MSc

Chemical pesticides provide significant benefit by controlling pests of agricultural crops. However, their use has increased at an alarming rate, along with proportionate increase in their adverse effect on the environment. In recent years, several insects have developed resistance towards a few pesticides due to their indiscriminate use. Several microbial agents have been used as an alternative for chemical pesticides and Bacillus thuringiensis is one among them. B. thuringiensis is a sporulating, Gram positive, facultative anaerobic soil bacterium, capable of synthesizing δ-endotoxins or Cry proteins during sporulation. Due to the insecticidal properties of these proteins, the bacterium is used against insect species of the orders Lepidoptera, Coleoptera and Diptera.
More than 500 isolates of B. thuringiensis obtained from the Western Ghats of Kerala, in a DBT funded project, are being maintained in the repository of the Department of Agricultural Microbiology, College of Horticulture, Thrissur. Several microbial inoculants such as Pseudomonas fluorescens, Trichoderma viride, Beauvaria bassiana, Lecanicillium lecanii have been developed by Kerala Agricultural University but there is no formulation of B. thuringiensis.
A study was conducted to develop a commercial formulation of B. thuringiensis and to evaluate its bio-efficacy against the pumpkin caterpillar, Diaphania indica (Saund.). Twenty native isolates with cent per cent mortality in previous studies were selected from the repository of the Department of Agricultural Microbiology. Morphological, biochemical and molecular characterisation of these native B. thuringiensis were carried out. The isolates showed only very slight variations in their cultural characteristics. Colonies appeared circular, creamy white with entire to undulate margin and flat elevation. The isolates showed positive reaction to starch and esculin hydrolysis, sucrose fermentation test and negative to Voges-Proskauer test.
The isolates were screened for the presence of lepidopteran specific insecticidal genes, using PCR. Among the 20 native B. thuringiensis isolates,
seven isolates yielded cry1 gene amplicons. None of the isolates produced cry2 and cry9 amplicons. The cry1 amplicons were further sequenced and when subjected to Blastn analysis showed homology towards cry1, cry1A, cry1Aa and cry1Ac. All the isolates have shown identity in the range of 93-96 per cent to the known cry1 genes. Thus, the presence of cry1 gene was confirmed.
Based on the abundance of crystal protein and cry1 gene, three native isolates (KAU-11, KAU-474 and KAU-2189) were further selected for laboratory bioassay against the lepidopteran pest, D. indica. Among the native isolates, KAU-2189 showed highest per cent morality and was further selected for liquid formulation studies. Suitability of three media (soy flour broth, coconut water broth and T3 broth) was assessed based on the population and spore count at 0 h, 72 h and 96 h after inoculation. Coconut water yielded higher population than the standard medium (T3 broth). Spore count of B. thuringiensis in coconut water and T3 were statistically on par. Thus, coconut water served as the best among the tested substrates for B. thuringiensis production and this was further used for the liquid formulation.
Bio-efficacy of the liquid formulation was evaluated in pot culture experiment against D. indica using little gourd (Coccinia indica) as the test crop. Btk (ABTEC), a commercial formulation was used as standard. The higher per cent mortality was recorded for the formulations containing KAU-2189 in coconut water broth and HD-1 in coconut water broth and both were statistically on par with each other. Minimum leaf damage was also recorded in treatments with these formulations and was statistically on par with each other. Shelf life studies of liquid formulations indicated that both population and spore count decreased from fourth month onwards.
The study revealed that the native isolates have the potential to be developed into a biopesticide. Coconut water could be used as an ingredient for low cost liquid formulation. Further evaluation under field conditions is required to confirm the efficiency of KAU-2189 as a biopesticide.