Mutational search for high temperature (60<sup>o</sup>C) tolerant variant of Rhizobium species CWP G34A—Mutation generates high temperature variant of Rhizobium species Cwp G34A

Abstract

This study focused on the development of thermophilic strain/s of a cowpea (Vigna unguiculata) compatible nitrogen fixing bacterium. A preliminary plant screening was carried out using some strains of tropical rhizobia and cowpea. Rhizobium species CWP G34A that formed Fix + nodules repeatedly was selected for further studies. First, it was tested for growth at high temperatures of 40˚C to 55˚C at 5˚C interval with 28˚C as the control temperature. Mutagenesis was conducted on the bacterium with ethylmethane sulphonate (EMS). The wild-type and mutants generated were tested for high temperature tolerance by growing them individually in nutrient broth at 60˚C for 24 hours. Optical density (670 nm) was read before and after incubation. The mutants were grouped into classes based on percentage difference in OD values obtained before and after exposure to 60˚C. Rhizobium species CWP G34A produced functional pink nodules on the cowpea consistently in three different plant tests. There was no growth at all the temperatures tested except at 28˚C and 40˚C after 24 hours of incubation. It grew better at former (51 × 10 10 Cfu/ml) than latter (11 Cfu/ml) temperature. Like the parental strain, all the mutants but one, did not grow after exposure to 60˚C. Sixty degree centigrade caused various reductions in optical density (OD) values of the variants. Eleven classes of the mutants were formed with membership percentage ranging from 1 to 22%. Class 1 contains only one member while class 11 has the highest mutant population of 22% with OD difference of 0 to 10% and −90 to −100% respectively. The high percentage reduction in the OD of variants in class 11 is similar to that of the unmutated cells (−94.56%). The only mutant that survived the 60 o C and grew was MU70. An increase of 1.67% in OD was obtained for MU70. Mutant MU70 therefore appeared a promising strain that can be further tested to inoculate cowpea in the dry and warm season for increased nitrogen fixation. This will provide encouraging information for farmers to grow the cowpea throughout the year particularly under high temperatures in summer in order to boost the yield of the legume.