Evaluation of Nitrogen Fixation Ability of Endophytic Bacteria in Kenyan Bananas (Musa Spp.) Using Biochemical and Molecular Techniques

Abstract

Nitrogen is a major nutrient element required by crops and its scarcity in soil adversely affects crop yield. Application of inorganic nitrogenous fertilizers is costly, causes environmental pollution and leads to poor quality and unsustainable banana yield. Bananas are staple food to a substantial population in the world and harbor endophytic bacteria. It is believed that endophytic bacteria, along with rhizospheric bacteria contribute to plant growth. Biochemical characterization the banana endophytic bacteria isolated on a nitrogen free media, quantification of the amount of nitrogen each can fix and molecular analysis of the nifH gene were the objectives of this study, with the view of developing biofertilizers to address the challenges attributed to the use of inorganic fertilizers.  Eighteen isolates were obtained from previous research that focused on isolation and molecular identification of the isolates by partial analysis of 16S rRNA gene. The isolates were subjected to morphological tests, biochemical assays for characterization, determination of quantities of nitrogen fixed by the bacteria through Acetylene Reduction Assay (ARA). Bergey's Manual of Determinative Bacteriology was used to identify the isolates based on biochemical assay results. For the molecular analysis, nested PCR approach was adopted to amplify nifH gene.  All the isolates showed the ability to fix nitrogen with varying quantities ranging from 0.244µl/ml to 7.03µl/ml. BLAST search yielded high scoring hits of nifH gene sequence in isolate no 5 identified as Raoultella terrigena. However, BLAST could not identify any annotated gene sequences for isolates 4, 15, 21, 27, and 29 of the sequenced isolates. Detection of nifH gene in Raoultella terrigena, quantification of nitrogen fixed by each isolate and the isolates' identities were the key findings this study. These findings are important in banana endophytes research that can lead to their exploitation for sustainable banana production and ecofriendly biofertilizers.