Plant growth-promoting rhizobacteria Shewanella putrefaciens and Cronobacter dublinensis enhance drought tolerance of pearl millet by modulating hormones and stress-responsive genes.

Drought is a major abiotic stress that affects crop productivity. Endophytic bacteria have been found to alleviate the adverse effects of drought on plants. In the present study, we evaluated the effects of two endophytic bacteria Shewanella putrefaciens strain MCL-1 and Cronobacter dublinensis strain MKS-1 on pearl millet (Pennisetum glaucum (L.) R. Br.) under drought stress conditions. Pearl millet plants were grown under three water levels: field capacity (FC), mild drought stress (MD), and severe drought stress (SD). The effects of inoculation on plant growth, physiological attributes, phytohormone content, and drought stress-responsive genes were assessed. The inoculation of pearl millet seeds with endophytes significantly improved shoot and root dry weight and root architecture of plants grown under FC and drought stress conditions. There was a significant increase in relative water content and proline accumulation in the inoculated plants. Among the phytohormones analyzed, the content of ABA and IAA was significantly higher in endophyte-treated plants under all moisture regimes than in uninoculated plants. C. dublinensis-inoculated plants had higher GA content than uninoculated plants under all moisture regimes. The expression level of genes involved in phytohormone biosynthesis (SbNCED, SbGA20oX, and SbYUC) and coding drought-responsive transcription factors (SbAP2, SbSNAC1 and PgDREB2A) was significantly higher under SD in endophyte-inoculated plants than in uninoculated plants. Thus, these endophytic bacteria presumably enhanced the tolerance of pearl millet to drought stress by modulating root growth, plant hormones, physiology and the expression of genes involved in drought tolerance.

Comparative response of Spodoptera litura challenged per os with Serratia marcescens strains differing in virulence.

Host plays an important role in influencing virulence of a pathogen and efficacy of a biopesticide. The present study was aimed to characterize the possible factors present in Spodoptera litura that influenced pathogenecity of orally ingested S. marcescens strains, differing in their virulence. Fifth instar larvae of S. litura responded differently as challenged by two Serratia marcescens strains, SEN (virulent strain, LC50 7.02 103 cfu/ml) and ICC-4 (non-virulent strain, LC50 1.19 1012 cfu/ml). Considerable increase in activity of lytic enzymes protease and phospholipase was recorded in the gut and hemolymph of larvae fed on diet supplemented with S. marcescens strain ICC-4 as compared to the larvae treated with S. marcescens strain SEN. However, a significant up-regulation of antioxidative enzymes SOD (in foregut and midgut), CAT (in the midgut) and GST (in the foregut and hemolymph) was recorded in larvae fed on diet treated with the virulent S. marcescens strain SEN in comparison to larvae fed on diet treated with the non-virulent S. marcescens strain ICC-4. Activity of defense related enzymes lysozyme and phenoloxidase activity were also higher in the hemolymph of larvae fed with diet treated with S. marcescens strain SEN as compared to hemolymph of S. marcescens strain ICC-4 treated larvae. More number of over-expressed proteins was observed in the gut and hemolymph of S. marcescens strains ICC-4 and SEN treated larvae, respectively. Identification of the selected differentially expressed proteins indicated induction of proteins involved in insect innate immune response (Immunoglobulin I-set domain, Apolipophorin III, leucine rich repeat and Titin) in S. marcescens strain SEN treated larvae. Over-expression of two proteins, actin related protein and mt DNA helicase, were noted in S. marcescens treated larvae with very high levels observed in the non-virulent strain. Up-regulation of homeobox protein was noted only in S. marcescens strain ICC-4 challenged larvae. This study indicated that ingestion of non-virulent S. marcescens strain ICC-4 induced strong immune response in insect gut while there was weak response to the virulent S. marcescens strain SEN which probably resulted in difference in their virulence.

Diversity and Tissue Preference of Osmotolerant Bacterial Endophytes Associated with Pearl Millet Genotypes Having Differential Drought Susceptibilities.

Genetic and functional diversity of osmotolerant bacterial endophytes colonizing the root, stem, and leaf tissues of pearl millet genotypes differing in their drought susceptibility was assessed. Two genotypes of pearl millet, viz., the drought tolerant genotype TT-1 and the drought susceptible genotype PPMI-69, were used in the present study. Diazotrophs were found to be the predominant colonizers, followed by the Gram positive bacteria in most of the tissues of both the genotypes. Higher proportion of bacterial endophytes obtained from the drought tolerant genotype was found to be osmotolerant. Results of 16S rRNA gene-ARDRA analysis grouped 50 of the highly osmotolerant isolates into 16 clusters, out of which nine clusters had only one isolate each, indicating their uniqueness. One cluster had 21 isolates and remaining clusters were represented by isolates ranging from two to four. The representative isolates from each cluster were identified, and Bacillus was found to be the most prevalent osmotolerant genera with many different species. Other endophytic bacteria belonged to Pseudomonas sp., Stenotrophomonas sp., and Macrococcus caseolyticus. High phylogenetic diversity was observed in the roots of the drought tolerant genotype while different tissues of the drought susceptible genotype showed less diversity. Isolates of Bacillus axarquiensis were present in all the tissues of both the genotypes of pearl millet. However, most of the other endophytic bacteria showed tissue/genotype specificity. With the exception of B. axarquiensis and B. thuringiensis, rest all the species of Bacillus were found colonizing only the drought-tolerant genotype; while M. caseolyticus colonized all the tissues of only the drought susceptible genotype. There was high incidence of IAA producers and low incidence of ACC deaminase producers among the isolates from the root tissues of the drought-tolerant genotype while reverse was the case for the drought-susceptible genotype. Thus, host played an important role in the selection of endophytes based on both phylogenetic and functional traits.

Characterization of putative virulence factors of Serratia marcescens strain SEN for pathogenesis in Spodoptera litura.

Two Serratia marcescens strains, SEN and ICC-4, isolated from diseased insect cadavers were observed to differ considerably in their virulence towards Spodoptera litura. The present study was aimed to characterize the possible virulence factors present in the virulent Serratia marcescens strain SEN. Both the S. marcescens strains were evaluated for the presence of various lytic enzymes such as chitinase, lipase, protease and phospholipase. The virulent S. marcescens strain SEN was observed to possess considerably higher activity of chitinase and protease enzymes; activity of phospholipase enzyme was also higher. Although, all the three toxin genes shlA, phlA and swr could be detected in both the S. marcescens strains, there was a higher expression of these genes in the virulent strain SEN. S. marcescens strain ICC-4 showed greater reduction in overall growth yield in the post-exponential phase in the presence of midgut juice and hemolymph of S. litura larvae, as compared to S. marcescens strain SEN. Proliferation of the S. marcescens strain SEN was also considerably higher in foregut, midgut and hemolymph of S. litura larvae, as compared to strain ICC-4. Peritrophic membrane treated with broth culture of the S. marcescens strain SEN showed higher damage as compared to strain ICC-4. The peritrophic membrane of larvae fed on diet treated with the virulent strain showed considerable damage while the peritrophic membrane of larvae fed on diet treated with the non-virulent strain showed no damage. This is the first report documenting the fate of ingested S. marcescens in S. litura gut and the relative expression of toxin genes from two S. marcescens strains differing in their virulence towards S. litura.

Induction of osmoadaptive mechanisms and modulation of cellular physiology help Bacillus licheniformis strain SSA 61 adapt to salt stress.

Bacillus licheniformis strain SSA 61, originally isolated from Sambhar salt lake, was observed to grow even in the presence of 25 % salt stress. Osmoadaptive mechanisms of this halotolerant B. licheniformis strain SSA 61, for long-term survival and growth under salt stress, were determined. Proline was the preferentially accumulated compatible osmolyte. There was also increased accumulation of antioxidants ascorbic acid and glutathione. Among the different antioxidative enzymes assayed, superoxide dismutase played the most crucial role in defense against salt-induced stress in the organism. Adaptation to stress by the organism involved modulation of cellular physiology at various levels. There was enhanced expression of known proteins playing essential roles in stress adaptation, such as chaperones DnaK and GroEL, and general stress protein YfkM and polynucleotide phosphorylase/polyadenylase. Proteins involved in amino acid biosynthetic pathway, ribosome structure, and peptide elongation were also overexpressed. Salt stress-induced modulation of expression of enzymes involved in carbon metabolism was observed. There was up-regulation of a number of enzymes involved in generation of NADH and NADPH, indicating increased cellular demand for both energy and reducing power.

Effects of lindane on lindane-degrading Azotobacter chroococcum; evaluation of toxicity of possible degradation product(s) on plant and insect.

The effects of lindane on growth and plant growth-promoting traits of two lindane-degrading Azotobacter chroococcum strains (JL 15 and JL 104) were determined. The potential of both A. chroococcum strains to degrade lindane was also determined. Lower concentrations of lindane had a stimulatory effect, and higher concentrations generally had an inhibitory effect on growth and plant growth-promoting activities. A high percentage (>90%) of lindane was degraded by both strains at a lindane concentration of 10 ppm. Lindane at 1,000 ppm decreased seed germination and reduced seedling fresh weight. However, the possible degradation products for a starting lindane concentration of 10 ppm was found to be non-phytotoxic. Toxicity studies with larvae of Spilarctia obliqua resulted in an LC50 estimate of 3.41 ppm for lindane solutions into which leaf discs were dipped. No toxicity was observed for possible degradation products.

Antioxidant, physiological and biochemical responses of drought susceptible and drought tolerant mustard (Brassica juncea L) genotypes to rhizobacterial inoculation under water deficit stress.

Response of drought susceptible (DS) genotype Pusa Karishma LES-39 and drought tolerant (DT) mustard genotype NPJ-124, to rhizobacterial inoculation under water deficit stress, was compared in the present study to determine the influence of inoculants on biochemical and physiological attributes of these two different genotypes. Inoculation was observed to improve root and shoot dry weight in both the genotypes, although better results were observed in the DS genotype. There was variation in the response of the two genotypes to rhizobacterial inoculation, under water deficit stress. Significant improvement in most of the physiological and biochemical parameters including antioxidative enzyme activities of the DS genotype; with concomitant decrease in starch content, accumulation of H2O2 and lipid peroxidation upon inoculation of rhizobacteria was observed. In contrast, there was improvement in only few physiological and biochemical parameters in the DT genotype in response to inoculation with rhizobacteria. There was significant increase in catalase enzyme activity along with concomitant decrease in lipid peroxidation. Thus, drought susceptibility of the mustard genotypes, NPJ-124 and Pusa Karishma LES-39, determined their physiological, biochemical and antioxidative responses to rhizobacterial inoculation under water deficit stress. Expression of drought stress responsive genes belonging to ABA-dependent (RD20 and RD26) and ABA-independent (DREB2 and DREB1-2) pathways was studied in the DS genotype. Expression of DREB2 and DREB1-2 genes was considerably enhanced due to inoculation under water deficit stress; indicating that in Bacillus-mediated priming for drought stress tolerance, in this genotype, ABA-independent pathway probably played key role in enhancing tolerance to drought stress.

Cloning and heterologous expression of cellulose free thermostable xylanase from Bacillus brevis.

Xylanase gene isolated from Bacillus brevis was expressed in E. coli BL21. Sequencing of the gene (Gen Bank accession number: HQ179986) showed that it belongs to family 11 xylanases. The recombinant xylanase was predominantly secreted to culture medium and showed mesophilic nature (optimum activity at 55°C and pH 7.0). The cell free culture medium exhibited 30 IU/ml xylanse activity. The enzyme did not show any cellulose activity and was active under wide range of temperature (40°C to 80°C) and pH (4 to 9). The enzyme showed considerable thermo stability and regained over 90% of activity, when returned to 55°C after boiling for 5 min. These physiochemical properties of B. brevis xylanse show high potential of its applications in paper and pulp industry.