Direct biostimulation of plants by Humic Substances

From the Speech “Biostimulation by Humic substances”, of Eduard Llaudó Avila, from Mediterranea de agroquimicos, presented at FShow Conferences – CAC Shangai 2019

I’m not a scientific researcher, but an agronomical engineer, and when I was requested by Citymax Group to speak about biostimulant action of the humic substances, I knew that it would not be easy, because our old friends are far from being that simple.

First overview

Taking a look to substances with biological effects on plants, in a first overview, we notice that, when we check a phytoregulator, for instance the indole acetic acid, it’s observed a specific growing stimulation.

or when checking an allelosubstance as the juglone, we also find a clear inhibition action in some plants.

These are examples of direct responses of plants to the application of specific substances.

But in case of humic and fulvic substances, we have supramolecular structures, with macro-molecules held together by non-covalent forces, also getting including complexed ions, so it becomes difficult to determine what causes which effect.

In tests conducted in growth chambers or under hydroponic conditions, Humic substances have been shown to cause morphological changes in some species of plants. In case of full season tests, increase of yield and yield quality is usually reported, but the most commonly reported direct effect of humic acids is the stimulation of root growth, specially at early stages of plant development.

The concentration, molecular weight and source of the humic fractions are significant in their effects. Additionally, Humic Substances are more effective in case of soils with poor fertility and low organic matter content.

In general terms, we know that humic + fulvic acids have direct effect on plants:

  • nutrition (by themselves, but also improving fertilizer action)
  • stimulation of plant metabolism

and they also have clear beneficial effects on soil structure, so indirectly on plants, too:

  • improvement of soil structure, by the clay-humic complex (CHC)
  • improvement of chemical properties of soil. :
    • CHC enhances buffering of water (increased resistance to drought and salinity),
    • also increases soil pH buffering, etc.
    • enhances the cation exchange capacity of soil
    • complexing interaction with metals
    • improves solubility of phosphorus by interfering with Ca-phosphate precipitation
  • improvement of rhyzosphere biological activity, which also causes crop stimulation.

We can understand that humic substances are plant biostimulants, but in order to be sure where we walk, before to deep in the effects of humic substances, let’s check the biostimulant meaning.

Biostimulant concept

We can find tens of definitions of plant biostimulant, from 1950’s to nowadays. One of the most accepted is the definition of Dr. Patrick Du Jardin (2015):

A plant biostimulant is any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content. By extension, plant biostimulants also designate commercial products containing mixtures of such substances and/or microorganisms.

The future European regulations point in the same way, classifying biostimulants as fertilizing products, which, regardless of their nutrient content, improve any of following agronomic features: nutrient use efficiency, tolerance to abiotic stress, crop quality, availability of confined nutrients in the soil and rhizosphere, humification and degradation of organic compounds in the soil.

In that way, upon the effects already described, Humic substances may be classified as biostimulants.

And discerning once again between direct and indirect actions on plants, despite their important chemical and biological effects on soil, which deserve a specific speech, let me concentrate on the direct effects on plants, more in accordance with Hankins, Dixon and Walsh definition of a bioestimulant (2004):

Natural or synthetic products of either mineral or organic composition that by their mode of action positively contribute to crop nutrition and the development of healthy plants”.

The gap between fertilizers and phytoregulators

Now, please, allow me to take the liberty of comparing biostimulants with a breakfast:

We notice that on the left there are the indispensable nutritious foods, whereas on the other extreme there is a stimulant beverage. And in the middle we can find a sort of commitment, which, depending on the proportions of the mixture, feeds and/or stimulates us.

To a certain extent, we could apply this simulation between feeding or stimulating if we were to draw a trajectory from fertilizers to phytoregulators. In the transition space we would find the bioestimulants.

As we know, biostimulants have undergone considerable development in last years, and they comprise a wide range of products. Excluding mixtures between them and biological products, we could make a very general classification, headed by the three great classic bioestimulants:

  • Amino Acids
  • Seaweed Extracts
  • Humic Acids, understood as the sum of Humic and Fulvic Acids.
  • Special mineral formulations
  • Organomineral formulations
  • other organic formulations

Approximately, and regardless of the different modes and degree of action of each product, we could classify them according to their properties, closer to nutritional or phytoregulatory action,

Understanding Humic substances

As we see, Humic Substances show a wide action spectrum, ranging from a mere nutritional capacity to an effect in plants closer to that of phytoregulators.

But determining this mode of action represents a quiet difficult task, not only because of the complex nature of such a multicomponent substance, but also due to the wide range of overlapping effects they show. Thus, I will try to make an approach focusing on features and effects.

Firstly to comment that, generally, the variations of composition and structure of Humic Substances according to their parent matter, formation process, etc. are less significant to physiological effects than concentration applied or molecular weight. And referring to molecular weight, Humic substances composition include three groups of molecules:

  • Humine, insoluble at any pH. They consist of large molecules with a molecular weight of 100,000 to 10,000,000 Da, which also aggregate not-humic materials.
  • Humic Acids, soluble at alkaline pH. There are big molecules of thousands of carbon rings, with a weight of 50,000 to 100,000 Da.
  • Fulvic Acids, water soluble. They are shorter organic structures of hundreds of carbon rings with a molecular weight of 500 to 10,000 Da.

One of the first things that we could infer is that the molecular weight is a relevant factor in the absorption by plants. Molecules of higher molecular weight are not easily absorbed, so their main effect occurs in the rhyzosphere:

– Molecular weight +

Consequently, Fulvic Acids, with shorter molecule structures, should be able to show a more significant metabolic activity than Humic Acids. But, in cases other than transportation of chelated elements, this rule is not always complied. For example: It has been shown that the physiological influence on root growth has no relation with molecular weight of humic substances, instead with bioactive molecules. These bioactive molecules could be pieces of the superstructure of Humic Substances, broked in the rhyzosphere, but may be also other kind of molecules, such as proteins, carbohydrates, aliphatic biopolymers, and lignin, often present in Humic Substances.

The difficulty to locate and describe these bioactive molecules and their pathways -talking in pesticide language-, leads us to focus mainly on effects, in the sense of European Specifications about biostimulants.

Direct biostimulation effects by Humic substances on plants

Herein, this could be a summary of the main direct effects of Humic Substances on plants:

  • + Fulvic acids enhance uptake of nutrients. It has been described enhanced uptake of N, P, K, Ca, Mg, Cu, Fe, and Zn when application of fulvic acids, but also related with concentration. The size of Fulvic Acid molecules and their chelating capacity suggest an action as natural providers of micronutrients. In case of Humic Acids, the effect would be the opposite, causing a micronutrient deficit due to a temporary binding to big organic structures, thus becoming less available to plants.
  • + Gene expression. Different studies show that the expression of hundreds of genes may be affected by Humic substances. Humic acids application affects biochemically up to 40 proteins related to energy pathways and cellular transport. Humic substances increase the ATPase activity in root cells, and thus the proton pump activity. They also stimulate nitrate assimilation through the upregulation of the target enzymes (NR, GDH, and GER) involved.
  • + Role on concentrations of Reactive Oxygen Species (ROS), such as O2, OH, or H2O2. It is suggested that, probably, ROS play a central role on Humic acids effects, which becomes specially relevant in case of abiotic stress. In studies of abiotic stress (such as induced drought or salinity ), it has been reported that humic substances induce peroxidase activity, thus decreasing the hydrogen peroxide content. It also increases the proline content (an indicator of abiotic stress), that stabilizes the membrane permeability.
  • + As a result of direct action on plant metabolism, or indirecty through ryzosphere activity, humic substances show auxin-like effects, such as enhanced lateral root development. It tends to be the main effect described when talking about main direct effects of humic substances, and it is directly related to applied concentrations.

Way of action

Now it would be suitable to speech about possible biochemical pathways, but unfortunately there are no clear related models. There are some researchers trying to define a pathway tree, steming from a main starting action, an initial biochemical event, and dealing to several effects, such as the action on ROS and the auxin-like role.

May be, this approach could be compared with a Billiard game:

Although specific effects may result in a cascade of subsequent effects, it also could happen that Humic substances actions be some parallel single effects overlapped.

Anyway, in order to get deeper into biostimulation effects and mechanisms, we’ll need the help of the biochemical researchers in order to find clues. And a first step is to determine that, when marking Humic substances with 14C isotopes, it can be seen that part of them binds tightly to roots cell wall within the first few hours of interaction. Also part of them can be absorbed and transferred to the shoots.

Focusing now specifically on root growth improving, please allow me to mention a recent assay, called “Lateral Inhibition by a Peptide Hormone-Receptor Cascade during Arabidopsis Lateral Root Founder Cell Formation”, carried out by a research group in Kobe University, in Japan.

It is known that the ubication of lateral roots depends on initiation sites induced by auxins. The domain of high auxin response, responsible for LR initiation, stretches out over several cells, but only a pair of the pericycle cells (LR founder cells) will give rise to Lateral Roots. These research team found out the role of a specific gene and peptide, the TOLS2 ones, on the stimulation/repression of lateral root growth, concluding that are responsible for the number of lateral or secondary roots.

This process takes place in the roots, where Humic Substances bind tightly and also are absorbed. So, let me to place the hypothesis that, maybe, Humic substances proven beneficial effect on lateral root growing could in some way have something to do with expression of TOLS2 gene, the action of TOLS2 peptide, or even with some kind of competition in the peptide receptors (although looking at the graphic root stimulation/humic acid concentration, we can understand that effect is not through the competence in receptors).

As we can see, as more we know, more new questions we have, but to find new clues is a work for scientific researchers, not for a field engineer like me.

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