GENETIC VARIATION IN GROUNDNUT (Arachis hypogaea L) NODULATING RHIZOBIA NATIVE TO PHOSPHORUS DEFICIENT SOILS OF WESTERN KENYA

Abstract

Soils in Western Kenya are characterized by high acidity with phosphorus (P) deficiency, affecting nodulation and nitrogen fixation of groundnut (Arachis hypogaea L.). The objectives of the study were to determine (1) the diversity of indigenous rhizobia strains nodulating groundnut using morphological and biochemical markers (2) identify indigenous groundnut rhizobia capable of nodulating groundnut under P deficiency, and (3) genotype the rhizobia isolates using polymerase chain-reaction-restriction fragment length polymorphism (PCR-RFLP) targeting 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) and 16S rRNA gene. Phenotypic diversity among the isolates was assessed by morphological and biochemical attributes. Sixty four isolates out of the 68 were confirmed to be rhizobia due to their ability to nodulate groundnut. Ninety six percent of the isolates exhibited semi-globose to globose colony shape on yeast extract mannitol agar (YEMA). Groundnut was nodulated by both fast and slow growing rhizobia isolates with 81% being fast growers. Fifty one isolates representing 75% of the bacteria isolates showed ability to produce acid on YEMA medium supplemented with bromothymol blue (BTB). The isolates varied in response to low pH with 39 and 61 growing at pH 4.0 and 5.5, respectively. All the isolates grew at pH 7.0 and 8.5. YEMA medium containing glucose, sucrose, starch and citrate supported growth of 64, 61, 56 and 5 isolates, respectively. Sixty four isolates showed a clear zone of solubilization on medium containing insoluble inorganic phosphate. Solubilization index (SI) varied from 1.1 to 6.8. Fast-growing rhizobia isolates N01, B02, I06, Q01, F05, C02, E01, Q03, I01 and B01 recorded the highest solubilization index of 3.8, 4.5, 4.6, 4.6, 4.7, 5.0, 5.1, 6.1, 6.1 and 6.8, respectively. Genetic diversity among the isolates was assessed by sequence analysis of 16S-23S ribosomal. Amplification of IGS and 16S rRNA for 54 isolates produced a single fragment of 750 and 1,500 bp, respectively. Restriction of amplified 16S-23S rDNA IGS region with four endonucleases Hinf l, Msp1, Taq1 and Csp61 produced 1-10 restriction fragments in all the isolates. These fragments varied in length from 50 to 500 bp, 130 to 600 bp, 130 to 610 bp and 30 to 600 bp, respectively. Ten different combinations of patterns representing 10 different IGS genotypes (A-J) were detected in the 54 isolates. Hinf I was found to be the most discriminative detecting 10 genotypes (A–J) among the 54 rhizobia isolates analyzed as compared to Msp1 (8), Taq1 (8) and Csp61 (9). Digestion of the 16S rRNA gene amplicons with endonucleases Hinf 1, Msp1, Taq1 and Csp61 each produced multiple fragments ranging between 1 and 8 depending on the isolate. Fragments ranged from 50 to 500 bp, 50 to 700, 150 to 1050 and 150 to 710 base pair in size, respectively. The number of genotypes per restriction enzyme varied (A-M) with Hinf 1, Msp1 and Taq1 delineating the largest at thirteen (13), and Csp61 representing the least (9) number of genotypes (A-I). Dendrogram from each cluster analysis with different enzymes and combined enzymes separated isolates from different sites of origin into two main groups. Both phenotypic and genotypic characterization showed great diversity among the 54 isolates with potential to solubilize inorganic phosphate and improve nitrogen fixation. The most promising rhizobia isolates from this study would be used as bio-fertilizer upon further validation in the greenhouse. The findings reported here if adopted will contribute to enhancing groundnut production on acid soils of western Kenya in particular and Africa in general.