Characterization of slow-growing root nodule bacteria from Inga oerstediana in organic coffee agroecosystems in Chiapas, Mexico

The leguminous tree genus Inga is thought to be critical for providing N to certified organic coffee shrubs that commonly grow beneath its shade in the state of Chiapas, Mexico. Organic certification prohibits use of synthetic N fertilizers, necessitating understanding of the Inga-rhizobia symbiosis. The objectives of this study were to: (1) establish a collection of isolates from Inga sp. nodules found on organic coffee farms in Chiapas, Mexico; (2) use phenotypic and genotypic approaches to characterize these isolates, and (3) examine cross-inoculation patterns of strains isolated from Inga sp. with other Inga species and N2-fixing trees. Eighty-three strains were isolated from root nodules of Inga oerstediana, Inga pavoniana and an unknown species of Inga. Aside from isolates of I. pavoniana, Inga isolates demonstrate characteristics consistent with Bradyrhizobium. Dendrograms generated using phylogenetic and phenotypic traits showed strains to exhibit significant diversity, even among strains originating from the same farm. Strains isolated from I. pavoniana were 95% similar, and displayed strikingly different characteristics from strains isolated from other species. Cross-inoculation experiments used three Inga strains (cowpea miscellany CB756, and isolates from I. oerstediana and I. sp.) as inoculants with I. edulis, I. fuellii, Acacia mangium, Acacia koa, Vigna unguculata and Gliricidia sepium. All three strains formed nodules containing leghaemoglobin with V. unguculata, while isolates from I. oerstediana, Inga sp. and cowpea miscellany formed nodules with I. feulli and I. edulis, but not with A. mangium and G. sepium. A. koa formed nodules containing leghaemoglobin when inoculated by cowpea miscellany strain CB756 and the strain from Inga sp., but nodules formed with the strain isolated from I. oerstediana did not contain leghaemoglobin. One and two-way ANOVA results showed that no mean biomass of inoculated seedlings was significantly greater than the −N control for each of A. koa, G. sepium, A. mangium (p < 0.001), supporting our hypothesis that inoculation with non-Inga strains would have no effect on biomass production. We suggest that great spatial variation exists among Inga isolates from organic coffee farms in the Chiapas highland region, both between and within farms, and that more research is needed to identify reasons for the genetic distribution and function of Inga symbionts.