Isolation, Evaluation and molecular characterisation of bacillus thuringiensis isolates against prostephanus truncatus, a major storage pest in maize
The study was initiated to investigate the potential of locally isolated Bacillus thuringiensis (Bt) isolates for the control of the larger grain borer (LGB) (Prostephanus truncatus), a major storage pest of maize and cassava in Kenya. Samples of soil, grain and dead insects were collected from various maize growing areas in Kenya and screened for the presence of Bt. The isolates were tested for toxic activity against P. truncatus in the laboratory. Bacillus thuringiensis var. tenebrio (Btt), toxic to the Colorado Potato Beetle (CPB) was included in the bioassays as a Standard control reference. Bt precipitate obtained from broth was mixed with maize flour at a ratio of 2:1 to make an artificial diet. Pellets of 2g were cut from the artificial diet and offered to insects placed in plastic containers. A total of 68 isolates of Bt were recovered out of 320 samples screened, and of which 85 % were from grains, 12 % from soils and 3 % from dead insects. Grains proved to be a better source of Bt isolates. The screening for Bt isolates in the laboratory revealed that different Bt isolates from different samples were either bipyramidal, irregular or square shaped. Ninety five (95 %) of the Bt isolates from grain samples were bi-pyramidal shaped and (5 %) from grains were square shaped. Analysis of soil samples showed that 12.5 % were square shaped and 87.5 % were bipyramidal shaped. The Bt isolates from insect samples were all irregular shaped (100 %). Among the Bt isolates, 82 were positive and 18 % were negative for Voges Paskeur test. Methyl Red tests revealed that 24 % of the Bt isolates were negative and 76 % were positive, whilst indole tests showed that 72 % of the Bt isolates were negative and 28 % were positive. The biochemical characteristics of isolated pathogens confirmed that these isolates were Bt. Mortality in the controls was 0 % and 3.3 %, 7 and 30 days after treatment, respectively Isolates of Bt were toxic to adult P. truncatus but mortality varied from 1.7 to 53 % and 3.3 to 85 %, 7 and 30 days after treatment between the isolates, respectively. Seven B. thuringiensis isolates (Bt 41, Bt 61, Bt 51, Bt 60, Bt 46, Bt 45 and Bt 44) were more toxic to adult P. truncatus than other isolates, causing mortalities of 50 and 80 %, 7 and 30 days after treatment. However, there was no significant difference between these isolates (P>0.05). The standard reference isolate, Bt tenebrio was also toxic, causing mortality of 37 % and 60 %, 7 and 30 days after treatment, and was significantly different (P<_0.05) from the most toxic isolates listed above. The lethal time 50 % mortality (LT50) values were calculated for isolates that achieved more than 50 % mortality after 30 days following treatment. The LT50ranged from 7 to 37 days and varied significantly (P<_0.05) among isolates. Bacillus thuringiensis isolates 41, 51, 61, 60, 45, 44, 46 and 23 had the shortest LT50 values of between 8 and 11 days. The standard reference strain, Bt tenebrio, had LT50 of 16 days, which was significantly different (P<0.05) from the most toxic isolates. Bacillus thuringiensis 41 and Bt 51 molecular sizes varied between 67 to 74 KDa while the Standard control isolate, Btt, had a molecular size of 67 KDa. There was a common protein band of 67 KDa in the two isolates (Bt 41 and 51). These results suggest that a further DNA study was necessary to ascertain the presence of cry 111 genes that are known to be toxic to coleopterans. These results demonstrated the potential of discovering isolates of B. thuringiensis that are toxic to adult P. truncatus and could be, therefore, developed as biopesticides. Preservation of these isolates in a germplasm bank for pathogen biodiversity should be considered since these pathogens could be utilized as sources of new cry genes for genetic transformation.