Population dynamics , stability and molecular ecology of rhizosphe reassociated Ochrobactrum species

Abstract

The genus Ochrobactrum has been recently described as containing legume-symbiotic species that are able to induce nodules on Acacia, Lupinus and Cytisus. The present study was planned to characterize an Ochrobactrum isolate from chickpea nodule, to study the ultrastructure of nodules induced by Ochrobactrum, confirmation of nodule occupancy using immunogold labeling, to analyze the effect of its inoculation on chickpea growth and yield, study the colonization potential and occurrence in the rhizosphere of chickpea as well as other crops. A Gram-staining-negative, aerobic, rod-shaped, non-spore forming bacterial strain Ca-34 isolated from root nodules of chickpea was characterized and studied for its taxonomic affiliation using polyphasic approach. Almost full-length 16S rRNA sequence (1492bp) showed similarities with those of the genus Ochrobactrum. The presence of common fatty acids and G+C ratio also supported the genus affiliation. Based on MALDI-TOF analysis and 16S rRNA sequence similarity (98.6%) the strains showed maximum similarities with O. intermedium (LMG 3301 T ). Ca-34 T showed an insertion of 46bp in the Helix 184 of 16S rRNA that forms an additional stem-loop structure. To our knowledge, this is the first report of 46bp insertion in any of the plant associated or nodule endophyte. Both strains (Ca-34 and O. intermedium LMG 3301 T ) are phylogenetic neighbors but DNA-DNA similarity value of 64% confirms their genome dissimilarity. Ca-34 T showed no amplification with primers reported to be specific for O. intermedium (LMG 3301 T ) or recA primers reported to amplify more than 100 Ochrobactrum species showing that Ca-34 T is different from other reported strains of the genus. The differences were further confirmed by different fingerprints obtained from RAPD, ARDRA and TP-RAPD profiles. The strain Ca-34 T was metabolically different from individual other type strains of Ochrobactrum in many reactions and from all type strains in positive gelatin hydrolysis, especially in negative assimilation of alanine amide and L-threonine. Based on phenotypic and genotypic data we concluded that strain Ca-34 T (DSM 22292, CCUG 57879) represents the type strain of a novel species for which we proposed the name Ochrobactrum ciceri sp. nov. xvUltrastructure observation of the nodules induced by Ca-34 T was carried out to see the infection process of the strain in chickpea. The electron microscopic observation showed that Ca-34 T has typical Meso/Rhizobium-like infection process confirmed by the infection thread, symbiosome and absence of PBH granules which is a characteristic of indeterminate chickpea nodules. Nodule occupancy was confirmed using immunogold labeling and nodule PCR using primers designed against the additional 46bp insertion to specifically amplify the Ca-34 T . Until now, there was no literature available on the ultrastructure of nodules induced by any Ochrobactrum specie. This study is the first report of the Ochrobactrum infection and host- ultrastructure. The nodulation and subsequent yield enhancement are important factors that determine the ecological success of any bacteria inoculated to the plant. The strain Ca-34 T was studied for its ecological behavior in the rhizosphere of ten chickpea genotypes (four kabuli and 6 desi) in field alone and/or along-with the Rhizobium in two different soil conditions (fertile and marginal). AT 60DAS (day after sowing) genotype and inoculation response was significant (P<0.05) at both sites but genotype x inoculation was non-significant at site 1 while highly significant at site 2. The data obtained showed that although Ca-34 T -inoculation has non-significant effect on early plant growth (60 DAS) and flowering (120DAS) but highly significant effect on yield (180DAS). Due to the difference in the fertility status and indigenous bacterial population of both soils, site 1 generally produced low biomass (3570-5161 Kg/ha) and grain yield (1317-2158 Kg/ha) as compared to site 2 which produced high biomass (6363- 6929 Kg/ha) as well as grain yield (3478-4265 Kg/ha). When compared to respective un-inoculated controls, maximum grain yield was obtained in Ca-34 T inoculated plants and inoculation response was significantly higher in kabuli genotypes as compared to desi genotypes. Ca-34 T -inoculation resulted in >100% increase in nodulation, biomass and grain yield and ≈72% increase in harvest index. Across bacterial inoculation treatments, grain yield was positively correlated to all the growth and yield parameters (r=0.262 to 0.856) e.g., nodule number and weight, plant weight, biomass, plant height, root length etc. The inoculation effect was more pronounced in marginal soil (Site 1) where no legume was grown previously as compared to the xvifertile soil where legumes were routinely grown (Site 2). PCA analysis clearly showed a site-specific response of genotype x bacterial inoculation. Although, comprehensive studies are available on Meso/Rhizobium-host inoculation and interaction but this kind of ecological data is being reported for the first time in literature for Ochrobactrum. The most important issue of bacterial inoculants is the rhizosphere competitiveness and colonization potential. The inoculated Ca-34 T strain showed very good colonization potential in chickpea roots, rhizosphere and nodules as confirmed by immunoblotting and specific PCR. In inoculated chickpea rhizosphere, the population of Ca-34 T was almost 1.5% while in nodules it was found to be 0.82% of total culturable population. Although the strain had also been isolated from non- inoculated chickpea rhizosphere, roots and nodules, but its presence in the rhizosphere of other crops could not be confirmed suggesting that the Ochrobactrum ciceri is mainly associated with chickpea and can be used for its nodulation and yield improvement in marginal lands.