Phenotypic and Genotypic Characterization of Cowpea Rhizobia in Western Kenya, Evaluation of the Symbiotic Efficiencies of Strains Tolerant to Abiotic Stresses and Identification of Symbiotically Efficient Strains

Abstract

Nitrogen is the most limiting nutrient for growth of most crop plants in sub-Saharan Africa and legume crops largely depend on fixed nitrogen from indigenous nitrogen-fixing bacteria. Providing nitrogen to a crop by application of synthetic nitrate fertilizers represents a significant cost both to the farmer and to the environment. The symbiotic characterization and identification of indigenous rhizobia is the basis for inoculants formulation for sustainable legume production in the face of climate change. The aim of this study was to evaluate the diversity of indigenous rhizobia responsible for cowpea nodulation in farmers’ fields across three Counties of Western Kenya. The symbiotic performance of abiotic stress-tolerant rhizobia in the K80 cowpea variety was also assessed. Cowpea root nodules and soil samples were collected from agricultural farms in western Kenya and the farms had a history of growing cowpea for the last two years. Pure bacterial cultures from cowpea root nodules were isolated on Yeast Extract Mannitol Agar (YEMA). The isolates were characterized based on morphological characteristics, abiotic stress tolerance (salinity at 0, 3 and 5% with control at 0.01%, pH 4, 6, 8 and 10 with control at pH 7 and temperature at 20 ºC, 25, 30, 35, 40 and 45 ºC, with control at 28 ºC) and 48 isolates were subjected to ERIC-PCR fingerprinting. All the 54 isolates exhibited typical characteristics of Rhizobium species on YEMA, YEMA supplemented with Congo red and YEMA supplemented with Bromothymol blue. On YEMA the colonies appeared white, elevated, circular with smooth margins, mucoid, glistening and 1-5 mm in diameter. On CR-YEMA the colonies were white to pale pink, elevated, gummy and shiny with entire margins. A dendogram on phenotypic characterization grouped the isolates into cluster A and B at 42% dissimilarity. Cluster A isolates are low to moderate mucus producers while cluster B are high mucus producers. The results showed high genetic variability among the rhizobia isolates based on also ERIC-PCR fingerprinting. A dendogram on ERIC-PCR fingerprinting grouped the isolates at 53% similarity level into cluster A with 42 isolates along with the CIAT 899 reference strain with 2-15 amplified fragments ranging from 75-5000 bp while cluster B had only six isolates with 1-3 small fragments amplified, sizes ranging from 75-700 bp. Nine indigenous rhizobia isolates tolerant to extreme temperature, salinity and pH were tested for symbiotic efficiency in a glasshouse experiment Treatments with nitrogenous fertilizer KNO3 (70 µg N mL-1) without inoculation were positive control and treatments without inoculation and lacking chemical nitrogen served as negative control group. The rhizobia isolates efficiently nodulated cowpea and showed wide variations in their symbiotic efficiency (SE). Five isolates (M2bii, B6ii, B2i, V5ii and V11i) showed significantly higher SE (p < 0.001) of (114.6 - 179.6%) compared to the commercial inoculant (CIAT 899) with SE of 109.3% and nitrogen supplemented treatment (100%) which were the positive controls. Three isolates B6ii, V11i and V5ii with the highest symbiotic efficiencies of 179.6%, 143.35% and 127.3% respectively and three isolates M7b, B2ii and V9i exhibiting SE of 98.46%, 93.3% and 83.17% respectively lower than the positive controls were subjected to nifH symbiotic gene sequencing and confirmed their identities as Bradyrhizobium and Rhizobium species. These isolates have potential for inoculant formulation. The results indicate the existence of indigenous rhizobia isolates which can tolerate environmental stresses and are symbiotically effective and thus can be developed into inoculants to improve the productivity of cowpea in Kenya.