistic effect) (Fig.  2 b). All treatments (Ca, Mg, K, CaMg, PCa, KCa, KMg, KCaMg, PCaMg, PKCa, PKCaMg, and, PCa) presented similar GSI values to the control treatment, indicating no-effect of nutrients compared to the control. Phosphorus alone presented the lowest GSI value (0.56, au), indicating an antagonistic effect comparatively to the control. The GSI reduction was 6.96 times in the GSI, comparatively to the control.

Germination speed index (GSI) of Conyza canadensis in response to the nutrients (alone or in combination) under solutions at pH 4.8 ( a ) and at pH 6.5 ( b ). Bars with the different colours, comparing means among treatments, are significantly different, according to the Tukey’ test ( p  < 0.05). Different letters represent a statistical difference for the same treatment between the two pH tested while ns represents not significant; The results in the Bars represent means, while error bar marks represent standard deviation ( n  = 4) from the mean value.

Germination speed index and pH levels

In the average of all nutrients, the GSI was not affected following the pH levels. In addition, pH interacted with KMg, Ca, PMg, K, and control treatment. At pH 4.8, the GSI values were higher for the KMg (4.69), Ca (3.8), K (5.2) presenting 1.20, 1.53, 1.78, and 1.34 times greater than pH 6.5, respectively. At pH 6.5, PMg (4.08) and control (3.9) treatments were higher compared to the pH 4.8, presenting 2.02 and 1.51 times greater, respectively.

Briefly, phosphorus alone had an antagonist effect, reducing GSI in both pH conditions by an average of ~ 5.6 times (Fig.  2 ).

Seedling length (SL) and nutrients

For SL, different nutrients had different effects follow the pH levels (Fig.  3 a,b). At low pH (4.8), nutrients produced three effects (i.e., synergistic, no-effect, and antagonistic effect) (Fig.  3 a). The PK combination and K alone presented the highest SL values (9.34 mm and 8.65 mm, respectively), comparatively with the control (5.35 mm). This indicated a synergistic effect of PK and K nutrients on SL compared to the control, increasing the values in 1.74 and 1.62 times, respectively. All other nutrients (Mg, Ca, K, PK, PCa, PMg, PKCa and, PCaMg, KMg, KCa, CaMg and PKCaMg) differed from the control, indicating an antagonistic effect. Phosphorus and PMg presented the lowest SL values, representing a 19.8 times reduction compared to the control treatment. At high pH (6.5), no-effect was observed among the treatments compared with the control (Fig.  2 b). All treatments presented similar SL values among them.

Seedling length in aerial part (SL) of Conyza canadensis in response to the nutrients (alone or in combination) under solutions at pH 4.8 ( a ) and at pH 6.5 ( b ). Bars with the different colours, comparing means among treatments, are significantly different, according to the Tukey’ test ( p  < 0.05). Different letters represent a statistical difference for the same treatment between the two pH tested while ns represents not significant; The results in the Bars represent means, while error bar marks represent standard deviation ( n  = 4) from the mean value.

Seedling length and pH levels

The pH had an interaction with some nutrients (Fig.  3 ). On average, the SL at low pH (2.76 mm) was higher than at pH 6.5 (1.22 mm). Comparing SL between both pH, the treatments control, PK, K and, KCa presented 8.05, 7.30, 6.93 and, 2.12 times greater at pH 4.8 than at pH 6.5, respectively. In opposition, the PMg treatment presented 4.47 times greater SL at pH 6.5 than at pH 4.8 (Fig.  3 ).

Briefly, K alone or in combination with P was synergistic for shoot length (SL) at low pH, while all other combinations were antagonistic at low pH. At pH 6.5, a wide range of combinations of nutrients ad no-effect on SL.

Root length (RL) and nutrients

The different nutrients affected the parameter in each pH level (Fig.  4 a,b). In both pH (i.e., low and high), two effects on RL were observed (i.e., synergistic and no-effect). At low pH (4.8), the PKCaMg treatment (combination from all nutrient tested) presented the highest RL value (4.32 mm) comparing to the control (1.28 mm), indicating a synergistic effect of nutrients. Furthermore, all other treatments: P, Mg, Ca, K, PK, PCa, PMg, KMg, KCaMg and, PCaMg presented similar SL values to the control, indicating no-effect of these nutrients (Fig.  4 a). At high pH (6.5), the treatment KMg presented the highest RL value (5.23 mm), comparing to the control (1.09 mm), indicating a synergistic effect of nutrients (Fig.  4 b). The KMg treatment presented RL 4.84 times greater than the control. All other nutrients: alone or in combination presented RL values similar to the control, indicating no-effect of nutrients.

Root length (RL) of Conyza canadensis in response to the nutrients (alone or in combination) under solutions at pH 4.8 ( a ) and at pH 6.5 ( b ). Bars with the different colours, comparing means among treatments, are significantly different, according to the Tukey’ test (p < 0.05). Different letters represent a statistical difference for the same treatment between the two pH tested while ns represents not significant; The results in the Bars represent means, while error bar marks represent standard deviation ( n  = 4) from the mean value.

Root length and pH levels

The pH presented an interaction with nutrients. KCa treatment, presenting higher RL values (3.32, times, respectively) greater at pH 4.8 than at pH 6.5. The KMg, PCa and PMg treatments presented higher RL values (1.72, 2.14, 8,72 times, respectively) greater at pH 6.5 than at pH 4.8.

Root:shoot ratio (RSR) and nutrients

At low pH (4.8), nutrients produced three effects (i.e., synergistic, no-effect, and antagonistic effect) on RSR (Fig.  5 a). The treatments PCa, PKCa, PCaMg, KCa, CaMg, and PKCaMg presented high RSR indicating a synergistic effect comparing with the control. Phosphorus presented the lowest RSR value, characterized as an antagonistic effect compared to the control treatment. All other treatment presented no-effect because did not differ to the control (5.35 mm). At high pH (6.5), no-effect was observed among the treatments (Fig.  2 b), except for the P treatment that demonstrates an antagonistic effect compared with the control.

Root:seedling ratio (RSR) of Conyza canadensis in response to the nutrients (alone or in combination) under solutions at pH 4.8 ( a ) and at pH 6.5 ( b ). Bars with the different colours, comparing means among treatments, are significantly different, according to the Tukey’ test ( p  < 0.05). Different letters represent a statistical difference for the same treatment between the two pH tested while ns represents not significant; The results in the Bars represent means, while error bar marks represent standard deviation ( n  = 4) from the mean value.

Root:shoot ratio and pH levels

The high pH (6.5) presented higher RSR values for KMg, PMg, PCa, PK and, K and control treatments compared with the same nutrients at low pH (Fig.  5 b).

The principal component analysis (PCA)

The PCA represented the association degree among studied variables. The first and the second principal components of PCA explained 66.9% and 26.2% of the data variations, for pH 4.8; and 76.6% and 22.0% for pH 6.5 (Fig.  6 ). Principal component analysis pointed that frequently K alone or in combination with Ca and Mg had a high positive association (vectors in the same directions) with seed final germination percentage, and root length and shoot length of C. canadensis . However, P resulted in an antagonistic response for these variables (vectors in opposite directions). Phosphorus associated with the K or Mg was the most prominent nutrients generating contrasting plant responses for germination speed index and seedling development (shoot length) depending on the pH level. Phosphorus showed a greater antagonistic effect, which was a low seed germination percentage; however, P allows a good development of C. canadensis seedling. Potassium had a positive correlation with the final germination percentage and root length of C. canadensis seeds, especially under low pH.

Principal component analyses considering nutrients (alone and in combination) under solution at pH 4.8 and 6.5. G = Final germination percentage; GSI = germination speed index; SL = seedling length in their aerial part; RL = root length; Data set used for each pH level (n = 60; treatments × replications). The principal components (i.e., axes PC1 and PC2) explain the magnitudes in percentage of data variability.

Nutrients and pH as factors to seed germination traits and horseweed seedling development

Overall, the studied species had important requirements to germinate, considering the germination traits (FGP and GSI) verified in the control treatment (~ 36%), suggesting an average ability to complete germination. In the same way, the seedlings may develop in a wide range of chemical conditions (pH and nutrients). Then, our study reinforces the literature which this species can easily get establish itself in several topsoil conditions 8 . Two agronomic/environmental reasons can be emphasized as major issues concerning the horseweed management: i) this species has a low control in no-till system and has a great capacity to reduce crop yield 3 , 8 and, ii) the no-till system has been receiving annually large amounts of fertilizers (nutrients) and lime on topsoil 28 , regardless the consequences for the weeds’ communities. The present work argues on the early plant development stage (germination and seedling development) because it seems to be a crucial plant stage for better understand the occurrence and establishment of C. canadensis in cropland areas. The results evidenced that nutrients, alone or in combination, play a role in germination traits and development of C. canadensis , while pH had an influence only under interaction with certain nutrients (Figs.  1 , 2 , 3 , 4 , 5 ). Below, we will present a discussion based on the synergistic effect, no-effect, or antagonistic effect that nutrients and pH had on seed germination and the development of C. canadensis. We will discuss also the finding implications on weed management at field conditions.

Nutrient as factor conditioning horseweed germination and seedling development

In the literature, nitrogen is one of the most studied nutrients that affect the horseweed germination process 31 , 32 , 33 , while studies on other nutrients are scarce. Here, the findings clearly show that the exposure of horseweed seeds to different nutrients affected the final germination percentage and initial development of C. canadensis (Figs.  1 , 2 , 3 , 4 , 5 ). Furthermore, a large range of nutrient combinations had no-effect on the germination traits and development of horseweed. Phosphorus had an antagonistic effect on the following parameters: FGP, GSI, SL (at pH 4.8), and RSR for both pH (4.8 and 6.5). The P alone seems to inhibit germination and early horseweed development, probably due to the imbalance of nutrients in the seeds 34 . However, P exposure in combination with K or Ca and Mg allowed a synergistic effect on C. canadensis seedling development (SL and RL). Our study evidenced that P inhibited germination process, but the seedling development was busted with the P presence in combination with other nutrients.

The exposure of certain nutrients such as N, or metals such as Al, promotes a competition of nutrients out and inside the seeds, degrading the cell’ seeds 34 . However, when salt stress (Cl - ) was tested with different cations (Mg, Na, and Ca), the germination traits were also affected 20 , 21 , 35 . As horseweed seeds do not undergo dormancy 33 , the germination process starts immediately when seeds fall out on topsoil in any crop season 14 , 36 . Thus, the conditions surrounding the seeds become determinants to germination trait responses 37 . In addition, the same nutrient may act equally or differently on the C. canadensis germination and seedling development processes depending on the pH, as clearly evidenced here for P alone or in combination with others ions (Figs.  1 , 2 , 3 , 4 , 5 ). As nutrients and pH conditions affected the horseweed germination, several physiological mechanisms may be involved as follow: seed nutrient balance, physiological seed deterioration, osmotic stress, and nutrient toxicity 34 , 38 . However, these mechanisms need to be better explored in future studies to answer how nutrients act in the germination process. The answer requires an understanding of the seed germination response beyond temperature and moisture substrate conditions, considering ion competition and pH conditions that influence seed deterioration/germination 34 . It is known that chemical osmotic effects from nutrient exposure easily led to changes in metabolic and physiological seed behavior 14 . Woodstock et al. 39 found that seed quality was associated with seed capacity for ion releases, as a response to adverse conditions. The mechanism is then associated with the physical integrity of seed membranes, which ensures high germination potential but may be altered by nutrient availability of the growing media.

In our study, seeds exposed to different ions varied the final germination percentage and seedling development, sometimes having no-effect on the germination and sometimes decreasing when compared to the control (distilled water only). Phosphorus exposure presented an antagonist effect on germination probably because the ion balance affected other mandatory ions of physiological importance, such as Zn inside the seed, which is an important nutrient for the seed’s germination process 40 . In contrast to the P, other nutrients alone or combined with Ca, Mg and K presented no-effect on germination. Yamashita and Guimarães (2011a) found Al at 1.5 cmolc Kg of soil (low Al contents) diminishes C. canadensis germination by 24%. In addition, calcium chlorate affects C. canadensis germination and GSI in concentrations higher than 6 cmolc L -1 and 2 cmolc L -1 , respectively 20 . These studies suggest that seed responses depend on the ion companion in the salt molecule, as reinforced here. Finally, our study provided a large range of nutrients which produced different responses on germination and development of horseweed species.

The synergistic effect on RSR was observed when several nutrients were combined at low pH. When a large nutrient availability (P, K, Ca, Mg) is offered to the horseweed the plant allocates nutrients in the root system. Kuchenbuch & Jung (1988) stated that a non-restrictive nutrient condition reaches a high RSR – high root system comparatively with shoots. Thus, adequate soil/substrate chemical conditions tents to increase RSR. On the other hand, P availability had an antagonistic effect on RSR. It seems that P availability promoted a shoot allocation comparatively to the root allocation 41 .

Even under unfavorable edaphic conditions, horseweed development was assured when a large variety of nutrient was available except for P, that had an antagonistic effect. It reinforces that horseweed is able for surviving in adverse and eutrophic soil conditions, as reported by Concenço and Concenço (2016) 10 .

Acidity as factor conditioning horseweed germination and seedling development

Few studies were produced on horseweed testing chemical conditions (nutrients vs pH) on germination process 20 , 21 , 40 . The literature shows that horseweed germination is favored by neutral pH conditions even under saline conditions 18 , 20 , 21 . Here, two pH levels were tested, which pH affected only SL values as a single factor. However, pH presented interaction with some nutrients alone or in combination (K, Ca, P, and Mg). Low pH (4.8) associated with Ca presented higher values for FGP and GSI. Furthermore, seedling elongation seemed to be benefited at pH 4.8, regardless of the nutrient combination studied. At pH (6.5), PMg nutrients combination presented higher values for FGP and RL, SL and RSR than a pH 4.8. pH 6.5 associated with the nutrients reached high values for the majority parameter evaluated, excepting SL. In the control treatment (only with distilled water), GSI and RSR values were higher at 6.5 than at pH 4.8 while for SL was the value was higher at pH 4.8.

In the literature, different pH levels (4.7, 5.7, 6.7, and 7.7) and nitrogen concentration in eight species at Spain 35 (covered the following families: Fabaceae, Onagraceae, Apiaceae, Poaceae, and Polygonaceae) were tested and it was found that the germination process is greatly dependent of N amounts and forms, but is not associated with pH levels. The pH influences the germination process but depends on the species 29 . Laghmouchi et al. (2017) 31 25 , did not find evidence of pH effects on the seed germination of Origanum compactum and Capsella bursa-pastoris in a large range of pH. However, Gentili et al. (2018) 32 studying an Asteraceae species ( Ambrosia artemisiifolia ) suggests that low soil pH (5.0) affects positively the growth and development while neutral pH limited it. Furthermore, contrasting pH effects have been described on germination dependence on the species studied 31 , 42 . In this sense, the germination of many species responds rather to salt stress and nutrient availability than pH levels 43 .

Our findings indicate that pH is not a critical factor for germination traits when taken into account as an isolated factor, but in combination with nutrients assumes a relevant role. Thus, this study provides good knowledge on the effect of ions on the C. canadensis germination and seedling development, which allows us to plan adequate soil management to reduce the weed pressure in croplands since the knowledge in the literature is scarce 14 , 18 , 22 , 36 .

Findings implication for horseweed management on field conditions

Here, it was demonstrated that different ions affected the germination traits of C. canadensis . Light, temperature, and water availability were controlled in our laboratory’s experiment, varying only nutrients availability and pH conditions. In addition, the doses used for the treatments are compatible with the doses of Ca, Mg, P, and K normally receipt as fertilizers on topsoil from no-till system cropped with soybean. Thus, we expected that the findings found here can be a subsidiary under the field situation, concerning only the pH and nutrients availability.

In this sense, the P nutrient stood out as the most important nutrient in germination trait and seedling growth, and secondly, the pH was determinant mainly in combination with nutrients. Despite this study has been conducted under controlled conditions, our results showed that P alone or in combination with Ca inhibited horseweed seed germination percentage around 7.28 and 1.64 times compared to the control. However, after seed germination, the combination of P with other nutrients (eutrophic conditions) had a synergistic effect on seedling development. In addition, P alone had a reduction of 4.28 times in GSI.

From the agronomic perspective, our study suggests that topsoil chemical conditions should be taken into account to develop effective and integrated alternatives for C. canadensis control. Tudela-Isanta et al. (2018) stated that low pH promoting increases in soil aluminum contents, but pH is micro factor stress controlling seed germination niche in habitat management 44 . It is well-known that liming regulates soil acidity and by consequence species ability for land invasion 27 . Thus, the occurrence, distribution, and hazardousness of weed species may be handling partially by soil chemical factors 37 .

Our approach became mandatory for horseweed control in no-till systems because (i) the ecological and physiological species characteristics favor their occurrence in croplands 15 , including high soil seed bank 8 (ii) the low chemical horseweed control efficiency due to the herbicide resistance mechanisms of C. canadensis 4 , 12 , 13 and, (iii) the low quality of a major no-till system practiced, including low crop residue 3 , topsoil P and K fertilization, which produces an intense soil eutrophication 28 .

As seed germination is one of the main mechanisms of alien species to invade and establish on agricultural areas 43 , handling ecological aspects of this mechanism may aid plant management 23 . The final germination percentage of horseweed varied from 6 to 35.5% following the nutrient available. This behavior may indicate the amplitude in the control effectiveness associated with fertilizer management in no-till system. In general, the species had a high capacity for land invasion when the seeds find favorable conditions (adequate temperature, moisture, and chemical environment). Although the species produce large quantities of seeds that are easily disseminated 15 , an adequate chemical environment may contribute to diminish the weed establishment. In this sense, the fertilization method commonly used in no-till system areas for furnish Ca and Mg (under lime product), P and K (under triple superphosphate and potassium chloride) transformed the topsoil in eutrophic condition 28 . This information may answer why in areas under no-till system in Brazil there is a high abundance of this weed species 4 . Thus, the manner of fertilization and liming should be revisited because it may favor the occurrence and establishment of horseweed in this system.

Although our results were obtained in laboratory conditions, they suggest that the topsoil with low nutrient contents and low pH level consists in an adequate edaphic condition for reaching low germination percentage and speed germination of C. canadensis . Furthermore, we state that adequate fertilization in no-till areas may improve crop nutrition efficiency and may assist in effective weed control, reducing pesticide use, and improving environmental quality. In addition, a good NT system must be properly conducted, observing the adequate straw quantity and quality, a good crop rotation to minimizes soil disturbance diminishing seed quantities in the soil seed bank, which follows recommendations of several authors 2 , 39 , 45 . These ideas are not novelty; classical studies in phytosociology, in which species ecology and edaphic conditions control the occurrence of species in environments, are readily available 6 , 8 , 23 , 24 , 27 , 39 , 44 .

Finally, our results contribute to an integrative strategy in weed management, in which nutrient management may be used to diminishing horseweed germination in no-till areas that present a high germination percentage 8 , 45 . Thus, we expected that low horseweed germination results in low horseweed occurrence and pressure on crops, reducing environmental pressure 9 .

Conclusions and implications

This work corroborates the hypothesis in which the seed exposure to nutrients, alone or in combination, and different pH levels (4.8 and 6.5) are important factors controlling seed germination traits and seedling development of horseweed seeds. Regarding the nutrient effect, the phosphorus alone has an antagonistic effect on the final germination percentage of horseweed in both pH tested. However, nutrient richness, including P, surrounding horseweed seeds shows a synergistic effect the seedling development (root length and shoot length). Medium acidity (pH level) is a secondary factor influencing the germination traits, but pH 4.8 promotes horseweed seedling development (increasing shoot length). The pH is a preponderant factor when in interaction with certain nutrients, presenting a high germination speed index at pH 6.5 in the control treatment and associated with K and at pH 4.8, when in association with Ca, KMg, and PMg.

These findings suggest that chemical topsoil conditions favor or inhibit seed germination traits and seedling development of horseweed species. Thus, chemical edaphic status aid to explain the weed occurrence in agricultural areas. The implication of our findings fills a scientific gap and fits as a useful approach to integrate future strategies of weed control, improving the substantiality in modern agriculture.

Materials and methods

Local of study.

The present study was carried out under controlled conditions at the University of Passo Fundo, State of Rio Grande do Sul, Brazil and complies with local and national regulations. The experiment used Canadian horseweed seeds ( Conyza canadensis (L.) Cronquist var. canadensis) bought from a commercial farmer and distributer (Agrocosmos 13,165–970, São Paulo, Engenheiro Coelho, Brazil). The seeds were originated from the owner production (the year of 2018) at São Paulo State (tropical climate regime). The seeds were managed at the Seed Analysis Laboratory by the Passo Fundo University, with Accreditation in the ISO/IEC 17,025—general requirements for the competence of testing and calibration laboratories, under the responsibility of Dr. Nadia Canali Lângaro. An aliquot of seeds used in this study (~ 10 g) was deposited in an herbarium of the Program of Post-graduation in Agronomy of the University of Passo Fundo (504–302,224—2019), for public access. The seeds were then submitted to the germination process in different nutrient solutions, in an environmentally controlled chamber.

Experimental design and assay conditions

We used 14 nutrient solutions containing P, K, Ca, and Mg, alone and in combination 46 . These elements were chosen because they are normally used in topsoil liming and fertilization under no-till crop system. The nutrient quantities used in topsoil follow in decreasing order Ca, Mg, K, and P. Each solution was divided into two aliquots: the pH of the first aliquot was adjusted to 4.8 with chloric acid (HCl), and the pH of the second aliquot was adjusted to 6.5 with sodium hydroxide (NaOH). Thus, the trial was divided into two parts according to pH, with 14 treatments each, plus the control (distilled water) (Table 2). We assume that pH levels were maintained or had low variation during the assay. Additionally, all material used in the experiment was sterilized with ethanol (70%, v:v).

We prepared nutrient solutions according to recommended lime/fertilizer doses used in the field for soybean crop 47 . In the field soybean crop, the fertilizer formulas and doses commonly used are: potassium chloride (KCl) at the dose of 80 kg/ha, or 25.6 kg of K per ha, and triple superphosphate at the dose of 40 kg/ha, or 35.2 kg of P per ha. For Ca and Mg, the formulas and doses were based on the lime practice at 2 ton/ha of dolomitic limestone applied on the topsoil. Thus, the doses per hectare of Ca is 992 kg, while Mg is 592.2 kg. A more detailed explanation of the doses for each treatment is given in Table 2.

In the laboratory, the nutrient solutions were prepared using distilled water combined with phosphoric acid (H 3 PO 4 ) as a P source, potassium sulfate (K 2 SO 4 ) as a K source, calcium hydroxide (Ca(OH) 2 ) as a Ca source, and magnesium oxide (MgO) as a source of Mg. High chemical purity reagents were used (P.A. Merck Co). The doses were calculated taken into account the surface area of plastic boxes for germination test—gerbox (10 cm × 10 cm × 3.5 cm).

Germitest paper (J. Prolab, São José dos Pinhais, PR, Brazil) placed in gerbox plastic boxes received 10 mL of nutrient solutions and 50 horseweed seeds per replicate. These composed the experimental units, which were incubated, in quadruplicate, in controlled climate chambers (volume capacity: 354 L) for 15 days at 24 °C (± 0.5 °C) in light 12/12-h photoperiod (Light characteristics: four OL T8, 8 W, 6500 k led lamps). The boxes were covered using plastic film to maintain the similar moisture during the assay.

Plant analyses

Over 10 days, the gerbox treatments were daily monitored to estimate the final germination percentage (FGP) and germination speed index (GSI). The seeds with root length over 2 mm were considered as a seed that accomplished their germination process and were recorded daily. The experiment was conducted based on the protocol proposed by the official seed analysis from the Brazilian government 48 .

The final germination percentage was calculated following Eq. ( 1 ), and is expressed as a percentage. The germination speed index (GSI) was evaluated using Eq. ( 2 ).

where FGP is the total seeds that accomplished their germination process (Ʃn 1 ) in relation to the total of seeds used in the assay (NS i );

where, G 1 , G 2 , G n are the number of seeds that accomplished their germination process for the day and N 1 , N 2 , N n are the number of days after the assay start.

Seedling shoot length (SL) and root length (RL) were evaluated at the end of the experiment (15 days after the start of the assay). The root:shoot ratio values were calculated using data of RL and SL.

Data analysis

A two-way analysis of variance, ANOVA ( P  < 0.05) was performed in a bi-factorial randomized design (14 nutrient treatments + control vs 2 pH) with post-hoc Tukey’s range test ( P  < 0.05) where all means of treatments were compared together 49 .

Statistical analysis was performed on R-Studio (version 3.6.1, 2019-07-05), an open-source software, and graphics were created on Sigmaplot (version 13). The normality of the distribution of residuals was tested by a P -value < 0.05 in a Shapiro–Wilk test, and the homogeneity of variances by a P- value < 0.05 in a Bartlett test. The Box-cox technique was employed to transform residuals from a non-normal distribution to a normal distribution. Principal component analysis (PCA) was performed in the dataset at P  < 0.1, for each pH level separately, using open-source software Past (version 3.23). The principal components (axes PC1 and PC2) explain the magnitudes in the percentage of data variability.

The data is presented to show which nutrients (alone or in combination) present a synergic, no effect, or antagonist effect compared with the control (distilled water). Thus, the synergistic effect is when average values were statistically higher than control treatment; no-effect means that there was no statistical difference, and antagonistic effect means low values compared with the control treatment.

Ethics approval

This manuscript consists of original research that has not been published before and is not currently being considered for publication elsewhere. Besides, the manuscript has been read and approved by all named authors.

Consent to participate and for publication

The authors consent for the participation in this publication.

Data availability

We supplied all data. All data used in this study were included in the Supplementary Information File.

Abbreviations

C. canadensis

Final germination percentage

Germination speed index

Root length

Seedling length in the aerial part

Shoot ratio

No-till system

Dainese, M. et al. A global synthesis reveals biodiversity-mediated benefits for crop production. Sci. Adv. 5 , eaax0121. https://doi.org/10.1126/sciadv.aax0121 (2019).

Article   PubMed   PubMed Central   ADS   Google Scholar  

Nunes, M. R. et al. Soil chemical management drives structural degradation of Oxisols under a no-till cropping system. Soil Res. 55 , 819–831 (2017).

Article   CAS   Google Scholar  

Bhowmik, P. C. & Bekech, M. M. Horseweed ( Conyza canadensis ) seed production, emergence and distribution in no-tillage and conventional-tillage corn ( Zea mays ). Agronomy 1 , 67–71 (1993).

Google Scholar  

Rizzardi, M. A., Schneider, T., Bianchi, M. A. & Rockenbach, A. P. Occurrence of horseweed biotypes with low susceptibility to glyphosate in the states of Rio Grande do Sul, Paraná and Mato Grosso do Sul, Brazil. Planta Daninha 37 , e019201666. https://doi.org/10.1590/s0100-83582019370100093 (2019).

Article   Google Scholar  

Bortoluzzi, E. C., dos Santos, D. R., Santanna, M. A. & Caner, L. Mineralogy and nutrient desorption of suspended sediments during a storm event. J. Soils Sedim. 13 , 1093–1105. https://doi.org/10.1007/s11368-013-0692-4 (2013).

Rejmánek, M., Richardson, D.M. & Pyšek, P. Plant invasions and invasibility of plant communities. In: Vegetation Ecology. Eddy van der Maarel & Janet Franklin (eds.) 387–424. https://doi.org/10.1002/9781118452592.ch13 (2013).

Dehnavi, A. R., Zahedi, M., Ludwiczak, A., Stefany Cardenas Perez, S. C. & Piernik, A. Effect of salinity on seed germination and seedling development of sorghum ( Sorghum bicolor (L.) Moench) genotypes. Agronomy 10 , 859. https://doi.org/10.3390/agronomy10060859 (2020).

Weaver, S. E. The biology of Canadian weeds. 115. Conyza canadensis. Can. J. Plant Sci. 81 , 867–875 (2001).

Trezzi, M. M. et al. Competitive ability of soybean cultivars with horseweed ( Conyza bonariensis ). Planta Daninha 31 (3), 543–550. https://doi.org/10.1590/S0100-83582013000300006 (2013).

Concenço, G. & Concenço, S. E. Conyza spp.: from ugly duckling to agriculture’s fittest swan—brief review. Pl. Dan. 34 (1), 183–189. https://doi.org/10.1590/S0100-83582016340100019 (2016).

Nandula, V. K., Poston, D. H., Koger, C. H., Reddy, K. N. & Reddy, K. R. Morpho-physiological characterization of glyphosate-resistant and-susceptible horseweed ( Conyza canadensis ) biotypes of US midsouth. Am. J. Plant Sci. 6 , 47–56. https://doi.org/10.4236/ajps.2015.61006 (2015).

Dinnelli, G. et al. Physiological and molecular insight on the mechanisms of resistance to glyphosate in Conyza canadensis (L.) Cronq. Biotypes. Pest. Biochem. Phys. 86 , 30–41 (2006).

Alcorta, M., Fidelibus, M. W., Steenwerth, K. L. & Shrestha, A. Competitive effects of glyphosate-resistant and glyphosate-susceptible horseweed ( Conyza canadensis ) on young grapevines ( Vitis vinifera ). Weed Sci. 59 , 489–494. https://doi.org/10.1614/WS-D-10-00186.1 (2011).

Yamashita, O. M. & Guimaraes, S. C. Germinação das sementes de Conyza canadensis e Conyza bonariensis em função da disponibilidade hídrica no substrato. Planta Daninha 28 , 309–317. https://doi.org/10.1590/S0100-83582010000200010 (2010).

Dauer, J., Mortensen, D. A. & VanGessel, M. Spatial and temporal dynamics governing long distance dispersal of Conyza canadensis . J. Appl. Ecol. 44 , 105–114 (2007).

Dauer, J., Mortensen, D. A. & Humston, R. Controlled experiments to predict horseweed ( Conyza canadensis ) dispersal distances. Weed Sci. 54 , 484–489. https://doi.org/10.1614/WS-05-017R3.1 (2006).

Buhler, D. D. & Owen, M. D. K. Emergence and survival of horseweed ( Conyza canadensis ). Weed Sci. 45 , 98–101 (1997).

Nandula, V. K., Eubank, T. W., Poston, D. H., Koger, C. H. & Reddy, K. N. Factors affecting germination of horseweed ( Conyza canadensis ). Weed Sci. J. 54 , 898–902 (2006).

Andersen, M. C. Diaspore morphology and seed dispersal in several wind dispersed Asteraceae . Am. J. Bot. 80 , 487–492 (1993).

Yamashita, O. M. & Guimarães, S. C. Efecto del estrés salino sobre la germinación de las semillas de Conyza canadensis y Conyza bonariensis . Bioagro 23 , 169–174 (2011).

Yamashita, O. M. & Guimarães, S. C. Germinação de sementes de Conyza canadensis e Conyza bonariensis em função da presença de alumínio no substrato. Ci. Rural. 41 , 599–601. https://doi.org/10.1590/S0103-84782011000400008 (2011).

Bewley, D. J. Seed germination and dormancy. Plant Cell 9 , 1055–1066 (1997).

Braun-Blanquet, J. Fitosociologia: bases para el estudio de las comunidades vegetales 3rd edn. (Aum. Blume, 1979).

Taylorson, R. B. Environmental and chemical manipulation of weed seed dormancy. Weed Sci. 3 , 135–154 (1987).

CAS   Google Scholar  

Kołodziejek, J. Effect of seed position and soil nutrients on seed mass, germination and seedling growth in Peucedanum oreoselinum (Apiaceae). Sci. Rep. 7 , 1959. https://doi.org/10.1038/s41598-017-02035-1 (2017).

Article   CAS   PubMed   PubMed Central   ADS   Google Scholar  

Kołodziejek, J. Growth and competitive interaction between seedlings of an invasive Rumex confertus and of co-occurring two native Rumex species in relation to nutrient availability. Sci. Rep. 9 , 3298. https://doi.org/10.1038/s41598-019-39947-z (2019).

Burke, M. J. W. & Grime, J. P. An experimental study of plant community invasibility. Ecology 77 , 776–790. https://doi.org/10.2307/2265501 (1996).

Rheinheimer, D. S., Gatiboni, L. C. & Kaminski, J. Fatores que afetam a disponibilidade do fósforo e o manejo da adubação fosfatada em solos sob sistema plantio direto. Ci. Rural 38 , 576–586 (2008).

Tudela-Isanta, M., Ladouceur, E., Wijayasinghe, M., Pritchard, H. W. & Mondoni, A. The seed germination niche limits the distribution of some plant species in calcareous or siliceous alpine bedrocks. Alp. Bot. 128 , 83–95. https://doi.org/10.1007/s00035-018-0199-0 (2018).

Leishman, M. R. & Thomson, V. P. Experimental evidence for the effects of additional water nutrients and physical disturbance on invasive plants in low fertility Hawkesbury Sandstone soils Sydney Australia. J. Ecol. 93 , 38–49. https://doi.org/10.1111/j.1365-2745.2004.00938.x (2005).

Laghmouchi, Y., Belmehdi, O., Bouyahya, A., Senhaji, N. S. & Abrini, J. Effect of temperature, salt stress and pH on seed germination of medicinal plant Origanum compactum . Bio. Agric. Biotec. 10 , 156–160. https://doi.org/10.1016/j.bcab.2017.03.002 (2017).

Gentili, R., Ambrosini, R., Montagnani, C., Caronni, S. & Citterio, S. Effect of soil pH on the growth, reproductive investment and pollen allergenicity of Ambrosia artemisiifolia L. Front. Plant Sci. 9 , 1335. https://doi.org/10.3389/fpls.2018.01335 (2018).

Article   PubMed   PubMed Central   Google Scholar  

Lazaroto, C. A., Fleck, N. G. & Vidal, R. A. Biologia e Ecofisiologia de buva ( Conyza bonariensis e Conyza canadensis ). Rev. Ci. R. 38 (3), 852–860 (2008).

Ebone, L. A., Caverzan, A. & Chavarria, G. Physiologic alterations in orthodox seeds due to deterioration processes. J. Plant Physiol. Biochem. 145 , 34–42. https://doi.org/10.1016/j.plaphy.2019.10.028 (2019).

Pérez-Fernández, M. A., Calvo-Magro, E., Montanero-Fernández, J. & Oyola-Velasco, J. A. Seed germination in response to chemicals: effect of nitrogen and pH in the media. J. Environ. Biol. 27 (1), 13–20 (2006).

PubMed   Google Scholar  

Wu, W. et al. Germination persistence and emergence of flaxleaf fleabane ( Conyza bonariensis [L.] Cronquist). Weed Biol Manag 7 , 192–199. https://doi.org/10.1111/j.1445-6664.2007.00256.x (2007).

Freijsen, A. H. J., Troelstra, S. R. & Van Kats, M. J. The effect of soil nitrate on the germination of Cynoglossum officinale L. (Borraginaceae) and its ecological significance. Acta Oecol./Oecol Plant. 1 , 71–79 (1980).

Walters, C., Ballesteros, D. & Vertucci, V. A. Structural mechanics of seed deterioration: standing the test of time. Plant Sci. 179 , 565–573. https://doi.org/10.1016/j.plantsci.2010.06.016 (2010).

Woodstock, L. W., Furman, K. & Leffler, H. R. Relationship between weathering deterioration and germination, respiratory metabolism, and mineral leaching from cotton seeds. Crop Sci. 25 , 459–466. https://doi.org/10.2135/cropsci1985.0011183X002500030008x (1985).

Ajouri, A., Asgedom, H. & Becker, M. Seed priming enhances germination and seedling growth of barley under conditions of P and Zn deficiency. J. Plant Nutr. Soil Sci. 167 , 630–636 (2004).

Kuchenbuch, R. & Jung, J. Changes in root-shoot ratio and ion uptake of maize ( Zea mays L.) from soil as influenced by a plant growth regulator. Plant Soil 109 , 151–157 (1988).

Rezvani, M. & Zaefarian, F. Hoary cress ( Cardaria draba (L.) Desv.) seed germination ecology, longevity and seedling emergence. P. Sp. Biol. 31 , 280–287. https://doi.org/10.1111/1442-1984.12113 (2016).

Rezvani, M., Zaefarian, F. & Amini, V. Effects of chemical treatments and environmental factors on seed dormancy and germination of shepherd’s purse ( Capsella bursa-pastoris (L.) Medic.). Acta Bot. Bras. 28 (4), 495–501. https://doi.org/10.1590/0102-33062014abb3337 (2014).

Tudela-Isanta, M., Ladouceur, E., Wijayasinghe, M., Pritchard, H. W. & Mondoni, A. The seed germination niche limits the distribution of some plant species in calcareous or siliceous alpine bedrocks. Alp. Bot. 128 (4), 83–95. https://doi.org/10.1007/s00035-018-0199-0 (2018).

Norsworthy, J. K. et al. Reducing the risks of herbicide resistance: best management practices and recommendations. Weed Sci. 60 , 31–62. https://doi.org/10.1614/WS-D-11-00155.1 (2012).

Hoagland, D.R. & Arnon, D.I. The water culture method for growing plants without soil. Berkeley: California Agricultural Experiment Station. Circular 347 (1950).

CQFS-Comissão de Química e Fertilidade do Solo Manual de calagem e adubação para os Estados de Rio Grande do Sul e de Santa Catarina. Viçosa, Sociedade Brasileira de Ciência do Solo (2016).

Brasil, Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Brasília: MAPA (2009).

Canteri, M. G., Althaus, R. A., Virgens Filho, J. S., Giglioti, E. A. & Godoy, C. V. SASM-Agri: sistema para análise e separação de médias em experimentos agrícolas pelos métodos Scoft-Knott, Tukey e Duncan. Revista Brasileira de Agrocomputação 1 (2), 18–24 (2001).

Derevyanchuk, M. et al. Seed germination, respiratory processes and phosphatidic acid accumulation in Arabidopsis diacylglycerol kinase knockouts—the effect of brassinosteroid, brassinazole and salinity. Steroids 147 , 28–36. https://doi.org/10.1016/j.steroids.2019.04.002 (2019).

Article   CAS   PubMed   Google Scholar  

Sardans, J. & Peñuelas, J. Potassium: a neglected nutrient in global change. Global Ecol. Biogeogr. 24 , 261–275 (2015).

Download references

Acknowledgements

We thank the National Council for Scientific and Technological Development—CNPq [Brasilia/Brazil: 304676/2019-5 for ECB] and Coordination of Superior Level Staff Improvement—CAPES by the PROSUC fellowships received by APH.

CNPq [Brasilia Brazil: 304676/2019-5; ECB fellowship] and CAPES by the fellowships received [CAPES/PROSUC fellowship].

Author information

Authors and affiliations.

University of Passo Fundo, Campus I, BR 285, km 292, Passo Fundo, Rio Grande do Sul, 99052-900, Brazil

Caroline Maldaner Follmer

Postgraduate Program in Agronomy, University of Passo Fundo, Campus I, BR 285, km 292, Passo Fundo, Rio Grande do Sul, 99052-900, Brazil

Ana Paula Hummes, Nadia Canali Lângaro & Claudia Petry

Federal Institute of Education, Science and Technology of Rio Grande Do Sul, Osvaldo Aranha, Bento Gonçalves, Rio Grande do Sul, 540, 995700-000, Brazil

Diovane Freire Moterle

Laboratory of Land Use and Natural Resources, University of Passo Fundo, Campus I, BR 285, km 292, Passo Fundo, Rio Grande do Sul, 99052-900, Brazil

Edson Campanhola Bortoluzzi

You can also search for this author in PubMed   Google Scholar

Contributions

C.M.F. and E.C.B. were responsible for the design of the work; C.M.F., A.P.H., and E.C.B. wrote the main manuscript text; A.P.H. contributed with the statistical analyses; N.C.L. gave supporting in data acquisition and, seed/seedling analysis; D.F.M. and C.P. prepared data, interpretation of data, and aid in discussion wrote. All authors reviewed the manuscript.

Corresponding author

Correspondence to Edson Campanhola Bortoluzzi .

Ethics declarations

Competing interests.

The authors declare no competing interests.

Additional information

Publisher's note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary information., rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Follmer, C.M., Hummes, A.P., Lângaro, N.C. et al. Nutrient availability and pH level affect germination traits and seedling development of Conyza canadensis . Sci Rep 11 , 15607 (2021). https://doi.org/10.1038/s41598-021-95164-7

Download citation

Received : 13 December 2020

Accepted : 19 July 2021

Published : 02 August 2021

DOI : https://doi.org/10.1038/s41598-021-95164-7

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

This article is cited by

Agro-industrial-residues as potting media: physicochemical and biological characters and their influence on plant growth.

• Pratibha Agarwal
• P. Hariprasad

Biomass Conversion and Biorefinery (2023)

By submitting a comment you agree to abide by our Terms and Community Guidelines . If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

WATER Latest WATER Information News

Exploring the relationship between water and plant growth, introduction.

Welcome to this article on how water affects plant growth! As we all know, water is one of the most important resources for plant growth and development. But have you ever wondered exactly how water impacts plants, and what happens when plants don’t get enough or get too much water?

In this article, we’ll explore the science behind plant-water relationships, the advantages and disadvantages of different watering techniques, and provide you with actionable tips for watering your plants to promote healthy growth. So, let’s dive right in!

Why is Water So Important for Plant Growth?

Water is one of the primary components in plant cells, and it’s needed for a variety of physiological processes, including photosynthesis, metabolism, and nutrient uptake.

In fact, water makes up about 90% of the weight of a plant, so it’s no surprise that it’s crucial for growth and survival.

Additionally, water helps maintain the turgor pressure in plant cells, which gives them the necessary stiffness to remain upright. Without enough water, plants can wilt and eventually die.

With that said, let’s explore exactly how water impacts plant growth.

How Does Water Affect Plant Growth?

Water and photosynthesis.

One of the primary ways that water affects plant growth is through photosynthesis. Photosynthesis is the process by which plants use light energy to convert carbon dioxide and water into glucose and oxygen.

Without enough water, plants can’t carry out photosynthesis efficiently, which can lead to stunted growth, yellowing leaves, and even death in severe cases.

On the other hand, too much water can also hinder photosynthesis by blocking the stomata (small openings on leaves that allow gases to enter and exit). When the stomata are blocked, gases can’t move in and out efficiently, which can prevent photosynthesis from occurring.

Water and Nutrient Uptake

Another way water impacts plant growth is through nutrient uptake. Plants require a variety of nutrients to grow, including nitrogen, phosphorus, and potassium.

Water plays a critical role in delivering these nutrients to the plant’s roots, where they can be absorbed and used. Without enough water, nutrient uptake can be limited, which can lead to stunted growth and nutrient deficiencies.

However, too much water can also be detrimental to nutrient uptake. When the soil is waterlogged, oxygen levels in the root zone can be reduced, which can lead to root damage and impaired nutrient uptake.

Water and Root Growth

Water is also essential for root growth and development. As plants absorb water, it creates a pressure gradient in the roots that helps push them deeper into the soil.

Additionally, water is needed for the formation of new root cells and the maintenance of existing ones. Without enough water, root growth can be stunted, which can limit overall plant growth and development.

However, too much water can also be harmful to root growth. When the soil is overly saturated, it can lead to root rot and damage, which can ultimately kill the plant.

Water and Transpiration

Finally, water is important for transpiration, which is the process by which water evaporates from the leaves of plants.

Transpiration helps regulate temperature within the plant, prevent wilting, and move nutrients and water up from the roots. Without enough water, transpiration rates can be reduced, which can lead to wilting and other issues.

However, excessive water can also hinder transpiration rates by increasing humidity levels around the plant and reducing the need for transpiration to cool the plant.

The Advantages and Disadvantages of Different Watering Techniques

Watering from above.

Watering from above is one of the most common methods of watering plants. This involves pouring water directly onto the soil and letting it soak in.

Advantages: This method is quick and easy, and it allows for a good amount of control over the amount of water the plant receives.

Disadvantages: With this method, it’s easy to overwater or underwater plants, and it can lead to water running off or evaporating before it has a chance to soak in. Additionally, watering from above can encourage the growth of fungal diseases, as the leaves and stems can stay wet for extended periods.

Drip Irrigation

Drip irrigation involves using a network of tubes and emitters to deliver water directly to the base of plants.

Advantages: This method is highly efficient, as it delivers water directly to the roots where it’s needed most. It also saves water by minimizing runoff.

Disadvantages: Drip irrigation systems can be expensive to install, and they require regular maintenance to ensure that they’re working properly. Additionally, they can be slow to deliver water, which may not be ideal for plants that need frequent watering.

Soaker Hoses

Soaker hoses are similar to drip irrigation systems, but they’re made of porous material that allows water to seep out along the entire length of the hose.

Advantages: Soaker hoses are relatively inexpensive and easy to install. They’re also highly efficient, as they deliver water directly to the roots where it’s needed.

Disadvantages: Soaker hoses can be prone to clogging, and they may not work well in areas with hard water or high mineral content. They also require regular cleaning and maintenance to prevent fungal growth.

Bottom Watering

Bottom watering involves placing plants in a tray filled with water and allowing them to soak up water through the root system.

Advantages: Bottom watering is a gentle and efficient way to water plants, and it ensures that the water is delivered directly to the roots where it’s needed most.

Disadvantages: This method can be time-consuming, as it requires waiting for the plants to soak up water. It also requires close monitoring to ensure that the plants aren’t sitting in water too long, which can lead to root rot and other issues.

Exploring the Relationship Between Water and Plant Growth: A Complete Table

Frequently Asked Questions

What happens if plants don’t get enough water.

Without enough water, plants can’t carry out photosynthesis efficiently, nutrient uptake can be limited, and root growth can be stunted. In severe cases, plants can wilt and die.

What happens if plants get too much water?

Excessive water can lead to blocked stomata, impaired photosynthesis, and reduced nutrient uptake. It can also lead to root rot and damage, which can ultimately kill the plant.

How often should I water my plants?

The frequency of watering will depend on the type of plant, the environment it’s in, and the type of soil. As a general rule, most plants prefer to be watered when the top inch of soil is dry.

Is it better to water plants in the morning or evening?

It’s generally better to water plants in the morning, as this gives them time to dry off before the cooler evening temperatures set in. Wet leaves and stems can encourage fungal growth, so it’s best to avoid watering in the evening if possible.

Can I reuse water from cooking and other household activities to water my plants?

Yes, you can reuse water from cooking and other household activities to water your plants. However, you should avoid using water that’s high in salt or other minerals, as this can be harmful to plants over time.

Can I water my plants too much?

Yes, it’s possible to overwater plants. This can lead to waterlogged soil, root rot, and other issues. It’s important to monitor soil moisture levels and avoid watering plants excessively.

Can I water my plants with cold water?

While it’s best to water plants with room temperature or slightly warm water, cold water won’t harm them as long as it’s not too cold. Extremely cold water can shock plants, so it’s best to avoid using water straight from the tap if it’s very cold.

Should I water my plants from above or below?

The best method of watering will depend on the type of plant and the environment it’s in. However, bottom watering and drip irrigation are generally considered to be the most efficient and effective methods.

How can I tell if my plants need water?

The easiest way to tell if your plants need water is to check the soil moisture levels. Stick your finger into the soil up to the second knuckle. If the soil feels dry at this depth, then it’s time to water.

Can I water my plants with tap water?

Yes, tap water is typically safe to use for watering plants. However, if your tap water is high in minerals or other contaminants, it’s best to use a water filter or choose a different water source.

How long should I water my plants for?

The duration of watering will depend on the type of plant and the watering method you’re using. As a general rule, water until the soil is moist but not waterlogged.

How much water do plants need?

The amount of water plants need will depend on their stage of growth, the type of plant, and the environment they’re in. As a general rule, most plants need about 1-2 inches of water per week.

Can I water my plants with ice cubes?

While it’s possible to water plants with ice cubes, it’s not the most efficient method. The ice cubes will take longer to melt and won’t distribute water evenly. It’s best to stick with traditional watering methods.

What should I do if I overwater my plants?

If you overwater your plants, you can try to remove excess water from the soil and move the plant to a more ventilated area. You may also need to cut back on watering until the soil dries out.

We hope that this article has given you a better understanding of the relationship between water and plant growth, as well as the advantages and disadvantages of different watering techniques. Remember to monitor soil moisture levels and avoid over- or under-watering your plants. With the right amount of water and care, your plants will thrive and grow strong!

Take Action Now

If you’re ready to start improving your plant’s growth today, start by assessing your current watering methods and making adjustments as needed. Take the time to understand your plant’s needs and the environment it’s in, and always be mindful of soil moisture levels.

While we’ve done our best to provide accurate and up-to-date information in this article, we cannot take responsibility for any damages or losses that may occur as a result of following the advice provided. Always consult with a professional if you’re unsure about any aspect of plant care.

Watch Video:Exploring the Relationship Between Water and Plant Growth

An official website of the United States government

Here's how you know

Official websites use .gov A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS A lock ( Lock Locked padlock ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

Plant Growth and Osmotic Potential

Water is a critical element for plant growth. All water used by land plants is absorbed from the soil by roots through osmosis. Osmosis is the movement of a solvent (e.g.water) across a semipermeable membrane from low solute (e.g.salt) concentration towards higher solute concentration. Excess levels of salts in soils makes soil water solute concentrations higher than in the plant root cells. This can limit plant water uptake, making it harder for plants to grow. (See Appendix A for more information)

About the Experiment

For this experiment, we’re going to test the effect that high salt soil concentrations have on plant growth and root development.

 What You'll Need

• 7 clear plastic cups (Solo cups)
• 7 non-clear plastic cups
• Potting soil (small bag)
• Wheatgrass or cat grass seed (250 seeds, can be found online or at local pet store)
• Baking soda
• Measuring spoons
• Drill & small bit

When using table salt (sodium chloride) and baking soda (sodium bicarbonate) to create saline and alkali soils, you can observe the germination and growth of grass leaves at increasing levels of salt and ph. Then you can treat the salt/alkali effected soils with "leaching" and observe plant growth.

Let's Do This!

1 . Drill 3 small holes in 7 clear plastic cups. Have an adult help with this step for safety.

2 . Fill 1 clear cup (with holes) with soil 1” from top of cup and place cup inside non-clear cup (without holes).

Pour ½ cup of water into the soil cup and allow to absorb. Pour another ½ cup of water into the soil cup.

Place 30 grass seeds on top of the wetted soil and cover with 1/8” of new soil and gently wet. Make sure seeds are covered with soil (Label cup “Control”).

3 . Fill 3 clear cups (with holes) with soil 1” from top. Add 1 teaspoon of salt to the soil of 1 cup (label cup “salt 1”). Add 1 tablespoon of salt to the 2nd cup (label cup “salt 2”). Add 3 tablespoons of salt to the 3rd cup (label cup “salt 3”).

Place each cup in a non-clear cup (no holes) and add ½ cup of water to each and let absorb. Add another ½ cup of water.

Place 30 grass seeds in each cup and cover with 1/8” of new soil and moisten new soil. Make sure seeds are covered with soil (Image 2).

4 . Fill 3 clear cups (with holes) ¼ full with soil. Add 1 tablespoon of baking soda to 1st cup and add more soil to fill cup 1” from the top. Hold your hand over the cup so soil does not spill and shake the cup to mix the baking soda with the soil (label cup “alkali 1”).

Add 2 tablespoons of baking soda to the 2nd cup and fill with soil 1" from top. Again, with hand over cup, shake to mix baking soda and soil (label cup “alkali 2”).

Add ½ cup of baking soda to the 3rd cup, fill with soil 1" from top and shake to mix (label cup “alkali 3”).

Place each cup in a non-clear cup (no holes). Add ½ cup of water to each and let absorb, then add another ½ cup of water. Place 30 grass seeds in each cup and cover with 1/8" of new soil and moisten new soil. Make sure seeds are covered with soil.

5 . Let grass germinate and grow for 1 week.

Let’s Look At The Results!

After 1 week count the number of plants in each cup and measure the tallest blades of grass in each cup. Record the numbers for each on the data sheet . Remove the clear cups and observe root growth.

After 1 week, remove “salt 2” and “alkali 2” clear cups from red cups and place in the sink or outside (where water can drain) and slowly pour 6 cups of water through each, making sure to not over-fill (pour ½ cup at a time and let drain).

Observe which cups drains fastest (alkali soils have poor drainage). Make sure seeds are still covered with soil (add some on top if necessary) and let them grow for 1 more week.

After 1 week (2 weeks total) observe if “leached” cups now have plants that are growing. Did leaching help the same for saline vs. alkali soils?

After 2 weeks , measure the height of plants in each cup and record the results. Again, observe the roots and record observations on the data sheet.

Summarize your data and observations.

• Why did plants grow or not grow in each cup?
• What effect did leaching have on plant growth and why?
• Did leaching work on both salt and baking soda equally and why?

• / Science
• / Physics
• / Science Projects

Intermediate-Level Science Projects: Does the pH of Water Affect the Growth of Bean Plants?

• Does the pH of Water Affect the Growth of Bean Plants?

Intermediate-Level Science Projects

• Creating New Plants Through Cross-Pollination
• Do Bean Plants Grow Better in Soil or in Water?

In This Section

• Understanding acids and bases
• The effects of positive and negative ions
• Different soils for different plants
• Finding the materials you'll need
• Guarding against contamination
• Considering other botany projects

Although this section is classified as a botany project, you'll notice as you work through it that it contains a fair amount of chemistry, as well.

It's not unusual for scientific areas, or disciplines, to cross over in the course of a project or experiments. This project is a good example of how that occurs.

You've probably heard of the pH scale, or heard someone talk about the pH factor of a particular material. But what is pH exactly, and how does it affect the growth of plants?

In this section, we'll explore the basics of pH, and experiment to learn how the pH factor of liquid affects the germination and growth of bean seeds. By the time you finish, you'll have had valuable lessons in both botany and chemistry, and have a better understanding of how branches of science overlap.

So What Seems to Be the Problem?

You know that plants need certain things to help them grow. They need some kind of growing medium, usually dirt. They need light, and they need water.

The problem you'll attempt to solve while doing this science fair project is whether the pH of the water with which plants are sprinkled affects the rate of growth.

To get a better idea of what you'll be doing, and to help you formulate a hypothesis, it's important that you have a general understanding of exactly what pH is.

The initials pH stand for percent hydronium ion. The pH scale is used as a measure of how acidic or basic a liquid is. But how do liquids become acidic or basic? Isn't a liquid just a liquid?

Basic Elements

An ion results from the loss or gain of one or more electrons from an atom, causing either positive or negative ions to form.

Water€”and distilled water, at that€”is the only liquid that is neutral. That means it's right in the middle of being acidic or basic€”and it's neither. It's just pure water.

The pH scale starts at zero and ends at 14. The more acidic a liquid is, the lower its number on the pH scale. The less acidic€”or more basic a liquid is€”the higher its number.

Most of the liquids you encounter on a daily basis are just around neutral. They might be a little above or a little below, but most liquids tend to be closer to neutral than at either end of the pH scale.

Liquids get their pH level as a result of molecules that split apart to form positive and negative ions. An ion is the loss or gain of electrons from an atom. When an atom loses electrons, it forms a positive ion. When an atom gains electrons, it forms a negative ion.

Liquids will be either acidic or basic (also called alkaline), depending on whether they contain positive or negative ions. If there are more positive ions in the water, the water is more acidic. If there are more negative ions in the water, the liquid is more basic.

In this experiment, you'll control the pH of the water you'll use on bean plants by adding certain substances to make distilled water either acidic or basic. You'll also control all other factors, such as how much water and light each plant gets.

Scientific Surprise

A negative ion, formed when an atom gains electrons, is also called an anion. A positive ion, formed when an atom loses electrons, is also known as a cation.

If you want to, you can use the name of this section, €œDoes the pH of Water Affect the Growth of Bean Plants?€ as the title for your project. Other names to consider might be:

• What Type of Soil Do Bean Plants Prefer?
• Acid or Alkaline€”What's Right for Bean Plants?
• To Grow or Not to Grow: Acidic vs. Alkaline Soil for Bean Plants

When you've finished with the experiment, you'll know whether bean plants prefer water that is acidic or basic.

What's the Point?

Some plants prefer acidic conditions. We call these acid-loving plants. Acid-loving plants include the following:

• Holly, pine, fir, spruce, birch, oak, magnolia, willow, and flowering crabapple trees
• Rhododendrons and azaleas
• Cranberry, strawberry and blueberry plants
• Mountain laurel

Other plants, however, such as the ones listed here, prefer alkaline soil:

• Yew, boxwood, and barberry shrubs
• Flowering plum and cherry trees
• Ash, beech, filbert, and maple trees
• Mock orange

Gardeners often help plants along by making the soil in which they grow either more acidic or more alkaline. There are products available, such as Miracid, that boost the acidity of soil. Garden lime (its chemical name is calcium carbonate) will help make soil alkaline.

In the experiment described below, you'll use distilled water as your control, and water with varying pH levels as your variables. This will allow you to observe the effects that liquids of varying pH levels have on the bean plants.

Who knows? You may end up increasing your interest in, or developing an interest in, gardening through this project. If nothing else, it will give you a better understanding of how plants grow and what types of factors affect them.

What Do You Think Will Happen?

If you've had experience with gardening and growing plants, you know that plants react differently to all sorts of factors.

Some plants like to be watered frequently, while others like to wait for a drink until the soil is completely dry. Some are much more susceptible to heat or cold than others. Some plants thrive in sunlight, while others like shady conditions. You've already read that some plants prefer an acidic soil, while others like basic soil.

You can do some research about growing bean plants to help you form a hypothesis. If you don't have much experience with plants, or don't have a good understanding of pH, it probably would be beneficial for you to learn more about growing plants, different types of soil, and so forth.

Or you can simply consider what you may already know about growing plants and make an educated guess about what will happen to the bean plants with which you'll be working.

Materials You'll Need for This Project

One thing about this experiment is that it's going to take some advance planning and a significant amount of time. You'll need almost a month from the time you plant the seeds until the time you draw final conclusions about the growth of the plants.

Standard Procedure

Online sources are available for discounted pet supply products.

You also will need a material or two with which you may not be familiar. Most of the materials you'll need though, are common household items. You'll need:

• A substance used to adjust the pH level of water. We suggest a set of products called pH Up and pH Down, a brand that's readily available in pet supply stores. You'll need a bottle of each pH Up and pH Down. Retail cost is about $3.50 a bottle.
• pH test strips or test kit. You also can purchase these supplies from your local pet supply store, hobby shop, or from online sources. A package of test strips in a hobby shop should run you somewhere about $3 or $4. You should have 50 strips to make sure you have enough for the experiment. Or, your science teacher may have extra test strips that you could get for this project. It doesn't hurt to ask, right?
• Seven large-size plastic drinking cups. Cups should be about 16 ounces to allow room for bean plants to grow.
• Soil to fill the cups about three-quarters full. You'll need to use the same soil for all the cups. Buying a bag of potting soil is recommended.
• Twenty-one bean seeds
• Distilled water
• Metric ruler
• Seven two-liter, plastic bottles, empty and washed well
• Small paper cups in which to measure water

If you're going to order supplies from an online provider, be sure to do so ahead of time so you don't get stuck, unable to begin your project.

Conducting Your Experiment

Explosion ahead.

Contaminating one bottle of water with any water from another bottle will affect the results of your experiment. Try as hard as you can to keep water with different pH levels completely separated.

Because you need seven individual bottles of water and seven cups, you'll need some space to set up this experiment.

It's very important that the cups containing the bean seeds are all kept in the same conditions. They each need to have the same amount of light, heat, and so forth.

And it's extremely important that each plant receives the same amount of water. If you give the plants different amounts of water, you'll be unable to determine whether the plant was affected by the pH of the water, or simply the varying amount.

Pour the water into the soil, not on the leaves of the plants. Plants take up water from their roots, not their leaves.

You can't mix water from any of the two-liter bottles, or use the same container to hold water from different bottles without washing it out in between. That's why it's recommended that when you water plants, you use small paper cups, pouring water from each two-liter bottle into its own paper cup, and then onto the bean plant.

If your plants don't need a full cup of water, measure up to the halfway mark of each cup and make a line. Fill the cup with water up to the line to assure that each plant gets the same amount.

While it's interesting to see how pH level affects plants, don't be tempted to drink any of the water yourself, and avoid splashing it on your skin, eyes, and so forth. Some of the treated waters you'll be using have very high or low pH levels, and should only be used for the purposes of this experiment.

Follow these steps to conduct the experiment:

1. Starting with seven two-liter bottles of distilled water, prepare each bottle so it has a specific pH value. Leave one bottle untreated (your control), with a pH level of 7. Add pH Up or pH Down to the other bottles so that one bottle has a pH level of 1, one has a pH level of 3, one has a pH level of 5, one has a pH level of 7, one has a pH level of 9, one has a pH level of 11, one has a pH level of 13. You'll raise or lower the pH level for each bottle from 7, depending on whether you're making the water acidic or alkaline. Cap the bottles tightly, label each one so you know which is which, and place them in an undisturbed location.

Do write down how many drops of either pH Up or pH Down you need to add to each two liter bottle. If you need to make more water at the different pH levels, you'll already know how much of the pH product to add, and the second round of water will have the exact same pH levels as the first round.

2. Plant three bean seeds into each of the seven large, plastic cups that have been filled about three-quarters of the way with potting soil.

3. Mark each cup and match one cup with one two-liter bottle of water. It's extremely important that each cup is watered from the same bottle each time, and not from any other bottle.

4. Making sure each cup gets the same amount of water, water the seeds in each cup so that the soil is moist, but not saturated. You might want to transfer water from the two-liter bottle to the cup with a tablespoon, allowing you to exactly measure how much each pot will get. Just be sure to wash the tablespoon between using it to handle water from different bottles.

5. Observe the cups every day, watering when the soil appears dry. Just be sure to always give the seeds in each cup the same amount of water. The plants will require more water as they get larger.

6. Using a metric ruler, measure the plants and record your observations every four days. Record the growth of the plants in a chart like the one found in the next section, €œKeeping Track of Your Experiment.€

Your experiment will be finished after 28 days, meaning you will have measured each plant seven times.

Keeping Track of Your Experiment

Make sure to note the height of each plant within the seven cups€”21 plants in all€”each time you measure. Keep track of how much water you've given the plants as well. The amount of water given to the plants in each cup should be the same.

Data charts to record water application and plant growth are provided, or you can make your own charts, if you prefer.

To calculate the average height for the three plants in each cup:

• Add the three heights together for each of the three plants in cup #1.
• Divide that total by 3.
• Record this number in data chart 2.

Repeat steps 1-3 for the remaining six cups.

Don't assume that all plants within a cup will grow equally. You can average the height of the three plants within each cup.

Putting It All Together

Use the information contained on your data charts to make some graphs, plotting the growth of each plant. Or, you can summarize your observations in written form, if you prefer.

Did some of the plants grow quickly at first, only to halt their growth later? Did any of the plants die? Did your hypothesis prove to be correct?

Look for trends and patterns, concluding which plants had the best overall growth, the fastest starts, and so forth.

Further Investigation

If you enjoyed this experiment and want to try another variation of it, you could try to grow plants hydroponically€”that is, in water instead of dirt€”while varying the pH level of the water.

It would be interesting to see if you get different results than you did when you used water of varying pH levels to water plants growing in dirt.

You also could put liquids other than water on plants to see how growth was affected. Or, you could work with acid-loving plants and base-loving plants, testing to see at what pH level they grow best.

Excerpted from The Complete Idiot's Guide to Science Fair Projects © 2003 by Nancy K. O'Leary and Susan Shelly. All rights reserved including the right of reproduction in whole or in part in any form. Used by arrangement with Alpha Books , a member of Penguin Group (USA) Inc.

To order this book direct from the publisher, visit the Penguin USA website or call 1-800-253-6476. You can also purchase this book at Amazon.com and Barnes & Noble .

• Science Projects for Beginners: Botany Projects

ORIGINAL RESEARCH article

Effects of low ph on photosynthesis, related physiological parameters, and nutrient profiles of citrus.

• 1 Institute of Plant Nutritional Physiology and Molecular Biology, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, China
• 2 Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, China
• 3 The Higher Educational Key Laboratory of Fujian Province for Soil Ecosystem Health and Regulation, Fujian Agriculture and Forestry University, Fuzhou, China

Seedlings of “Xuegan” ( Citrus sinensis ) and “Sour pummelo” ( Citrus grandis ) were irrigated daily with a nutrient solution at a pH of 2.5, 3, 4, 5, or 6 for 9 months. Thereafter, the following responses were investigated: seedling growth; root, stem, and leaf concentrations of nutrient elements; leaf gas exchange, pigment concentration, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and chlorophyll a fluorescence; relative water content, total soluble protein level, H 2 O 2 production and electrolyte leakage in roots and leaves. This was done ( a ) to determine how low pH affects photosynthesis, related physiological parameters, and mineral nutrient profiles; and ( b ) to understand the mechanisms by which low pH may cause a decrease in leaf CO 2 assimilation. The pH 2.5 greatly inhibited seedling growth, and many physiological parameters were altered only at pH 2.5; pH 3 slightly inhibited seedling growth; pH 4 had almost no influence on seedling growth; and seedling growth and many physiological parameters reached their maximum at pH 5. No seedlings died at any given pH. These results demonstrate that citrus survival is insensitive to low pH. H + -toxicity may directly damage citrus roots, thus affecting the uptake of mineral nutrients and water. H + -toxicity and a decreased uptake of nutrients (i.e., nitrogen, phosphorus, potassium, calcium, and magnesium) and water were likely responsible for the low pH-induced inhibition of growth. Leaf CO 2 assimilation was inhibited only at pH 2.5. The combinations of an impaired photosynthetic electron transport chain, increased production of reactive oxygen species, and decreased uptake of nutrients and water might account for the pH 2.5-induced decrease in CO 2 assimilation. Mottled bleached leaves only occurred in the pH 2.5-treated C. grandis seedlings. Furthermore, the pH 2.5-induced alterations of leaf CO 2 assimilation, water-use efficiency, chlorophylls, polyphasic chlorophyll a fluorescence (OJIP) transients and many fluorescence parameters, root and leaf total soluble proteins, H 2 O 2 production, and electrolyte leakage were all slightly greater in C. grandis than in C. sinensis seedlings. Hence, C. sinensis was slightly more tolerant to low pH than C. grandis . In conclusion, our findings provide novel insight into the causes of low pH-induced inhibition of seedling growth and leaf CO 2 assimilation.

Introduction

Acidic soils that limit crop growth and productivity are often observed all over the world, especially in the tropics and subtropics. Approximately 30% of the world's ice-free land is acidic, and approximately 12% of crops are cultivated on acidic soils ( von Uexküll and Mutert, 1995 ). What is worse, soil acidification is becoming an increasingly major problem due to the improper application of chemical fertilizers—particularly the overuse of nitrogen (N) fertilizers—alongside acid rain and intensive agriculture and monoculture ( Wu et al., 2013 ; Yang et al., 2013 ). The effects of aluminum (Al)-toxicity—a major factor limiting crop productivity on acidic soils—on plants have drawn widespread attention, but few studies have investigated the damage to plants from low pH ( Yang et al., 2015 ).

Poor crop growth and yield on acidic soils is usually due to the combination of toxicities of H + , Al, and manganese (Mn) and a lack of nutrients—namely phosphorus (P), calcium (Ca), magnesium (Mg), potassium (K), and molybdenum (Mo)— and a reduced uptake of water ( von Uexküll and Mutert, 1995 ; Bian et al., 2013 ). In tropical America, over 70% of the acidic soils display Al-toxicity and Mg and Ca deficiencies, and almost all the acidic soils are P-deficient or have a high P-fixation capacity ( George et al., 2012 ). For example, Zhang et al. (2014) showed that pH 3.0 decreased the uptake and utilization efficiency of P in Juglans regia seedlings. Forest ecosystems with acidic soils are often restricted by low Ca and Mg availability ( St Clair and Lynch, 2005 ). Schubert et al. (1990) showed that transferring Vicia faba plants from pH 7 to pH 4 led to the reduced uptake of N, P, K, Ca, Mg, and sulfur (S). Malkanthi et al. (1995) observed that the levels of K, Ca, Mg, Mn, and Zn in the roots and tops of wheat, barley, and chili plants were lower at pH 3.8 than at pH 5.5. Similarly, the K, Ca, Mg, and Mn levels in Pinus pinaster roots and needles were lower at pH 3.5 than at pH 4.5, 5.5, and 6.5, whereas the levels of P and Fe were higher at pH 3.5 and 4.5 than at pH 5.5 and 6.5 ( Arduini et al., 1998 ). However, Anugoolprasert et al. (2012) reported that the uptake of N, P, K, Ca, and Mg, and their concentration in roots, leaflets, petioles and whole plant, were not altered over the range of pH 3.6 to 5.7 for 4.5 months; this possibly explains the normal growth of sago palm seedlings at pH 3.6. Kidd and Proctor (2001) have suggested that the direct toxicity of H + was the primary cause of the poor growth in H + -intolerant plants growing in very acidic soils.

Low pH can affects plant water uptake. Kamaluddin and Zwiazek (2004) observed that low pH caused a large and rapid decrease in both the water flow rate and the hydraulic conductivity in seedling roots of paper birch ( Betula papyrifera ). A pH 4.5 decreased the whole-root water conductivity in the H + -sensitive maize cultivar Adour 250 , but it did not in the H + -tolerant maize cultivar BR 201 F ( Gunsé et al., 1997 ). Tournaire-Roux et al. (2003) showed that the inhibition of water hydraulic conductivity (water uptake) in Arabidopsis roots by anoxia was primarily caused by cytosol acidosis, while changing the pH between 5.5 and 8.0 of a root-bathing solution did not affect the cytosol pH nor the root water hydraulic conductivity. Finally, Yang M. et al. (2011) observed that a low pH decreased the water content in Eucalyptus roots, stems, and leaves.

Low pH also inhibits CO 2 assimilation in some plant species, including J. regia ( Zhang et al., 2014 ), Eucalyptus ( Yang et al., 2015 ), sugar maple ( Acer saccharum ) and red maple ( Acer rubrum ) ( Ellsworth and Liu, 1994 ; St Clair and Lynch, 2005 ). St Clair and Lynch (2005) also reported that the base cation stimulation of photosynthesis in sugar maple on acidic soils was correlated with its foliar nutrient status. Ellsworth and Liu (1994) had earlier suggested that photosynthesis in sugar maple on acidic soils might be co-limited by N and Ca, or by Ca × Mg interactions. Yang M. et al. (2011) observed that a low pH decreased the chlorophyll (Chl) level in Eucalyptus leaves. Yang et al. (2015) further investigated the effects of low pH on leaf gas exchange and Chl in four vegetatively-propagated Eucalyptus clones (G9, G12, G3, and G4); they found that pH 3.0 decreased leaf photosynthesis, transpiration, and Chl level in the four clones as well as the leaf water-use efficiency (WUE) in the G4 leaves, but pH 3.0 did not affect WUE in the G9, G12, and G3 leaves. Zhang et al. (2014) reported that pH 3.0 decreased the leaf net photosynthetic rate, transpiration rate, actual quantum yield of the photosystem II (PSII) electron transport (Φ PSII ), whereas it increased leaf non-photochemical quenching (NPQ); however, pH 3 had no effect upon leaf stomatal conductance, photochemical quenching (qP), and the maximum PSII efficiency of dark-adapted leaves (F v /F m ), thus leading the authors to conclude that non-stomatal factors played a role in the low pH-induced inhibition of photosynthesis. Nonetheless, pH 4.0 did not influence spatial heterogeneity of Chl fluorescence, F v /F m , Φ PSII , and quantum yields of regulated (Φ NPQ ) and nonregulated (Φ NO ) energy dissipation in the leaves of Plantago algarbiensis and P. almogravensis ( Martins et al., 2013a , c ). Altering the pH between 5.7 and 3.6 did not reduce the Chl concentration, photosynthetic rate, stomatal conductance, and transpiration rate in sago palm leaves ( Anugoolprasert et al., 2012 ). However, to our best knowledge, little is still known about the effects of low pH on PSII photochemistry (i.e., absorption flux, trapped energy flux, electron flux, and dissipated energy flux) of leaves.

Low pH can induce oxidative stress and electrolyte leakage via the enhanced production of active oxygen species (ROS). Martins et al. (2013b) found that lipid peroxidation (malondialdehyde, MDA) was elevated in the pH 4.0-treated P. algarbiensis shoots, but not in the pH 4.0-treated P. almogravensis ones, and that the activities of antioxidant enzymes were enhanced or not affected in the shoots of the two Plantago species—suggesting that the higher antioxidant enzyme activities were insufficient to protect the low pH-treated P. algarbiensis shoots against oxidative damage. In another experiment, Martins et al. (2011) observed that pH 4.5 led to an increase in the MDA level in P. algarbiensis roots and shoots and P. almogravensis roots, but not in P. almogravensis shoots. Yang M. et al. (2011) reported that low pH increased membrane permeability in Eucalyptus leaves. Hydroponic experimentation showed that pH 3.5 led to an accumulation of H 2 O 2 and severe lipid peroxidation that was accompanied by an increased activity of ascorbate peroxidase (APX) and decreased activities of superoxide dismutase (SOD) and catalase (CAT) in the roots of two rice cultivars ( Zhang et al., 2015 ). Cucumber roots treated with pH 4.5 had a higher level of MDA and activities of monodehydroascorbate reductase (DHAR), guaiacol peroxidase (GPX), APX, and glutathione reductase (GR), but had lower activities of Cu/Zn-SOD, than did the pH 6.5-treated roots ( Shi et al., 2006 ). However, pH 4.0 did not affect H 2 O 2 , MDA and the total soluble protein levels, electrolyte leakage, protein oxidation, and the SOD, CAT, APX, and GPX activities in the roots and leaves of P. algarbiensis and P. almogravensis ( Martins et al., 2013c ).

Citrus plants are considered insensitive to acidic soils ( Yuda and Okamoto, 1965 ). Fang et al. (2011) used a solution culture approach to investigate the effects of pH 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 on several citrus rootstock seedlings. At pH 1.0, all seedlings died within 10 days after treatment, but the pH 4-treated seedlings showed normal growth except for a yellow tip that occurred in some leaves within 30 days. Using sand and solution cultures, Guest and Chapman (1944) found that Citrus sinensis seedlings died within a few days at pH 2.0, but they were not killed for months at pH 2.5 and 3.0 though their growth was limited or negligible. Nevertheless, citrus do not thrive in trongly acidic soils, because serious problems may arise when the soil pH is 5.0 or lower ( Chapman, 1968 ). Citrus will often display poor growth and have a shortened lifespan when cultivated on soil with a low pH and high active Al ( Lin and Myhre, 1990 ). In China, most of the citrus are grown in acidic and strongly acidic soils. Li et al. (2015) reported that the pH values of 319 soils sampled from pummelo ( Citrus grandis ) orchards in Pinghe, Zhangzhou, China had an average value of 4.34 and ranged from 3.26 to 6.22, with up to 90.0% of the orchard soils having a pH lower than 5.0. So far, however, only a handful of reports have empirically investigated the effects of low pH on citrus growth ( Yuda and Okamoto, 1965 ), mineral nutrient uptake ( Randhawa and Iwata, 1968 ; He et al., 1999 ; Li et al., 2015 ), and ROS metabolism alongside a few other physiological parameters ( Fang, 2011 ). Randhawa and Iwata (1968) reported that the N, Ca, and Mg (Ca, Mg, and P) levels decreased in the leaves (roots), whereas the K level increased in the roots and leaves of Citrus natsudaidai seedlings, as the pH decreased from 7.0 to 4.0. He et al. (1999) observed that Fe, Zn, and Mn (Ca) in grapefruit ( Citrus paradisi ) leaves increased (decreased) with decreasing soil pH. The concentration of P and Ca in pummelo leaves decreased with decreasing soil pH ( Li et al., 2015 ). Fang (2011) found that the activities of SOD, GPX, and CAT and the level of total soluble proteins displayed an upward trend, as a whole, as the pH decreased from 6.0 to 2.0; in contrast, the level of MDA decreased first to reach its lowest value at pH 4, but then increased as the pH decreased further.

The objectives of this work were ( a ) to determine how low pH affects gas exchange, related physiological parameters, and the mineral nutrient profiles in citrus seedlings; and ( b ) to understand the mechanisms by which low pH may lead to a decrease in leaf CO 2 assimilation.

Materials and Methods

Plant materials and culture conditions.

This study was conducted at the Fujian Agriculture and Forestry University (FAFU) in Fuzhou, China. Seedling culture was performed according to Han et al. (2008) and Peng et al. (2015) , with some modifications. Briefly, seeds of “Sour pummelo” ( C. grandis ) and “Xuegan” ( C. sinensis ) were germinated in plastic trays filled with clean river sand. Four weeks after germination, uniform seedlings that had a single stem were chosen and transplanted into 6-L terracotta pots (two seedlings per pot) containing clean river sand. Seedlings were grown in a greenhouse under a natural photoperiod at FAFU. One week after transporting, each pot was irrigated every other day with 500 mL of a nutrient solution containing 2.5 mM Ca(NO 3 ) 2 , 2.5 mM KNO 3 , 1 mM MgSO 4 , 0.5 mM KH 2 PO 4 , 20 μM Fe-EDTA, 10 μM H 3 BO 3 , 2 μM MnCl 2 , 2 μM ZnSO 4 , 0.5 μM CuSO 4 , and 0.065 μM (NH 4 ) 6 Mo 7 O 24 . Seven weeks after transplanting, each pot was fertilized daily until saturated with the same nutrient solution (approximately 500 mL), except that the pH of the nutrient solution was adjusted to 2.5, 3, 4, 5, or 6 with 1 M HCl. There were 20 replicates (20 pots, 40 seedlings) per treatment in a completely randomized design. In this experiment, the pH 5 treatment served as the control because seedling growth and many physiological parameters reach their maximum at pH 5. Nine months after the pH treatment began, recent fully-expanded (approximately 7-week-old) leaves and approximately 5-mm-long white root apices were used for all measurements except that for root mineral element concentrations. After leaf gas exchange and Chl a fluorescence were measured, leaf disks (0.2826 cm 2 in size) and approximately 5-mm-long white root apices from the same seedlings were harvested from randomly selected seedling at noon on a sunny day and immediately frozen in liquid N 2 , then stored at −80°C until they were used for the assays of ribulose-1,5-bisphosphate carbohylase/oxygenase (Rubisco), total soluble proteins, and pigments. The remaining seedlings that were not sampled were selected randomly to measure plant biomass, root and leaf relative water content (RWC), and electrolyte leakage, and the root, stem and leaf mineral element concentrations.

Measurements of Leaf, Stem and Root Dry Weight (DW), and Specific Leaf Weight

Nine months after the pH treatment began, 10 seedlings per treatment from 10 pots were collected. The seedlings were divided into leaves, stems, and roots. Their DW was measured after being dried at 70°C for 48 h. Specific leaf weight was calculated as the ratio of leaf weight to leaf area.

Leaf Pigments, and Root and Leaf Total Soluble Proteins

Leaf pigments were extracted with 80% (v/v) acetone. The Chl, Chl a and Chl b, and carotenoids (Car) in the extract were determined according to Lichtenthaler (1987) .

Root and leaf total soluble proteins were extracted with 50 mM KH 2 PO 4 -Na 2 HPO 4 (pH 7.0) and 5% (v/v) insoluble polyvinylpyrrilodone (PVP), and assayed according to Bradford (1976) .

Electrolyte Leakage, RWC, and H 2 O 2 Production

Root and leaf electrolyte leakage was assayed according to Han et al. (2008) . Briefly, 20 fresh leaf disks (0.2826 cm 2 in size) from the same leaf or 20 approximately 5-mm-long white root apices taken at midday under full sun, were immediately transferred to a 50-mL tube filled with 15 mL of distilled water. The tubes were placed at room temperature in the dark for 24 h and the first electrical conductance (C 1 ) was measured. Then the tubes were incubated in a boiling water bath for 15 min and the second electrical conductance (C 2 ) was measured after being cooled. The electrolyte leakage was calculated as: electrolyte leakage (%) = (C 1 /C 2 ) × 100.

Root and leaf RWC were gravimetrically determined ( Panković et al., 1999 ). After fresh weight (FW) was measured, approximately 0.2 g of roots and 0.5 g of leaves were floated on distilled water in Petri dishes in the dark. After reaching a constant turgid weight (ca. 6 h), the roots and leaves were dried. The RWC was calculated as: RWC (%) = (FW − DW)/(turgid weight − DW) × 100.

Root and leaf H 2 O 2 production were determined according to Chen et al. (2005b) . About 100 mg of roots and 15 leaf disks (0.2826 cm 2 in size) were incubated in 2 mL of a 50 mM phosphate buffer (pH 7.0), 5 U horseradish GPX, and 0.05% (w/v) guaiacol for 2 h at room temperature in the dark. Then the absorbance was measured at 470 nm.

Measurements of Mineral Elements, and the Calculation of Nutrient Uptake and Element Distribution in Roots, Stems, and Leaves

Fibrous roots, the middle sections of stems, and approximately 7-week-old leaves (midribs and petioles removed) were collected and dried at 70°C for 48 h. Dried samples were ground in a mortar to pass through a 40-mesh sieve and stored for later analysis.

To measure the root, stem, and leaf concentrations of P, K, Fe, Mn, Cu, Zn, Ca, and Mg, approximately 0.3-g samples were digested in a 7 mL mixture of HNO 3 :H 2 O 2 (5:2 v/v). P was determined colorimetrically as the blue molybdate-phosphate complexes according to Lu (1999) . K was assayed using FP640 Flame Photometry (Shanghai Precision Scientific Instrument Co., Ltd, Shanghai, China). Fe, Mn, Cu, Zn, Ca, and Mg were determined using a PinAAcle 900F Atomic Absorption Spectrometer (Perkinelmer Singapore Pte Ltd, Singapore). N was measured using a Kjeltec 8200 Auto Distillation (FOSS Analytical AB, Höganäs, Sweden) after samples had been digested with H 2 SO 4 and H 2 O 2 ( Lu, 1999 ). B was determined by the curcumin method after samples were ashed at 500°C for 5 h and dissolved in 0.1 M HCl ( Kowalenko and Lavkulich, 1976 ). S was assayed using the simple turbidimetric method based on the formation of the BaSO 4 precipitate in its colloid form after approximately 0.3-g samples were digested with a 6-mL mixture of HNO 3 :HClO 4 (4:1 v/v; Lu, 1999 ).

Nutrient uptake per plant was the sum of the element content (element concentration × tissue DW) in the roots, stems, and leaves. Element distributions in roots, stems, or leaves (%) were calculated as: (element content in roots, stems, or leaves/the sum of element content in roots, stems, and leaves) × 100.

Leaf Gas Exchange and Rubisco Measurements

Leaf gas exchange was measured by a CIARS-2 portable photosynthesis system (PP Systems, Herts, UK) at an ambient CO 2 concentration under a controlled light intensity of 996–1004 μmol m −2 s −1 between 9:30 and 12:30 on a clear day. During all of these measurements, the leaf temperature and relative humidity were 30.0 ± 0.2°C and 64.5 ± 0.6%, respectively. Leaf Rubisco was extracted and assayed according to Chen et al. (2005a) and Lin et al. (2009) , respectively.

Measurements of Leaf OJIP Transients by Handy PEA and the JIP Test

The polyphasic Chl a fluorescence (OJIP) transients were measured by a Handy Plant Efficiency Analyzer (Handy PEA, Hansatech Instruments Limited, Norfolk, UK). The transient was induced by a saturating red light of approximately 3,400 μmol m −2 s −1 , which was provided by an array of three light-emitting diodes (peak 650 nm) that were focused on the leaf surface to provide homogenous illumination over the exposed area of the leaf. All the measurements were performed on 3-h dark-adapted plants at room temperature.

The OJIP transients were analyzed according to the JIP test ( Strasser et al., 2004 ; Jiang et al., 2008 ; Chen and Cheng, 2009 ). The following data from the original measurements were extracted and used: fluorescence intensities at 20 μs (F 20μ s , considered as the minimum fluorescence F o ), 50 μs (F 50μ s ), 300 μs (F 300μ s ), 2 ms (J-step, F J ), 30 ms (I-step, F I ), and P-step (considered as the maximum fluorescence F m ). The following parameters that refer to “time 0” (start of fluorescence induction) are: ( a ) fluorescence parameters derived from the extracted data, i.e., the maximum variable fluorescence F v = F m − F o and the approximated initial slope (in ms −1 ) of the fluorescence transient V = f(t) [M o = 4(F 300μ s −F o )/(F m −F o )]; ( b ) the specific energy fluxes per reaction center (RC) for energy dissipation (DI o /RC) and absorption (ABS/RC); ( c ) the yields of the flux ratios, i.e., quantum yield for energy dissipation (φ Do = DI o /ABS = F o /F m ), maximum quantum yield of primary photochemistry (φ Po = TR o /ABS = F v /F m ), quantum yield for the reduction of the end acceptors of photosystem I (PSI) per photon absorbed (φ Ro = RE o /ABS), and quantum yield for electron transport (φ Eo = ET o /ABS); ( d ) the overall grouping probability (P 2G ); and ( e ) the total performance index (PI tot, abs ).

Measurements of Conventional Fluorescence Parameters by FMS-2

Conventional fluorescence parameters were determined by a pulse-modulated fluorometer FMS-2 (Hansatech Instruments, Norfolk, UK). Both F m and F o were measured after the leaves were dark-adapted for 40 min. Steady-state fluorescence (F s ) and the maximum (F m ′) and minimum (F o ′) fluorescences were measured under natural light at midday in full sun. For this determination, the F s was monitored to ensure it was stable before a reading was taken; the F m ′ was obtained by imposing a 1-s saturating flash of approximately 6,000 μmol m −2 s −1 at the leaf surface to reduce all the PSII centers. To measure the F o ′, a black cloth covered the leaf when a far-red light was switched on to rapidly oxidize the PSII centers by drawing electrons from PSII to PSI. The NPQ was calculated as: F m /F m ′−1. The photochemical quenching coefficient, qP, was expressed as: (F m ′−F s )/(F m ′−F o ′). The non-photochemical quenching coefficient, qNP, was defined as: (F m −F m ′)/(F m −F o ′). The Φ PSII was calculated as: (F m ′−F s )/F m ′. The efficiency of excitation transfer to PSII RCs under natural light (F m ′/F v ′) was defined as: (F m ′−F o ′)/F m ′. Finally, the electron transport rate through PSII was estimated from (F m ′−F s )/F m ′ × 0.5 × LA × photosynthetic photon flux (PPF), for which the PSI photochemistry was assumed equivalent to that of PSII ( Genty et al., 1990 ), and where LA is the leaf absorbance (0.84; Baker, 2008 ).

Statistical Analysis

There were 10 replicates for plant biomass; three replicates for Rubisco; four replicates for gas exchange, pigments, H 2 O 2 production, RWC, electrolyte leakage, total soluble proteins, specific leaf weight, and mineral nutrients; and 7–15 replicates for the OJIP transients and the fluorescence parameters. The results are presented using the mean ± SE of 3–15 replicates. For a given dependent variable or parameter above, significant differences among the means of 10 treatment combinations were tested by a two (species) × five (pH levels) factorial ANOVA; the 10 means were compared on a pairwise basis by the Duncan's new multiple range test at P < 0.05. Linear and nonlinear regression was performed with the corresponding equations from SigmaPlot software (SigmaPlot 10.0, Systat Software Inc., USA).

Effects of pH on Seedling Growth

Overall, the pH-2.5 treatment greatly decreased root, stem, leaf, and whole plant DW; pH 3 slightly inhibited seedling growth; pH 4 had almost no influence on seedling growth; and seedling growth reached a maximum at pH 5 (Figures 1 , 2 ). At pH 2.5, many rotted fibrous roots were observed, and the living roots had turned abnormally dark brown (Figures 2A,D ). Mottled bleached leaves were found in four C. grandis seedlings treated with pH 2.5 (Figure 2B ). No seedling death was observed for the two citrus species at each given pH.

Figure 1. Effects of pH on root (A), stem (B), leaf (C), and whole plant (D) DW of Citrus sinensis and Citrus grandis seedlings . Bars represent means ± SE ( n = 10). Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

Figure 2. Effects of pH on the growth of Citrus grandis (A,B) and Citrus sinensis (C,D) seedlings .

Effects of pH on Leaf Gas Exchange, Rubisco Activity, and Pigment Levels

As shown in Figure 3 , leaf CO 2 assimilation, stomatal conductance, transpiration, and Rubisco activity were little changed as the pH decreased from 6 to 3, but they greatly decreased at pH 2.5. Leaf WUE was lower at pH 2.5 than at pH 5. All five parameters were similar between the two citrus species at each given pH. Intercellular CO 2 concentration did not significantly differ among the 10 treatment combinations, but there was a slight increase observed in the pH 2.5-treated C. grandis leaves.

Figure 3. Effects of pH on CO 2 assimilation (A), stomatal conductance (B), intercellular CO 2 concentration (C), transpiration rate (D), water-use efficiency (WUE, E), and Rubisco activity (F) in Citrus sinensis and Citrus grandis leaves . Bars represent means ± SE ( n = 3 for Rubisco or n = 4 for the other parameters). Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

As shown in Figure 4 , leaf Chl a, Chl b, Chl a+b, and Car concentrations greatly increased as the pH increased from 2.5 to 3, after which they remained unchanged or were only slightly altered with increasing pH. These concentrations did not differ significantly between the two citrus species at pH 3, 4, 5, and 6, but they were lower in C. sinensis leaves than in C. grandis leaves at pH 2.5. Moreover, there was little difference in the ratios of leaf Chl a/b and Car/Chl among the 10 treatment combinations. The only exception was the lower Car/Chl ratio in the pH 2.5-treated C. sinensis leaves when compared with the other nine treatment combinations.

Figure 4. Effects of pH on Chl a (A), Chl b (B), Chl a+b (C), Chl a/b (D), Car (E), and Car/Chl (F) in C. sinensis and C. grandis leaves . Bars represent means ± SE ( n = 4). Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

Leaf CO 2 assimilation increased with increasing leaf stomatal conductance, the activity of Rubisco, and the concentration of Chl a, Chl b, or Chl a+b, but it decreased with an increasing intercellular CO 2 concentration (Figure 5 ).

Figure 5. Leaf CO 2 assimilation in relation to stomatal conductance (A), intercellular CO 2 concentration (B), Rubisco activity (C), Chl a (D), Chl b (E), and Chl a+b (F) . Points represent means ± SE for the independent variable ( n = 4) and the dependent variables ( n = 3 or 4). Data for CO 2 assimilation, stomatal conductance, intercellular CO 2 concentration, and Rubisco activity are from Figure 3 . Data for Chl a, Chl b, and Chl a+b are from Figure 4 . Data for the two citrus species were pooled together.

Effects of pH on Chl a Fluorescence and Related Parameters

Our results showed that pH 2.5 caused an increased O-step and P-step in C. sinensis and C. grandis leaves compared with pH 5, and that the pH 2.5-treated C. sinensis and C. grandis leaves had positive ΔI-, ΔJ-, ΔK-, and ΔL-bands around 30 ms, 2 ms, 300 μs, and 130 μs as compared with the pH 5-treated leaves, respectively. The pH 2.5-induced alterations of the OJIP transients and the ΔI- and ΔL-bands were greater in the leaves of C. grandis than in those of C. sinensis . Little, if any, differences were observed in the OJIP transients among the pH 3-, 4-, 5-, and 6-treated leaves (Figure 6 ).

Figure 6. Effects of pH on the mean chlorophyll a fluorescence (OJIP) transients (A,F) and the different expressions derived from the transients in dark-adapted leaves: (B,G) between F o and F m : V t = (F t −F o )/(F m −F o ) and (C,H) the differences of the five samples to the reference sample treated with pH 5.0 (ΔV t ); (D,I) between F o and F 300μ s : W K = (F t −F o )/(F 300μ −F o ) and (E,J) the differences of the five samples to the reference sample treated with pH 5.0 (ΔW K ) . Each point was the mean of 8–15 replicates.

As shown in Figure 7 , the F o , F m , M o , ABC/RC, DI o /RC, DI o /ABS, qNP, and NPQ all increased, and whereas the F v /F m , ET o /ABS, RE o /ABS, P 2G , PI tot, abs , qP, F m ′/F v ′, Φ PSII , and ETR all decreased as the pH increased from 2.5 to 3, with further increasing pH there was hardly any change in all these parameters. Nonetheless, the F v did not greatly change in response to pH. All these parameters were similar between the two citrus species at pH 3, 4, 5, or 6, but the pH 2.5-induced changes in F o , F v , F m , M o , ABC/RC, DI o /RC, DI o /RC, F v /F m , RE o /ABS, P 2G , PI tot, abs , and ETR were slightly greater in C. grandis than in C. sinensis leaves.

Figure 7. Effects of pH on F o (A), F m (B), F v (C), M o (D), ABS/RC (E), DI o /RC (F), DI o /ABS (G), F v /F m (H), ET o /ABS (I), RE o /ABS (J), P 2G (K), PI tot, abs (L), qP (M), qNP (N), NPQ (O), F m ′ / F v ′ (P), Φ PSII (Q), and ETR (R) in dark-adapted C. grandis and C. sinensis leaves . Bars represent means ± SE ( n = 7–15). Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

Leaf CO 2 assimilation decreased with increasing F o , F m , F v , M o , ABC/RC, DI o /RC, DI o /ABS, qNP, or NPQ, whereas it increased with increasing F v /F m , ET o /ABS, RE o /ABS, P 2 G , PI tot, abs , qP, F m ′/F v ′, Φ PSII , or ETR (Figure 8 ).

Figure 8. Leaf CO 2 assimilation in relation to F o (A), F m (B), F v (C), M o (D), ABS/RC (E), DI o /RC (F), DI o /ABS (G), F v /F m (H), ET o /ABS (I), RE o /ABS (J), P 2G (K), PI tot, abs (L), qP (M), qNP (N), NPQ (O), F m ′ / F v ′ (P), Φ PSII (Q), and ETR (R) . Points represent means ± SE for the independent variable ( n = 4) and the dependent variables ( n = 7–15). Data for CO 2 assimilation are from Figure 3 . Data for the 18 fluorescence parameters are from Figure 7 . Data for the two citrus species were pooled together.

Effects of pH on RWC, H 2 O 2 Production, Electron Leakage, Total Soluble Proteins in Roots and Leaves and Specific Leaf Weight

Both pH 2.5 and 3 decreased the root RWC, while only pH 2.5 lowered the leaf RWC. Root and leaf RWCs were similar between the two citrus species at each given pH (Figures 9A,F ).

Figure 9. Effects of pH on root (A–D) and leaf (F–I) relative water content (RWC, A,F), H 2 O 2 production (B,G), electrolyte leakage (C,H), concentrations of total soluble proteins (D,I), and specific leaf weight expressed on a fresh weight (FW, E) or dry weight (DW, J) basis in the C. sinensis and C. grandis seedlings . Bars represent means ± SE ( n = 4). Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

Both pH 2.5 and 3 increased the root H 2 O 2 production, while only pH 2.5 enhanced the leaf H 2 O 2 production. Root (Leaf) H 2 O 2 production was significantly higher in C. grandis than in C. sinensis at pH 2.5 (2.5, 3, 4, and 6), but similar between the citrus species at pH 3–6 (pH 5; Figures 9B,G ).

Root and leaf electrolyte leakage increased as the pH increased from 2.5 to 3, after which leakage remained relatively stable under increasing pH. Root and leaf electrolyte leakage was higher in C. grandis than in C. sinensis at pH 2.5, but it was similar between the citrus species at pH 3–6 (Figures 9C,H ).

For C. grandis , the total soluble protein level in roots increased as the pH increased from 2.5 to 4, after which it remained unchanged with increasing pH. For C. sinensis , the total soluble protein level in roots was lowest at pH 2.5, intermediate at pH 3 and 6, and highest at pH 4 and 5. The total soluble protein level in leaves of the two citrus species increased as the pH increased from 2.5 to 3, but these levels were little changed with increasing pH. The total soluble protein levels in roots and leaves were higher in C. grandis than in C. sinensis , or they were statistically similar between the two species at each given pH (Figures 9D,I ).

The specific leaf weight was decreased at pH 2.5 and it was higher in C. grandis than in C. sinensis , or it was similar between the two species at each given pH irrespective of how the data were expressed (Figures 9E,J ).

Leaf CO 2 assimilation decreased with increasing root and leaf H 2 O 2 production or electrolyte leakage, but it increased with increasing root and leaf RWC (Figure 10 ).

Figure 10. Leaf CO 2 assimilation in relation to root (A,C,E) and leaf (B,D,F) H 2 O 2 production (A,B), RWC (C,D), and electrolyte leakage (E,F) . Points represent means ± SE for the independent variable ( n = 4) and the dependent variables ( n = 4). Data for CO 2 assimilation came from Figure 3 . Data for H 2 O 2 production, RWC, and electrolyte leakage came from Figure 9 . Data for the two citrus species were pooled together.

Effects of pH on Element Concentrations, Uptake, and Distributions

The leaf N level was lower at pH 2.5 than at pH 3–6, but the stem and root N levels remained little changed over the range of pH 2.5–6. The P level in C. grandis ( C. sinensis ) leaves and stems increased as the pH increased from 2.5 to 4 (3), but it went unchanged with increasing pH. The root P level increased as the pH increased from 2.5 to 5, but it then kept stable with increasing pH. The K concentration in the C. sinensis leaves and stems and in the C. grandis leaves displayed little change in the range of pH 2.5–6; however, the K level in the C. sinensis roots and in the C. grandis stems and leaves was lower at pH 2.5 than at pH 3–6. Generally viewed, the Ca levels in the leaves, stems, and roots all increased as the pH increased from 2.5 to 4, after which they were relatively stable with increasing pH. The Mg level in the C. grandis leaves and stems and in the C. sinensis leaves decreased with decreasing pH, but the Mg level in the C. sinensis stems did not change in response to pH. The Mg level in the C. sinensis roots was reduced at pH 2.5, 3, and 4, but especially at pH 2.5 and 3, while its level in the C. grandis roots was elevated at pH 2.5 and pH 3, though especially at pH 3. Leaf and root S decreased with increasing pH, while the stem S level was higher at pH 2.5 than at the other pH treatments. Leaf P, K, Ca, and S, stem P, K, and S, and root P levels were all higher in C. sinensis than in C. grandis seedlings; or similar between the two citrus species at each given pH. Conversely, the leaf Mg, stem Ca and Mg, and root N, K, Ca, Mg, and S levels were all lower in C. sinensis than in C. grandis seedlings, or they were similar between the two citrus species at each given pH (Figure 11 ).

Figure 11. Effects of pH on the N (A,G,M), P (B,H,N), K (C,I,O), Ca (D,J,P), Mg (E,K,Q), and S (F,L,R) concentrations in C. sinensis and C. grandis leaves, stems, and roots . Bars represent means ± SE ( n = 4). Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

The Fe level in the C. grandis leaves was lower at pH 2.5 and 3 than at pH 4–6, while the Fe level in the C. sinensis leaves did not differ among the five pH treatments. The Fe level in the C. sinensis ( C. grandis ) stems increased as the pH increased from 2.5 to 3 (4), but it then kept relatively stable with increasing pH, though it decreased at pH 6. The root Fe concentration decreased with increasing pH. Leaf and stem Mn levels decreased with increasing pH. The root Mn level increased as the pH decreased from 6 to 3, then it decreased or went unchanged at pH 2.5. Leaf B concentration in the two citrus species was decreased only at pH 2.5. The B level in the C. sinensis ( C. grandis ) stems increased as the pH increased from 2.5 to 4 (3), but then it went unchanged with increasing pH, though it decreased at pH 6. Although the root B concentration increased as the pH increased from 2.5 to 5, it decreased at pH 6. The Cu level in the C. grandis leaves increased as the pH decreased from 6 to 4, after which it was little changed with decreasing pH; the Cu level in the C. sinensis leaves was highest at pH 5 and lowest at pH 6. Root Cu level in the two citrus species decreased as the pH increased from 2.5 to 4, but it then remained stable with increasing pH. The Zn level in the C. sinensis leaves and stems were lower at pH 5 and 6 than at pH 2.5, 3, and 4, while its level in the C. grandis leaves and stems were lower at pH 6 than at pH 2.5–5. The Zn level in the C. sinensis roots increased as the pH decreased from 6 to 3, but it then decreased at pH 2.5; the Zn level in C. grandis roots was highest at pH 3 and lowest at pH 6. Generally viewed, the leaf Fe, Mn, B and Cu, stem Fe, Mn, B, Cu and Zn, root Fe, B, Mn, and Zn concentrations all were higher in C. grandis than in C. sinensis , or they were similar between the two citrus species at each given pH. The exceptions to this generalization were that the Mn (Cu) level was higher in C. sinensis than in C. grandis leaves at pH 2.5 (5), and the Fe level was higher in C. sinensis than in C. grandis stems at pH 2.5. By contrast, the leaf Zn and root Cu concentrations were higher in the C. sinensis than in those of C. grandis , or they were similar between the two citrus species at pH 2.5–5, albeit leaf Zn lower was lower in the C. sinensis vs. C. grandis at pH 6 (Figure 12 ).

Figure 12. Effects of pH on the Fe (A,F,K), Mn (B,G,L), B (C,H,M), Cu (D,I,N), and Zn (E,J,O) concentrations in the C. sinensis and C. grandis leaves, stems, and roots. Bars represent means ± SE ( n = 4) . Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

For C. sinensis , the N, P, K, Ca, Mg, and B uptake per plant increased as the pH increased from 2.5 to 5, then continued to rise or kept unchanged with increasing pH; For C. grandis , these elemental uptake per plant increased as the pH increased from 2.5 to 5, but then it went unchanged or decreased with increasing pH. The Mn uptake per plant in the two citrus species increased as the pH increased from 2.5 to 3, but it then decreased with increasing pH. Treatment with pH 2.5 decreased the S, Fe, Cu, and Zn uptake per plant compared with the corresponding uptake at pH 5 (Figures 13A–F,M–Q ).

Figure 13. Effects of pH on mineral element uptake per plant (A–F,M–Q) and per root DW (G–L,R–V) . Bars represent means ± SE ( n = 4). Differences among the 10 treatments were analyzed by two (species) × five (pH) factorial ANOVA. Different letters above the bars indicate a significant difference at P < 0.05.

Compared with pH 5, treatment with pH 2.5 decreased the N, P, K, Ca, Mg, and B uptake per root DW, whereas it increased the S, Fe, Mn, and Zn uptake per root DW; however, pH 2.5 did not influence Cu and Zn uptake per root DW (Figures 13G–L , R–V ).

Leaf CO 2 assimilation increased with increasing leaf N, P, Ca, Mg, Fe, or B, whereas it decreased with increasing leaf S, Mn, Cu, or Zn—it did not display a significant relationship with leaf K. Except for the Mn uptake per plant, the leaf CO 2 assimilation increased with increasing uptake per plant of the other elements (Figure 14 ).

Figure 14. Leaf CO 2 assimilation in relation to the mineral element concentrations in leaves (A–F,M–Q) and their uptake per plant (G–L,R–V) . Points represent means ± SE for the independent variable ( n = 4) and the dependent variables ( n = 4). Data for CO 2 assimilation came from Figure 3 . Data for the mineral element concentrations (mineral element uptake per plant) came from Figures 11 – 13 ). Data for the two citrus species were pooled together.

Compared with pH 5, treatment with pH 2.5 lowered all the element distributions in the C. sinensis leaves and the S, Fe, and Cu distributions in the C. sinensis stems; it increased, or did not affect, the 11 element distributions in the C. sinensis roots and the N, P, K, Mg, Mn, B, and Zn distributions in the C. sinensis stems. Compared with pH 5, pH 2.5 decreased or did not influence the K distribution in the stems and roots and the distributions of the other 10 elements in the leaves and stems; pH 2.5 increased or did not influence the K distribution in the leaves and the distributions of the other 10 elements in the roots of the C. grandis seedlings (Figures S1, S2).

Low pH very often affects the uptake of nutrients and water by plants ( Kamaluddin and Zwiazek, 2004 ; Bian et al., 2013 ). As expected, pH 2.5 lowered the water uptake in citrus, as indicated by the reduced root and leaf RWC (Figures 9A,F ). This result is supported by the finding that the water content was decreased in low pH-treated Eucalyptus roots, stems, and leaves ( Yang M. et al., 2011 ). As shown in Figures 9 – 13 , the uptakes of mineral nutrients were greatly altered at pH 2.5. Compared with pH 5, the pH 2.5 lowered N, P, K, Ca, and Mg uptake per plant or root DW, and the S uptake per plant. Low pH (4.0 relative to 7.0) induced decreases in the N, P, K, Ca, and Mg uptake per plant in V. faba ( Schubert et al., 1990 ). Similarly, Malkanthi et al. (1995) observed that a pH 3.8 (relative to 5.5) decreased the K, Ca, and Mg uptake per plant in wheat, barley, and chili, and likewise in cowpea for Ca and Mg uptake per plant. However, the uptake of N, P, K, Ca, and Mg in sago palm seedlings was not changed in the range of pH 3.6–5.7 over a 4.5-month period ( Anugoolprasert et al., 2012 ). Thus, it appears that the effects of low pH on macronutrient uptake per plant depend on both the plant species identity and the H + strength (i.e., pH value).

On the whole, apart from a few exceptions, the concentrations of N, P, K, Ca, and Mg were decreased in the pH 2.5-treated C. grandis and C. sinensis roots, stems, and leaves (Figure 11 ). This agrees with the report that pH 3 decreased Ca and Mg levels in Picea abies roots and needles ( George et al., 2012 ); that P, K, and Mg levels were lowered in the low pH-treated V. faba roots and shoots ( Schubert et al., 1990 ); that the levels of K, Ca, and Mg in the roots and tops of wheat, barley, and chili were lower at pH 3.8 than at pH 5.7 ( Malkanthi et al., 1995 ); and that P and Ca levels in pummelo leaves increased linearly with increasing soil pH ( Li et al., 2015 ). However, the levels of N, P, K, Ca, and Mg in the roots, leaflets, petioles, and whole plant of sago palm seedlings did not differ among pH 3.6, 4.5, and 5.7 ( Anugoolprasert et al., 2012 ). The concentrations of N, P, K, Ca, and Mg might have been reduced in sago palm seedlings if the pH was lower than pH 3.6, because the concentration of N, P, K, Ca, and Mg in citrus roots, stems, and leaves were greatly reduced at pH 2.5 but little affected at pH 4 relative to pH 5 (Figure 11 ). In contrast, the S level was increased in the low pH-treated C. grandis and C. sinensis roots, stems, and leaves (Figures 11F,L,R ), which is consistent with the finding that the S concentration in the tops of ginger, maize, wheat, French bean, and tomato plants was higher at pH 3.3 than at pH 4.0 ( Islam et al., 1980 ).

So far, however, there is little published information available on the effects of low pH on plant micronutrients. H + -toxicity is thought to inhibit the uptake of cations ( George et al., 2012 ). However, treatment with pH 2.5 did not lower Fe, Cu, Mn, and Zn uptake per root DW in the two citrus species, or the Mn uptake per plant in C. sinensis , when compared with pH 5 (Figures 13N,R,S,U,V ). This result may be related to the insensitivity of citrus plants to acidic soils, as reported previously by Yuda and Okamoto (1965) . Interestingly, the B uptake per plant or per root DW was reduced by a low pH (Figures 13O,T ). This result is supported by a work showing that B could alleviate low pH-induced damage in Arabidopsis roots ( Koyama et al., 2001 ).

The Fe, Mn, Cu, and Zn concentrations in the C. grandis and C. sinensis roots, stems, and leaves were all higher at pH 2.5 than at pH 5, or they were similar between the two treatments, though there was a lower level of Fe detected in the C. grandis leaves at pH 2.5 than at pH 5 (Figures 12A,B,D–G,I–L,N–Q ). The observed higher Fe, Mn, Cu, and Zn concentrations in the pH 2.5-treated C. grandis and C. sinensis roots, stems, and leaves might be associated with a reduced dilution due to decreased growth (Figure 1 ) and with higher uptake per root DW (Figures 13R,S,U,V ). As shown in Figures 12A,D,F,I,K,N , the root Fe and Cu concentrations were higher at pH 2.5 than those at the other treatment levels of pH, while no such results were observed for the leaf and stem Fe and Cu concentrations; this may be explained by the increased Fe and Cu distributions in the roots, and the decreased or unchanged Fe and Cu distributions in the leaves and stems, at pH 2.5 (Figures S2A,D,F,I,K,N). By contrast, the B level was decreased in the pH 2.5-treated C. grandis and C. sinensis roots, stems, and leaves (Figures 12C,H,M ) likely due to the decreased B uptake per plant or root DW (Figures 13O,T ).

In this experiment, many of the fibrous roots became rotten and the living roots turned abnormally dark brown when exposed to pH 2.5 (Figures 2A,D ). Thus, it is reasonable to presume that H + -toxicity may directly damage citrus roots, thus affecting the uptake of vital mineral nutrients and water.

Our results showed that pH 2.5 lowered the root, stem, leaf, and whole plant DW (Figures 1 , 2 ). The low pH-induced poor growth of citrus seedlings may be due to the combined interplay of direct H + -toxicity—as shown by the damaged roots (Figures 2A,D )—deficiencies of macronutrients—as indicated by the decreased N, P, K, Ca, and Mg concentrations (Figures 11A–E )—and uptake per plant or root DW (Figures 13A–E,G–K) , and the decreased water uptake—as indicated by the decreased root and leaf RWC (Figures 9A,F ).

In spite of the reduced growth at pH 2.5, no seedling deaths occurred in the two citrus species at each given pH during the entire experiment. Similar results have been obtained for several citrus rootstocks ( Fang, 2011 ; Fang et al., 2011 ), as well as for C. sinensis seedlings ( Guest and Chapman, 1944 ). Based on the present results, we conclude that the two citrus species studied were insensitive to low pH. This above conclusion is supported by the fact that most of physiological parameters monitored in Figures 3 , 4 , 7 , 9 were altered only at pH 2.5, and that pH 4 had almost no influence on these parameters and the OJIP transients (Figure 6 ).

As shown in Figure 2B , mottled bleached leaves were observed only in the pH 2.5-treated C. grandis seedlings (Figure 2B ). Furthermore, the pH 2.5-induced alterations of many physiological parameters shown in Figures 3 , 4 , 7 , 9 , and of the JIP transients (Figure 6 ), were slightly greater in C. grandis than in C. sinensis leaves. Evidently, when the results are taken together, seedlings of C. sinensis had a slightly higher tolerance to a low pH than did those of C. grandis . However, the difference in low pH tolerance between the C. grandis and C. sinensis species is apparently lower than the difference between them in their Al-tolerance ( Yang L. T. et al., 2011 ; Jiang et al., 2015 ; Li et al., 2016 ). This latter discrepancy is supported by a study showing that plant races were separately adapted to Al 3+ or low pH- (H + -) toxicity ( Kidd and Proctor, 2001 ).

We found that pH 2.5 greatly inhibited the CO 2 assimilation in C. grandis and C. sinensis leaves, and that this inhibition was slightly greater in C. grandis than in C. sinensis leaves (Figure 3A ). The pH 2.5-induced decrease in leaf CO 2 assimilation could not be explained only by decreased stomatal conductance, because the intercellular CO 2 concentration increased and stayed unchanged in pH 2.5-treated C. granddis and C. sinensis leaves, respectively (Figure 3C ), and because leaf CO 2 assimilation decreased with the increasing intercellular CO 2 concentration (Figure 5B ). Thus, the pH 2.5-induced decrease in leaf CO 2 assimilation in citrus may be primarily driven by non-stomatal factors.

In this context, the pH 2.5-induced decreases in Chl a, Chl b, and Chl a+b were probably not the main factor inhibiting leaf CO 2 assimilation because their reductions were much lower than that for leaf CO 2 assimilation (Figures 3A , 4A–C ). This conclusion is supported by our results showing that DI o /RC, DI o /ABS, NPQ, and qNP were all elevated in the pH 2.5-treated C. grandis and C. sinensis leaves (Figures 7F,G,N,O ).

The observed positive ΔL-band at ca. 130 μs in the OJIP transients from the pH 2.5-treated leaves (Figures 6E,J ) suggested that the grouping (stability) of the PSII units and the energy exchange between the independent PSII units were both reduced ( Strasser et al., 2004 ; Liao et al., 2015 ). This interpretation is further supported by our result that P 2G was decreased in the pH 2.5-treated leaves (Figure 7K ). The appearance of a positive ΔK-band at 300 μs in the OJIP transients from the pH 2.5-treated leaves (Figures 6C,H ) indicated that the oxygen evolving complex (OEC) had been damaged ( Srivastava et al., 1997 ). The observed positive ΔJ- and ΔI-bands at 2 ms and 30 ms, respectively, in the OJIP transients from the pH 2.5-treated leaves (Figures 6C,H ) suggested that the reduction of the PSII acceptor side had been elevated ( Strasser et al., 2004 ). The amount of electrons from the RCs at the acceptor side depends on both the capacity of electron donation to the RCs and the capacity of the electron transport chain from the RCs to the electron acceptors. Based on these results, we conclude that at pH 2.5, the PSII acceptor side was more severely damaged than was the PSII donor side. We observed that pH 2.5 led to increased DI o /RC, decreased F v /F m and ET o /ABS (Figures 7F,H,I ), and altered the OJIP transients (Figure 6 ) in leaves, together indicating that photoinhibition damaged the PSII complexes in these citrus leaves ( Maxwell and Johnson, 2000 ; Force et al., 2003 ). In the present study, the pH 2.5-induced decrease in F v /F m was caused by an increased F o , since the F m slightly increased with decreasing pH (Figures 7A,B ). The observed higher F o in the pH 2.5-treated leaves was likely associated with an increased inactivation of the PSII RCs, as increased by the decreased qP (Figure 7M ), and with the enhanced damage to OEC, as indicated by the positive ΔK-band (Figures 6C,H ). Furthermore, the higher F o may have arisen from the pH 2.5-induced accumulation of reduced Q A ( Bukhov et al., 1990 ), as indicated by the increased M o (Figure 7D ). In addition, the pH 2.5-treated leaves displayed decreased RE o /ABS, PI tot, abs , F m ′/F v ′, Φ PSII , and ETR (Figures 7J,L,P–R ). Obviously, treatment with pH 2.5 impaired the whole electron transport chain from the donor side of PSII to the reduction of the PSII end acceptors, thus decreasing ETR. Regression analysis showed that leaf CO 2 assimilation increased with increasing F v /F m , ET o /ABS, RE o /ABS, P 2G , PI tot, abs , qP, F m ′/F v ′, Φ PSII , or ETR, (Figures 8H–M,P–R ). Based on these results, we conclude that pH 2.5 damaged the whole photosynthetic electron transport chain, thus inhibiting leaf CO 2 assimilation in seedlings of these two citrus species.

Light-driven ROS production can cause oxidative damage to vital photosynthetic components and thereby inhibit photosynthesis ( Foyer and Shigeoka, 2011 ). We observed that pH 2.5 greatly increased the H 2 O 2 production and the electrolyte leakage in C. grandis and C. sinensis leaves, though more so in the C. grandis leaves (Figures 9G,H ), and that leaf CO 2 assimilation decreased with increasing leaf H 2 O 2 production and electrolyte leakage (Figures 10B,F ). Hence, the observed higher ROS production may be responsible for the pH 2.5-induced inhibition of photosynthesis in citrus leaves.

The leaf photosynthetic rate decreases with decreasing leaf RWC ( Lawlor, 2002 ). However, the relative importance of stomatal and non-stomatal limitations to photosynthesis under water stress is not yet fully understood. Typically, as the RWC decreases, the stomatal limitation of photosynthesis will also decrease and the metabolic limitation will increase ( Lawlor, 2002 ; Zhou et al., 2007 ), which entails limitations to ribulose-1,5-disphosphate (RuBP) regeneration ( Gunasekera and Berkowitz, 1993 ), photophosphorylation ( Tezara et al., 1999 ), and Rubisco activity ( Maroco et al., 2002 ; Parry et al., 2002 ). Zhou et al. (2007) observed that water stress decreased photosynthetic rate, Rubisco activity, the energy flux via linear electron transport, and increased ΔpH- and xanthophyll-mediated thermal dissipation. Our results showed that the pH 2.5-induced decrease in leaf CO 2 assimilation (Figure 3A ) was accompanied by decreases in root and leaf RWC (Figures 9A,F ), leaf Rubisco activity (Figure 3F ) and ETR, and by an increase in NPQ (Figures 7O,R ). Furthermore, leaf CO 2 assimilation decreased with decreasing root RWC, leaf RWC (Figures 10C,D ), Rubisco activity (Figure 5C ), or ETR (Figure 8R ), and with increasing NPQ (Figure 8O ); leaf Rubisco activity ( y ) increased with increasing leaf RWC ( y = −61.1653 + 0.8351 x, r 2 = 0.9174, P < 0.0001). Thus, it is reasonable to assume that a low pH lowered the water uptake and induced water stress, thus inhibiting photosynthesis in the C. grandis and C. sinensis leaves.

A study has shown that the base cation-induced increase in sugar maple photosynthesis on acid soils was associated with an improved foliar nutrient status ( St Clair and Lynch, 2005 ). Ellsworth and Liu (1994) suggested that leaf photosynthesis of sugar maple on acidic soils was co-limited by N and Ca, or by interactions of Ca with other nutrients, such as Mg. We observed that leaf CO 2 assimilation decreased with increasing leaf N, P, Ca, or Mg concentrations (N, P, Ca, or Mg uptake per plant) (Figures 14A,B,D,E,G,H,J,K ). Thus, the pH 2.5-induced decreases in these nutrients might be responsible for the observed lower leaf CO 2 assimilation.

Our results also showed that the growth of seedlings (Figures 1 , 2 ) and the status of many of their physiological parameters (Figures 3 , 4 , 7 , 9 , 11 , 12 ) reached their maximum at pH 5. This seems to contradict the early view that serious problems for citrus might arise when the soil pH was 5.0 or lower ( Chapman, 1968 ). In our study, citrus seedlings were grown under favorable conditions of mineral nutrients and the direct toxicity of H + might be the primary cause for the poor seedling growth. However, a significant difference might also occur when citrus are grown on acidic soils due to the increased solubility of Al and Mn, and/or decreased availability of P, Ca, Mg, and Mo ( George et al., 2012 ; Kochian et al., 2015 ; Li et al., 2015 ). Thus, the optimum pH for citrus might be higher in a soil culture than when grown in solution or a sand culture ( Yuda and Okamoto, 1965 ). These findings indicate that suitable fertilizers might alleviate the toxicity of acidic soils upon citrus. Adjusting the soil nutrients via careful fertilization should contribute to greater harvest yield and the sustainable management of citrus across a range of acidic soils.

Our results demonstrate that citrus seedlings were insensitive to low pH, and that C. sinensis is slightly more tolerant to this low pH than is C. grandis . H + -toxicity could directly damage the citrus roots, thus affecting their uptake of mineral nutrients and water. The results suggest that the low pH-induced inhibition of growth was caused by the combination of H + -toxicity, deficiencies of nutrients, and decreased water uptake. Here, only pH 2.5 noticeably inhibited leaf CO 2 assimilation, which was probably due to the combination of an impaired photosynthetic electron transport chain, increased ROS production, and decreased uptake of water and nutrients. In sum, these findings increase our understanding of the factors by which a low pH can decrease citrus growth, and of the mechanisms by which low pH inhibits leaf CO 2 assimilation.

Author Contributions

AL performed most of the experiment and drafted the manuscript; JZ participated in the measurements of photosynthesis and fluorescence; LY participated in the direction of this study; XY and NL participated in the analysis of the nutrient elements; LT and DL participated in the cultivation of the experimental seedlings; LC designed and directed the study and also revised the manuscript. All authors have read and approved the final manuscript.

This work was financially supported by an earmarked fund for the China Agriculture Research System (No. CARS27).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00185/full#supplementary-material

Anugoolprasert, O., Kinoshita, S., Naito, H., Shimizu, M., and Ehara, H. (2012). Effect of low pH on the growth, physiological characteristics and nutrient absorption of sago palm in a hydroponic system. Plant Prod. Sci. 15, 125–131. doi: 10.1626/pps.15.125

CrossRef Full Text | Google Scholar

Arduini, I., Kettner, C., Godbold, D. L., Onnis, A., and Stefani, A. (1998). The influence of pH on root morphology and mineral content of Pinus pinaster Ait. seedlings. Plant Biosyst. 132, 3–9. doi: 10.1080/11263504.1998.10654185

Baker, N. R. (2008). Chlorophyll fluorescence: a problem of photosynthesis in vivo . Annu. Rev. Plant Biol. 59, 89–113. doi: 10.1146/annurev.arplant.59.032607.092759

PubMed Abstract | CrossRef Full Text | Google Scholar

Bian, M., Zhou, M., Sun, D., and Li, C. (2013). Molecular approaches unravel the mechanism of acid soil tolerance in plants. Crop J. 1, 91–104. doi: 10.1016/j.cj.2013.08.002

Bradford, M. M. (1976). A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. doi: 10.1016/0003-2697(76)90527-3

Bukhov, N. G., Sabat, S. C., and Mohanty, P. (1990). Analysis of chlorophyll a fluorescence changes in weak light in heat treated Amaranthus chloroplasts. Photosynth. Res. 23, 81–87. doi: 10.1007/BF00030066

Chapman, H. D. (1968). “The mineral nutrition of citrus,” in The Citrus Industry, Vol. 2 , eds W. Reuther, H. J. Webber, and L. D. Batchelor (Berkeley, CA: Division of Agricultural Sciences, University of California), 127–189.

Chen, L. S., and Cheng, L. (2009). Photosystem 2 is more tolerant to high temperature in apple ( Malus domestica Borkh.) leaves than in fruit peel. Photosynthetica 47, 112–120. doi: 10.1007/s11099-009-0017-4

Chen, L. S., Qi, Y. P., and Liu, X. H. (2005b). Effects of aluminum on light energy utilization and photoprotective systems in citrus leaves. Ann. Bot. 96, 35–41. doi: 10.1093/aob/mci145

Chen, L. S., Qi, Y. P., Smith, B. R., and Liu, X. H. (2005a). Aluminum-induced decrease in CO2 assimilation in citrus seedlings is unaccompanied by decreased activities of key enzymes involved in CO 2 assimilation. Tree Physiol. 25, 317–324. doi: 10.1093/treephys/25.3.317

Ellsworth, D. S., and Liu, X. (1994). Photosynthesis and canopy nutrition of four sugar maple forests on acid soils in northern Vermont. Can. J. For. Res. 24, 2118–2127. doi: 10.1139/x94-272

Fang, Z. J. (2011). Evaluation of Acid Tolerance for Citrus Rootstock Seedlings and Their Differences in Physiological Characteristics . Master thesis, Jiangxi Agricultural University, Nanchang.

Fang, Z. J., Yang, L. L., Huang, C. H., Gu, Q. Q., and Xu, X. B. (2011). Analysis on acid tolerance of several citrus rootstock seedlings. South China Fruits 40, 8–11.

Google Scholar

Force, L., Critchley, C., and van Rensen, J. J. S. (2003). New fluorescence parameters for monitoring photosynthesis in plants. 1. The effect of illumination on the fluorescence parameters of the JIP-test. Photosynth. Res . 78, 17–33. doi: 10.1023/A:1026012116709

Foyer, C. H., and Shigeoka, S. (2011). Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiol. 155, 93–100. doi: 10.1104/pp.110.166181

Genty, B., Harbinson, J., and Baker, N. R. (1990). Relative quantum efficiencies of the two photosystems of photorespiratory and non-respiratory conditions. Plant Physiol. Biochem. 28, 1–10.

George, E., Horst, W. J., and Neumann, E. (2012). “Adaptation of plants to adverse chemical soil conditions,” in Marschner's Mineral Nutrition of Higher Plants, 3rd Edn. , ed P. Marschner (London: Academic Press), 409–472.

Guest, P. L., and Chapman, H. D. (1944). Some effects of pH on the growth of citrus in sand and solution cultures. Soil Sci. 58, 455–466. doi: 10.1097/00010694-194412000-00005

Gunasekera, D., and Berkowitz, G. A. (1993). Use of transgenic plants with Rubisco antisense DNA to evaluate the rate limitation of photosynthesis under water stress. Plant Physiol. 103, 629–635. doi: 10.1104/pp.103.2.629

Gunsé, B., Poschenrieder, C., and Barceló, J. (1997). Water transport properties of roots and root cortical cells in proton- and Al-stressed maize varieties. Plant Physiol. 113, 595–602. doi: 10.1104/pp.113.2.595

Han, S., Chen, L. S., Jiang, H. X., Smith, B. R., Yang, L. T., and Xie, C. Y. (2008). Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings. J. Plant Physiol. 165, 1331–1341. doi: 10.1016/j.jplph.2007.11.002

He, Z. L., Alva, A. K., Calvert, D. V., Li, Y. C., and Banks, D. J. (1999). Effects of nitrogen fertilization of grapefruit trees on soil acidification and nutrient availability in a Riviera fine sand. Plant Soil 206, 11–19. doi: 10.1023/A:1004364805789

Islam, A. K. M. S., Edwards, D. G., and Asher, C. J. (1980). pH optima for crop growth. Results of a flowing solution culture experiment with six species. Plant Soil 54, 339–357. doi: 10.1007/BF02181830

Jiang, H. X., Chen, L. S., Zheng, J. G., Han, S., Tang, N., and Smith, B.R. (2008). Aluminum-induced effects on photosystem II photochemistry in citrus leaves assessed by the chlorophyll a fluorescence transient. Tree Physiol. 28, 1863–1871. doi: 10.1093/treephys/28.12.1863

Jiang, H. X., Yang, L. T., Qi, Y. P., Lu, Y. B., Huang, Z. R., and Chen, L. S. (2015). Root iTRAQ protein profile analysis of two citrus species differing in aluminum-tolerance in response to long-term aluminum-toxicity. BMC Genomics 16:949. doi: 10.1186/s12864-015-2133-9

Kamaluddin, M., and Zwiazek, J. J. (2004). Effects of root medium pH on water transport in paper birch ( Betula papyrifera ) seedlings in relation to root temperature and abscisic acid treatments. Tree Physiol. 24, 1173–1180. doi: 10.1093/treephys/24.10.1173

Kidd, P. S., and Proctor, J. (2001). Why plants grow poorly on very acid soils: are ecologists missing the obvious? J. Exp. Bot. 52, 791–799. doi: 10.1093/jexbot/52.357.791

Kochian, L. V., Piñeros, M. A., Liu, J., and Magalhaes, J. V. (2015). Plant adaptation to acid soils: the molecular basis for crop aluminum resistance. Annu. Rev. Plant Biol. 66, 571–598. doi: 10.1146/annurev-arplant-043014-114822

Kowalenko, C. G., and Lavkulich, L. M. (1976). A modified curcumin method for boron analysis of soil extracts. Can. J. Soil Sci. 56, 537–539. doi: 10.4141/cjss76-068

Koyama, H., Toda, T., and Hara, T. (2001). Brief exposure to low-pH stress causes irreversible damage to the growing root in Arabidopsis thaliana : pectin-Ca interaction may play an important role in proton rhizotoxicity. J. Exp. Bot. 52, 361–368. doi: 10.1093/jexbot/52.355.361

Lawlor, D. W. (2002). Limitation to photosynthesis in water-stressed leaves: stomata vs. metabolism and the role of ATP. Ann. Bot. 89, 871–885. doi: 10.1093/aob/mcf110

Li, H., Yang, L. T., Qi, Y. P., Gu, P., Lu, Y. B., and Chen, L. S. (2016). Aluminum-toxicity-induced alterations of leaf proteome in two citrus species differing in aluminum-tolerance. Int. J. Mol. Sci. 17:1180. doi: 10.3390/ijms17071180

Li, Y., Han, M. Q., Lin, F., Ten, Y., Lin, J., Zhu, D. H., et al. (2015). Soil chemical properties, ‘Guanximiyou’ pummelo leaf mineral nutrient status and fruit quality in the southern region of Fujian province, China. J. Soil Sci. Plant Nutr. 15, 615–628. doi: 10.4067/s0718-95162015005000029

Liao, X. Y., Yang, L. T., Lu, Y. B., Ye, X., and Chen, L. S. (2015). Roles of rootstocks and scions in aluminum-tolerance of citrus. Acta Physiol. Plant. 37:1743. doi: 10.1007/s11738-014-1743-1

Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. doi: 10.1016/0076-6879(87)48036-1

Lin, Z. H., Chen, L. S., Chen, R. B., Zhang, F. Z., Jiang, H. X., and Tang, N. (2009). CO 2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply. BMC Plant Biol. 9:43. doi: 10.1186/1471-2229-9-43

Lin, Z., and Myhre, D. L. (1990). Citrus root growth as affected by soil aluminum level under field conditions. Soil Sci. Soc. Am. J. 54, 1340–1344. doi: 10.2136/sssaj1990.03615995005400050023x

Lu, R. K. (1999). Methods of Soil and Agrochemistry Analysis. Beijing: China Agriculture Science and Technology Press.

Malkanthi, D. R. R., Yokoyama, K., Yoshida, T., Moritsugu, M., and Matsushita, K. (1995). Effects of low pH and Al on growth and nutrient uptake of several plants. Soil Sci. Plant Nutr. 41, 161–165. doi: 10.1080/00380768.1995.10419571

Maroco, J. P., Rodrigues, M. L., Lopes, C., and Chaves, M. M. (2002). Limitations to leaf photosynthesis in field-grown grapevine under drought-metabolic and modelling approaches. Funct. Plant Biol. 29, 451–459. doi: 10.1071/PP01040

Martins, N., Gonçalves, S., Palma, T., and Romano, A. (2011). The influence of low pH on in vitro growth and biochemical parameters of Plantago almogravensis and P. algarbiensis . Plant Cell Tiss. Organ Cult. 107, 113–121. doi: 10.1007/s11240-011-9963-1

Martins, N., Gonçalves, S., and Romano, A. (2013b). Metabolism and aluminum accumulation in Plantago almogravensis and P. algarbiensis in response to low pH and aluminum stress. Biol. Plant. 57, 325–331. doi: 10.1007/s10535-012-0271-3

Martins, N., Osório, M. L., Gonçalves, S., Osório, J., Palma, T., and Romano, A. (2013a). Physiological responses of Plantago algarbiensis and P. almogravensis shoots and plantlets to low pH and aluminum stress. Acta Physiol. Plant. 35, 615–625. doi: 10.1007/s11738-012-1102-z

Martins, N., Osório, M. L., Gonçalves, S., Osório, J., and Romano, A. (2013c). Differences in Al tolerance between Plantago algarbiensis and P. almogravensis reflect their ability to respond to oxidative stress. Biometals 26, 427–437. doi: 10.1007/s10534-013-9625-3

Maxwell, K., and Johnson, G. N. (2000). Chlorophyll fluorescence -a practical guide. J. Exp. Bot. 51, 659–668. doi: 10.1093/jexbot/51.345.659

Panković, D., Sakač, Z., Kevrešan, S., and Plesničar, M. (1999). Acclimation to long-term water deficit in the leaves of two sunflowe hybrids: photosynthesis, electron transport and carbon metabolism. J. Exp. Bot. 50, 127–138. doi: 10.1093/jexbot/50.330.127

Parry, M. A., Andralojc, P. J., Khan, S., Lea, P. J., and Keys, A. J. (2002). Rubisco activity: effects of drought stress. Ann. Bot. 89, 833–839. doi: 10.1093/aob/mcf103

Peng, H. Y., Qi, Y. P., Lee, J., Yang, L. T., Guo, P., Jiang, H. X., et al. (2015). Proteomic analysis of Citrus sinensis roots and leaves in response to long-term magnesium-deficiency. BMC Genomics 16:253. doi: 10.1186/s12864-015-1462-z

Randhawa, S. S., and Iwata, M. (1968). Effects of pH, calcium concentrations and sources of nitrogen on the growth and inorganic compositions of citrus seedlings in solution culture. J. Jap. Soc. Hortic. Sci. 37, 23–31. doi: 10.2503/jjshs.37.319

Schubert, S., Schubert, E., and Mengel, K. (1990). Effect of low pH of the root medium on proton release, growth, and nutrient uptake of field beans ( Vicia faba ). Plant Soil 124, 239–244. doi: 10.1007/BF00009266

Shi, Q. H., Zhu, Z. J., Juan, L. I., and Qian, Q. Q. (2006). Combined effects of excess Mn and low pH on oxidative stress and antioxidant enzymes in cucumber roots. Agri. Sci. China 5, 767–772. doi: 10.1016/S1671-2927(06)60122-3

Srivastava, A., Guisse, B., Greppin, H., and Strasser, R. J. (1997). Regulation of antenna structure and electron transport in photosystem II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP. Biochim. Biophys. Acta 1320, 95–106. doi: 10.1016/S0005-2728(97)00017-0

St Clair, S. B., and Lynch, J. P. (2005). Base cation stimulation of mycorrhization and photosynthesis of sugar maple on acid soils are coupled by foliar nutrient dynamics. New Phytol . 165, 581–590. doi: 10.1111/j.1469-8137.2004.01249.x

Strasser, R. J., Tsimilli-Micheal, M., and Srivastava, A. (2004). “Analysis of the chlorophyll a fluorescence transient,” in Chlorophyll a Fluorescence: A Signature of Photosynthesis , eds G. C. Papageorgiou and Govindjee (Berlin: Springer), 321–362.

Tezara, W., Mitchell, W. J., Driscoll, S. D., and Lawlor, D. W. (1999). Water stress inhibits plant photosynthesis by decreasing coupling factor and ATP. Nature 401, 914–917. doi: 10.1038/44842

Tournaire-Roux, C., Sutka, M., Javot, H., Gout, E., Gerbeau, P., Luu, D. T., et al. (2003). Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. Nature 425, 393–397. doi: 10.1038/nature01853

von Uexküll, H. R., and Mutert, E. (1995). Global extent, development and economic impact of acid soils. Plant Soil 171, 1–15. doi: 10.1007/BF00009558

Wu, D. M., Fu, Y. Q., Yu, Z. W., and Shen, H. (2013). Status of red soil acidification and aluminum toxicity in south China and prevention. Soils 45, 577–584.

Yang, L. T., Jiang, H. X., Tang, N., and Chen, L. S. (2011). Mechanisms of aluminum-tolerance in two species of citrus: secretion of organic acid anions and immobilization of aluminum by phosphorus in roots. Plant Sci. 180, 521–530. doi: 10.1016/j.plantsci.2010.11.011

Yang, L. T., Qi, Y. P., Jiang, H. X., and Chen, L. S. (2013). Roles of organic acid anion secretion in aluminium tolerance of higher plants. BioMed Res. Int. 2013:173682. doi: 10.1155/2013/173682

Yang, M., Huang, S. X., Fang, S. Z., and Huang, X. L. (2011). Response of seedling growth of four Eucalyptus clones to acid and aluminum stress. Plant Nutr. Fert. Sci. 17, 195–201.

Yang, M., Tan, L., Xu, Y., Zhao, Y., Cheng, F., Ye, S., et al. (2015). Effect of low pH and aluminum toxicity on the photosynthetic characteristics of different fast-growing Eucalyptus vegetatively propagated clones. PLoS ONE 10:e0130963. doi: 10.1371/journal.pone.0130963

Yuda, E., and Okamoto, S. (1965). The effect of soil reaction on the growth of young citrus plants. I. Forms of nitrogen fertilizer and kinds of pH adjusting agent. J. Jap. Soc. Hortic. Sci. 34, 177–186. doi: 10.2503/jjshs.34.177

Zhang, C. P., Meng, P., Li, J. Z., and Wan, X. C. (2014). Interactive effects of soil acidification and phosphorus deficiency on photosynthetic characteristics and growth in Juglans regia seedlings. Chin. J. Plant Ecol. 38, 1345–1355. doi: 10.3724/SP.J.1258.2014.00129

CrossRef Full Text

Zhang, Y. K., Zhu, D. F., Zhang, Y. P., Chen, H. Z., Xiang, J., and Lin, X. Q. (2015). Low pH-induced changes of antioxidant enzyme and ATPase activities in the roots of rice ( Oryza sativa L.) seedlings. PLoS ONE 10:e0116971. doi: 10.1371/journal.pone.0116971

Zhou, Y., Lam, H. M., and Zhang, J. (2007). Inhibition of photosynthesis and energy dissipation induced by water and high light stresses in rice. J. Exp. Bot. 58, 1207–1217. doi: 10.1093/jxb/erl291

Keywords: chlorophyll a fluorescence, Citrus grandis , Citrus sinensis , low pH, OJIP transient, photosynthesis, uptake of nutrient and water

Citation: Long A, Zhang J, Yang L-T, Ye X, Lai N-W, Tan L-L, Lin D and Chen L-S (2017) Effects of Low pH on Photosynthesis, Related Physiological Parameters, and Nutrient Profiles of Citrus . Front. Plant Sci. 8:185. doi: 10.3389/fpls.2017.00185

Received: 26 October 2016; Accepted: 30 January 2017; Published: 21 February 2017.

Reviewed by:

Copyright © 2017 Long, Zhang, Yang, Ye, Lai, Tan, Lin and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Li-Song Chen, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Skip to content

Refine Your Search

Water quality: ph and alkalinity.

Recently, some growers have expressed concern about the "high pH" of their irrigation water and its potential adverse effects on plants. The purpose of this article is to allay some of these concerns by pointing out the difference between "high pH" and "high alkalinity".

Alkalinity and pH are two important factors in determining the suitability of water for irrigating plants. pH is a measure of the concentration of hydrogen ions (H+) in water or other liquids. In general, water for irrigation should have a pH b etween 5.0 and 7.0. Water with pH below 7.0 is termed "acidic" and water with pH above 7.0 is termed "basic"; pH 7.0 is "neutral". Sometimes the term "alkaline" is used instead of "basic" and often "alkaline" is confused with "alkalinity". Alkalinity is a measure of the water's ability to neutralize acidity. An alkalinity test measures the level of bicarbonates, carbonates, and hydroxides in water and test results are generally expressed as "ppm of calcium carbonate (CaCO3)". The desirable range f or irrigation water is 0 to 100 ppm calcium carbonate. Levels between 30 and 60 ppm are considered optimum for most plants.

Irrigation water tests should always include both pH and alkalinity tests. A pH test by itself is not an indication of alkalinity. Water with high alkalinity (i.e., high levels of bicarbonates or carbonates) always has a pH value ÷7 or above, but water with high pH doesn't always have high alkalinity. This is important because high alkalinity exerts the most significant effects on growing medium fertility and plant nutrition.

High pH and High Alkalinity Effects on Plant Nutrition

Potential adverse effects. In most cases irrigating with water having a "high pH" ( 7) causes no problems as long as the alkalinity is low. This water will probably have little effect on growing medium pH because it has little ability to neutralize acidity. This situation is typical for many growers using municipal water in Massachusetts, including water originating from the Quabbin Reservoir.

Of greater concern is the case where water having both high pH and high alkalinity is used for irrigation. In Massachusetts this situation is most common in Berkshire county. One result is that the pH of the growing medium may increase signifi cantly with time. This increase may be so large that normal lime rates must be reduced by as much as 50%. In effect the water acts as a dilute solution of limestone! The problem is most serious when plants are grown in small containers because small volum es of soil are poorly buffered to pH change. Therefore, the combination of high pH and high alkalinity is of particular concern in plug seedling trays. Trace element deficiencies and imbalances of calcium (Ca) and magnesium (Mg) can result from irrigating with high alkalinity water.

It is much more difficult to predict the effects of irrigating outdoor flower crops, gardens, and landscape plants with water having high pH and high alkalinity. On the one hand, in some parts of the United States, long-term irrigation of crops with wa ter high in bicarbonates and carbonates has led to yield-limiting trace element deficiencies which must be corrected with special fertilizers. On the other hand, in New England, several factors probably act together to partially offset the effects of high alkalinity water. First, rainfall levels are relatively high and historically this has caused Ca and Mg ions to leach from the soil. These are replaced with H+ and the result is acidic soil. Second, this acidification may be helped along by the rather ac idic rainfall common in this region in more recent times. Third, acid-forming fertilizers also help counteract high pH and alkalinity.

Potential beneficial effects. For some greenhouse operators, water with moderate levels of alkalinity (30-60 ppm) can be an important source of Ca and Mg. With the exception of Peter's EXCEL and a few other fertilizers, most water soluble fertil izers do not supply Ca and Mg. Also, the Ca and Mg from limestone may be inadequate for some plants. Moderately alkaline water could be beneficial as a source of extra Ca and Mg for crops prone to Ca and Mg deficiencies (e.g., poinsettia).

Other Effects of High pH and High Alkalinity

In addition to nutritional disorders of plants, water with high alkalinity can cause other problems. Bicarbonates and carbonates can clog the nozzles of pesticide sprayers and drip tube irrigation systems with obvious effects. The activity of some pestici des, floral preservatives, and growth regulators is markedly reduced by high alkalinity. When some pesticides are mixed with water they must acidify the solution to be completely effective. Additional acidifier may be needed to neutralize all of the alkal inity. To determine if a chemical is affected by high alkalinity, carefully review the product's label. Unfortunately this potentially important information is not always printed on the label, so considerable extra effort may be necessary to find the inf ormation. A call to the manufacturer will probably be needed for most chemicals.

Acidification of High Alkalinity Water

Many greenhouse operators inject acid (e.g., phosphoric, nitric, or sulfuric acid) into water with problematic high levels of alkalinity. Acidification of water having high pH but low alkalinity is rarely necessary. The use of acid injection sh ould be considered very carefully for several reasons. First, it is an extra step in production which will require additional materials and equipment. Second, acids are dangerous to handle and may damage some injectors and piping systems. Third, phosphori c or nitric acid are sources of P and NO3, so the regular fertilizer program may need to be modified to take into account the addition of these nutrients. This would depend on how much acid must be used to neutralize the alkalinity and reduce pH. Fourth, sometimes acid injection causes the solubilization of normally precipitated (unavailable) forms of trace elements resulting in levels toxic to plants.

The amount of acid required to reach the desired pH (i.e., neutralize alkalinity) is determined by laboratory titration of a water sample with the appropriate acid or by a calculation procedure. Some "fine-tuning" may be needed later when actual inject ion is started. Acid is always injected prior to the addition of fertilizer or other chemicals.

Resource Mattson N. Substrate pH: Getting it Right for Your Greenhouse Crops . Cornell University.  

Center for Agriculture, Food, and the Environment

Civil Rights and Non-Discrimination Information

Our Covid-19 Response & Resources

College of Natural Sciences

Mass. Agricultural Experiment Station

UMass Extension

UMass Research and Education Center Farms

UMass Cranberry Station

Water Resources Research Center

Interest Areas

Agriculture

Commercial Horticulture

Environmental Conservation

Food Science

Youth Development & 4-H

Pesticide Education

Plant Diagnostics Laboratory

Soil and Plant Nutrient Testing Laboratory

Hot Water Seed Treatment

Water Testing / Environmental Analysis Laboratory

Conservation Assessment Prioritization System (CAPS)

Extension Risk Management/Crop Insurance Education

Mass. Envirothon

Mass. Herp Atlas

Mass. Keystone

North American Aquatic Connectivity Collaborative

UMass Design Center in Springfield

Extension Sales Portal

Agriculture & Commercial Horticulture Resources

Community & Economic Vitality

Disaster Preparedness

Food Safety

Home Lawn & Garden

Integrated Pest Management (IPM)

Land Conservation Tools

Pollinators

Tick testing

Resources for Faculty and Staff

Extension Outreach Programs

4-H Youth Development

Crops, Dairy, Livestock and Equine

Greenhouse Crops and Floriculture

Landscape, Nursery and Urban Forestry

Clean Energy

Climate Change

Nutrition Education

Value-Added Food

Does the Type of Water Affect Plant Growth?

Plants need light, air and nutrients to thrive. They derive their nutrients from soil or, in hydroponics, nutrient-rich water. In soil gardens, you need to water the garden frequently so the good bacteria, fungi and microbes can add nutrients to the soil for the plants to use. Plants also need water for cellular function. The type of water used, both in soil gardening and in hydroponics, can affect plant growth.

Advertisement

Video of the Day

Too much water in a soil garden can drown the roots of a plant. Too little water and the plants cannot draw enough oxygen from the soil to "breathe". The basic way to tell if your soil garden needs water is to stick your finger into the soil. If you feel damp soil, wait a day. If the soil feels dry, water the garden. In hydroponics, if the water level is too low in your system, you need to add water so the plant roots can draw their nutrients.

Water aids in transporting nutrients into plants via their roots. It also provides support on the cellular level; without water, plants wilt. But if the water is too acidic or too alkaline, plants may be unable to properly process the water for use.

Water with equal parts acid and alkaline is considered to be neutral, or have a pH balance of 7.0. When water is more acidic, that number goes below seven. For water that is more alkaline, the number goes above seven. The pH balance of water affects the acid and alkaline content of soil. If water is too acidic, calcium, magnesium and potassium levels are reduced. Calcium is required for cell growth, magnesium for chlorophyll formation and potassium for synthesizing proteins. If water is too alkaline, calcium builds up, effectively cutting off the flow of nutrients to plants' roots.

Should the water you use for your plants have a perfectly balanced pH of seven? Not necessarily. Both soil and water have a pH balance, and in soil gardening, you need to reach the right pH balance for the plants you grow. Most herbs and vegetables prefer more acidic growing conditions, with a pH of between 5.5 and 6.5. The pH balance of your water will affect the pH balance of your soil, ultimately affecting the growth and health of your plants.

Considerations

In both soil gardening and hydroponics, the pH balance of the water you use for your plants is significant. Whether you use tap water, water from the hose or distilled water, you need to check the pH balance before adding it to your garden for your plants. In soil gardening, check the pH of your soil, as well, and amend the soil as needed to reach an optimum balance for your plants. In hydroponics gardening, check the pH balance after adding nutrients, as well, and then amend the water. There are products available for adjusting the pH of water in hydroponics systems.

Intermediate-Level Science Projects: Does the pH of Water Affect the Growth of Bean Plants?

• Does the pH of Water Affect the Growth of Bean Plants?

Intermediate-Level Science Projects

• Creating New Plants Through Cross-Pollination
• Do Bean Plants Grow Better in Soil or in Water?

In This Section

• Understanding acids and bases
• The effects of positive and negative ions
• Different soils for different plants
• Finding the materials you'll need
• Guarding against contamination
• Considering other botany projects

Although this section is classified as a botany project, you'll notice as you work through it that it contains a fair amount of chemistry, as well.

It's not unusual for scientific areas, or disciplines, to cross over in the course of a project or experiments. This project is a good example of how that occurs.

You've probably heard of the pH scale, or heard someone talk about the pH factor of a particular material. But what is pH exactly, and how does it affect the growth of plants?

In this section, we'll explore the basics of pH, and experiment to learn how the pH factor of liquid affects the germination and growth of bean seeds. By the time you finish, you'll have had valuable lessons in both botany and chemistry, and have a better understanding of how branches of science overlap.

So What Seems to Be the Problem?

You know that plants need certain things to help them grow. They need some kind of growing medium, usually dirt. They need light, and they need water.

The problem you'll attempt to solve while doing this science fair project is whether the pH of the water with which plants are sprinkled affects the rate of growth.

To get a better idea of what you'll be doing, and to help you formulate a hypothesis, it's important that you have a general understanding of exactly what pH is.

The initials pH stand for percent hydronium ion. The pH scale is used as a measure of how acidic or basic a liquid is. But how do liquids become acidic or basic? Isn't a liquid just a liquid?

Basic Elements

An ion results from the loss or gain of one or more electrons from an atom, causing either positive or negative ions to form.

Water—and distilled water, at that—is the only liquid that is neutral. That means it's right in the middle of being acidic or basic—and it's neither. It's just pure water.

The pH scale starts at zero and ends at 14. The more acidic a liquid is, the lower its number on the pH scale. The less acidic—or more basic a liquid is—the higher its number.

Most of the liquids you encounter on a daily basis are just around neutral. They might be a little above or a little below, but most liquids tend to be closer to neutral than at either end of the pH scale.

Liquids get their pH level as a result of molecules that split apart to form positive and negative ions. An ion is the loss or gain of electrons from an atom. When an atom loses electrons, it forms a positive ion. When an atom gains electrons, it forms a negative ion.

Liquids will be either acidic or basic (also called alkaline), depending on whether they contain positive or negative ions. If there are more positive ions in the water, the water is more acidic. If there are more negative ions in the water, the liquid is more basic.

In this experiment, you'll control the pH of the water you'll use on bean plants by adding certain substances to make distilled water either acidic or basic. You'll also control all other factors, such as how much water and light each plant gets.

Scientific Surprise

A negative ion, formed when an atom gains electrons, is also called an anion. A positive ion, formed when an atom loses electrons, is also known as a cation.

If you want to, you can use the name of this section, “Does the pH of Water Affect the Growth of Bean Plants?” as the title for your project. Other names to consider might be:

• What Type of Soil Do Bean Plants Prefer?
• Acid or Alkaline—What's Right for Bean Plants?
• To Grow or Not to Grow: Acidic vs. Alkaline Soil for Bean Plants

When you've finished with the experiment, you'll know whether bean plants prefer water that is acidic or basic.

What's the Point?

Some plants prefer acidic conditions. We call these acid-loving plants. Acid-loving plants include the following:

• Holly, pine, fir, spruce, birch, oak, magnolia, willow, and flowering crabapple trees
• Rhododendrons and azaleas
• Cranberry, strawberry and blueberry plants
• Mountain laurel

Other plants, however, such as the ones listed here, prefer alkaline soil:

• Yew, boxwood, and barberry shrubs
• Flowering plum and cherry trees
• Ash, beech, filbert, and maple trees
• Mock orange

Gardeners often help plants along by making the soil in which they grow either more acidic or more alkaline. There are products available, such as Miracid, that boost the acidity of soil. Garden lime (its chemical name is calcium carbonate) will help make soil alkaline.

In the experiment described below, you'll use distilled water as your control, and water with varying pH levels as your variables. This will allow you to observe the effects that liquids of varying pH levels have on the bean plants.

Who knows? You may end up increasing your interest in, or developing an interest in, gardening through this project. If nothing else, it will give you a better understanding of how plants grow and what types of factors affect them.

What Do You Think Will Happen?

If you've had experience with gardening and growing plants, you know that plants react differently to all sorts of factors.

Some plants like to be watered frequently, while others like to wait for a drink until the soil is completely dry. Some are much more susceptible to heat or cold than others. Some plants thrive in sunlight, while others like shady conditions. You've already read that some plants prefer an acidic soil, while others like basic soil.

You can do some research about growing bean plants to help you form a hypothesis. If you don't have much experience with plants, or don't have a good understanding of pH, it probably would be beneficial for you to learn more about growing plants, different types of soil, and so forth.

Or you can simply consider what you may already know about growing plants and make an educated guess about what will happen to the bean plants with which you'll be working.

Materials You'll Need for This Project

One thing about this experiment is that it's going to take some advance planning and a significant amount of time. You'll need almost a month from the time you plant the seeds until the time you draw final conclusions about the growth of the plants.

Standard Procedure

Online sources are available for discounted pet supply products. You can order pH Up and pH Down for $2.73 a bottle from PETdiscounters.com. It's on the web at www.petdiscounters.com .

You also will need a material or two with which you may not be familiar. Most of the materials you'll need though, are common household items. You'll need:

• A substance used to adjust the pH level of water. We suggest a set of products called pH Up and pH Down, a brand that's readily available in pet supply stores. You'll need a bottle of each pH Up and pH Down. Retail cost is about $3.50 a bottle.
• pH test strips or test kit. You also can purchase these supplies from your local pet supply store, hobby shop, or from online sources. A package of test strips in a hobby shop should run you somewhere about $3 or $4. You should have 50 strips to make sure you have enough for the experiment. Or, your science teacher may have extra test strips that you could get for this project. It doesn't hurt to ask, right?
• Seven large-size plastic drinking cups. Cups should be about 16 ounces to allow room for bean plants to grow.
• Soil to fill the cups about three-quarters full. You'll need to use the same soil for all the cups. Buying a bag of potting soil is recommended.
• Twenty-one bean seeds
• Distilled water
• Metric ruler
• Seven two-liter, plastic bottles, empty and washed well
• Small paper cups in which to measure water

If you're going to order supplies from an online provider, be sure to do so ahead of time so you don't get stuck, unable to begin your project.

Conducting Your Experiment

Explosion ahead.

Contaminating one bottle of water with any water from another bottle will affect the results of your experiment. Try as hard as you can to keep water with different pH levels completely separated.

Because you need seven individual bottles of water and seven cups, you'll need some space to set up this experiment.

It's very important that the cups containing the bean seeds are all kept in the same conditions. They each need to have the same amount of light, heat, and so forth.

And it's extremely important that each plant receives the same amount of water. If you give the plants different amounts of water, you'll be unable to determine whether the plant was affected by the pH of the water, or simply the varying amount.

Pour the water into the soil, not on the leaves of the plants. Plants take up water from their roots, not their leaves.

You can't mix water from any of the two-liter bottles, or use the same container to hold water from different bottles without washing it out in between. That's why it's recommended that when you water plants, you use small paper cups, pouring water from each two-liter bottle into its own paper cup, and then onto the bean plant.

If your plants don't need a full cup of water, measure up to the halfway mark of each cup and make a line. Fill the cup with water up to the line to assure that each plant gets the same amount.

While it's interesting to see how pH level affects plants, don't be tempted to drink any of the water yourself, and avoid splashing it on your skin, eyes, and so forth. Some of the treated waters you'll be using have very high or low pH levels, and should only be used for the purposes of this experiment.

Follow these steps to conduct the experiment:

1. Starting with seven two-liter bottles of distilled water, prepare each bottle so it has a specific pH value. Leave one bottle untreated (your control), with a pH level of 7. Add pH Up or pH Down to the other bottles so that one bottle has a pH level of 1, one has a pH level of 3, one has a pH level of 5, one has a pH level of 7, one has a pH level of 9, one has a pH level of 11, one has a pH level of 13. You'll raise or lower the pH level for each bottle from 7, depending on whether you're making the water acidic or alkaline. Cap the bottles tightly, label each one so you know which is which, and place them in an undisturbed location.

Do write down how many drops of either pH Up or pH Down you need to add to each two liter bottle. If you need to make more water at the different pH levels, you'll already know how much of the pH product to add, and the second round of water will have the exact same pH levels as the first round.

2. Plant three bean seeds into each of the seven large, plastic cups that have been filled about three-quarters of the way with potting soil.

3. Mark each cup and match one cup with one two-liter bottle of water. It's extremely important that each cup is watered from the same bottle each time, and not from any other bottle.

4. Making sure each cup gets the same amount of water, water the seeds in each cup so that the soil is moist, but not saturated. You might want to transfer water from the two-liter bottle to the cup with a tablespoon, allowing you to exactly measure how much each pot will get. Just be sure to wash the tablespoon between using it to handle water from different bottles.

5. Observe the cups every day, watering when the soil appears dry. Just be sure to always give the seeds in each cup the same amount of water. The plants will require more water as they get larger.

6. Using a metric ruler, measure the plants and record your observations every four days. Record the growth of the plants in a chart like the one found in the next section, “Keeping Track of Your Experiment.”

Your experiment will be finished after 28 days, meaning you will have measured each plant seven times.

Keeping Track of Your Experiment

Make sure to note the height of each plant within the seven cups—21 plants in all—each time you measure. Keep track of how much water you've given the plants as well. The amount of water given to the plants in each cup should be the same.

Data charts to record water application and plant growth are provided, or you can make your own charts, if you prefer.

To calculate the average height for the three plants in each cup:

• Add the three heights together for each of the three plants in cup #1.
• Divide that total by 3.
• Record this number in data chart 2.

Repeat steps 1-3 for the remaining six cups.

Don't assume that all plants within a cup will grow equally. You can average the height of the three plants within each cup.

Putting It All Together

Use the information contained on your data charts to make some graphs, plotting the growth of each plant. Or, you can summarize your observations in written form, if you prefer.

Did some of the plants grow quickly at first, only to halt their growth later? Did any of the plants die? Did your hypothesis prove to be correct?

Look for trends and patterns, concluding which plants had the best overall growth, the fastest starts, and so forth.

Further Investigation

If you enjoyed this experiment and want to try another variation of it, you could try to grow plants hydroponically—that is, in water instead of dirt—while varying the pH level of the water.

It would be interesting to see if you get different results than you did when you used water of varying pH levels to water plants growing in dirt.

You also could put liquids other than water on plants to see how growth was affected. Or, you could work with acid-loving plants and base-loving plants, testing to see at what pH level they grow best.

Excerpted from The Complete Idiot's Guide to Science Fair Projects © 2003 by Nancy K. O'Leary and Susan Shelly. All rights reserved including the right of reproduction in whole or in part in any form. Used by arrangement with Alpha Books , a member of Penguin Group (USA) Inc.

To order this book direct from the publisher, visit the Penguin USA website or call 1-800-253-6476. You can also purchase this book at Amazon.com and Barnes & Noble .

• Science Projects for Beginners: Botany Projects

Here are the facts and trivia that people are buzzing about.

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings

Preview improvements coming to the PMC website in October 2024. Learn More or Try it out now .

• Advanced Search
• Journal List
• PMC11344539

Plant growth promoting bacteria (PGPB)-induced plant adaptations to stresses: an updated review

Awmpuizeli fanai.

1 Department of Biotechnology, Mizoram University, Aizawl, Mizoram, India

Beirachhitha Bohia

Felicia lalremruati, nancy lalhriatpuii.

2 Department of Biotechnology/Life Sciences, Pachhunga University College, Aizawl, Mizoram, India

Rosie Lalmuanpuii

3 Department of Botany, Mizoram University, Aizawl, Mizoram, India

Prashant Kumar Singh

Associated data.

The following information was supplied regarding data availability:

This is a literature review; there is no raw data.

Plants and bacteria are co-evolving and interact with one another in a continuous process. This interaction enables the plant to assimilate the nutrients and acquire protection with the help of beneficial bacteria known as plant growth-promoting bacteria (PGPB). These beneficial bacteria naturally produce bioactive compounds that can assist plants’ stress tolerance. Moreover, they employ various direct and indirect processes to induce plant growth and protect plants against pathogens. The direct mechanisms involve phytohormone production, phosphate solubilization, zinc solubilization, potassium solubilization, ammonia production, and nitrogen fixation while, the production of siderophores, lytic enzymes, hydrogen cyanide, and antibiotics are included under indirect mechanisms. This property can be exploited to prepare bioformulants for biofertilizers, biopesticides, and biofungicides, which are convenient alternatives for chemical-based products to achieve sustainable agricultural practices. However, the application and importance of PGPB in sustainable agriculture are still debatable despite its immense diversity and plant growth-supporting activities. Moreover, the performance of PGPB varies greatly and is dictated by the environmental factors affecting plant growth and development. This review emphasizes the role of PGPB in plant growth-promoting activities (stress tolerance, production of bioactive compounds and phytohormones) and summarises new formulations and opportunities.

Introduction

Plants and bacteria have a continuous and dynamic co-evolutionary relationship where they interact and communicate through the release of bioactive chemical signals. This interaction can be positive or negative ( Wille et al., 2019 ). Positive interactions benefit the plants as they assist in obtaining minerals, phytohormones, and other nutrients. These beneficial bacterial species can also act against phytopathogen by releasing several bioactive compounds thereby helping plants to endure several stressful conditions. On the contrary, harmful interactions are inimical, since pathogens colonize the plant tissues resulting in the death of the host plants ( Dolatabadian, 2021 ; Adedayo et al., 2022 ). Therefore, exploiting these beneficial microorganisms can help sustain the plants against stress that hinders their productivity.

The regulations of soil fertility, the nutrient cycle, and the preservation of plant diversity are all significantly influenced by microorganisms as a component of the soil ecosystem. Zhou et al. (2020) state that the rhizosphere, a small region surrounding the plant roots serves as a vital zone for significant biological interaction occurring between the plant and microorganisms. It is a productive area where microorganisms like bacteria, actinobacteria, fungi, algae, and protozoa actively battle for nutrition and space to thrive ( Manghwar et al., 2023 ). The plant growth promoting microorganism (PGPM) can inhabit and interact with the roots of plants, which is advantageous to both the host and microorganisms; a population of rhizospheric fungi and bacteria has the potential to provide a habitat for other microbes as well ( dos Lopes, Dias-Filho & Gurgel, 2021 ). Among all the beneficial microorganisms bacteria are the most abundant, followed by fungi and actinobacteria ( Poria et al., 2021 ). They create a positive influence on the plant through nutrient assimilation and acquisition by direct or indirect mechanisms ( Kumari, Meena & Upadhyay, 2018 ).

The expansion of the global population puts food security at threat which leads to a rise in the increasing application of inorganic chemical-based fertilizers, detrimental to human health and the environment ( Mitter et al., 2021 ), The various environmental stress factors further contribute to the low yielding of crops; therefore, organic farming reliant on microflora like PGPM ensures food availability, enhancing crop productivity, quality, and better environment-friendly agricultural techniques ( da Silva Oliveira et al., 2023 ). Hence, crop production utilizing PGPM offers sustainability and safeguards soil biodiversity by minimizing the use of chemical fertilizers ( dos Lopes, Dias-Filho & Gurgel, 2021 ).

The different ways of plant growth promotion by bacteria are illustrated in Fig. 1 . Among all the bacteria, proteobacteria comprise most plant growth-promoting bacteria (PGPB). It includes genera like Pantoea, Thiobacillus, Pseudomonas, Micrococcus, Rhodococcus, Azospirillum , Azotobacter , Acinetobacter, Acetobacter Klebsiella , Enterobacter , Alcaligenes , Arthrobacter , Burkholderia , Azorhizobium, Achromobacter , Serratia, Bradyrhizobium, Flavobacterium, Mesorhizobium, Microrhizobium, Streptomyces, Bacillus, Azoarcus, Aeromonas, Azoarcus, Caulobacter, Chromobacterium, Delftia, Frankia, Flavobacterium, Gluconacetobacter, Paenibacillus, Rhizobium and Streptomyces dos Lopes, Dias-Filho & Gurgel (2021) , have all demonstrated that bacteria can boost plant development. Following Oldroyd et al. (2011) , the Fabaceae, Poaceae, Asteraceae, Solanaceae, Brassicaceae, and Crassulaceae were the most abundant family connected with the PGPB host plant, they are depicted in Table 1 .

The impact of biotic stress (caused by living organisms) and abiotic stress (environmental factors) on plants highlighting the role of PGBP in mitigating stress effects. PGBP enhance plant growth, nutrient uptake, and stress tolerance through mechanisms such as siderophores, lytic enzyme,HCN, antibiotics & nitrogen fixation, hormone production, etc .

The PGPB benefits the plants in several ways, which include aiding several biotic and abiotic stress tolerances, inducing plant growth promotion by solubilizing different inorganic mineral nutrients, nitrogen fixation, the release of plant growth regulators, and several other biochemicals that directly or indirectly favor the plant productivity.

Search methodology

The main purpose of this review is to emphasize the significance of plant growth-promoting organisms, especially bacteria in the mitigation of various biotic and abiotic factor-induced stress on plants, and their mechanisms employed to improve the quality and quantity of plant products to achieve sustainable agriculture. To ensure the comprehensive and unbiased coverage of the literature, we focus on the latest publications in each of their particular area, between 2000 to 2024 including research articles, review articles, and case studies using Google as a search engine. To elucidate some points, terms like bioinoculants, bioformulants, and bioactive compounds were searched. Some properties of PGPB including bioremediating potential, Pharmaceutical potential, and genetics involved in the plant growth-promoting ability are excluded in this review which may be diverted from this review’s goals and confuse the readers.

Rationale and intended audience

Agricultural system using beneficial bacteria as bioinoculants is a promising way of achieving a sustainable future. It is a good alternative to chemical fertilizer as an efficient, eco-friendly, productive fertilizer that may aid provide the food demand of the growing global population. They not only enhanced the plant growth but also maintained the soil fertility and enhanced the soil microbiome.

Plant growth promoting bacteria induced stress tolerance

The continual exposure of plants to various stresses (both biotic and abiotic) adversely impacts their growth and development, resulting in compromised yield and quality ( Singh et al., 2021 ). As a result, plants develop specific types of defense mechanisms for stress response, which is assisted by a naturally occurring PGPB boosting the resistance against various phytopathogens via producing biochemicals and enhancing the soil fertility ( Ramakrishna, Yadav & Li, 2019 ; Leontidou et al., 2020 ). The different stress factors and mechanisms of bacterial stress tolerance are depicted in Fig. 2 .

The impact of biotic stress (caused by living organisms) and abiotic stress (environmental factors) on plants highlighting the role of PGBP in mitigating stress effects. PGBP enhance plant growth, nutrient uptake, and stress tolerance through mechanisms such as nitrogen fixation, hormone production, and biocontrol.

Abiotic stress factor

Climate change induces several abiotic stresses such as drought, salinity, temperature, and heat. These along with nutrient limitations and the presence of heavy metals are crucial players behind compromised yield by crop plants ( Naing, Maung & Kim, 2021 ). In the following points, we will discuss the abiotic stress tolerance mechanisms offered by PGPB.

According to Ahluwalia, Singh & Bhatia (2021) , drought stress is divided into four types, a hydrological drought, a socio-economic drought, a meteorological drought and agricultural drought. A hydrological drought arises when there is scarcity and limited water supply in a particular place; a socio-economic drought occurs when water resources are insufficient to meet the demand, meteorological drought happens in dry weather places, and an agriculture drought results due to the reduction of water level in the soil. Drought stress affects the plant by developing reactive oxygen species, which negatively influence the plant structure and mechanism ( Nautiyal et al., 2013 ). Therefore, certain mitigation processes like the application of biochar, nanoparticles, film farming, drought resistant plant cultivars, however, provide limited advantages to the agricultural system ( Fadiji et al., 2022 ).

PGPB can provide the plant with a better adaptation and tolerance to drought by regulating water absorption, modifying the root structure, and producing phytochemicals ( Khan & Bano, 2019 ). Maize plants treated with Bacillus pumilus and Pseudomonas putida demonstrated tolerance to drought stress and nutrient limitations ( Kálmán et al., 2024 ). Likewise, in another study by Ilyas et al. (2020) , bacteria such as B. subtilis and A. brasilense release a distinct amount of osmolytes that increase the drought tolerance in wheat, enhancing seed and plant growth germination. This plant growth-promoting bacteria also adapts to water stresses by releasing various bioactive compounds like indole-3-acetic acid, salicylic acid, dihydroxybenzoicacid (DHBA), 1-aminocyclopropane-1-carboxylic acid deaminase, and exopolysaccharides and regulate plant growth ( Ahmed et al., 2021 ). According to Danish et al. (2020) , the soil of water-stress maize was inoculated with the drought tolerating bacteria, and a tremendous improvement in the nutrient uptake by shoot and root elongation and increasing the diameter, dry biomass, and chlorophyll content was observed in the plant. The drought-tolerating bacterial strains, Pseudomonas lini , and Serratia plymuthica inoculation lowers the drought-induced harm and increases the soil aggregrate stability ( Zhang et al., 2020 ). Moreover, inoculation of Bacillus albus , and Bacillus cereus increases the seed vigor index ( Ashry et al., 2022 ). All these tolerant strain improves the pigment concentration in the plants increasing the photosynthetic efficiency and the antioxidant properties as well ( Saleem et al., 2021 ).

Other drought-tolerating bacteria include Achromobacter xylosoxidans , B. pumilus ( Castillo-Lorenzo et al., 2018 ), P. aeruginosa, L. adecarboxylate, E. cloacae, P. putida, A. xylosoxidans ( Danish et al., 2020 ), Zobellalle denitrificans, Endophyticans, P. fluorescens S3X, Staphylococcus sciuri ( Khalilpour, Mozafari & Abbaszadeh-Dahaji, 2021 ).

Temperature stress

A rise in the mean temperature of the climate is one of the most serious abiotic stresses endured by plants ( Desaint et al., 2021 ) Bacterial species such as Bacillus cereus, Serratia liquefacience, Pseudomonas putida, P. fluorescens ( Mitra et al., 2021 ), Burkholderia phytofirmans, Curvularia proturberata ( Rana et al., 2021 ), Parabulkholderia phytofirmans ( Issa et al., 2018 ), Bacillus sp ., and Pseudomonas sp. ( Ahmad et al., 2023 ) are identified to have a heat tolerance by balancing plant regulators like cytokinins, ACC deaminase, and antioxidant enzymes which control plant absorption of water and induce the expression of heat shock proteins ( Moumbock et al., 2021 ).

A diverse amino acid and its compound have been reported to reduce the harmful effect, and also aid plants in response to heat stress ( Santos et al., 2022 ). The application of B. cereus increased overall plant biomass, chlorophyll content, and expression of heat shock protein ( Khan et al., 2020b ). A study done by Park et al. (2017) reveals that Enterobacter SA187 inoculation increases the heat endurance in the wheat and Arabidopsis plants, thereby increasing the grain yield, height of the plant, and weight of the seed. Furthermore, B. cereus increased the carotenoid, protein, ascorbate peroxidase, chlorophyll content, and superoxide dismutase level in the plant ( Bisht & Mishra, 2020 ).

Other bacterial strain like B. thuringiensis, B. subtilis, P. brassicacearum ( Ashraf, Bano & Ali, 2019 ), B. velenzensis ( Abd El-Daim, Bejai & Meijer, 2019 ), B. cereus ( Khan et al., 2020a ).

The salinity of soil is caused by the scarcity of water ( Reints, Dinar & Crowley, 2020 ) and a high concentration of NaCl from the compost fertilizer, which is used for sewage and domestic waste treatment ( Gondek et al., 2020 ). Soil salinization also occurs when there is a high concentration of soluble ions like bicarbonate, magnesium, sodium, chloride, sulfate, carbonate, and calcium ( Shahid, Zaman & Heng, 2018 ). Excessive Na + concentration can completely change the soil composition, reduce the fertility of the soil ( Manishankar et al., 2018 ) germination rate, and decrease the photosynthetic pigment production by changing the structure of chloroplasts ( Ahmed et al., 2020 ), moreover inducing ion toxicity and osmotic imbalance ( Krishnamoorthy et al., 2022 ). A study by Taïbi et al. (2016) shows that salinity stress decreases the overall plant development, fruit yield, weight, and total number of fruits in a single strawberry plant. Ansari, Ahmad & Pichtel (2019) also state that a plant’s water movement and stomata conductivity declined due to saline stress. The plant growth-promoting bacteria can counteract the adverse effect of salinity by stimulating the stress response, reducing the ROS production, production of Na-binding exopolysaccharides ( Talebi Atouei, Pourbabaee & Shorafa, 2019 ) and also producing phytohormones which promote the growth of root cells, enhancing the water intake ( Subramaniam et al., 2020 ). According to Del Rosario Cappellari et al. (2020) , a salt-tolerating strain Bacillus amyloliquifacience GB03 produces a volatile organic compound that significantly increases the stress mitigation by sixfold to the plant, compared to the one that is not exposed to the compounds. The salt-stress-tolerating bacteria include Streptomyces sp. ( Tolba et al., 2019 ), Aneurinibacillus aneurinilyticus , Paenibacillus sp . ( Gupta & Pandey, 2019 ), Pseudomonas azotoformance ( Liu et al., 2021 ). Also, a bacterial strain like Pseudomonas, Bacillus, Enterobacter, Klebsiella, Agrobacterium, Streptomyces , and Ochromobactrum can tolerate sodium chloride up to 150 g/L ( Zhang et al., 2018 ).

Heavy metal

Heavy metals are inorganic soil pollutants that negatively impact plants. Even though heavy metals are toxic in higher concentrations, they are also an essential source of micronutrients ( Ayangbenro & Babalola, 2017 ). Industrial effluent, farm, agrochemical, and domestic waste are anthropological sources of heavy metals in the soil ( Kamran et al., 2020 ). Heavy metal enters the plant system, adversely affecting the environment and human beings. Therefore, applying phytohormone-producing bacteria is a sustainable way of removing heavy metals from the soil. The bacteria produce plant hormones that alter the root structure, aiding the plant system to tolerate heavy metal stress ( Ashraf et al., 2017 ). Bacillus sp. is an efficient cadmium accumulator that lowers the availability of H 2 O 2 , O − 2 , and malondialdehyde (MDA) which in turn can trigger heavy metal induced reactive oxygen species stress to the plant ( Zhang et al., 2022 ). Although heavy metal are considered harmful for ecological health, they can be tailored and employed for plants over all development. Bacillus subtilis, Azospirillum brasilens , and Pseudomonas fluorescens integrated with nano zinc increase the concentration of nitrogen, phosphorus, and zinc which in turn enhances the over all productivity of wheat crop ( Jalal et al., 2023c ). Also, co-inoculation of Rhizobium tropici and Bacillus subtilis along with nano-zinc foliar spray enhanced the chlorophyll content, zinc concentration and the grain yield ( Jalal et al., 2023a ). Plant regulators like IAA ( Jalmi et al., 2018 ) and gibberelins ( Sytar et al., 2019 ) enhanced the plants’ stress tolerance ( Abdelaal et al., 2021 ). Bacteria develop mechanisms like biomolecules and biochemical production by modifying heavy metal mobilization in response to heavy metal.

Heavy metal tolerating bacteria includes B. subtilis, P. brassicacearum , B. Thuringeansis ( Ashraf, Bano & Ali, 2019 ), B. cereus ( Asaf et al., 2017 ), Raoultella ornithinolytica , Brevibacterium, Aspergillus sp., Trichoderma sp., Pseudomonas flourescens ( Bhatt et al., 2019 ), Enterobacter sp. ( Naveed et al., 2020 ), Bacillus sp. ( Khan et al., 2017 ), B. megaterium MCR-8 ( Hansda, Kumar & Anshumali, 2017 ), Variovorax sp., Bacteroidetes bacterium, P. putida ( Kamran et al., 2020 ), Alcaligenes faecalis , and P. syringae ( Okpara-Elom et al., 2024 ).

Biotic stresses

PGPM can potentially inhibit biotic stress in plants, induced by pathogenic fungi, bacteria, nematodes, insects, and weeds ( Gupta et al., 2021 ), this type of resistance is known as Systemic acquired resistance (SAM), also when the plant growth-promoting bacteria elicit the biotic stress by producing elicitors like volatile organic compounds, microbe associated molecular patterns (MAMPs) in, and bioactive secondary metabolites, it is called induced systemic resistance (ISR) ( Romera et al., 2019 ; Dubey et al., 2020 ).

According to Migunova & Sasanelli (2021) , the PGPB inhibits pathogenic nematodes directly by releasing different lytic enzymes, antibiotics, and volatile organic compounds indirectly through nitrogen fixation, siderophores, and solubilizing phosphate phytohormones production. Bacterial strains such as Pasteuria penetrans ( Mohan et al., 2020 ) and Brevibacillus laterosporus produce proteases that inhibit the nematode Heteroderaglycines ( Abd-Elgawad & Askary, 2018 ). Bacillus megaterium also produces proteases against M. graminocula ( Liang et al., 2019 ). Also, P. aeruginosa , P. cepacia , and P. fluorescens have anthelmintic, antimicrobial, antiviral, cytotoxic, and antitumor properties that can fight against phytopathogens ( Bhavya & Geetha, 2021 ) Moreover, bacteria like B. subtilis , and B. pumilus produce chitinase that combats nematodes like M. hapla and Meloidogyne sp. ( Kohli et al., 2018 ) by causing hydrolysis, disrupting chitin synthesis, producing volatile compounds acting as antifungal ( Xie et al., 2020 ). B. amyloliquefaciens FZB42 produces bacteriocins inhibiting M. incognita ( Liang et al., 2019 ). According to Aeron et al. (2019) , Anthrobacter nicotianae and Bacillus sp. release volatile compounds that exhibit activity against M. graminicola and M. incognita . Lyseni bacillus, Staphyococcus, Pseudomonas , and Enterobacter also have antibacterial, and antifungal properties ( Mamonokane, Eunice & Mahloro, 2018 ).

According to Oleńska et al. (2020) , the interactions between plants and various bacteria have an array of effects on plant productivity and soil fertility by producing various chemicals like siderophores, phytohormones, and antibiotics, which inhibit the efficacy of various pathogenic fungi, dissolve phosphate in the soil, and produce indole acetic acid, which promotes plant growth. Moreover, the PGPB must possess inherent rhizospheric competency which enables it to colonize the rhizosphere in addition to the previously stated properties.

PGPB also reduces or stops the effect of specific pathogens that compete for nutrients and interact with certain beneficial microorganisms and indirectly encourages plant productivity ( Benizri et al., 2021 ). PGPB synthesizes antimicrobials like bacteriocins, antibacterial proteins, and enzymes that induce both narrow- and broad-spectrum inhibition of bacteria by altering the structural membrane and damaging the cell wall ( Nazari & Smith, 2020 ; Mak, 2018 ) The antagonistic effect of PGPR can occur by producing cell wall hydrolases such as chitinase, glucanase, proteases, and ligases that can destroy the pathogenic cell ( Pérez-Montaño et al., 2014 ). Other than directly acting as an anti-pathogenic activity, the antibiotic also triggers the induced systematic resistance (ISR) in plants, suppressing the disease and acting as a biocontrolling agent. The pathogenic bacteria mainly belong to bacterial genera like Erwinia , Pectobacterium , Pantoea , Agrobacterium , Pseudomonas , Ralstonia, Burkholderia, Acidovorax, Xanthomonas, Clavibacter, Streptomyces, Xylella, Spiroplasma , and Phytoplasma . They cause various diseases, including wilting of leaves, spots, galls, blights, and root rot ( Nabila & Kasiamdari, 2021 ). Some examples of the most common disease-causing plant pathogens and the PGP strain that suppresses them are listed in Table 2 .

Classification of pgpb

Based on their interactions with plants.

Based on interactions, PGPB can be categorized into two types, namely free-living rhizobacteria and symbiotic bacteria. The free-living rhizobacteria are present outside the plant cells, while the symbiotic bacteria, also called endophytes, reside in the intercellular spaces of the plant allowing them direct access to the exchange of metabolites ( Turan et al., 2016 ).

Following Djaya et al. (2019) , endophytic bacteria are the organisms that colonize the internal tissues of plants at least once in any part of their lifetime. Various endophytic bacteria colonize different plant parts such as leaves, stems, roots, and flowers ( Santoyo et al., 2016 ). The density of culturable bacterial cells per gram retrieved from the root is higher than that of stem and leaves, then flowers and fruits ( Amend et al., 2019 ). According to Afzal et al. (2019) , the diversity of bacteria depends upon the conducive conditions, genetic composition, and physiology of the plant parts they colonize. The relationship between plants and endophytes is considered to be symbiotic and interacts with the root more efficiently. Still, recent studies reveal that the microorganism’s mutualism or pathogenicity depends on the genetic composition, environmental factors, and co-colonization of bacteria. Therefore, the term endophytes also includes the pathogen colonizing the plant tissue ( Compant et al., 2021 ). The endophytic bacteria can be pre-inoculated in the seeds, enhancing the seed quality, increasing the shelf life, and boosting the plant’s endurance to specific stresses ( Zapata-Sarmiento et al., 2020 ). Besides, the rhizospheric bacteria can penetrate the plant tissues through the cracks in the roots and cause various tissue injuries to the plant due to the continuous growth of the plant ( Sørensen & Sessitsch, 2007 ). Endophytic bacteria can also be used as bio-controlling agents; they help in plant growth directly and indirectly ( Hernández-León et al., 2014 ), such as the production of antibiotics, cell wall degrading enzymes, and pathogen-resistant volatile compounds. They induce systemic resistance and reduce ethylene production ( Santoyo et al., 2016 ). Furthermore, endophytic actinobacteria also generate secondary metabolites, improving the growth and resilience to various environmental stresses ( Girão et al., 2019 ).

The study performed by Pitiwittayakul, Wongsorn & Tanasupawat (2021) shows the endophytes isolated such as Nguyenibacter vanlangensis , Acidomonas methanolica , Asaia bogorensis , Tanticharoeniaaidae , Burkholderia gladioli , and Bacillus altitudinis from the stem of sugarcane from the Nakhon Ratchasima Province in Thailand effectively inhibit the mycelial growth of F.moniliforme AITO1. These isolates also exhibit plant growth promotion properties through ammonia production, zinc, phosphate solubilization, and biosynthesis of auxin and siderophores. This result highlights that endophytes can be potentially used as PGP and antifungal.

Plant growth-promoting rhizobacteria (PGPR) refers to the bacteria that colonize the rhizospheric region, and aid in plant growth and development by producing beneficial metabolites ( Santoyo et al., 2021 ). The bacteria must have the ability to induce plant growth, suppress or stop the pathogen, and should be invasive ( More et al., 2022 ). PGPR affects the plant by producing and releasing secondary metabolites, which can be employed for crop nutrition and protection, increasing the availability and uptake of different micronutrients from the soil and replacing chemical pesticides ( Ramakrishna, Yadav & Li, 2019 ; Zhao et al., 2021 ). The rhizospheric microbial diversity is determined by the exudates and metabolites secreted by the roots that provide the optimum environment for microbial growth regarding nutrient availability ( Zhao et al., 2021 ; Vives-Peris et al., 2020 ).

A study by Dörr, Moynihan & Mayer (2019) and Lee et al. (2022) reported that the tomato seeds inoculated with a PGPR Rhodopseudomonas palustris enhance the overall plant post-harvest quality and increase the nutrient availability in the fruits.

Based on their cell wall composition

According to Dörr, Moynihan & Mayer (2019) , the PGPB can also be classified based on the composition of their cell wall; the bacteria that consist of a thick peptidoglycan wall can retain a Gram dye are called Gram-positive, while the ones that have a thin peptidoglycan wall and cannot keep the Gram’s dye are known as Gram-negative.

The Gram-positive PGPB includes B. alveli , B. thuringeansis , Clostridium novyi , C.limosum , Symbiobacterium thermophillum, etc ., Gram-negative PGPB includes Citrobacter sp ., C. freundii , C. intermedius , C. koseri , E. coli , Enterobacterderogenes , Flavobacterium sp . ( Rodrigues et al., 2016 ), Azotobacter chroococcum, A. insignis, A. nigricans, A. brasilense, Az. salinestris , and Az. vinelandii ( Shelat, Vyas & Jhala, 2017 ).

Mechanism of plant growth promoting activity by bacteria

Antagonistic activity (indirect mechanism), siderophores production.

Siderophores are the ferric-specific ligands produced by bacteria to combat low iron stress and improve plant growth ( Sayyed et al., 2013 ). They are classified as hydroxamates, phenolates, and carboxylates on the basis of their iron-binding component ( Nosrati et al., 2018 ). Over 250 types of siderophores have been structurally characterized ( Boukhalfa et al., 2003 ). Iron is an essential micronutrient for plant and microorganism growth, metabolism, and survival ( de Souza et al., 2015 ). Siderophore-producing bacteria have an iron-regulated protein on their cell surface which transports the ferric iron complex thus, iron becomes available for the metabolic process. Siderophores produced by bacteria are the primary source of iron in events of inefficient iron present in plants ( Perez et al., 2019 ).

According to Loaces, Ferrando & Scavino (2011) , the rhizobacterial ability to release siderophore has conferred various advantages to endophytic bacteria for colonizing the plant roots and excluding other microorganisms from the same environment. The bacteria that produce siderophores are mostly Bacillus, Chryseobacterium Phyllobacterium ( Bhatt et al., 2019 ), Pseudomonas sp . like Pseudomonas fluorescens, P. putida , P. aeruginosa , and P. aureofaciens .

The siderophore produced by the bacterial isolates can be tested by the method determined by Passari et al. (2015) . A bacterial colony is inoculated in the blue agar plates containing chrome azurol S (CAS) agar medium and incubated at 27 °C for 5 days. The colonies with a yellow-orange halo zone were considered positive for siderophore production.

The siderophore-based drugs and siderophores isolated from microbes can be used efficiently to treat beta-thalassemia and certain anemia, iron overload diseases like hemochromatosis and hemosiderosis, iron poisoning ( Pietrangelo, 2003 ), antimalarial, desferrioxamine-B is produced by Streptomyces piosus which is active against P.falcipararum which causes the depletion of iron. This type of siderophore also inhibits the growth of parasites that cause sleeping sickness in humans ( Nagoba & Vedpathak, 2011 ) and cancer treatment ( Petrik et al., 2017 ; Ribeiro & Simões, 2019 ).

Production of lytic enzymes

Plant growth-promoting bacteria serve as defendants of other bacterial pathogens by producing several enzymes, they are elaborated in the following.

Protease production

Proteases produced by microorganisms account for two-thirds of all commercial proteases worldwide ( Younes & Rinaudo, 2015 ). The proteases from microbes are desirable since they produce a greater yield and are rapid, space-saving, and cost-effective ( Nisha & Divakaran, 2014 ; Ali et al., 2016 ). Proteases can be classified into alkaline, acidic, and neutral. Bacillus sp . is the most commonly commercially exploited microbe for protease production. An antifungal metabolite from B. subtilis subsp. natto purified by Castaldi et al. (2021) shows that different proteolytic enzymes such as serine protease, and subtilisin act as an antifungal, showing a high active peak when analyzed with liquid chromatography coupled with tandem mass spectrometry. These show the production of protease as a potential defense system to protect plant aginst a threatening pathogen, which in turn indirectly aids the plant development. Several protease-producing bacteria include B. clausei, B. licheniformis, B. lentus, A. salinivibrio , and Cryptococcus aureus . Streaming processes purify extracellular alkaline proteases like Subtilisin Carlsberg and Subtilisin Novo to obtain end products ( Kalaiarasi & Sunitha, 2009 ).

The production of proteases can be screened using a well plate assay method ( Masi, Gemechu & Tafesse, 2021 ). The bacterial strains were inoculated on a 1% Skim milk agar plate. The proteolytic activity was confirmed when a clear halo zone was formed. It was expressed in terms of a millimeter. The PSI for protease activity was calculated using the following.

The proteases produced from different bacterial sources can be purified using ion exchange and gel filtration chromatography ( Kanmani et al., 2011 ; Sa et al., 2012 ). Other than being an antagonizing agent, alkaline protease is also involved in formulations of ointment, gauze, and non-woven tissues ( Awad et al., 2013 ). It also treated lytic enzyme deficiency syndrome ( Gupta & Khare, 2007 ; Palanivel, Ashokkumar & Balagurunathan, 2013 ). Moreover, bandages immobilized with elastomers are used for burns, wounds, carbuncles, and furuncles ( Palanivel, Ashokkumar & Balagurunathan, 2013 ). Intracellularly-produced proteases have contributed to protein turnover, hydrolysis, hormone regulation, and cell differentiation ( Adrio & Demain, 2014 ). Industrial sectors have extensively explored numerous bacterial species for synthesizing products like detergent, food and brewing, silk degumming, denture cleaner, and waste management ( Razzaq et al., 2019 ).

Catalase production

Bacteria with catalase activity are critical for the self-defense and protection of the plant roots against hydrogen peroxide. This type of bacteria indirectly assists plant growth during oxidative stress ( Bumunang & Babalola, 2014 ). Bacteria such as B. marinus, B. insolitus, B. sphaericus, B. pasteurii, B. laterosporus, B. badiu , and Staphylococcus aureus are positive for catalase activity ( Talaiekhozani, 2022 ). The bacterial catalase activity can be screened using the tube method described by Kumar et al. (2012) . Bacterial colonies incubated for 18–24 h were inoculated in a test tube containing 3% H 2 O 2 and placed in a dark room to observe the bubble formation. The tubes showing a bubble formation were regarded as bacteria having a catalase activity.

Amylase production

There are three types of amylase: alpha, beta, and gamma. Among these, alpha-amylase is produced mainly by bacteria, fungi, and actinobacteria. These amylases hydrolyse the cell wall of pathogen thereby guarding the host plant against phytopathogen. The majority of these enzymes are produced by endophytes of medicinal plants and crops ( Ismail et al., 2021 ). The bacteria that are known to produce a high amount of alpha-amylase are Bacillus amyloliquefaciens , Bacillus licheniformis , Bacillus strearothermophilus , and Geobacilus bacterium ( Far et al., 2020 ).

Amylase producers can be observed after spot inoculation of bacterial isolates in the starch Agar medium, incubated at 28 ± 2 °C for 7 days. Iodine solution was splashed onto plates, and after 5–10 min of reaction, a definite halo zone was observed ( Mishra & Behera, 2008 ).

The alkaline amylase is an essential constituent of liquid and solid detergents. They are mainly used to remove starch-containing stains ( Niyonzima & More, 2014 ).

Urease production

Some soil bacteria can degrade urea in the form of ammonium and nitrate, which plants can later utilize as a source of nitrogen ( Witte, 2011 ). Brink (2010) can determine urea hydrolysis. The urea-buffer solution (1% urea at pH 6 with 0.00025% phenol red was added to Stuart’s urea broth that contained 5 ml of bacterial cultures. Production of ammonia due to increased pH leads to a change of color. Tubes were incubated for 3 to 5 days at 37 °C and 120 rpm on an orbital shaker. The appearance of red or pink from yellow indicates the breakdown of urea by the bacteria.

The ureolytic bacteria can precipitate calcite by increasing pH and producing carbonate ions. This property is exploited for soil nutrient enrichment, concealment of concrete cracks, and various biomineralization approaches ( Cui et al., 2022 ).

Hydrogen cyanide production

Hydrogen cyanide is a highly toxic volatile compound capable of cellular respiration disruption ( Alemu, 2016 ). They inhibit pathogenic fungi, nematodes, insects, and termites ( Sehrawat, Sindhu & Glick, 2022 ). The HCN produced by rhizospheric bacteria also acts as a controlling agent for weeds by colonizing the plant roots and hindering their growth. It has no adverse effect on the plant host.

Hydrogen cyanide production can be screened according to a method described by Lorck (1948) . According to him, bacteria were streaked in an agar media containing 4.4 g L −1 of glycine. The Whatman filter paper was soaked in an alkaline prate solution and put on the lid of the culture plate and then inoculated at 28 °C for 3 days. The color change was observed and considered hydrogen cyanide production.

The HCN-producing bacteria mainly belong to Pseudomonas and Bacillus species ( Voisard et al., 1989 ; Lieberei, Fock & Biehl, 1996 ; Damodaran et al., 2013 ), Bacillus pumilus , and Bacillus subtilis ( Damodaran et al., 2013 ).

Direct mechanism

Phytohormone production.

Plant hormones are indispensable chemical messengers that direct the plant’s ability to react to the environment ( Gutiérrez-Mañero et al., 2001 ; Vejan et al., 2016 ). Both plants and microorganisms can carry out the biosynthesis of plant hormones like cytokinins and auxin. The study carried out by Daud et al. (2019) , Swarnalakshmi et al. (2020) and Mekureyaw et al. (2022) . Certain bacteria like Paenibacillus polymyxa , Rhizobium leguminosarum and Pseudomonas fluorescens are known to be cytokine producers. However, the production of cytokinins is not well studied and investigated due to their diverse compound groups, usually present in minute amounts, making them difficult to identify and quantify.

Moreover, studies regarding the gibberellic acid’s production as a plant growth promoter are limited; only a few studies were conducted during the last 20 years; from the latest study performed by Gutiérrez-Mañero et al. (2001) , bacterial strains B. pumilus and Bacillus licheniformis produced four forms of gibberellic acid. Among all the plant hormones, IAA is most commonly investigated and regarded as one of the critical PGB traits for plant growth promotion. It is a heterocyclic compound with a carboxymethyl group that induces leaf formation, embryo development, root initiation and growth, phototropism, geotropism, and fruit development ( Chandra et al., 2019 ). A carboxymethyl group, acetic acid, is responsible for all the functions performed by IAA ( Mike-Anosike, Braide & Adeleye, 2018 ). Certain studies indicated that some rhizospheric bacteria can produce physiologically active IAA, which inspires root elongation, cell division, and plant growth ( Rehman et al., 2020 ). The bacteria that produced phytohormone include Azospirillum ( Pedraza et al., 2020 ; Raffi & Charyulu, 2020 ), Arthrobacter spp. Bradyrhizobium, Bacillus, Pantoea, Rhanella, Burkholderia, Arthrobacter, Herbaspirillum, Pseudomonas, Enterobacter, Mesorhizobium , and Brevundimonas ( Prasad et al., 2019 ). Plants and microbes synthesized IAA through several interrelated pathways. One of them is the dependent pathway. Microbes’ IAA varies by physiological parameters like pH, temperature, carbon, and nitrogen sources ( Chandra, Askari & Kumari, 2018 ). The bacterial strains like Bacillus paenibacillus polymyxa, Bacillus subtilis, Camamonas acidovorans, Bacillus megatarium, B. simplex , and Enterobacter cancerogenus , produce IAA, which enable the plant to absorb more nutrients from the soil, resulting in the overall enhancement of growth and development in plants ( Goswami et al., 2013 ).

IAA production by rhizobacteria can be analyzed as per the method described by Ahmed & Hasnain (2010) . Here, the bacterial isolates were grown in nutrient broth supplemented with 0.5% of L-tryptophan at 27 °C for 3 days. The suspension was then centrifuged at 11,000 rpm for 10 min, and collect the supernatant. Further, 1 ml of the supernatant was added to 2 ml of Salkwoski reagent (1 ml of 0.5 M FeCl 3 + +50 ml of 35% perchloric acid) and incubated at room temperature in the dark for 30 min. The formation of a pink color indicated that the bacteria produced IAA. Production of IAA is quantitatively determined by taking OD at 530 nm, and the concentration was expressed in μg/ml. This is an efficient protocol commonly used for qualitative and quantitative estimation of IAA production.

Phosphate solubilization

According to Satyaprakash et al. (2017) , phosphorus is considered the second most essential nutrient for the plant; inadequate phosphorus eventually hinders the growth of the plant. Studies have reported the ability of bacteria to solubilizes the inorganic phosphate compounds like tricalcium phosphate, dicalcium phosphate, hydroxyapatite, as well as rock phosphate into a soluble organic phosphate by releasing organic acids ( Verma et al., 2017 ) like citric acid and gluconic acid which chelate the cations of phosphate using the hydroxyl and carboxyl groups present in them ( Youssef, 2014 ).

Several studies involving phosphorus-solubilizing bacteria for plant growth improvement report the treatment of maize, wheat, and lettuce seeds with phosphate-solubilizing bacteria (PSB) such as Pseudomonas putida, Azospirillum lipoferum , Bacillus firmus and Bacillus polymyxa , enhance the solubility of phosphorus in the soil ( Mohamed et al., 2019 ). Other species like P. chlororaphis , Serratia marcescens , B.subtilis , B. megaterium , Arthrobacter aureofaciens , Phyllobacterium myrsinacearum , Rhodococcus erythropolis ( Kaymak, 2011 ), Burkholderia, flavobacterium, Rhizobium, Erwinia, Acetobacter, Micrococcus, Agrobacterium and Achromobacterium ( Youssef, 2014 ) were identified as phosphate solubilizing bacteria.

According to studies conducted by Lin et al. (2023) in the potato plant, the phosphate-solubilizing bacteria strain Bacillus megaterium activated the gene expression responsible for salinity, drought, and heat stress. Furthermore, the bacteria also trigger different metabolic processes in the plant.

Moreover, the application of B. subtilis, A. brasilense , and P. fluorescens as a single and combined or coupled with different application rates of phosphorous pentoxide increases the overall sugarcane yields ( Rosa et al., 2022 , 2023 ).

Screening for phosphate solubilization by bacteria can be done by following the method described by Kesaulya, Zakaria & Syaiful (2015) .

Ammonia production

Ammonia production is a remarkable trait of PGPR for plant growth promotion. When ammonia produced by bacteria is accumulated in the soil, it resulted in alkalinity conditions that repress many phytopathogen. Moreover, ammonia supplies nitrogen to the plant, resulting in root and shoot elongation and biomass growth with increased plant biomass, eventually enhanced the plant growth indirectly ( Bhattacharyya et al., 2020 ; Gohil et al., 2022 ). The bacterial isolates can be screened for ammonia production by a method described by Bhattacharyya et al. (2020) . The bacteria incubated for 5 days at 30 °C in a broth containing peptone, NaCl, and yeast extract were centrifuged at 10,000 rpm for 15 min, and subsequently, 0.5 ml Nessler reagent was added. The development of a brown and yellow color indicated ammonia production, and the light absorbance was determined using a Spectrophotometer at 450 nm. The ammonia-producing microbe includes Pseudomonas putida ( Ahemad & Khan, 2012 ), Klebsiella sp. ( Ahemad & Khan, 2010 ), Enterobacter asburiae ( Wickramasinghe et al., 2021 ).

Nitrogen fixation

Nitrogen is considered the most essential nutrient for plant development since it is required for the overall growth and the production of fruits and seeds ( Mahmud et al., 2020 ). Plants cannot directly utilize atmospheric nitrogen; therefore, bacteria assist in nutrient uptake by a symbiotic relationship with plant roots or by non-symbiotic bacteria ( Batista et al., 2018 ).

Bacterial genera that form a symbiotic relationship with the plant roots include Bradyrhizobium, Mesorhizobium, Sinorhizobium, Azhorhizobium, Pararhizobium, Neorhizobium , and Pseudomonas ( Nascimento et al., 2019 ). A non-symbiotic bacteria includes Achromobacter, Herbespirillum, Azoarcus ( Turan et al., 2016 ) Glucanoacetobacter, Azoarcus, Azotobacter, Azospirillum, Acetobacter, Enterobacter, Burkholderia, Pseudomonas, Cyanobacteria and Diazotrophicus ( Basile & Lepek, 2021 ).

A study by Galindo et al. (2024) shows that the application of microbial consortia such as A. subtilis and A. brasilense combined with different nitrogen application rates upregulate the root and shoot development, carbon dioxide uptake, transpiration and leaf chlorophyll index. Also, these microbial consortia inoculated in the seed improved the grain yield and nitrogen accumulation of wheat ( Gaspareto et al., 2023 ). Moreover, Bradyrhizobium sp. and Bacillus sp. co-inoculation improve nodule formation thereby enhancing the nitrogen fixation resulting in the overall plant yield of Vigna unguiculata ( Galindo et al., 2022 ).

Zinc solubilization

Zinc serves as an essential co-factor for enzyme activity that is involved in plant growth promotion by the microbes; the siderophore production and zinc ion production are also correlated ( Eshaghi et al., 2019 ). The optimum zinc concentration in plants is about 30 to 100 mg/kg; below this level results in deficiency ( Fasim et al., 2002 ). According to Singh et al. (2005) , zinc deficiency resulted in the slow growth and arising of necrotic marks in the plants. Zinc solubilizing bacteria play an essential role in overcoming inadequate zinc availability. The rhizobacteria mostly solubilizes zinc by producing organic acid metabolites, which lowers the pH of the soil where it is produced and iron chelating enzymes ( Fasim et al., 2002 ). The plant enzymes like carbonic anhydrase and superoxide dismutase are bound structurally by the zinc.

The microbes like P.aeruginosa, Gluconacetobacter diazotrophicus, P. striata, P.fluorescense, Burkholderia cenocepacia, S. liquifaciens, S. marcescens, B. thurigeansis, B.aryabhattai ( Kamran et al., 2017 ), B. subtilis, Thiobacillus thioxidans , and cyanobacteria ( Hussain et al., 2015 ) are reported to solubilize zinc. Also, genera like Rhizobium, Pseudomonas , and Bacillus promote the zinc translocation towards plants from soil, thereby improving the grain yield and zinc biofortification ( Jalal et al., 2022 ) These zinc-solubilizing microbes improve the overall quality and productivity of wheat by producing exopolysaccharides and siderophores ( Jalal, Júnior & Teixeira Filho, 2024 ). Moreover, co-inoculation of B.subtilis and foliar zinc oxide, P. fluorescence and foliar zinc oxide improved the the chlorophyll content, an amino acids, grains glutelin and prolamin in maize ( Jalal et al., 2023b ).

The zinc solubilization potential of bacteria can be screened using a modified Pikovskaya Agar containing insoluble zinc oxide. On the medium, 5 µL of bacterial culture was inoculated, then incubated at 28 ± 2 °C; the result can be observed after the 2, 4, and 7 days. The potential for zinc solubilization of the isolate was indicated by developing a distinct halo zone around the bacterial growth spot ( Sharma et al., 2012 ). The zinc solubilizing index can be calculated by the ratio of Halo zone formed + colony/colony diameters ( Saravanan, Madhaiyan & Thangaraju, 2007 ).

According to a study by Wu et al. (2013) , zinc inhibited biofilm production by A.pleuropneumoniae, Salmonella typhymurium, E.coli, S.aureus , and Streptococcus suis efficiently. The zinc nanoparticles obtained from the bacteria can also be used to enhance the antimicrobial and biocidal activity of the human oral microbiome ( Lallo da Silva et al., 2019 ).

Potassium solubilization

Potassium is naturally present in the soil but they are not readily absorbed by it since it exists in an insoluble form, therefore plant growth-promoting bacteria solubilize potassium by proffering a variety of organic acids including citric acid, oxalic acid, and tartaric acid ( Olaniyan et al., 2022 ). Potassium has several critical functions, as it can alter enzymes physical structures and expose the active site for the reactions. Furthermore, it activates at least 60 enzymes involved in plant growth ( Prajapati & Modi, 2012 ). According to Rawat, Pandey & Saxena (2022) , plants depend on potassium to open and close stomata. The high level of potassium content in plants resulted in improved disease resistance, fiber quality in cotton, and durability of fruit and vegetables and their physical quality ( Prajapati & Modi, 2012 ).

Potassium concentration in the plants regulates water retained in the plant, and low potassium content results in sensitivity to water stress. Bacteria including Acidithiobacillus, Burkholderia and Pseudomonas ( Sharma, Shankhdhar & Shankhdhar, 2016 ), Bacillus megaterium, Arthrobacter ( Keshavarz Zarjani et al., 2013 ), Pantoea ananatis,Rahnella aquatilis, Enterobacter sp. ( Bakhshandeh, Pirdashti & Lendeh, 2017 ), Bacillus mucilaginosus, Paenibacillus mucilaginosus ( Hu, Chen & Guo, 2006 ), Bacillus licheniformis, Pseudomonas azotoformans ( Maurya et al., 2016 ), Bacillus edaphicus ( Sheng, Huang & YongXian, 2000 ), and Pseudomonas putida ( Bagyalakshmi, Ponmurugan & Marimuthu, 2012 ) have been identified as a potent K solubilizer.

The bacteria’s ability to saturate the potassium can be studied by spot inoculation in Aleksandrow agar medium, the halo zone formed around the bacteria after seven days suggests the potential to solubilize potassium ( Sood et al., 2023 ). This protocol is time-friendly, and commonly used for quality testing.

Bioformulations of plant growth promoting bacteria

PGPRs significantly interest the agro-industrial sector. They have been utilized and produced primarily for global crops ( Tabassum et al., 2017 ). The plant growth-promoting bacteria are formulated to increase their survival rate while stored and applied to the plants. They are developed into liquid or solid-based wet and dry products ( Berger et al., 2018 ). Liquid formulation is regarded as the most effective among them, and it is further divided into seed inoculation, soil inoculation, and shoot inoculation ( Lopes, Dias-Filho & Gurgel, 2021 ). According to Lee et al. (2022) , liquid inoculants are a mixture of a whole culture and a compound like oil, water, and other polymeric compounds that can enhance the stability, adhesion as well as capacity of dispersion. According to Nosheen, Ajmal & Song (2021) , these inoculants inhabit the soil environment and the interior part of the plant tissues, enhancing growth and development. In addition to the aforementioned inoculants, Kaur & Kaur (2018) elaborate different types of bioformulants, both carrier-based and encapsulated. Carrier-based inoculants employ, clay, sawdust, straw, charcoal, and other biodegradable materials as carriers which can aid in the survival of inoculants. Also, the encapsulated bioformulation recruits a natural encapsulator like agar, agarose, cellulose, biochar, and synthetic encapsulator including polyvinylpyrrolidone (PVP), polystyrene, and polyacrylamides to extend the shelf life of inoculants. A good inoculant must have a long shelf life with high endurability in harsh environments and be compatible with other agrochemical products, in addition to that, they must possess the ability to be introduced to the plant via foliar spray, seed treatment, soil application, bio priming, and seed dip ( Ahmad et al., 2022 ). These inoculants are biofertilizers and can be classified based on their function.

i) Nitrogen fixer: According to Nosheen, Ajmal & Song (2021) bacterial species like Klebsiella, Desulfovbrio, Anabaena, Rhodospirillum, Rhizobium, Frankia, Aulosira bejerinkic, sligonema, Nostoc, Trichodesmium, Acetobacterdiazotrophicus, Clostridium, Azospirillum spp, Alkaligenes, Azoarcus spp, and Enterobacter are commonly formulated as a nitrogen-fixing biofertilizer. Among PGPB, Azospirillum is an industrially notable microbe developed as a biofertilizer with strong efficacy ( Etesami & Emami, 2017 ; Raffi & Charyulu, 2020 ).

ii) Potassium solubilizers: Even though there is a significant number of potential potassium solubilizing bacteria, only a few are on the market due to failed survival during the different stages of formulations. According to Etesami & Emami (2017) bacteria like Mucilaginosus, B.circulanscan, Arthrobacter spp, B.edaphicus , and Bacillus are being formulated as potassium solubilizers.

iii) Plant growth promoter: The bacteria like A grobacterium, Eewinia, Pseudomonas fluorescens, Xanthomonas, Enterobacter, Rhizobium, Streptomyces, Arthrobacter and Pseudomonas sp. ( Nosheen, Ajmal & Song, 2021 ) are being effectively used.

Biopesticides are another formulation of beneficial bacteria with targeted activity against pathogens, contrary to a chemical pesticide that is non-targeted and causes significant harm to other beneficial organisms. The Environmental Protection Agency (EPA) highly promotes biopesticides since they are environmentally harmless. One of the most commonly exploited bacteria for biopesticide production is Bacillus thuringiensis ( DeJong, 2020 ).

However, formulation of efficacious bioinoculants is a great challenge, since many of the potential candidates failed to thrive within the formulation as well as after application in the agriculture field. Therefore, Cell-free supernatant (CFSs) with a secondary metabolite is also a desirable biostimulant and biofertilizer for the achievement of sustainable agriculture. According to Pellegrini et al. (2020) , Bacillus sp. is a capable genus for CFSs. Moreover, Azospirillum brasilense ( Berbel et al., 2020 ), Bradyrhizobium diazoefficiens, R.tropici CIAT889 ( Gustavo Moretti et al., 2019 ), Bradyrhizobium sp IC-4059 ( Tewari, Pooniya & Sharma, 2020 ), and Lactobacillus rahmnosus ( Caballero et al., 2020 ), are all known for their effective biostimulation. They offer a high potential to act against phytopathogen as they possess a variety of bioactive molecules including, surfactin, subtilin, subtilisin, mycosubtilin, and rhizoctocins ( Pellegrini et al., 2020 ).

In today’s world of agriculture system, the culturable land is being contaminated and eroded, however, demands for crops increase to feed the growing population, therefore, adoption of sustainable farming is highly essential. Plant growth-promoting microorganisms, specifically bacteria, have been extensively studied for their potential ability to produce essential metabolites that directly or indirectly assist plant growth and development. Furthermore, the PGPM can naturally release essential biochemicals like plant growth regulators, siderophores, hydrogen cyanide, lytic enzymes, ammonia, etc ., and can solubilize inorganic phosphate, zinc, and potassium. They also have the potential to fix the atmospheric nitrogen and convert it into soluble form using enzymes called nitrogenase. Therefore, these beneficial bacteria are essential in attaining a sustainable agriculture system, enabling us to obtain a high quality and high quantity of plant products in a much safer and environmentally friendly way. However, limited reports have been made on the formulation of these beneficial bacteria for biofertilizers and biocontrolling agents to enhance crop yield under different biotic and abiotic stress.

Plant growth-promoting bacteria hold the most promising way of sustainable agriculture, accordingly further research and exploration of potential plant growth-promoting bacteria for specific stresses and plants, also having a broad spectrum activity can be investigated to extend the productivity of desired crops. Moreover, study on the compatibility of PGPB and the host plants needed more attention to aid eliminating the possible loss of active plant growth promoting traits of PGPB while adapting into the new host plant environment. This can also help in selection of a right microbial strains for a particular crop of a particular environment. In addition to that, further study on the co-existing potential of different PGPBs can be done, a strain which can coinhabit the same environment without lowering each of their potential active traits to produce more effective microbial consortia.

Acknowledgments

Authors are thankful to Dr. Laldinpuii, Assistant Professor, Department of English, Pachhunga University College, Aizawl, Mizoram for her meticulous proofreading and invaluable English corrections significantly enhanced the quality of this article.

Funding Statement

This work was supported by the Department of Science and Technology, Science and Engineering Research Board (DST-SERB), Government of India, vide project sanction no.: EEQ/2022/000878; Indian Council of Medical Research (ICMR) under sanction no.: ECD/NER/5/2022-23; UGC SRG F.30-555/202t(BSR) and RPG (11/1-349/2022/FIN-B/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Additional Information and Declarations

Zothan Puia is an Academic Editor for PeerJ.

Awmpuizeli Fanai conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Beirachhitha Bohia analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Felicia Lalremruati analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Nancy Lalhriatpuii analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Lalrokimi analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Rosie Lalmuanpuii analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Prashant Kumar Singh analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Zothanpuia conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Using Conductivity (EC) and pH Measurements to Control Hydroponic Solutions

Robert Pavlis

Hydroponics can be a great way to grow plants but it is important to make sure the nutrient solution contains enough food for the whole growth cycle. If either pH or EC (electrical conductivity) is out of whack, plants grow poorly or stop growing all together. It is therefore important to measure and control the nutrient solution. This post will look at what the numbers mean and what you should do about them to keep plants growing well.  

Key Takeaways

• Keeping the pH and EC in the preferred range is important for plant growth.
• Use EC and not TDS for measurements and online discussions.
• Nutrient lockout does not really exist, but is important to understand.

This post uses  affiliate links

Table of Contents

How do Hydroponic Solutions Work?

Hydroponic solutions are mixtures of water and salts that the plant needs, including nutrients such as nitrate, calcium, phosphate, potassium etc. The amount of each nutrient at the start of the growing process is formulated to match the amounts used by plants.

In a perfect world, the plant would absorb the correct amount of nutrients and water and over time the level of hydroponics solution would decrease without any change in the concentration of nutrients. Life is not that perfect. In the real world the species of plant, its growth stage as well as the growing condition influences the amount of nutrients the plant absorbs. Over time the level can become unbalanced resulting in poor plant growth.

Why is this a bigger problem in hydroponics than soil? Soil has a natural ability to buffer both pH and EC, which means that soil keeps these values in a narrow range compared to the water in hydroponics. Soil takes care of many changes automatically to keep values in the range that plants prefer. Hydroponics does not have this buffering capability and the gardener has to more closely monitor and adjust the values.

It is the gardeners job to keep the solution balanced so the level of nutrients never changes much from the original formulation. This is done by monitoring both pH and EC.

What is EC?

EC stands for electrical conductivity which is an indirect measurement of the total amount of dissolved ions in a solution. All of the nutrients in fertilizer are ions in water . Tap water and a very dilute fertilizer solutions have a very low EC value while a concentrated fertilizer solution has a high EC value.

EC is measured with a meter, which can be as simple as a probe, or it can consist of an actual metering device with an attached probe. The common units for EC are millisiemens per centimeter (mS/cm).

It is common to have people state an EC value without including units which can cause confusion. The EC of hydroponic solutions are normally between 0.5 and 4.0 mS/cm. If someone talks about EC values that are over 500 then they are using µS/cm (microsiemens per centimeter).

1 mS/cm = 1,000 µS/cm

The Difference Between EC and TDS?

To complicate things some people like to use TDS instead of EC for measuring fertilizer. EC measures only particles that allow the conductance of electricity and these include all of the nutrients in a fertilizer solution.

TDS is short Total Dissolved Solids and includes all particles, not just the ones that conduct electricity. In the case of water and fertilizer solutions, almost all the particles conduct electricity. This means EC and TDS are essentially the same thing but there are two important facts that complicate things.

• TDS meters do not measure total dissolved solids . They measure conductivity (EC) and convert that number to a TDS number (mg/l = ppm).
• The conversion factor from EC to TDS is not a standard value because it is non-linear and depends on the actual particles being measured.

TDS meters may be referred to as TDS testers or PPM (parts per million) testers , but these are just different names for the same thing.

Most TDS meters use one of two conversion factors, either 0.7 or 0.5 to convert EC to TDS. If the EC measurement is 1,000 µS/cm, the meter either reports 500 or 700. Some meters allow you to change this conversion factor.

The 0.5 factor is based on sodium chloride and the 0.7 factor is based on the 442 Natural Water Conversion system. If someone online reports a ppm value (TDS) without stating the conversion factor used, you don’t know which factor is used and the number is not very useful.

What does this all mean? If you are using a TDS meter, find out the conversion factor for the meter and convert the reading to EC. If your meter does both TDS and EC, ignore the TDS completely and only use EC. If you are buying a new meter, buy one that reports the EC value.

Work in EC units – not TDS units.

Some TDS Myths

You will find quite a bit of misinformation about TDS online.

• “EC is the preferred method for measuring the nutrient concentration in hydroponics because it is more accurate and reliable”. This is not true. Since only EC is measured with meters and the number is then converted to TDS by a specific factor, both values are equally accurate and reliable.
• “µS/cm and ppm (TDS) should be used because they are more accurate than mS/cm.” Not true. Just because you see more digits in a number does not mean it is more accurate. The accuracy depends on how clean the meter is, how well it has been calibrated and the accuracy of the electronics. Both µS/cm and mS/cm are equally accurate.
• “Total dissolved solids (TDS) measures both the EC-generating particles AND the particles that are unable to conduct electricity in the water.” This can be true, but in the gardening world it is not true. If TDS is measured properly in a laboratory, using gravimetric methods, it does measure all particles. However, if TDS is measured using a meter, it is really measuring conductivity (i.e. EC) and so both TDS and EC are measuring only the ionic particles (i.e. nutrients).

The Right EC for Hydroponics

Most plants do best with EC between 0.5 and 2.5 mS/cm. Values below 0.5 or above 2.5 are almost always detrimental to plant growth. However, each type of plant has a preferred range and the closer you are to its requirements, the better it will grow.

You would normally start with an EC that is close to the middle of the plants referred range. If the plant absorbs water and nutrients at the same rate, the EC will not change, but this is usually not the case. Water and nutrients can be absorbed at different rates.

If the plant absorbs water faster than the nutrients, the EC will go up since the nutrient solution is becoming more concentrated. If the plant absorbs nutrients faster than the water, the solution gets more diluted and the EC value drops.

It is important that the EC is checked on a regular basis and that any large changes in EC are corrected. You want to ensure that the plant is always growing in its preferred range.

How Often Should You Check EC?

Professional growers check daily or even more frequently. Checking daily for homeowners is overkill.

I suggest you check it daily if you are new to measuring EC. See how much it is changing. If the change is very small, check twice a week. If it is still not changing much go to once a week.

Keep in mind that when you start new plants they are small and use very little water or nutrients. So the EC change will be small. As the plant gets bigger it uses more which results in faster EC changes. Ideally you check EC often enough so that you catch it before the value is outside of the preferred range.

What to Do if the EC Level Is Too High or Too Low?

If your nutrient solution EC is too high you can add some water to bring it back into range.

If the EC is too low you can add a more concentrated nutrient solution. This could be a fertilizer solution that is made with twice the amount of nutrients as usual.

In both cases add some of the adjusting solution and retest the mixture. If it is still out of range, add some more and retest.

Preferred EC Ranges for Different Plants

The following table of plants is from Oklahoma State University .

The above requirements also change for different growth phases. They are lower during the growth stage and higher by about 0.5 mS/cm during flowering and fruiting stage. Each brand of fertilizer will also have a different starting EC depending on both the form and amount of each nutrient.

The idea that a higher phosphorus level is needed to increase blooming is a myth.

What is Nutrient Lockout?

The term Nutrient Lockout is popular in the cannabis growing world and in hydroponics. It is rarely used by gardeners growing in soil and is almost never used in the scientific community. As such, it does not have a good definition and many online definitions are incomplete or only partially correct.

Nutrient lockout does not really exist because nutrients are not locked out of the plant. There are however conditions that make it more difficult for roots to absorb nutrients. Here are some situations that can cause this issue.

• Insoluble nutrients. When nutrients are mixed incorrectly or get too concentrated, they start to form insoluble compounds and precipitate out of solution. Plants can’t use these nutrients.
• Incorrect pH. Nutrients are less available to plants at extreme pH.
• High osmotic pressure. When nutrients become too concentrated, the osmotic pressure of the solution is higher than inside the plant which causes water to leave the roots. In effect they become hydrated. When this happens, plants can’t absorb water or most nutrients.

Statements such as “your plants are starving although they’re surrounded by food” are not really correct since nutrients are only food for plants when they are in the correct chemical form.

How do you prevent nutrient lockout? Check the EC and pH regularly and keep the values in the normal range for the plant.

The Right pH for Hydroponics

pH is a measure of the acidity or alkalinity of a substance and provides a number between 0 to 14. The availability of nutrients is affected by pH and most plants grow well in a range of 5.6 to 6.5. When pH drifts outside of this range some nutrients become less available and others can become toxic. Keeping the value between 6.0 and 6.5 will ensure that pH does not create a nutrient issue.

Here is an example of what happens when the pH gets too high. A higher pH causes iron to be converted from the Fe2+ form which plants can use to the Fe3+ form which is harder for plants to absorb. Iron also combines with carbonate and phosphate to form precipitates that leave the solution. You will see this as white powder in the bottom of the hydroponic unit. Without adequate iron, plants show interveinal chlorosis and don’t grow very well. This is not really an iron deficiency but a pH imbalance.

How to adjust pH

If pH is too low, an alkaline solution is added to raise the pH and an acid is added if the pH is too high. You can use commercial “pH up” and “pH down” products (Amazon affiliate link) for this. The pH up products usually contain potassium hydroxide or potassium carbonate, while pH down products contain phosphoric acid. Mild organic acids such as household vinegar or citric acid can also be used but their effect is not long lasting.

Be careful using commercial adjusting products since a small amount can result in a large pH change.

Hard Water in Hydroponics

Hard water tends to have high levels of calcium, magnesium, sodium and/or carbonate. These can cause several issues in hydroponics.

• Too much sodium is toxic to plants.
• Hard water commonly has a high pH.
• The EC is naturally high.
• Most hydroponic fertilizer adds extra calcium and magnesium, which is not needed for hard water.

Most hydroponic nutrients are formulated based on “pure water”. For this reason many people use softer water ( RO, distilled, rain water ) to make up the hydroponic fertilizer solution. Alternatively, look for a fertilizer that does not include calcium and magnesium . Keep in mind that nitrogen should be provided in a nitrate form, not a ammonium form , which is more common in non-hydroponic fertilizers.

Alkalinity vs Harness

It is also important to understand the difference between alkalinity and hardness.

Alkalinity is a  measure of the total carbonates  (CO3), bicarbonates (HCO3) and hydroxyl ions (OH) and is usually expressed as the equivalent of CaCO3 , e.g. 100 ppm CaCO3.

Water hardness is the amount of dissolved calcium and magnesium in the water . High harness is anything over 150 ppm, but values up to 200 ppm usually don’t cause growth problems for plants because neither calcium nor magnesium are not very toxic to plants.

High levels of calcium, magnesium and carbonate can all combine with nutrients and precipitate out of solution forming a white powder at the bottom of the tank. This can reduce micronutrients to levels that are too low for proper plant growth.

The Limitation of EC Measurements

Using EC to monitor hydroponic solutions has a serious limitation that few people talk about. Remember that EC is the total electrical conductivity of a solution. Consider these three solutions:

• A complete Masterblend fertilizer solution with an EC = 2.0 mS/cm.
• A solution of potassium nitrate with an EC = 2.0 mS/cm.
• A solution of sodium chloride (table salt) with an EC = 2.0 mS/cm.

Each solution has the same EC and is a good EC value for growing strawberries. The first one will grow good strawberries because it contains all the needed nutrients. The second solution will grow very poor plants because it has no phosphorus and the third will kill the plants because sodium is toxic. All three solutions have the same EC value.

EC is only valuable if the ratio of nutrients in the solution are correct. It tells you nothing about the actual ratio of these nutrients. This is important because the ratio changes over time.

You might start with the perfect ratio of nutrients, but plants will take what they want from the mixture. Some nutrients are absorbed by active transport and others are absorbed at the same rate as water. In any mixture, the ratio of nutrients changes over time and EC gives you no insight into what they are.

What can a gardener do?

Short of doing a lot of lab tests, all a gardener can do is assume the ratio is acceptable for a while and after that point, throw out the mixture and start with a fresh one. How often do you need to do that? I really don’t know because I have not found any information about how fast the solution changes. If you have some data please add a link to it in the comments.

The rate of replacement does depend on the number and size of plants in the nutrient solution, but I think it would be a good idea to replace the solution every 6-8 weeks. It should certainly be replaced if you suspect nutrient issues with the leaves. The old solution can be poured in the garden.

Related Posts You Will Enjoy

• 10 Top Gardening Myths of 2017
• Podcast With Joe Gardener – Microbe Science for Gardeners

I have been gardening my whole life and have a science background. Besides writing and speaking about gardening, I own and operate a 6 acre private garden called Aspen Grove Gardens which now has over 3,000 perennials, grasses, shrubs and trees. Yes--I am a plantaholic!

1 thought on “Using Conductivity (EC) and pH Measurements to Control Hydroponic Solutions”

Once someone has an EC meter, and a pH meter, those two tools can be used to test soil. Lots of commercial greenhouse growers use this method to watch for EC or pH changes. The Pour-Thru method of soil testing: https://www.css.cornell.edu/courses/260/Media%20testing.pdf

Also, maybe a post about maintaining a pH meter would help people. As you know, a pH meter is a perishable instrument. Proper care makes them somewhat less perishable.

Thanks for all the help you give your readers!

Leave a Comment Cancel reply

Information

• Author Services

Initiatives

You are accessing a machine-readable page. In order to be human-readable, please install an RSS reader.

All articles published by MDPI are made immediately available worldwide under an open access license. No special permission is required to reuse all or part of the article published by MDPI, including figures and tables. For articles published under an open access Creative Common CC BY license, any part of the article may be reused without permission provided that the original article is clearly cited. For more information, please refer to https://www.mdpi.com/openaccess .

Feature papers represent the most advanced research with significant potential for high impact in the field. A Feature Paper should be a substantial original Article that involves several techniques or approaches, provides an outlook for future research directions and describes possible research applications.

Feature papers are submitted upon individual invitation or recommendation by the scientific editors and must receive positive feedback from the reviewers.

Editor’s Choice articles are based on recommendations by the scientific editors of MDPI journals from around the world. Editors select a small number of articles recently published in the journal that they believe will be particularly interesting to readers, or important in the respective research area. The aim is to provide a snapshot of some of the most exciting work published in the various research areas of the journal.

Original Submission Date Received: .

• Active Journals
• Find a Journal
• Proceedings Series
• For Authors
• For Reviewers
• For Editors
• For Librarians
• For Publishers
• For Societies
• For Conference Organizers
• Open Access Policy
• Institutional Open Access Program
• Special Issues Guidelines
• Editorial Process
• Research and Publication Ethics
• Article Processing Charges
• Testimonials
• Preprints.org
• SciProfiles
• Encyclopedia

Article Menu

• Subscribe SciFeed
• Recommended Articles
• Google Scholar
• on Google Scholar
• Table of Contents

Find support for a specific problem in the support section of our website.

Please let us know what you think of our products and services.

Visit our dedicated information section to learn more about MDPI.

JSmol Viewer

How do plant growth-promoting bacteria use plant hormones to regulate stress reactions.

1. Introduction

2. pgpb and phytohormones in the rhizosphere, 2.1. auxins, 2.2. cytokinins, 2.3. gibberellins, 2.4. salicylic acid, 2.5. abscisic acid, 2.6. volatile organic compounds, 2.7. ethylene and acc deaminase, 3. synergistic effects of pgpb on plant growth through the interaction of multiple pathways, 3.1. effect of iaa on acc deaminase and ethylene synthesis, 3.2. interactions among phytohormones, 4. strategies for assessing the ability of pgpb to synthesize phytohormones, 4.1. determination of the potential for iaa synthesis, 4.2. detection of acc deaminase activity, 5. conclusions, author contributions, data availability statement, conflicts of interest.

• Cui, M.; Guo, Y.; Chen, J. Influence of Transfer Plot Area and Location on Chemical Input Reduction in Agricultural Production: Evidence from China. Agriculture 2023 , 13 , 1794. [ Google Scholar ] [ CrossRef ]
• Devi, P.I.; Manjula, M.; Bhavani, R.V. Agrochemicals, Environment, and Human Health. Annu. Rev. Environ. Resour. 2022 , 47 , 399–421. [ Google Scholar ] [ CrossRef ]
• Akinnawo, S.O. Eutrophication: Causes, Consequences, Physical, Chemical and Biological Techniques for Mitigation Strategies. Environ. Chall. 2023 , 12 , 100733. [ Google Scholar ] [ CrossRef ]
• Chataut, G.; Bhatta, B.; Joshi, D.; Subedi, K.; Kafle, K. Greenhouse Gases Emission from Agricultural Soil: A Review. J. Agric. Food Res. 2023 , 11 , 100533. [ Google Scholar ] [ CrossRef ]
• Ortiz-Monasterio, J.I.; Raun, W. Paper Presented At International Workshop On Increasing Wheat Yield Potential, Cimmyt, Obregon, Mexico, 20–24 MARCH 2006 Reduced Nitrogen and Improved Farm Income for Irrigated Spring Wheat in the Yaqui Valley, Mexico, Using Sensor Based Nitrogen Manageme. J. Agric. Sci. 2007 , 145 , 215–222. [ Google Scholar ] [ CrossRef ]
• Majeed, A.; Abbasi, M.K.; Hameed, S.; Imran, A.; Rahim, N. Isolation and Characterization of Plant Growth-Promoting Rhizobacteria from Wheat Rhizosphere and Their Effect on Plant Growth Promotion. Front. Microbiol. 2015 , 6 , 198. [ Google Scholar ] [ CrossRef ]
• Timofeeva, A.M.; Galyamova, M.R.; Sedykh, S.E. Plant Growth-Promoting Soil Bacteria: Nitrogen Fixation, Phosphate Solubilization, Siderophore Production, and Other Biological Activities. Plants 2023 , 12 , 4074. [ Google Scholar ] [ CrossRef ]
• Montoya-Martínez, A.C.; Parra-Cota, F.I.; de los Santos-Villalobos, S. Beneficial Microorganisms in Sustainable Agriculture: Harnessing Microbes’ Potential to Help Feed the World. Plants 2022 , 11 , 372. [ Google Scholar ] [ CrossRef ]
• Timofeeva, A.M.; Galyamova, M.R.; Sedykh, S.E. Bacterial Siderophores: Classification, Biosynthesis, Perspectives of Use in Agriculture. Plants 2022 , 11 , 3065. [ Google Scholar ] [ CrossRef ]
• Timofeeva, A.; Galyamova, M.; Sedykh, S. Prospects for Using Phosphate-Solubilizing Microorganisms as Natural Fertilizers in Agriculture. Plants 2022 , 11 , 2119. [ Google Scholar ] [ CrossRef ]
• Gurska, J.; Glick, B.R.; Greenberg, B.M. Gene Expression of Secale Cereale (Fall Rye) Grown in Petroleum Hydrocarbon (PHC) Impacted Soil With and Without Plant Growth-Promoting Rhizobacteria (PGPR), Pseudomonas putida . Water Air Soil Pollut. 2015 , 226 , 308. [ Google Scholar ] [ CrossRef ]
• Chet, I.; Inbar, J. Biological Control of Fungal Pathogens. Appl. Biochem. Biotechnol. 1994 , 48 , 37–43. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Penrose, D.M.; Glick, B.R. Methods for Isolating and Characterizing ACC Deaminase-containing Plant Growth-promoting Rhizobacteria. Physiol. Plant. 2003 , 118 , 10–15. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Asif, R.; Yasmin, R.; Mustafa, M.; Ambreen, A.; Mazhar, M.; Rehman, A.; Umbreen, S.; Ahmad, M. Phytohormones as Plant Growth Regulators and Safe Protectors against Biotic and Abiotic Stress. In Plant Hormones—Recent Advances, New Perspectives and Applications ; IntechOpen: Rijeka, Croatia, 2022. [ Google Scholar ]
• Egamberdieva, D.; Wirth, S.J.; Alqarawi, A.A.; Abd_Allah, E.F.; Hashem, A. Phytohormones and Beneficial Microbes: Essential Components for Plants to Balance Stress and Fitness. Front. Microbiol. 2017 , 8 , 2104. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Santoro, M.V.; Zygadlo, J.; Giordano, W.; Banchio, E. Volatile Organic Compounds from Rhizobacteria Increase Biosynthesis of Essential Oils and Growth Parameters in Peppermint ( Mentha piperita ). Plant Physiol. Biochem. 2011 , 49 , 1177–1182. [ Google Scholar ] [ CrossRef ]
• Nascimento, F.X.; Glick, B.R.; Rossi, M.J. Multiple Plant Hormone Catabolism Activities: An Adaptation to a Plant-associated Lifestyle by Achromobacter Spp. Environ. Microbiol. Rep. 2021 , 13 , 533–539. [ Google Scholar ] [ CrossRef ]
• Rashid, M.I.; Mujawar, L.H.; Shahzad, T.; Almeelbi, T.; Ismail, I.M.I.; Oves, M. Bacteria and Fungi Can Contribute to Nutrients Bioavailability and Aggregate Formation in Degraded Soils. Microbiol. Res. 2016 , 183 , 26–41. [ Google Scholar ] [ CrossRef ]
• Novo, L.A.B.; Mahler, C.F.; González, L. Plants to Harvest Rhenium: Scientific and Economic Viability. Environ. Chem. Lett. 2015 , 13 , 439–445. [ Google Scholar ] [ CrossRef ]
• Goswami, D.; Thakker, J.N.; Dhandhukia, P.C. Portraying Mechanics of Plant Growth Promoting Rhizobacteria (PGPR): A Review. Cogent Food Agric. 2016 , 2 , 1127500. [ Google Scholar ] [ CrossRef ]
• Arkhipova, T.N.; Evseeva, N.V.; Tkachenko, O.V.; Burygin, G.L.; Vysotskaya, L.B.; Akhtyamova, Z.A.; Kudoyarova, G.R. Rhizobacteria Inoculation Effects on Phytohormone Status of Potato Microclones Cultivated In Vitro under Osmotic Stress. Biomolecules 2020 , 10 , 1231. [ Google Scholar ] [ CrossRef ]
• Pankievicz, V.C.S.; do Amaral, F.P.; Ané, J.-M.; Stacey, G. Diazotrophic Bacteria and Their Mechanisms to Interact and Benefit Cereals. Mol. Plant-Microbe Interact. 2021 , 34 , 491–498. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Ma, Y.; Zhang, C.; Oliveira, R.S.; Freitas, H.; Luo, Y. Bioaugmentation with Endophytic Bacterium E6S Homologous to Achromobacter piechaudii Enhances Metal Rhizoaccumulation in Host Sedum plumbizincicola . Front. Plant Sci. 2016 , 7 , 75. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Iqbal, N.; Khan, N.A.; Ferrante, A.; Trivellini, A.; Francini, A.; Khan, M.I.R. Ethylene Role in Plant Growth, Development and Senescence: Interaction with Other Phytohormones. Front. Plant Sci. 2017 , 8 , 475. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Ma, Y.; Oliveira, R.S.; Freitas, H.; Zhang, C. Biochemical and Molecular Mechanisms of Plant-Microbe-Metal Interactions: Relevance for Phytoremediation. Front. Plant Sci. 2016 , 7 , 918. [ Google Scholar ] [ CrossRef ]
• Spence, C.; Bais, H. Role of Plant Growth Regulators as Chemical Signals in Plant–Microbe Interactions: A Double Edged Sword. Curr. Opin. Plant Biol. 2015 , 27 , 52–58. [ Google Scholar ] [ CrossRef ]
• Dodd, I.C.; Zinovkina, N.Y.; Safronova, V.I.; Belimov, A.A. Rhizobacterial Mediation of Plant Hormone Status. Ann. Appl. Biol. 2010 , 157 , 361–379. [ Google Scholar ] [ CrossRef ]
• Taylor, J.L.; Zaharia, L.I.; Chen, H.; Anderson, E.; Abrams, S.R. Biotransformation of Adenine and Cytokinins by the Rhizobacterium Serratia proteamaculans . Phytochemistry 2006 , 67 , 1887–1894. [ Google Scholar ] [ CrossRef ]
• Ullah, A.; Bano, A.; Khan, N. Climate Change and Salinity Effects on Crops and Chemical Communication Between Plants and Plant Growth-Promoting Microorganisms Under Stress. Front. Sustain. Food Syst. 2021 , 5 , 618092. [ Google Scholar ] [ CrossRef ]
• Yen, K.-M.; Serdar, C.M.; Gunsalus, I.C. Genetics of Naphthalene Catabolism in Pseudomonads. CRC Crit. Rev. Microbiol. 1988 , 15 , 247–268. [ Google Scholar ] [ CrossRef ]
• Sazonova, O.I.; Izmalkova, T.Y.; Kosheleva, I.A.; Boronin, A.M. Salicylate Degradation by Pseudomonas putida Strains Not Involving the “Classical” Nah2 Operon. Microbiology 2008 , 77 , 710–716. [ Google Scholar ] [ CrossRef ]
• Nascimento, F.X.; Glick, B.R.; Rossi, M.J. Isolation and Characterization of Novel Soil- and Plant-Associated Bacteria with Multiple Phytohormone-Degrading Activities Using a Targeted Methodology. Access Microbiol. 2019 , 1 , e000053. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Fukami, J.; Nogueira, M.A.; Araujo, R.S.; Hungria, M. Accessing Inoculation Methods of Maize and Wheat with Azospirillum brasilense . AMB Express 2016 , 6 , 3. [ Google Scholar ] [ CrossRef ]
• Sahoo, R.K.; Ansari, M.W.; Pradhan, M.; Dangar, T.K.; Mohanty, S.; Tuteja, N. Phenotypic and Molecular Characterization of Native Azospirillum Strains from Rice Fields to Improve Crop Productivity. Protoplasma 2014 , 251 , 943–953. [ Google Scholar ] [ CrossRef ]
• Fukami, J.; Cerezini, P.; Hungria, M. Azospirillum : Benefits That Go Far beyond Biological Nitrogen Fixation. AMB Express 2018 , 8 , 73. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Cohen, A.C.; Bottini, R.; Piccoli, P.N. Azospirillum brasilense Sp 245 Produces ABA in Chemically-Defined Culture Medium and Increases ABA Content in Arabidopsis Plants. Plant Growth Regul. 2008 , 54 , 97–103. [ Google Scholar ] [ CrossRef ]
• Weijers, D.; Wagner, D. Transcriptional Responses to the Auxin Hormone. Annu. Rev. Plant Biol. 2016 , 67 , 539–574. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Spaepen, S.; Vanderleyden, J.; Remans, R. Indole-3-Acetic Acid in Microbial and Microorganism-Plant Signaling. FEMS Microbiol. Rev. 2007 , 31 , 425–448. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Korasick, D.A.; Enders, T.A.; Strader, L.C. Auxin Biosynthesis and Storage Forms. J. Exp. Bot. 2013 , 64 , 2541–2555. [ Google Scholar ] [ CrossRef ]
• Patten, C.L.; Glick, B.R. Bacterial Biosynthesis of Indole-3-Acetic Acid. Can. J. Microbiol. 1996 , 42 , 207–220. [ Google Scholar ] [ CrossRef ]
• Khalid, A.; Tahir, S.; Arshad, M.; Zahir, Z.A. Relative Efficiency of Rhizobacteria for Auxin Biosynthesis in Rhizosphere and Non-Rhizosphere Soils. Soil Res. 2004 , 42 , 921. [ Google Scholar ] [ CrossRef ]
• Cassán, F.; Coniglio, A.; López, G.; Molina, R.; Nievas, S.; de Carlan, C.L.N.; Donadio, F.; Torres, D.; Rosas, S.; Pedrosa, F.O.; et al. Everything You Must Know about Azospirillum and Its Impact on Agriculture and Beyond. Biol. Fertil. Soils 2020 , 56 , 461–479. [ Google Scholar ] [ CrossRef ]
• Spaepen, S.; Vanderleyden, J. Auxin and Plant-Microbe Interactions. Cold Spring Harb. Perspect. Biol. 2011 , 3 , a001438. [ Google Scholar ] [ CrossRef ]
• Zúñiga, A.; Poupin, M.J.; Donoso, R.; Ledger, T.; Guiliani, N.; Gutiérrez, R.A.; González, B. Quorum Sensing and Indole-3-Acetic Acid Degradation Play a Role in Colonization and Plant Growth Promotion of Arabidopsis thaliana by Burkholderia Phytofirmans PsJN. Mol. Plant-Microbe Interact. 2013 , 26 , 546–553. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Leveau, J.H.J.; Gerards, S. Discovery of a Bacterial Gene Cluster for Catabolism of the Plant Hormone Indole 3-Acetic Acid. FEMS Microbiol. Ecol. 2008 , 65 , 238–250. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Calatrava, V.; Hom, E.F.Y.; Guan, Q.; Llamas, A.; Fernández, E.; Galván, A. Genetic Evidence for Algal Auxin Production in Chlamydomonas and Its Role in Algal-Bacterial Mutualism. iScience 2024 , 27 , 108762. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Calatrava, V.; Hom, E.F.Y.; Llamas, Á.; Fernández, E.; Galván, A. OK, Thanks! A New Mutualism between Chlamydomonas and Methylobacteria Facilitates Growth on Amino Acids and Peptides. FEMS Microbiol. Lett. 2018 , 365 , fny021. [ Google Scholar ] [ CrossRef ]
• Olesen, M.R.; Jochimsen, B.U. Identification of Enzymes Involved in Indole-3-Acetic Acid Degradation. Plant Soil 1996 , 186 , 143–149. [ Google Scholar ] [ CrossRef ]
• Mole, B.M.; Baltrus, D.A.; Dangl, J.L.; Grant, S.R. Global Virulence Regulation Networks in Phytopathogenic Bacteria. Trends Microbiol. 2007 , 15 , 363–371. [ Google Scholar ] [ CrossRef ]
• Duca, D.; Lorv, J.; Patten, C.L.; Rose, D.; Glick, B.R. Indole-3-Acetic Acid in Plant–Microbe Interactions. Antonie Van Leeuwenhoek 2014 , 106 , 85–125. [ Google Scholar ] [ CrossRef ]
• Kudoyarova, G.; Arkhipova, T.; Korshunova, T.; Bakaeva, M.; Loginov, O.; Dodd, I.C. Phytohormone Mediation of Interactions Between Plants and Non-Symbiotic Growth Promoting Bacteria Under Edaphic Stresses. Front. Plant Sci. 2019 , 10 , 1368. [ Google Scholar ] [ CrossRef ]
• Keswani, C.; Singh, S.P.; García-Estrada, C.; Mezaache-Aichour, S.; Glare, T.R.; Borriss, R.; Rajput, V.D.; Minkina, T.M.; Ortiz, A.; Sansinenea, E. Biosynthesis and Beneficial Effects of Microbial Gibberellins on Crops for Sustainable Agriculture. J. Appl. Microbiol. 2022 , 132 , 1597–1615. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Morales-Cedeño, L.R.; Orozco-Mosqueda, M.d.C.; Loeza-Lara, P.D.; Parra-Cota, F.I.; de los Santos-Villalobos, S.; Santoyo, G. Plant Growth-Promoting Bacterial Endophytes as Biocontrol Agents of Pre- and Post-Harvest Diseases: Fundamentals, Methods of Application and Future Perspectives. Microbiol. Res. 2021 , 242 , 126612. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Bianco, C.; Defez, R. Medicago truncatula Improves Salt Tolerance When Nodulated by an Indole-3-Acetic Acid-Overproducing Sinorhizobium meliloti Strain. J. Exp. Bot. 2009 , 60 , 3097–3107. [ Google Scholar ] [ CrossRef ]
• Pii, Y.; Crimi, M.; Cremonese, G.; Spena, A.; Pandolfini, T. Auxin and Nitric Oxide Control Indeterminate Nodule Formation. BMC Plant Biol. 2007 , 7 , 21. [ Google Scholar ] [ CrossRef ]
• Camerini, S.; Senatore, B.; Lonardo, E.; Imperlini, E.; Bianco, C.; Moschetti, G.; Rotino, G.L.; Campion, B.; Defez, R. Introduction of a Novel Pathway for IAA Biosynthesis to Rhizobia Alters Vetch Root Nodule Development. Arch. Microbiol. 2008 , 190 , 67–77. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Baudoin, E.; Lerner, A.; Mirza, M.S.; El Zemrany, H.; Prigent-Combaret, C.; Jurkevich, E.; Spaepen, S.; Vanderleyden, J.; Nazaret, S.; Okon, Y.; et al. Effects of Azospirillum brasilense with Genetically Modified Auxin Biosynthesis Gene IpdC upon the Diversity of the Indigenous Microbiota of the Wheat Rhizosphere. Res. Microbiol. 2010 , 161 , 219–226. [ Google Scholar ] [ CrossRef ]
• Salomé, P.A.; Merchant, S.S. A Series of Fortunate Events: Introducing Chlamydomonas as a Reference Organism. Plant Cell 2019 , 31 , 1682–1707. [ Google Scholar ] [ CrossRef ]
• Zhang, C.; Wang, M.-Y.; Khan, N.; Tan, L.-L.; Yang, S. Potentials, Utilization, and Bioengineering of Plant Growth-Promoting Methylobacterium for Sustainable Agriculture. Sustainability 2021 , 13 , 3941. [ Google Scholar ] [ CrossRef ]
• Calatrava, V.; Hom, E.F.Y.; Llamas, Á.; Fernández, E.; Galván, A. Nitrogen Scavenging from Amino Acids and Peptides in the Model Alga Chlamydomonas reinhardtii . The Role of Extracellular l-Amino Oxidase. Algal Res. 2019 , 38 , 101395. [ Google Scholar ] [ CrossRef ]
• Zhao, Y. Auxin Biosynthesis: A Simple Two-Step Pathway Converts Tryptophan to Indole-3-Acetic Acid in Plants. Mol. Plant 2012 , 5 , 334–338. [ Google Scholar ] [ CrossRef ]
• Wang, X.; Zeng, L.; Liao, Y.; Zhou, Y.; Xu, X.; Dong, F.; Yang, Z. An Alternative Pathway for the Formation of Aromatic Aroma Compounds Derived from L-Phenylalanine via Phenylpyruvic Acid in Tea ( Camellia sinensis (L.) O. Kuntze) Leaves. Food Chem. 2019 , 270 , 17–24. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Osugi, A.; Sakakibara, H. Q&A: How Do Plants Respond to Cytokinins and What Is Their Importance? BMC Biol. 2015 , 13 , 102. [ Google Scholar ] [ CrossRef ]
• Kieber, J.J.; Schaller, G.E. Cytokinin Signaling in Plant Development. Development 2018 , 145 , dev149344. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Kieber, J.J.; Schaller, G.E. The Perception of Cytokinin: A Story 50 Years in the Making. Plant Physiol. 2010 , 154 , 487–492. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Orozco-Mosqueda, M.d.C.; Santoyo, G.; Glick, B.R. Recent Advances in the Bacterial Phytohormone Modulation of Plant Growth. Plants 2023 , 12 , 606. [ Google Scholar ] [ CrossRef ]
• Auer, C.A. Cytokinin Inhibition of Arabidopsis Root Growth: An Examination of Genotype, Cytokinin Activity, and N6-Benzyladenine Metabolism. J. Plant Growth Regul. 1996 , 15 , 201–206. [ Google Scholar ] [ CrossRef ]
• Strnad, M. The Aromatic Cytokinins. Physiol. Plant. 1997 , 101 , 674–688. [ Google Scholar ] [ CrossRef ]
• Tarkowská, D.; Doležal, K.; Tarkowski, P.; Åstot, C.; Holub, J.; Fuksová, K.; Schmülling, T.; Sandberg, G.; Strnad, M. Identification of New Aromatic Cytokinins in Arabidopsis thaliana and Populus × Canadensis Leaves by LC-(+)ESI-MS and Capillary Liquid Chromatography/Frit–Fast Atom Bombardment Mass Spectrometry. Physiol. Plant. 2003 , 117 , 579–590. [ Google Scholar ] [ CrossRef ]
• Spíchal, L.; Rakova, N.Y.; Riefler, M.; Mizuno, T.; Romanov, G.A.; Strnad, M.; Schmülling, T. Two Cytokinin Receptors of Arabidopsis thaliana , CRE1/AHK4 and AHK3, Differ in Their Ligand Specificity in a Bacterial Assay. Plant Cell Physiol. 2004 , 45 , 1299–1305. [ Google Scholar ] [ CrossRef ]
• Hönig, M.; Plíhalová, L.; Husičková, A.; Nisler, J.; Doležal, K. Role of Cytokinins in Senescence, Antioxidant Defence and Photosynthesis. Int. J. Mol. Sci. 2018 , 19 , 4045. [ Google Scholar ] [ CrossRef ]
• Mok, D.W.; Mok, M.C. CYTOKININ METABOLISM AND ACTION. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001 , 52 , 89–118. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lomin, S.N.; Krivosheev, D.M.; Steklov, M.Y.; Arkhipov, D.V.; Osolodkin, D.I.; Schmülling, T.; Romanov, G.A. Plant Membrane Assays with Cytokinin Receptors Underpin the Unique Role of Free Cytokinin Bases as Biologically Active Ligands. J. Exp. Bot. 2015 , 66 , 1851–1863. [ Google Scholar ] [ CrossRef ]
• Li, Y.; Baldauf, S.; Lim, E.-K.; Bowles, D.J. Phylogenetic Analysis of the UDP-Glycosyltransferase Multigene Family of Arabidopsis thaliana . J. Biol. Chem. 2001 , 276 , 4338–4343. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Bajguz, A.; Piotrowska, A. Conjugates of Auxin and Cytokinin. Phytochemistry 2009 , 70 , 957–969. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Hluska, T.; Hlusková, L.; Emery, R.J.N. The Hulks and the Deadpools of the Cytokinin Universe: A Dual Strategy for Cytokinin Production, Translocation, and Signal Transduction. Biomolecules 2021 , 11 , 209. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Frebort, I.; Kowalska, M.; Hluska, T.; Frebortova, J.; Galuszka, P. Evolution of Cytokinin Biosynthesis and Degradation. J. Exp. Bot. 2011 , 62 , 2431–2452. [ Google Scholar ] [ CrossRef ]
• Wei, X.; Moreno-Hagelsieb, G.; Glick, B.R.; Doxey, A.C. Comparative Analysis of Adenylate Isopentenyl Transferase Genes in Plant Growth-Promoting Bacteria and Plant Pathogenic Bacteria. Heliyon 2023 , 9 , e13955. [ Google Scholar ] [ CrossRef ]
• Nester, E.W.; Gordon, M.P.; Amasino, R.M.; Yanofsky, M.F. Crown Gall: A Molecular and Physiological Analysis. Annu. Rev. Plant Physiol. 1984 , 35 , 387–413. [ Google Scholar ] [ CrossRef ]
• Zboralski, A.; Filion, M. Pseudomonas Spp. Can Help Plants Face Climate Change. Front. Microbiol. 2023 , 14 , 1198131. [ Google Scholar ] [ CrossRef ]
• Frébortová, J.; Greplová, M.; Seidl, M.F.; Heyl, A.; Frébort, I. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc Sp. PCC 7120. PLoS ONE 2015 , 10 , e0138468. [ Google Scholar ] [ CrossRef ]
• Arkhipova, T.N.; Prinsen, E.; Veselov, S.U.; Martinenko, E.V.; Melentiev, A.I.; Kudoyarova, G.R. Cytokinin Producing Bacteria Enhance Plant Growth in Drying Soil. Plant Soil 2007 , 292 , 305–315. [ Google Scholar ] [ CrossRef ]
• Großkinsky, D.K.; Tafner, R.; Moreno, M.V.; Stenglein, S.A.; García de Salamone, I.E.; Nelson, L.M.; Novák, O.; Strnad, M.; van der Graaff, E.; Roitsch, T. Cytokinin Production by Pseudomonas fluorescens G20-18 Determines Biocontrol Activity against Pseudomonas syringae in Arabidopsis . Sci. Rep. 2016 , 6 , 23310. [ Google Scholar ] [ CrossRef ]
• Akhtar, S.S.; Mekureyaw, M.F.; Pandey, C.; Roitsch, T. Role of Cytokinins for Interactions of Plants With Microbial Pathogens and Pest Insects. Front. Plant Sci. 2020 , 10 , 1777. [ Google Scholar ] [ CrossRef ]
• De Rybel, B.; Mähönen, A.P.; Helariutta, Y.; Weijers, D. Plant Vascular Development: From Early Specification to Differentiation. Nat. Rev. Mol. Cell Biol. 2016 , 17 , 30–40. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Brenner, W.G.; Ramireddy, E.; Heyl, A.; Schmülling, T. Gene Regulation by Cytokinin in Arabidopsis . Front. Plant Sci. 2012 , 3 , 8. [ Google Scholar ] [ CrossRef ]
• Aremu, A.O.; Fawole, O.A.; Makunga, N.P.; Masondo, N.A.; Moyo, M.; Buthelezi, N.M.D.; Amoo, S.O.; Spíchal, L.; Doležal, K. Applications of Cytokinins in Horticultural Fruit Crops: Trends and Future Prospects. Biomolecules 2020 , 10 , 1222. [ Google Scholar ] [ CrossRef ]
• AlAli, H.A.; Khalifa, A.; Almalki, M. Plant Growth-Promoting Rhizobacteria from Ocimum basilicum Improve Growth of Phaseolus vulgaris and Abelmoschus esculentus . South African J. Bot. 2021 , 139 , 200–209. [ Google Scholar ] [ CrossRef ]
• Al-Tammar, F.K.; Khalifa, A.Y.Z. Plant Growth Promoting Bacteria Drive Food Security. Brazilian J. Biol. 2022 , 82 , e267257. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Forni, C.; Duca, D.; Glick, B.R. Mechanisms of Plant Response to Salt and Drought Stress and Their Alteration by Rhizobacteria . Plant Soil 2017 , 410 , 335–356. [ Google Scholar ] [ CrossRef ]
• Arkhipova, T.N.; Veselov, S.U.; Melentiev, A.I.; Martynenko, E.V.; Kudoyarova, G.R. Ability of Bacterium Bacillus subtilis to Produce Cytokinins and to Influence the Growth and Endogenous Hormone Content of Lettuce Plants. Plant Soil 2005 , 272 , 201–209. [ Google Scholar ] [ CrossRef ]
• Xu, J.; Li, X.-L.; Luo, L. Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress. Appl. Environ. Microbiol. 2012 , 78 , 8056–8061. [ Google Scholar ] [ CrossRef ]
• Palberg, D.; Kisiała, A.; Jorge, G.L.; Emery, R.J.N. A Survey of Methylobacterium Species and Strains Reveals Widespread Production and Varying Profiles of Cytokinin Phytohormones. BMC Microbiol. 2022 , 22 , 49. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Mekureyaw, M.F.; Pandey, C.; Hennessy, R.C.; Nicolaisen, M.H.; Liu, F.; Nybroe, O.; Roitsch, T. The Cytokinin-Producing Plant Beneficial Bacterium Pseudomonas fluorescens G20-18 Primes Tomato ( Solanum lycopersicum ) for Enhanced Drought Stress Responses. J. Plant Physiol. 2022 , 270 , 153629. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Van De Velde, K.; Ruelens, P.; Geuten, K.; Rohde, A.; Van Der Straeten, D. Exploiting DELLA Signaling in Cereals. Trends Plant Sci. 2017 , 22 , 880–893. [ Google Scholar ] [ CrossRef ]
• Zaidi, A.; Ahmad, E.; Khan, M.S.; Saif, S.; Rizvi, A. Role of Plant Growth Promoting Rhizobacteria in Sustainable Production of Vegetables: Current Perspective. Sci. Hortic. (Amsterdam). 2015 , 193 , 231–239. [ Google Scholar ] [ CrossRef ]
• Khan, A.L.; Waqas, M.; Hussain, J.; Al-Harrasi, A.; Hamayun, M.; Lee, I.-J. Phytohormones Enabled Endophytic Fungal Symbiosis Improve Aluminum Phytoextraction in Tolerant Solanum lycopersicum : An Examples of Penicillium janthinellum LK5 and Comparison with Exogenous GA3. J. Hazard. Mater. 2015 , 295 , 70–78. [ Google Scholar ] [ CrossRef ]
• You, Y.-H. Fungal Diversity and Plant Growth Promotion of Endophytic Fungi from Six Halophytes in Suncheon Bay. J. Microbiol. Biotechnol. 2012 , 22 , 1549–1556. [ Google Scholar ] [ CrossRef ]
• Kozaki, A.; Aoyanagi, T. Molecular Aspects of Seed Development Controlled by Gibberellins and Abscisic Acids. Int. J. Mol. Sci. 2022 , 23 , 1876. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Hedden, P.; Thomas, S.G. Gibberellin Biosynthesis and Its Regulation. Biochem. J. 2012 , 444 , 11–25. [ Google Scholar ] [ CrossRef ]
• Olanrewaju, O.S.; Glick, B.R.; Babalola, O.O. Mechanisms of Action of Plant Growth Promoting Bacteria. World J. Microbiol. Biotechnol. 2017 , 33 , 197. [ Google Scholar ] [ CrossRef ]
• Nelson, S.K.; Steber, C.M. Gibberellin Hormone Signal Perception: Down-regulating DELLA Repressors of Plant Growth and Development. In Annual Plant Reviews ; Wiley: Hoboken, NJ, USA, 2016; Volume 49, pp. 153–188. [ Google Scholar ]
• Yamaguchi, S. Gibberellin Metabolism and Its Regulation. Annu. Rev. Plant Biol. 2008 , 59 , 225–251. [ Google Scholar ] [ CrossRef ]
• Liu, J.; Qiu, G.; Liu, C.; Li, H.; Chen, X.; Fu, Q.; Lin, Y.; Guo, B. Salicylic Acid, a Multifaceted Hormone, Combats Abiotic Stresses in Plants. Life 2022 , 12 , 886. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Koo, Y.M.; Heo, A.Y.; Choi, H.W. Salicylic Acid as a Safe Plant Protector and Growth Regulator. Plant Pathol. J. 2020 , 36 , 1–10. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Riaz, A.; Rafique, M.; Aftab, M.; Qureshi, M.A.; Javed, H.; Mujeeb, F.; Akhtar, S. Mitigation of Salinity in Chickpea by Plant Growth Promoting Rhizobacteria and Salicylic Acid. EURASIAN J. SOIL Sci. 2019 , 8 , 221–228. [ Google Scholar ] [ CrossRef ]
• Mehrasa, H.; Farnia, A.; Kenarsari, M.J.; Nakhjavan, S. Endophytic Bacteria and SA Application Improve Growth, Biochemical Properties, and Nutrient Uptake in White Beans Under Drought Stress. J. Soil Sci. Plant Nutr. 2022 , 22 , 3268–3279. [ Google Scholar ] [ CrossRef ]
• de Andrade, W.L.; de Melo, A.S.; Melo, Y.L.; da Silva Sá, F.V.; Rocha, M.M.; da Silva Oliveira, A.P.; Fernandes Júnior, P.I. Bradyrhizobium Inoculation Plus Foliar Application of Salicylic Acid Mitigates Water Deficit Effects on Cowpea. J. Plant Growth Regul. 2021 , 40 , 656–667. [ Google Scholar ] [ CrossRef ]
• Lefevere, H.; Bauters, L.; Gheysen, G. Salicylic Acid Biosynthesis in Plants. Front. Plant Sci. 2020 , 11 , 338. [ Google Scholar ] [ CrossRef ]
• Torrens-Spence, M.P.; Bobokalonova, A.; Carballo, V.; Glinkerman, C.M.; Pluskal, T.; Shen, A.; Weng, J.-K. PBS3 and EPS1 Complete Salicylic Acid Biosynthesis from Isochorismate in Arabidopsis . Mol. Plant 2019 , 12 , 1577–1586. [ Google Scholar ] [ CrossRef ]
• Rekhter, D.; Lüdke, D.; Ding, Y.; Feussner, K.; Zienkiewicz, K.; Lipka, V.; Wiermer, M.; Zhang, Y.; Feussner, I. Isochorismate-Derived Biosynthesis of the Plant Stress Hormone Salicylic Acid. Science (80-.). 2019 , 365 , 498–502. [ Google Scholar ] [ CrossRef ]
• Grobelak, A.; Hiller, J. Bacterial Siderophores Promote Plant Growth: Screening of Catechol and Hydroxamate Siderophores. Int. J. Phytoremediation 2017 , 19 , 825–833. [ Google Scholar ] [ CrossRef ]
• Kjærbølling, I.; Mortensen, U.H.; Vesth, T.; Andersen, M.R. Strategies to Establish the Link between Biosynthetic Gene Clusters and Secondary Metabolites. Fungal Genet. Biol. 2019 , 130 , 107–121. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Mishra, A.; Baek, K.-H. Salicylic Acid Biosynthesis and Metabolism: A Divergent Pathway for Plants and Bacteria. Biomolecules 2021 , 11 , 705. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Weber, T.; Kim, H.U. The Secondary Metabolite Bioinformatics Portal: Computational Tools to Facilitate Synthetic Biology of Secondary Metabolite Production. Synth. Syst. Biotechnol. 2016 , 1 , 69–79. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Zeevaart, J.A.D.; Creelman, R.A. Metabolism and Physiology of Abscisic Acid. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1988 , 39 , 439–473. [ Google Scholar ] [ CrossRef ]
• Melcher, K.; Xu, Y.; Ng, L.-M.; Zhou, X.E.; Soon, F.-F.; Chinnusamy, V.; Suino-Powell, K.M.; Kovach, A.; Tham, F.S.; Cutler, S.R.; et al. Identification and Mechanism of ABA Receptor Antagonism. Nat. Struct. Mol. Biol. 2010 , 17 , 1102–1108. [ Google Scholar ] [ CrossRef ]
• Liu, S.; Lv, Z.; Liu, Y.; Li, L.; Zhang, L. Network Analysis of ABA-Dependent and ABA-Independent Drought Responsive Genes in Arabidopsis thaliana . Genet. Mol. Biol. 2018 , 41 , 624–637. [ Google Scholar ] [ CrossRef ]
• Nambara, E.; Marion-Poll, A. ABSCISIC ACID BIOSYNTHESIS AND CATABOLISM. Annu. Rev. Plant Biol. 2005 , 56 , 165–185. [ Google Scholar ] [ CrossRef ]
• Chen, K.; Li, G.; Bressan, R.A.; Song, C.; Zhu, J.; Zhao, Y. Abscisic Acid Dynamics, Signaling, and Functions in Plants. J. Integr. Plant Biol. 2020 , 62 , 25–54. [ Google Scholar ] [ CrossRef ]
• Hartung, W.; Sauter, A.; Turner, N.C.; Fillery, I.; Heilmeier, H. Abscisic Acid in Soils: What Is Its Function and Which Factors and Mechanisms Influence Its Concentration? Plant Soil 1996 , 184 , 105–110. [ Google Scholar ] [ CrossRef ]
• Forchetti, G.; Masciarelli, O.; Alemano, S.; Alvarez, D.; Abdala, G. Endophytic Bacteria in Sunflower ( Helianthus annuus L.): Isolation, Characterization, and Production of Jasmonates and Abscisic Acid in Culture Medium. Appl. Microbiol. Biotechnol. 2007 , 76 , 1145–1152. [ Google Scholar ] [ CrossRef ]
• Cohen, A.C.; Travaglia, C.N.; Bottini, R.; Piccoli, P.N. Participation of Abscisic Acid and Gibberellins Produced by Endophytic Azospirillum in the Alleviation of Drought Effects in Maize. Botany 2009 , 87 , 455–462. [ Google Scholar ] [ CrossRef ]
• Sgroy, V.; Cassán, F.; Masciarelli, O.; Del Papa, M.F.; Lagares, A.; Luna, V. Isolation and Characterization of Endophytic Plant Growth-Promoting (PGPB) or Stress Homeostasis-Regulating (PSHB) Bacteria Associated to the Halophyte Prosopis Strombulifera. Appl. Microbiol. Biotechnol. 2009 , 85 , 371–381. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Hartung, W. The Evolution of Abscisic Acid (ABA) and ABA Function in Lower Plants, Fungi and Lichen. Funct. Plant Biol. 2010 , 37 , 806. [ Google Scholar ] [ CrossRef ]
• Crocoll, C.; Kettner, J.; Dörffling, K. Abscisic Acid in Saprophytic and Parasitic Species of Fungi. Phytochemistry 1991 , 30 , 1059–1060. [ Google Scholar ] [ CrossRef ]
• Belimov, A.A.; Dodd, I.C.; Safronova, V.I.; Dumova, V.A.; Shaposhnikov, A.I.; Ladatko, A.G.; Davies, W.J. Abscisic Acid Metabolizing Rhizobacteria Decrease ABA Concentrations in Planta and Alter Plant Growth. Plant Physiol. Biochem. 2014 , 74 , 84–91. [ Google Scholar ] [ CrossRef ]
• Zhang, J.; Schurr, U.; Davies, W.J. Control of Stomatal Behaviour by Abscisic Acid Which Apparently Originates in the Roots. J. Exp. Bot. 1987 , 38 , 1174–1181. [ Google Scholar ] [ CrossRef ]
• Vacheron, J.; Desbrosses, G.; Bouffaud, M.-L.; Touraine, B.; Moënne-Loccoz, Y.; Muller, D.; Legendre, L.; Wisniewski-Dyé, F.; Prigent-Combaret, C. Plant Growth-Promoting Rhizobacteria and Root System Functioning. Front. Plant Sci. 2013 , 4 , 356. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Zhou, S.; Hu, W.; Deng, X.; Ma, Z.; Chen, L.; Huang, C.; Wang, C.; Wang, J.; He, Y.; Yang, G.; et al. Overexpression of the Wheat Aquaporin Gene, TaAQP7, Enhances Drought Tolerance in Transgenic Tobacco. PLoS ONE 2012 , 7 , e52439. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Hasegawa, S.; Poling, S.M.; Maier, V.P.; Bennett, R.D. Metabolism of Abscisic Acid: Bacterial Conversion to Dehydrovomifoliol and Vomifoliol Dehydrogenase Activity. Phytochemistry 1984 , 23 , 2769–2771. [ Google Scholar ] [ CrossRef ]
• Montes-Osuna, N.; Cernava, T.; Gómez-Lama Cabanás, C.; Berg, G.; Mercado-Blanco, J. Identification of Volatile Organic Compounds Emitted by Two Beneficial Endophytic Pseudomonas Strains from Olive Roots. Plants 2022 , 11 , 318. [ Google Scholar ] [ CrossRef ]
• Kloepper, J.W.; Ryu, C.-M.; Zhang, S. Induced Systemic Resistance and Promotion of Plant Growth by Bacillus Spp. Phytopathology® 2004 , 94 , 1259–1266. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Kai, M.; Effmert, U.; Piechulla, B. Bacterial-Plant-Interactions: Approaches to Unravel the Biological Function of Bacterial Volatiles in the Rhizosphere. Front. Microbiol. 2016 , 7 , 108. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Choub, V.; Won, S.-J.; Ajuna, H.B.; Moon, J.-H.; Choi, S.-I.; Lim, H.-I.; Ahn, Y.S. Antifungal Activity of Volatile Organic Compounds from Bacillus velezensis CE 100 against Colletotrichum Gloeosporioides. Horticulturae 2022 , 8 , 557. [ Google Scholar ] [ CrossRef ]
• Tahir, H.A.S.; Gu, Q.; Wu, H.; Raza, W.; Hanif, A.; Wu, L.; Colman, M.V.; Gao, X. Plant Growth Promotion by Volatile Organic Compounds Produced by Bacillus subtilis SYST2. Front. Microbiol. 2017 , 8 , 171. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Venneman, J.; Vandermeersch, L.; Walgraeve, C.; Audenaert, K.; Ameye, M.; Verwaeren, J.; Steppe, K.; Van Langenhove, H.; Haesaert, G.; Vereecke, D. Respiratory CO 2 Combined With a Blend of Volatiles Emitted by Endophytic Serendipita Strains Strongly Stimulate Growth of Arabidopsis Implicating Auxin and Cytokinin Signaling. Front. Plant Sci. 2020 , 11 , 544435. [ Google Scholar ] [ CrossRef ]
• Sharifi, R.; Ryu, C.-M. Revisiting Bacterial Volatile-Mediated Plant Growth Promotion: Lessons from the Past and Objectives for the Future. Ann. Bot. 2018 , 122 , 349–358. [ Google Scholar ] [ CrossRef ]
• del Carmen Orozco-Mosqueda, M.; Macías-Rodríguez, L.I.; Santoyo, G.; Farías-Rodríguez, R.; Valencia-Cantero, E. Medicago truncatula Increases Its Iron-Uptake Mechanisms in Response to Volatile Organic Compounds Produced by Sinorhizobium meliloti . Folia Microbiol. (Praha) 2013 , 58 , 579–585. [ Google Scholar ] [ CrossRef ]
• Lemfack, M.C.; Nickel, J.; Dunkel, M.; Preissner, R.; Piechulla, B. MVOC: A Database of Microbial Volatiles. Nucleic Acids Res. 2014 , 42 , D744–D748. [ Google Scholar ] [ CrossRef ]
• Yasmin, H.; Rashid, U.; Hassan, M.N.; Nosheen, A.; Naz, R.; Ilyas, N.; Sajjad, M.; Azmat, A.; Alyemeni, M.N. Volatile Organic Compounds Produced by Pseudomonas pseudoalcaligenes Alleviated Drought Stress by Modulating Defense System in Maize ( Zea mays L.). Physiol. Plant. 2021 , 172 , 896–911. [ Google Scholar ] [ CrossRef ]
• Sudha, A.; Durgadevi, D.; Archana, S.; Muthukumar, A.; Suthin Raj, T.; Nakkeeran, S.; Poczai, P.; Nasif, O.; Ansari, M.J.; Sayyed, R.Z. Unraveling the Tripartite Interaction of Volatile Compounds of Streptomyces rochei with Grain Mold Pathogens Infecting Sorghum. Front. Microbiol. 2022 , 13 , 923360. [ Google Scholar ] [ CrossRef ]
• Poveda, J. Beneficial Effects of Microbial Volatile Organic Compounds (MVOCs) in Plants. Appl. Soil Ecol. 2021 , 168 , 104118. [ Google Scholar ] [ CrossRef ]
• Lemfack, M.C.; Gohlke, B.-O.; Toguem, S.M.T.; Preissner, S.; Piechulla, B.; Preissner, R. MVOC 2.0: A Database of Microbial Volatiles. Nucleic Acids Res. 2018 , 46 , D1261–D1265. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Giorgio, A.; De Stradis, A.; Lo Cantore, P.; Iacobellis, N.S. Biocide Effects of Volatile Organic Compounds Produced by Potential Biocontrol Rhizobacteria on Sclerotinia sclerotiorum . Front. Microbiol. 2015 , 6 , 1056. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Rybakova, D.; Rack-Wetzlinger, U.; Cernava, T.; Schaefer, A.; Schmuck, M.; Berg, G. Aerial Warfare: A Volatile Dialogue between the Plant Pathogen Verticillium longisporum and Its Antagonist Paenibacillus polymyxa . Front. Plant Sci. 2017 , 8 , 1294. [ Google Scholar ] [ CrossRef ]
• Kai, M.; Effmert, U.; Berg, G.; Piechulla, B. Volatiles of Bacterial Antagonists Inhibit Mycelial Growth of the Plant Pathogen Rhizoctonia solani . Arch. Microbiol. 2007 , 187 , 351–360. [ Google Scholar ] [ CrossRef ]
• Rojas-Solís, D.; Zetter-Salmón, E.; Contreras-Pérez, M.; Rocha-Granados, M.d.C.; Macías-Rodríguez, L.; Santoyo, G. Pseudomonas stutzeri E25 and Stenotrophomonas maltophilia CR71 Endophytes Produce Antifungal Volatile Organic Compounds and Exhibit Additive Plant Growth-Promoting Effects. Biocatal. Agric. Biotechnol. 2018 , 13 , 46–52. [ Google Scholar ] [ CrossRef ]
• Riyazuddin, R.; Verma, R.; Singh, K.; Nisha, N.; Keisham, M.; Bhati, K.K.; Kim, S.T.; Gupta, R. Ethylene: A Master Regulator of Salinity Stress Tolerance in Plants. Biomolecules 2020 , 10 , 959. [ Google Scholar ] [ CrossRef ]
• Burg, S.P.; Burg, E.A. Molecular Requirements for the Biological Activity of Ethylene. Plant Physiol. 1967 , 42 , 144–152. [ Google Scholar ] [ CrossRef ]
• Guzmán, P.; Ecker, J.R. Exploiting the Triple Response of Arabidopsis to Identify Ethylene-Related Mutants. Plant Cell 1990 , 2 , 513–523. [ Google Scholar ] [ CrossRef ]
• Ali, S.; Charles, T.C.; Glick, B.R. Amelioration of High Salinity Stress Damage by Plant Growth-Promoting Bacterial Endophytes That Contain ACC Deaminase. Plant Physiol. Biochem. 2014 , 80 , 160–167. [ Google Scholar ] [ CrossRef ]
• Gómez-Cadenas, A.; Tadeo, F.R.; Primo-Millo, E.; Talon, M. Involvement of Abscisic Acid and Ethylene in the Responses of Citrus Seedlings to Salt Shock. Physiol. Plant. 1998 , 103 , 475–484. [ Google Scholar ] [ CrossRef ]
• Arraes, F.B.M.; Beneventi, M.A.; Lisei de Sa, M.E.; Paixao, J.F.R.; Albuquerque, E.V.S.; Marin, S.R.R.; Purgatto, E.; Nepomuceno, A.L.; Grossi-de-Sa, M.F. Implications of Ethylene Biosynthesis and Signaling in Soybean Drought Stress Tolerance. BMC Plant Biol. 2015 , 15 , 213. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Gupta, R.; Min, C.W.; Kim, S.W.; Yoo, J.S.; Moon, A.-R.; Shin, A.-Y.; Kwon, S.-Y.; Kim, S.T. A TMT-Based Quantitative Proteome Analysis to Elucidate the TSWV Induced Signaling Cascade in Susceptible and Resistant Cultivars of Solanum lycopersicum . Plants 2020 , 9 , 290. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Gupta, R.; Min, C.W.; Kim, Y.-J.; Kim, S.T. Identification of Msp1-Induced Signaling Components in Rice Leaves by Integrated Proteomic and Phosphoproteomic Analysis. Int. J. Mol. Sci. 2019 , 20 , 4135. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Glick, B.R. Modulation of Plant Ethylene Levels by the Bacterial Enzyme ACC Deaminase. FEMS Microbiol. Lett. 2005 , 251 , 1–7. [ Google Scholar ] [ CrossRef ]
• Munns, R.; Tester, M. Mechanisms of Salinity Tolerance. Annu. Rev. Plant Biol. 2008 , 59 , 651–681. [ Google Scholar ] [ CrossRef ]
• Siddikee, M.A.; Chauhan, P.S.; Sa, T. Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper ( Capsicum annuum L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-Producing Halotolerant Bacteria. J. Plant Growth Regul. 2012 , 31 , 265–272. [ Google Scholar ] [ CrossRef ]
• Ma, W.; Charles, T.C.; Glick, B.R. Expression of an Exogenous 1-Aminocyclopropane-1-Carboxylate Deaminase Gene in Sinorhizobium Meliloti Increases Its Ability To Nodulate Alfalfa. Appl. Environ. Microbiol. 2004 , 70 , 5891–5897. [ Google Scholar ] [ CrossRef ]
• Nascimento, F.X.; Brígido, C.; Glick, B.R.; Oliveira, S. ACC Deaminase Genes Are Conserved among Mesorhizobium Species Able to Nodulate the Same Host Plant. FEMS Microbiol. Lett. 2012 , 336 , 26–37. [ Google Scholar ] [ CrossRef ]
• Ma, W.; Sebestianova, S.B.; Sebestian, J.; Burd, G.I.; Guinel, F.C.; Glick, B.R. Prevalence of 1-Aminocyclopropane-1-Carboxylate Deaminase in Rhizobium Spp. Antonie Van Leeuwenhoek 2003 , 83 , 285–291. [ Google Scholar ] [ CrossRef ]
• Sun, Y.; Cheng, Z.; Glick, B.R. The Presence of a 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Deletion Mutation Alters the Physiology of the Endophytic Plant Growth-Promoting Bacterium Burkholderia Phytofirmans PsJN. FEMS Microbiol. Lett. 2009 , 296 , 131–136. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Ali, S.; Charles, T.C.; Glick, B.R. Delay of Flower Senescence by Bacterial Endophytes Expressing 1-Aminocyclopropane-1-Carboxylate Deaminase. J. Appl. Microbiol. 2012 , 113 , 1139–1144. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Nascimento, F.X.; Rossi, M.J.; Soares, C.R.F.S.; McConkey, B.J.; Glick, B.R. New Insights into 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Phylogeny, Evolution and Ecological Significance. PLoS ONE 2014 , 9 , e99168. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Glick, B.R. Bacteria with ACC Deaminase Can Promote Plant Growth and Help to Feed the World. Microbiol. Res. 2014 , 169 , 30–39. [ Google Scholar ] [ CrossRef ]
• Sheehy, R.E.; Honma, M.; Yamada, M.; Sasaki, T.; Martineau, B.; Hiatt, W.R. Isolation, Sequence, and Expression in Escherichia coli of the Pseudomonas Sp. Strain ACP Gene Encoding 1-Aminocyclopropane-1-Carboxylate Deaminase. J. Bacteriol. 1991 , 173 , 5260–5265. [ Google Scholar ] [ CrossRef ]
• Jacobson, C.B.; Pasternak, J.J.; Glick, B.R. Partial Purification and Characterization of 1-Aminocyclopropane-1-Carboxylate Deaminase from the Plant Growth Promoting Rhizobacterium Pseudomonas putida GR12-2. Can. J. Microbiol. 1994 , 40 , 1019–1025. [ Google Scholar ] [ CrossRef ]
• Hontzeas, N.; Zoidakis, J.; Glick, B.R.; Abu-Omar, M.M. Expression and Characterization of 1-Aminocyclopropane-1-Carboxylate Deaminase from the Rhizobacterium Pseudomonas putida UW4: A Key Enzyme in Bacterial Plant Growth Promotion. Biochim. Biophys. Acta Proteins Proteomics 2004 , 1703 , 11–19. [ Google Scholar ] [ CrossRef ]
• Khalifa, A. ACC Deaminase-Containing Rhizobacteria from Rhizosphere of Zygophyllum coccineum Alleviate Salt Stress Impact on Wheat ( Triticum aestivum L.). Basic Appl. Sci. Sci. J. King Faisal Univ. 2020 , 21 , 89–101. [ Google Scholar ] [ CrossRef ]
• Ali, S.; Glick, B.R. Plant–Bacterial Interactions in Management of Plant Growth under Abiotic Stresses. In New and Future Developments in Microbial Biotechnology and Bioengineering ; Elsevier: Amsterdam, The Netherlands, 2019; pp. 21–45. [ Google Scholar ]
• Araya, M.A.; Valenzuela, T.; Inostroza, N.G.; Maruyama, F.; Jorquera, M.A.; Acuña, J.J. Isolation and Characterization of Cold-Tolerant Hyper-ACC-Degrading Bacteria from the Rhizosphere, Endosphere, and Phyllosphere of Antarctic Vascular Plants. Microorganisms 2020 , 8 , 1788. [ Google Scholar ] [ CrossRef ]
• Bal, H.B.; Adhya, T.K. Alleviation of Submergence Stress in Rice Seedlings by Plant Growth-Promoting Rhizobacteria With ACC Deaminase Activity. Front. Sustain. Food Syst. 2021 , 5 , 606158. [ Google Scholar ] [ CrossRef ]
• Cheng, Z.; Park, E.; Glick, B.R. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas putida UW4 Facilitates the Growth of Canola in the Presence of Salt. Can. J. Microbiol. 2007 , 53 , 912–918. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Belimov, A.A.; Dodd, I.C.; Hontzeas, N.; Theobald, J.C.; Safronova, V.I.; Davies, W.J. Rhizosphere Bacteria Containing 1-aminocyclopropane-1-carboxylate Deaminase Increase Yield of Plants Grown in Drying Soil via Both Local and Systemic Hormone Signalling. New Phytol. 2009 , 181 , 413–423. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Moon, Y.S.; Ali, S. Possible Mechanisms for the Equilibrium of ACC and Role of ACC Deaminase-Producing Bacteria. Appl. Microbiol. Biotechnol. 2022 , 106 , 877–887. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Ma, W.; Guinel, F.C.; Glick, B.R. Rhizobium Leguminosarum Biovar Viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants. Appl. Environ. Microbiol. 2003 , 69 , 4396–4402. [ Google Scholar ] [ CrossRef ]
• Nukui, N.; Minamisawa, K.; Ayabe, S.-I.; Aoki, T. Expression of the 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Gene Requires Symbiotic Nitrogen-Fixing Regulator Gene NifA2 in Mesorhizobium Loti MAFF303099. Appl. Environ. Microbiol. 2006 , 72 , 4964–4969. [ Google Scholar ] [ CrossRef ]
• Alemneh, A.A.; Zhou, Y.; Ryder, M.H.; Denton, M.D. Is Phosphate Solubilizing Ability in Plant Growth-promoting Rhizobacteria Isolated from Chickpea Linked to Their Ability to Produce ACC Deaminase? J. Appl. Microbiol. 2021 , 131 , 2416–2432. [ Google Scholar ] [ CrossRef ]
• Gamalero, E.; Glick, B.R. Bacterial Modulation of Plant Ethylene Levels. Plant Physiol. 2015 , 169 , 13–22. [ Google Scholar ] [ CrossRef ]
• Barnawal, D.; Bharti, N.; Maji, D.; Chanotiya, C.S.; Kalra, A. 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-Containing Rhizobacteria Protect Ocimum sanctum Plants during Waterlogging Stress via Reduced Ethylene Generation. Plant Physiol. Biochem. 2012 , 58 , 227–235. [ Google Scholar ] [ CrossRef ]
• Glick, B.R.; Cheng, Z.; Czarny, J.; Duan, J. Promotion of Plant Growth by ACC Deaminase-Producing Soil Bacteria. In New Perspectives and Approaches in Plant Growth-Promoting Rhizobacteria Research ; Springer Netherlands: Dordrecht, The Netherlands, 2007; pp. 329–339. [ Google Scholar ]
• Pierik, R.; Tholen, D.; Poorter, H.; Visser, E.J.W.; Voesenek, L.A.C.J. The Janus Face of Ethylene: Growth Inhibition and Stimulation. Trends Plant Sci. 2006 , 11 , 176–183. [ Google Scholar ] [ CrossRef ]
• van Loon, L.C.; Geraats, B.P.J.; Linthorst, H.J.M. Ethylene as a Modulator of Disease Resistance in Plants. Trends Plant Sci. 2006 , 11 , 184–191. [ Google Scholar ] [ CrossRef ]
• Robison, M.M.; Griffith, M.; Pauls, K.P.; Glick, B.R. Dual Role for Ethylene in Susceptibility of Tomato to Verticillium Wilt. J. Phytopathol. 2001 , 149 , 385–388. [ Google Scholar ] [ CrossRef ]
• Glick, B.R.; Penrose, D.M.; Li, J. A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-Promoting Bacteria. J. Theor. Biol. 1998 , 190 , 63–68. [ Google Scholar ] [ CrossRef ]
• Prayitno, J.; Rolfe, B.G.; Mathesius, U. The Ethylene-Insensitive Sickle Mutant of Medicago Truncatula Shows Altered Auxin Transport Regulation during Nodulation. Plant Physiol. 2006 , 142 , 168–180. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Stearns, J.C.; Woody, O.Z.; McConkey, B.J.; Glick, B.R. Effects of Bacterial ACC Deaminase on Brassica Napus Gene Expression. Mol. Plant-Microbe Interact. 2012 , 25 , 668–676. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Adie, B.; Chico, J.M.; Rubio-Somoza, I.; Solano, R. Modulation of Plant Defenses by Ethylene. J. Plant Growth Regul. 2007 , 26 , 160–177. [ Google Scholar ] [ CrossRef ]
• Robert-Seilaniantz, A.; Navarro, L.; Bari, R.; Jones, J.D. Pathological Hormone Imbalances. Curr. Opin. Plant Biol. 2007 , 10 , 372–379. [ Google Scholar ] [ CrossRef ]
• Cao, H.; Li, X.; Dong, X. Generation of Broad-Spectrum Disease Resistance by Overexpression of an Essential Regulatory Gene in Systemic Acquired Resistance. Proc. Natl. Acad. Sci. USA 1998 , 95 , 6531–6536. [ Google Scholar ] [ CrossRef ]
• Koornneef, A.; Pieterse, C.M.J. Cross Talk in Defense Signaling. Plant Physiol. 2008 , 146 , 839–844. [ Google Scholar ] [ CrossRef ]
• Grant, M.; Lamb, C. Systemic Immunity. Curr. Opin. Plant Biol. 2006 , 9 , 414–420. [ Google Scholar ] [ CrossRef ]
• Pieterse, C.M.J.; Zamioudis, C.; Berendsen, R.L.; Weller, D.M.; Van Wees, S.C.M.; Bakker, P.A.H.M. Induced Systemic Resistance by Beneficial Microbes. Annu. Rev. Phytopathol. 2014 , 52 , 347–375. [ Google Scholar ] [ CrossRef ]
• van Wees, S.C.; Luijendijk, M.; Smoorenburg, I.; van Loon, L.C.; Pieterse, C.M. Rhizobacteria-Mediated Induced Systemic Resistance (ISR) in Arabidopsis Is Not Associated with a Direct Effect on Expression of Known Defense-Related Genes but Stimulates the Expression of the Jasmonate-Inducible Gene Atvsp upon Challenge. Plant Mol. Biol. 1999 , 41 , 537–549. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Pieterse, C.M.J.; van Wees, S.C.M.; van Pelt, J.A.; Knoester, M.; Laan, R.; Gerrits, H.; Weisbeek, P.J.; van Loon, L.C. A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis . Plant Cell 1998 , 10 , 1571–1580. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Ahn, I.-P.; Lee, S.-W.; Suh, S.-C. Rhizobacteria-Induced Priming in Arabidopsis Is Dependent on Ethylene, Jasmonic Acid, and NPR1. Mol. Plant-Microbe Interact. 2007 , 20 , 759–768. [ Google Scholar ] [ CrossRef ]
• Tran, H.; Ficke, A.; Asiimwe, T.; Höfte, M.; Raaijmakers, J.M. Role of the Cyclic Lipopeptide Massetolide A in Biological Control of Phytophthora Infestans and in Colonization of Tomato Plants by Pseudomonas fluorescens . New Phytol. 2007 , 175 , 731–742. [ Google Scholar ] [ CrossRef ]
• Verhagen, B.W.M.; Glazebrook, J.; Zhu, T.; Chang, H.-S.; van Loon, L.C.; Pieterse, C.M.J. The Transcriptome of Rhizobacteria-Induced Systemic Resistance in Arabidopsis . Mol. Plant-Microbe Interact. 2004 , 17 , 895–908. [ Google Scholar ] [ CrossRef ]
• Cartieaux, F.; Contesto, C.; Gallou, A.; Desbrosses, G.; Kopka, J.; Taconnat, L.; Renou, J.-P.; Touraine, B. Simultaneous Interaction of Arabidopsis thaliana with Bradyrhizobium Sp. Strain ORS278 and Pseudomonas syringae Pv. Tomato DC3000 Leads to Complex Transcriptome Changes. Mol. Plant-Microbe Interact. 2008 , 21 , 244–259. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Yan, Z.; Reddy, M.S.; Ryu, C.-M.; McInroy, J.A.; Wilson, M.; Kloepper, J.W. Induced Systemic Protection Against Tomato Late Blight Elicited by Plant Growth-Promoting Rhizobacteria. Phytopathology® 2002 , 92 , 1329–1333. [ Google Scholar ] [ CrossRef ]
• De Vleesschauwer, D.; Djavaheri, M.; Bakker, P.A.H.M.; Höfte, M. Pseudomonas Fluorescens WCS374r-Induced Systemic Resistance in Rice against Magnaporthe Oryzae Is Based on Pseudobactin-Mediated Priming for a Salicylic Acid-Repressible Multifaceted Defense Response. Plant Physiol. 2008 , 148 , 1996–2012. [ Google Scholar ] [ CrossRef ]
• Segarra, G.; Van der Ent, S.; Trillas, I.; Pieterse, C.M.J. MYB72, a Node of Convergence in Induced Systemic Resistance Triggered by a Fungal and a Bacterial Beneficial Microbe. Plant Biol. 2009 , 11 , 90–96. [ Google Scholar ] [ CrossRef ]
• Glazebrook, J. Contrasting Mechanisms of Defense Against Biotrophic and Necrotrophic Pathogens. Annu. Rev. Phytopathol. 2005 , 43 , 205–227. [ Google Scholar ] [ CrossRef ]
• Van der Ent, S.; Van Wees, S.C.M.; Pieterse, C.M.J. Jasmonate Signaling in Plant Interactions with Resistance-Inducing Beneficial Microbes. Phytochemistry 2009 , 70 , 1581–1588. [ Google Scholar ] [ CrossRef ]
• Glick, B.R.; Jacobson, C.B.; Schwarze, M.M.K.; Pasternak, J.J. 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Mutants of the Plant Growth Promoting Rhizobacterium Pseudomonas putida GR12-2 Do Not Stimulate Canola Root Elongation. Can. J. Microbiol. 1994 , 40 , 911–915. [ Google Scholar ] [ CrossRef ]
• Cheng, Z.; Duncker, B.P.; McConkey, B.J.; Glick, B.R. Transcriptional Regulation of ACC Deaminase Gene Expression in Pseudomonas putida UW4. Can. J. Microbiol. 2008 , 54 , 128–136. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Duan, J.; Müller, K.M.; Charles, T.C.; Vesely, S.; Glick, B.R. 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Genes in Rhizobia from Southern Saskatchewan. Microb. Ecol. 2009 , 57 , 423–436. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Bouffaud, M.-L.; Renoud, S.; Dubost, A.; Moënne-Loccoz, Y.; Muller, D. 1-Aminocyclopropane-1-Carboxylate Deaminase Producers Associated to Maize and Other Poaceae Species. Microbiome 2018 , 6 , 114. [ Google Scholar ] [ CrossRef ]
• Jha, B.; Gontia, I.; Hartmann, A. The Roots of the Halophyte Salicornia brachiata Are a Source of New Halotolerant Diazotrophic Bacteria with Plant Growth-Promoting Potential. Plant Soil 2012 , 356 , 265–277. [ Google Scholar ] [ CrossRef ]
• Sorty, A.M.; Ntana, F.; Hansen, M.; Stougaard, P. Plant-Root Exudate Analogues Influence Activity of the 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Gene in Pseudomonas hormoni G20-18T. Microorganisms 2023 , 11 , 2504. [ Google Scholar ] [ CrossRef ]
• Vega-Celedón, P.; Bravo, G.; Velásquez, A.; Cid, F.P.; Valenzuela, M.; Ramírez, I.; Vasconez, I.-N.; Álvarez, I.; Jorquera, M.A.; Seeger, M. Microbial Diversity of Psychrotolerant Bacteria Isolated from Wild Flora of Andes Mountains and Patagonia of Chile towards the Selection of Plant Growth-Promoting Bacterial Consortia to Alleviate Cold Stress in Plants. Microorganisms 2021 , 9 , 538. [ Google Scholar ] [ CrossRef ]
• Heydarian, Z.; Gruber, M.; Coutu, C.; Glick, B.R.; Hegedus, D.D. Gene Expression Patterns in Shoots of Camelina Sativa with Enhanced Salinity Tolerance Provided by Plant Growth Promoting Bacteria Producing 1-Aminocyclopropane-1-Carboxylate Deaminase or Expression of the Corresponding AcdS Gene. Sci. Rep. 2021 , 11 , 4260. [ Google Scholar ] [ CrossRef ]
• Guo, D.-J.; Singh, R.K.; Singh, P.; Li, D.-P.; Sharma, A.; Xing, Y.-X.; Song, X.-P.; Yang, L.-T.; Li, Y.-R. Complete Genome Sequence of Enterobacter roggenkampii ED5, a Nitrogen Fixing Plant Growth Promoting Endophytic Bacterium With Biocontrol and Stress Tolerance Properties, Isolated From Sugarcane Root. Front. Microbiol. 2020 , 11 , 580081. [ Google Scholar ] [ CrossRef ]
• Fernández-Llamosas, H.; Ibero, J.; Thijs, S.; Imperato, V.; Vangronsveld, J.; Díaz, E.; Carmona, M. Enhancing the Rice Seedlings Growth Promotion Abilities of Azoarcus Sp. CIB by Heterologous Expression of ACC Deaminase to Improve Performance of Plants Exposed to Cadmium Stress. Microorganisms 2020 , 8 , 1453. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Goswami, D.; Thakker, J.N.; Dhandhukia, P.C. Simultaneous Detection and Quantification of Indole-3-Acetic Acid (IAA) and Indole-3-Butyric Acid (IBA) Produced by Rhizobacteria from l-Tryptophan (Trp) Using HPTLC. J. Microbiol. Methods 2015 , 110 , 7–14. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Arund, J.; Luman, M.; Uhlin, F.; Tanner, R.; Fridolin, I. Is Fluorescence Valid to Monitor Removal of Protein Bound Uremic Solutes in Dialysis? PLoS ONE 2016 , 11 , e0156541. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Dworkin, M.; Foster, J.W. EXPERIMENTS WITH SOME MICROORGANISMS WHICH UTILIZE ETHANE AND HYDROGEN. J. Bacteriol. 1958 , 75 , 592–603. [ Google Scholar ] [ CrossRef ]
• Honma, M.; Shimomura, T. Metabolism of 1-Aminocyclopropane-1-Carboxylic Acid. Agric. Biol. Chem. 1978 , 42 , 1825–1831. [ Google Scholar ] [ CrossRef ]

Click here to enlarge figure

Share and Cite

Timofeeva, A.M.; Galyamova, M.R.; Sedykh, S.E. How Do Plant Growth-Promoting Bacteria Use Plant Hormones to Regulate Stress Reactions? Plants 2024 , 13 , 2371. https://doi.org/10.3390/plants13172371

Timofeeva AM, Galyamova MR, Sedykh SE. How Do Plant Growth-Promoting Bacteria Use Plant Hormones to Regulate Stress Reactions? Plants . 2024; 13(17):2371. https://doi.org/10.3390/plants13172371

Timofeeva, Anna M., Maria R. Galyamova, and Sergey E. Sedykh. 2024. "How Do Plant Growth-Promoting Bacteria Use Plant Hormones to Regulate Stress Reactions?" Plants 13, no. 17: 2371. https://doi.org/10.3390/plants13172371

Article Metrics

Article access statistics, further information, mdpi initiatives, follow mdpi.

Subscribe to receive issue release notifications and newsletters from MDPI journals

• Open access
• Published: 24 August 2024

Exploring the impact of plant growth-promoting bacteria in alleviating stress on Aptenia cordifolia subjected to irrigation with recycled water in multifunctional external green walls

• Mansoure Jozay   ORCID: orcid.org/0000-0002-2513-6794 1 ,
• Hossein Zarei   ORCID: orcid.org/0000-0002-2792-9480 1 ,
• Sarah Khorasaninejad   ORCID: orcid.org/0000-0002-2786-4015 1 &
• Taghi Miri   ORCID: orcid.org/0000-0002-2428-1332 2  

BMC Plant Biology volume  24 , Article number:  802 ( 2024 ) Cite this article

189 Accesses

1 Altmetric

Metrics details

Rapid urbanization and population growth exert a substantial impact on the accessibility of drinking water resources, underscoring the imperative for wastewater treatment and the reuse of non-potable water in agriculture. In this context, green walls emerge as a potential solution to augment the purification of unconventional waters, simultaneously contributing to the aesthetic appeal and enjoyment of urban areas. This study aims to optimize water management in green walls by investigating the impact of bacterial strains on the biochemical properties and performance of the ornamental accumulator plant, Aptenia cordifolia , grown with various unconventional water sources. The experiments were designed as split plots based on a completely randomized block design with three replications. The main factor was recycled water with three levels (gray water, wastewater from the Kashfroud region of Mashhad, and urban water (control)). The sub-factor included different bacterial strains at four levels, composed of various bacteria combinations, (B1: Psedoumonas flucrecens  +  Azosporillum liposferum  +  Thiobacillus thioparus  +  Aztobactor chorococcum , B2: Paenibacillus polymyxa  +  Pseudomonas fildensis  +  Bacillus subtilis  +  Achromobacter xylosoxidans  +  Bacillus licheniform , B3: Pseudomonas putida  +  Acidithiobacillus ferrooxidans  +  Bacillus velezensis  +  Bacillus subtilis  +  Bacillus methylotrophicus  +  Mcrobacterium testaceum , and the control level without bacterial application (B0).

The findings revealed significant differences at the 5% probability level across all morphophysiological traits, including plant height, the number and length of lateral branches, growth index, and plant coverage. Moreover, superior morphophysiological traits were observed in plants cultivated in substrates inoculated with wastewater irrigation. Substrates inoculated with bacteria exhibited the highest relative water content (RWC) and chlorophyll levels, coupled with the lowest relative saturation deficit (RSD), electrolyte leakage (EL), and carotenoid levels. Furthermore, plant growth-promoting bacteria (PGPB), from a biochemical perspective, were associated with increased carbohydrates, total protein, and anthocyanin. They also contributed to controlling oxidative stress caused by free radicals by enhancing the activity of antioxidant enzymes, such as guaiacol peroxidase (GPX), polyphenol oxidase (PPO), ascorbate peroxidase (APX), and peroxidase (POD), while reducing catalase enzyme (CAT) activity. This led to increased resistance to stress, as evidenced by a decrease in malondialdehyde and proline levels. The study concludes that the MIX B3, being both ecofriendly and economical, represents an effective strategy for mitigating the adverse effects of wastewater on plants.

This study showed that plant irrigation using wastewater increases the levels of proline, phenols and oxidative stress. However, the application of plant growth promoting bacteria (PGPB) reduced oxidative damage by increasing antioxidant activity and decreasing proline and phenol levels. These findings show the potential of bacterial treatments to improve plant growth and reduce adverse effects of recycled water irrigation.

Peer Review reports

Urban environments, with their current unsustainable developments, pose significant challenges that require fundamental environmental reforms. The primary reasons for these challenges include the purification of pollutants, urban heat islands, the expansion of impervious surfaces, climate change, loss of biodiversity, and the aesthetic damage associated with current urban transformations [ 1 ]. However, natural and non-built urban environments have the capacity for ecological restoration and improvement. Unfortunately, the unsustainable approaches of urban development have deprived us of these options [ 2 ]. To address this dilemma, Artmann and Sartison [ 3 ] proposed a nature-based solution. One of these solutions is sustainable green infrastructure in cities, including exterior green walls.

Considering that horizontal expansion of green spaces may not be feasible due to limited vacant land, vertical development of gardens remains the only viable solution in theory to fulfill this objective. The severe shortage of space in today's densely populated urban environments underscores the need for future cities to explore the expansion of rooftops and green walls [ 4 ]. Vertical green walls can be considered as sustainable environmental technologies that offer numerous economic, environmental, and social benefits and are also regarded as urban lungs [ 5 ]. However, the fundamental potential of these infrastructures, in terms of sustainable building elements, has received less attention in research with the aim of enhancing their environmental efficiency and multifunctionality [ 6 ].

The high water demand of green systems is a limiting factor for their development in water-scarce regions. To minimize water requirements, it is advisable to opt for drought-tolerant and heat-resistant plant species [ 7 ]. For example, Aptenia cordifolia is a CAM (Crassulacean Acid Metabolism) plant that is drought-resistant and also capable of phytoremediation. Aptenia cordifolia belonging to the Aizoaceae family, is a succulent and perennial species widely employed in green spaces in southern Iran. It is known as a hardy, flowering, perennial, trailing plant. In Iran it is also introduced as Ice Flower. A. cordifolia is resistant to heat and drought. It has light pink, white and red flowers that bloom throughout the year. The solitary to clustered flowers arise in leaf axils and generally open during the day. This plant needs full sun exposure.

Another solution can be the utilization of alternative water sources such as graywater and wastewater [ 5 ]. Since green walls are considered an environmental innovation, it is necessary to incorporate the possibility of rainwater harvesting and the reuse of recycled water [ 8 ].

Soil pollution with heavy metals, originating from fertilizers and non-conventional water sources used in urban green spaces, presents a significant issue due to the potential transfer of these contaminants to agricultural products in cities. Therefore, multiple challenges hinder the sustainability of this concept in urban environments, requiring effective planning to address them. Important factors such as agricultural and horticultural practices, including growth-promoting bacteria, design elements, and plant characteristics, play a crucial role in soil filtration [ 9 ] and the removal of pollutants. Recently, it has been reported that planting crops in Substrate containing certain growth-promoting bacteria can efficiently fulfill their nutrient requirements and reduce the need for chemical fertilizers [ 10 ]. Research on biofertilizers includes various types of microorganisms that can convert less accessible forms of essential nutrients into accessible forms through biological processes. This leads to the development of better root systems and seed germination [ 11 ]. Liu et al., [ 12 ] isolated forty-one bacterial strains from the rhizosphere soil and root tissues of five plant species ( Artemisia argyi Levl.، Gladiolus gandavensis Vaniot Houtt، Boehmeria nivea L.، Veronica didyma Tenore و Miscanthus floridulonizing Lab). Their results showed that among the bacteria, two strains, Klebsiella michiganensis TS8 and Lelliottia jeotgali MR2, exhibited higher tolerance to cadmium and were highly successful in cadmium phytoremediation of the soil. TS8 increased plant height and the dry weight of leaves from 9.39 to 1.99. It appears that strains of Pseudomonas, Mycobacterium, Staphylococcus, Micrococcus, Bacillus, Paenibacillus, and Klebsiella were widely used solely for phytoremediation, and the combined application of these strains were more successful than their individual application [ 13 ]. Some native plants and grasses also have the potential for phytoremediation in metal-contaminated water. Phytoremediation through biostimulation is a promising approach that can enhance the synergistic effects of microorganisms and plants [ 14 ].

On one hand, the most significant challenge is population growth and the increasing water demand for economic activities, particularly agriculture. Water scarcity is a major concern in densely populated urban areas. On the other hand, the disposal of graywater and urban wastewater can contaminate surface and groundwater sources. In this regard, green walls can improve the treatment of non-conventional waters, enhance the hydrological cycle, and increase the beauty and enjoyment of urban areas [ 15 ]. The benefits of using ornamental plant coverings with distinct aesthetic features are evident in satisfying the visual preferences of the community and providing pleasant landscape value [ 16 ]. Using alternative ornamental species in constructed wetlands (CWs), represents an effective system for pollutant removal [ 17 ]. Additionally, the use of these systems can significantly improve the visual quality of the landscape, which is often undervalued but has positive social and psychological impacts on people's daily lives [ 18 ].

One of the important aspects of non-conventional water is graywater and wastewater. Graywater refers to wastewater generated from laundry, toilets, showers, baths (also known as light graywater), and, in some cases, kitchen sinks and dishwashers (known as dark graywater). Light graywater is produced in significant amounts (45% to 60% of domestic wastewater) and contains a lower pollutant load compared to mixed domestic wastewater [ 19 ]. For this reason, considerable efforts have been made to reuse it on-site. Additionally, treated wastewater has good nutrient value that can enhance plant growth, reduce fertilizer consumption, and increase the productivity of nutrient-depleted soils [ 20 , 21 ].

Given the challenges posed by population growth and increased water demand for economic activities, particularly in agriculture, urban areas face significant hurdles. Freshwater scarcity emerges as a primary concern in these regions, where the disposal of graywater and urban wastewater poses a potential threat to surface and groundwater sources, exacerbating the issue. While green walls can effectively treat polluted water, enhance the hydrological cycle, and beautify urban areas, they may also introduce stress conditions. These stressors arise from microclimate factors and the use of unconventional water for plant growth, potentially increasing the transfer of heavy metals and other non-biological stresses Additionally, the use of wastewater in urban agriculture requires additional caution. Therefore, the primary objective of this study is to investigate the feasibility of utilizing organic biological fertilizers to improve soil organic matter content, providing a sustainable alternative to chemical fertilizers. The goal is to mitigate existing stresses and promote environmentally friendly soil management practices.

The study area and site

This research was conducted in Mashhad, located in northeastern Iran. Mashhad is the capital of Khorasan Razavi province and the second largest and most populous city in Iran. It has a semi-arid climate with cold winters and hot, dry summers (elevation of 995 m above sea level, geographical coordinates 36 degrees 18 min north, 59 degrees 36 min east). The average annual precipitation is approximately 255 mm. The average minimum and maximum temperatures annually are -4 and 22 degrees Celsius, respectively, and the relative humidity is reported to be about 40% [ 22 ]. The precise location of the experiment was on a 15 m wall in the outdoor area of the Armgan Greenhouse, located in the northern part of Mashhad (Fig.  1 ).

Location of the study site in Mashhad

The green wall systems

To conduct this research, vertical cultivation panels (mesh made of 5 mm steel wire) were installed at a suitable distance from shading factors, facing east–west. The vertical cultivation system used in this project was called the "Almich" system (initially developed in Malaysia). The experimental units consisted of Almich green wall pots, plastic pots made of new or recycled polypropylene, with dimensions of approximately 20 × 20 and a depth of about 21 cm. Leca was used as a drainage layer at the bottom of the pots, followed by a layer of geotextile as a soil filter.

In each plot, two plants of each tested species were cultivated. This study was conducted on an exterior green wall from March to December 2022. Each wall consisted of two panels measuring 44 × 106 cm, and each set of three walls constituted one replication of the experiment. Each wall had 4 vertical rows and 4 horizontal rows, resulting in a total of 16 plots (experimental units) per wall. Considering A. cordifolia and the mentioned treatments with three replications, the first experiment included 48 experimental units (Fig.  2 ).

A view of the experiment’s external green walls

The experiment

This experiment aimed to investigate the impact of irrigation water quality and different bacterial strains on the growth and performance of selected ornamental plants with phytoremediation characteristics, while also measuring the water consumption of these species under green wall conditions. This research was implemented as split-plot layout, based on a randomized complete block design with three replications from March to December 2022. The organic matter content in this experiment was 20%, which is very close to the maximum recommended 30% organic matter content (OC) by FLL (2018) [ 23 ].

The growth medium used in all experimental units was the same and consisted of the following components: 25% cocopeat [ 24 ], 5% vermicompost [ 25 ], 55% perlite [ 26 ], 10% vermiculite [ 27 ], and 5% zeolite [ 28 ].

Unconventional water treatments

The main-factor included graywater collected from rainwater, a twin sink designed for fruit and vegetable washing, wastewater from the Kashafrood region and urban water (control). The main factor was applied in three main tank reservoirs and irrigated to the plants in the form of drip irrigation, with irrigation levels at around 80% of the field capacity, adjusted based on the flow rate of the drip emitters.

Treatment of plant growth-promoting bacteria

In this research, growth-promoting bacteria were utilized. Apart from fulfilling the plant's requirements for chemical fertilizers, these bacteria also absorbed heavy metals from the soil [ 29 , 30 ]. Previous research suggests that the combined application of these bacterial strains has been more effective than their individual application [ 30 ]. The sub-factor of different biological strains of bacteria at four levels: Mix1 ( Psedoumonas flucrecens  +  Azosporillum Liposferum  +  Thiobacillus thioparus  +  Aztobactor chorococcum ), Mix2 ( Paenibacillus polymyxa  +  Pseudomonas fildensis  +  Bacillus subtilis  +  Achromobacter xylosoxidans  +  Bacillus licheniformis ), Mix3 ( Pseudomonas putida  +  Acidithiobacillus ferrooxidans  +  Bacillus velezensis  +  Bacillus subtilis  +  Bacillus methylotrophicus  +  Mcrobacterium testaceum ) and B0- control (without bacterial inoculation).

Each plant received 20 cc of the biofertilizer. The bacteria utilized in this experiment were procured from the Soil Biology and Biotechnology Laboratory at the Golestan Agricultural and Natural Resources Research Center LBSG, specifically identified by isolate number 041011 in the laboratory bank. The bacteria were extracted from the rhizosphere of agronomy plants like soybean and wheat using the method of Ju et al. [ 31 ]. The full scientific name and strains of the bacterial treatments in this study are given in Abbreviation. Except for the non-inoculated controls, the substrates around each plant specimen were watered and inoculated with 20 ml of bacterial suspension to obtain a bacterial concentration of 108 CFU/ml. The liquid was injected evenly around the plant root zone and the substrate surface around each plant using a syringe two weeks after planting. This method was selected to ensure an even distribution of the bacterial treatment in the substrate.

Selected accumulator ornamental plant

This plant, especially in phytoremediation, has effective applications for pollution extraction. They may act as a phytostabilizer, particularly in areas affected by metals [ 32 ] (Fig.  3 ). The selection of A. Cordifolia for the experiment was based on its ornamental qualities and accumulation characteristics. Additionally, this plant belongs to the CAM family, and in accordance with the traits of the Crassulaceae pathway, it exhibits rapid growth, high biomass production, robust root development, and high water use efficiency. These attributes enable effective pollutant removal, making it suitable for use in urban green walls. The Aptenia cordifolia seedlings utilized in this experiment were sourced from regions conducive to their growth within the country (Iran). Specifically, they were obtained from the Shandiz greenhouse in Mashhad, where such seedlings are abundantly produced.

Trends in Temperature, Relative Humidity, Precipitation, and Wind Speed in the Experimental Months of the Year 2022 in Mashhad City

The substrate was first passed through a 2 mm sieve after air drying. Physical and chemical characteristics were determined using standard laboratory methods as described in the subsequent sentences. Acidity and conductivity were measured using the extract and saturated mud [ 33 ], and the numbers were red using an Electrical Conductivity (E.C.) meter model JENWAY4510, and a pH meter model METROHM691. Particle density, bulk density, and total porosity were measured based on Chen et al. [ 34 ]. The field capacity and permanent wilting point of the substrate was measured according to the method of Salter and Haworth [ 35 ]. Organic carbon and organic matter measurements were based on Walkley and Black [ 36 ]. The digestion method determined macro and microelements. An instrument Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES) Model 76,004,555 made in Germany, was used to take these measurements (Table  1 ). Throughout the study period, data on relative humidity, wind speed, precipitation, and air temperature at the experiment site were meticulously recorded (Fig.  4 ).

The appearance conditions of the four experimental plants studied in the under B3 and I2 treatments A , B , C and D during spring/summer/autumn and winter season!

Measurements

Morphological traits.

The measured morphological traits in this experiment included plant height, internode distance (measured using a ruler), stem diameter, leaf diameter, leaf length, leaf width (measured using a digital caliper), number of lateral branches, number of nodes, and number of leaves on lateral branches. These measurements were taken on a monthly basis [ 37 , 38 ]. The growth index (plant width × plant length × plant height) was also assessed on a monthly basis.

Plant coverage

The plant surface coverage, also known as the horizontal and vertical coverage of plants, was calculated using quadrat constructed to the size of 20 × 20 square centimeters for each experimental unit in each green wall. Each quadrat consisted of 100 chambers, with each chamber measuring 2 × 2 in width and length.

Physiological and Biochemical Traits

All physiological and biochemical traits were evaluated at the end of the experiment during the autumn season.

Total chlorophyll and carotenoid

The total chlorophyll concentration was assessed using the method described by Dere [ 39 ]. Fresh leaves weighing 0.2 g were thoroughly ground and homogenized in a mortar with 10 mL of 96% methanol. The grinding and homogenization process should be carried out in a cool and low-light environment. The resulting mixture was filtered through filter paper, and then subjected to centrifugation at 2500 rpm for 10 min. The supernatant was immediately collected, and the light absorption at wavelengths of 666 nm, 653 nm, and 470 nm was measured using a spectrophotometer for chlorophyll a, chlorophyll b, and carotenoids, respectively. Finally, the carotenoid and total chlorophyll concentrations were determined using the Eqs. ( 3 ) and ( 4 ):

Relative Water Content (RWC)

The calculation of leaf relative water content (RWC) was performed using the method described by Hossain et al., [ 40 ]. Initially, a leaf sample was weighed using a balance to obtain the initial weight. Then, to obtain the turgid weight, the samples were placed in closed containers containing distilled water for 12 h at a temperature of 21 °C (19 to 23 °C). After removing excess water from the leaf surface, the turgid weight of the samples was measured. To determine the dry weight, the samples were transferred to an oven at a temperature of 70 °C for 48 h (Eq.  5 ).

In which Fw represents the fresh weight, Dw represents the dry weight, and Tw represents the turgid weight of the leaf.

Relative Saturation Deficit (RSD)

The relative saturation deficit was calculated using the method described by Samar Raza et al., [ 41 ]. After collecting the leaves and weighing wet weight them (Fw), they are submerged in distilled water at room temperature for 5 h. Then, the leaves were removed from the water, re-weighed, and their turgid weight (Tw) is obtained. The relative saturation deficit is calculated using Eq.  6 .

Electrolyte Leakage (EL)

The stability of the cell membrane was measured using the method described by Sairam and Srivastava [ 42 ]. Leaf segments measuring 2 cm in size were prepared and washed. These segments were then placed in test tubes along with 10 ml of distilled water. At this stage, the electrical conductivity of the test samples (E1) was measured using a JENWAY conductivity meter. Then, the test tubes were transferred to an autoclave and subjected to a temperature of 121 degrees Celsius for 15 min to kill the leaf cells. After cooling down, the electrical conductivity was measured again (E2) in this stage. Finally, the electrolyte leakage values were calculated using Eq.  7 .

Soluble carbohydrates

To measure the soluble carbohydrates, 2.0 ml of methanol extract were mixed with 3 ml of anthrone reagent (0.15 g anthrone in 100 ml of 72% sulfuric acid). The mixture was then placed in a hot water bath at a temperature of 100 degrees Celsius for 20 min to allow the reaction to occur. The absorbance of the samples was measured at a wavelength of 620 nm using a spectrophotometer after cooling [ 43 ].

Total Protein Content

To measure the protein concentration, 5 ml of urine reference solution were added to a test tube, followed by the addition of 100 µl of protein extract, and it was quickly mixed. After 5 min, the absorption was read at a wavelength of 595 nm using a spectrophotometer. The protein concentration was calculated using the standard curve of albumin [ 44 ].

Anthocyanins

To measure anthocyanins in the leaves, the method described by Nadernejad et al., [ 45 ] was used. Fresh plant tissue weighing 0.1 g was ground in a mortar and pestle with 10 ml of methanol acid solution (pure methanol and pure hydrochloric acid in a volumetric ratio of 1:99). The extract was poured into a screw-capped test tube and kept in darkness at a temperature of 25 °C for 24 h. Then, it was centrifuged at 4000 rpm for 10 min, and the absorbance of the supernatant was measured at a wavelength of 550 nm using a spectrophotometer. The concentration was calculated using Eq.  8 , considering the extinction coeffici of 33,000 (ε) in cm/mol (Eq.  8 ).

where A represents absorbance, b is the cell width, and c is the concentration of the solution under investigation.

Total phenols

Determination of total phenols was performed using the Folin-Ciocalteu reagent at a wavelength of 765 nm, following the method described by Singleton and Rossi [ 46 ]. The measurement was carried out by calibrating the standard curve with gallic acid, and the amount of total phenolic compounds was expressed as milligrams of gallic acid equivalents per 100 g of dry weight.

Malondialdehyde (MDA)

To measure malondialdehyde (MDA) levels, approximately 0.2 g of fresh leaf tissue (the youngest leaves at the tip of the stem) were ground in a mortar containing 5 ml of 0.1% tri-chloroacetic acid (TCA). To the resulting centrifuged solution (1 ml), 5 ml of 20% TCA solution containing 5.0% thiobarbituric acid (TBA) were added. The concentration of malondialdehyde was measured at a wavelength of 532 nm. As other compounds besides malondialdehyde in the solution exhibit non-specific absorption, their absorption at a wavelength of 600 nm was also measured [ 47 ] (Eq.  9 ).

In Eq. ( 9 ), A represents the absorption of the sample of interest, £ represents the molar absorptivity coefficient, which is equal to 1.55 × 10 –5 Mcm −1 , and C represents the concentration of malondialdehyde.

Assays of antioxidant activities

To measure the antioxidant activity, the methanolic extract was first diluted at a ratio of 1:10. Then, to deactivate the free radicals, 4 ml of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) solution was added to each sample [ 48 ]. The samples were kept in darkness for 30 min, and the absorbance of the resulting solutions and the absorbance of the control sample were measured at a wavelength of 517 nm using a spectrophotometer. The inhibition percentage of DPPH was obtained by comparing the absorbance of the extract sample with the absorbance of the control sample using Eq.  10 .

Assay GPX activity

To evaluate GPX activity, initially, 2 ml of 0.05 M sodium phosphate buffer with a pH of 5.6 were mixed with 2 ml of 3% hydrogen peroxide and 2 ml of 5-micromolar ascorbate in an ice bath. Immediately, 1.0 ml of enzyme extract from leaf tissue was added, and the absorbance changes at 256 nm were monitored by a spectrophotometer for 2 min with 10 s intervals. The enzyme activity is defined as the volume of enzyme required to hydrolyze one millimole of substrate per minute at 25 °C. Then, the enzyme activity was calculated in units per minute per milligram of protein [ 49 , 50 ].

Assay PPO activity

To measure the activity of the PPO enzyme, pyrogallol was used as the enzyme substrate. The reaction mixture consisted of 2.5 ml of 50 mM potassium phosphate buffer (pH 7), 200 µl of 0.02 M pyrogallol, and 100 µl of enzyme extract. The absorbance of the samples was read at a wavelength of 420 nm after three minutes using a spectrophotometer. The enzyme activity was calculated using the molar absorptivity coefficient of 6.2 Mm −1 Cm −1 [ 51 ].

Assay CAT activity

The CAT activity was measured by Kendal and Scandellius [ 52 ] method. Initially, 5.2 ml of 0.05 M potassium phosphate buffer with a pH of 7 and 0.3 ml of 3% hydrogen peroxide were mixed together in an ice bath. Immediately, 0.2 ml of enzyme extract were added to the mixture, and the absorbance changes at a wavelength of 240 nm were monitored for 4–3 min. Enzyme unit was defined per H 2 O 2 µmol ml- 1 decomposed per minute at 25 °C and then the enzyme activity was calculated in terms of unit changes per minute per mg of protein.

Assay APX activity

To evaluate APX activity, according to this method, initially, 2 ml of 0.05 M sodium phosphate buffer with a pH of 5.6 were mixed with 2 ml of 3% hydrogen peroxide and 2 ml of 5-micromolar ascorbate in an ice bath. Immediately, 1.0 ml of enzyme extract from leaf tissue was added, and the absorbance changes at 256 nm were monitored by a spectrophotometer for 2 min with 10 s intervals. The enzyme activity is defined as the volume of enzyme required to hydrolyze one millimole of substrate per minute at 25 °C. Then, the enzyme activity was calculated in units per minute per milligram of protein [ 50 ].

Assay POX activity

Measurement of POX activity was done according to Holley's method [ 53 ]. In this regard, initially, 2 ml of 0.2 M acetate buffer with a pH of 5, 0.2 ml of 3% hydrogen peroxide, and 0.1 ml of a 0.02 M benzidine solution in 50% methanol are mixed in an ice bath. Then, 0.1 ml of leaf enzyme extract is added to this mixture, and the absorbance curve of the samples is plotted using a spectrophotometer at a wavelength of 530 nm, at room temperature, every 30 s for 3 min. The specific enzyme activity is calculated by using the standard curve and determining the change in enzyme unit per minute per milligram of protein.

Statistical analysis

JMP 8 software was used for statistical analysis. Data analysis was performed using analysis of variance (ANOVA) and mean comparison with the Tukey test at a significance level of at least 5%. All graphs were plotted using Excel software.

According to the analysis of variance, the simple effects of recycled water treatments showed significant differences at the 5% probability level in the growth index characteristics of A. cordifolia . For plant height, only the simple effects of bacterial strains were statistically significant at the 5% level. When evaluating the number of lateral branches, significant differences were observed in both the interaction effects of the two treatments and the individual effects of bacterial strains (p ≤ 0.05). The simple effects of irrigation water type and bacterial strain showed significant differences at 5% probability in surface coverage and RSD. Additionally, in the case of total chlorophyll, RWC, carotenoids, and EL, not only the simple effects of irrigation and bacteria but also their interaction effects were significant at 5% level (p ≤ 0.05) (Table  2 ).

Type of irrigation water and bacteria on morphophysiological traits of Aptenia cordifolia

The impact of varied irrigation water and bacterial strains on the growth traits and surface coverage.

According to the analysis of variance, all morphological traits of the plant species were statistically significant at 5% probability level.

Plant height and growth index

As shown in Fig.  5 , the simple effects of bacterial strains on the height of the studied plant species in ice plant indicate that inoculation of the substrates with Mix B3 and Mix B2 resulted in an increase in plant height. Regarding the growth index in the ice plant, irrigation with wastewater and gray water resulted in a higher growth index compared to urban water.

Simple effects of recycled water and bacterial strains on growth traits, Plant height: (standard error: 2.95). Growth index: (standard error: 3575.99) and coverage level, the data are shown A and B (standard error: 1.06, 2.34). Data analysis was used JMP 8 software and analysis of variance (ANOVA) as split plots based on a completely randomized block design

As evident from the analysis of variance (Table  3 ), in relation to the biochemical data of A. cordifolia , the simple effects of bacterial strain treatments showed significant differences at 1% probability level in proline, GPX activity, and CAT. However, the simple effects of irrigation water type and bacterial strain showed significant differences at 1% level in POX activity. In the evaluation of carbohydrates and antioxidant activity, apart from the interactive effect of the two treatments, there were significant differences in the simple effects of bacterial strains (p ≤ 0.05). Additionally, in leaf phenolic, besides the simple effects of irrigation water type (p ≤ 0.01), the block effect and their interaction effects also showed significant differences (p ≤ 0.01). According to the analysis of variance (Table  3 ), regarding the total protein, anthocyanins, MDA, PPO enzyme, and APX, apart from the simple effects of irrigation water type and bacterial strain ( p  ≤ 0.01), their interaction effects also showed significant differences (p ≤ 0.05).

Surface coverage

As observed in Fig.  3 , the treatment involving the inoculation of the substrates with Mix B3 and irrigation with wastewater led to increased surface coverage (262.67 cm2) in the ice plant. It is noteworthy that the Mix B1 resulted in less surface coverage compared to the control (without bacterial application), suggesting that the B1 combination was not particularly successful in A. cordifolia .

Impact of varied irrigation water and bacterial strains on the number and length of lateral branches

Figure  6 demonstrates that the highest number of lateral branches in A. cordifolia . (11 branches) was observed in the presence of Mix B3 and irrigation with wastewater. The lowest number of lateral branches in the ice plant was observed in tap water in the presence of Mix B3 and B0, with an average of approximately 3 branches (Fig.  6 ). Additionally, the maximum lateral branch length in A. cordifolia . in all three water type treatments was related to the presence of the combined strains B3, B2, and B1, and the lowest was related to the control.

Interaction effects of recycled water and bacterial strains on n the number and length of lateral branches data are shown (standard error: 1.06, 2.34). Data analysis was used JMP 8 software and analysis of variance (ANOVA) as split plots based on a completely randomized block design

According to Fig.  7 , the highest total chlorophyll content was observed in Mix B1 in plants irrigated with wastewater (2.07 mg/g, FW), while the lowest total chlorophyll content was associated with the B0 of plants irrigated with urban water (0.62 mg/g, FW). Regarding carotenoid content in the A. cordifolia , the control treatment (without bacterial application) using urban water exhibited the highest carotenoid levels. Plants treated with the combined bacterial strains showed less leaf yellowing across various irrigation water types.

Interaction effects of recycled water and bacterial strains on photosynthetic pigments data are shown total chlorophyll: (standard error: 0.11). Carotenoid: (standard error: 0.54). Data analysis was used JMP 8 software and analysis of variance (ANOVA) as split plots based on a completely randomized block design

Impact of varied irrigation water and bacterial strains on RWC

According to Fig.  8 , in A. cordifolia , inoculation with Mix B3 in the substrate and irrigation with gray water resulted in the highest leaf water content (65.68%). The lowest leaf water content was observed in the control treatment (without bacterial application) and irrigation with urban water (26.69%).

Interaction effects of recycled water and bacterial strains on RWC, (standard error: 2.57). Data analysis was used JMP 8 software and analysis of variance (ANOVA) as split plots based on a completely randomized block design

Impact of varied irrigation water and bacterial strains on RSD

As evident in Fig.  9 , the highest relative water deficit in A. cordifolia was associated with the control treatment (without bacterial application). It is noteworthy that the highest RSD in the experiment was also observed in plants irrigated with urban water. It appears that recycled waters resulted in a lower RSD.

Interaction effects of recycled water and bacterial strains on RSD, data are shown A and B : (standard error: 1.41, 2). Data analysis was used JMP 8 software and analysis of variance (ANOVA) as split plots based on a completely randomized block design

Biochemical characteristics of ornamental plant

Different small Latin letters in each column of the table indicate a significant difference based on Tukey's test at a probability level of at least 5%. Data represent ± 1 standard error (SE). Data analysis was used JMP 8 software and analysis of variance (ANOVA) as split plots based on a completely randomized block design.

GPX: (guaiacol peroxidase), POD: (peroxidase), CAT: (catalase), B1:) Sedoumonas flucrecens  +  Azosporillum Liposferum  +  Thiobacillus thioparus  +  Aztobactor chorococcum ), B2: ( Paenibacillus polymyxa  +  Pseudomonas fildensis  +  Bacillus subtilis  +  Achromobacter xylosoxidans  +  Bacillus licheniformis ), B3: ( Pseudomonas putida  +  Acidithiobacillus ferrooxidans  +  Bacillus velezensis  +  Bacillus subtilis  +  Bacillus methylotrophicus  +  Mcrobacterium testaceum ) and B0: without the use of bacteria.

The impact of varied irrigation water and bacterial strains on soluble carbohydrates

According to Table  4 , in A. cordifolia , the highest soluble carbohydrates content was associated with Mix B3 and irrigation with wastewater (62.82 mg/g FW). On the other hand, urban water in the control treatment (without bacterial application) and Mix B1 in gray water and urban water exhibited similar behavior and resulted in the lowest soluble carbohydrates content (4.18 mg/g FW).

The impact of varied irrigation water and bacterial strains on total protein

In A. cordifolia , the highest total protein content was observed the substrate inoculated with Mix B3 and plants irrigated with wastewater (81.70 mg/g FW). The lowest total protein content was found in the control treatment (without bacterial application) and irrigation with gray water (0.16 mg/g FW). It appears that gray water was not very successful in enhancing cellular sap thickening in A. cordifolia.

The impact of varied irrigation water and bacterial strains on total phenol

The highest total phenolic content was observed in the substrate inoculated with Mix B2 and plants irrigated with wastewater in A. cordifolia (5.11 mg/g FW). However, the lowest content was found in the control treatment (without bacterial application) and irrigation with tap water (1.33 mg/g FW) (Table  4 ).

The impact of varied irrigation water and bacterial strains on anthocyanin

In A. cordifolia , the highest anthocyanin was observed in the substrate inoculated with Mix B3 and plants irrigated with gray water (7.93 μmol/g FW), while the lowest content was found in the control treatment (without bacterial application) and irrigation with urban water (0.52 μmol/g FW). It appears that the inoculation of the substrate with Mix B3 leads to an increase in anthocyanins in Plants of the CAM family.

The impact of varied irrigation water and bacterial strains on MDA

The MDA content was significantly affected by the main effects and interaction effects of different irrigation water types and bacterial strains (Table  3 ). The application of wastewater led to an increase in the MDA content in plants under irrigation (18.90 μmol/g FW), indicating a degree of tissue damage in the plant. In most cases, the MDA content decreased with bacterial treatments. The highest reduction in MDA content was observed, in order, with Mix B3, Mix B2, and irrigation with wastewater (0.82 μmol/g FW, 0.45 μmol/g FW).

The impact of varied irrigation water and bacterial strains on proline

In A. cordifolia , the control treatment (without bacterial application) and Mix B1 resulted in higher proline levels. The presence of Mix B3 and Mix B2 performed better in reducing proline. According to Table  5 , statistically, the three types of water sources had a similar effect and did not show any specific statistical differences. It is important to note that Mix B1 acted similarly to the control treatment in reducing proline stress indicators. It appears that Mix B1 may not be effective in reducing the stress caused by unconventional water sources in A. cordifolia (Table  5 ).

The impact of varied irrigation water and bacterial strains on antioxidant activity

The results obtained from ANOVA indicated that different bacterial strains had a significant effect (at a minimum level of 5%) on the activity of GPX, PPO, CAT, APX, and POX enzymes (Table  3 ). The application of different bacterial strains increased the activity of GPX, PPO, CAT, APX, and POX enzymes (Tables  4 and 5 ). Table 4 showed that in the ice plant, the highest antioxidant activity was observed in Mix B2 and Mix B3 and irrigation with wastewater (53.12% and 54.20%, respectively). In contrast, irrigation with municipal wastewater and tap water in the control treatment (without bacteria) exhibited the lowest antioxidant activity (22.19%).

Bacterial inoculation, in most cases, increased the activity of the four antioxidant enzymes in different irrigation water types (Tables  4 and 5 ). The highest increase in PPO and APX activity was observed, respectively, with Mix B3 and irrigation with wastewater (0.14 unit −1  mg protein) and (4.21 unit −1  mg protein). The lowest level of PPO activity was observed in the control treatment (without bacteria) and Mix B2 and Mix B1 in all three water types. Regarding APX, the lowest activity was associated with the control treatment (without bacteria) and irrigation with wastewater and urban water (Table  4 ).

Comparing the mean data shows that the substrate inoculated with Mix B2 and Mix B3 was associated with the highest GPX activity (0.012 and 0.01 unit −1  mg protein, respectively), while the lowest activity of this enzyme was observed in Mix B1 and the control medium (B0) (0.005 unit −1  mg protein). CAT activity exhibited an opposite trend compared to the other enzymes in this study, where Mix B1 and the control medium (B0) had the highest CAT activity (0.65 unit −1  mg protein) (Table  5 ). Additionally, the highest increase in POX activity was observed with Mix B3 (1.75 unit −1  mg protein), while other bacterial combinations did not significantly improve POX activity and were similar to the control. On the other hand, irrigation of plants with graywater resulted in increased POX activity (1.53 unit −1  mg protein) (Table  5 ).

Water scarcity has necessitated the use of unconventional water sources for agricultural. The issue of water crisis is a significant concern globally, particularly in Middle Eastern countries, where climate change and global warming are exacerbating the problem [ 54 ]. Overcoming this scarcity, farmers resort to irrigating their crops with treated or untreated wastewater due to its low cost and availability.

The impact of varied irrigation water and bacterial strains on morphological factors of Aptenia cordifolia in external green wall

The persistent application of recycled water has the potential to deteriorate soil quality and contribute to the accumulation of toxic heavy metals in the food chain [ 55 ]. This decline in soil quality caused by unconventional water sources results in reduced quality and quantity of crops grown in these soils and can be considered a form of stress for plants [ 56 ]. However, as observed in the present experiment, the presence of the mentioned microbial strains in wastewater not only did not reduce the quality and quantity of the cultivated plants compared to tap water but also improved certain traits (height, growth index, number and length of lateral branches, chlorophyll, leaf content, RWC, RSD and EL). These results are consistent with the findings of Urbano et al. (2017) [ 57 ] regarding increased plant growth due to higher nutrient content compared to freshwater, which can serve as a nutrient source for agriculture and reduce the demand for chemical fertilizers. Furthermore, studies have shown that plant growth-promoting bacteria enhance productivity in various environmental conditions, especially under stressful situations, to improve the detrimental effects of unconventional water on the quantitative and qualitative traits of plants [ 58 ]. In the present study, it was also demonstrated that the application of bacteria in the substrate resulted in improvement of the adverse effects of these unconventional waters, although the performance of all bacterial treatments was not identical. Mix B3 took the top position, followed by Mix B2 in the subsequent ranking. These bacteria appear to stimulate plant growth through nutrient provision, secretion of plant growth hormones, and various other mechanisms associated with PGPR [ 59 ]. In this study, an increase in chlorophyll content in all tested plants resulted in higher photosynthetic productivity, ultimately leading to better growth, increased plant organs, and improved surface coverage by the plant. The extent of surface coverage depends on the growth characteristics of plants, such as height, length, and width, which are influenced by nutrient conditions and substrate moisture [ 60 ]. Jozay et al., [ 61 ] also stated that the composition and content of substrate significantly affect the growth and coverage of plants on external green surfaces. Rapid coverage in vertical green structures, such as green walls, is a desirable trait. The utilization of bacterial combinations has proven effective in enhancing this characteristic. Kumar et al., [ 62 ] reported that the combined use of sewage sludge and plant growth-promoting rhizobacteria resulted in improved performance ( Luffa acutangula (L.) Roxb), with the highest fresh biomass (9.6 ± 0.3 g), growth rate (1.4 ± 0.1 g/day), plant length (15.5 ± 0.3 cm), root length (10.4 ± 0.3 cm) and total chlorophyll (3.2 ± 0.1 mg/g). The findings of this study regarding the increase in growth indices of the studied plants in the presence PGPB and irrigation align with the results of the mentioned researchers.

It can be said that A. cordifolia (Mesembryanthemum crystallinum) has been studied in favorable conditions in all four seasons and due to its greenness, trailing habit, and sufficient coverage to create a desirable visual landscape, it can be used for its visual attractiveness and appeal in combination substrates used in exterior green walls. Kazemi et al., [ 63 ] examined the growth and performance of four plant species in various substrate types in indoor green wall systems, and the results indicated that A. cordifolia is a desirable species for indoor green wall systems. One of the influential factors in the aesthetic performance of green wall systems is the percentage of plant coverage, which is dependent on plant growth indices, and this finding aligns with the study by Kazemi et al., [ 63 ] regarding the application of A. cordifolia and the current research results.

The leaf chlorophyll content has a close relationship with leaf nitrogen content in plants and can serve as an indicator of nitrogen availability in the soil [ 64 ]. Lubbe et al., [ 65 ] highlighted a decrease in chlorophyll content in the leaves of Amarantus dubius and Solanum nigrum under greywater irrigation treatments, suggesting that greywater irrigation may jeopardize nutrient accessibility compared to tap water irrigation. Vajpayee et al., [ 66 ] assessed the chl a/b ratio among plants exposed to different concentrations of tannery effluents and reported a greater reduction in chlorophyll-a compared to chlorophyll-b. The maximum reduction in carotenoid content in Spirodela polyrhiz a was observed after 7 days at a concentration of 75% tannery effluent. The present results support the degradation of carotenoids due to increased effluent in the soil and metal toxicity. In this study, an increase in pigments was observed in wastewater and greywater in the presence of PGPB, a trend that contradicted the significant reduction in photosynthetic pigments observed with effluent application. In present research, greywater and effluent irrigation resulted in increased plant greenness, which may be attributed to the presence of PGPB.

Yadav and Pandey [ 67 ] also observed a positive correlation between leaf relative water content and the concentration of chlorophyll, protein, and ribisco activity. Leaf water content in plants helps maintain physiological water balance under unfavorable environmental conditions. Jozay et al., [ 68 ] stated that there is a direct relationship between leaf water content and resistance to environmental stress conditions in PGPR (Plant Growth-Promoting Rhizobacteria). Plants inoculated with bacterial strains have the ability to modify lateral root system architecture and increase RWC [ 69 ]. Increased relative water content (RWC) in stressed leaves of plants inoculated with plant growth-promoting rhizobacteria (PGPR) has also been reported in other studies [ 70 ]. In general, an increase in the number of lateral roots and root hairs adds surface area for nutrient and water uptake. Enhanced water and nutrient uptake by inoculated roots improve the water status of plants, which in turn can be a primary factor in promoting plant growth [ 71 ].

The RSD impacts the water relations of plants, including the water content of plant tissues and gas exchange in leaves, leading to an increase in the relative water content (RWC) of leaves and a decrease in transpiration and leaf stomatal conductance [ 72 ]. Optimal growth conditions, along with efficient nutrient absorption and transport to plants, enhance the accumulation of ions and organic molecules in leaf vacuoles. This contributes to maintaining the water balance by reducing leaf osmotic potential [ 73 ]. Vishnupradeep et al., [ 74 ] found that MST-PGPB inoculation increased RWC under various stress conditions. PGPB can maintain water potential to prevent water loss by reducing root surface drying. Likewise, Woo et al., [ 75 ] suggested that PGPB inoculation improves RWC through various PGP metabolites, including siderophore production, ACC deaminase activity, phosphate solubilization, and IAA synthesis, resulting in enhanced plant tolerance to abiotic stress, biomass production, and protein content, particularly under non-biological stress conditions [ 76 ]. Substrate inoculated with Mix B3 and Mix B2, leading to an increase in pigments and RWC, as well as a reduction in RSD, confirming the findings of the mentioned researchers.

Impact of varied irrigation water and bacterial strains on Biochemical factors of Aptenia cordifolia in external green wall

Previous studies have reported an increase in growth associated with PGPB and an increase in secondary metabolites and anthocyanins [ 77 ]. The microbial population has a positive correlation with plant biomass, antioxidant enzyme activity, and anthocyanins, while it has a negative correlation with the production of free radicals [ 78 ], indicating the contribution of rhizosphere microbial population in reducing oxidative stress through increased anthocyanin production and enhanced antioxidant enzyme activity. In plants, the greatest decrease in proline and MDA content and the highest increase in antioxidant enzyme activity were observed substrate inoculated with Mix B3 and Mix B2. Singh and Malaviya [ 79 ] reported that due to acute toxicity of chromium present in wastewater, anthocyanins in the effluent were below detectable levels after 4 days. The increase in anthocyanins in this study, despite repeated use of wastewater and graywater, could be attributed to the presence of bacterial strains. The results of this study, which showed an increase in anthocyanins through substrate inoculated with Mix B3 in irrigated vegetable plants with recycled water, are consistent with the reports of Pagnani et al., [ 80 ] and Kumar et al., [ 62 ].

The result indicates that the increase in proline was in response to watering the plants with municipal wastewater. Proline may act as an osmolyte under stress conditions and increase proline activities thereby minimizing the side effects of stress [ 81 ]. In other findings, Islam et al., [ 82 ] reported that proline acts as a growth regulator and also protects cells against ROS accumulation. It is thought that proline is able to reduce the negative effects of cadmium toxicity on plant growth in plant tissues and reduce oxidative stress [ 83 ]. In this study, the use of growth-promoting bacteria reduced the adverse effects of wastewater irrigation, and it is worth noting Mix B3 and Mix B2 was more successful in reducing the amount of proline than the other two treatments. As a result, it can be said the use of growth-promoting bacteria has put the plant in a favorable condition by increasing the growth index and biomass and has reduced stress and proline content.

Proline and free phenols are non-enzymatic antioxidants that support organisms in unfavorable conditions by reducing the undesirable effects of reactive oxygen species (ROS) [ 84 ]. Environmental stresses, such as heavy metal exposure in unconventional water sources and urban gardening, have been documented to increase ROS production. Antioxidant molecules and enzymes play a crucial role in detoxifying ROS in plant cells. Antioxidant compounds, including proline and phenols, inhibit oxidation and play vital roles in stress responses [ 85 ]. After irrigating plants with wastewater in the control treatment without bacteria, the levels of these compounds significantly increased, confirming the findings of the aforementioned researchers [ 86 ]. It has been reported that the content of phenolic compounds tends to rise when plants are subjected to heavy metal stress, as phenolic compounds act as scavengers of reactive oxygen species and metal chelators. In contrast, the content of free proline and total phenols considerably decreased in response to our treatments compared to plants irrigated with water in the presence of growth-promoting bacteria. The highest reduction was observed in free proline when substrate inoculated with Mix B3 and Mix B2. These results indicate the role of these treatments in reducing the toxic effects of wastewater on plants. Our findings align with previous studies by Liu et al., [ 12 ], which reported that wastewater treatment with bacteria significantly reduces the stress caused by metal contaminants in wastewater. Regarding total phenols, the control treatment in urban water showed the highest reduction. The decrease in phenolic content may be attributed to oxidative stress, while a further increase in the content of phenolic compounds (as non-enzymatic antioxidants) was observed when substrate inoculated with Mix B3 and wastewater. These results are consistent with the reports of Khodamoradi et al., [ 87 ] regarding the reduction of phenols under stress conditions.

Malondialdehyde (MDA) is a byproduct of lipid oxidation and is responsible for cellular membrane damage, inducing alterations in membrane radical properties. These changes ultimately culminate in cell death [ 88 ]. In this study, the MDA content increased in response to wastewater irrigation in the control treatment B0. This may be attributed to the oxidative system's inability to reduce ROS levels, thereby failing to prevent damage to the cell membrane. This result is also supported by Yildirim et al., [ 89 ], who demonstrated that irrigation with contaminated water generally increased the MDA in plants. In contrast, the MDA content significantly decreased in response to treatments B3 and B2 compared to plants irrigated with wastewater in treatment B0. This may be due to the reduction in ROS production and the enhancement of the antioxidant system and repair mechanisms. These results are supported by a recent study by Malik et al., [ 90 ].

Some studies have found that PGPB strains can enhance plant growth and mitigate the negative effects of various stresses on plant growth. Enhanced growth associated with PGPB and increased secondary metabolites, carbohydrates, total protein, and antioxidant potential have been reported in Astragalus mongholicus [ 91 ]. Plant antioxidant enzymes GPX, PPO, CAT, APX, POX act as the first line of defense for tolerating unfavorable conditions. These enzymes stimulate the detoxification of ROS and reduce the detrimental effects of non-biological stress [ 91 ]. Another finding is that in most cases, bacterial treatments increased the activity of antioxidant enzymes. The greatest increase in biomass production and antioxidant enzyme activity was observed in substrate inoculated with Mix B3 and Mix B2 in wastewater, indicating a significant contribution of bacterial inoculation in activating antioxidant enzymes and promoting plant growth. The further increase in GPX, PPO, APX, and POX activities resulting from the most effective bacterial inoculation may indicate the major role of these enzymes in improving plant biomass through substrate inoculated with Mix B3.

Under wastewater stress, plants exposed to a stronger antioxidant system are less exposed to free radicals, leading to lower MDA production. High antioxidant activity in stress-tolerant plants may indicate their ability to neutralize harmful oxidants and maintain their growth and productivity at a normal level. Therefore, it can be concluded that these bacteria, in addition to promoting growth, provide new perspectives for the development of biofertilizers to alleviate environmental stresses. It can be inferred that greater protection was achieved under wastewater irrigation when bacterial strains were applied. Ultimately, Mix B3 and Mix B2 appear to be recommendable to farmers due to their economic and environmental compatibility, aiding in mitigating the adverse effects of recycled water use in urban gardening.

Phytoremediation is an environmentally friendly and cost-effective alternative to removing pollution from soil. Due to the non-degradable chemical nature of heavy metals in soil, we need to understand the function of antipollution facilitators such as plant growth promoting bacteria to improve or facilitate the removal of heavy metals by plants [ 92 , 93 ]. Heavy metal tolerant PGPR have also increased plant growth in heavy metal contaminated soil in recent years. These PGPR have successfully played an important role in promoting plant growth while reducing toxicity or damage to plants exposed to stress produced by various heavy metals in soil [ 94 ]. PGPR produce plant growth regulators, phytohormones, and various secondary metabolites that promote plant growth and reduce heavy metal toxicity. Various mechanisms are employed by PGPR to enhance plant growth under heavy metal stress. Many of them lead to the reduction of heavy metal toxicity [ 95 ].

Known mechanisms by which PGPR can benefit plants under a variety of stresses include: (1) bioremediation of heavy metal-contaminated soils by sequestering toxic heavy metal species and improving soil structure (by bacterial exopolysaccharides); (2) the synthesized enzyme ACC (1aminocyclopropane-1-carboxylate) deaminase, an enzyme that is involved in the reduction of stress-induced ethylene levels in the roots of growing plants; (3) supply of N2 to the plant through biological nitrogen fixation. (4) Production of siderophores. (5) production of phytohormones (such as ABA (abscisic acid), GA (gibberellic acid), auxin, for example, indole-3-acetic acid (IAA) and CK (cytokinin); (6) control of plant pathogens by various mechanisms such as the production of extracellular enzymes that hydrolyze the fungal cell wall, competition for nutrients in the rhizosphere, induction of systemic resistance (ISR), and production of antibiotics and siderophores; (7) dissolution and mineralization of nutrients, especially inorganic phosphate; and (8) improving resistance to abiotic stresses [ 92 , 96 ].

Therefore, growth-promoting bacteria, as living organisms, play an important role in plant nutrition balance. They can help improve their nutrition by reducing environmental stress and increasing the digestion and absorption of nutrients in plants. Using growth-promoting bacteria as an alternative to chemical fertilizers for soil repair and plant nutrition can be an effective method. These bacteria are commonly known as biologicals or biofertilizers. The use of microorganisms can reduce the stresses in various plants in stressed soils, thus opening a potential and promising strategy for sustainable agriculture [ 97 ].

In a recent report by Jozay [ 92 ], it was suggested that PGPR could be used as a bioremediation method for soils contaminated with toxic metals. PGPR contain bacteria that are rhizospheric and endophytic and facilitate bioremediation. Plants accumulate heavy metals in the roots and reduce their transfer to other parts of the plant. These microorganisms provide benefits to plants by providing nutrients and reducing the harmful effects of pollutants.

As pointed out by FAO [ 98 ], promoting safe and healthy agricultural practices in urban environments is essential to achieve sustainable urban development. At the same time, environmental pollutants in cities should be controlled while using city resources and inputs. The purpose of this study is to investigate the potential of green wall systems to produce horticultural materials that are aligned with the goals of sustainable urban development.

The results of this study indicated that plant irrigation using wastewater significantly increased the content of free proline, total phenol, GPX, PPO, CAT, APX, POX and MDA compared to the control. On the other hand, the PGPB reduced the oxidative damage effects of wastewater irrigation by increasing antioxidant activity and enhancing GPX, PPO, APX, POX. As a result, the content of free proline, total phenol, and MDA decreased significantly, while carbohydrates, proteins, and anthocyanins increased in the combined bacterial treatments. It can be concluded that better protection under wastewater irrigation was achieved using our treatments. Ultimately, Mix B3 and Mix B2 economically and environmentally friendly, can be recommended to farmers for mitigating the adverse effects of reclaimed water on urban gardening. Substrate inoculated with Mix B3, wastewater irrigation, and subsequent greywater irrigation, affects water availability, nutrient availability, and physiological traits of ornamental plants, including RWC, RSD, and plant pigment properties, leading to freshness and increased greening of the plant. It also has a considerable effect on morphological traits such as height, growth index, lateral branches, and surface coverage, and there are significant differences in plant growth improvement among the combined substrate inoculated with different bacteria types. This treatment can be used to improve the qualitative and morphological traits of ornamental plants used in external green walls under similar climatic conditions to Mashhad city. Water scarcity is expected to transform the reuse of reclaimed water for irrigation into a widespread and common practice globally. The incorporation of growth-promoting bacteria can serve as aneffective amendment for mitigating the toxicity associated with wastewater, facilitating improved plant growth in areas irrigated with wastewater and greywater. As a sustainable solution, green walls have the potential to mitigate water, soil, and air pollution, thereby enhancing environmental sustainability. By incorporating technology into multifunctional green walls, we can take a significant step towards sustainable urban development. Living green walls are not only introduced as effective tools for urban space management but also as instruments for enhancing the climate resilience of cities. The outcome of this research is the registration of a water recycling system utilizing nature-based methods, with Patent number 110287.

Future Prospects

Future research should focus on understanding the molecular mechanisms underlying these beneficial interactions and exploring the application of PGPB in other plant species and environmental conditions. Additionally, long-term field studies are needed to evaluate the sustainability and economic viability of using PGPB in urban green wall systems.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon ‎reasonable request.‎

Abbreviations

Strain bacteria 0–3

Without the use of bacteria

Psedoumonas flucrecens  +  Azosporillum liposferum  +  Thiobacillus thioparus  +  Aztobactor chorococcum

Paenibacillus polymyxa  +  Pseudomonas fildensis  +  Bacillus subtilis  +  Achromobacter xylosoxidans  +  Bacillus licheniform

Pseudomonas putida  +  Acidithiobacillus ferrooxidans  +  Bacillus velezensis  +  Bacillus subtilis  +  Bacillus methylotrophicus  +  Mcrobacterium testaceum

Organic carbon

Organic matter

Field capacity

Permanent wilting poin

V´asquez A, Giannotti E, Gald´amez E, Vel´asquez P, Devoto, C. Green Infrastructure planning to tackle climate change in Latin American cities. In C. Henriquez, & H. Romero (Eds.), Urban Climate in Latin-American Cities. 2019;32–354. Springer.

Langemeyer J, Connolly JJT. Weaving notion of justice into urban ecosystem service research and practice. Environ Sci Policy. 2020;109:1–14.

Article   Google Scholar  

Artmann M, Sartison K. The role of urban agriculture as a nature-based solution: A review for developing a systemic assessment framework. Sustainability. 2018;10:1937.

Cojocariu M, Chelariu EL, Chiruţă C. Study on Behavior of Some Perennial Flowering Species Used in Vertical Systems for Green Facades in Eastern European Climate. Appl Sci. 2022;12(1):474.

Article   CAS   Google Scholar  

Mahmoudi Jabri K, Nolde E, Ciroth A, Bousselmi L. Life cycle assessment of a decentralized greywater treatment alternative for non-potable reuse application. Int J Environ Sci Tech. 2020;17(1):433–44.

Jozay M, Rabbani Khairkhah M, Kazemi F. The impact of humic acid solutions and types of growing media on some morphophysiological and biochemical features of Syngonium sp and Pothos sp plants in interior green wall conditions. Plant Arshive. 2021;21:2240–52.

Google Scholar  

Savi T, Casolo V, Luglio J, Bertuzzi S, Trifilo P, Lo Gullo M.A, ardini, A. Species-specific reversal of stem xylem embolism after a prolonged drought correlates to endpoint concentration of soluble sugars. Plant Physiology and Biochemistry. 2016; 106:198–207.

Jozay M. Rabbani Khairkhah SM. Vertical green systems (green wall). Iran Publishing House (Aristo Publications). 2019.

Li X, Chang Z, Lian X, Meng G, Ma J, Guo R, Wang Y. Phytoremediation of cadmium contaminated alkaline soil using the ornamental hyperaccumulator Mirabilis jalapa L. enhanced by double harvesting: a field study. Environmental Science and Pollution Research. 2022; 1–8.

Chouhan S, Agrawal L, Prakash A. Amelioration in traditional farming system by exploring the different plant growth-promoting attributes of endophytes for sustainable agriculture. Arch Microbiol. 2022;204:151.

Article   CAS   PubMed   Google Scholar  

Singh P, Singh RK, Zhou Y, Wang J, Jiang Y, Shen N, Wang Y, Yang L, Jiang M. Unlocking the strength of plant growth promoting Pseudomonas in improving crop productivity in normal and challenging environments: a review. Journal of Plant Interactions. 2022;17(1):220–38.

Liu Sh, Liu H, Chen R, Ma Y, Yang B, Chen Zh, Liang Y, Fang J, Xiao Y. Role of Two Plant Growth-Promoting Bacteria in Remediating Cadmium-Contaminated Soil Combined with Miscanthus floridulus (Lab). Plants. 2021;10:912.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Xiao Y, Liu H, Chen R, Liu S, Hao X, Fang J. Heteroauxin-producing bacteria enhance the plant growth and lead uptake of Miscanthus floridulus (Lab.). International Journal of Phytoremediation. 2022; 1–8.

Gavrilescu M. Enhancing phytoremediation of soils polluted with heavy metals. Curr Opin Biotechnol. 2022;74:21–31.

Zanin G, Bortolini L, Borin M. Assessing stormwater nutrient and heavy metal plant uptake in an experimental bioretention pond. Land. 2018;7:150.

Zhuang J, Qiao L, Zhang X, Su Y, Xia Y. Effects of visual attributes of flower borders in urban vegetation landscapes on aesthetic preference and emotional perception, Int. J. Environ. Res. Publ. Health. 2021; 18(17):9318. https://doi.org/10.3390/ijerph18179318 , 3.

Brix H, Arias CA. The use of vertical flow constructed wetlands for on-site treatment of domestic wastewater: new Danish guidelines. Ecol Eng. 2005;25:491–500. https://doi.org/10.1016/j.scitotenv.2015;09.154 .

Silvestri G, Aliata F. El paisaje como cifra de armonía, Ediciones Nueva Visi´on, Buenos Aires. 2001.

Noutsopoulos C, Andreadakis A, Kouris N, Charchousi D, Mendrinou P, Galani A, Mantziaras I, Koumaki E. Greywater characterization and loadings, physicochemical treatment to promote onsite reuse. J Environ Manage. 2018;216:337–46.

Soudi B. Appui a La Promotion De La Reutilisation Des Eaux Usees Par Le Renforcement Des Aspects Institutionnels, Reglementaires Et Financieres, Ainsi Que Des Démarches Participatives, Des Mesures Incitatives Et La Sensibilisation (Activité n_EFS-MO-2). 2018. Available online: https://www.swim-h2020.eu/wp-content/uploads/2018/09/SWIM-H2020-EFS-MO-2 .

Rosa PAL, Galindo FS, Oliveira CE dS, Jalal A, Mortinho ES, Fernandes GC, Marega EMR, Buzetti S, Teixeira Filho MCM. Inoculation with Plant Growth-Promoting Bacteria to Reduce Phosphate Fertilization Requirement and Enhance Technological Quality and Yield of Sugarcane. Microorganisms. 2022;10:192.

National Centers for Climatology. Climate information of the city of Mashhad, 2019. available on: https://www.ncdc.noaa.gov/climate-information , access date: 2019.07.25.

F.L.L. Guidelines for the planning, construction and maintenance of green roofing, Forschungsgesellschaft Landschaftsentwic. Bonnklung Landschaftsbau e.V.-FLL, Bonn. 2018.

Khorshid M, Oustan S, Najafi N, Khataee A. Kinetic characterization of hexavalent chromium stabilization in contaminated soils amended with cocopeat. Arab J Geosci. 2020;13(11):1–12.

Wang F, Miao L, Wang Y, Zhang M, Zhang H, Ding Y, Zhu W. Using cow dung and mineral vermireactors to produce vermicompost for use as a soil amendment to slow Pb2+ migration. Appl Soil Ecol. 2022;170: 104299.

Gong Y, Zhang G, Hao Y, Nie L. Enrichment Evaluation of Heavy Metals from Stormwater Runoff to Soil and Shrubs in Bioretention Facilities. Water. 2022;14(4):638.

Bai G, Luo F, Zou Y, Liu Y, Wang R, Yang H, Liu Z, Chang J, Wu Z, Zhang Y. Effects of vermiculite on the growth process of submerged macrophyte Vallisneria spiralis and sediment microecological environment. J Environ Sci. 2022;118:130–9.

Yu G, Wang G, Chi T, Du C, Wang J, Li P, Zhang Y, Wang S, Yang K, Long Y, Chen H. Enhanced removal of heavy metals and metalloids by constructed wetlands: A review of approaches and mechanisms. Sci Total Environ. 2022;821:153516.

Ekhlasi Nia A. Evaluation of arsenic and iron transport from sediments of a potable water treatment plant wastewater stabilization pond system. M.Sc. Thesis, In the Department of Civil, Geological and Environmental Engineering, Canada. 2021.

Huang Q, Qiu W, Yu M, Li S, Lu Z, Zhu Y, Kan X, Zhuo R. Genome-Wide Characterization of Sedum plumbizincicola HMA Gene Family Provides Functional Implications in Cadmium Response. Plants. 2022;11(2):215.

Ju W, Liu L, Fang L, Cui Y, Duan Ch, Wu H. Impact of co-inoculation with plant-growth-promoting rhizobacteria and rhizobium on the biochemical responses of alfalfa-soil system in copper contaminated soil. Ecotoxi & Environl Safety. 2019;167:218–26.

Esfandiari M, Hakimzadeh Ardakani MA, Sodaiezadeh H. Investigation of heavy metal Contamination in the ornamental plants of green spaces in the city of Yazd. Journal of Ornamental Plants. 2020;10(4):213–22.

Allison LE, Moodie CD. Carbonate. p. 1379–1400. In C.A. Black et al. (Ed.) Methods of Soil Analysis. Part 2. 2nd Ed. Agron. Monogr. 9. ASA, CSSA, and Soil Science Society of America, Madison, WI. 1965.

Chen Y, Inbar Y, Hadar Y. Composted agricultural wastes as potting media for ornamental plants. Soil Sci. 1988;145(4):298–303.

Salter PJ, Haworth F. The available water capacity of a sandy loam soil: I. A critical comparison of methods of determining the moisture content of soil at field capacity and at the permanent wilting percentage. Journal of Soil Science. 1961;12:326–34.

Walkley A, Black TA. An examination of Deglijareff method for determining soil organic matter and a proposed modification of the choromic acid titration method. Soil Sci. 1934;37:29–38.

Mozaffari S, Khorasaninejad S, Gorgini SH. The effects of irrigation regimes and humic acid on some of physiological and biochemical traits of Common Purslane in greenhouse. Journal of Crops Improvement (Journal of Agriculture). 2017;19(2):401–16.

Gorgini Shabankareh H, Khorasaninejad S, Sadeghi M, Tabasi AR. The effects of irrigation periods and humic acid on morpho- physiological and biochemical traits of Thyme ( Thymus vulgaris ). Journal of Plant Ecophysiological Research. 2018;13(51):67–82.

Dere S, Günes T, Sivaci R. Spectrophotometric determination of chlorophyll-a, b and total carotenoid contents of some algae species using different solvents. Turk J Bot. 1998;22:7–13.

Hossain MI, Khatun A, Talukder MSA, Dewan MMR, Uddin MS. Effect of drought on physiology and yield contributing characters of sunflower. Bangladesh J Agric Res. 2010;35(1):113–24.

Samar Raza MA, Saleem MF, Khan IH, Jamil M, Ijaz M, Khan MA. Evaluating the drought stress tolerance efficiency of wheat (Triticum aestivum L) Cultivars. Russ J  Agri Socio-Econ Sci. 2012;12(12):41–6.

Sairam RK, Srivastava GC. Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress. Plant Sci. 2002;162:897–904.

Sadasivam S, Manickam A. In: Biochemical Methods for Agricultural Sciences. New Delhi: Wiley Eastern Ltd.; 1992. p. 184–5.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1–2):248–54.

Nadernejad N, Ahmadimoghadam A, Hossyinifard J, Poorseyedi S. Study of the rootstock and cultivar effect in PAL activity, production of phenolic and flavonoid compounds on flower, leaf and fruit in Pistachio ( Pistacia vera L). Journal of Plant Biology. 2013;15:95–110.

Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am J Enol Vitic. 1965;16:144–58.

Heath RL, Packer L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem  Biophys. 1968;125(1):189–98.

Moon JH, Terao J. Antioxidant activity of caffeic acid and dihydrocaffeic acid in lard and human lowdensity lipoprotein. J Agric Food Chem. 1998;46:5062–5.

Jia L, Xu W, Li W, Ye N, Liu R, Shi L, Bin Rahman AR, Fan M, Zhang J. Class III peroxidases are activated in proanthocyanidin deficient Arabidopsis thaliana seeds. Ann Bot. 2013;111:839–47.

Asada K, Takahashi M. Production and scavenging of active oxygen in chloroplasts, In: D.J. Kyle, B. Osmond & C.J. Arntzen (Eds), photoinhibition, Elsevier Amsterdam. 1987;227–287.

Jebara S, Jebara M, Limam F, Aouani ME. Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress. J Plant Physiol. 2005;162(8):929–36.

Chandlee JM, Scandalios JG. Analysis of variants affecting the catalase developmental program in maize scutellum. Theor Appl Genet. 1984;69:71–7.

Holy MC. Indole acetic acid oxidase: a dual catalytic enzyme. Plant Phisiology. 1972;50:15–8.

Rezapour S, Kouhinezhad P, Samadi A. The potential ecological risk of soil trace metals following over five decades of agronomical practices in a semi-arid environment. Chem Ecol. 2017;34:68–78.

Zemanová V, Trakal L, Ochecova P, Száková J, Pavlíková D. A model experiment: competitive sorption of Cd, Cu, Pb and Zn by three different soils. Soil and Water Research. 2014;9:97–103.

Kanwal A, Farhan M, Sharif F, Hayyat MU, Shahzad L, Ghafoor GZ. Effect of industrial wastewater on wheat germination, growth, yield, nutrients and bioaccumulation of lead. Sci Rep. 2020; 10:11352–11361. https://doi.org/10.1038/s41598-020-68208-7 .

Urbano VR, Mendonça TG, Bastos RG, Souza CF. Effects of treated wastewater irrigation on soil properties and lettuce yield. Agric Water Manag. 2017;181:108–15.

Ramakrishna W, Yadav R, Li K. Plant growth promoting bacteria in agriculture: two sides of a coin. Appl Soil Ecol. 2019;138:10–8.

Chiappero J, Cappellari Ld. R Sosa Alderete LG, Palermo TB, Banchio E. Plant growth promoting rhizobacteria improve the antioxidant status in Mentha piperita grown under drought stress leading to an enhancement of plant growth and total phenolic content. Industrial Crops and Products. 2019;139:111553.

Rabbani Khairkhah SM, Kazemi F. The effect of the type of cultivation substrate and humic acid on some morphophysiological traits and water consumption of scorpion tail sedum ( Carpobrotus edulis L) on a green roof system. Journal of Plant Environmental Physiology. 2022;17(66):1–23.

Jozay M, Kazemi F, Fotovat A. The performance of cover plant ( Frankenia thymifolia ) and its effect on the physicochemical properties of the recycled substrates in four different seasons in external green wall systems. Journal of Plant Environmental Physiology. Ready to print publishing. 2022.

Kumar V, Eid EM, Al-Bakre DA, Abdallah SM, Širić I, Andabaka Ž, Kumar P, Goala M, Adelodun B, Singh J, Kumari S. Combined use of sewage sludge and plant growth-promoting rhizobia improves germination, biochemical response and yield of ridge gourd ( Luffa acutangula (L) Roxb) under field conditions. Agriculture. 2022;12(2):173.

Kazemi F, Rabbani M, Jozay M. Investigating plant and air-quality performances of an internal greenwall system under hydroponic conditions. Environmental Management. 2020;275:111230.

Marenco R, Antezana-vera S, Nascimento H. Relationship between specific leaf area, leaf thickness, leaf water content and SPAD-502 readings in six Amazonian tree spe¬cies. Photosynthetica. 2009;47:184–90.

Lubbe E, Rodda N, Naidoo S. Effects of greywater irrigation on germination, growth and photosynthetic characteristics in selected African leafy vegetables. Water SA. 2016;42(2):203–12.

Vajpayee P, Sharma SC, Tripathi RC, Rai UN, Yunus M. Bioaccumulation of chromium and toxicity to photosynthetic pigments, nitrate reductase activity and protein content of Nelumbo nucifera Gaertin. Chemosphere. 1999;39:2159–69.

Yadav R, Pandey P. Assessment of air pollution tolerance index (APTI) and anticipated performance index (API) of roadside plants for the development of greenbelt in urban area of bathinda city, punjab, India. Bull Environ Contam Toxicol. 2020;105:906–14.

Jozay M, Kazemi F, Fotovat A. Evaluating the environmental performance of the growing media in a green wall system in a dry climate region. Desert. 2019;20:217–30.

Bertrand A, Gatzke C, Bipfubusa M, Levesque V, Chalifour FP, Claessens A, Rocher S, Tremblay GF, Beauchamp CJ. Physiological and biochemical responses to salt stress of alfalfa populations selected for salinity tolerance and grown in symbiosis with salt-tolerant rhizobium. Agronomy. 2020;10(4):569.

Zilaie MN, Arani AM, Etesami H, Dinarvand M, Dolati A. Halotolerant plant growth-promoting rhizobacteria-mediated alleviation of salinity and dust stress and improvement of forage yield in the desert halophyte seidlitzia rosmarinus. Environ Exp Bot. 2022;201: 104952.

Chieb M, Gachomo EW. The role of plant growth promoting rhizobacteria in plant drought stress responses. BMC Plant Biol. 2023;23(1):407.

Article   PubMed   PubMed Central   Google Scholar  

Anjum SA, Xie XY, Wang L, Saleem MF, Man C, Lei W. Morphological, physiological and biochemical responses of plants to drought stress. Afr J Agric Res. 2011;6:2026–32.

Aeron A, Khare E, Jha CK, Meena VS, Aziz SMA, Islam MT, Kim K, Meena SK, Pattanayak A, Rajashekara H, Dubey RC. Revisiting the plant growth-promoting rhizobacteria: Lessons from the past and objectives for the future. Arch Microbiol. 2020;202(4):665–76.

Vishnupradeep R, Bruno LB, Taj Z, Karthik C, Challabathula D, Kumar A, Freitas H, Rajkumar M. Plant growth promoting bacteria improve growth and phytostabilization potential of Zea mays under chromium and drought stress by altering photosynthetic and antioxidant responses. Environ Technol Innov. 2022;25: 102154.

Woo OG, Kim H, Kim JS, Keum HL, Lee KC, Sul WJ, Lee JH. Bacillus subtilis strain GOT9 confers enhanced tolerance to drought and salt stresses in Arabidopsis thaliana and Brassica campestris. Plant Physiol Biochem. 2020;148:359–67.

Rajkumar M, Bruno LB, Banu R. Alleviation of environmental stress in plants: The role of beneficial Pseudomonas spp. Crit Rev Environ Sci Technol. 2017;47:372–407.

Lin Y, Zhang H, Li P, Jin J, Li Z. The bacterial consortia promote plant growth and secondary metabolite accumulation in Astragalus mongholicus under drought stress. BMC Plant Biol. 2022;22(1):1–14.

Mohsin SM, Hasanuzzaman M, Parvin K, Fujita M. Pretreatment of wheat (Triticum aestivum L) seedlings with 2,4-D improves tolerance to salinity-induced oxidative stress and methylglyoxal toxicity by modulating ion homeostasis, antioxidant defenses, and glyoxalase systems. Plant Physiol Biochem. 2020;152:221–31.

Singh A, Malaviya P. Chromium phytoaccumulation and its impact on growth and photosynthetic pigments of Spirodela polyrrhiza (L) Schleid on exposure to tannery effluent. Environmental Sustainability. 2019;2:157–66.

Pagnani G, Pellegrini M, Galieni A, D’Egidio S, Matteucci F, Ricci A, Stagnari F, Sergi M, Lo Sterzo C, Pisante M, Del Gallo M. Plant growth-promoting rhizobacteria (PGPR) in Cannabis sativa ‘Finola’ cultivation: an alternative fertilization strategy to improve plant growth and quality characteristics. Ind Crops Prod. 2018;123:75–83.

Khamsuk O, Sonjaroon W, Suwanwong S, Jutamanee K, Suksamrarn A. Effects of 24-epibrassinolide and the synthetic brassinosteroid mimic on chili pepper under drought. Acta Physiol Plant. 2018;40:1–12.

Islam MM, Hoque MDA, Okuma E, Banu MNA, Shimoishi Y, Nakamura Y, Murata Y. Exogenous proline and glycine betaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells. J Plant Physiol. 2009;166:1587–97.

Radic S, Babic M, Skobic D, Roje V, Pevalek-Kozlina B. Ecotoxicological effects of aluminum and zinc on growth and antioxidants in Lemna minor L. Ecotoxicol Environ Saf. 2010;73:336–42.

Zandi P, Schnug E. Reactive oxygen species, antioxidant responses and implications from a microbial modulation perspective. Biology (basel). 2022;11:1–30. https://doi.org/10.3390/biology11020155 .

Racchi M. 2013) Antioxidant defenses in plants with attention to prunus and citrus spp. Antioxidants 2:340–369. https://doi.org/10.3390/antiox2040340 .

Pratyusha S. Phenolic compounds in the plant development and defense: an overview. In: Plant stress physiology-perspectives in agriculture. Plant Stress Physiology-Perspectives in Agriculture. 2022;125–185.

Khodamoradi S, Sagharyan M, Samari E, Sharifi M. Changes in phenolic compounds production as a defensive mechanism against hydrogen sulfide pollution in Scrophularia striata. Plant Physiol Biochem. 2022; 177:23–31. https://doi.org/10.1016/j.plaphy.2022.02.013 .

Sharma P, Jha AB, Dubey RS, Pessarakli M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot. 2012;1–26. https://doi.org/10.1155/2012/217037 .

Yildirim E, Ekinci M, Turan M, Agar G, Ors S, Dursun A, Kul R, Balcl T. Impact of cadmium and lead heavy metal stress on plant growth and physiology of rocket (Eruca sativa L). J Agric Nat. 2019; 22:843–850. https://doi.org/10.18016/ksutarimdoga.vi.548626 .

Malik Z, Afzal S, Dawood M, Abbasi GH, Khan MI, Kamran M, Zhran M, Hayat MT, Aslam MN, Rafay M. Exogenous melatonin mitigates chromium toxicity in maize seedlings by modulating antioxidant system and suppresses chromium uptake and oxidative stress. Environ Geochem Health. 2022; 44:1451–1469. https://doi.org/10.1007/s10653-021-00908-z .

Rai GK, Mushtaq M, Bhat BA, Kumar RR, Singh M, Rai PK. Reactive oxygen species: friend or foe. In: Thermotolerance in crop plants. Springer Nature Singapore, Singapore. 2022;129–162.

Jozay M. Combining Green Space and Urban Gardening in External Green Wall under the Influence of Growth-Promoting Bacteria and Types of Recycled Water. Gorgan, Iran: Gorgan University of Agricultural Sciences and Natural Resources; 2024.

Jozay M, Zarei H, Khorasaninejad S, Miri T. Maximising CO2 Sequestration in the City: The Role of Green Walls in Sustainable Urban Development. Pollutants. 2024;4(1):91–116.

Etesami H. Bacterial mediated alleviation of heavy metal stress and decreased accumulation of metals in plant tissues: mechanisms and future prospects. Ecotoxicol Environ Saf. 2018;147:175–91.

Etesami H, Maheshwari DK. Use of plant growth promoting rhizobacteria (PGPR) with multiple plant growth promoting traits in stress agriculture: Action mechanisms and future prospects. Ecotoxicol Environ Saf. 2018;156:225–46.

Glick BR. Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res. 2014;169:30–9.

Jozay M, Zarei H, Khorasaninejad S, Miri T. The environmental function of external green elements in facade architecture. 8th.International Conference on Researches in Science & Engineering & 5th.International Congress on Civil, Architecture and Urbanism in Asia 24 August. 2023, Kasem Bundit University, Bangkok, Thailand.

FAO. New Standards to Curb the Global Spread of Plant Pests and Diseases; UN Food and Agriculture Organization: Rome, Italy. 2019.

Download references

Acknowledgements

Not applicable.

This work was supported by Gorgan University of Agricultural sciences and Natural Resources] for their support and resources in conducting this research under a grant number 480. The facilities and expertise provided by the university have been instrumental in the successful completion of this study.

Author information

Authors and affiliations.

Horticultural Sciences Department, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

Mansoure Jozay, Hossein Zarei & Sarah Khorasaninejad

School of Chemical Engineering, University of Birmingham, Birmingham, UK

You can also search for this author in PubMed   Google Scholar

Contributions

Mansoure Jozay: Contributed to the original writing and formatting of the article. Hossein Zarei: Assisted in manuscript discussion, review and corrections. Sarah Khorasaninejad: Provided input on article formatting. Taghi Miri: Reviewed the manuscript.

Corresponding authors

Correspondence to Hossein Zarei or Taghi Miri .

Ethics declarations

Ethics approval and consent to participate.

This research meets all the ethical guidelines, including adherence to the legal requirements of my country. The authors voluntarily agree to participate in this research study.

Consent for publication

Competing interests.

The authors declare no competing interests.

Additional information

Publisher’s note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Jozay, M., Zarei, H., Khorasaninejad, S. et al. Exploring the impact of plant growth-promoting bacteria in alleviating stress on Aptenia cordifolia subjected to irrigation with recycled water in multifunctional external green walls. BMC Plant Biol 24 , 802 (2024). https://doi.org/10.1186/s12870-024-05511-9

Download citation

Received : 12 February 2024

Accepted : 12 August 2024

Published : 24 August 2024

DOI : https://doi.org/10.1186/s12870-024-05511-9

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Antioxidant activity
• External green wall
• Ornamental plant
• Oxidative stress

BMC Plant Biology

ISSN: 1471-2229

• General enquiries: [email protected]

Mulching techniques impact on soil chemical and biological characteristics affecting physiology of lemon trees

• Research Article
• Open access
• Published: 29 August 2024

Cite this article

You have full access to this open access article

• Rafael Olmos-Ruiz 1 ,
• María Hurtado-Navarro 1 , 2 ,
• Jose Antonio Pascual 2 &
• Micaela Carvajal   ORCID: orcid.org/0000-0001-7321-4956 1  

The lemon cultivation methods and techniques are crucial to ensure maximum productivity in the face of climate change. Mulching with plastic is commonly used in citrus production for saving water, but some side effects need to be investigated. In our study, we investigated different plastic and biological mulching on lemon trees determining growth and physiological parameters in relation to soil chemical and biological composition.

The experiment was divided into four different lines with ten trees per treatment, the effect of mulching with white and black plastic film, dry pruning mulching respect to a non-mulched treatment of lemon tree orchard during a crop season. The impact of these treatments on vegetative growth, stomatal gas exchange and mineral nutrition on plant and soil bacterial communities were evaluated.

Our results showed that the type of mulching significantly influenced in the parameters studied. All mulching treatments increased temperature and soil moisture levels; plastic mulching treatments had significantly higher values in terms of intrinsic water use efficiency; while mulching with dry pruning showed higher soil microbial activity, leading to increased water use efficiency and productivity.

The results showed that different methods of mulching affected the physiology of lemon trees interacting in a complex way to determine their growth. Specifically, mulching using dry pruning improved the exchange of gases in the plant and plant nutrition which was related to the biological soil health.

Explore related subjects

• Environmental Chemistry

Avoid common mistakes on your manuscript.

Introduction

The lemon tree ( Citrus x limon ) (L.) Osbeck is one of the most important commercial plants of the Rutaceae family, with a worldwide distribution. The main producers are India, Mexico, China, Argentina, Brazil, and Spain (Smilanick et al. 2019 ). The primary citrus cultivation areas in Spain are in semi-arid regions of the east and south, namely the Region of Murcia and the Valencian Community, where cultivation methods and techniques are crucial to ensuring maximum productivity (Georgiou and Gregoriou 1999 ). These regions suffer from desertification, a complex and dynamic process of land degradation caused by the interaction between climate change and intensive farming (Prăvălie 2021 ).

Climate change in the Mediterranean area is causing alterations in precipitation, resulting in lower amounts due to an increase in intensity but shorter durations (Nicholson et al. 2018 ). Consequently, water availability for irrigation will be drastically reduced. Low water availability has been reported to reduce leaf gas exchange, leaf expansion and nutrient uptake (Bista et al. 2018 ) leading to reductions in crop growth and yields (Aliche et al. 2018 ).

The availability of water for agriculture will decrease, and the increase in temperatures will imply a greater need for water, along with an extension of the crop irrigation period (Torelló-Sentelles and Franzke 2022 ). Currently, several soil moisture conservation techniques are employed in agriculture, such as mulching, generally considered to prevent water loss, weed suppression, that improve and increase crop yield in agricultural production (Lamont 2005 ; Kader et al. 2017 ). Mulching techniques used materials such as plastic film, paper, straw, mineral-based material or woodchips for ground cover (Lamont 2005 ). However, the most commonly used material is plastic, as it is highly resistant compared to others (Crawford and Quinn 2017 ). Different colours of plastic have various effects on the hydrothermal environment of the soil and crop growth due to their distinct physical properties (Zhang et al. 2023 ). Black plastic mulching, for instance, absorbed more than 90% of solar radiation, warming the soil (Strik et al. 2006 ), while white plastic mulching reflected a high proportion of solar radiation, decreasing soil temperature and increased canopy light intensity and air temperature (Andreotti et al. 2010 ). On the other hand, organic mulching, made of any bulk material placed on the soil surface, has been also used for the purpose to retain and minimize water loss, and they also improved soil physical characteristics, enhancing canopy microclimate (Iqbal et al. 2020 ). However, little is known about the effect of mulching on soil microbiological and mineral availability changes.

Soil microorganisms drives many aspects of biogeochemical carbon and nutrient cycling, water holding capacity, water purification, pathogen control and climate change mitigation, key aspects of plant development (Dangi 2014 ). Most soil processes are mediated by the biodiversity of soil microorganisms in direct relation to the physic-chemical properties of their environment (Lemanceau et al. 2015 ).

Soil bacteria play a crucial role in cultivated soils and are related to crop production (Davison 1988 ), since the interactions between plants and bacteria in the rhizosphere, the area surrounding plant roots, significantly influence plant health and soil fertility. Rhizobacteria also participate in the process of mineral nutrient solubilization (Hayat et al. 2010 ), contributing to increased resistance to environmental stress, the stability of soil aggregates and the enhancement of soil structure and organic matter content. Rhizobacteria are effective at retaining more organic nitrogen and various nutrients within the plant-soil system which, in turn, reduces the dependency on nitrogen and phosphorus fertilizers and promotes the release of these essential nutrients (Richardson 2001 ).

In our study, we investigated the effects of different mulches on drip irrigated lemon trees of the variety 'Fino' ( Citrus x limon ) with fertirrigation and grown in organic modality. We compared mulch with white/black plastic film and mulch with dry pruning crushed, with no mulching as control. The objective of the research was to determine their effects on the soil temperature, water content and nutrient availability in relation to leaf gas exchange and mineral concentration in lemon leaves on growth. Also, the soil bacteria community was determined and its relationship with the other parameters studied.

Materials and Methods

Location and plant growth conditions.

The experimental farm “Cañada Honda” is located near the village of Librilla (Region of Murcia, Spain). It is a semi-arid area (Mediterranean climate), with an average annual rainfall of 300–350 l/m 2 and average annual temperatures of 18.8 ºC (Elvira-Rendueles et al. 2019 ).

Seven-year-old lemon trees of the variety "Fino" ( Citrus x limon ) with drip irrigation were used. This variety is characterized by a large harvest from November to March. The lemon trees are grown with organic methods; being certified 100% organic in 2021. The soil showed a pH of 8.66 ± 0.02 and an electrical conductivity (EC) of 139.33 ± 23.26 µS cm −1 . Each tree was irrigated with 2 drippers twice a week, for 90–120 min and the water flow rate was set at 4 l/h per dripper. The crop was organic with manure and organic fertilization as reported previously (Olmos et al. 2024 ).

The experiment was divided into four different lines with ten trees per treatment, with a 3 × 5 crop frame, comparing soil cover with white/black plastic film mulch, mulching with crushed dry pruning and an outdoor control over a period of six months. Samples were collected between March-July 2023. To make the sampling representative of the crop, samples were randomly taken from five different trees, avoiding crop edge.

The white and black plastic (130 g m −2 ) were composed of semi-impermeable polypropylene geotextile; while the dry pruning was obtained from the pruning of lemon trees. Dry pruning was used after open air dried, crushed with a tract, and stored for 3 months in the open air before being used as mulch.

Plant and Physiological analysis

Vegetative growth.

Ten measurements of the new leaf area were made with an interval of 15 days between each measurement from March to July, selected as points represented on the graph in the months of March, May and July. Three leaves on each plant from each of the treatments were previously selected and marked. This procedure was carried out in situ by drawing the outline of the leaf on a sheet of paper and subsequently using the ImageJ program (Tsung-Luo 2017 ) to calculate the area, and then obtain the relative growth rate with the following equation: RGR = (Ln A2-Ln A1) / (t1-t2), (cm 2 cm −2  day −1 ). The measurement of lemon fruit growth was calculated by measuring the length and diameter with a caliper.

Stomata content

Stomatal printing on the new leaves of the tree was carried out to count how many stomata were open or closed on the leaf per unit area. To do this, the surface of the underside of the leaf was impress on a slide with adhesive tape (composed of cellulose acetate), digested with a drop of acetone. In this way, the entire surface of the leaf was impressed on the adhesive tape. Subsequently, the impressions were observed under an optical microscope (OLYMPUS U-CMAD3, Olympus Corporation, Tokyo, Japan) and a counted with an ImageJ analysis program (Jinn 2017 ). Five plates of new leaves were obtained for each of five trees per treatment.

Photosynthetic parameters

Photosynthetic capacity (An, μmol CO 2 m −2  s −1 ), stomatal conductance (Gs, mol H 2 O m −2  s −1 ), internal content of CO 2 (Ci, μmol mol⁻ 1 ) were measured in fully-expanded new leaves using a TPS-2 Portable Photosynthesis System (PP Systems, Inc., Amesbury, MA, USA). Intrinsic water use efficiency (WUE i μmol CO 2 mol −1 H 2 O) was calculated by dividing the net photosynthetic rate and the stomatal conductance. Five new leaves were measured for each of the five trees in each treatment.

Leaves macro and micro mineral content

The macro and micro mineral contents were analysed using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) on a Thermo ICAP 6500 Duo instrument (Thermo Fisher Scientific, Waltham, MA, USA). Leaves were collected, dried, and ground into a fine powder. A total of 200 mg of each sample was added to a 25 mL tube along with a mixture of 4 mL of 68% purity HNO 3 and 1 mL of 33% purity H 2 O 2 for digestion. Additionally, a Teflon reactor contained 300 mL of high-purity de-ionized water, 30 mL of 33% purity H 2 O 2 , and 2 mL of 98% purity H 2 SO 4 was added. The microwave heating digestion program consisted of three steps: starting at 20 ºC and 40 bar, increasing by 10 bar per minute for30 min until reaching 220 ºC, and maintaining the temperature at 220 ºC for 20 min. After cooling, the mineralized samples were transferred to 10 mL (for micro minerals) and 25 mL (for macro minerals) double gauge tubes, and the volume was adjusted using high-purity de-ionized water. Calibration standards were prepared using a multi-mineral standard solution supplied by SCP Science (Quebec, Canada).

Soil analysis

Soil macro and micro mineral content

It was obtained by using the same above method that the one for macro and micro mineral content of leaves.

Soil available Carbon, Nitrogen and Phosphorus

Available carbon and nitrogen in the soil samples was determined through elemental analyzer (C/N Flash EA 112 Series- Leco Truspec). Eight grams of soil samples previously sieved through 2 mm mesh were extracted with 100 mL of distilled water for 1 h in an automatic shaker. After that it was centrifuged at 3500 rpm for 10 min and the supernatant was filtered through Whatman No. 2 filter paper to obtain a clear filtrate that it was used to do the determination of available carbon and nitrogen.

Available phosphorus in the soil samples was determined using the Olsen method. Two grams of sieved air-dried soils was extracted with 100 mL of 0.5 M sodium bicarbonate (NaHCO₃) solution at pH 8.5. The mixture was shaken for 30 min, followed by filtration through Whatman No. 2 filter paper. The phosphorus concentration in the clear filtrate was quantified colorimetrically using the molybdenum blue method, with absorbance measured at 880 nm. Calibration was performed using standard phosphorus solutions to ensure accurate quantification.

pH and apparent densit

Soil pH was measured in a 1:10 (w/v) water-soluble extract, after soil shaking for 60 min, using a pH meter (Crison pHmeter, micropH 2001). The dry apparent density was determined from the Spanish standard UNE-EN 13041–2012, by means of gravimetry and particle density was measured by calcination at 540ºC.

Soil temperature and moisture

The soil temperature was measured using a precision thermometer (Precision Plus, ETI Ltd, Worthing, West Sussex, United Kingdom) obtaining six measurements (three being in the superficial part of the soil under the tarp and the other three at 15 cm from depth) from each of the selected sampling point of each of the five selected trees in each treatment, at 15 day-intervals between each measurement from March to July.

To determine the moisture content, a determined quantity of soil was weighted and placed in an oven with temperature range of 110 ± 5 ºC for about 24 h. After that, the difference in the wet mass and dry mass of the soil was the water content of the soil. It was determined by triplicate per treatment.

Soil biological parameters

For this purpose, dehydrogenase activity and Bacterial community was conducted exclusively at the sampling time of July, coinciding with the culmination of the growing season and the harvest of the lemon fruits, a period that marks the end of the primary physiological activities of the trees. Additionally, July typically experiences the highest temperatures, which can induce significant stress on both soil biological parameters and tree physiology. These conditions were expected to provide the most informative snapshot of the microbial dynamics under peak environmental and physiological stress conditions.

Dehydrogenase activity

Dehydrogenase activity in the soil was determined using a colorimetric procedure according to (von Mersi and Schinner 1991 ). Briefly, 2 mL of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT, 0.015 M) were added to 2 g of soil and then homogenized and incubated at 25 ºC for 4 h in the dark. Subsequently, 8 mL of acetone were added to all samples and put them on an orbital shaker (250 rpm) for 1 h in the dark. Iodonitrotetrazolium formazan (INTF) was determined in the centrifuged extracts by measurement at 485 nm spectrophotometrically. The dehydrogenase activity was expressed as nmol INTF g −1  h −1 .

Bacteria community

This parameter comprises the soil DNA extraction, amplification of the representative bacterial sequences and the further deep analysis of them by taxonomic characterization and grouping them by using through alpha and beta diversity and Principal Coordinates Analysis (PCoA) analysis. Their relationship with the rest of physical, physico-chemical and chemical soil parameters were analyzed by using the Distance-based redundancy analysis (db-RDA).

DNA was extracted from soil samples (500 mg) using the DNeasy Power Soil Pro Kit (Qiagen) following the manufacture’s protocol. The quantity and quality of the DNA extracts were quantified using a Nano Drop 2000 fluorospectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

As stablished in the Molecular Biology Service at the University of Murcia, purified DNA was used as the template for generating a 16S rRNA gene library. The oligonucleotide primers used for this experiment were 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′ and 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3′, where the underlined regions are the Illumina adapter overhang nucleotide sequences, while the non-underline sequences are locus-specific sequences targeting conserved regions within the V3 and V4 domains of prokaryotic 16S rRNA genes (Klindworth et al. 2013 ). The amplified fragments were quantified with the Qubit dsDNA HS Assay Kit (Invitrogen, Merelbeke, Belgium) on a Qubit 2.0 Fluorometer prior to sequencing. Paired-end sequencing of the library was performed on an Illumina MiSeq sequencer (San Diego, CA, USA) using the MiSeq Reagent Kit (v3) with the longest read length set to 2 × 300 base pairs (bp). Library qualities were estimated using the Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent).

The 16S-V4 sequencing library was first reviewed with FastQC for overall quality assessment, and the libraries were processed in R package DADA2 (v.1.8.0) (Callahan et al. 2016 ). Reads were quality trimmed with the “filterAndTrim” function with “maxEE (2,5)”. Reads below 165 bp after the trimming process were discarded. Errors learned from all samples were used for sample inference with the dada2 algorithm by employing an evaluation of 1E8 bases. Forward and reverse reads are merged below to generate a table of sequences, and the resulting Amplicon Sequence Variants (ASVs) were subjected to de novo chimera detection, using DADA2 and any artifacts were removed.

For bacteria taxonomic assignment, ASVs were queried against the SILVA database v.132 using IDTAXA (Murali et al. 2018 ) implemented in the R package DECIPHER (Wright 2016 ) with a threshold 40. Sequences identified as non-bacterial were discarded. Similarly, to numerous recently published studies, we chose to forego rarefaction of our samples as it increases uncertainty in relative abundances (McMurdie and Holmes 2013 ).

The abundance matrix, the taxonomy assignment and the metadata obtained from samples were merged and imported with the phyloseq v3.12 package (McMurdie and Holmes 2013 ) to produce the alpha and beta bacterial diversity. The "prune_taxa" function was used to keep only subsystems with absolute abundance > 0.01%. Counts were normalized in each sample using the median sequencing depth, and phylum and class level plots were created using the ggplot2 and ggpubr packages. Alpha diversity was calculated in R using the phyloseq package, and several alpha indices were generated, such as Shannon and Simpson, using the function “plot_richness”. Beta diversity was calculated using weighted and unweighted Unifrac distances (Lozupone and Knight 2005 ). To test for significant differences in community composition among different seasons, permutational multivariate analysis of variance using distance matrices (PERMANOVA) was conducted using the Adonis function in the R package vegan with 999 permutations, and the results were visualized by Principal Coordinates Analysis (PCoA).

UpSet plots were created using the upsetR package version 1.4.0. This was done by transforming the data frame of average counts for each soil sample into a data frame that exclusively contained 0 and 1 values. Subsequently, the data was arranged based on the frequency of intersection size.

Finally, db-RDA analysis was conducted to identify soil physicochemical properties with a significant impact on soil bacterial communities across different factors using the dbrda function of the R vegan package vegan v2.6–1. Parameters that significantly explained variation in the bacterial community were identified using forward selection (the ordistep function of the vegan package) with p value < 0.05.

Statistical analysis

Statistical analyses were performed using the SPSS 29.0.0.0 (241). Significant differences between the values from all parameters were determined at p  ≤ 0.05, according to a one-way ANOVA followed by Duncan’s test. For the studies of alpha diversity, also Student test was used and for the beta diversity, also Adonis was used. All the results are presented as the mean ± SE.

The Relative Growth Rate (RGR) of leaves in March did not show significant differences between treatments (Fig.  1 a), but in May, the RGR of black plastic mulching showed a significant increase respected to the dry pruning and control, but not of white plastic. In any time, dry pruning mulching did not show significant differences respect to the control. However, in July, RGR for both plastic treatments black and also white showed a significant decrease compared with control and dry pruning treatments.

a Leaf Relative Growth Rate (RGR) (cm 2 cm −2  day −1 ) in the different treatments (control, black plastic, white plastic and dry pruning) in March, May and July, and ( b ) number of fruits per tree in July. The statistics in RGR were performed at each time, for study the mulching (represented with lowercase letters) and for study the time on a specific treatment (represented with uppercase letters). Significant differences were determined between the values of the treatment parameters at p  ≤ 0.05, according to a one-way ANOVA followed by Duncan's test

The number of fruits per tree harvested in July (Fig.  1 b ) showed a significant increase in dry pruning and black plastic compared with the white plastic and control treatments, which exhibited similar lower values. Furthermore, when the fruit relative growth was measured in July, no differences between treatment were found (data not shown).

Stomata content and WUE i

The total stomata per mm 2 , percentage of open-closed stomata and leaf humidity were measured. The number of stomata (Fig.  2 a) did not show significant differences in March; but in May, a significant increase was observed in the dry pruning treatment compared with the black plastic treatment, obtaining no significant differences between black plastic, white plastic treatment and control. In July, the number of stomata was significantly increased in the treatments with black and white plastic compared with control, but no significant differences were observed with dry pruning.

Results of ( a ) total stomata mm −2 , b % leaf humidity, c % open and % closed stomata in the different treatments (control, black plastic, white plastic and dry pruning) in March, May and July. The statistics were performed at each time, for study the mulching (represented with lowercase letters) and at each treatment, for study the time (represented with uppercase letters); Significant differences were determined between the values of the treatment parameters at p  ≤ 0.05, according to a one-way ANOVA followed by Duncan's test

Regarding to leaf humidity (Fig.  2 b), no significant differences in the months of March and May were observed. However, some significant differences were observed among all treatments in July. In general, white plastic mulching exhibited the highest value of % leaf moisture while the black plastic treatment displayed the lowest value.

Respected to the percentage of open stomata respect to the total (Fig.  2 c) different behaviour was observed attending to the sampling time. In March, it showed a significant decrease in black plastic compared with the other treatments, including control, which did not show differences between themselves, In May, itincreased in dry pruning compared to the others treatments, which did not show differences among them. In July, dry pruning and white plastic mulching showed a significant increase on this parameter compared with control and black plastic treatments, which did not show differences between them. Finally, regarding the closed stomata as calculated from the same microscope frame, the values were the opposite than open stomata (Fig.  2 c).

Leaf gas exchange parameters

The photosynthesis (An), stomatal conductance (Gs), CO 2 internal concentration (Ci) were measured and intrinsic water use efficiency (WUE i ) was calculated. In March, the An (Fig.  3 a) showed a significant increase in the dry pruning mulching respect to the other three treatments which did not show differences between black plastic and control. In May, all treatments showed a significant increase respect to March, and a different trend was observed where the higher values were obtained in black and white plastic compared with control and dry pruning treatments. In July, a significant decrease was observed in control compared with dry pruning treatment, obtaining no significant differences between white and black plastic treatments.

Results of ( a ) net photosynthetic rate An (μmol CO 2 m −2  s −1 ), b stomata conductance Gs (mol m −2  s −1 ), c internal CO 2 content Ci (μmol mol⁻ 1 ) and ( d ) intrinsic water use efficiency (WUE i ) (μmol CO 2 mol −1 H 2 O) in the different treatments (control, black plastic, white plastic and dry pruning) in March, May and July. The statistics were performed at each time, for study the mulching (represented with lowercase letters) and at each treatment, for study the time (represented with uppercase letters); Significant differences were determined between the values of the treatment parameters at p  ≤ 0.05, according to a one-way ANOVA followed by Duncan's test

Regarding Gs, it was observed a significant increase in March respect to the other sampling times, showing control and white plastic treatment, similar An significant lower values. In May, Gs did not show significant differences between all treatments. In July, a significant increase was observed in dry pruning compared with the rest. In March, the Ci (Fig.  3 c) showed a significant decrease in the dry pruning, white and black plastic treatments, compared to control. In May, control maintained the higher value with significant decreases with dry pruning and while plastic (being this later one the lowest value). In July, only significant decreases were observed in white plastic compared with the rest. Finally, the WUE i in March and May, did not show significant values, while in July a significant increase was observed in black plastic compared to control (Fig.  3 d).

Soil pH and apparent density

These two parameters showed expected values from the Mediterranean soil (Rashid and Ryan 2004 ), for the case of the pH ranged from 7.69 to 7.7 in the soil covered with plastic that it was a slightly higher than control with 7.87 and the soil with dry pruning mulching was 8.55 but in any of the cases were significantly different. The apparent density was a usual value for a Mediterranean and the treatments did not produce any significantly change ranged from 1.16 to 1.26 g cm 3 .

Temperature and moisture of soil

Temperature and soil moisture were determined (Table  1 ). The temperatures measured in the different distances from the mulching (under 15 cm into soil, on top of the mulching and 1 m over the mulching) were increasing from March to July, being similar in March and May. All the temperatures were higher in black plastic compared with the rest of mulching treatments. Furthermore, comparing the other treatments, in the month of March, the temperature on soil surface or mulching was similar between white plastic, dry pruning mulching and control; but in May, white plastic showed significantly higher temperatures than control and dry pruning; and in July, the white plastic and dry pruning were significantly lower than control. The temperature at 15 cm into soil was stable at March and May respecting to all treatments. However, in July, white plastic and dry pruning, temperature was significantly lower than control and black plastic. Temperature at 1 m above soil or mulching surface was similar in March and May for control, white and dry pruning. In July, a gradual increase was observed from black plastic, dry pruning, white plastic and control, being the highest significant value for the black plastic treatment, followed by white plastic, dry pruning and control treatments.

The soil moisture, in March, showed no significant differences between treatments, while in May and July the mulching treatments showed significantly higher soil moisture than control. In July, a significant decrease was observed compared to the other months for the four treatments.

Soil and leaves mineral content

In Table  2 , the significant soil macronutrients and micronutrients are shown Values are shown in the same units than in leaves for easier comparation. In March, Ca showed a significant decrease when dry pruning mulching was compared to control, while no differences were found for both plastic treatments. For white plastic, potassium exhibited a decrease in May and an increase in July. Magnesium showed an increase with dry pruning in March and July and with black plastic in May. Phosphorus showed only an increase with all treatments in July. Total N level was significantly increased across all treatments in July, being also remarkable the significant higher N in white plastic mulching in March. Total organic carbon (TOC) showed significantly higher values in the black plastic and dry pruning treatments compared to control for the three sampling times. However, the white plastic treatment did not exhibit significant differences. According to micronutrients, they showed slight alterations in certain times with any of the treatments as B that showed an increase in May and July with dry pruning, Cu increased in March with white plastic and in May with black plastic. Also, Fe exhibited only an increased in July with dry pruning, Mn showed an increase in May and July with black plastic and Zn increased in March with white plastic and in May and July with black plastic. Macronutrients and micronutrients not listed show no significant differences.

In Table  3 , the significant leaf macronutrients and micronutrients are shown. In May, Ca showed a significant increase with dry pruning compared to the rest of mulching treatments. July displayed a significant increase in black plastic, a decrease in white plastic, both compared to control and dry pruning treatments. Potassium exhibited a significant increase in May with dry pruning compared to the control, black and white plastic treatments, and a significant increase in July with white plastic compared to the control, black and dry pruning treatments, along with a decrease in black plastic compared to the same treatments. In March, Mg showed a significant increase with dry pruning compared to black and white plastic treatments; while only in May and July showed this increase also respect to whole treatments. Phosphorus showed a significant increase with dry pruning in May, and white plastic, and dry pruning in July. Sulphur had a significant increase in May with dry pruning and in July with black plastic. Total N had a significant increase in May with black plastic compared to white plastic, and no significant differences with control and dry pruning. In July, a significant increase was observed with white plastic compared to control, black plastic, and dry pruning, with similar values for control, black plastic, and dry pruning. Carbon did not show significant differences with any of the treatments.

In terms of micronutrients, B showed a significant increase in March with dry pruning, and in July, a significant increase was observed in black plastic. Cupper had a significant increase in May with dry pruning and in July with white plastic, while Fe had only in July a significant increase with black plastic and Mn in May with dry pruning and in July, in black plastic white plastic, and dry pruning. Zinc showed a significant increase in May with dry pruning and in July with black plastic Macronutrients and micronutrients not listed show no significant differences.

Dehydrogenase activity was measured once the lemon fruit were harvested at the last sampling time in July (Fig.  4 ). This enzymatic activity of the different mulching treatments showed a significant increase respect to the control. The dry pruning treatment showed a significant difference respect to the black plastic but not to the white one, while no significant differences were observed between both white and black plastic.

Results of dehydrogenase activity (nmol INTF g −1 dry soil h −1 ) in the different treatments (control, black plastic, white plastic and dry pruning) in July. The statistics were performed individually for each parameter; significant differences between the values from all parameters were determined at p  ≤ 0.05, according to a one-way ANOVA followed by Duncan’s test

In Table  4 , the water soluble organic carbon and nitrogen and the available phosphorus in July are shown. Soil organic carbon (SOC) showed a significant increase when dry pruning mulching was compared to control, white and black plastic treatments. Available nitrogen (TN) did not show significant differences among the treatments. Available P showed a significant increase when white plastic, dry pruning and black plastic mulching were compared to control. Apparent density and pH did not show significant differences.

Effect of different mulches on bacterial phyla or Bacterial community composition of different mulching treatments

In the same way than the above soil biological parameter, the bacterial community was analysed from the soil sampling in July (Fig.  5 ). The predominant identified bacterial phyla, classes and orders are presented in Fig.  5 a, b and c respectively. The prevailing phylum under the different covers was Proteobacteria, accounting for an average of 39%, succeeded by Actinobacteria (26%) and Choloroflexi (now named Chloroflexota, 11%). At the class level, Alphaproteobacteria exhibited the highest relative abundance (27.2% in average), encompassing detected orders such as Rhizobiales, Rhodospirillaes (also known as Azospirillales), Rhodobacterales and Sphingomonadales. Actinobacteria (12.62%) constituted the second most abundant class in this bacterial community, featuring identified orders like Micrococcales, Gaiellales and Solirubrobacterales. Lastly, Gammaproteobacteria (12.85%) stood as the third most abundant class, with Pseudomonadales being one of the identified order.

Relative abundance (%) of bacterial community at the phylum ( a ), class ( b ) and order ( c ) in soil samples of the treatments (control, black plastic, white plastic and dry pruning) in July

Alpha diversity indices, including richness and Shannon, are presented in Fig.  6 a as regards the first two indices, the dry pruning treatment showed significant higher values compared to the control group, while no significant changes were observed between the white and black plastic treatments and the control.

Alpha diversity indices ( a ) and beta diversity PCoA ( b ) based on weighted UniFrac distances of bacterial community found in the soil samples of the different treatments (control, black plastic, white plastic and dry pruning) in July. For the studies of alpha diversity, also Student test was used and for the beta diversity, also Adonis was used

In terms of beta diversity, bacterial PCoA based on weighted (Fig.  6 b) UniFrac distances showed that bacterial community structure was altered depending on the applied mulching. As regards the weighted UniFrac data, the two principal PCoA coordinates explained 46.6% of the variations (32.2% and 14.4%, respectively) and exhibited significant changes between the different mulching treatments and the control group (PERMANOVA, p  ≤ 0.05).

Bacterial sequences were assigned to 2483 ASVs (Fig.  7 ), with 1123 of the ASVs, corresponding to 45.23% of the total, shared amongst all treatments. The two groups covered by plastic and the dry pruning treatment shared 308 ASVs with each other, which represents 12.40% of the total. The control group harboured the lowest proportion of unique ASVs (2), corresponding to 0.08%, while the other three groups showed the same number of unique ASVs (8), that corresponded to 0.32% of the total of bacterial sequences.

UpSet diagram illustrating the distribution of ASVs in the bacterial community found in the soil samples of the treatments (control, black plastic, white plastic and dry pruning) in July. The total size of each treatment is represented on the left barplot

Redundancy analysis (RDA) was performed to determine the relationship between physicochemical properties of the soil of the different treatments and the bacterial community (Fig.  8 ). The first and second axis (RDA1 and RDA2) explained 72% of the variation in the bacterial community composition in the soil analysed. While the white plastic treatment did not show a correlation with any examined parameter, the black plastic and dry pruning treatments showed correlation with subsoil temperature and N content, the soil control only showed correlation with soil water content.

Redundancy analysis (RDA) of bacterial community and physicochemical properties (arrows) of the different treatments (control, black plastic, white plastic and dry pruning)

The environment as temperature, radiation and humidity influenced the physiological processes of the roots, such as the absorption of water and mineral nutrients (Dodd et al. 2000 ). In our experiments, in March, none of the mulching treatments affected significantly vegetative growth of the trees because at early spring the trees did not start its vegetative development in accordance with low temperatures and short photoperiods (Kozlowski and Pallardy 2002 ). In the following sampling corresponding to May, the ambient temperature increased in the Mediterranean area and favoured vegetative growth (Camarero et al. 2021 ), which showed significant differences according to the type of mulch used. In this time, the black and white plastic treatment showed the highest significant values of vegetative growth probably related to the higher water retention of soil covered that it would avoid evaporation (Sinmidele et al. 2015 ). Accordingly, a mulching with grass clippings, was reported to present lower soil moisture than plastic mulching (Kader et al. 2017   2013 ). In our experiment, in July, there was a general growth decrease, probably due to the stress caused by the high temperatures recorded, which negatively affect the vegetative growth of lemon trees (Pérez-Pérez et al. 2009 ). According to the treatments, in July, the opposite results were obtained compared to May, showing control and dry pruning higher vegetative growth values than black and white mulching. This fact was not related to the number of fruits per tree where the higher number was obtained in black and dry pruning mulching. This, revealed the complexity of the citrus growth that not only were affected by ambient temperatures, but also on soil factors as we will be discuss in this section.

Leaf humidity of the different studied treatments (Fig.  2 ) increased in May and July compared to March which could indicate that seasonal strategy for saving water in leaves affected by temperature and light (Ribeiro and Machado 2007 ). The reduction of stomata has been reported as an adaptive mechanism used by plants to reduce water loss (Karimi et al. 2015 ), but the number of open and closed stomata indicated a short term regulation. In this way, the changes in the different seasons were not high, but appeared with treatments in May and July. Hence, in the month of July, black and white plastic mulching had higher number of stomata than control and dry pruning, but this was not corresponding with % leaf humidity or % stomata closed. As higher leaf humidity and higher number of stomata open appeared in white plastic and dry pruning mulching, should correlate with higher water transport through leaves indicating the need of transport either water and/or nutrients. In other way, the WUE i has been associated to crop productivity in agricultural ecosystems (Ono et al. 2013 ). As the decreased observed from March to July in our results, this parameter could be related to the need of fruit production.

It has been reported that plant photosynthesis is regulated by several climate factors, such as temperature, solar radiation and water availability (Nemani et al. 2003 ). In our experiments, a considerable increase in May and a decrease in the July was observed, but in a different relationship between treatments; pointing to soil influencing parameter rather than to only photosynthetic seasonality due to temperature variation (Garonna et al. 2018 ). The most significant changes should have appeared in the month of May and July, where the temperature was moderate and excessively high respectively, but only slight changes occurred with no relation to growth. Furthermore, there were only small increases of stomatal conductance from March to May in control, black and white plastic. The observed changes during season bring the assumption that this parameter was not dependent on temperature or light intensity as it was previously described (Allakhverdiev et al. 2008 ). Also, the fact that the values were higher in March and July in dry pruning mulching, bring the possibility that changes were occurred by other non-studied mechanisms. Under these conditions, plants altered leaves internal concentration of CO 2 during different seasons at similar rate than Gs and An. They showed decreases in white plastic compared to black plastic that seemed to be not related to the rest of the parameter but that could show a higher water diffusion in through membranes (Martinez et al. 2011 ) as it occurred in white plastic trees.

Temperature and humidity of soil

The soil temperature recorded in both the upper and lower soil layers, as it was expected, increased in July respect to May and March (Costa et al. 2023 ). It is a factor that depended on the climatic conditions during the season and it was also affected to the physical soil properties, it has been reported that higher amplitude of the daily temperature has been related to the sand and clay composition in the soil ( 2012 ; Sremac et al. 2021 ). Accordingly, as our soils had similar composition, the differences were only related to mulching treatment. Therefore, the black plastic showed the highest soil temperature as it absorbs more solar radiation, which has been reported to turn into plant growth, while the white plastic mulches reflected a high proportion of solar radiation (Andreotti et al. 2010 ), which decreased soil temperature as significantly lower values growth values obtained in May and July compared to black plastic.

The highest significant moisture records were obtained in the month of May in all the treatments studied due to the accumulated rainfall in that period (100 L m −2 ), while the lowest moisture data were recorded in the month of July due to a decrease of rainfall (2 L m −2 ). However, soil moisture was significantly higher in the white plastic and dry pruning treatments than in the black plastic and control treatments. Water availability for plants is closely related to the efficiency of photosynthesis (Cheng et al. 2011 ) as it regulated stomatal conductance, affecting both the entry of CO 2 into the mesophyll and the release of H 2 O by leaf transpiration (Ribeiro and Machado 2007 ). As soil water content has been positively and directly correlated with photosynthetic rate and growth (Lamptey et al. 2020 ), we attempted to relate these two parameters in our experiments but we found no direct relation. Therefore, other parameters should be influencing gas such as exchange parameters. Either, on the contrary as reported, there was a lack of relation between WUE i and soil moisture (Pandey et al. 2015 ) and temperature (Robinson et al. 2020 ). It has been also reported that mulching is one of the water management practices for increasing water use efficiency in crops located in semi-arid regions (Yaghi et al. 2013 ). However, we only found increases in black plastic mulching compared to control because higher temperatures provide more kinetic energy to water molecules for evaporating (Zeppetello et al. 2019 ).

The results obtained in the mineral concentration of the lower soil layer of the different treatments studied indicated that in May, the greatest significant increase in the concentrations of macronutrients and micronutrients of all the treatments studied appeared. This could be due to the high values of both temperature and humidity (Kader et al. 2017 ) since it has been related to the greater decomposition of added organic matter fertilization and consequently the greater the release of nutrients available to plants (Lenka et al. 2019 ). However, in July, the opposite occurred, with significantly lower concentrations. This tendency could be due to the increase in temperature and decrease soil humidity. It should be noted that in terms of differentiation between treatments, there is a significant increase of the macronutrients N, Ca and Mg; and of the micronutrients B, Cu and Fe for the dry pruning treatment, followed by the white and black plastic treatments, with respect to the control treatment, in the month of July. This fact could be due to the occurred pruning residue mineralization (Blagodatskaya and Kuzyakov 2008 ), favouring nutrient release (Manzoni et al. 2008 ) as a key component of nutrient availability and plant productivity (Kaspari et al. 2008 ). The fact that also white and black mulching also showed higher reported mineral content than control fits with the high values of both temperature and humidity that could be related to greater decomposition of added organic matter fertilization as alluded previously (Lenka et al. 2019 ).

Regarding the mineral content of leaves (Table  4 ), first of all, the nutrient levels in plant tissues of the plant vary over time, depending on the growth stage of the plant and the part analysed (Hu et al. 2023 ). Citrus trees change mineral nutrient absorption during growth and development processes (Bui et al. 2020 ). Therefore, increasing root zone temperature, affects water and nutrient uptake by accelerating metabolic activity (Lee et al. 2005 ) and promoting increased root volume and absorption area (Hussain and Maqsood 2011 ). However, these parameters were not related to the treatments that favoured the high leaf concentration of nutrients. In fact, higher N, Ca, Fe and Mn were found in leaves of trees grown under black plastic and dry pruning mulching which were the treatments that provide higher number of fruit.

Soil biological activity

The determination of soil microbiology was done in July once lemon crop season occurred and the decrease in water availability and the high sun irradiation were mostly affecting growth and photosynthetic processes (Flexas et al. 2012 ). Also, soluble organic carbon (SOC), available nitrogen (TN) available phophorus (P Olsen) apparent density and pH were also determined in July for correlation. In this way, firstly, soil dehydrogenase activity was determined as an indicator of the microbial redox system (Yang et al. 2003 ). Dehydrogenase activity of the mulching treatments was significantly higher to the control, being the dry pruning group which obtained the higher values. This may be attributed to the input of C and N resulting from the decomposition of crop residues (as related to SOC results) added to the soil as mulch, probably due to the whole season of pruning residues in contact to the soil that it should be increased due to temperature, humidity and microbial activity (Boyero et al. 2011 ). In the treatment involving white and black plastic mulch, the significant increase observed compared to the control, but lower compared to the dry pruning mulching, could be attributed to the documented rise in temperature and humidity, that it would also increase soil mineralization from the natural organic matter, as previously reported (Luo et al. 2019 ). Accordingly, the composition of the soil microbial community has been reported to be temperature dependent (Creamer et al. 2015 ), and related to mineralisation of soil organic matter (Huygens et al. 2011 ), which could be related to the high availability reported in July of our study. Therefore, the fact that higher values of Gs and An were observed in trees grown under dry pruning could be related to this increase in mineralization (Yessoufou et al. 2023 ; Geng et al. 2017 ).

According to Lauber et al. ( 2008 ), soil microbial community composition is significantly correlated with changes in soil chemical properties. In this study, the C and N soil content play important roles in changes in the bacterial community structure. The dominant taxonomic groups identified in the soil assayed were Proteobacteria, Actinobacteria, Chloroflexi and Acidobacteria, all reported by several other studies related to agricultural ecosystems (Smit et al. 2001 ; Valinsky et al. 2002 ). Proteobacteria phylum is one of the most abundant in soil ecosystems, which members occupied the highest richness across all soil samples. The Alphaproteobacteria, comprising orders Rhizobiales, Rhodospirillales, Rhodobacterales and Sphingomonadales, play crucial roles in degradation of inorganic compounds and nitrogen fixation (Fallah et al. 2021 ) and stimulating plant growth, underscoring their significance from an agricultural perspective (Ceja-Navarro et al. 2010 ). Actinobacteria is a diverse phylum of Gram-positive bacteria which some of them participate in carbon cycling and have been linked with soil organic matter production (Trinchera et al. 2022 ). On the other hand, Acidobacteria is a phylum of Gram-negative bacteria which members are physiologically diverse and ubiquitous. They are particularly abundant in soil habitats and can degrade complex and recalcitrant carbon sources (Fierer et al. 2003 ). Less copious phyla such as the Planctomycetota phylum comprises widely distributed bacteria, with many species capable of anaerobic ammonium oxidation (Kallscheuer and Jogler 2021 ), which is why they play an important role in global nitrogen and carbon cycles. The occurrence of members within the Cyanobacteria phylum was unexpected, given their aquatic inclination. Nevertheless, recent research, exemplified in the study led by Mhete et al. ( 2020 ), has revealed their presence in soil crusts within arid ecosystems, where they are documented as primary producers, demonstrating the ability to fix nitrogen.

The higher species richness observed through alpha diversity analysis in the dry pruning treatment compared to control can be attributed to the augmented availability of carbon for rhizosphere microorganisms. This suggests favourable soil health conditions, potentially leading to positive effects on crop productivity (Nielsen and Winding 2002 ). The absence of significant values in bacterial communities involving black and white plastic mulches may be attributed to the potential creation of an anaerobic environment, reducing oxygen exchange (Liao et al. 2021 ).

This could be due to the addition of organic matter from the pruning residues, which might provide more diverse habitats and resources for different bacterial species where dry pruning materials may create a more conducive environment for microbial diversity compared to plastic mulches being affected by numerous various factors, where one of the most important one is organic and nutrient incorporation more than moisture or temperature. Therefore, mulching produce higher fruit production, but if we want to preserve soils quality, pruning residue mulching would be a good choice. The impact of these changes on long-term soil fertility and plant productivity would be an important avenue for further research.

Conclusions

The study explored how mulching treatments influenced growth and physiological processes of lemon trees across different seasons. Mulching enhanced fruit production by the positive effect on tree gas exchange and water and nutrient uptake while preserving soil quality. However, depending on the environmental temperature, as it has been studied from the different sampling times, it would be concluded that rising ambient temperatures favoured vegetative growth, with black and white plastic mulching. This was related to the higher temperature and moisture than in dry pruning mulching and control. According to the mineral nutrients in soil, interestingly, the dry pruning treatment showed a significant increase in mineral concentrations in July, particularly for macronutrients and micronutrients, possibly due to increased mineralization of organic malemotter from crop pruning residue inputs. The soil microbial activity, as it was pointed out by dehydrogenase activity, and soil bacterial communities and diversity, also pointed out how the dry pruning mulching showed higher values, since it could be inhibited in the other plastic mulching by the high temperature. Accordingly, the higher number of fruits was related to the dry mulching. Overall, the study highlights the intricate interplay between environmental factors, mulching treatments, and physiological responses in lemon tree growth, but revealed that the use of dry pruning could be helpful to improve both the lemon crop and soil health, that it would be converted in a sustainable way of lemon production.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

References   

Aliche EB, Oortwijn M, Theeuwen T et al (2018) Drought response in field grown potatoes and the interactions between canopy growth and yield. Agric Water Manag 206:20–30. https://doi.org/10.1016/j.agwat.2018.04.013

Article   Google Scholar  

Allakhverdiev SI, Kreslavski VD, Klimov VV et al (2008) Heat stress: An overview of molecular responses in photosynthesis. Photosynth Res 98:541–550. https://doi.org/10.1007/s11120-008-9331-0

Article   CAS   PubMed   Google Scholar  

Andreotti C, Ravaglia D, Costa G (2010) Effects of fruit load and reflective mulch on phenolic compounds accumulation in nectarine fruit. Eur J Hortic Sci 75:53–59 ( https://hdl.handle.net/10863/35952 )

CAS   Google Scholar  

Bista DR, Heckathorn SA, Jayawardena DM et al (2018) Effects of drought on nutrient uptake and the levels of nutrient-uptake proteins in roots of drought-sensitive and -tolerant grasses. Plants 7:28. https://doi.org/10.3390/plants7020028

Article   CAS   PubMed   PubMed Central   Google Scholar  

Blagodatskaya E, Kuzyakov Y (2008) Mechanisms of real and apparent priming effects and their dependence on soil microbial biomass and community structure: Critical review. Biol Fertil Soils 45:115–131. https://doi.org/10.1007/s00374-008-0334-y

Boyero L, Pearson RG, Gessner MO et al (2011) A global experiment suggests climate warming will not accelerate litter decomposition in streams but might reduce carbon sequestration. Ecol Lett 14:289–294. https://doi.org/10.1111/j.1461-0248.2010.01578.x

Article   PubMed   Google Scholar  

Bui T, Vu B, Vy NTT et al (2020) Estimation of the soil to plant transfer factor and the annual organ equivalent dose due to ingestion of food crops in Ho Chi Minh city, Vietnam. Chemosphere 259:127432. https://doi.org/10.1016/j.chemosphere.2020.127432

Article   CAS   Google Scholar  

Callahan BJ, McMurdie PJ, Rosen MJ et al (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. https://doi.org/10.1038/nmeth.3869

Camarero JJ, Rubio-Cuadrado Á, Gazol A (2021) Climate windows of intra-annual growth and post-drought recovery in Mediterranean trees. Agric For Meteorol 308:108606. https://doi.org/10.1016/j.agrformet.2021.108606

Ceja-Navarro JA, Rivera-Orduña FN, Patiño-Zúñiga L et al (2010) Phylogenetic and multivariate analyses to determine the effects of different tillage and residue management practices on soil bacterial communities. Appl Environ Microbiol 76:3685–3691. https://doi.org/10.1128/AEM.02726-09

Cheng T, Rivard B, Sánchez-Azofeifa A (2011) Spectroscopic determination of leaf water content using continuous wavelet analysis. Remote Sens Environ 115:659–670. https://doi.org/10.1016/j.rse.2010.11.001

Costa JM, Egipto R, Aguiar FC et al (2023) The role of soil temperature in mediterranean vineyards in a climate change context. Front Plant Sci 14:1145137. https://doi.org/10.3389/fpls.2023.1145137

Article   PubMed   PubMed Central   Google Scholar  

Crawford CB, Quinn B (2017) Physiochemical properties and degradation. Microplastic Pollutants. https://doi.org/10.1016/B978-0-12-809406-8.00004-9

Creamer CA, de Menezes AB, Krull ES et al (2015) Microbial community structure mediates response of soil C decomposition to litter addition and warming. Soil Biol. Biochem. 80:175–188. https://doi.org/10.1016/j.soilbio.2014.10.008

Dangi SR (2014) Soil ecology and ecosystem services. Soil Sci Soc Am J 78:335–335. https://doi.org/10.2136/sssaj2013.0005br

Davison J (1988) Plant beneficial bacteria. Nat Biotechnol 6:282–286 https://doi.org/10.1038/nbt0388-282

Dodd IC, He J, Turnbull CG et al (2000) The influence of supra-optimal root-zone temperatures on growth and stomatal conductance in Capsicum annuum L. J Exp Bot 51:239–248. https://doi.org/10.1093/jexbot/51.343.239

Elvira-Rendueles B, Moreno JM, Costa I et al (2019) Pollen calendars of Cartagena, Lorca, and Murcia (Region of Murcia), southeastern Iberian Peninsula: 2010–2017. Aerobiologia 35:477–496. https://doi.org/10.1007/s10453-019-09578-y

Fallah N, Yang Z, Tayyab M et al (2021) Depth-dependent influence of biochar application on the abundance and community structure of diazotrophic under sugarcane growth. PLoS ONE 16:1–19. https://doi.org/10.1371/journal.pone.0253970

Fierer N, Schimel JP, Holden PA (2003) Variations in microbial community composition through two soil depth profiles. Soil Biol Biochem 35:167–176. https://doi.org/10.1016/S0038-0717(02)00251-1

Flexas J, Barbour MM, Brendel O (2012) Mesophyll diffusion conductance to CO2: An unappreciated central player in photosynthesis. Plant Sci. 193–194:70–84. https://doi.org/10.1016/j.plantsci.2012.05.009

Garonna I, De Jong R, Stöckli R et al (2018) Shifting relative importance of climatic constraints on land surface phenology. Environ Res Lett 13:024025. https://doi.org/10.1088/1748-9326/aaa17b

Geng YJ, Chen L, Yang C et al (2017) Dry-season deficit irrigation increases agricultural water use efficiency at the expense of yield and agronomic nutrient use efficiency of Sacha Inchi plants in a tropical humid monsoon area. Ind Crops Prod 109:570–578. https://doi.org/10.1016/j.indcrop.2017.09.022

Georgiou A, Gregoriou C (1999) Growth, yield and fruit quality of “Shamouti” orange on fourteen rootstocks in Cyprus. Sci Hortic (amsterdam) 80:113–121. https://doi.org/10.1016/S0304-4238(98)00232-5

Hayat R, Ali S, Amara U et al (2010) Soil beneficial bacteria and their role in plant growth promotion: A review. Ann Microbiol 60:579–598. https://doi.org/10.1007/s13213-010-0117-1

Hu Y, Zhao M, Liu Z et al (2023) Seasonal changes in gas exchange, water and macro-nutrient content differ between Citrus cultivars. Environ Exp Bot 213:105432. https://doi.org/10.1016/j.envexpbot.2023.105432

Huygens D, Schouppe J, Roobroeck D et al (2011) Drying-rewetting effects on N cycling in grassland soils of varying microbial community composition and management intensity in south central Chile. Appl. Soil Ecol. 48:270–279. https://doi.org/10.1016/j.apsoil.2011.04.012

Hussain S, Maqsood MA (2011) Root zone temperature influences nutrient accumulation and use in maize. Pakistan J Bot 43:1551–1556

Google Scholar  

Iqbal R, Raza MAS, Valipour M et al (2020) Potential agricultural and environmental benefits of mulches—a review. Bull Natl Res Cent 44:1–16. https://doi.org/10.1186/s42269-020-00290-3

Jinn TL (2017) Using silicon polymer impression technique and scanning electron microscopy to measure stomatal apertures. Bio-Protocol, vol 7. https://doi.org/10.21769/bioprotoc.2449

Kader MA, Senge M, Mojid MA (2017) Recent advances in mulching materials and methods for modifying soil environment. Soil Tillage Res 168:155–166. https://doi.org/10.1016/j.still.2017.01.001

Kallscheuer N, Jogler C (2021) The bacterial phylum Planctomycetes as novel source for bioactive small molecules. Biotechnol Adv 53:107818. https://doi.org/10.1016/j.biotechadv.2021.107818

Karimi S, Yadollahi A, Arzani K et al (2015) Gas-exchange response of almond genotypes to water stress. Photosynthetica 53:29–34. https://doi.org/10.1007/s11099-015-0070-0

Kaspari M, Garcia MN, Harms KE et al (2008) Multiple nutrients limit litterfall and decomposition in a tropical forest. Ecol Lett 11:35–43. https://doi.org/10.1111/j.1461-0248.2007.01124.x

Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41(1):e1–e1. https://doi.org/10.1093/nar/gks808

Kozlowski TT, Pallardy SG (2002) Acclimation and adaptive responses of woody plants to environmental stresses. Bot Rev 68:270–334. https://doi.org/10.1663/0006-8101(2002)068[0270:AAAROW]2.0.CO;2

Lamont WJ (2005) Plastics: Modifying the microclimate for the production of vegetable crops. Horttechnology 15:477–481. https://doi.org/10.21273/horttech.15.3.0477

Lamptey S, Li L, Xie J et al (2020) Tillage system affects soil water and photosynthesis of plastic-mulched maize on the semiarid Loess Plateau of China. Soil Tillage Res 196:104479. https://doi.org/10.1016/j.still.2019.104479

Lauber CL, Strickland MS, Bradford MA et al (2008) The influence of soil properties on the structure of bacterial and fungal communities across land-use types. Soil Biol Biochem 40:2407–2415. https://doi.org/10.1016/j.soilbio.2008.05.021

Lee SH, Ahn SJ, Im YJ et al (2005) Differential impact of low temperature on fatty acid unsaturation and lipoxygenase activity in figleaf gourd and cucumber roots. Biochem Biophys Res Commun 330:1194–1198. https://doi.org/10.1016/j.bbrc.2005.03.098

Lemanceau P, Maron PA, Mazurier S et al (2015) Understanding and managing soil biodiversity: a major challenge in agroecology. Agron Sustain Dev 35:67–81. https://doi.org/10.1007/s13593-014-0247-0

Lenka S, Trivedi P, Singh B et al (2019) Effect of crop residue addition on soil organic carbon priming as influenced by temperature and soil properties. Geoderma 347:70–79. https://doi.org/10.1016/j.geoderma.2019.03.039

Liao Y, Cao HX, Xue WK, Liu X (2021) Effects of the combination of mulching and deficit irrigation on the soil water and heat, growth and productivity of apples. Agric Water Manag 243:106482. https://doi.org/10.1016/j.agwat.2020.106482

Lozupone C, Knight R (2005) UniFrac: A new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71:8228–8235. https://doi.org/10.1128/AEM.71.12.8228-8235.2005

Luo S, Wang S, Yao P et al (2019) Soil microbial communities under film mulching and N fertilization in semiarid farmland. Nutr Cycl Agroecosyst 114:157–170. https://doi.org/10.1007/s10705-019-09998-9

Manzoni S, Jackson RB, Trofymow JA, Porporato A (2008) The global stoichiometry of litter nitrogen mineralization. Science 321:684–686. https://doi.org/10.1126/science.1159792

Martínez-Ballesta MC, Rodríguez-Hernández MC, Alcaraz-López C (2011) Plant hydraulic conductivity: In: Lakshmanan Elango (ed) hydraulic conductivity - issues, determination and applications, In: Tech, pp 103–121.  https://doi.org/10.5772/18580

McMurdie PJ, Holmes S (2013) Phyloseq: An R Package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8:e61217. https://doi.org/10.1371/journal.pone.0061217

Mhete M, Eze PN, Rahube TO et al (2020) Soil properties influence bacterial abundance and diversity under different land-use regimes in semi-arid environments. Sci Afr 7:e00246. https://doi.org/10.1016/j.sciaf.2019.e00246

Murali A, Bhargava A, Wright ES (2018) IDTAXA: A novel approach for accurate taxonomic classification of microbiome sequences. Microbiome 6:1–15. https://doi.org/10.1186/s40168-018-0521-5

Nemani RR, Keeling CD, Hashimoto H et al (2003) Climate-driven increases in global terrestrial net primary production from 1982 to 1999. Science 300:1560–1563. https://doi.org/10.1126/science.1082750

Nicholson SE, Funk C, Fink AH (2018) Rainfall over the African continent from the 19th through the 21st century. Glob Planet Change 165:114–127. https://doi.org/10.1016/j.gloplacha.2017.12.014

Nielsen MN, Winding A (2002) Microorganisms as indicators of soil health. Natl Environ Res Inst 388:82

Olmos R, Garcia P, Carvajal M et al (2024) Exploring membrane vesicles in citrus fruits: a comparative analysis of conventional and organic farming approaches. J Sci Food Agric 104:235–248. https://doi.org/10.1002/jsfa.12903

Ono K, Maruyama A, Kuwagata T et al (2013) Canopy-scale relationships between stomatal conductance and photosynthesis in irrigated rice. Glob Chang Biol 19:2209–2220. https://doi.org/10.1111/gcb.12188

Pandey S, Singh J, Maurya IB (2015) Effect of black polythene mulch on growth and yield of winter dawn strawberry (Fragaria × ananassa) by improving root zone temperature. Indian J Agric Sci 85:1219–1222. https://doi.org/10.56093/ijas.v85i9.51634

Pérez-Pérez JG, Robles JM, Tovar JC et al (2009) Response to drought and salt stress of lemon “Fino 49” under field conditions: Water relations, osmotic adjustment and gas exchange. Sci Hortic (amsterdam) 122:83–90. https://doi.org/10.1016/j.scienta.2009.04.009

Prăvălie R (2021) Exploring the multiple land degradation pathways across the planet. Earth-Sci Rev 220:103689. https://doi.org/10.1016/j.earscirev.2021.103689

Rashid A, Ryan J (2004) Micronutrient constraints to crop production in soils with Mediterranean-type characteristics: a review. J Plant Nutr 27(6):959–975

Ribeiro RV, Machado EC (2007) Some aspects of citrus ecophysiology in subtropical climates: Re-visiting photosynthesis under natural conditions. Braz J Plant Physiol 19:393–411. https://doi.org/10.1590/S1677-04202007000400009

Richardson A (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Funct. vol 28Plant Biol, pp 897–906 https://doi.org/10.1071/PP01093

Robinson JM, Barker SLL, Arcus VL et al (2020) Contrasting temperature responses of soil respiration derived from soil organic matter and added plant litter. Biogeochemistry 150:45–59. https://doi.org/10.1007/s10533-020-00686-3

Sinmidele KO, Babatunde IJ, Omolola OE (2015) Response of tomato variety (Roma F) yield to different mulch materials and staking in Kabba, Kogi State, Nigeria. J Agric Stud 3:61. https://doi.org/10.5296/jas.v3i2.7568

Smilanick JL, Erasmus A, Palou L (2019) Citrus fruits. Postharvest pathol fresh hortic prod 3–53. https://doi.org/10.12987/9780300242546-011

Smit E, Leeflang P, Gommans S et al (2001) Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods. Appl Environ Microbiol 67:2284–2291. https://doi.org/10.1128/AEM.67.5.2284-2291.2001

Sremac AF, Lalic B, Cuxart J et al (2021) Maximum, minimum, and daily air temperature range in orchards: What do observations reveal? Atmosphere (basel) 12:1–22. https://doi.org/10.3390/atmos12101279

Strik B, Righetti T, Rempel H (2006) Black plastic mulch improved the uptake of 15nitrogen from inorganic fertilizer and organic prunings in summer-bearing red raspberry. HortScience 41:272–274. https://doi.org/10.21273/hortsci.41.1.272

Torelló-Sentelles H, Franzke CLE (2022) Drought impact links to meteorological drought indicators and predictability in Spain. Hydrol Earth Syst Sci 26:1821–1844. https://doi.org/10.5194/hess-26-1821-2022

Trinchera A, Migliore M, Warren Raffa D et al (2022) Can multi-cropping affect soil microbial stoichiometry and functional diversity, decreasing potential soil-borne pathogens? A study on European organic vegetable cropping systems. Front Plant Sci 13:952910. https://doi.org/10.3389/fpls.2022.952910

Tsung-Luo J (2017) Using silicon polymer impression technique and scanning electron microscopy to measure stomatal apertures. Bio-Protocol 7:e2449–e2449. https://doi.org/10.21769/bioprotoc.2449

Valinsky L, Vedova G, Scupham AJ et al (2002) Analysis of bacterial community composition by oligonucleotide fingerprinting of rRNA genes. Appl Environ Microbiol 68:3243–3250. https://doi.org/10.1128/AEM.68.7.3243-3250.2002

Von Mersi W, Schinner F (1991) An improved and accurate method for determining the dehydrogenase activity of soils with iodonitrotetrazolium chloride. Biol Fertil Soils 11:216–220. https://doi.org/10.1007/BF00335770

Wright ES (2016) Using DECIPHER v2.0 to analyze big biological sequence data in R. R J 8:352–359. https://doi.org/10.32614/rj-2016-025

Yaghi T, Arslan A, Naoum F (2013) Cucumber (Cucumis sativus, L.) water use efficiency (WUE) under plastic mulch and drip irrigation. Agric Water Manag 128:149–157. https://doi.org/10.1016/j.agwat.2013.06.002

Yang YJ, Dungan RS, Ibekwe AM et al (2003) Effect of organic mulches on soil bacterial communities one year after application. Biol Fertil Soils 38:273–281. https://doi.org/10.1007/s00374-003-0639-9

Yessoufou MWIA, Tovihoudji PG, Zakari S et al (2023) Hill-placement of manure and fertilizer for improving maize nutrient- and water-use efficiencies in the northern Benin. Heliyon 9. https://doi.org/10.1016/j.heliyon.2023.e17823

Zeppetello LR, Battisti DS, Baker MB (2019) The origin of soil moisture evaporation “regimes.” J Clim 32:6939–6960. https://doi.org/10.1175/JCLI-D-19-0209.1

Zhang Y, Huang C, Zhao L et al (2023) Effects of different colors of film mulch on soil temperature and rice growth in a non-flooded condition. J Sci Food Agric 103:6352–6361. https://doi.org/10.1002/jsfa.12709

Download references

Acknowledgements

The authors thank Dr Lucía Yepes and Dr. Gloria Barzana for their help with statistics.

Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was funded by Spanish Ministerio de Ciencia e Innovación and CDTI (MIP-243413).

Author information

Authors and affiliations.

Aquaporins Group. Plant Nutrition Department, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Campus Universitario de Espinardo, Edificio 25, 30100, Murcia, Spain

Rafael Olmos-Ruiz, María Hurtado-Navarro & Micaela Carvajal

Enzymology and Bioremediation of Soils and Organic Waste Department, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Campus Universitario de Espinardo, Edificio 25, 30100, Murcia, Spain

María Hurtado-Navarro & Jose Antonio Pascual

You can also search for this author in PubMed   Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Rafael Olmos-Ruiz, María Hurtado-Navarro, José Antonio Pascual, and Micaela Carvajal. The first draft of the manuscript was written by Rafael Olmos-Ruiz and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Micaela Carvajal .

Ethics declarations

Competing interest.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Responsible Editor: Juan Barcelo.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Olmos-Ruiz, R., Hurtado-Navarro, M., Pascual, J.A. et al. Mulching techniques impact on soil chemical and biological characteristics affecting physiology of lemon trees. Plant Soil (2024). https://doi.org/10.1007/s11104-024-06894-2

Download citation

Received : 02 May 2024

Accepted : 02 August 2024

Published : 29 August 2024

DOI : https://doi.org/10.1007/s11104-024-06894-2

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Soil-microbiota
• Citrus x limon
• Gas exchange
• Mineral-nutrient
• Relative-growth-rate
• Find a journal
• Publish with us
• Track your research