Azospirillum, a bacteria of well-established use in extensive crops

With 20 million doses of commercial products applied in the continent, there is no discussion about the effectiveness of the inoculation results of this microorganism, mainly in cereals and legumes. This is stated by Dr. Fabricio Cassán, who reviews the technological and scientific developments that have allowed great advances, such as the massive co-inoculation with rhizobia in soybeans and significant savings in nitrogen fertilizers.

Azospirillum corresponds to a genus of bacteria that promotes plant growth. It is found in soils in various parts of the world and is a good colonizer: there are reports for more than 100 plant species. Associated with the root system of plants, these microorganisms contribute to crop production by acting on both aerial and root growth.

One of the leading experts on the subject is the Argentine Fabricio Cassán, microbiologist and doctor in biological sciences, Assistant Professor in the ​​plant physiology area at the National University of Río Cuarto, Argentina, and independent researcher at the Institute of Agrobiotechnology, INIAB , of the CONICET. Author and editor of numerous scientific publications, his work in the Plant Physiology and Plant-Microorganism Interaction Laboratory focuses on plant growth-promoting rhizobacteria and phytohormones.

-What is the current situation of the Azospirillum bacterium? Azospirillum?

In recent years there has been a very significant increase in the use of Azospirillum in commercial products. The number of doses used throughout the continent, but mainly in Argentina and Brazil, is estimated at 20 million per year (1 to 1.5 doses per hectare are used), which places this bacterium as the second most widely used microorganism, mainly in extensive crops. We are talking about corn, we are talking about co-inoculation in legumes: in Brazil, beans, soybeans; in Argentina, mainly soybeans. Co-inoculation, the combination of Rhizobium with Azospirillum, has improved the format of products for legumes. Rhizobium with Azospirillum, has improved the format of products for legumes.

-The first in number of doses is Rhizobium?

-Of course, it is in the order of 80 million doses between Argentina and Brazil. If you add Paraguay and probably Uruguay, you may be close to 100 million doses a year.

Según Fabricio Cassán, en el caso de Azospirillum the frequency of success is between 75 and 80% of cases.

According to Cassán, the change has been driven by the increase in companies focused on producing inoculants based on Azospirillum, and an important role of the State.

The most transcendental event was Brazil entering the equation –he comments–, because when they start, the numbers increase significantly. Today the companies in that country see that inoculation technology with Azospirillum shows results and have incorporated it into the product packages that they normally offer. In Argentina, the INTA [National Institute of Agricultural Technology] and in Brazil, the Embrapa [Brazilian Agricultural Research Company], have generated information and have presented numbers, as a product of metadata analysis of many trials where the robustness of this technique has been verified.

One of the difficulties for the massification of Azospirillum, says Dr. Cassán, is that, unlike rhizobia, visible to the naked eye in the root nodules, its presence and effect is demonstrated through measurements and statistical models.

–If with rhizobia 80-85% of the tests obtain results with some differences against a control, in the case of Azospirillum the frequency of success is between 75 and 80% of the cases. Yield differentials vary depending on the crop, for example in winter cereals, such as wheat, it increases around 14% on average, in summer cereals, such as corn, 9.5%; in soybean, in interaction with rhizobia and together with its contribution, it adds 6 to 8%.

THE PRESENCE OF THE BACTERIA MAKES THE SYSTEM WORK DIFFERENTLY

The academic notes that there is an evolution in the field of research. Initially Azospirillum was associated with nitrogen fixation, but the amount it could fix was not enough to explain the changes. Then, they thought of an action related to the phytohormones that make the roots grow. However, the trials attempting to emulate that effect with the sole application of hormones, without the microorganisms, did not show the same results. Today, it is postulated that there is an interaction with the plant that increases the efficiency in the use of nutrients, but it is not clear how.

–When you incorporate the bacteria, you need less fertilizers to match a productive response, so there is a cost saving. The root system is impacted by the presence of the microorganism, it grows more and increases its absorption of nutrients from the soil. What is not known is the way in which the plant knows of the presence of the bacterium, that is the model’s gray area. Possibly the plant takes the presence of the bacteria as a positive signal from the environment. When that signal is high, it spawns a build program X; if not, the development program is Y.

–But does Azospirillum affect plant hormones?

–There are reports of production of almost all hormones by this bacterium: ethylene, abscisic acid, it synthesizes various forms of cytokinins, there are many studies regarding auxins, of which it is a great generator. It also produces nitric oxide, an important regulator of root development.

However, he adds, there is a lack of bacterial research related to phytohormones, there is little information regarding its metabolic pathway.

The most common use of Azospirillum involves seed treatment, since it is the most economic and efficient practice in extensive crops, indicates the specialist. But there is also technical information regarding other practices, such as foliar application in soybeans, corn and wheat.

– Products that are recommended for foliar treatment are already being offered. We know that the production of hormones, of auxins in particular, have an important role in foliar use. However, I repeat, the presence of the bacteria makes the system work differently. You can apply auxins to the foliage and a completely different effect takes place compared to when you incorporate the bacteria alone or together with said hormone.

CONTRIBUTION FOR A GREATER EFFICIENCY IN THE USE OF FERTILIZERS

–Regarding the use with fertilizers, what is the current proposal?

–The idea is to lower the fertilization dose by incorporating a biological product, because there is already a general agreement in the world about the impact of chemical fertilizers on the environment, especially global warming. Making their use more efficient does not mean to stop using them completely, because there are crops whose production, realistically, cannot be imagined without them. In legumes like soybeans, nitrogen comes entirely from biological fixation, but in other crops, if you don't add it, the plant won't produce unless it finds it in the soil. And agricultural soils usually do not have the amount necessary to solve the producer's commercial equation. The numbers indicate that lower fertilization doses, with inoculation, end up giving a similar result compared to higher fertilization doses without inoculation. Fertilizer savings means money savings. A producer who uses 60 kg less N/ha obtains an ecological as well as an economic benefit.

– What is your research team focusing on?

-Some time ago it was thought that there was no communication between the plant and the bacterium. Then it was suggested that there could be some kind of communication, and today we are trying to understand how this dialogue occurs, how the plant can perceive the microorganism.

THE NEW CERTAINTIES THAT OMICS DEVELOPMENT HAS BROUGHT

What leads are you following?

–There are particular structures, secretion systems of a specific type of protein that allow anchoring to other cells (they can be, for example, other bacterial cells or eukaryotic cells [cells with a true nucleus typical of organisms such as animals, plants and fungi]) and we understand that they have a role in the communication process. Omics technologies [genomics, metabolomics, transcriptomics, etc.] have also made it possible to look inside the genome and understand its organization, which has generated many lines of work. Thanks to this, a very significant advance in recent years has been the ability to generate tools for the unequivocal identification of the bacterium. Azospirillum has a marked pleomorphism [appearance of multiple structural forms during its life cycle], which made it extremely difficult to trace it from the time you put it in the seed until it became a plant, due to the difficulty of recognizing it by observation or in the laboratory. Now it is possible to follow the bacteria in this process, there are tools with 100% efficiency that allow us to say: yes, it is the bacteria that I inserted.

-What are the practical implications of this?

–It allows us to shed light on issues that were previously difficult to understand or that were misunderstood, limited by technology. In other words, when you wanted to know how long the microorganism survived in the seed, you recovered it and counted it on a plate, but in many cases there was no certainty that the bacteria you were counting was the one you had put. With the new tools you know unequivocally and precisely how much of what you put in survived, and based on that you have the possibility of correcting variables such as the formulation or the way of cultivating the bacteria to achieve greater survival in unfavorable conditions. This had a great impact on the improvement of products such as those for foliar application, for example, which would be very difficult to develop without understanding the effect of light on the bacteria.

Another rapidly evolving matter concerns the identification of new species:

-There are an increasing number of works on the subject. There has been a whole taxonomic modification, a repositioning. Today a new family, Azospirillaceae, was established, which was previously considered part of another family.

Likewise, the use of the concept of microbiome, referring to all the microorganisms present in a specific environment, has intensified.

–The idea is understanding why, in agriculture, if you put a growth-promoting bacterium in a system with a biological balance in which there are millions of bacteria with different capacities, even in a numerically lower amount, you can obtain a change in the plants and you can get proven benefits.

THE LIMITS OF THE MAP OF AZOSPIRILLUM SPECIES AND STRAINS ARE CHANGING

Survival of Azospirillum in agricultural systems has been found to be quite low:

–It enters the ground floor system, its effect is evident, but then the signal is lost, as if it had never been applied. Unlike bacteria aimed at biological control, whose antibiosis mechanisms behave more aggressively with the environment, Azospirillum does not have negative effects on other microorganisms, except for a possible exception not yet fully confirmed. And it is also different from Rhizobium, because, in Argentina, naturalized populations have established in the soil over time. On the other hand, even in research it is not easy to find and isolate Azospirillum from the soil.

–So you have to apply it over and over again?

-Of course. Having found a bacterium with the ability to interact with plants but without being recalcitrant on the system was a great coincidence at the time they began to select microorganisms for agriculture.

–Regarding the description of new species, are there any that “compete” with Azospirillum brasilense commercially?

–Until 2020, I would say 100% of the products were formulated based on Azospirillum brasilense. But there are more and more species. Some strains that were thought to be the Brazilian type are no longer so. For example, the Sp245 strain is now part of A. baldaniorumand the Az39, which has been used in Argentina for more than 40 years, is very close to being recognized as another species, which will be called A. argentinense. Obviously, they have characteristics that differentiate them. However, I don't know if there are differences in functional terms. The same has happened with Rhizobia, in the case of the USDA110 strain, which was previously included in Bradyrhizobium japonicum and is now B. diazoefficiens.

–Will advances like the ones you described help improve the 75-80% results obtained in the trials, which you mentioned before?

–In the refinement of the tools that are being generated for evaluation, yes. Not only in terms of identifying new species, but also the ability to trace the bacteria and attribute the effect that correspond to that bacteria alone. The variability that existed has been reduced. It annoys me when I evaluate scientific papers where they continue to use the phrase “the results of Azospirillum inoculation are still under discussion…” With 20 million doses of commercial product there is not much to discuss. To improve, yes. The impact needs to be better understood, which is associated with having more powerful measurement tools. "Sharpen the pencil’s point", especially at levels as sensitive as the combined use with fertilizers, where you need numbers that help. Today the number is between 40 and 80 kg less N/ha, depending on the crop. As the data becomes more precise, it impacts and improves the practice.