Ramesh Chand Dubey

Rhizosphere competent Mesorhizobiumloti MP6 induces root hair curling, inhibits Sclerotinia sclerotiorum and enhances growth of Indian mustard (Brassica campestris)

The bacterial strain Mesorhizobium loti MP6, isolated from root nodules of Mimosa pudica induced growth and yield of Brassica campestris. The isolate MP6 secreted hydroxamate type siderophore in Chrom-Azurol Siderophore (CAS) agar medium. Production of hydrocyanic acid (HCN), indole acetic acid (IAA) and phosphate solubilizing ability was also recorded under normal growth conditions. Root hair curling was observed through simple glass-slide technique. In vitro study showed a significant increase in population of M. loti MP6 in rhizosphere due to root exudates of B. campestris. In dual culture technique the strain showed a strong antagonistic effect against Sclerotinia sclerotiorum, a white rot pathogen of Brassica campestris. The growth of S. sclerotiorum was inhibited by 75% after prolonged incubation. Efficient root colonization of mustard seedlings was confirmed by using a streptomycin-resistant marker M. loti MP6strep+. The M. loti MP6 coated seeds proved enhanced seed germination, early vegetative growth and grain yield as compared to control. Also, a drastic decline (99%) in the incidence of white rot was observed due to application of M. loti MP6.

Effect of Chemical Fertilizer-adaptive Variants, Pseudomonas aeruginosa GRC2 and Azotobacter chroococcum AC1, on Macrophomina phaseolina Causing Charcoal Rot of Brassica juncea

Pseudomonas aeruginosa GRC2, siderophore-producing strain, inhibited growth of Macrophomina phaseolina in vitro and reduced charcoal rot in seeds of Brassica juncea in field when coated with adaptive strains. P. aeruginosa GRC2 and Azotobacter chroococcum AC1 produced indole-3-acetic acid and solubilized insoluble phosphate. A. chroococcum AC1 fixed nitrogen asymbiotically. Urea and diammonium phosphate-adaptive variant strains of P. aeruginosa and A. chroococcum strongly inhibited M. phaseolina in comparison to parental strains. Bacterization of seeds induced seed germination, seedling growth, and enhanced yield of B. juncea by 10.87% as compared to full doses of urea and diammonium phosphate. Both adaptive strains of chemical fertilizers aggressively colonized roots, showing effectiveness to growth and developments of B. juncea.

Root nodule bacteria from Clitoria ternatea L. are putative invasive nonrhizobial endophytes.

In this study, bacteria (8 species and 5 genera) belonging to the classes Betaproteobacteria, Gammaproteobacteria, and Sphingobacteria were isolated from root nodules of the multipurpose legume Clitoria ternatea L. and identified on the basis of partial 16S rRNA sequencing. The root nodule bacteria were subjected to phenotypic clustering and diversity studies using biochemical kits, including Hi-Media Carbokit™, Enterobacteriaceae™ identification kit, ERIC-PCR, and 16S ARDRA. All the strains showed growth on Ashbys N-free media over 7 generations, indicative of presumptive nitrogen fixation and further confirmed by amplification of the nifH gene. None of the strains showed the capability to renodulate the host plant, neither alone nor in combination with standard rhizobial strains, which was further confirmed by the absence of nodC bands in PCR assay. The results clearly indicate the common existence of nonrhizobial microflora inside the root nodules of legumes, which were thought to be colonized only by rhizobia and were responsible for N2 fixation in leguminous crops. However, with the recent discovery of nodule endophytes from a variety of legumes, as also observed here, it can be assumed that symbiotic rhizobia are not all alone and that these invasive endophytes belonging to various bacterial genera are more than just opportunistic colonizers of specialized nodule niche.

Differential antagonistic responses of Bacillus pumilus MSUA3 against Rhizoctonia solani and Fusarium oxysporum causing fungal diseases in Fagopyrum esculentum Moench

Chitinase and surfactin-mediated biocontrol of Rhizoctonia solani and Fusarium oxysporum causing wilt and root rot of Fagopyrum esculentum respectively has been studied in this communication. Bacillus pumilus MSUA3 as a potential bacterial strain strongly inhibited the growth of R. solani and F. oxysporum involving the chitinolytic enzymes and an antibiotic surfactin. Plant growth promoting attributes seem to be involved in plant growth promotion and yield attributes. The action of cell-free culture supernatant (CFCS) was found deleterious to F. oxysporum and R. solani even in the heat-treated (boiled/autoclaved) CFCS. The possible involvement of surfactin in disease control was revealed by colony PCR amplification of SrfA. Chitinolytic enzyme and antibiotic surfactin evidenced differential biocontrol of F. oxysporum and R. solani by B. pumilus MSUA3. A significant reduction in disease index under gnotobiotic conditions and productivity enhancement of F. esculentum using vermiculite-based bioformulation revealed B. pumilus MSUA3 as a successful potential biocontrol agent (BCA) and an efficient plant growth promoting rhizobacterium (PGPR) for disease management and productivity enhancement of buckwheat crop.

Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation

Strain RT1 was isolated from root nodules of Lens culinaris (a lentil) and characterized as Rhizobium etli (a Gram-negative soil-borne bacterium) by 16S rDNA sequencing and phylogenetic analysis. The signaling molecules produced by R. etli (RT1) were detected and identified by high-performance liquid chromatography coupled with mass spectrometry. The most abundant and biologically active N-acyl homoserine lactone molecules (3-oxo-C8-HSL and 3-OH-C14-HSL) were detected in the ethyl acetate extract of RT1. The biological role of 3-oxo-C8-HSL was evaluated in RT1. Bacterial motility and biofilm formation were affected or modified on increasing concentrations of 3-oxo-C8-HSL. Results confirmed the existence of cell communication in RT1 mediated by 3-oxo-C8-HSL, and positive correlations were found among quorum sensing, motility and biofilm formation in RT1.

Evaluation of Probiotic Potential and Safety Assessment of Lactobacillus pentosus MMP4 Isolated From Mare’s Lactation

Lactic acid bacteria isolated from indigenous milk of different animals were investigated for their efficacy, safety, and probiotic potential. The most potential isolate MMP4 was screened from mare’s milk, which was further identified as Lactobacillus pentosus by using 16S rRNA gene sequencing and phylogeny. The probiotic potential of strain MMP4 was assessed by its ability to survive under acidic environment and in presence of bile salts along with the ability to inhibit food-borne as well as clinical pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Salmonella typhi. The phenol tolerance with cogent hydrophobicity to different hydrocarbons was demonstrated. Bile salt hydrolase activity of L. pentosus MMP4 was confirmed by detecting the Bsh gene by using colony PCR. The presence of Mub, Map, and EF-Tu genes involved in adhesion conferred the behavior of passage and adherence to gastrointestinal tract. Scanning electron microscopy of intestinal autopsy from albino mice revealed the attachment of bacterial cells on the mucus-lined intestinal walls against pathogens and further proved in vivo adhesion ability. The presence of intrinsic antibiotic resistance and lack of DNase, gelatinase, and hemolytic activity in MMP4 support its safety as probiotic traits. Thus, MMP4 bears an excellent and pragmatic properties for being used as probiotic and may be exploited in dairy industry.