The rhizosphere, which is the narrow zone of soil surrounding plant roots, can comprise up to 1011 microbial cells per gram of root and above 30,000 prokaryotic species that, in general, improve plant productivity. The collective genome of the rhizosphere microbial community enveloping the plant roots is larger compared to that of plants and is referred to as microbiome, whose interactions determine the crop health in natural agro-ecosystems by providing numerous services to crop plants viz., organic matter decomposition, nutrient acquisition, water absorption, nutrient recycling*,* weed control and biocontrol.
The metagenomic study provides the individual, the core rhizosphere and endophytic microbiomes activity in Arabidopsis thaliana using 454 sequencing (Roche) of 16S rRNA gene amplicons. It has been proposed that exploiting tailor-made core microbiome transfer therapy in agriculture can be a potential approach in managing plant diseases for different crops. Rhizosphere microbial communities as an alternative to chemical fertilizers has become a subject of great interest in sustainable agriculture and biosafety programmes.
A major focus in the coming decades would be on safe and eco-friendly methods by exploiting the beneficial microorganisms in sustainable crop production. Such microorganisms, in general, consist of diverse naturally occurring microbes whose inoculation into the soil ecosystem advances soil physicochemical properties, soil microbial biodiversity, soil health, plant growth and development and crop productivity. The agriculturally useful microbial populations cover plant growth-promoting rhizobacteria, N2-fixing cyanobacteria, mycorrhiza, plant disease suppressive beneficial bacteria, stress-tolerant endophytes and biodegrading microbes. Biofertilizers are a supplementary component to soil and crop management traditions, viz. crop rotation, organic adjustments, tillage maintenance, recycling of crop residue, soil fertility renovation and the biocontrol of pathogens and insect pests, whose operation can be significantly useful in maintaining the sustainability of various crop productions. Azotobacter, Azospirillum, Rhizobium, cyanobacteria, phosphorus- and potassium-solubilizing microorganisms and mycorrhizae are some of the PGPRs that have been found to increase in the soil under no tillage or minimum tillage treatment. Efficient strains of Azotobacter, Azospirillum, Phosphobacter and Rhizobacter can provide significant amount of nitrogen to Helianthus annus and to increase the plant height, number of leaves, stem diameter percentage of seed filling and seed dry weight. Similarly, in rice, addition of Azotobacter, Azospirillum and Rhizobium promotes the physiology and improves the root morphology.
Azotobacter plays an important role in the nitrogen cycle in nature, as it possesses a variety of metabolic functions. Besides playing a role in nitrogen fixation, Azotobacter has the capacity to produce vitamins, such as thiamine and riboflavin, and plant hormones, viz. indole acetic acid (IAA), gibberellins (GA) and cytokinins (CK). A. chroococcum improves the plant growth by enhancing seed germination and advancing the root architecture by inhibiting the pathogenic microorganisms around the root systems of crop plants. This genus includes diverse species, namely, A. chroococcum, A. vinelandii, A. beijerinckii, A. nigricans, A. armeniacus and A. paspali.
It is used as a biofertilizer for different crops, viz. wheat, oat, barley mustard, sesame, rice, linseeds, sunflower, castor, maize, sorghum, cotton, jute, sugar beets, tobacco, tea, coffee, rubber and coconuts. Azospirillum is another free-living, motile, Gram-variable, aerobic bacterium that can thrive in flooded conditions and promotes various aspects of plant growth and development. Azospirillum has been shown to exert beneficial effects on plant growth and crop yields both in greenhouse and in field trials. Diverse species of the Azospirillum genus, including A. lipoferum, A. brasilense, A. amazonense, A. halopraeferens and A. irakense have been reported to improve the productivity of various crops. Interestingly, it was observed that Azospirillum inoculation can change the root morphology via producing plant growth-regulating substances via siderophore production. It also increases the number of lateral roots and enhances the formation of root hairs to provide more root surface area to absorb sufficient nutrients. This improves the water status of the plant and aids the nutrient profile in the advancement of plant growth and development. Co-inoculation of Azospirillum brasilense and Rhizobium meliloti plus 2,4-D had a positive effect on the grain yield and N, P, K content of Triticum aestivum. Rhizobium has been used as an efficient nitrogen fixer for many years. It plays an important role in increasing yields by converting atmospheric nitrogen into usable forms. Being resistant to different temperature ranges, Rhizobium normally enters the root hairs, multiplies there and forms nodules. Rhizobium inoculants in different locations and soil types have been reported to significantly increase the grain yields of Bengal gram and lentil and enhance the rhizosphere of pea, alfalfa and sugar beet, berseem, ground nut and soybean. Rhizobium isolates obtained from wild rice have been reported to supply nitrogen to the rice plant to promote growth and development. A Rhizobiaceae species, Sinorhizobium meliloti 1021, infects plants other than legumes, e.g. rice, to promote growth by enhancing the endogenous level of plant hormone and photosynthesis performance to confer plant tolerance to stress. In groundnut, the IRC-6 rhizobium strain has resulted in the enhancement of several useful traits such as increased number of pink coloured nodules, nitrate reductase activity and leghaemoglobin content in 50 DAI (days after inoculation). Rhizobial symbiosis provides defence to plants against pathogens and herbivores, such as, Mexican bean beetle and the greenhouse whitefly Trialeurodes vaporariorum.
The beneficial soil microorganisms sustain crop production either as biofertilizers or as symbionts. They perform nutrient solubilization, which facilitates the nutrient availability and thereby uptake. This improves the plant growth by advancing the root architecture. Their activity provides several useful traits to plants such as increased root hairs, nodules and nitrate reductase activity, and efficient strains of Azotobacter, Azospirillum, Phosphobacter and Rhizobacter can provide a significant amount of available nitrogen through nitrogen cycling. Biofertilizers produce plant hormones, which include indole acetic acid (IAA), gibberellins (GA) and cytokinins (CK). Biofertilizers improve photosynthesis performance to confer plant tolerance to stress and increase the resistance to pathogens, thereby resulting in crop improvement.
A key advantage of beneficial microorganisms is to assimilate phosphorus for their own requirements, which in turn, becomes available in its soluble form in sufficient quantities in the soil. Pseudomonas, Bacillus, Micrococcus, Flavobacterium, Fusarium, Sclerotium, Aspergillus and Penicillium have been reported to be active in the solubilization process. A phosphate-solubilizing bacterial strain NII-0909 of Micrococcus sp. has polyvalent properties, including phosphate solubilization and siderophore production. Similarly, two fungi, Aspergillus fumigatus and A. niger, isolated from decaying cassava peels have been found to convert cassava wastes by the semi-solid fermentation technique to phosphate biofertilizers. Burkholderia vietnamiensis, a species of stress tolerant bacteria, produces gluconic and 2-ketogluconic acids, which are involved in phosphate solubilization. Enterobacter and Burkholderia isolated from the rhizosphere of sunflower produce siderophores and indolic compounds (ICs) which can solubilize phosphate. Potassium-solubilizing microorganisms (KSM), such as the genera Aspergillus, Bacillus and Clostridium, are efficient in potassium solubilization in the soil and mobilization in different crops. Mycorrhizal mutualistic symbiosis with plant roots satisfies the plant nutrients demand, which leads to enhanced plant growth and development, and protects plants from pathogen attacks and environmental stress. It leads to the absorption of phosphate by the hyphae from outside to the internal cortical mycelia, which finally transfer phosphate to the cortical root cells. Nitrogen-fixing cyanobacteria, such as Aulosira, Tolypothrix, Scytonema, Nostoc, Anabaena and Plectonema, are commonly used as biofertilizers. Besides the contribution of nitrogen, growth-promoting substances and vitamins liberated by these algae, Cylindrospermum musicola increases the root growth and yield of rice plants. Interestingly, genetic engineering was used to improve the nitrogen-fixing potential of Anabaena sp. strain PCC7120. Constitutive expression of the hetR gene driven by a light-inducible promoter enhanced HetR protein expression, leading to higher nitrogenase activity in Anabaena sp. strain PCC7120 as compared with the wild-type strain. This, in turn, caused better growth of paddy when applied to the fields.
Abiotic and biotic stresses are the major constraints that affect the productivity of crops. Many tools of modern science have been extensively applied for crop improvement under stress, of which the role of PGPRs as bioprotectants has become of paramount importance in this regard. Trifolium alexandrinum inoculated with Rhizobium trifolii showed higher biomass and increased nodulation under salinity stress conditions. Pseudomonas aeruginosa has been shown to withstand biotic and abiotic stresses. Paul and Nair found that P. fluorescens MSP-393 produces osmolytes and salt-stress induced proteins that overcome the negative effects of salt. P. putida Rs-198 enhanced the germination rate and several growth parameters, viz, plant height, fresh weight and dry weight, of cotton under alkaline and high-salt conditions via increasing the rate of uptake of K+, Mg2+ and Ca2+, and by decreasing the absorption of Na+. A few strains of Pseudomonas reportedly confer plant tolerance via 2,4-diacetylphloroglucinol (DAPG). Interestingly, systemic response was found to be induced against P. syringae in Arabidopsis thaliana by P. fluorescens DAPG. Calcisol produced by PGPRs, viz. P. alcaligenes PsA15, Bacillus polymyxa BcP26 and Mycobacterium phlei MbP18, provides tolerance to high temperatures and salinity stress. It has been demonstrated that inoculation of plants with AM fungi also improves plant growth under salt stress. Achromobacter piechaudii was also shown to increase the biomass of tomato and pepper plants under 172 mM NaCl and water stress. Interestingly, a root endophytic fungus Piriformospora indica was found to defend its host plants against salt stress. It has been found that inoculation of PGPR alone or along with AM like Glomus intraradices or G. mosseae resulted in better nutrient uptake and improvement in the normal physiological processes in Lactuca sativa under stress conditions. The same plant treated with P. mendocina increased its shoot biomass under salt stress. Studies on the mechanisms involved in osmotic stress tolerance employing transcriptomic and microscopic strategies have revealed a considerable change in the transcriptome of Stenotrophomonas rhizophila DSM14405T in response to salt stress*.* A combination of AM fungi and N2-fixing bacteria helped the legume plants in overcoming drought stress. The effect of A. brasilense along with AM can be seen in other crops such as tomato, maize and cassava. A. brasilense and AM in combination improved the plant tolerance to various abiotic stresses. The additive effect of Pseudomonas putida or Bacillus megaterium and AM fungi was effective in alleviating drought stress. Application of Pseudomonades sp*.* under water stress improved the synthesis of antioxidant and photosynthetic pigments in basil plants. Interestingly, a combination of three bacterial species caused the highest CAT, GPX and APX activity and chlorophyll content in leaves under water stress. Pseudomonas spp. was found to have a positive effect on the seedling growth and seed germination of A. officinalis L. under water stress. The photosynthetic efficiency and the antioxidant response of rice plants subjected to drought stress have been found to increase after inoculation of arbuscular mycorrhiza. The beneficial effects of mycorrhizae have also been reported under both drought and saline conditions. Heavy metals such as cadmium, lead and mercury from hospital and factory waste accumulate in the soil and enter plants through the roots. Azospirillium spp., Phosphobacteria spp. and Glucanacetobacter spp. isolated from the rhizosphere of rice fields and mangroves have been found to be more tolerant to heavy metals, especially iron. P. potida strain 11 (P.p.11), P. potida strain 4 (P.p.4) and P. fluorescens strain 169 (P.f.169) can protect canola and barley plants from the inhibitory effects of cadmium via IAA, siderophore and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). It has been reported that rhizoremediation of petroleum contaminated soil can be expedited by adding microorganisms in the form of effective microbial agent (EMA) to different plant species such as cotton, ryegrass, tall fescue and alfalfa.
PGPRs as biological agents proved to be one of the alternatives of chemical agents to provide resistance to various pathogen attacks. Apart from acting as growth-promoting agents, they can provide resistance against pathogens by producing metabolites. Bacillus subtilis GBO3 can induce defense-related pathways, viz. salicylic acid (SA) and jasmonic acid (JA). Application of PGPR isolates, viz. B. amyloliquefaciens 937b and B. pumilus SE-34, provides immunity against tomato mottle virus. B. megaterium IISRBP 17 characterized from black pepper stem acts against Phytophthor capsici. Bacillus subtilis N11 along with mature composts was found to control Fusarium infestation on banana roots. Similarly, B. subtilis (UFLA285) was found to provide resistance against R. solani and also to induce foliar and root growth in cotton plants. In another interesting study, Paenibacillus polymyxa SQR-21 was identified as a potential agent for the biocontrol of Fusarium wilt in watermelon. Further, the exploitation of PGPRs was found to be effective to manage the spotted wilt viruses in tomato, cucumber mosaic virus of tomato and pepper, and banana bunchy top virus in banana. In some cases, along with bacteria, mycorrhizae can also confer resistance to fungal pathogens and inhibit the growth of many root pathogens, such as R. solani, Pythium spp., F. oxysporum, A. obscura and H. annosum, by improving the plant nutrient profile and thereby the productivity. For instance, Glomus mosseae is effective against Fusarium oxysporum f. sp. basilica, which causes root-rot disease of basil plants. Medicago tranculata also showed induction of various defense-related genes with mycorrhizal colonization. It was shown that addition of arbuscular mycorrhizal fungi and Pseudomonas fluorescens to the soil can reduce the development of root-rot disease and enhance the yield of Phaseolus vulgaris L.
Mycorrhiza is the association of fungi with the roots of higher plants. While it remains an enigma, it serves as a model system to understand the mechanism behind stimulation of growth in the root cells as a result of mycorrhizal inhabitation. The genome sequencing of two EM fungi (ectomycorrhizae), L. bicolor 13 and T. melanosporum (black truffle) 14, has helped in the identification of factors that regulate the development of mycorrhiza and its function in the plant cell. Fifteen genes up-regulated during symbiosis have been identified as putative hexose transporters in L. bicolor. Its genome lacks genes encoding invertases, making it dependent on plants for glucose. However, T. melanosporum possesses one invertase gene, and unlike L. bicolor, it can directly use the sucrose of the host. The up-regulation of transporter genes during symbiosis indicated the role of transportation of useful compounds like amino acids, oligopeptides and polyamines through the symbiotic interface from one organism to the other. Free-living mycelium can take nitrate and ammonium from the soil. Subsequently, these compounds reach the mantle and Hartig net and are then transferred to the plants. Cysteine-rich proteins (MISSP7) of the fungus play an important role as effectors and facilitators in the formation of symbiotic interfaces. Many genes related to auxin biosynthesis and root morphogenesis showed up-regulation during mycorrhizal colonization. Further, G. versiforme possesses inorganic phosphate (Pi) transporters on its hyphae, which help in the direct absorption of phosphate from the soil, and a glutamine synthase gene was found in G. intraradice, which strengthens the possibility that nitrogen metabolized in the fungal hyphae can be transported later to the plant. Bioactive compounds called Myc factors similar to the Nod factors of Rhizobium are suggested to be secreted by mycorrhiza and Rhizobium and to be perceived by host roots for the activation of signal transduction pathways or the common symbiosis (SYM) pathway. The pathways that prepare the plant for both AM and Rhizobium infection have some common points. The common SYM pathway prepares the host plant to bring about changes at the molecular and anatomical level with the first contact of fungal hyphae. So far, calcium is supposed to be the hub of secondary messengers via Ca2+ spiking in the nuclear region of root hairs. Rhizobium leguminosarum biovar viciae can induce various genes in plants like pea, alfalfa and sugar beet, as evident from the microarray studies. PGPRs produce IAA which, in turn, induces the production of nitric oxide (NO), which acts as a second messenger to trigger a complex signaling network leading to improved root growth and developmental processes.
Expression of ENOD11 and many defense-related genes and root-remodelling genes get up-regulated during entry. Subsequently, this allows the formation of a pre-penetration apparatus (PPA). Although the biology behind the development of arbuscules is unknown, a gene called Vapyrin, when knocked down, causes a decline in the growth of arbuscules. Many other genes, including those encoding subtilisin protease, phosphate transporter or two ABC transporters, are known to be involved in arbuscule formation. Nitrogen-fixation genes are popularly used by scientists today to create engineered plants that can fix atmospheric nitrogen. The induction of nif genes in case of nitrogen-fixing bacteria takes place under low concentration of nitrogen and oxygen in the rhizosphere. Interestingly, sugarcane plantlets inoculated with a wild strain of G. diazotrophicus, have demonstrated fixation of radioactive N2 when compared with the G. diazotrophicus mutant that has a mutant nifD gene, which proved the significance of nif genes. The efficiency of nitrogen fixation is dependent on the utilization of carbon. Bacteria like Bacillus subtilis (UFLA285) can differentially induce 247 genes in cotton plants as compared to controls where no PGPR was supplied to the cotton plant. Many disease-resistance genes that work via jasmonate/ethylene signaling as well as osmotic regulation via proline synthesis genes were differentially expressed with UFLA285 induction. Various differentially expressed genes were identified, including ones encoding metallothionein-like protein type 1, a NOD26-like membrane integral protein, ZmNIP2-1, a thionin family protein, an oryzain gamma chain precursor, stress-associated protein 1 (OsISAP1), probenazole-inducible protein PBZ1, as well as auxin- and ethylene-responsive genes. The expression of the defense-related proteins PBZ1 and thionins have been found to get repressed in the rice–H. seropedicae association, suggesting the modulation of plant defense responses during colonization.
Among the PGPR species, Azospirillum has been suggested to secrete gibberellins, ethylene and auxins. Some plant-associated bacteria can also induce phytohormone synthesis. For example, lodgepole pine, when inoculated with Paenibacillus polymyxa, had elevated levels of IAA in the roots. Rhizobium and Bacillus were found to synthesize IAA at different cultural conditions such as pH, temperature and in the presence of agro-waste as a substrate. Ethylene, unlike other phytohormones, is responsible for the inhibition of growth of dicot plants. It was found by Glick et al. that PGPR could enhance the growth of the plant by suppressing the expression of ethylene. Interestingly, a model has been suggested in which ethylene synthesis from 1-aminocyclopropane-1-carboxylate (ACC), an immediate precursor of ethylene, which is hydrolyzed by bacterial ACC-deaminase enzyme in the need of nitrogen and carbon source is also one of the mechanisms of induction of conditions suitable for growth. ACC-deaminase activity has also been found in bacteria such as Alcaligenes sp*., Bacillus pumilus, Pseudomonas* sp. and Variovorax paradoxus. The involvement of ACC deaminase in the indirect influence on the growth of plants was proved in canola, where mutations in the ACC deaminase gene caused the loss of effect of growth-promoting Pseudomonas putida. Interestingly, the potential of PGPRs was further enhanced by introducing genes involved in the direct oxidation (DO) pathway and mineral phosphate solubilisation (MPS) into some useful strains of PGPRs. The gene encoding glucose dehydrogenase (gcd) involved in the DO pathway was cloned and characterized from Acinetobacter calcoaceticus and E. coli and Enterobacter asburiae. Moreover, a gene encoding a soluble form of GCD has been cloned from Acinetobacter calcoaceticus and G. oxydans. Furthermore, there are reports of site-directed mutagenesis of glucose dehydrogenase (GDH) and gluconate dehydrogenase (GADH) that has improved the activity of this enzyme. Mere substitution of S771M provided thermal stability to E. coli, whereas mutation of glutamate 742 to lysine improved the EDTA tolerance of E. coli PQQGDH. The application of this technology was achieved by transferring genes involved in the DO pathway, viz. GDH, GADH and pyrroloquinoline quinine (PQQ), to rhizobacteria and phosphoenolpyruvate carboxylase (PPC) to P. fluorescens, providing the MPS trait.
To recapitulate briefly, excess nutrients are accumulated in soils, particularly phosphorus, as a result of over-application of chemical fertilizers by farmers during intensive agricultural practices. The major research focus is and should be on the production of efficient and sustainable biofertilizers for crop plants, wherein inorganic fertilizer application can be reduced significantly to avoid further pollution problems.
Finally, let us reiterate the most important and specific points, as defined by Swapna Latha Aggani from Kakatiya University, on which the research on biofertilizers should focus:
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