Biochemical and Molecular Approaches to Study Microbial Colonization in Legume

Abstract

In this study, initially mung bean nodulating strain, Bradyrhizobium sp. MN-S, was phylogenetically identified on the basis of sequence analysis of 16S rRNA and nod gene. 16S rRNA gene sequence did not generate sufficient polymorphism, as MN-S showed 100% similarity with three species of Bradyrhizobium. Later, symbiotic genes, nodCD1VW, were sequenced, and two of the four genes generated sufficient polymorphism to assign the taxonomic status of MN-S. The nodC sequence showed 99% similarity to the nodC sequence of B. yuanmingense, while low similarity with other Bradyrhizobium spp. Similarly, nodD1 sequence showed 99% similarity to B. yuanmingense nodD1, while only 93% with B. liaoningense. However, nodV and nodW could not be amplified on a variety of PCR conditions, that showed that these genes might be absent in B. yuanmingense MN-S. The molecular keys of colonization and infection, ‘Nod factors’ of B. yuanmingense were identified to be in the mass range of 1178 to 1211 Da, which was significantly less than the size of comparable Nod factors from B. japonicum 532C. B. yuanmingense MN-S was also able to form biofilm, colonize root nodules and promote the mung bean growth, which designate it a potential plant growth promoting agent. Moreover, comparative assessment of Nod factor production inducing flavonoids, quercetin and genistein, revealed that both the flavonoids induce a similar type of Nod factor family in B. yuanmingense MN-S. It is suggested that quercetin might be a stronger inducer of Nod factors production than genistein, as the inoculation of pre-incubated culture of B. yuanmingense MN-S in quercetin showed improve root colonization, biofilm formation, nodulation and seedling growth. Furthermore, endophytic bacteria from root nodules of mung bean and pea were isolated and characterized for plant growth promoting attributes. Potential bacterial isolates were selected on the basis of indole acetic acid production, phosphate solubilization, nitrogen fixation, root colonization and biofilm formation. These bacterial strains were identified through 16S rRNA gene sequence analysis, and found that the bacterial strains isolated from mung bean nodule belong to species of Bacillus, Ochrobactrum and Agrobacterium while strains isolated from pea nodules belong to xOchrobactrum and Enterobacter spp. These nodule endophytic bacteria were unable to nodulate their respective hosts. Moreover, nodC and nodD1 genes could not be amplified in these isolates. Co-inoculation efficacy of these non-rhizobial nodule endophytic bacteria with rhizobia was also evaluated. Mung bean non-rhizobial bacteria were co- inoculated with B. yuanmingense MN-S, while pea bacterial strains with Rhizobium leguminosarum PS-I. Mung bean bacterial strains, Bacillus subtilis M2, Agrobacterium tumefaciens M5 and Bacillus subtilis M6 upon co-inoculation with B. yuanmingense MN- S significantly improved nodulation and seedling growth, while the rest of co-inoculation combination showed a non-significant effect. The enhancement due to co-inoculation in nodule number and nodule dry weight was 78% and 127%, respectively, compared with the B. yuanmingense MN-S alone. Co-inoculation combination of B. yuanmingense MN- S with Bacillus subtilis M6 performed best by increasing 22% grain yield while the rest of combinations marginally benefited the plant. The results signify that non-rhizobial plant growth promoting bacteria improve nodulation and grain yield of legumes upon co- inoculation with crop specific rhizobia. Moreover, the effect of two systemic fungicides, carbendazim and thiophanate methyl, on the growth of nodulating bacteria, colonization, nodulation and grain yield of mung bean and pea was also evaluated. Recommended application rate of fungicides did not significantly inhibit the growth of tested bacteria. In field trials, maximum mung bean grain yield was achieved in the combined treatments of B. japonicum USDA110 & carbendazim and B. yuanmingense MN-S alone, which is 50% and 49% higher than control, respectively. A significant increase of 34-43% in peas pod yield was achieved in the combined treatments of R. leguminosarum & fungicides compared with the un- inoculated control. It is concluded that rational application of fungicides and bioinoculants demands a deep understanding of fungicide-bioinoculant-plant compatibility.