Investigation results:

Keys to the effective foliar application of biostimulants

Although foliar aspersion is the most common mechanism used for applying nutrients on crops, its high variability reflects the lack of knowledge of the factors that determine the retention and penetration levels in the leaf. German scientist Heiner Goldbach explains how recent research has shed light on this subject.

Patricio Trebilcok and Francisco Fabres

“Spray and pray” says an old saying in agriculture. It refers to the uncertainty caused by foliar spraying of biostimulants on crop leaves. This practice faces a complex obstacle: the anatomy of the leaves, precisely designed to prevent the entry of external elements to avoid potential damage that can be transmitted to the rest of the plant.

Although biostimulants manage to penetrate the protective barriers of the foliage, and for this reason continue to be used, they do so with great variability: at times a significant percentage of the product manages to penetrate, at others the penetration is insignificant. This has precisely been one of the research fields of the German agrobiologist Heiner Goldbach, Ph.D. in plant nutrition and professor at the University of Bonn, who has sought to clarify the mysterious factors that favor or hinder the absorption of bio-stimulants through foliage.

Goldbach points out that many of the biostimulants are applied on the leaves by spraying. In fact, a study by Coherent Market Insights indicates that 78% of the global biostimulant market corresponds to foliar products, well above those applied to the soil (almost 9%) and to seeds (12%). However, the academic points out that its effectiveness has two major obstacles: the structural design of the leaves and the heterogeneous characteristics of the world of biostimulants, where one can find from simple substances, to complex mixtures, and even microorganisms. 

Heiner Goldbach says that there are two key aspects in the foliar application of biostimulants: the retention of what is applied and the penetration in the leaf.

.

"Everything in the leaf’s morphology is designed to go against this absorption, and yet it happens. Foliar application really works", points out Professor Goldbach in his presentation "Foliar application of bio-stimulants: how to make them reach the apoplast", held at the World Congress of Bio-stimulants.

Unfortunately, it does not always work the same way and sometimes the results can be contradictory. Goldbach explains this with past examples in the field of fertilizers: in different foliar application trials of urea in broad beans (Eichert & Goldbach, 2008) carried out under almost identical conditions, the absorption varied from 6.9% to 49.3% of the dose used. This is a fairly typical phenomenon in foliar spraying, and this is where the idea “spray and pray” comes from. The high variability in the results indicates that the process is still not well understood and therefore it is not possible to fully control it. The expert clarifies that “foliar application really works, sometimes better, sometimes worse”.

THE CHALLENGE OF RETENTION

In his analysis of the factors that allow a greater efficacy in the use of foliar biostimulants, Goldbach analyzes two variables: the retention of what is applied and the level of penetration in the leaf.

The first has to do with the biostimulant’s ability to remain on the leaf’s surface which is quite hydrophobic and therefore the liquids that fall on it tend to drain. It is a characteristic that increases or decreases depending on the species, the variety, the production conditions, the environmental humidity, or the nutritional status of the plant.

This situation makes an important aspect of the biostimulant application process more difficult: the need to uniformly cover the leaf surface, avoid excessive runoff and maintain a moist coating for as long as possible, enabling the "transportation" of the compounds.

In order for the foliage to retain a significant volume of the sprayed solution, certain products are used (surfactants and other adjuvants) that collaborate to spread the solution on the leaf surface, as well as to increase the adhesion and retention of the drops to properly wet the leaves (figure one). But one has to be careful not to go overboard: if an excessive volume is applied or if the tension is reduced too much, there can be runoff that not only reduces the amount of biostimulant retained in the leaf, but also produces a disproportionate distribution. When it travels, some parts of the leaf are left without the product while others —for example, certain folds— suffer from over saturation (figure 2).

Figure 01. Wettability and surface tension determine the volume of the solution that remains on the leaf. The wettability increases as the contact angle approaches 0° and the liquid is more spread out.

Figure 02. Problems resulting from two extremes: not enough wettability with too much retention (left) or too much wettability with poor retention that runs off and collects in the folds of the leaf (right).

Taking these aspects into consideration, Goldbach analyzes the main factors that can influence retention processes on these surfaces.

Type of compounds: very polar ones (molecules with bonds in which the distribution of atoms is not symmetrical), such as magnesium chloride, are hygroscopic and absorb water with very low relative humidity in the air. On the contrary, other salts, like most sulfates, only absorb water when there are high relative humidity levels. The deliquescence point (DRH) is the relative humidity level at which a compound begins to liquefy.

Species: there are noticeable differences in the leaf’s surface properties in different plant species, and therefore in their permeability.

Cultivation: two varieties of the same species can be very different in their leaf structure in certain aspects like the shape of the wax that covers its cuticle.

Environmental conditions: plants of the same species and variety can be affected by the conditions of the air. To give an example, microscopic images of the surface of outdoor plants’ leaves exposed to "normal German air" show polluting elements that clog the pores through which the products should enter, while leaves of similar plants in a greenhouse with filtered air have fully operational pores.

Hygroscopy/deliquescence of the bio-stimulant (formulation): In order to enter the leaves, the products must be dissolved in a liquid (at least the polar or semi-polar substances). With very few exceptions, dried remains on the leaf’s surface will never enter the leaf. Therefore, the surface must be kept moist for as long as possible for the absorption of the biostimulant to take place. Some intervening factors are: a high relative humidity in the air helps keep the leaf moist, a substance with a low deliquescence point will be more easily preserved in liquid form, and at night there is less risk of the product drying out as opposed to daytime when temperatures rise and relative humidity drops.

LEAF PENETRATION

The second great challenge of the foliar application process is the transfer of bio-stimulant compounds from the plant’s surface to the interior of the tissues. This can happen in two ways: one, through the waxy layers and cuticles, the other, through the stomata, which are adjustable openings in the epidermis or "skin" of the plants. For a long time there’s been a discussion on whether it is really feasible to penetrate the leaf using the second option. It’s a debate that Goldbach settles with the evidence that some stomata –but not all – do allow a transfer from the exterior, entering through the pores, towards the apoplast. "While the pores in the cuticles have dimensions of a few nanometers (one millionth of a millimeter), the pores in the stomata let larger compounds and microorganisms come through" he says. 

There are other factors that also contribute to the variability of penetration, such as the leaf’s morphology. For example, plants adapted to a dry environment (xeromorphic) are very different from those adapted to a humid environment (hygromorphic). On the other hand, in most species, the stomata are concentrated on the underside of the leaf, and only a few have stomata on the upper side. 

Figure 3 shows the noticeable differences in the penetration of an element such as boron in a xeromorphic plant, like the lychee (just over 7 micrograms during the experimental period), and another hygromorphic plant, like the soybean (400 micrograms in the same period). But it also shows that the underside presents a much higher level of absorption than the upper part: it triples in the case of the soybean leaf and is 7 times greater in the case of the lychee.

Figure 03. Foliar boron uptake in two species: lychee and soybean.
Will, Eicher, Fernández et al. (2012), J. Plant Nutr. Soil Sci. 175, 180-188.

The main factors that influence penetration – some of which coincide with those that influence retention – are:

–The chemical properties of the components that are applied: their polarity and the distribution of the electrical charge of the molecules.

–The polarity of a substance plus the formulation technique. For example, if it contains any type of salt with its corresponding deliquescence point (low in the case of chlorides or high in sulphates).

–The species: there are fundamental differences in the surface of the leaf and in the properties of the cuticle.

–The concentration gradient,which will be explained later.

When talking about the entry of biostimulants in the leaf, we can broadly divide the process into three types: polar molecules, ranging from small to large –for example, the amino acids, serine, cysteine ​​​​and threonine–, non polar molecules, also from small to large –such as the amino acids glycine, alanine and leucine–, and particulate matter/microorganisms –such as Bacillus and Pseudomonas–.

PENETRATION OF POLAR SUBSTANCES IN THE LEAF

How do hydrophilic compounds penetrate the hydrophobic surface of the leaf?

It is a passive process called diffusion, which is to say that it is driven by the concentration gradient between the outside and the inside of the leaf. Thus, the highly concentrated particles of the applied solution tend to move towards the area of ​​lower concentration, the apoplast.

The cuticle is composed of crystalline and amorphous type waxes. The penetration of polar substances occurs through these materials and in an indirect way. Since the molecules have different sizes, the rate of penetration is also different (figure 4)

Figure 04. Absorption of polar substances (for example salts) through the cuticle, and selection according to molecular size. Simplified model.

Environmental humidity also plays an important role. In fact, there is practically no penetration at relative humidity levels below 70-80% (figure 5). This is explained because the cuticular layer does not only contain waxes, but also hydrophilic compounds or domains generally made up of cell wall polysaccharides. Under dry conditions, these hydrophilic elements remain well covered by the cuticle, but with higher humidity the water is absorbed by these polymers, which expand. The expansion of the cuticles can even be measured. (figure 6).

Figure 05. Effect of relative humidity on the permeability of isolated cuticles.

Figure 06. “Dynamic” pore model.

Modificado de: Eichert & Fernández (2012), en: Marschner 3ª ed.

The activity of the stomata also plays a role, because when they open there is an increase in relative humidity on the leaf’s surface, close to 90% or more (figure 7). When they close, the humidity drops. It is a very dynamic process, since not all the stomata open at the same time.

Figure 07. Relative humidity on the surface of the leaf with open stomata (diagram of a plant, such as broad bean, with stomata on both sides of the leaf).

Based on a schematic by E.H. Rub, completed and modified using a representation of https://www.slideshare.net/safa-medaney/what-are-plants-2.

Another relevant issue is the size of the pores. For a long time the estimated size was 1 nanometer (nm), one millionth of a millimeter, for isolated cuticles without waxes. But cuticle isolation may result in a risk of bias because only certain cuticles are selected, which undergo a lengthy process that can also change their properties. Therefore, Professor Goldbach together with Professor Thomas Eichert prefer the use of intact leaves, finding pore sizes even above 40 nm in surfaces with stomata.

It is also important to consider that the increased permeability of the cuticle in high humidity conditions is added to the deliquescence point of polar substances on the outside, which makes them absorb water and generate a high concentration. A high concentration means a high gradient of diffusion into the leaf’s interior. This implies a dual influence of relative humidity (Fernández and Eichert, 2009). However, the maximum penetration is not achieved with the maximum humidity, but with levels of 70 to 80%. Also, there is a great difference in the absorption by the lower and upper surface of the leaf (figure 8).

Figure 08. Absorption of calcium nitrate Ca(NO3) in coffee leaves.

It is possible to adjust to the expected relative humidity level by selecting a product with a higher or lower deliquescence point. However, it should be noted that deliquescence decreases when two different salts or components are mixed together. For example, ammonium nitrate (deliquescence 62%) combined with urea (deliquescence 79%) has less than 20% deliquescence. "For this reason, many of our growers use this combination in spring for their first foliar applications, as it maintains a good humidity level that favors the absorption of compounds," says Goldbach.

PARTICULATE MATTER / MICROORGANISMS

For a long time it was believed that these kinds of materials could not pass through the stomatal surface, given its ability to repel water (hydrophobicity) and its particular structure. Goldbach admits that this is true in the case of a droplet, because a large force is required to push it through the stomata. However, together with Eichert and Burkhart (1998), they ran tests using a series of compounds and, using fluorescent dye, were able to verify that some of them did penetrate the stomatal surface.

They demonstrated that not all stomata are as hydrophobic as publications indicate. In Photo 1 of a leek stoma, the low contact angles of a stoma can be seen, indicating that its inner surface is hydrophilic. New tests with 40nm of fluorescent particles penetrated through the stomata and some of them even reached the apoplast (figure 9).

Photo 1. Hydrophilic inner surface of a leek (Allium porrum) stoma.

Figure 09. Distribution of fluorescent Nanospheres® at different depths.

Ultimately, the particles can enter the leaf’s apoplast, there is absorption through the stomatal pores and the transport mechanism is usually the concentration of gradients, that is, by diffusion. There are no indications of penetration by infiltration of the stomata, as there was a very low concentration found inside the leaf (the apoplast). It was also observed that particles larger than one micrometer (one thousandth of a millimeter) did not pass through the stomata.

Humic acids do not commonly enter through the upper leaf cuticle but through the stomata on the underside.

On the other hand, as long as the stomata are open there is a higher level of penetration of the compounds. The evaluations made with different forms of nitrogen have shown that open stomata can contribute up to 75% or sometimes 80% of total absorption (figure 10).

Figure 10. Relative contribution of stomata and cuticle to the penetration of different nitrogenous fertilizers

Therefore, it’s been verified that there is more than one route of entry: the cuticular route and the stomatal route. The stomata are probably the only possible entrance route for the particles, if there are no major cracks or breaks in the leaf’s surface.

NON POLAR SUBSTANCES

The absorption rate is strongly influenced by the molecular volume. At higher volumes the absorption decreases significantly, especially in lipophilic species (non-polar substances attracted by lipids, generally hydrophobic) in which the alterations in molecular size make a great difference in terms of penetration.

SUMMARY

Two limiting processes in foliar uptake were addressed in Dr. Heiner Goldbach's presentation: retention and penetration.

-Retention and penetration can be modified with the use of adjuvants, such as detergents, adherents and wetting agents, in addition to the appropriate choice of salts or other complexes.

-Most, if not all, of the transfer from the leaf’s surface to the apoplast occurs by diffusion. It is not an active process.

-There are three main routes:

Non-polar substances:these dissolve in the cuticular layer and move through it in the lipophilic domains. In this case there is a noticeable separation by size.

Polar substances: their movement through the polar domains of the cuticle depends on relative humidity.

The permeability through the cuticle can lead to a separation by molecular size and charge.

The stomatal route allows the penetration of whole microorganisms, requiring sufficient penetrable stomata, cracks, etc., and is free for particles smaller than one micron.

GLOSSARY:

Adsorption: adhesion of a gas, liquid or dissolved solid (adsorbate) to a surface (of the adsorbent). While in absorption that gas, liquid or solid dissolves or penetrates the absorbent, adsorption is a surface phenomenon.

Adjuvant: substance that is added to another to improve its application or enhance its effect.

Apoplast: the apoplast of the leaf is a space found between the cells, including the cell wall and the xylem. Water, nutrients and other substances flow through it.

Cuticle: a protective layer that is found in the outermost surface of the plant and that interacts with the environment. The cuticle is formed and secreted by the cells of the epidermis.

Deliquescence: a phenomenon in which a substance absorbs moisture from the air until it assumes a liquid form (aqueous solution). In addition to the waxes that are frequently found on the leaf, a great barrier between the exterior and the apoplast corresponds to the cuticle: first the cuticle itself, and then a cuticular layer that has a certain continuity with the cell wall of polysaccharides partially intermingled with it.

Epidermis: the outer layer of the skin. It is the living shield that covers the surface of almost the entire plant.

Stomata: cells that are part of the epidermis plant and give shape to a pore or opening called an ostiole. They are a passageway for most of the oxygen and carbon dioxide, two gasses used by the cells inside the plant during photosynthesis and cellular respiration. They are also the main route by which the plant loses the water absorbed by the roots, in the form of vapor.

Hydrophilic: a substance that has an affinity for water, which easily captures it.

Hydrophobicity: an ability of a material to repel or be repelled by water.

Hygromorphism: a set of morphological and physiological characteristics that allow a successful survival in soils or humid environments.

Hygroscopy: the ability to absorb moisture from the environment.

Lipophilic: a compound that has the ability to dissolve in fats, vegetable oils, and lipids in general. In other words, a lipophilic substance is one that has affinity and is soluble in lipids.

Micron (µm): one-thousandth of a millimeter.

Nanometer (nm): one millionth of a millimeter.

Polarity: a property that represents the separation of electrical charges in the same molecule. This characteristic is related to aspects such as solubility, melting point, boiling point and intermolecular forces, among others. For example, the solvent capacity of water is a result of it being a polar molecule, and oil and water do not mix because the former is nonpolar while the latter is polar. Molecules with polar bonds in which the distribution of atoms is not symmetrical are polar, while molecules bonded to identical atoms distributed symmetrically, are nonpolar..

Deliquescence Point (DRH): the relative humidity level at which a compound begins to liquefy.

Solute: a substance (for example, salt) dissolved in another called a solvent (for example, water) to form a mixture called a solution (for example, brine).

Surfactant: a compound that reduces the surface tension of the liquid to which it is added.

Xeromorphism: a set of morphological and physiological characters that provide plants with protection against drought.