Water stress amelioration and plant growth promotion in wheat plants by osmotic stress tolerant bacteria.

Soil microorganisms with potential for alleviation of abiotic stresses in combination with plant growth promotion would be extremely useful tools in sustainable agriculture. To this end, the present study was initiated where forty-five salt tolerant bacterial isolates with ability to grow in high salt medium were obtained from the rhizosphere of Triticum aestivum and Imperata cylindrica. These bacteria were tested for plant-growth-promoting rhizobacteria traits in vitro such as phosphate solubilization, siderophore, ACC deaminase and IAA production. Of the forty-five isolates, W10 from wheat rhizosphere and IP8 from blady grass rhizosphere, which tested positive in all the tests were identified by morpholological, biochemical and 16SrDNA sequencing as Bacillus safensis and Ochrobactrum pseudogregnonense respectively and selected for in vivo studies. Both the bacteria could promote growth in six varieties of wheat tested in terms of increase in root and shoot biomass, height of plants, yield, as well as increase in chlorophyll content. Besides, the wheat plants could withstand water stress more efficiently in presence of the bacteria as indicated by delay in appearance of wilting symptoms increases in relative water content of treated water stressed plants in comparison to untreated stressed ones, and elevated antioxidant responses. Enhanced antioxidant responses were evident as elevated activities of enzymes such as catalase, peroxidase, ascorbate peroxidase, superoxide dismutase and glutathione reductase as well as increased accumulation of antioxidants such as carotenoids and ascorbate. Results clearly indicate that the ability of wheat plants to withstand water stress is enhanced by application of these bacteria which also function as plant growth promoting rhizobacteria.

Evaluation of Ochrobactrum anthropi TRS-2 and its talc based formulation for enhancement of growth of tea plants and management of brown root rot disease.

AIM: To evaluate Ochrobactrum anthropi TRS-2 isolated from tea rhizosphere and its talc based formulation for growth promotion and management of brown root rot disease of tea. METHODS AND RESULTS: Ochrobactrum anthropi TRS-2, isolated from tea rhizosphere could solubilize phosphate, produce siderophore and IAA in vitro and also exhibited antifungal activity against six test pathogens. Application of an aqueous suspension of O. anthropi to the rhizosphere of nursery grown tea seedlings of five varieties of tea (TV-18, T-17, HV-39, S-449, UP-3 and) led to enhanced growth of the treated plants, as evidenced by increase in height, in the number of shoots and number of leaves per shoot. Treatment with O. anthropi also decreased brown root rot of tea, caused by Phellinus noxius. Multifold increase in activities of chitinase, beta-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in tea plants was observed on application of O. anthropi to soil followed by inoculation with P. noxius. A concomitant increase in accumulation of phenolics was also obtained. Further, talc based formulation of O. anthropi was prepared and its survival determined every month up to a period of 12 months. Ochrobactrum anthropi could survive in the formulation up to a period of 9 months with a concentration of 7.0 log(10) CFU g(-1), after which there was a decline. Talc formulation was as effective as aqueous suspensions in both plant growth promotion and disease suppression. CONCLUSION: Ochrobactrum anthropi, either in aqueous suspension or as talc formulation induced growth of tea plants and suppressed brown root rot disease. It induced defense responses in tea plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochrobactrum anthropi and its talc based formulation can be considered as an addition to available plant growth promoting rhizobacteria (PGPR) currently being used for field application. The present study offers a scope of utilizing this bacterium for growth promotion and disease management which would help in reduction of the use of chemicals in tea plantations.

Serological detection and immunogold localization of cross-reactive antigens shared by Camellia sinensis and Exobasidium vexans.

AIMS: Pathogenicity of Exobasidium vexans, causal agent of blister blight of tea, was studied in 30 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen using immunological techniques. METHODS AND RESULTS: Whole plant inoculation of tea varieties with E. vexans showed that T-78 and T-17/1/54 were most susceptible and most resistant respectively. Antigen preparations from tea varieties, pathogen, nonpathogen (Fusarium oxysporum) and of nonhosts (Glycine max, Leucaena leucocephala and Oryza sativa) were compared by indirect enzyme-linked immunosorbent assay and dot-immunobinding assay using polyclonal antibodies raised against the pathogen, nonpathogen, susceptible and resistant tea varieties. Cross-reactive antigens (CRA) were found among susceptible varieties and E. vexans isolates but not in resistant varieties, nonhosts or nonpathogen. Indirect staining of antibodies using fluorescein isothiocyanate indicated CRA were concentrated mainly around epidermal and mesophyll cells in compatible host (T-78). This was substantiated by ultrastructural studies using gold-labelled antibodies through transmission electron microscopy which showed specific localization in the chloroplasts and host cytoplasm. CONCLUSION: Pathogenicity of E. vexans to different tea varieties is therefore related to the level of antigenic similarity between host and pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunological methods proved to be valuable in screening commercially cultivated tea varieties against E. vexans.

Detection of cross-reactive antigens shared by Fusarium oxysporum and Glycine max by indirect ELISA and their cellular location in root tissues.

Pathogenicity test of Fusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium of F. oxysporum. Polyclonal antisera were raised against the mycelial suspension of F. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.

Sequence repeat-induced disruption of the major heat-inducible HSP70 gene of Neurospora crassa.

The process of repeat-induced point mutation (RIP) was used to disrupt hsps-1, the gene encoding the major heat-inducible member of the HSP70 family of Neurospora crassa. A plasmid DNA, containing an incomplete copy of hsps-1 and the selectable marker qa-2+, was introduced into germinated conidia. The sexual progeny of transformants with ectopically integrated hsps-1 DNA was examined for RIP by Southern-blot analysis of MboI- and Sau3A-digested genomic DNA. Progeny strains, showing RIP, were tested for heat shock-responsive expression of hsps-1, by RNA-blot hybridization and Western-blot analysis, as well as for thermotolerance. Isolates with RIP showed low levels of hsps-1 mRNA and a lack of induction of HSP70 protein by heat shock, accompanied by only a marginal decrease in the acquisition of thermotolerance.

Accumulation of antifungal compounds in tea leaf tissue infected with Bipolaris carbonum.

Varietal resistance of tea towards Bipolaris carbonum was tested following detached leaf inoculation technique. Among the fourteen varieties tested, three were found to be highly susceptible, while other three were resistant. Leaf exudates and diffusates collected from the resistant varieties were more fungitoxic than those from the susceptible ones. Two antifungal compounds isolated from healthy and B. carbonum-infected tea leaves exhibited clear inhibition zones at RF 0.8 and 0.65, respectively, in a chromatographic bioassay. On the basis of their color reaction on TLC and UV-spectra these were identified to be catechin and pyrocatechol. Resistant and susceptible varieties accumulated 439-510 and 187-212 micrograms/g fresh mass tissue of pyrocatechol, respectively, 2 d after inoculation with B. carbonum, while a low concentration (45-58 micrograms/g) of this compound was detected in healthy leaf tissue.

Antagonism of Erwinia herbicola towards Leptosphaeria maculans causing blackleg disease of Brassica napus.

Phyllosphere micro-organisms of Brassica napus were isolated and their antagonism against Leptosphaeria maculans, causal agent of blackleg disease, was tested in vitro. In paired culture, Erwinia herbicola was found to be highly antagonistic to L. maculans. Bioassay of the culture filtrate of the bacterium against the test fungus revealed that Erw. herbicola secretes an antifungal substance into the culture medium. This substance was partially thermolabile and markedly reduced the germination and germ tube length of L. maculans. Aqueous bacterial suspensions and cold-sterilized culture filtrates, when applied to the seedlings prior to inoculation, significantly reduced the severity of blackleg disease.

An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.

A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.

Biochemical studies following ovulation induction with clomiphene citrate and human menopausal gonadotropin in infertile Indian women.

Biochemical studies were done in 65 anovulatory women before and following induction of ovulation with ethinyl estradiol/clomiphene citrate/human chorionic gonadotropin and also clomiphene citrate/human menopausal gonadotropin/human chorionic gonadotropin to determine the metabolic adverse effects, if any, of such therapy. Significant increases occurred in lipids and lipoprotein levels, while minor changes were seen in liver function enzymes and fasting blood sugar. Sialic acid decreased significantly after therapy in both the regimens, and both induction therapies seem to be free of adverse metabolic effects of hormones. On comparison with normal ovulatory women, this study suggests that some of the biochemical parameters can serve as reliable indicators of ensuing ovulation.