ENVIRONMENTAL MICROBIOLOGY

Terrestrial Environment Neeru Narula and Manjula Vasudeva

CONTENTS Introduction Rhizosphere Phyllosphere Brief account of microbial interactions Competition Rumen microbiology Biofertilizers Biological N2 fixation Nitrogenase enzyme The nif genes Symbiotic N2 fixation Rhizobium Frankia Non-symbiotic N2 fixation Azotobacter and Azospirillum Mycorrhizae

Keywords
Terrestrial Environment, Rhizosphere, Phyllosphere, Brief account of microbial interactions, Competition, Rumen microbiology, Biofertilizers, Biological N2 fixation, Nitrogenase enzyme, The nif genes, Symbiotic N2 fixation, Rhizobium, Frankia, Non-symbiotic N2 fixation, Azotobacter and Azospirilum, Mycorrhizae.

Introduction Terrestrial environment is the process occurring within the soil and on or near the plants that influence the functioning of the ecosystem. The process of soil development involves complex interactions among the parent material (rock, sand, glacial drift etc.), topography, climate and living organisms. The term soil refers to the outer, loose material of the earths surface, a layer distinctly different from the underlying bedrock. Agriculturally, it is the region supporting plant life and from which plants obtain their mechanical support and many of their nutrients. Chemically, the soil contains a multitude of organic substances not present in the underlying strata. For microbiologists, the soil environment is unique in many ways. It contains a vast population of bacteria, actinomycetes, fungi, algae and protozoa. It is one of the most dynamic sites of biological interactions in nature; and it is the region where occur many of the biochemical reactions concerned in the destruction of organic matter, in the weathering of rocks and in the nutrition of agricultural crops. The physical and chemical characteristics of soil determine the nature of the environment in which microorganisms are found. These environmental characteristics in turn affect the composition of the microscopic population both qualitatively and quantitatively. It is from the soil that the water, air and the inorganic and organic nutrients are obtained. The soil serves as a buffer to the drastic changes that occur above the ground. The organisms like algae, lichens or mosses remain dormant on the dry rock and grow when moisture is present. They are phototrophic and produce organic matter which supports the growth of chemoorganotrophic bacteria and fungi. The number of chemoorganotrophs increase directly with the degree of plant cover of the rocks. CO2 produced during respiration by chemoorganotrophs is converted to carbonic acid (CO2 + H2O ------ H2CO3), which is involved in the dissolution of lime stone rocks. Many chemoorganotrophs excrete organic acids which further promote dissolution of rocks into smaller particles. Freezing and thawing also cause cracks in the rocks. In these crevices, raw soil forms and pioneering higher plants can develop. Plant roots penetrate into crevices and increase the fragmentation of the rock and hence develop rhizosphere (soil that surrounds plant roots) microflora. When the plants die, their remains are added to the soil and become nutrients for microbial development. Minerals are further rendered soluble and as water percolates, it carries some of these chemical substances deeper. As weathering proceeds, the soil increases in depth, thus permitting the development of large plants and trees. Soil animals play an important role in keeping the upper layers of the soil mixed and aerated. The movement of materials downwards results in the formation of many layers known as the soil profile which is dependent on climatic and other factors and takes hundreds of years to be formed. The soil is a complex habitat with numerous microenvironments and niches. Microorganisms are present in the soil primarily attached to soil particles. The most important factor influencing microbial activity in surface soil is the availability of water, whereas in deep soil nutrient availability plays a major role. The organic fraction of soil, often termed humus, contains the organic carbon and nitrogen which is needed for microbial development, is the dominant food reservoir. The greatest microbial activity is in the organic-rich surface layers, especially in and around the rhizosphere. The numbers and activity of soil microorganisms depend to a great extent on the balance of nutrients present. In some soils carbon is not the limiting nutrient, but instead the availability of inorganic nutrients such as phosphorous and nitrogen limit microbial productivity. 2

The deep soil surface, which can extend for several hundred meters below the soil surface, is not a biological wasteland. A variety of microorganisms, primarily bacteria, are present in most deep underground soils. In samples collected aseptically from bore holes drilled down to 300 m, a diverse array of bacteria have been found including anaerobes such as sulphate reducing bacteria, methanogens and homoacetogens and various aerobes and facultative aerobes. Microorganisms in the deep subsurface presumably have access to nutrients because groundwater flows through their habitats, but activity measurements suggest that metabolic rates of these bacteria are rather low in their natural habitats. Compared to microorganisms in the upper layers of soil, the biogeochemical significance of deep subsurface microorganisms may thus be minimal. However, there is evidence that the metabolic activities of these buried microorganisms may over very long periods be responsible for some mineralization of organic compounds and release of products into the ground water (Fig. 1).

Fig. 1: Profile of mature soil (Biology of Microorganisms)

Rhizosphere Rhizosphere is the region immediately outside the root. It is a zone where microbial activity is usually high. Hiltner in 1904 observed the zone of intense microbial activity around the roots and named it as rhizosphere. The influence of the root on soil microorganisms starts immediately after seed germination which increases as the plant grows and reaches a maximum when plants have reached the peak of their vegetative growth. The bacterial count is almost always higher in the rhizosphere than it is in region of the soil devoid of roots, often many times higher. This is because roots excrete significant amounts of sugars, amino acids, hormones and vitamins, which promote such an extensive growth of bacteria and fungi that these organisms often form microcolonies on the root surface. Roots initially have little or nomicrobial colonization but as the plants grow in the soil, the root exudates composed of a mixture of nearly 18 amino acids, 10 sugars, 10 organic acids, mucilage and other substances together with sloughed-off root cap and other cells and exerts influence on microbial colonization. These nutrients allow the dormant spores to germinate. The rhizosphere microorganisms influence plant growth by controlling the availability and uptake of nutrients.

Phyllosphere Phyllosphere is the surface of the plant leaf, and under conditions of high humidity, as in wet forests in tropical and temperate zones, the microbial flora of leaves may be quite high. Leaf surface carries a heterogenous population of microbes, which grow, reproduce and multiply on leaves in dynamic equilibrium with the existing micro- and macro-environment. Many of the bacteria on leaves fix nitrogen and nitrogen fixation presumably aids these organisms in growing with the predominantly carbohydrate nutrients provided by leaves. The leaf surface microbes are important in several ways. For instance, some of them are known to fix atmospheric nitrogen for the benefit of higher plants, have antagonistic action against fungal parasites, degrade plant surface waxes and cuticles, produce plant hormones, decomposes plant material after leaf fall, activate plants to produce phytoalexins, have toxic effects on cattle, act as a source of allerginic air-borne spores and influence the growth behaviour and root exudation of plants. The age and position of a leaf on the plant is an important factor for microbial colonization of its surface. The physiological and biochemical status of the leaf greatly influences the population and composition of the microflora. The earliest colonizers on newly formed leaves have to face almost no competition as they are devoid of any microbes, and in fact, they receive a potential supply of surface nutrients. But as they get established, they face a relatively hostile environment because of widely fluctuating temperatures and the incidence of UV radiations. They may immediately grow utilizing the fresh supply of substrates present on the leaf surface or lie dormant and inactive until the leaf becomes senescent. The leaf surface medium comprises exudates, chemical compounds resulting from biological activity of various microbes including nitrogen fixers and components resulting from atmospheric pollution. The structure and the chemistry of the leaf surface influences the occurrence of the plant surface colonizers apart from physical factors like temperature, relative humidity, light and wind velocity which also interact with the leaf surface community in various ways. Leaves at the seedling stage of plants usually harbour the least number of microbes which increases as the plants age, reaching the maximum population only on leaves which start yellowing at maturity.

Brief account of Microbial Interactions Plants are exposed to very large numbers of microorganisms that are present in the soil and are deposited on leaves and stems. Plants are the prime source of nutrients for microorganisms because they are the main source of organic matter in the environment. They provide nutrients indirectly from plant exudates, the shedding of leaves, pollen, etc. and also from dead plant matter. In some cases, nutrients are provided directly to microorganisms that form close associations with plants. Associations with plants can range from those that are extremely detrimental to the plant, such as those with virulent pathogens, through interactions which do not appear to influence plant growth, to beneficial ones such as those formed with mycorrhizal fungi or nitrogen fixing bacteria. For most microorganisms, interactions with growing plants extend no further than the colonization of the surfaces of stems, leaves and roots because these are regions where exudates are available. Symbionts have developed methods that permit them to enter the host and obtain direct access to nutrients. For symbionts, such as Rhizobium and mycorrhizal fungi, the plant benefits is usually positively related to the amount of root that becomes invaded and fairly high levels of infection being most beneficial. A number of possible interactions may occur between two species. Odum has proposed the following relations: a) Neutralism in which the two microorganisms behave entirely independently b) Symbiosis, the two symbionts relying upon one another and both benefiting the relationship c) Protocooperation, an association of mutual benefit to the two species but without the cooperation being obligatory for their existence or for their performance of some reaction d) Commensalisms, in which only one species derives benefit while the other is unaffected e) Competition, a condition in which there is a suppression of one organism as the two species struggle for limiting quantities of nutrients, O2, space, or other common requirements f) Amensalism, in which one species is suppressed while the second is not affected, often the result of toxin production g) Parasitism and Predation, the direct attack of one organism upon another; h) Synergism, in field situations is the possible synergistic effect in the plant between inducing virus and other non related viruses which could be brought to those plants from outside sources e.g. TMV and cucumber mosaic virus together cause a more severe disease than either of them alone. It would thus seem unwise to introduce TMV into field grown tomatoes where the aphid-borne cucumber mosaic virus might be present in surrounding areas and eventually be transmitted to the tomato plants. Symbiosis is the result of interaction of the two partners and both partners are modified in some way to achieve this. This must arise by the interchange of molecules between the partners, which may act as signals which cause the partner to modify itself or which may themselves cause modifications. In the symbiosis, modifications to the host enable the partners to form the symbiosis, and the structure and physiology of both host and microorganism are adapted for the aerobic fixation of nitrogen. Two members are required for the association, a plant and microorganism. The classical example of such a symbiosis is between leguminous plants and bacteria of the genus 5

Rhizobium. The seat of the symbiosis is within the nodules that appear on the plant roots. Legumes, the most important plant group concerned in symbiotic N2 fixation, are dicotyledonous plants of the family Lesuminosae, having species such as Trifolium, Melilotus, Medicago, Lotus, Phaseolus, Dalea, Crotalaria, Vicia, Vigna, Pisum and Lathyrus. Rhozibia grow readily in culture media containing a carbon source such as mannitol or glucose, ammonium or nitrate to supply the required nitrogen and several inorganic salts. None of the bacteria in culture solution utilize N2; the fixation reaction is thus the result of a true symbiosis as neither symbiont can carry out the process alone. Of particular importance to the development of the symbiotic relationship is the presence of a large population of rhizobia. Symbiosis is the living together in close physical association of two or more different organisms. There are three types of symbiotic relationships: Commensalism: (Latin com, together and mensa table) is a relationship in which one symbiont, the commensal, benefits while the other (sometimes called the host) is neither harmed or helped. Often both the host and the commensal eat at the same table. The spatial proximity of the two partners permits the commensal to feed on substances captured or ingested by the host. The commensal also obtains shelter by living either on or in the host. The commensal is not directly dependent on the host metabolically and causes it no particular harm. When the commensal is separated from its host experimentally, it can survive without being provided some factor or factors of host origin e.g. the common, nonpathogenic strain of E. coli lives in the human clone. E. coli flourishes in the colon, but also grows quite well outside the host and this is a typical commensal. These relationships can be very complex. When O2 is used up by the facultative anaerobic E. coli, obligate anerobes such as Bacteroids are able to grow in the colon. The anaerobes benefit from their association with the host and E. coli but E. coli derives no obvious benefit from the anaerobes. In this case, the commensal E. coli contributes to the welfare of other symbionts. Mutualism: (Latin mutus, borrowed or reciprocal) defines the relationship in which some reciprocal benefit accrues to both partners. In this relationship the mutualist and the host are metabolically dependent on each other. Symbiosis involves intimate interactions based on mutual benefit, which is a good definition of mutualism. Under nitrogen-limiting conditions legumes nodulated with active N2-fixing strains of Rhizobium benefit from the interaction and the growth of the legume plants stimulates the growth of rhizobia and other microorganisms in the soil. However, strains of Rhizobium exist that fix N2 inefficiently, or not at all and such strains are either of little benefit to plant growth or are detrimental because they are utilizing the plants energy without providing reciprocal benefit. The interaction between rhizobia and leguminous plants has been studied in great detail for many years. Rhizobia are able to nodulate only a small proportion of the very large number of species in the family Leguminosae and one non legume, Parasponia. Within this range of host plants, specificity in the ability of particular plant and Rhizobium species to form effective symbioses is observed e.g. R. trifolii nodulates clovers (Trifolium spp.) which in turn are not usually nodulated by other rhizobia. Symbioses involving Rhizobium are only one example of the type of interaction between plants and N2 fixing microorganisms than can occur. The actinomycetes Frankia nodulates a range of dicotyledonous plants, as does the cyanobacterium Nostoc on the cycad Macrozamia. In each case there is a degree of specificity, which implies that there are mutual recognition systems. The range of plants with which cyanobacteria can form a symbiosis is 6

very wide indeed. It encompasses diatoms, fungi, mosses, liverworts, ferns, cycads and angiosperms. The proportion of heterocysts to vegetative cells is much higher in the symbiotic form than in the free-living cyanobacteria. Studies on the differentiation of heterocysts in the filaments has shown that this is determined by the nitrogen status. N2 is fixed by the heterocysts and diffuses into the vegetative cells. In the symbiosis where the products of N2 fixation are excreted for use by the host, combined nitrogenlevels along the filament become depleted sooner than in free-fixing forms, thus resulting in a greater proportion of heterocysts in symbiotic systems. As with other symbioses, there are morphological adaptations of the host and also physiological adaptations to cater for the special demands of N2 fixation. Lichens are symbioses of fungi, ascomycetes and basidiomycetes with algae. By far the highest proportion of lichen species are associations of fungi with green algae, but about 25 genera have cyanobacteria as the algal symbiont. In the lichens, Azolla and most cycad systems, the cyanobacteria exist outside the hosts cells, but in one species of cycad Macrozamia communis, the cyanobacteria have been found inside the cells. This is similar to the symbiosis with Gunnera. The cyanobacterial symbiont is Nostoc, and this symbiosis is of interest as it is the only known cyanobacterial association with an angiosperm. In Azolla the cyanobacteria (Anabaena) exist in pockets within the leaf, and in Gunnera, Nostoc is contained in glands at the base of leaves. Both are examples of containment that has resulted from the development of a complex interaction. The cyanobacterial symbioses have a wide range, and from a consideration of some of the associations it can be seen that there is a great diversity of interactions, ranging from the lichens where the cyanobacterial partner photosynthesizes and provides the host with both carbon and nitrogen, through the association with Azolla where photosynthesis takes place but carbon is supplemented by the host, to the situation in cycads and in Gunnera where the endophyte is dependent wholly upon the host for carbon. N2 has to be given up to the host if the symbiosis is to be mutualistic and the high proportion of heterocysts in all these associations show that it is. Mycorrhizae represent particularly interesting plant-microbe interactions because they are so wide spread and grown on lettuce can infect maize, grasses, beans, citrus and almost any other plant species that can form mycorrhizal associations of the VA type. Mycorrhizae are symbiotic because the plant provides the fungus with organic nutrients and in return fungus facilitates the uptake of mineral nutrients and in particular phosphate. Mycorrhizal fungi can be so important that some species of host plant are almost dependent upon them to be able to grow in soils low in PO4; citrus and cassava are particularly good examples. Mycorrhizae are thus something of an enigma. They are wide spread and show little sign of specificity, yet they involve complex interactions, and possibly most plants are partially dependent upon them for their PO4 nutrition. These symbioses have evolved to enable the fungus to invade roots and routinely colonize from 10 to 90% of the root length with no obvious harmful effect on the plant. In soil, many microorganisms occur in close proximity and they interact in a unique way that is in marked contrast to the behaviour of pure cultures studied by the microbiologists in the laboratory. Members of the microflora rely upon one another for certain growth substances, but at the same time they exert detrimental influences so that both beneficial and harmful effects are evident. The three beneficial relationships, symbiosis, protocooperation and commensalism are found to operate among the soil inhibitor. Microorganisms in time develop certain relations that are 7

beneficial and others that are detrimental. Sometimes the benefit is mutual, but commensal relationships are quite frequent. One of the more important beneficial associations is that involving two species, one of which can attack a substrate not available to the second organism, but the decomposition results in the formation of products utilized by the second. This type of commensalism is not infrequent in nature and it is the way many polysaccharides are transformed to nutrients supporting non-specialized microorganisms; e.g. cellulolytic fungi produce from cellulose a number of organic acids that serve as carbon sources for noncellulolytic bacteria and fungi. A second beneficial association arises from the need of many microorganisms for accessory growth substances. These growth factors are synthesized by certain microorganisms, and their excretion permits the proliferation of nutritionally complex soil inhabitants. The microbial decomposition of biologically produced inhibitors that prevent the proliferation of sensitive species is another instance of a beneficial relationship. Aerobes may permit the growth of obligate anaerobes by consuming the O2 in the environment. In addition to these instances of commensalism and protocooperation, several well documented example of true symbiosis are in evidence, particularly those concerned with N2 fixation.

Competition Microbial Competition The categories of deleterious interactions are summarized by the terms competition, amensalism, parasitism and predation, i.e. (a) the rivalry for limiting nutrients, (b) the release by one species of products toxic to its neighbours and (c) the direct feeding of one organism upon a second. Because the supply of nutrients in soil is perennially inadequate, competition for carbon, minerals or oxygen is quite common. Alteration of the environment to the detriment of certain microbial species may occur through the synthesis of metabolic products that are bacterostatic or bactericidal by the utilization of oxygen which leads to the suppression of obligate aerobes, or by the autotrophic formation of nitric and sulphuric acid which affects the proliferation of acid-sensitive microorganisms. Predatory and parasitic activities likewise are not rare. Predation and parasitism are observed in the feeding upon bacteria by protozoa and myxobacteria, the attack on nematodes by predacious fungi, the digestion of fungal hyphae by bacteria and the lysis of bacteria and actinomycetes by bacteriophages. In mixed cultures of several microorganisms in laboratory media or in partially sterilized soil, some species are suppressed while others survive, multiply and assume dominance. The usual cause of this phenomenon is the competition for nutrients, space or oxygen. In competition, certain microorganisms dominate through their capacity to make most effective use of the limiting factors in the environment. Therefore, when large populations of alien bacteria are added to soil, the invaders do not establish and soon die out. The habitat is foreign and the invader fails to find a niche. The disappearance itself is probably the result of competitive effects since specific toxic substances active against the alien bacteria are difficult to demonstrate. Microbiological competition for available carbon, however, is probably one of the more important interactions between organisms. It is likely that the role of an element in modifying the biological equilibrium is determined by the demand of the microflora and the supply in the soil. As a first approximation, the ability of an organism to compete is probably governed by its capacity to utilize the carbonaceous substrates found in soil, its growth rate, and its nutritional complexity. A simple nutrition could be advantageous, but the presence in soil of growth factors suggests the effective competitors need not be nutritionally independent, as they can develop at the expense of growth factors obtained from the environment.

Amensalism It is the suppression of the growth of one organism by the products of growth of a second organism. This may result from a situation as simple as the alteration of the soil pH or the production of the growth-inhibiting or lethal biological product. Thiobacillus spp. commonly reduces soil pH through the oxidation of sulphide to sulfate. Because the pH may reach values as low as 2, the growth of any pH sensitive microbes is inhibited. Two major types of biological inhibitors or toxins are produced by soil microbes: those effective at high concentrations (organic acids, chelators) and those that are effective at low concentrations (antibiotics). The growth-controlling impact of the former compounds in soil has been reasonably well accepted, because the substances can be quantified easily in soil samples and their interactions with soil microbial populations can be shown. The role of antibiotics within the soil ecosystem is more problematic. Another group of biologically synthesized compounds that appear to be useful in reducing plant disease through antagonism of pathogens are siderophores. These substances appear to be active at higher concentrations than is characteristic of antibiotics, but when they result in suppression of microbial growth at low concentrations, they can be classified as antibiotics. Siderophores are extra cellular, lowmolecular weight (500 to 1000 dalltons) iron-transporting compounds synthesized by a variety of microorganisms growing under low iron conditions. These substances selectively complex ferric ion with a high affinity, thereby reducing iron availability to competing organisms. Most commonly studied siderophore-synthesizing microbes from the view of controlling plant pathogens are members of the fluorescent pseudomonad group.

Parasitism and Predation Predators and parasites, organisms that feed upon living biomass, play a key role in the soil ecosystem. Parasites and predators maintain the soil bacterial and fungal populations in an active state and enhance nutrient movement between soil reservoirs through consumption of microbial biomass. The feeding activity of predators and infectivity of parasites maintains a younger, more active, soil microbial population. Essentially all types of predators or parasites are present in the soil ecosystems. Bacteria, which prey on other bacteria, bolellovibrios, bacteriophages, protozoa, as well as nematodes are all active in soil ecosystems. These organisms may ingest their nutrients by consuming intact cells (holozoic feeding), as is commonly described for protozoa, or extra cellular enzymes that lyse other bacteria, fungi or algae may be produced. In the predator prey relationship between protozoa and bacteria, a change in either group will bring about a qualitative and quantitative change in the other. The presence of a nutrient supply in the form of bacteria is essential for the development of soil protozoa and large numbers of bacteria must be ingested for one protozoan cell division. In well-mannered fields, the daily increases in bacteria and protozoa seem to be inversely related, one group increases as the other decreases. Protozoa, therefore are undoubtedly a key factor in limiting the size of the bacterial population, probably reducing the abundance of edible cells and serving as a biological antagonist in maintaining the equilibrium. Myxobacteria and myxomycetes also affect the true bacteria by feeding directly upon them. Fungi are capable of parasitizing one another, and the parasitized species is thereby often eliminated. The parasitism may entail a penetration into the hosts mycelium or a coiling around the hosts hyphae. The virulence of individual fungi varies greatly even in a single species. Certain fungi are predacious, capturing and consuming nematodes or amoebae, and the study of the nematode-trapping fungi may prove of practical value in the control of plant diseases caused by nematodes. A key consideration in evaluating predator or parasite behaviour in any ecosystem parasitic relates to the observation that both the host and parasites or prey and predators coexist in the same ecosystem. 9

Rumen Microbiology Ruminants are the herbivorous mammals. Their digestive system includes four compartments, the large pouch, small honey comb like, Omasum and abomasums (the stomach). Domestic animals such as cows, sheep, goat, buffalo, camel etc. and the wild animals such as deer, giraffe are the ruminants. Rumen is a special organ within which the digestion of cellulose and other plant polysaccharides occurs through the activity of special microbial populations. Mammals lack the enzymes needed to digest cellulose, so the rumen containing a large number of microorganisms (mainly bacteria and protozoa) which play an essential role in ruminant nutrition, break down plant material ingested by the host animal and provide the animal with protein, vitamins and assimilable carbon and energy yielding substrates. The rumen has a large size (100-150 litres in a cow, 6 litres in a sheep) and its position in the alimentary tract as the organ where ingested food goes first. The rumen contents are anaerobic, pH varies with diet but generally it is between 6-6.5. The rumen temperature is about 39-40C in the cow due to the (exothermic) microbial fermentation. The reduction potential in rumen is 30 mV. Food enters the rumen mixed with saliva containing bicarbonate and is churned in a rotary motion during which the microbial fermentation occurs. This peristallic action grinds the cellulose into a fine suspension, which assists in microbial attachment. The food mass then passes gradually into the reticulum where it is formed into small clumps called cuds, which are regurgitated into the mouth where they are chewed again. Now finely divided solids, well mixed with saliva, are swallowed again, but this time the material passes to the omasum, finally ending in the abomasum, an organ more like a true (acidic) stomach. Here chemical digestive processes begin that continue in the small and large intestine. (In the suckling animal, the rumen and reticulum are not fully developed and ingested food passes from the oesophagus via the oesophageal groove to the omasum and abomasum thus by passing the rumen). The number and type of microorganisms depend upon the nature of animals diet and on the period of time since the last intake of food; the rumen contents contain approximately 1010 cells bacteria ml-1 rumen fluid. Food remains in the rumen for about 9-12 hrs. During this period, cellulolytic bacteria and protozoa hydrolyze cellulose to the disaccharide cellobiose and to free glucose units. Released glucose then undergoes bacterial fermentation with the production of volatile fatty acids (VFAs), primarily acetic, propionic and butyric and the gases CO2 and methane. The host animal absorbs the fatty acids from the rumen and from the omasum and abomasums and eliminates the gases by erutation (the ruminant uses fatty acids rather than glucose as primary sources of energy and carbon). The acidity of the fermentation products is counteracted by the buffering action of the ruminants saliva which is produced in copious amounts and contains sodium bicarbonate and sodium hydrogen phosphate. Many microbial cells formed in the rumen are digested in the gastrointestinal tract and serve as a major source of proteins and vitamins for the animal. Since many of the microorganisms of the rumen are able to grow on urea as a sole nitrogen source, it is often supplied in cattle feed in order to promote microbial protein synthesis. The bulk of this protein ends up in the animal itself. A ruminant is thus nutritionally superior to a non-ruminant when subsisting on foods that are deficient in protein, such as gases (Fig. 2a & b).

Rumen bacteria Biochemical reactions taking place in rumen are complex and hence involve a large number of microorganisms where anaerobic bacteria dominate. Several different bacteria hydrolyze 10

cellulose to sugars and ferment sugars to VFAs. Fibrobacter succinogens and Ruminococcus albus are cellulolytic anaerobes. If a ruminant is gradually switched from cellulose to a diet of high in starch (grains), then starch digesting bacteria Ruminobacter amylophilus and Succinomonas amylolytica develop. If an animal is fed legume hay, which is high in pectin, then pectin digesting bacterium Lachnospira multiparus is in the rumen flora. In fermentation process, succinate is converted to propionate and CO2 and lactate is fermented to acetic and other acids by Selenomonas and Megaphaera. A number of rumen bacteria produce ethanol which is fermented to acetate + H2. H2 quickly reduces CO2 to CH4 by methanogens. In the rumen 65% CO2 and 35% CH4 are present and which leave the rumen by belching. In addition to prokaryotes, rumen also has protozoal fauna (about 106/ml) which are obligate anaerobes as well as anaerobic fungi that ferment cellulose to VFAs. Rumen fungi also degrade plant polysaccharides as well as partially degrades lignin, hemicellulose and pectins.

(a)

(b) Fig.2: (a) Schematic diagram of the rumen and gastrointestinal system of a cow. (b) Biochemical reactions in the rumen. (Biology of Microorganisms) 11

Sometimes changes in the microbial composition of the rumen is fatal i.e. death of the animal e.g. if a cow is changed abruptly from forage to grain diet, an explosive growth of Streptococcus bovis from normal growth of 107 cell/ml to 1010 cells/ml takes place. Grains contain high level of starch whereas grasses contain cellulose. S. bovis is a lactic acid bacterium, ferments starch to lactate which acidifies the rumen which is called acidosis, killing off normal rumen flora. Such carbohydrates lead to a proliferation of acid producing bacterium which cause a fall in pH and consequent loss of protozoans and many species of bacteria. As a result, acidophilic Lactobacillus spp. predominate and cause a further fall in pH. Other animals Buffalo, deer, reindeer, caribon and elk are also ruminants. Beleen whales also have a rumen like fermentation. They contain multichambered stomach whose fore stomach is similar to rumen and show abundant volatile fatty acids similar to cattle.

Biofertilizers Biofertilizers can be defined as a microbial preparation containing N2 fixing or PO4solubilizing or celluoylic or such other useful microorganisms which by virtue of special biochemical processes can increase the availability of a certain important nutrients in the vicinity of the root system leading to better plant growth and crop productivity. Biofertilizers are also called microbial inoculants. They are products containing living microorganisms which have the ability to mobilize nutritionally important elements from nonusable to usable form through biological process. Normally, the microorganisms are evolved after intensive researches and are included in certain carriers such as charcoal, lignite or peat. Biofertilizers have an important role to play in improving the nutrient supplies to crop plants as well as trees in Indian agriculture as an alternate source of soil fertility building through renewable sources. These can help in increasing the biologically fixed atmospheric N or increase the native P availability to crop plants. Among the biofertilizers useful in increasing N supply, N2 fixing bacteria Rhizobium, Azotobacter, Azospirillum, blue green algae and Azolla are important. Among those associated with increased P availability, different P solubilizing bacteria and mycorrhizae are of significance. Thus, industrial production of biofertilizers has come to help farmers to economize on chemical fertilizer inputs. Biofertilizers can be defined as preparations containing live efficient microbes performing various functions like nitrogen fixation, phosphorus solubilization or mobilization. They are cheap, economical sources of nutrients and are ecofriendly i.e. nonpolluting in nature. Biofertilizers are known to make a number of positive contributions in soil thereby improving soil health in general and crop health in particular by various mechanisms as under: a) They fix atmospheric nitrogen and provide to various crops. b) They produce and liberate various plant growth promoting substances, vitamins and help in better and quick growth. c) They produce certain antimicrobial agents and suppress the incidence of pathogens. d) They solubilize or mobilize phosphorus in soil. e) They improve soil physical, chemical and biological health of soil. Following are the various groups of biofertilizers for different crops which are being popularly used:

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Nitrogen fixing biofertilizers 1. Rhizobial Biofertilizers: Fix atmospheric nitrogen in symbiosis with leguminous crops. Bacteria provide nitrogen to plants and plants in turn provide carbon sources and other nutrients to bacteria e.g. Rhizobium. 2. Azotobacter Biofertilizers: Fix atmospheric nitrogen a symbiotically in soil. Produce unspecified plant growth promoting substances thereby induce profuse root and shoot growth. They also produce certain antimicrobial agents which keep away pathogens. Azotobacter biofertilizers are used for non leguminous crop like cereals millets and oil seed. 3. Azospirillum Biofertilizers: Azospirillum form a loose symbiosis with nonleguminous crops and are known as associative symbionts. These bacteria are benefited by root exudates of plants and help the plants by fixing atmospheric nitrogen and producing plant growth promoting substances. 4. Cyanobacterial Biofertilizers or BGA Biofertilizers: These are useful in rice fields. They fix atmospheric nitrogen and produce plant growth promoting substances and Vitamins. 5. Phosphate solubilizing biofertilizer: Many times phosphate becomes a imiting factor for plant growth because much of it in the soil is bound as insoluble calcium, iron or aluminium phosphates. The availability of phosphates therefore depends on the degree of solubilization of insoluble phosphates by various organic and iorganic acids produced by the microorganisms thereby solubilize insoluble phosphates and make it available to the plant. Bacillus mregaterium, B. polymyxa, Predomonas, Aspergillus, Mycorrhiza are commonly used phosphate solubilizing biofertilizers. 6. Plant growth promoting rhizobacteria (PGPR): PGPR are also being used a biofertilizer as they are able to produce various phytohormones like, IAA, Cytokinin and gibberellins etc which are important for plant growth and productivity. Popularly used PGPR are Pseudomonas, Bacillus, Agrobacterium, Cellomonas, Arthrobacter, Alcaligenes, Actinoplane

Biological N2 fixation The utilization of atmospheric N2 gas as a source of nitrogen is called nitrogen fixation. Prokaryotes both anaerobic and aerobic fix N2. No eukaryotic organisms fix N2. There are some bacteria called symbiotic fix N2 only in association with certain plants. Biological N2 fixation is brought about by free-living bacteria or blue-green algae, which make use of N2 by non-symbiotic means and by symbiotic associations composed of a microorganisms and a higher plant. N2 fixation, the reduction of N2 to NH3 involves a complex enzyme system called nitrogenase, which consists of dinitrogenase and dinitrogenase reductase, metalcontaining enzyme found only in certain prokaryotic organisms. Most nitrogenase contain molybdenum or vanadium and iron as metal cofactors and the process of N2 fixation is highly energy-demanding. Nitrogenase and associated regulatory proteins are encoded by the nif regulation. Certain artificial substrates that are structurally similar to N2, such as acetylene and cyanide are also reduced by nitrogenase (Fig. 3). Nitrogenase is a functional enzyme which reduces N2 to ammonia and depends on energy source from ATP. The nitrogenase has two components: one containing Mo-Fe, designated as Mo-Fe protein (nitrogenase) and the other iron protein (nitrogenase reductase). Both the components are essential for nitrogenase activity.

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ATP N2 + 3H2 2 NH3 For this reaction, for every two electron transfer by nitrogenase, four ATP moles are required.

Fig. 3: Pathway of nitrogen fixation (Agricultural Microbiology)

Nitrogenase enzyme The reduction of N2 to ammonia is catalysed by the enzyme nitrogenase. Reaction has a high activation energy because molecular N2 is an unreactive gas with a triple bond between the two N2 atoms. Therefore, N2 reduction requires at least 8 electrons and 16 ATP moles, 4 ATPs per pair of electrons. N2 + 8H+ + 8e- + 16 ATP 2NH3 + H2 + 16 ADP + 16 P; The electrons come from ferredoxin that has been reduced in a variety of ways: i) by photosynthesis in cyanobacteria. ii) Respiratory processes in aerobic N2 fixers, iii) Fermentations in anaerobic bacteria e.g. Clostridium pasteurianum (an anaerobic bacterium) reduces ferredoxin during pyruvate oxidation, whereas the aerobic Azotobacter uses electrons from NADPH to reduce ferredoxin. Nitrogenase is a complex system consisting of two major protein components a MoFe protein joined with one or two Fe proteins. The MoFe protein contains 2 atoms of molybdenum and 28 to 32 atoms of iron; the Fe protein has 4 iron atoms. Fe protein is first reduced by ferredoxin, then it binds ATP. ATP binding changes the conformation of the Fe protein and lowers its reduction potential, enabling it to reduce the MoFe protein. ATP is hydrolyzed 14

when this electron transfer occurs. Finally, reduced MOFe protein donates electrons to atomic nitrogen. Nitrogenase is quite sensitive to O2 and must be protected from O2 inactivation within the cell. The reduction of N2 to NH3 occurs in 3 steps, each of which requires an electron pair. Six electron transfers take place and this requires a total 12 ATPs per N2 reduced. The overall process actually requires at least 8 electrons and 16 ATPs because nitrogenase also reduces protons to H2. The H2 reacts with diimine (HN=NH) to form N2 and H2 (Fig 4).

Fig. 4: Mechanism of Nitrogenase Action (Prescott- Harley-Klein) Nitrogenase can reduce a variety of molecules containing triple bonds (e.g. acetylene, cyanide and azide) 15

HC = CH + 2H+ + 2e- H2C = CH2 The rate of reduction of acetylene to ethylene is even used to estimate nitrogenase activity. Once molecular N2 has been reduced to ammonia, the ammonia can be incorporated into organic compounds.

The nif genes The genes for nitrogen fixation, called nif genes are found in both symbiotic and free living systems, but in the Rhizobium-legume system, the nif genes are distinct. The symbiotic activation of nif-genes in the Rhizobium is dependent on low oxygen concentration, which in turn is regulated by another set of genes called fix-genes which are common for both symbiotic and free living nitrogen fixation systems. The nif-genes have been investigated most thoroughly in Klebsiella. Work with Klebsiella pneumoniae has shown that there are 17 nif-genes. Proteins have been identified and in some cases functions for these genes are known. If one gene codes for the synthesis of one polypeptide, then several genes will be necessary to code for the nitrogen-fixing system. The Fe protein is composed of two sub-units but, as each of these is the same, one gene will code for this protein. The MoFe protein has two different sub-units, each of which will require one gene. The molybdenum cofactor will require a gene and further genes will be necessary to code for any special electron donors in the system. The genes that code for these proteins are all adjacent to one another on the Klebsiella chromosomes. Functions of the nif genes of Klebsiella pneumonia Gene nif H nif D nif K nif M nif B nif N nif E nif V nif F nif J nif A nif L nif Q nif S nif U nif X nif Y Function of the gene or gene product Codes fore the sub-unit of the Fe protein Codes for the sub-unit of the FeMo protein Codes for the -sub-unit of the FeMo protein Activation of the Fe protein Involved in the synthesis and insertion of the iron molybdenum cofactor, FeMoCo As for B As for B As for B Codes for a flavodoxin Codes for a pyruvate: flavodoxin oxidoreductase Codes for an activator mole for the other nif genes Codes for a repressor molecule for the other nif genes Possibly concerned with molybdenum uptake Possibly concerned with processing the FeMo protein As S Unknown Unknown 16

The nif H, D and K genes code for the polypeptides of the Fe and MoFe proteins. These genes are readily identified by the lack of, or alteration of the particular proteins in mutants. Mutations in the nif M gene results in an inactive Fe protein, so that the product of the nif M gene must be involved in modifying the protein in some way, perhaps incorporating the Fe-S cluster. Similarly, mutations in several genes affect the activity of the MoFe protein. Mutations of nif V give an altered substrate specificity. These mutants are unable to reduce N2 but can reduce acetylene. Carbon monoxide, which does not inhibit hydrogen evaluation from normal nitrogenase, inhibits hydrogen evaluation from nif V mutants. When FeMoCo was obtained from the nif V mutant protein and was combined with protein of a nif B mutant, a protein from which FeMoCo is absent, the nif V-phenotype was obtained. However, when FeMoCo from a normal protein was added to the nif B mutant protein a normal protein resulted. Thus it was concluded that the nif V product modifies FeMoCo in order to produce effective nitrogenase. From studies of nif V- mutants it has been concluded that FeMoCo contains the binding site for N2 and CO. Three other genes, nif B, N and E have been identified with the synthesis of FeMoCo and the nif Q products action is thought to be the acquisition of molybdenum. Thus five of the genes nif Q, B, N, E and V are connected with the synthesis of the molybdenum cofactor. The products of the genes nif S and U are thought to modify the MoFe protein, although there is no hard evidence for this as yet. If this is true, then nine genes are needed to produce the complete active MoFe protein. Two genes are concerned with electron transport to nitrogenase: nif F and J. Extracts of mutants of both of these genes can fix nitrogen if they are provided with the artificial electron donor, sodium dithionite. Extracts of nif F- mutants can be rendered active by providing Azotobacter flavodoxin. It is thus assumed that the product of nif F is a flavodoxin. The nif J product has been shown to be the enzyme pyruvate : flavodoxin oxidoreductase, which catalyses the oxidation of pyruvate to produce reduced flavodoxin: Pyruvate + CoA + flavodoxin ----- acetyl CoA + flavodoxin + CO2 ox red With flavodoxin and the pure enzyme in the reaction mixture, the reduction of nitrogenase and then acetylene can be achieved, when the flavodoxins from Azotobacter is used, the activity is one third of that with the flavodoxin from Klebsiella, which demonstrates that there is some specificity for the reductant and that flavodoxins from different species may differ. The genes which control the synthesis of the nitrogenase proteins will be present in all the species that fix nitrogen. However, the genes concerned with electron transport will differ, as the provision of electrons depends upon the metabolism of pyruvate. It is interesting to note that the genes nif F and J for electron transport to nitrogenase are transcribed in the opposite direction to the other nif genes. The genes nif X and nif Y have been identified by means of their polypeptide products from cloned fragments of the nif region. The function of these genes has however not been established. The remaining two genes are nif A and nif L. The purpose of these genes is to control the expression of the other genes in the nif region (Fig. 5a & 5b). 17

Fig. 5: The nitrogenase system (a) Steps in nitrogen fixation: reduction of N2 to 2 NH3. (b) The genetic structure of the nif regulation in Klebsiella pneumoniae, the best studied nitrogen fixing organism. (Biology of Microorganisms)

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Symbiotic N2 fixation Two members are required for the association, a plant and a microorganism. The classical example of such a symbiosis is that between leguminous plants and bacteria of the genus Rhizobium. The seat of the symbiosis is within the nodules that appear on the plant roots. Legumes, the most important plant group concerned in symbiotic N2 fixation, are dicotyledonous plants of the family Leguminosae. For successful symbiotic N2 fixation, a healthy plant growing in sufficient light and an effective nodule forming bacterium are required. The nodule fixes N2 only for a short duration when it is in the highest symbiotic relationship with the plant. The energy requirement for biological N2 fixation appears to be high and this becomes the limiting factor in the quantity of N2 fixed in different legumeRhizobium combinations. Fifty percent of natural nitrogen fixation is accomplished by the Rhizobium legume association. Different symbiotic associations are Rhizobium-legumes; Rhizobium non legumes, frankia and Angiosperm and cyanobacterial associations.

Rhizobium The rhizobia are soil organisms that inhabit the rhizosphere of legumes and other plants. There are two main types of rhizobia, fast growers and slow growers. The division of Rhizobium into species is based on the interaction with plants; those bacteria which nodulate clovers, are put in R. trifolii and those that nodulate peas and vetches are put in R. leguminosarum. The bacteria are Gram negative, non-spore forming, aerobic rods, 0.5 to 0.9 wide and 1.2 to 3.0 long. They are typically motile and utilize several carbohydrates, sometimes with the accumulation of acid but never of gas. Rhizobium bacteria stimulate leguminous plants to develop root nodules, which the bacteria infect and inhabit. The nodules develop in a complex series of steps (Fig. 6). (i) (ii) (iii) (iv) (v) Recognition of the correct partner on the part of both plant and bacterium and attachment of the bacterium to root hairs. Invasion of the root hair by the bacterial formation of an infection thread. Travel to the main root via the infection thread. Formation of deformed bacterial cells, bacteroids, within the plant cells and development of the nitrogen-fixing state. Continued plant and bacterial division and formation of the mature root nodule.

The roots of leguminous plants secrete a variety of organic materials that stimulate the growth of a rhizosphere microflora. If there are rhizobia in the soil, they grow in the rhizosphere and build up to high population densities. Attachment of bacterium to plant in the legume Rhizobium symbiosis is the first step in the formation of nodules. A specific adhesion protein called rhicadhesin which is present on the surface of Rhizobium is a calcium binding protein and binds calcium complexes on the root hair surface. Rhizobium cells penetrate into the root hair via the root hair tip. Following binding, the root hair curls as a result of the action of substances excreted by the bacterium called Nod factors and the bacteria enter the root hair and induce formation by the plant of a cellulosic tube, called the infection thread, which spreads down the root hair. Root cells adjacent to the root hairs subsequently become infected by rhizobia and Nod factors stimulate plant cell division, eventually leading to formation of the nodule. The bacteria multiply rapidly within the plant cells and are transformed into swollen, misshapen and branch forms called bacteroids. Bacteroids become surrounded simply or in small groups by portions of the plant cell membrane. Only after the formation of bacteroids does nitrogen fixation begins. N2 fixation takes place in these nodules and the effective nodules are pink in color. Atmospheric N2 gets 19

reduced to NH3 by the nitrogenase system and then to amino acids in the root nodules. Legume-Rhizobium symbiosis is influenced by a variety of factors like host, bacterial strains, temperature, light, soil pH, phosphorus, combined N2, micronutrients and interaction with other soil organisms.

Fig. 6: Steps in the formation of a root nodule in legume infected by Rhizobium (Biology of Microorganisms)

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Symbiotic process is controlled by a number of nod genes of which some are host specific and the other are common nod genes. Some of the nod genes induce the host plant to react by producing nodulins which are flavanoids. Some nod genes are required for root-hair curling and for cell division. The genes for N2 fixation, called nif genes, are found in both symbiotic and free living systems, but in the Rhizobium-legume system, the nif genes are distinct. The plant supplies carbon compounds, derived from photosynthesis in the shoot, which helps the bacteria to produce ATP, which is required in large quantities for N2 fixation. The leghaemoglobin (Lb) present in the nodule binds O2 so as to facilitate N2 fixation by the bacterium, since presence of O2 inhibits nitrogenase enzyme of the bacterium. The bacteria in the nodule undergo limited DNA replication and division and then transform into bacteroids. The plant-derived peribacteroid membrane (PBM) forms the envelop for the bacteroids. There is specific gene-controlled interaction taking place between the bacteroids and the PBM. The symbiotic activation of nif-genes in the Rhizobium is dependent on low O2 concentration, which in turn is regulated by another set of genes called fix genes which are common for both symbiotic and free-living N2 fixation systems. The Rhizobium bacterial cells and the host cells cooperate intimately in respect of cellular metabolism with the required energy and growth regulation, accompanied by the genetic transcription and translation through DNA. The exchange of carbon sources with that of the nitrogenous substances is balanced as to make the bacterium a symbiont instead of a pathogen i.e. the bacterium through infective, should ultimately come under the control of the host (Fig. 6).

Frankia The Alder tree (genus alnus) has N2-fixing root nodules that harbor a filamentous, streptomycete like, N2 fixing organism called Frankia. The members of this genus are actinomycetes : most of these bacteria at sometime in their life cycle have a filamentous habit which often superficially bears some morphological resemblance to the fungi. They are however, prokaryotes with hyphae of smaller dimensions in Frankia typically less than 2 m diameter-than fungi. An important feature of Frankia is that many strains can fix N2 at normal O2 concentration at rates sufficient to support growth in culture. although when assayed in cell extracts the nitrogenase of Frankia is sensitive to molecular O2, like intact cells of Azotobacter. Intact cells of Frankia fix N2 at full O2 tensions. This is because Frankia protects its nitrogenase by localizing it in terminal swellings on the cells called Vesicles. The vesicles contain thick walls of laminated structure that act as a barrier to O2 diffusion, thus maintaining the O2 tension within vesicles at levels compatible with nitrogenase activity. Frankia vesicles resemble the heterocysts produced by some filamentous cyanobacteria as localized sites of N2 fixation. N2 fixation in such cultures is inhibited by the addition of combined N2. Like Alder, Frankia nodulates a number of other small woody plants. This root nodule symbiosis has been reported in at least 8 families of plants, many of which show no evolutionary relationships to one another. This suggests that the nodulation process in the Frankia symbiosis is more of a generalized phenomenon than the highly specific process observed in the Rhizobium-legume symbiosis and this holds promise for experimental attempts to expand the Frankia symbiosis to agriculturally important plants (Fig. 7).

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Fig. 7(a&b): Frankia nodules and Frankia cells. (a) Root nodules of the common alder Alnus glutiosa (b) Frankia culture purified from nodules of Componia pergrina. (Biology of Microorganism)

Non-symbiotic N2 fixation Biological N2 fixation is brought about by free-living bacteria or blue-green algae, which make use of N2 by non-symbiotic means. A number of environmental factors govern the rate and magnitude of non-symbiotic N2 fixation and the transformation is markedly affected by the physical and chemical characteristics of their habitat: Microorganisms that assimilate N2 have the ability to utilize ammonium and sometimes nitrate and other combined forms of nitrogen. In fact, ammonium salts are used preferentially and often at a greater rate than molecular N2 so that the presence of ammonium, ineffect, inhibits the fixation, i.e. the bacteria use the N2 salt rather than N2 from the atmosphere.

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Many inorganic nutrients are necessary for the development of the microorganisms. Molybdenum, calcium and iron are critical for the fixation reaction. Molybdenum is required for the metabolism of N2, but microorganisms do not use nitrate unless molybdate is present although the molybdenum requirement for nitrate utilization is less than for N2 fixation. In like manner, Fe salts are implicated in the N2 metabolism of Azotobacter, Clostridium, Algae, Aerobacter and Achromobacter, but the specific requirement for N2 metabolism is often difficult to establish because Fe is required, to a lesser extent, for growth upon fixed compounds of N2. A requirement for Ca has been demonstrated during N2 assimilation by blue-green algae and some Azotobacter spp., but the need for calcium can sometimes be replaced by strontium. Azotobacter is characteristically sensitive to high hydrogen ion concentrations. Their absence is associated directly with pH. As a rule, environments more acid than pH 6.0 are free of the organism or contain very few Azotobacter cells. Similarly, the bacteria generally, will neither grow nor fix N2 in culture media having a pH below 6.0. Beijerinckia spp. do not possess the acid sensitivity like Azotobacters and they develop and fix N2 from pH 3 to 9. Blue-green algae bacteria, however, develop poorly in media and are sparse in soils more acid than approximately pH 6.0 whereas the acid tolerance of Clostridium falls between Azotobacter and Bcijerinckia. There is some evidence that the occurrence of Azotobacter is also related to the available PO4 content of soils. About 1 mg of phosphorus must be assimilated by Azotobacter for each 5 to 10 mg of N2 fixed. The distribution of blue-green algae in wet paddy fields is likewise associated with the PO4 content of the soil.

Azotobacter and Azospirillum Azotobacter is a free living heterotrophic nitrogen fixing bacterium encountered in neutral to alkaline soil conditions. The bacterium not only provides the nitrogen but produces a variety of growth promoting substances. These include indole acetic acid (IAA), gibberellic acid (GA), vitamin-B and anti fungal substances. Another important characteristic of Azotobacter associated crop improvement is excretion of ammonia in the rhizosphere in the presence of root exudates and help in modification of nutrient uptake by the plants. These strains are better competitors than the non excreting strains. The genus Azotobacter is highly versatile in utilizing carbon sources therefore, application of organic carbon containing sources to the soil improves the asymbiotic N2 fixation capacity by the diazotroph. The benefits of Azotobacter inoculation are: enhanced branching of roots, production of plant growth hormones, enhancement of uptake of NO3, NH4+, H2PO4-, K+, Rb++ and Fe++, improved water status of plants, increased nitrate reductase activity and antifungal compounds. Members of this genus are strict aerobes: O2 is required for metabolism and also to fix N2. N2 fixation therefore occurs in an aerobic environment and there must be a mechanism to prevent the access of O2 to the O2-sensitive proteins. Azotobacter has a very high rate of respiration and when the organism is deprived of respirable substrate, as when it is grown on a medium low in carbon, the nitrogenase is more susceptible to O2. N2 fixation is inhibited when the organism is suddenly exposed to an O2 concentration higher than that in which it has been grown. Azotobacter therefore, grows better at O2 concentration lower than atmospheric when fixing N2. Where there is sufficient substrate it is likely that the O2 23

concentration will be lower than that of atmospheric because of the respiration of both Azotobacter and of other microorganisms in the vicinity. The genus Azotobacter comprises large, Gram-negative, obligately aerobic rods capable of fixing N2 non-symbiotically. The tropical soils have much higher Azotobacter populations than those found under temperate climates. In soil, their numbers vary from a few to a few hundred per gm of soil. Application of nitrogenous fertilizers drastically reduces the Azotobacter population in soil. Inoculation of soil or seed with Azotobacter is effective in increasing yields of crops in well manured soil with high organic matter content. Azospirillum: an associative micro aerophilic N2 fixer, commonly found in association with roots of cereals and grasses has received great interest as a biofertilizer. Its useful characters include high N2 fixation capacity, low energy requirement and tolerance to high soil temperature for its suitability under tropical conditions. Azospirillum is a mesophyllic bacterium and is reported to occur in association with crops grown in acidic to alkaline pH range. Azospirilla are metabolically versatile and can grow vigorously in the presence of nitrogenous compounds present in the soil but as soon as the external N2 supply is exhausted the bacteria shift to diazotrophy. Use of Azospirillum inoculum under saline alkaline conditions is possible because strains adapted to these stress conditions maintained high N2 activity. Crops grown to pre-treated seed give increased yields up to about 10 to 30%. Azospirillum participates in all steps of the N2 cycle except nitrification. It can fix atmospheric N2 in pure culture and under microaerophilic conditions. Azospirillum spp. have been isolated from the rhizosphere of a large number of monocotyledons and a few dicotyledon plants. Azospirillum lipoferum has been observed to fix atmospheric N2 in the cortical cells of the roots of maize. Substantial increases in yield were reported following the inoculation of sorghum and pearl millet with Azospirillum brasilense under several agro-climatic conditions in India. In addition to N2 fixation, hormonal effects have also been shown to be responsible for at least part of yield increase following inoculation with Azospirillum. It has also been shown that Azospirillum and Azotobacter, besides enhancing N2 uptake by plants, increase the number of root hairs and root hormone exudation. This genus of spirally curved Gram negative bacteria is interesting as its members not only live in the rhizosphere of grasses but can also enter the root cortex. These organisms use root exudates for their carbon and energy source while fixing N2. Azospirillum is a O2-sensitive and can fix N2 only at low O2 concentrations. It is a tropical bacterium and has a high optimum temperature so that it does not occur to any great extents in temperature latitude. It has a wide host range.

Mycorrhizae Mycorrhizae are fungus root associations, first discovered by Albert Bernhard Frank in 1885. The word mycorrhizae comes from the Greek words meaning fungus and roots. These microorganism contribute to plant functioning in natural environments, agriculture and reclamation. The roots of 95% of all kinds of vascular plants are normally involved in symbiotic associations with mycorrhizae (Fig 8). Five types of mycorrhizae can be recognized: (i) Ectomycorrhizae which form a sheath around roots but lack intracellular penetration of the cortical cells; three types of Endomycorrhizae: (ii) ericoid, (iii) orchid and (iv)

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vesicular-arbuscular mycorrhiza which colonize the root cortical cells intracellularly and (v) Ectendomycorrhizae which form sheath and produce intracellular penetrations (Fig 9).

Fig. 8: Components of the mycorrhizal symbiosis. Phosphate enters the plant, along with other mineral nutrients, both directly from the soil and through the fungus. (Advances in Agricultural Microbiology) Ectomycorrhizae: are found mainly in forest-trees, especially conifers, beeches and oaks and are most highly developed in temperate forests. In a forest, almost every root of every tree is mycorrhizal. The root system of a mycorrhizal tree is composed of both long and short roots. The short roots, which are characteristically dichotomously branched, show the typical fungal sheath, whereas long roots are usually uninfected. Most mycorrhizal fungi do not attack cellulose and leaf litter but instead use simple carbohydrates for growth and usually have one or more vitamin requirements; they obtain their nutrients from root secretions. The mycorrhizal fungi are never found in nature except in association with roots and hence can be considered obligate symbionts. These fungi produce plant growth substances that induce morphological alterations in the roots, causing characteristically short dichotomously branched mycorrhizal roots to be formed. Despite the close relationship between fungus and root, there is little species specificity involved, a single species of pine can form mycorrhizae with over 40 species of fungi. Ectomycorrhizal fungi penetrate intra cellularly and partially replaces the middle lamellae cortical cells of feeder roots. These fungi form a dense mycelial net around and between the plant cells termed Hartig net. Ectomycorrhizal associations are also characterized by a dense, generally continuous hyphal network over the feeder root surface called a fungal mantle. This fungal mantle varies from one to two hyphal diameters to as many as 30 or 40 depending upon the fungal associate, the host and the environmental conditions. Example of plant spp. forming ectomycorrhizal associations are spp. in the families Pinaceae, Salicaceae, Betulaceae and Fugaceae. Most ectomycorrhizal fungi are 25

basidomycetes (primarily of the families Amanitaceae, Boletaceae, Cortinariaceae, Russulaceae, Tricholomataceae, Rhizopogonaceae and Sclerodermataceae). Mycorrhizal hyphae have been found to be very efficient in the uptake of phosphorus from the soil, which would otherwise be unavailable to the plant. The ectomycorrhizal fungi help in the phosphorus nutrition of plants through increased surface area of absorption, offer protection against some of the soil-borne plant pathogens and enhance rooting and survival of cuttings through production of growth hormones. Endomycorrhizae: are distinguished by the fact that the fungus penetrates the cortical cells of feeder roots and may form large vesicles and arbuscles (hence the term vesicular-arbuscular mycorrhizae (VAM). These fungi do not form dense fungal mantles, but they do develop a loose, intermittent arrangement of mycelium on the root surface. Endomycorrhizae are formed by most agronomic, horticultural and ornamental crops, as well as some forest tree spp. that do not form ectomycorrhizae. The fungal spp. are phycomyces many of which are in the genus Endogone. VAM colonization originates from hyphae arising from soil borne propagules. On reaching the cortex, hyphae grow into cells by tree like dichotomous branching to give arbuscules. When colonization is well established, oval structures called vesicles may form which have storage functions. Vesicles appear to be organelles for the storage of lipid and energy reserves and arbuscules, which resemble the haustoria of rust fungi and mildews are complex ramifications of small branches of the fungus that provide sites for nutrient exchange. VA mycorrhizal fungus grown on lettuce can infect maize, grasses, beans, citrus and almost any other plant spp. that can form mycorrhizal associations of the VA type. Ectomycorrhizal fungi have more limited host ranges and there are plant spp. which are not infected by endomycorrhizal fungi. Endomycorrhizae are of particular interest, as it has not been possible to grow these fungi, usually members of the zygomycetes, without the plant. In this association the fungal hyphae penetrate the outer cortical cells of the plant root, where they grow intracellularly and form coils, swellings, or minute branches. Endotrophic mycorrhizae are found in wheat, corn, beans, tomatoes, apples, oranges and many other commercial crops, as well as most pasture and rangeland grasses. Recent studies show that plant flavonoids may stimulate spore germination and this could lead to the development of plant-free cultures of these mycorrhizae. Mycorrhizal fungi have been observed to improve plant growth through better uptake of P and Zn from soil. The VAM fungi penetrates the outermost cortex region, when the plant is well supplied with phosphorus, but in phosphorus deficient plants they penetrate deep into the cortex and help the plant to obtain the nutrient from the soil. Recent studies have shown that they stimulate beneficial organisms like Rhizobium, Azotobacter and phosphate solubilizers in the rhizosphere and suppress the growth of root pathogenic fungi and nematodes. In addition, the mycorrhizal fungi are reported to increase the availability of water to plants, resulting in more vigorous growth under drought conditions. The ericoid mycorrhiza is seen in members of heath family like blueberry and Erica. Pezizella ericae, an ascomycete, is the most common fungal symbiont, which can be cultured. Currently, several laboratories are studying this association, as blueberry is an important cash crop. All orchids are infected at some stage in their life cycle by the Orchidaceous mycorrhizal fungi. A recent study has shown that artificial inoculation of orchids with the mycorrhizal fungus is not necessary as the fungus is abundantly present in nature. 26

Ectodomycorrhizae: These mycorrhizae resemble ectomycorrhizae in forming a Hartig net and a fungal mantle. A resemblance with the endomycorrhizae is associated with their penetration of cortical cells. This mycorrhizal grouping is the least studied and the nature of the fungal symbionts has not been totally elucidated. A mycorrhizae is a mutualistic symbiosis between plant and fungus localized in a root or root-like structure in which energy moves primarily from plant to fungus and inorganic resources move from fungus to plant. The formation of mycorrhizae is particularly pronounced in land low in phosphorus and N2 and high nutrient levels are correlated with poor mycorrhizal development. N2 fixing microorganisms can increase soil N2 while mycorrhizal fungi effectively augment the absorbing surface of the roots. Many tropical soils are so phosphorus deficient that they cannot respond to N2 until this deficiency is corrected. It is here that endomycorrhiza takes on added significance, as it enhances the absorption of phosphorus. Mycorrhizae are symbiotic because the plant provides the fungus with organic nutrients and the fungus facilitates the uptake of mineral nutrients and in particular PO4. Mycorrhizal fungi can be so important that some species of host plant are almost dependent upon them to be able to grow in soils low in PO4, citrus and cassava are particularly good example. Mycorrhizae are thus something of an enigma. They are wide spread and show little sign of specificity, yet they involve complex interactions and possibly most plants are partially dependent upon them for their PO4 nutrition. These symbioses have evolved to enable the fungus to invade roots and routinely colonize from 10% to 90% of the root length with no obvious harmful effect on the plant. Depending on the environment of the plant, mycorrhizae can increase a plants competitiveness. In wet environments they increase the availability of nutrients, especially phosphorus. In arid environments, where nutrients do not limit plant functioning to the same degree, the mycorrhizae aid in water uptake, allowing increased transpiration rates in comparison with nonmycorrhizal plants. These benefits have distinct energy costs for the plant in the form of photosynthate required to support the plants mycorrhizal habit. Under certain conditions the plant is apparently willing to trade photosynthate produced with the increased water acquisition for H2O. The beneficial effect on the plant of the mycorrhizal fungus is best observed in poor soils, where trees that are mycorrhizal, thrive but non mycorrhizal one do not. When trees are planted in prairie soils, which ordinarily lack a suitable fungal inoculum, trees that were artificially inoculated at the time of planting grow much more rapidly than uninoculated trees. It is well established that the mycorrhizal plant is able to absorb nutrients from its environment more efficiently than does a nonmycorrhizal one. This improved nutrient absorption is probably due to the greater surface area provided by the fungal mycelium.

Suggested Reading
1. 2. 3. 4. 5. 6. 7. Avances in Agricultural Microbiology (1982) by Subba Rao. Agricultural Microbiology (1996) by G. Rangaswamy and D.J. Bagayaraj. Brock Biology of Microorganisms (1997) by Michael T. Madigan, John M. Martinko, Jack Parker. Ecology of Microbial Communities (1987) by M. Fletcher, T.R.G. Gray & J.G. Jones. Introduction to Soil Microbiology (1961) by Martin Alexander. Microbiology (1999) by Lansing M. Prescott, John P. Harley & Donald A. Klein. Nitrogen Fixation in Plants by (1986) R.O.D. Dixon & C.T. Wheeler.

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8. 9. 10.

Plant-Microbe Interactions. Molecular & Genetic Perspectives (1984) Vol. 1 by Tsune Kosnge & Eugene W. Nester. Plant Microbe Interaction in Sustainable Agriculture (1995) by R.K. Behl, A.L. Khurana & R.C.Dogra. Soil Microbiology by (1994) Robert L. Tate III.

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