Nitrogen Fixing Bacteria: Rhizobium, Azotobacter & Azospirillum !
Organisms capable of fixing atmospheric nitrogen into available organic form are known as nitrogen fixers. Nitrogen is essential to all plants and is a macronutrient. There are organisms like bacteria and algae capable of fixing atmospheric nitrogen.
These bacteria can exist in free-living form like Azotobacter, Beijerinkia, Clostridium, Klebsiella in soil, or as associative symbionts like Azospirillum in plant roots or as symbionts like Rhizobium in leguminous plant roots and Frankia in actinorhizal plants.
The Actinomycete genus Frankia is Gram-positive bacteria that nodulate about eight plant families representing about 25 genera of dicotyledonous collectively called actinorhizal plants. The term actinorhiza is given to nitrogen-fixing root nodules that are formed by Frankia.
Rhizobium is most often seen in leguminous plants forming root nodules. Rhizobium infects the legume root to form root nodules in which molecular nitrogen is reduced to ammonia.
The list of nitrogen fixing bacteria associated with non-legumes includes species of Acetobacter, Azomonas, Beijerinckia, Bacillus, Clostridium, Enterobacter, Erwinia, Klebsiella, Mycobacterium, Rhodospirillum, Rhodo-pseudomonas, Xanthobacter, and Mycobacterium.
Rhizobium acts as a wonder biofertilizer for legumes like pulses, gram, chickpeas, soyabeans and lentils etc. whereas Azospirillum and Azotobacter are good biofetilizers for cereals like wheat, millet, sorghum and maize etc. These biofertilizers are also shown to produce the LAA, gibberellin and cytokine- like substances which promote plant growth.
Rhizobium- Characteristic, Features and Functions:
Rhizobium is a soil habitat bacterium, which can colonize the legume roots and fix the atmospheric nitrogen symbiotically. These bacteria are rod shaped, motile aerobic and gram positive.
Association of the bacteria in the plant roots causes nodule formation. Rhizobia move from the root surface to the inner root tissue where they grow and divide inside a tubule called an infection thread. Rhizobia inside the thread grow and divide, thereby keeping the tubule filled with bacteria.
In the root nodule a special protein called leg-hemoglobin is found which imparts a red color and maintains the oxygen levels in the cells. The globin protein is encoded by the plant genes but the heme cofactor is formed by the associated bacteria. The bacteriods get organic acids from the plant cells and in exchange give free ammonia for their utilization.
In the laboratory they can be grown in CRYEMA – Congo Red Yeast Extract Mannitol Agar culture medium and for mass production the inoculum is cultured in flasks or fermenters in a liquid medium without agar.
All these bacteria are classified into 6 genera – Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium and Allorhizobium which forms nitrogen fixing root nodulation in leguminous plants. Each legume plant requires a specific species of Rhizobium to form effective nodules and nitrogen fixation.
One effective Rhizobium strain may bring about similar results in different legume plants in terms of nodulation and nitrogen fixation. Further, Rhizobium population in soil is dependent on the type of legume plants growing. The Rhizobial population may be lost in the absence of legume plants and in such cases artificial seed inoculation is required to restore the Rhizobial population.
The following criteria should be used for collection of Rhizobial strains for inoculating leguminous crops:
i. Ideal inoculants strain which can form nodules in the target legume.
ii. The Rhizobial strain should be able to show a high percentage of nodule formation.
iii. The strain should be able to fix nitrogen across a range of environmental conditions.
iv. The strain should be able to persist in soil even in the absence of a legume host.
Rhizobium as a Bio fertilizer:
The symbiotic association of plants with the bacterium leads to improvement in root uptake of nitrogen and it is further modified for balanced nutrient uptake in terms of Nitrogen, Phosphate and Potassium (NPK) through co-inoculation of bacterial mixture with plants.
In Chickpea, when Rhizobium spp co-inoculated with some Pseudomonas and Bacillus strains, resulted in enhanced growth, nodulation and nitrogen fixation. Similar results are observed in Chickpea on combined application of Rhizobium, Phosphate solubilising bacteria (PSB) and Trichoderma fungus. In Beans, increased nodulation and Acetylene reduction activity recorded on application of Rhizobium leguminosarum and Pseudomonas putida.
Azotobacter- Characteristic, Features and Functions:
In 1901, Martinus Beijerinck discovered the genus Azotobacter. Azotobacter is a gram- negative, free-living and nitrogen-fixing soil bacteria. This genus comprises seven species: A. chroococcum, A. vinelandii, A. beijerinckii, A. paspali, A. armeniacus, A. nigricans and A. salinestri. Azotobacter species cannot survive under acidic pH.
Azotobacter is often deficient in Indian soils because of a lack of organic matter and antagonistic action of other microorganisms present in the rhizosphere.
Azotobacter is considered as a good biofertilizer because it plays a significant function in the nitrogen fixation in the soil. Besides this the bacterium produces abundant slime containing polysaccharides which helps in soil aggregation. Therefore, they provide increased resistance to environmental stress such as low and high temperatures, water deficit and salt stress conditions.
Beside this, Azotobacter also produces a number of plant growth enhancer molecules including thiamine, riboflavin, nicotine, Indole Acetic Acid (IAA), gibberellins and cytokinins. Therefore, it brings improvement in the seed germination, root development and plant growth and thereby increases crop productivity. Further, Azotobacters make ease of heavy metals mobility in the rhizosphere and consequently improving bioremediation of the rhizosphere from heavy metals like cadmium, mercury and lead. Overall, they have been considered key players in improving soil fertility and conserving soil ecology.
Azotobacter as a Bio fertilizer:
Azotobater sp. are being exploited as a bio fertilizer to improve diverse crop species including wheat, oat, barley mustard, sea sum, rice, linseeds, sunflower, castor, maize, sorghum, cotton, jute, sugar beets, tobacco and tea. It is advantageous to cereals, vegetables and fruit crops. It is more effective in vegetable crops where farmers apply adequate amounts of manure as it require about 1000 kg of organic carbon to fix 30 kg of N/ha. Its application as a bio fertilizer enhances the crop yield by 15-20%.
Azospirillum- Characteristic features and functions:
Azospirillum is nitrogen fixing gram-negative, curved-rod shape and motile bacteria. First isolated and purified Azospirillum was initially named as Spirillum lipoferum. It is found to exhibit oxidase positive function and acetylene-reduction action. They perform the associative symbiotic relation with the graminaceous plants.
Azospirillum sp. colonizes the rhizosphere and is also found to reside within the roots of sorghum, bajra and ragee. The A. lipoferum, A. brasilense and A. amazonense are extremely effective nitrogen-fixing bacteria in the rice rhizosphere. Azospirillum sp. is chemo-heterotrophic bacteria which show associative symbiosis through secretion of growth regulatory substances like (IAA).
It directly provides plant benefits by improving the shoot and root development, rate of water absorption and mineral uptake by roots. Additionally Azospirillum inoculation provides disease resistance and drought tolerance.
Azospirillum- Associative Effect with Different Microorganisms:
Its associative effects have been reported in different crops after inoculation. The associative symbiosis of A. chroococcum with higher plants was reported. The various associative effects of Azospirillum along with other microorganism were also discovered like: Paenibacillus polymyxa 1465 and A. brasilense in wheat; A. lipoferum and Sinorhizobium melilot in alfalfa plants; A. brasilense and Phyllobacterium myrsinacearum in microalga. Chlorella vulgaris and A. brasilense with Rhizobium japonicum on nodulation and yield in Glycine max plants.
Phosphate Solubilising Bacteria (PSB):
In soil, phosphorous is majorly present as a component of organic matter constituting 30-50% of total phosphorous. It is in the form of inositol phosphate (Soil phytate). Secondly, soil phosphorous is accumulated by regular application of phosphorous fertilizers which are soluble inorganic phosphorous unavailable to the plants due to immobilization.
These phosphates occur in nature in the form of insoluble and inorganic compounds like tricalcium phosphate, dicalcium phosphate, rock phosphate and hydroxyapatite. These forms of phosphate are not absorbed by plants.
Many of these phosphorous compounds are of higher molecular weight which needed to be converted into low molecular weight organic phosphorous or soluble ionic phosphorous before being assimilated by plants. The number of bacteria like Pseudomonas, Bacillus, Burkholderia, Achromobacter, Agrobacterium, Microccocus, Aereobacter, Flavobacterium and Erwinia which do solubilize them into absorbable form by producing many organic and inorganic acids.
In addition to this Pseudomonas and Bacillus produces plant growth hormones helping plants to grow faster and healthier.
Pseudomonas belongs to the family Pseudomonadaceae and was first classified by Walter Migula. It is a gram negative, rod shaped, flagellate and aerobic bacteria. There are various species of Pseudomonas known of which Pseudomonas fluorescents and Pseudomonas protegens are the plant growth promoting bacteria. These bacteria are also used as biocontrol agents. Today with genome sequencing, various species are well identified.
Bacillus is a gram positive, rod shaped bacteria. It is ubiquitous in nature. There are many species of Bacillus known for promoting plant growth through secretion of phytohormones, solubilization and mobilization of phosphates, siderophore production for induced systemic resistance and antibiosis.
Therefore, these bacteria help in biofertilization and are also useful as biocontrol mechanisms. Bacillus subtilis is used as a model organism.
Potassium Mobilising Bacteria (KMB):
Potassium is required by plants for synthesis and transfer of amino acids, proteins and carbohydrates. Potassium regulates membrane permeability, hydration of protoplast and plays an important role in making plants drought, pest and disease resistant.
Potassium is present as soil clay minerals like illite, leucite, microcline etc. and is made available to the plant roots for absorption by Potassium Mobilizing Bacteria (KMB) like Frateuria aurantia and Bacillus mucilaginous through the action of secreted organic acids.
Frateuria aurantia belongs to the family Pseudomonaceae and is a rod shaped gram negative bacteria which can grow in a wide range of temperature (15-42°C) and pH range from 3.5- 11. They are free-living soil bacteria and can be easily isolated from the rhizosphere of the plants. Frateuria aurantia is also considered to be Plant Growth Promoting Rhizobacteria (PGPR).
In India vast areas show potassium deficiency. The bacteria act on clay minerals and mobilize silica along with potassium. Besides KMB the phosphate solubilising bacteria also improve potassium mobility from the soil towards plant roots. Also silicate solubilising bacteria like Bacillus circulars release potassium from potassium bearing silicate minerals.
Micronutrient Solubilising Bacteria:
Micronutrients are as important as NPK for plant growth. The Bacillus mucilaginous is an example of potassium solubilizer which release silicon and potassium from different silicate minerals. The important micro nutrients like zinc, iron, copper, molybdenum and sulphur etc. can be made available in the soil with the help of many bacteria. Most of these micro nutrients are unavailable forms in soil.
Bacterial Biofertilizers from Bacillus subtilis and Thiobacillus thioxidans solubilizes zinc and makes it available to the crop plants. These are known as Zinc Solubilizing Bacteria (ZSB). Also important are the Silicate Solubilizing Bacteria (SSB) such as Bacillus subtilis and Bacillus circulars. Thus, micronutrient requirements are also fulfilled by bacterial Biofertilizers.