Read this article to learn about the growth-promoting microorganisms in plants with respect to three aspects.
The three aspects are: (1) Biological Nitrogen Fixation (2) Bio-control of Phytopathogens and (3) Bio-fertilizers.
Certain beneficial microorganisms, present in the soil, are known to influence the plant growth, development and yield. These bacteria and fungi may provide growth-promoting products to plants or inhibit the growth of soil pathogenic microorganisms (phytopathogens), which hinder the plant growth. The former is the direct effect while the latter is the indirect effect of growth- promoting bacteria in plants.
The growth-promoting activity of microorganisms and the biotechnological approaches are described briefly with respect to the following aspects:
1. Biological nitrogen fixation.
2. Bio-control of phytopathogens.
3. Bio-fertilizers.
1. Biological Nitrogen Fixation:
Nitrogen is an essential element of many biomolecules, the most important being nucleic acids and amino acids. Although nitrogen is the most abundant gas (about 80%) in the atmosphere, neither animals nor plants can use this nitrogen to synthesize biological compounds. However, there are certain microorganisms on which the living plants (and animals) are dependent to bring nitrogen into their biological systems.
The phenomenon of fixation of atmospheric nitrogen by microorganisms is known as diazotrophy and these organisms are collectively referred to as diazotrophs. Diazotrophs are biological nitrogen fixers, and are prokaryotic in nature.
Nitrogen Cycle:
An outline of the nitrogen cycle is depicted in Fig. 52.1. Nitrogen enters the soil with the deposits of dead animals and plants, and urea of urine. These waste materials (proteins, urea) are decomposed by soil bacteria into ammonia and other products. The ammonia is converted to nitrite (NO2) and then nitrate (NO3) by certain bacteria belonging to the genera Nitrosomonas and Nitrobacter.
The nitrate is degraded by various microorganisms to release nitrogen that enters atmosphere. This atmospheric nitrogen is taken up by the nitrogen fixing bacteria (present on the roots of leguminous plants) and used for the synthesis of biomolecules (e.g. amino acids). As the animals consume the leguminous plants as food, the nitrogen cycle is complete.
Nitrogen Fixing Bacteria:
It is estimated that about 50% of the nitrogen needed by the plant comes from nitrogen fixing bacteria. These are two types of nitrogen fixing microorganisms-asymbiotic and symbiotic.
Asymbiotic nitrogen fixing microorganisms:
The gaseous nitrogen of the atmosphere is directly and independently utilized to produce nitrogen-rich compounds. When these non- symbiotic organisms die, they enrich the soil with nitrogenous compounds. Several species of bacteria and fungi can do this job e.g. Clostridium pasturianum, Azatobacter chrooccum. The mechanism of nitrogen fixation by asymbiotic bacteria is not clearly understood. It is believed that nitrogen is first converted to hydroxylamine or ammonium nitrate, and then incorporated into biomolecules.
Symbiotic nitrogen fixing microorganisms:
These microorganisms live together with the plants in a mutually beneficial relationship, phenomenon referred to as symbiosis. The most important microorganisms involved in symbiosis belong to two related genera namely Rhizobium and Brady-rhizobium. These symbiotic bacteria also referred to as nodule bacteria are Gram negative, flagellated and rod-shaped. The host plants harbouring these bacteria are known as legumes e.g. soybean, peas, beans, alfalfa, peanuts, and clover.
Each one of the species of Rhizobium and Bradyrizobium are specific for a limited number of plants, which survive as the natural hosts (Table 52.1). It is now clearly known that these bacteria do not interact with plants other than the natural hosts.
The relationship between the symbiotic bacteria and the legumes is well recognized. On the roots of legumes, there are a number of nodules (swellings) in which Rhizobium sp thrive. These bacteria trap atmospheric nitrogen and synthesize nitrogen-rich compounds (amino acids, proteins etc.) used by the legumes. At the same time, the legumes supply important nitrogen compounds for the metabolism of Rhizobia.
The growth of legumes has been known to enrich the soil fertility. This is due to the fact that the concentration of nitrogen compounds in the soil increases as a result of the presence of symbiotic bacteria. For this reason normally, nitrogen fertilizers are not needed in the fields cultivated legumes.
Mechanism of Nitrogen Fixation:
Inside the root nodules of leguminous plants, the bacteria proliferate. These bacteria exist in a form that has no cell wall. The bacteria of the nodules are capable of fixing nitrogen by means of the specific enzyme namely nitrogenase.
Nitrogenase:
Nitrogenase is a complex enzyme containing two oxygen sensitive components. Component I has two α-protein subunits and two β-protein subunits, 24 molecules of iron, two molecules of molybdenum and an iron molybdenum cofactor (FeMoCo). Component II possesses two a-protein subunits (different from that of component I) and a large number of iron molecules. Component I of nitrogenase catalyses the actual conversion of N2 to ammonia while component II donates electrons to component I (Fig. 52.2).
Leg-hemoglobin:
A protein comparable of hemoglobin in animals has been identified in the nodules of leguminous plants. Leg-hemoglobin (LHb) contains iron and is red in colour. It is an oxygen binding protein. The heme part of leg-hemoglobin is synthesized by the bacterium while the protein (globin) portion is produced by the host plant. Leg-hemoglobin is absolutely necessary for nitrogen fixation. The nodules that lack LHb are not capable of fixing nitrogen.
It is LHb that facilitates the appropriate transfer of oxygen (by forming oxyLHb) to the bacteria for respiration to produce ATP. And energy in the form of ATP is absolutely required for nitrogen fixation. Another important function of LHb is that it prevents the damaging effects of direct exposure of O2 on nitrogenase.
In Fig. 52.2, the fixation of nitrogen by symbiotic bacteria is depicted. As the oxyLHb supplies O2, bacterial respiration occurs. The ATP generated is used for fixing nitrogen to produce ammonia. The complex reaction is summarized below.
N2 + 8H+ + 8e– + 16 ATP
↓
2NH3 + H2↑ + 16 ADP + 16 Pi
Hydrogenase:
During the course of nitrogen fixation by nitrogenase, an undesirable reaction also occurs. That is reduction of H+ to H2 (hydrogen gas). For the production of hydrogen, ATP is utilized, rather wasted. Consequently the efficiency of nitrogen fixation is drastically lowered. It is possible theoretically to reduce the energy wastage by recycling H2 to form H+.
In fact, some strains of Brady rhizobium japonicum in soybean plants were found to use hydrogen as the energy source. These strains were found to possess an enzyme namely hydrogenase. Recycling of the hydrogen gas that is formed as a byproduct in nitrogen fixation is shown in Fig. 52.3.
It is advantageous for nitrogen fixation if the symbiotic bacteria possess the enzyme hydrogenase. However, the naturally occurring strains of Rhizobium and Bradhyrhizobium do not normally possess the gene encoding hydrogenase.
Genetic Manipulations for Nitrogen Fixation:
The nitrogen-fixing bacteria that are closely associated with the world’s food supply are among the favoured organisms for genetic manipulations. Biotechnologists consider the following possibilities for effective utilization of diazotrophs (or the process of diazotrophy) as natural biological fertilizers.
i. Gene alterations in Rhizobium sp to improve nitrogen fixing efficiency, and bacteria-host plant interactions
ii. Genetic engineering of Rhizobium sp so that it can form a symbiotic relationship with non- leguminous plants such as wheat, rice and corn.
iii. Transfer of genes for nitrogen fixation from Rhizobium sp to other bacteria such as Agrobacterium tumefaciens. The so developed A. tumefaciens can infect several important crops (e.g. tomato, tobacco, petunia) and fix nitrogen.
Some other considerations include the insertion of nitrogen-fixing genes into plants or even animals including humans. This approach, which enables the plants, animals and humans to directly trap and utilize nitrogen from the atmosphere, is rather theoretical. Life would be certainly different if introduction of nitrogen-fixing genes in man can dispense with the habit of eating daily! Despite several possibilities and optimistic predictions, the success in the genetic engineering in relation to nitrogen-fixation is very limited, and briefly described below.
Genetic Engineering of Nitrogenase Gene:
It is absolutely necessary to identify, isolate and characterize the nitrogen-fixing genes, nif genes to undertake any kind of genetic manipulations. Genetic complementation is the common technique used for the isolation nif genes. The procedure basically involves the identification and characterization of clones from a wild-type library to restore nitrogen fixation in the various mutants of the original organism.
The nif genes were first isolated from the clone banks of the diazotroph Klebsiella pneumonia. This organism is found in soil, water and human intestine, and its molecular biology is well known. The isolation of nif genes by genetic complementation from K. pneumonia is depicted in Fig. 52.4, and involves the following steps.
1. The wild-type DNA of K. pneumonia (Nif+) was cut with restriction endonucleases.
A clone bank was constructed with a vector and maintained in E.coli.
2. K. pneumonia cells are exposed to a mutagenic agent. This may result in the mutation of nif genes to form Nif– cells.
3. The Nif– K. pneumonia cells are then conjugated with E.coli cells, carrying the clone bank on a vector.
4. The trans-formants of K. pneumonia possessing Nif+ phenotype can be selected by growing the cells on a minimal medium that does not contain fixed nitrogen.
5. The DNA fragment in the plasmid that contains nif genes, which complements Nif– mutation in K. pneumonia can be isolated.
The isolation of nif genes will be more effective if a series of independently derived Nif– mutants are employed in genetic complementation experiments. This approach increases the likelihood of different nif genes isolation. Further, by using DNA hybridization probes of nif genes, DNA clone bank of K. pneumonia can be screened. This allows the identification of additional genes that are involved in nitrogen fixation.
Nitrogenase gene cluster:
The entire set of nitrogenase genes from K. pneumonia has been identified. Some highlights are listed:
i. The nitrogenous (nif) genes are located as a single cluster, occupying approximately 24kb of the bacterial genome.
ii. There are seven distinct operons that encode 20 different proteins.
iii. All the nif genes transcribe and translate in a well-coordinated fashion.
iv. The nif genes are under the regulatory control of two genes namely nifA and nifL.
Although the nif genes have been characterized from K. pneumonia, for technical reasons, the contribution of this organism for biological nitrogen fixation is almost insignificant. However, the nif genes of K. pneumonia have been used as hybridization probes to identify nif genes from the DNA clone banks of diazotrophic organisms (particularly Rhizobium sp). It is now known that almost all the nitrogen-fixing bacteria possess similar type of nif gene clusters.
Manipulation of nif genes:
Some workers have been successful in introducing extra copies of nitrogenase regulatory genes namely nifA and nifL into diazotrophs. For instance, insertion of more nifA genes into Rhizobium meliloti resulted in an increased biomass production in alfalfa plants.
This is due to the fact that nitrogen fixation in these plants is much increased. But the major limitation of this approach is that the genetically engineered bacteria have a diminished growth rate. As a result, these newly developed bacteria loss their efficiency as plant-growth promoting agents.Despite continuous efforts by several groups of workers, no significant success has been reported in the transfer of nif genes into plants.
The major limitations are listed:
i. Transfer of 24 kb nif gene cluster has not been effective since the normal cellular concentration of oxygen would inactivate nitrogenase enzyme. Any reduction in O2 results in cell death.
ii. Transfer of one or two genes of nif gene cluster is useless.
iii. There are no plant promoters that can respond to nifA regulatory protein. Consequently, it is not possible to turn on the nif genes in transgenic plants.
iv. The plant cells cannot process nif gene transcripts, which are multi-genic in nature.
For the various reasons given above, it has not been so far possible to introduce functional nitrogen fixation capability into plants.
Genetic Engineering of Hydrogenase Gene:
The role of the enzyme hydrogenase in promoting nitrogen fixation has already been described Hydrogenase is synthesized by hup (hydrogen uptake) genes, which are not present in the naturally occurring Rhizobial strains. Considerable variations have been identified in hydrogenases from different organisms. There are different types of hydrogenases, which usually contain subunits. Different genes code these subunits.
Isolation of hydrogenase genes:
The technique of genetic complementation can be successfully employed for this isolation of hydrogenase (hup) genes. In fact, hup genes were first isolated from E.coli by this approach. Later, hup genes from B.japonicum have also been isolated.
Manipulation of hup genes:
The hydrogenase (hup) genes have been introduced into R.leguminosarum. These Hup+ strains of bacteria, when inoculated into legumes, resulted in higher nitrogen fixation.
Genetic Engineering of Nodulation Genes:
Establishment of nodules on the roots of leguminous plants is a prerequisite for nitrogen fixation. Certain genes involved in nodulation namely nod genes have been identified in Rhizobium melitoti. The technique of genetic complementation has been used to isolate nod genes from R. melitoti. A large number of nod genes (about 20 nodA — nodX) have been identified in diazotrophs.
The nod genes are broadly divided into three groups:
i. Common genes
ii. Host-specific genes
iii. Regulatory genes.
The functions of each one of the nod genes in nodulation have not been clearly identified. Further, many more new nod genes are being discovered every year.
Manipulation of nod genes?
The process of nodulation is complex through the participation of a large number of nod genes, besides various other factors-concentration of nutrients, soil temperature, light, CO2 concentration etc. Despite attempts by several workers, no success has been reported to enhance the ability of Rhizobium sp for nodulation through genetic manipulations.
2. Bio-control of Phytopathogens:
Phytopathogens can drastically reduce the crop yield, which may be in the range of 25-100%. Chemical agents are commonly used to control them. This is associated with ill-health affects on humans, besides environmental pollution. There are certain bacteria that can lessen or prevent the deleterious effects of plant pathogens- fungi or bacteria. And thus, they promote plant growth by indirect means.
Plant growth-promoting bacteria are capable of producing a wide range of substances that can restrict the damage caused by phytopathogens to plants. The important plant growth-promoting substances are siderophores, antibiotics and certain enzymes.
Siderophores:
There are some bacteria in the soil that can synthesize a low molecular weight (400-1,000 Daltons) iron-binding peptides, collectively referred to as siderophores. Siderophores have high affinity to bind to iron in the soil and transport it to the microbial cell. This is required since iron is essential, and cannot directly enter the bacteria due to a very low solubility.
The growth-promoting bacteria, through siderophores can take up large quantities of iron from the soil, and make it unavailable for the growth and existence of fungal pathogens. This is possible since the siderophores produced by fungi have a very low affinity when compared to siderophores of bacteria. There is no harm to the plants since they can grow at a much lower concentration of iron in the soil.
Genetic manipulations for siderophores:
It is now clearly established that siderophores can prevent the proliferation of phytopathogens. It is therefore logical to think of siderophore genes for more effective bio-control of plant pathogens. Pseudobactin is a siderophore synthesized by plant growth-promoting bacteria Pseudomonas putida. By genetic complementation and other techniques of molecular biology, at least five separate gene clusters involved in pseudobactin production have been identified.
Genetic manipulation for improved synthesis of siderophore is not an easy job, since it is a complex process involving a large number of genes. Some success, however, has been reported in cloning the genes for iron siderophore receptors from certain plant growth-promoting bacteria, and introducing them into other bacteria.
Antibiotics:
Certain antibiotics produced by plant growth- promoting bacteria, can prevent the growth and proliferation of plant pathogens. Some of the antibiotics synthesized by pseudomonads are agrocin 84, agrocin 434, herbicolin, phenazine, oomycin, pyrrolinitrin.
Genetic manipulations for antibiotics:
It is possible to enhance the growth-promoting activity of the bacteria by inserting genes encoding the synthesis of antibiotics. In fact, a genetically engineered bio-control bacterium of Agrobacterium radiobacter has been developed, and is being marked commercially since 1989. This transgenic organism produces the antibiotic agrocin 84 that is toxic to crown gall disease-causing Agrobacterium tumefaciens. By this biocontrol approach, the crop yield of almond trees and peach trees can be improved by preventing crown gall disease.
Enzymes:
Certain plant growth-promoting bacteria are capable of synthesizing some enzymes that can degrade fungal cell walls and lyse them. These enzymes include chitinase, β-1,3-glucanase, protease and lipase.
Genetic manipulation for enzymes:
The genes responsible for encoding the enzyme namely chitinase and β-glucanase have been isolated and characterized. Chitinase gene has been isolated from Serralia mercescens and introduced into Rhizobium meliloti and Trichoderma harzianum. The genetically engineered organisms displayed an increased antifungal activity. By this bio-control approach, the phytopathogen Rhizoctonia solani has been effectively controlled.
3. Bio-fertilizers:
It is estimated that more than 100 million tons of fixed nitrogen are needed for global food production. The use of chemical/synthetic fertilizers is the common practice to increase crop yields. Besides the cost factor, the use of fertilizers is associated with environmental pollution.
Scientists are on a constant look for alternate, cheap and environmental-friendly sources of nitrogen and other nutrients for plants. The term bio-fertilizers is used to refer to the nutrient inputs of biological origin to support plant growth. This can be achieved by the addition of microbial inoculants as a source of bio-fertilizers.
Bio-fertilizers broadly includes the following categories:
i. Symbiotic nitrogen fixers
ii. Asymbiotic nitrogen fixers
iii. Phosphate solubilizing bacteria
iv. Organic fertilizers.
The most important microorganisms used as bio-fertilizers along with the crops are listed in Table 52.2.
Some of the important features of these bio-fertilizers are briefly described.
Symbiotic Nitrogen Fixers:
The diazotropbic microorganisms are the symbiotic nitrogen fixers that serve as bio-fertilizers. e.g. Rhizobium sp and Brady rhizobium sp. The details on these bacteria with special reference to nitrogen fixation must be referred now. Many attempts are being made (although the success has been limited) to genetically modify the symbiotic bacteria for improving nitrogen fixation.
Green manuring:
It is a farming practice wherein the leguminous plants which are benefited by the symbiotic nitrogen fixing bacteria are ploughed into the soil and a non-leguminous crop is grown to take benefits from the already fixed nitrogen. Green manuring has been in practice in India for several centuries. It is a natural way of enriching the soil with nitrogen, and minimizing the use of chemical fertilizers. Rhizobium sp can fix about 50-150 kg nitrogen/hectare/annum.
Asymbiotic Nitrogen Fixers:
The asymbiotic nitrogen-fixing bacteria can directly convert the gaseous nitrogen to nitrogen- rich compounds. When these asymbiotic nitrogen fixers die, they enrich the soil with nitrogenous compounds, and thus serve as bio-fertilizers e.g. Azobacfer sp, Azospirillum sp.
Blue-green algae (cynobacteria):
Blue-green algae multiply in water logging conditions. They can fix nitrogen in the form of organic compounds (proteins, amino acids). The term algalization is used to the process of cultivation of blue-green algae in the field as a source of bio-fertilizer.
Blue-green algae, besides fixing nitrogen, accumulate biomass, which improves the physical properties of the soil. This is useful for reclamation of alkaline soils besides providing partial tolerance to pesticides. Cynobacteria are particularly useful for paddy fields. The most common blue-green algae are Azobacter sp and Azospirillum sp.
Azolla:
Azolla is an aquatic fern, which contains an endophytic cynobacterium Anabaena azollae in the leaf cavities providing a symbiotic relationship. Azolla with Anaebaena is useful as biofertilizer.
But due to certain limitation (listed below), the use of Azolla has not become popular:
i. Azolla plant requires adequate supply of water.
ii. It can be easily damaged by pest diseases.
iii. Azolla cultivation is labour intensive.
Phosphate Solubilizing Bacteria:
Certain bacteria (e.g. Thiobacillus, Bacillus) are capable of converting non-available inorganic phosphorus present in the soil to utilizable (organic or inorganic) form of phosphate. These bacteria can also produce siderophores, which chelates with iron, and makes it unavailable to pathogenic bacteria. Thus, besides making phosphate available, the plants are protected from disease – causing microorganisms.
Mycorrhizas:
Mycorrhizas are the fungus roots (e.g. Glomus sp) with distinct morphological structure. They are developed as a result of mutual symbiosis between certain root-inhabiting fungi and plant roots. Mycorrhizas are formed in plants, which are limited with nutrient supply. These plants may be herbs, shrubs and trees. For the development of mycorrhizas, the fungus may be located on the root surface (ectomycorrhizas) or inside the root (endomycorrhizas).
In recent years, an artificially produced inoculum of mycorrhizal fungi is used in crop fields. This practice improves plant growth and yield, besides providing resistance against biotic (pathogens) and abiotic (climatic changes) stress. Mycorrhizas also produce plant growth-promoting substances.
Organic Fertilizers:
There are several organic wastes, which are useful as fertilizers. These include animal dung, urine, urban garbage, sewage, crop residues and oil cakes. A majority of these wastes remain unutilized as organic fertilizers. There exists a good potential for the development of organic manures from these wastes.
Benefits of Bio-fertilizers:
i. Low cost and easy to produce. Small farmers are immensely benefited.
ii. Fertility of the soil is increased year after year.
iii. Free from environmental pollution.
iv. Besides nutrient supply, some other compounds, which promote plant growth, are also produced e.g. plant growth hormones, antibiotics.
v. Bio-fertilizers increase physicochemical properties of the soil, soil texture, and water holding capacity.
vi. Reclaimation of saline or alkaline soil is possible by using bio-fertilizers.
vii. Bio-fertilizers improve the tolerance of plants against toxic heavy metals.
viii. Plants can better withstand biotic and abiotic stresses and improve in product yield.
Limitations of Bio-fertilizers:
i. Bio-fertilizers cannot meet the total needs of the plants for nutrient supply.
ii. They cannot produce spectacular results, as is the case with synthetic fertilizers.
Considering the advantages and disadvantages of bio-fertilizers, a realistic and pragmatic approach is to use combination of bio-fertilizers and synthetic fertilizers for optimum crop yield.