Enhanced Survival and Nodule Occupancy of Pigeon pea Nodulating Rhizobium sp. ST1 expressing fegA Gene of Bradyrhizobium japonicum 61A152

Abstract

Problem statement: Rhizobial isolates belonging to genera (Rhizobium sp. and Mesorhizobium sp.) in our laboratory produced only catecholate type of siderophores. Although FhuA and FegA (ferrichrome receptors) homologs were found to be present in the sequenced genomes of few rhizobia (e.g., 1 in R. etli and 2 in Mesorhizobium sp. BNC1), laboratory isolates of the corresponding genera failed to utilize ferrichrome, a siderophore which is present in nanomolar concentrations in the soil. This inability was considered as a negative fitness factor with respect to rhizospheric colonization by these rhizobia. Approach: The 2.4 kb fegA gene (encoding ferrichrome receptor) was amplified along with its native promoter from Bradyrhizobium japonicum 61A152 and cloned in a broad host range plasmid vector pUCPM18. The plasmid construct pFJ was transferred by conjugation into Rhizobium sp. ST1 to give transconjugant ST1pFJ12. The consequence of FegA expression on the transconjugant was tested under lab and soil conditions, using physiological experiments. Results: Ability of the transconjugant ST1pFJ12 to utilize ferrichrome and expression of a 79 kD protein band on the outer membrane of the transconjugant confirmed FegA expression. Transconjugant ST1pFJ12 exhibited increased growth rate as compared to the parent strain ST1, in minimal media containing ferrichrome as the sole iron source, confirming the positive effect of FegA expression. Inoculation of pigeon pea seedlings with transconjugant ST1pFJ12 led to a marked increase in plant growth parameters as compared to plants inoculated with the parent strain ST1, the effect being more pronounced when Ustilago maydis, a ferrichrome producer was co-inoculated in the systems. Nodule occupancy on pigeon pea plant when inoculated with the transconjugant ST1pFJ12 alone was 57% which increased to 66% when co-inoculated with U. maydis as compared with 37 and 30% respectively, seen with parental strain ST1 inoculation. Conclusion: The clear increase in nodule occupancy and higher rhizospheric colonization by the fegA transconjugants, presented in this study together with the previous research reported from our laboratory, led us to conclude that ferrichrome utilization ability played an important role in the rhizospheric colonization of the bioinoculant strains. Testing the ability to utilize hydroxamate siderophores therefore, holds prime importance in selecting an efficient biofertilizer strain.