In this article we will discuss about the microorganisms in relation to plant growth.
What are Rhizosphere and Rhizoplane?
Rhizosphere refers to the region in the vicinity of roots in which the maximum microbial growth and activities operate (Fig. 34.1). The term ‘rhizosphere’ was first used by Lorenz. Hiltner a German scientist, in 1904.
Greater microbial growth and activities take place in rhizosphere because the roots release a wide variety of materials including various alcohols, sugars, ethylene, amino and organic acids, vitamins, nucleotides, polysaccharides, and enzymes that create unique environments for the soil microorganisms.
The intensity of the microbial growth and activities depends on the distance to which the root exudates can migrate. The rhizosphere microorganisms not only increase their numbers when the root exudates become available, but their composition and function also change.
Rhizosphere microorganisms, as they respond to root exudates, also serve as labile sources of nutrients for other organisms, thus creating a soil microbial loop in addition to playing critical roles in organic matter synthesis and degradation.
Rhizoplane refers to the ‘root surface’ together with closely adhering soil particles. In sampling the system for rhizoplane studies, soil adhering to roots is removed and roots subjected to serial washing by sterilized water (10-12 times) until the clean root surface is exposed.
When such washed roots are plated, characteristic fungi and bacteria appear on agar plates, thereby indicating that there are certain microorganisms intimately associated with the root surface.
Some fungi inhabit the root surface in a mycelial state. They belong to the genera Mortierella, Cephalosporium, Trichoderma, Penicillium, Gliocladium, Gliomastix, Fusarium, Cylindrocarpon, Botrytis, Coniothyrium, Mucor, Phoma, Pythium, and Aspergillus.
Fine structure studies on the epithelial layer of plant roots after inoculation with specific bacteria have shown that bacteria get embedded on the surface of the root with the help of the mucilagenous external layer on the ‘mucigel’ normally present on actively growing root system.
Rhizosphere effect is the direct influence exerted by plant roots on the microorganisms within the rhizosphere. Likewise, the microbial populations in the rhizosphere considerably effect the growth of the plant. As a result of these interactions there is qualitative effect and microbial populations reach much higher densities in the rhizosphere than in the non-rhizosphere soil.
It is now clearly established that greater number of bacteria fungi and actinomycetes are present in the rhizosphere soil than in non-rhizosphere soil and there are innumerable reports in literature to substantiate this fact (Table 34.1). Several factors such as soil type, its moisture, pH and temperature, and the age and condition of plants are known to influence the rhizosphere effect.
Apart from the numerical preponderance of microorganisms in the rhizosphere, the rhizosphere effect is also manifested in the occurrence and distribution of bacteria characterized by specific requirements (Table 34.2) of amino acids, B- vitamins, and specialized growth factors (nutritional groups).
However, greater rhizosphere effect is seen With bacteria than with actinomycetes or with fungi. It is almost negligible with regard to algae and protozoa.
Root Exudates and their Influence on Rhizosphere Microorganisms:
Variety of Root Exudates:
A variety of organic substances available at the root region by way of exudates from roots are one of the most important factors responsible for rhizosphere effect. These substances directly or indirectly influence the quality and quantity of microorganisms in the rhizosphere. The substances exuded by plant roots include amino acids, sugars, organic acids, vitamins, nucleotides, and many other unidentified substances.
The nature of substances exuded by roots of plants has been summarized in Table 34.3. The nature and amount of substances exuded are dependent on the species of the plant, age, and environmental conditions under which they grow.
By the use of 14CO2, it has been shown that products of photosynthesis are translocated to the root system and find their way into the rhizosphere in less than 12 hours, clearly indicating the influence of the metabolism of plants in determining the extent of the rhizosphere effect.
The root cap and active growth areas are primary regions of root exudation and one of major sites of carbon release from seminal wheat roots into the soil happens to be the zone of the root elongation. It has been advocated that exudation is either from root tips or regions at which lateral roots emerge from the main root.
Following are the important influences exerted by root exudates on soil micro-flora:
1. Root exudates influence the survival and proliferation of dormant structures of various soil surviving pathogens. Spores or sclerotia of many pathogenic fungi such as Rhizoctonia, Fusarium, Selerotium, Aphanomyces, Pythium, Colletotriclium, Verticillium, Phytophthora, and Plasmodiophora have been shown to germinate by the stimulus provided by the root exudates of susceptible cultivars of the host plants. The stimulus for germination has been attributed to compounds exuded by plant roots which help to overcome, in some manner, the static nature of dormant reproductive structures in soil.
2. Root exudates may provide a food base to result in the growth of antagonists which could suppress the growth of pathogenic microorganisms in soil. Many instances have been reported where the rhizosphere of resistant varieties harboured more numbers of Streptomyces and Trichoderma than that of the susceptible varieties.
3. One of the attributes of root exudates is the possible role they play in neutralizing the soil pH and altering the microclimate of rhizosphere through liberation of water and carbon dioxide. Such changes may influence infections of roots by pathogenic fungi.
Changes in Rhizosphere Micro-flora:
Changes in the rhizosphere micro-flora are reported to take place by:
(1) Soil amendments,
(2) Foliar application of nutrients, and
(3) Artificial inoculation of seed or soil with preparations containing live microorganisms, especially bacteria (bacterization).
Microbial seed inoculants such as Azotobacter, Beijerinckia, Rhizobium, or P-solubilizing microorganisms may help in the establishment of beneficial microorganisms in the rhizosphere or in the immediate vicinity of growing roots.
Field experiments have shown that counts of Azotobacter in wheat rhizosphere increased upon artificial seed inoculation indicating the efficiency of bacterization as a means of altering and improving the rhizosphere micro-flora.
Many experiments have been done to find out the effects of N, P, and K additions on rhizosphere micro-flora. Also, extensive studies have been done on induced changes in the rhizosphere micro-flora by foliar sprays of antibiotics, growth regulators, pesticides, and inorganic nutrients in the hope that such an approach may serve as a new tool in biological control of root diseases. However, no definite conclusions or guidelines have emerged from such studies to merit their application under field conditions.
Nitrogen Fixation by Free Living Microorganisms in Rhizosphere:
Many free living microorganisms in the rhizosphere are found to fix atmospheric nitrogen. These microorganism are Azotobacter, Azospirillum, Klebsiella, Bacillus, Beijerinckia, Pseudomonas, Clostridium, Enterobacter, Erwinia, Derxia, Frankia, etc.
Colonization by Azotobacter has been observed to be limited in the rhizosphere and practibly negligible on rhizosphere. Colonization of rhizoplane by Azospirillum has been noticed with rather extensive intrusion within root tissues. Sporangia of Frankia have been observed in the rhizosphere of Casuarina seedlings.
Associative and Antagonistic Activities:
Many microorganisms depend on in rhizosphere for extracellular products, mainly amino acids and growth promoting factors. This represents their associate functioning in the soil. Co-inoculation of nitrogen fixing Azotobacter and Azospirillum isolates with Rhizobium appears to have beneficial influence in increasing, nodule number, nitrogen fixation, and yield of soybean, pea, and clover.
Russian workers have demonstrated an increase in amino acid content in plants grown in soil inoculated with specific microorganisms. Secretion of antibiotics by microorganisms and the resultant biological inhibition of growth of other susceptible microorganisms are demonstrable in soil as well as in pure cultures.
Such antagonistic effects are natural to expect even in uncultivated soil and from the agronomic point of view excessive inhibition of Azotobacter or Rhizobium in the root region may lead to decreased nitrogen fixation or nodulation.
Plant Growth Promoting Rhizobacteria (PGPR) in Rhizosphere:
The term ‘rhizobacteria’ refers to those non-symbiotic (free living) bacteria which colonize the rhizosphere very aggressively. These free-living rhizobacteria affect the plant growth favourably and, in broad sense, are called plant growth promoting rhizobacteria (PGPR).
PGPR have been discovered by Kloepper et at. in 1980 and belong to genera Pseudomonas, Bacillus, Serratia, Arthrobacter, and Streptomyces. Pseudomonas spp. exhibit fluorescence under ultra-violet light and hence are also known as fluorescent pseudomonads.
Two possible mechanisms have been suggested to explain the beneficial effects of PGPRs in enhancing production.
(i) Competition for substrate and niche exclusion, and
(ii) Production of siderophores and antibiotics. However, more than one mechanism may operate for mediating a biological control.
Fluorescent pseudomonads ‘mop up’ nutrients in the rhizoshpere because of their versatility in growth and nutrient absorption. The points of emergence of lateral roots are favourite spots where DRBs and PGPRs appear to complete for these spots very effectively.
PGPRs are being commercially produced and marketed. A product by name QUANTUM 4000 is being marketed by Gustafson Inc. Dallas, Texas as a growth promoter on peanut (groundnut) and cotton. The product contains Bacillus subtilis strain GBO3, which is a derivative of strain A13.
Other products may follow once procedures are standardized for mass multiplication together with strategies for carriers and quality control. Prior to this the repeatability of success under field conditions have to be established with regard to fluorescent pseudomonads; quite often, strains that succeed under green house conditions fail to do so in the field.
The use of multiple strains of fluorescent pseudomonads has shown success in the control of diseases such as take all of wheat and Fusarium wilt of radish.
Encouraging results in the control of take-all diseases of wheat have come in northwest China from field trails covering 4000 ha during 1991-94 by using P. fluoresceins CN12 and Tn5 derivatives, in different sites exhibiting varying environmental conditions; the yield increases of wheat due to rhizobacterial inoculation varied from about 16 to 65 percent.
Phyllosphere and Phylloplane:
Phyllosphere refers to the zone on leaves inhabited by microorganisms and the phylloplane represents the leaf surface. The term ‘phyllosphere’ was coined by Ruinen, a Dutch microbiologist, from her observations on Indonesian forest vegetation, where thick microbial epiphytic associations exist on leaves.
Plant parts, especially leaves are exposed to dust and air currents resulting in the establishment of a typical flora on their surface aided by the cuticle, waxes and appendages, which help in the anchorage of microorganisms. These microorganisms may die, survive or proliferate on leaves depending on the extent of influence of the materials in leaf diffusates or exudates.
Leaf diffusates or leachates have been analysed for their chemical constituents. The principal nutritive factors are amino acids, glucose, fructose, and sucrose. If the catchment areas on leaves or leaf sheaths arc significantly substantial, such specialized habitats may provide niches for nitrogen fixation and secretion of substances capable of promoting the growth of plants.
Under conditions of high humidity, as in wet forests in tropical and temperate zones, the microflora in phyllosphere may be quite high.
The dominant and useful bacteria that have been identified are Azotobacter, Beijerinckia, Pseudomonas, Pseudobacterium, Phytomonas, Erwinia, Sarcina. Nitrogen fixing cyanobacteria (e.g., Anabaena, Calothrix, Nostoc, Scytonerna, Tolypothrix) have been encountered in phyllosphere in various moss forests.
Some of the fungi and actinomycetes recorded in phyllosphere are Podospora, Uncinula, Sporobolomyces, Cryptococcus, Rliodotorula, Cladosporium, Altemaria, Cercospora, Helmintliosporium, Erysiphe, Sphaerotheca, Torula, Torulopsis, Oidium, Rhytichosporium, Spermospora, Aureobasidium, Metarrhizium, Myrothecium, Verticilliuni, Melanospora, Saccliaromyces, Candida, Tilletia, Tilletiopsis, Penicillium, Cephalosporium, Fusarium, Periconia, Pithomyces, Mucor, Cunninghamella, Fusarium, Trichoderma, Heterosporium, Stachybotrys, Aspergillus, Curvularia, Rhizopus, Syncephalastrum, Actinomyces, and Streptomyces.
Many bacteria in phyllosphere are considered to fix nitrogen, while the leaves in turn provide carbohydrates and other nutrients to them. Bacteria of the genera Bacillus, Achromobacter, Pseudomonas, Cellulomonas have been isolated from the phyllosphere of pea and wheat and have been proved to be potential nitrogen fixers (Table 34.4).
Phyllosphere microorganisms are thought to effectively control air-borne pathogens from disease causation. These microbes or their propagates induce plants to synthesize ‘phytoalexins’ as defence weapon to counter disease causing pathogens.
Notwithstanding the above observations, the exact role of phyllosphere microorganisms still remains conjectural. Experiment done under laboratory conditions to demonstrate nitrogen fixation in the phyllosphere of several plants by the use of 15N and the quantitative data obtained arc so divergent that one is led to believe that fixation of nitrogen is a very variable phenomenon in the phyllosphere.
Therefore, experiments have to be designed to study the fate of biologically fixed nitrogen in the phyllosphere. In recent part, spraying of leaves of crop plants with aqueous solutions of sucrose or with bacterial suspensions has resulted in enhanced growth and yield of certain legumes and cereals in pot during experimental trials.
Apparently, such sprays sometimes have intensified the biochemical events on the phyllosphere towards the beneficial side. These observations require to be necessarily evaluated under field conditions so as to exploit the phyllosphere phenomenon towards improvement of agricultural output.
3. Mycorrhizae (Sing. Mycorrhiza):
Mycorrhizae represent a mutualistic symbiosis between the root system of higher plants and fungal hyphae and contribute variously in plant growth promotion.
4. Cyanobacteria (Oxygenic Phototrophs):
The cyanobacteria or the blue green bacteria are amongst the oldest organisms evolved on earth. They represent the only group of oxygen-evolving photosynthetic prokaryotes which possess ability to fix atmospheric nitrogen and promote plant growth.
Frank (1889) first reported their ability of nitrogen-fixation. However, most of the cyanobacteria produce heterocysts that act as the site of nitrogen-fixation.
5. Phosphate Solubilizing Microorganisms (PSM):
Phosphorus occurs in soil in two forms, organic phosphates and inorganic phosphates but deficiency of phosphorus may take place in crop plants growing in soils containing adequate phosphates. This may be partly due to the fact that plants are able to absorb phosphorus only in its available form.
Phosphates present in soil are made available to plants mainly by the activities of some soil microorganisms, which are called phosphate solubilizing microorganisms (PSM).
The latter not only play important role in reducing phosphorus deficiency in plants by way of altering unavailable phosphate into available form, but also they release soluble inorganic phosphate (H7SO4) into soil through decomposition of phosphate-rich organic compounds.
In addition, certain soil inhabiting microorganisms, through assimilation, may immobilize available phosphates in their cellular material and such immobilization processes in soil may contribute to phosphorus deficiency of crop plants.
Although bacteria are used in the large scale preparations of phosphate solubilizing cultures to promote plant growth, fungi such as Aspergillus, Penicillium, Cladosporium, Paecilomyces, Fusarium, Rhizoctonia, etc. appear more advantageous agents in the solubilisation of phosphates. Some fungi (e.g. Glomus, etc.) form associations with plant roots. These associations are called mycorrhizae.
Mycorrhizal fungi convert non- available phosphorus into an available form, produce growth promoting substances, and also protect plants against soil pathogens. Phosphate solubilizing microorganisms usually reduce the pH of the substrate by secretion of various organic acids such as formic acid, acetic acid, propionic acid, lactic acid, organic acid, fumaric acid, and succinic acid.
Phosphate Solubilisation in Different Soils:
Phosphate solubilisation in neutral or alkaline soil is apparently not rare since one-tenth to one-half of the bacterial isolates (e.g. isolates of Pseudomonas, Mycobacterium, Bacillus, Micrococcus, Flavobacterium, etc.) tested usually are capable of solubilizing calcium phosphates, and counts of bacteria solubilizing insoluble phosphates may range from 105 to 107 per gram. Such bacteria arc often especially abundant on rhizoplanes (root surfaces).
On the contrary, acid soils are generally poor in calcium ions and, therefore, phosphates are precipitated in the form of ferric or aluminium compounds which are not so easily amenable to solubilisation by soil micro-organisms.
In acid soils, however, the deficiency of phosphorus may occur due to the earlier mentioned reason, and this deficiency can be overcome by inoculating seed or soil with phosphate solubilizing microorganisms along with phosphatic fertilizers.
Some bacteria produce iron chelating substances, called siderophores. The siderophores transport iron into bacterial cells. Florescent pseudomonads produce yellow-green, fluorescent siderophores which specifically recognize and sequester the limited supply of iron in the rhizosphere and thereby reduce the availability of this trace element for the growth of the pathogenic micro-organisms. The availability of iron in soil decreases with increase in pH and therefore PSM function better in neutral and alkaline soils than in acid soils.
Ferric Phosphate Mobilization:
Although solubilisation of phosphate usually requires acid production as stated earlier other mechanisms may account for ferric phosphate mobilization. In flooded soil, the iron in the form of insoluble ferric phosphate is reduced resulting in the formation of soluble iron with a concomitant release of phosphorus into solution.
Such increases in the availability of phosphorus on Hooding may explain why rice cultivated under water often requires less amount of fertilizer phosphorus than the same crop grown in dry-lands. Phosphorus may also be made more available for plant uptake by certain bacteria that release hydrogen sulphide (H2S). a product that reacts with ferric phosphate to yield ferrous sulphide, liberating the phosphorus.
Commercialization of PSM:
Phosphate solubilizing microorganisms (PSM) have been found more beneficial in case of vegetable than cereal crops. A commercial preparation, namely, phosphobacterin was widely used for the first time in USSR. This preparation contained bacterial cells of Bacillus megatherium. But, the usefulness of phosphobacterin in soil was rather overemphasized despite the fact that increase in grain yield was of the order of 5-10%.
However, various field trials have been conducted by Indian Agricultural Research Institute (IARI) with wheat, maize, arhar, rice, potato, groundnut, gram. etc. to test the efficiency of phosphate solubilizing bacteria on the crop yield. It was found that significant increases where limited to 13 out of 38 experiments. These results demonstrated that there was no consistent response with respect to increase in yield.
Structure and Characteristics:
Azotobacter is a soil-inhabiting bacterium and comprises large, gram-negative, obligately aerobic rods (Fig. 34.2A). This bacterium freely lives in soil and fixes atmospheric nitrogen nonsymbiotically. The first species of Azotobacter was discovered by the Dutch microbiologist M. Beijerinck in the beginning of 20th century, and was named by him Azotobacter chroococcum.
Subsequently, many other species of Azotobacter were isolated from different soils of the world and some important ones are: A. agilis, A. vinelandii, A. beinjerinckii, A. insignis, A. macrocytogenes, A. paspali, etc. Azotobacter cells are large, many isolates being almost the size of yeasts, with diameters of 2-4 μm or more. Pleomorphism is common and a variety of cell shapes and sizes have been described. Some strains possess peritrichous flagella.
Although the Azotobacter is an obligate aerobe, its enzyme callcd nitrogenasc that catalyzes atmospheric nitrogen fixation is oxygen-sensitive. It has been studied that the high respiratory rate characteristic of Azotobacter and the abundant capsular slime help protect nitrogenase from oxygen.
This bacterium grows on a wide variety of carbohydrates, alcohols, organic acids, amonia, urea, and nitrate. Azotobacter forms cysts (Fig. 34.2B) the resting structures, which arc resistant to desiccation, mechanical disintegration, and ultraviolet and ionizing radiation. Each cyst measures about 3 μm in diameter.
Mechanism of N2 Fixation:
Azotobacter (A. chroococcum and A. vinelandii) is one of the most extensively investigated member amongst free-living nitrogen fixing bacteria. The use of 15N tracer and acetylene reduction method have however enriched our knowledge regarding the biochemical pathway between atmospheric nitrogen (dinitrogen; N2) and ammonia (NH3) but the exact nature of intermediate products have eluded even critical investigators.
Nevertheless, the overall reaction in the enzymic reaction of N2 to NH3 can be postulated as under:
Nitrogenase of Azotobacter:
Nitrogenase, the enzyme that catalyzes atmospheric nitrogen fixation, consists of two protein fractions:
(i) The Mo-Fe containing protein (molecular weight 220,000-2,70,000) and
(ii) Fe containing protein (molecular weight 55,000-66,800).
In Azotobacter (A. vinelandii), two additional nitrogenases have been investigated. One of these possesses vanadium (V) instead of molybdenum (Mo) and the other has neither molybdenum nor vanadium.
The characterization of these nitrogenases has generated fresh problems in pinpointing evidences to demonstrate the essentiality of molybdenum for N2-fixation and characterization of the site at which nitrogen binds to nitrogenase.
Effect of Azotobacter Inoculation:
Azotobacter inoculated seed or soil effectively increase crop yield if the soil is well-manured and contains high organic matter. In addition to fix atmospheric nitrogen, Azotobacter synthesizes substances like B-vitamins. indole acetic acid and gibberellins in pure culture during experimental trails.
Also, the bacterium possesses fungistatic properties to some pathogenic fungi such as Altemaria and Fusarium. These properties of Azotobacter, however, favour the fact that these bacteria promote seed germination and plant growth.
Generally low population of Azotobacter is found in the rhizosphere of crop plants and in uncultivated soil. Often inoculation of soil or seed docs not improve the situation. To overcome this limitation, repeated application of Azotobacter during different stages of growth of a crop is now being recommended with the object of increasing the number of bacteria in soil.
Some experiments on inoculation of soil with Azotobacter with different doses of inorganic N fertilizer have revealed the possibility of saving considerable amount of N fertilizer while still attaining desired yields of rice. Field trials with new and efficient cultures of Azotobacter have shown that the yields of some important crop plants can be substantially increased by Azotobacter inoculation (Table 34.5).
Azospirillum is a rod to spirillum-shaped nitrogen fixing bacterium and freely lives in soil forming nonspecific symbiotic associations with various plants (Fig. 34.3), in particular, corn. This genus consists of species, namely, A. lipoferum, A. brasilense, A. amazonense, A. halopraeferans, A. nitrocaptans, and A. seropedica. Azospirillum lipoferum was originally described and named Spirillum lipoferum by Beijerinck in 1922.
Although this species of the genus was known since 1963 as a nitrogen fixer, it was Dobereiner and colleagues in Brazil who in 1975 highlighted and attributed the nitrogen fixation potential of some tropical forage grasses (e.g., Digitaria, Panicum, maize, sorghum, wheat and rye) to the activity of S. lipoferum in their roots.
Azospirillum characteristically develops white, dense, and undulating pellicles on a semi-solid malate containing enrichment medium. The pellicle is formed 2 mm below the surface of the medium indicating the microaerophilic nature of the bacterium. Azospirillum in gram-negative contains poly-β-hydroxy butyrate (PHB) granules, and shows polymorphism and spirillar movement.
It fixes atmospheric nitrogen (dinitrogen) in microaerophilic surroundings (low oxygen conditions) but possesses ability to grow profusely in ammonium- rich environment without fixing nitrogen. The bacterium also produces growth substances such as indole acetic acid (IAA), kinetins, and gibberellins. Table 34.6 shows the species of Azospirillum and their association partners investigated since 1974.
Effect of Azospirillum Inoculation:
Indian Agricultural Research Institute carried out field experiments to know that effect of Azospirillum inoculation in different parts of India.
These experiments revealed that seed inoculation of sorghum (Sorghum bicolor), bajra (Pennisetum americanum) and ragi (Eleusine corocana) increased grain and fodder yields in different agro-climatic conditions of India (Table 34.7). Similar results have been obtained by scientists in Israel who also find responses of millets to Azospirtilum inoculation.
M.W. Beijerinck, a Dutch microbiologist, was the first to isolate and cultivate a microorganism from the nodules of legumes in 1888. He named it Bacillus radicicola which ensured its place in Bergey’s Manual of Determinative Bacteriology under the genus Rhizobium.
Rhizobia represent the most well known group of symbiotic nitrogen fixers and all rhizobia were previously included in the genus Rhizobium, but later this genus has been split into six genera whose taxonomic position is shown in Table 34.8.
Each of the six genera of rhizobia consists of many recognized species the important ones of which are shown in Table 34.9.
Structure and Characteristics:
Rhizobia are soil inhabiting, free-living heterotropic bacteria which show locomotion with the help of peritrichous or sub-polar flagella. The different strains of rhizobia are attracted by different flavonoids, chemical substance secreted by hosts, and reach the host’s root zone to form symbiotic association to fix atmospheric nitrogen. However, the structure and characteristics of different genera of rhizobia are the following.
Rhizobium is a rod-shaped, 0.5-0.9 x 1.2-3.0 μm long, motile, gram-negative, non-spore forming bacterium. It utilizes organic acids salts as carbon sources without gas formation and grows optimum at 27°C temperature and 6.8 pH. This bacterium for root nodules with its leguminous hosts and fixes atmospheric nitrogen.
The colonics of Rhizobium appear as circular, convex, semitranslucent, raised, mucilaginous, and usually 2-4 mm in diameter. The strains of Rhizobium are fast-growing, where generation time lasts about 6 hours besides showing some other differences with rest of the members of family Rhizobiaceae.
Sinorhizobium, like Rhizobium, is fast growing. It is rod-shaped, usually contains poly-β-hydroxybutyric acid (PBHA) granules, non-spore forming, gram-negative, motile, and aerobic. Most of its strains grow at 35°C temperature and 6.8 pH. Sinorhizobium is a new genus.
It has been observed recently that some rhizobial strains, which are fast growers, nodulate soybean (generally, slow growing bradyrhizobia nodulate soybean). These fast growers were identified as a seperate genus Sinorhizobium.
Bradyrhizobium strains are slow growers with a generation time usually about 12 hours or even more. They move in the soil with the help of one polar or subpolar flagcllum. The growth on carbohydrate medium is accompanied by exopolysaccharide (EPS) slime.
Some strains can grow chemolithotrophically (inorganic salt users) in the presence of H2, CO2, and low level of oxygen. The bacteroids in root nodules are slightly swollen rods with rare branching or coccus forms. The main symbiotic partner of Bradyrhizobium is soybean, while other plants (e.g. Lotus, Vigna, Lupinus, Cicer, Mimosa, Lablab, Acacia, Dalbergia) also are the symbiotic partner.
Mesorhizobium is raised as a new genus of the family Rhizobiaceae only recently and has been named on the basis of whole sequence studies of 165 rRNA. Some of the species of Rhizobium, namely, R. loti, R. haukuii, R. ciceri, R. mediterraneum, R. tianslianense arc now known as the species of genus Mesorhizobium.
Azorhizobium strains bear flagella and are motile. They bear peritrichous flagella on solid medium but one lateral flagellum in liquid medium. They are oxidase and catalase positive and cannot oxidize mannitol. However, Azorhizobium, in contrast to other rhizobia, is a stem-nodulating bacterium.
A caulinodans develops nodules on the stem of tropical aquatic legume Sesbania and fixes atmospheric nitrogen. Stem-nodulated leguminous plants are quite widespread in tropical regions where soils are often nitrogen-deficient because of leaching and intense biological activity.
Root Nodule Formation and N2-Fixation:
The rhizobia grow free-living in soil, infect leguminous plants, and establish a symbiotic existence. Infection of the roots of a legume with the appropriate species of one of five genera of rhizobia leads to the formation of root nodules that fix atmospheric nitrogen. Nitrogen fixation by these symbionts are of considerable agricultural significance as it leads to considerable increases in combined nitrogen in the soil. Since nitrogen deficiencies often occur in unfertilized bare soils, nodulated legumes can grow well in areas where other plants cannot.
9. Actinorhizae (Frankia-Induced Nodulation):
Apart from legumes nodulated by rhizobia, roots of the some non-leguminous plants are nodulated by an actinomycete named Frankia. These actinomycete associations with plant roots are called actinorhizae (sing, actinorhiza). Actinorhizae fix considerable amounts of nitrogen and are important, particularly in trees and shrubs.
There are 25 genera from 8 angiosperm families which have been described to possess actinorhizal root nodules. These families with genera mentioned in parentheses are Casurinaceae (Casurina, Allocasuarina, Centhostoma, Gymnostoma), Coriariaceae (Coriaria), Dasticaceae (Datisca), Betulaceae (Alnus), Myricaeeae (Comptonia, Myrica), Elaeagnaceae (Elaeagnus, Hippophae, Shepherdia), Rhamnaceae (Adolphia, Ceanothus, Colletia, Discaria, Kentrothamnus, Retanilla, Talguenea, Trevoa) and Rosaceae (Cercocarpus, Chaemabatia, Cowania, Dryas, Purshia).
The genera Casuarina for tropical and sub-tropical regions and Ainus (A. rubra. A. glutinosa, A. crispa, A. jorullensis, A. acuminata) for temperate regions stand out as excellent examples for the benefits they provide to the ecosystems by way of nitrogen inputs.
They can adapt themselves to grow under most diverse environmental conditions and geographical zones. Casuarina species (C. equisetifolia, C. cunninghamiana, C. littoralis, C. stricta, C. junghuniana, C. glauca and C. torulosa) provide substantial fuel and building materials in tropical countries while alders provides the most utilised hard wood as well as bark for paper industries in temperate regions.
Frankia is the actinomycete classified under family Frankiaceae, suborder Frankineae, order Actinomycetales, class Actinobacteria. This genus was named after its discoverer Frank in the 1880s. Frankia is filamentous, strcptomycete-like, possesses multilocular sporangia (Fig. 34.4), and forms clusters of spores when a hypha divides both transversely and longitudinally.
It is microaerophilic, grows slowly, forms non- motile spores, and grows in symbiotic association with the roots of earlier mentioned variety of nonleguminous angiosperms.
Entry of Frankia to the Host Plant:
Frankia cells get embedded in a mucilage layer in the root region or the spores may get attached to root hairs the root hairs get deformed or curled. The actual entry of Frankia into root hairs has not been seen but hyphae are seen as simple or multiple threads often branching inside the deformed hair in a host derived cell wall material that is continuous with the root hair cell wall (encapsulation).
The threads could be seen penetrating the cortex and in some root sections, pre-nodule formation can be seen within 10-14 days.
Sooner or later, lateral roots in the vicinity of the primary nodule primordium appear, their meristems undergo branching and progressively get infected with Frankia resulting in the formation of a typical adult nodular structure referred to as a ‘rhizothamnion’. In a sense, actinorhizal root nodule is essentially a modified lateral root.
Structural Organization of Actinorhizal Nodule:
Actinorhizal nodules of Alnus and Casuarina occur in clusters attaining a diameter of 5 to 6 cm somewhat resembling a tennis ball (Fig. 34.5) often weighing up to 444 kg dry weight of nodules/ha. There are two types of structural organization in actinorhizal root nodules: Alnus type and Casuarina type. Alnus-type of root nodules possess many lenticels on nodules that provide ventilation.
Internally, there is a central vascular bundle surrounded by a cortex in which several pockets of Frankia inhabiting zones can be seen containing vesicles that are the sites of nitrogenase activity. Casuarina-type of root nodules possess suberized cells containing the hyphal endophyte with swollen tips.
The suberized cells in Casuarina type of root nodules are impervious to air and hence provide protection to nitrogenase and to the swollen hyphal tips which are belived to be the sites of nitrogen fixation. However, the structural differences in both types are diagrammatically depicted in Fig. 34.6.
Both the types of actinorhizal root nodules differ from legume root nodules formed by rhizobia. Legume root nodules are characterized by a central uniformly infected bacteroid and leghaeinoglobin containing zone surrounded by a tight inner cortex that limits gas diffusion with the vascular bundles lying outside the inner cortex.
Nitrogen Fixation and Ammonia Assimilation by Frankia:
Nitrogenase activity in actinorhizal nodules is host as well as Frankia dependent espeon the morphological state of Frankia whether in the form of spores or hyphae. Nitrogenase has been detected in vesicles as well as hyphae but abundance of vasicles coincides with high nitrogenase activity.
The vesicles contain thick wall that retard O2 diffusion thus protecting oxygen sensitive nitrogenase. Two hypotheses are given to understand the possible mechanism of ammonia assimilation on lines similar in cyanobacterial heterocysts. One hypothesis assumes that glutamine is produced in vesicles and could be transported to vegetative hyphae through the constricted stem cell of the vesicles.
In the hyphae, glutamine would be converted by the enzyme GOGAT to glutamate with one of the resulting glutamates going back to the vesicles to function as an ammonia acceptor for repeating the reaction.
In the second hypothesis, it is considered that the enzyme GS (glutamine synthetase) is not active in ammonia assimilation in vesicles which leads to accumulation of the fixed product in the hyphae and surroundings where it would be assimilated by the GS-GOGAT system, presumably aided by the high affinity ammonia perm-ease present in nitrogen starved hyphae which helps in mopping up all free ammonia.
Reafforestation with Frankia-Inoculated Trees:
In developing countries, deforestation for fuel has rendered the land barren and continuous deforestation of the same land in overpopulated regions of such countries has resulted in soils which remain deficient in nitrogen, the most important element for the normal growth of plants.
One of the least expensive and non-polluting ways to replenish the lost soil nitrogen is reafforestation by planting self-supporting nitrogen-fixing trees.
In Pennsylvania, U.S.A. reafforestation of mine spoils have been done by planting nitrogen, fixing red alder (Alnus rubra) inoculated with nodule forming Frankia. In New Quebec, Canada, alder plants (Alnus spp.) have been planted on a large scale to fill dam dykes. In Senegal, Egypt, and the coastal region of India and China nitrogen-fixing Casuarina spp. have been planted on a large scale to contain and stabilise sandy tracts which have made inroads to agricultural land.