Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Plant Growth-Promoting Bacteria
Nitrogen Fixation

Under natural conditions, plant growth rate and yield depend on their genotype, nutrient availability, the presence of beneficial soil microorganisms, and the absence of pathogens (so-called phytopathogens, from phyto — plant). Some species of beneficial natural soil Bacteria and Fungi exert a direct effect, while others act indirectly. The former provide plants with growth-promoting compounds, whereas the latter suppress the proliferation of pathogenic soil microorganisms, preventing their negative impact on the plant.

The primary mechanisms of Plant Growth Promotion by "direct-acting" microorganisms include: 1) Fixation of Atmospheric nitrogen, which is subsequently utilized by the plant; 2) formation of readily assimilable forms of iron and phosphorus and/or uptake from the soil and delivery of these beneficial minerals to the plants; 3) synthesis of phytohormones that induce plant Cell proliferation. Indirect plant growth promotion by a beneficial microbial strain occurs by preventing the growth of phytopathogenic soil microorganisms that could otherwise adversely affect normal Plant GROWTH AND DEVELOPMENT. This action is termed antibiosis and may involve either the depletion of a limiting substrate by the beneficial microorganism or the Synthesis and Secretion of a compound that inhibits phytopathogen growth.

Recent genetic experiments aimed at developing microbial strains capable of more effectively promoting plant growth have focused primarily on addressing the following four challenges.

✵ MOLECULAR MECHANISMS OF nitrogen fixation. The goal of all research has been to evaluate the potential for increasing nitrogen fixation levels by microorganisms, thereby reducing The amount of chemical fertilizers applied to the soil.

✵ ROOT nodule formation by symbiotic bacteria. The objective of these studies was to engineer recombinant bacteria capable of competing with naturally occurring symbiotic bacteria.

✵ Microbial synthesis of iron-chelating compounds (siderophores). It is hoped that microbial strains capable of suppressing phytopathogen growth can be obtained.

✵ Microbial synthesis of phytohormones. These studies were conducted to develop bacterial strains that synthesize and secrete specific amounts of phytohormones to accelerate plant growth.

Research in this field is conducted primarily on bacteria rather than fungi. This is partly because beneficial fungi are difficult to culture, making them challenging to work with under laboratory conditions, and furthermore, it is impossible to obtain these organisms in quantities sufficient for inoculation.

Nitrogen (N₂) is a gas that accounts for approximately 80% (by volume) of the air we breathe. Neither plants nor animals can utilize it directly to synthesize the essential biological nitrogen-containing compounds they require, such as Amino Acids and NUCLEOTIDES; nitrogen must first be incorporated into ammonia (fixed). This is an energy-intensive process because the triple bond in the N₂ molecule (N = N), which must first be broken, is extremely strong. The energy for Biological Nitrogen Fixation is released during the Hydrolysis of large amounts of adenosine triphosphate (ATP). High temperatures and pressures are required for the chemical (industrial) conversion of N₂ into ammonia.

To meet the food industry's demand for agricultural products, more than 100 million tons of fixed nitrogen are required annually. Approximately half of this amount is supplied by synthetic (chemically synthesized) fertilizers, while plants obtain most of the remaining half from nitrogen-fixing (diazotrophic) bacteria such as Rhizobium, Frankia, Azospirillum, Azotobacter, and cyanobacteria. No eukaryotic Organism is capable of fixing nitrogen.

While the application of chemical fertilizers has significantly increased crop yields, their long-term use leads to soil pollution and nutrient depletion. Moreover, chemical fertilizers are becoming increasingly expensive. All of this has stimulated the search for alternative sources of fixed nitrogen, particularly The Development of diazotrophic microbial strains that could serve as "bacterial fertilizers."

A wide variety of bacteria possess The ability to fix nitrogen, and many of them could, in principle, be used as fertilizers. However, until bacterial fertilizers are shown to be as effective as chemical ones, it is unlikely that the conservatism of agricultural producers can be overcome and current practices changed. For example, soybean—the second most economically important and widely grown crop in the US—establishes a symbiotic relationship with the bacterium Bradyrhizobium japonicum. Through this Symbiosis, the bacteria provide the plant with fixed nitrogen, while receiving readily assimilable forms of carbon produced during Photosynthesis. Following plant inoculation with certain B. japonicum strains, the final yield of plant biomass can increase by 25–50%, eliminating The Need for any chemical nitrogen supplements. Approximately 50% of soybeans are grown in a few US regions, generally utilizing similar technology. Yet, only a small fraction of this crop is currently treated with B. japonicum. Farmers still rely on native B. japonicum strains and chemical fertilizers.

The reputation of bacterial fertilizers is highly controversial. In the 1950s, more than 10 million hectares of agricultural land in the USSR were treated with a mixture of diazotrophic bacteria, primarily consisting of Azotobacter chroococcum and Bacillus megaterium. In approximately 60% of cases, the yields of various grain crops increased by 10–20%. However, these field trials proved to be flawed and irreproducible; many researchers questioned the validity of the results, and The Use of bacterial inoculants as fertilizers did not gain traction. Nevertheless, economic concerns, the need to prevent environmental pollution, and The Emergence of new technologies have prompted scientists to re-examine the potential of bacterial fertilizers.

The microorganisms currently used in agriculture belong primarily to two genera: Rhizobium and Bradyrhizobium. These are Gram-negative, rod-shaped, flagellated bacteria that form symbiotic relationships with legumes. Each species of Rhizobium and Bradyrhizobium is specific to only a small number of plant species and does not interact with non-host plants (Table 14.1). At a specific stage of its life cycle, Rhizobium penetrates the plant root Cells and initiates a cascade of changes leading to root nodule formation. Within the root nodule, the bacteria proliferate rapidly and exist in a form lacking a Cell wall. These nodule bacteria fix atmospheric nitrogen using the enzyme Nitrogenase. The Structural and Biochemical interactions between the symbionts—Rhizobium and the host plant—are highly complex and mutually beneficial. Within the nodule, nitrogenase is protected from the Toxic effects of atmospheric oxygen in two ways. First, oxygen barely penetrates the nodule. Second, the oxygen level inside the nodule is regulated by the protein leghemoglobin. The heme component of this oxygen-binding protein is synthesized by the bacterium, while the globin part of the molecule is encoded by the plant genome. The plant provides the bacteria with the fixed carbon sources necessary for growth, produced during photosynthesis, while the plant benefits from this symbiotic relationship by receiving fixed nitrogen from the bacterium.

Class="center">Table 14.1. Specificity of Rhizobium and Bradyrhizobium species toward different plants