Shekhar Chandra Bisht

Characterisation of a psychrotolerant plant growth promotingPseudomonas sp. strain PGERs17 (MTCC 9000) isolated from North Western Indian Himalayas

A psychrotolerant, Gram negative, rod shaped, plant growth promoting bacterium (PGPB) was isolated from high altitude of North Western Indian Himalayas. The identity of the bacterium was confirmed by morphological, biochemical and sequencing of the 16S rRNA gene. The sequence analysis revealed maximum similarity withPseudomonas vancouverensis. It exhibited tolerance to a wide pH range (5–12; optimum 7.0) and salt concentrations up to 5% (w/v). The isolate produced 8.33 and 1.38 μg/ml of IAA at 15°C and 4°C respectively, on the third day after incubation. It solubilised 42.3, 66.3 and 74.1 μg/ml of tricalcium phosphate at 4, 15 and 28°C respectively after seven days of incubation. The strain also possessed HCN and siderophore production abilities at 4°C. It exhibited inhibitory activity against several phytopathogenic fungi in three different bioassays. The maximum relative growth inhibition was recorded againstSclerotium rolfsii andRhizoctonia solani (100%), followed byPythium sp. (73.1%) andFusarium oxysporum (19.7%), in volatile compound assays. Seed bacterization with the isolate enhanced the germination of wheat seedlings grown at 18±1°C by 20.3%. Bacterized seeds also recorded 30.2 and 27.5% higher root and shoot length respectively, compared to uninoculated controls.

Isolation, molecular characterization and growth-promotion activities of a cold tolerant bacterium Pseudomonas sp. NARs9 (MTCC9002) from the Indian Himalayas

A bacterium that grows and expresses plant growth promotion traits at 4 degrees C was isolated from the rhizospheric soil of Amaranth, cultivated at a high altitude location in the North Western Indian Himalayas. The isolate was Gram negative and the cells appeared as rods (2.91 x 0.71 microm in size). It grew at temperatures ranging from 4 to 30 degrees C, with a growth optimum at 28 degrees C. It exhibited tolerance to a wide pH range (5-10; optimum 8.0) and salt concentrations up to 6% (wt/vol). Although it was sensitive to Rifampicin (R 20 microg mi-1), Gentamicin (G 3 microg mi-1), and Streptomycin (S 5 microg mi-1), it showed resistance to higher concentrations of Ampicillin (A 500 microg mi-1), Penicillin (P 300 microg mi-1), Polymixin B sulphate (Pb 100 microg mi-1) and Chloramphenicol (C 200 microg mi-1). The 16S rRNA sequence analysis revealed maximum identity with Pseudomonas lurida. The bacterium produced indole Acetic Acid (IAA) and solubilizes phosphate at 4, 15 and 28 degrees C. It also retained its ability to produce rhamnolipids and siderophores at 15 degrees C. Seed bacterization with the isolate enhanced the germination, shoot and root lengths of thirty-day-old wheat seedlings by 19.2, 30.0 & 22.9% respectively, as compared to the un-inoculated controls.

COINOCULATION OF RHIZOBIUM LEGUMINOSARUM-PR1 WITH A COLD TOLERANT PSEUDOMONAS SP. IMPROVES IRON ACQUISITION, NUTRIENT UPTAKE AND GROWTH OF FIELD PEA (PISUM SATIVUM L.)

The effect of a cold tolerant Pseudomonas sp. Strain, PGERs17, on nodulation, iron acquisition and nutrient uptake of field pea (Pisum sativum L. variety VL Matar 42) seedling was determined on the basis of iron acquisition and growth promotion, chlorophyll content, physiologically available iron, Leghaemoglobin from nodules, nitrogen (N) uptake, phosphorus (P) uptake, potassium (K) uptake, iron (Fe), and zinc (Zn) uptake of shoots. Coinoculation of PGERs17 with Rhizobium leguminosarum-PR1 significantly (P > 0.05) increased nodulation (156.2%) and 57.1% higher plant biomass. Coinoculation enhanced total chlorophyll content (31.5%), physiologically available iron (106.7%), total iron (95.9%) and 17.5-fold higher leghaemoglobin concentration in root nodules over uninoculated control plants. Coinoculation also enhance N uptake (66.3%), P uptake (23.3%), K uptake (47.1%), and 2.75-fold higher Zn uptake of shoots compare with uninoculated control. Hence, cold tolerant Pseudomonas sp. strain PGERs17 can be employed as a bioinoculant along with Rhizobium leguminosarum-PR1 to enhance plant growth, iron acquisition and nutrient uptake of field pea enhance plant growth, iron acquisition and nutrient uptake of field pea seedlings at cold temperature conditions.

Cold-Tolerant PGPRs as Bioinoculants for Stress Management

Cold stress is a major environmental constraint to plant productivity. Cold-induced losses in yield probably exceed those from all other causes, since both the severity and duration of the stress are critical. Plants have evolved special mechanisms to overcome the life-endangering influence of low temperature and to survive freezing. Plant-growth-promoting rhizobacteria (PGPRs) have a high potential in agriculture because they can improve plant growth, especially under limiting or stress (cold/chilling) conditions. The agricultural importance of cold-tolerant microbes stems from the fact that the world over temperate agro-ecosystems is characterized by low temperatures and short growing seasons that subject both plant and microbial life to cold-temperature induced stress. Hence, there is a need to identify a group of potential PGPRs that could retain their functional traits under cold (low)-temperature conditions. Such microbes can be profitably used as bioinoculants in agricultural production systems in the temperate regions of the world. This chapter deals with the effect of temperature on plants and management of cold stress by using cold-tolerant PGPRs in improving soil quality and productivity of agricultural crops.

Cold-Tolerant Agriculturally Important Microorganisms

Cold-tolerant microorganisms are endowed with the ability to grow at 0°C, though their growth optima lie in the mesophilic range. To overcome the stress induced by low temperatures they have evolved a variety of adaptive responses at the cellular and molecular levels. Multiple cell membrane modifications ensure that solute transport is not impaired at low temperatures. Other mechanisms include the synthesis of cold-shock proteins (Csps), cold acclimation proteins (Caps), cryoprotectants, ice nucleation factors, cold-adapted enzymes, and RNA degradosomes. The agricultural importance of such microbes stems from the fact that the world over temperate agro-ecosystems are characterized by low temperatures and short growing seasons that subject both plant and microbial life to cold temperature induced stress. Hence, there is a need to identify potential microbes that retain their functional traits under low temperature conditions. Such microbes can be profitably used as inoculants in agricultural production systems in the temperate regions of the world. This chapter deals with the cold tolerance/resistance mechanisms operating in microorganisms and the utility of cold-tolerant microbes in improving soil quality and productivity of agricultural crops.

CspA Encodes a Major Cold Shock Protein in Himalayan Psychrotolerant Pseudomonas Strains

The major cold-shock protein (CspA) encoding gene cspA were detected in three Himalayan psychrotrophic Pseudomonad strains, by PCR amplification. Partial sequencing of three Pseudomonas strains cspA gene and BLAST search confirmed the high similarity with putative bacterial cspA gene and bacterial CspA protein. Bioinformatics analysis of these partial CspA amino acid sequences showed presence of putative conserved region for DNA/RNA-binding motifs RNP-1 and RNP-2. Protein homologies of all three bacterial CspA proteins belong to S1 like protein (Ribosomal protein S1-like RNA-binding domain). Presence of cspA gene and its high similarity with Bacillus cereus group demonstrating uniqueness of cspA gene in these Pseudomonas strains and suggesting strong evolutionary relationship between these two groups to survive in cold environments. Probable CspA protein expression levels were checked after cold shock (28°C to 4°C) and cold acclimation (4°C and 15°C) experiment. SDS-PAGE analysis revealed a small protein of approximate size of 7.5 kDa was expressed after cold shock (28°C to 4°C) and continuously over-expressed with the incubation time at cold temperature (4°C). Therefore it was predicted this protein would be product of cspA gene and suggesting this protein aids survival in Himalayan environments.

Synergistic effect of inoculating plant growth-promoting Pseudomonas spp. and Rhizobium leguminosarum-FB1 on growth and nutrient uptake of rajmash (Phaseolus vulgaris L.)

Plant growth-promoting bacteria (PGPB) Pseudomonas lurida-NPRp15 and Pseudomonas putida-PGRs4 possessing multiple plant growth-promoting traits were isolated from rhizoplane of pea and rhizosphere of garlic, respectively. The effects of individuals and combinations of Pseudomonas spp. with effective root nodulating symbiotic nitrogen fixing Rhizobium leguminosarum-FB1 on plant growth, nutrient uptake and yield of the rajmash plant were studied under greenhouse conditions. Bacterial inoculation resulted in significantly higher values for plant dry biomass, N, P, K, Zn and Fe contents as compared to the uninoculated control. Furthermore, dual inoculation of P. lurida-NPRp15 with R. leguminosarum-FB1 significantly increased root and shoot dry weight, nodulation, nutrient uptake, pod yield, and nutrient content of pods of rajmash VL63 compared to controls, single and triple inoculation. The results of the study indicate the potential of harnessing the benefit of plant growth-promoting and nitrogen-fixing microorganisms to improve the growth and yield of rajmash.