Evaluation of entomopathogenic Bacillus sphaericus isolated from Lombok beach area against mosquito larvae

Objective: To isolate, characterize and evaluate toxicity of Bacillus sphaericus(B. sphaericus) from beach area of Lombok Island.Methods: Soil was collected from determined locations and suspended in sterile physiological saline water. After heat shock was applied, suspension was spread on NYSM agar medium. Colonies grown were then observed and isolated. Colony, cell morphology,and biochemical/physiological characteristics were tested and compared to B. sphaericus2362 as standard. Initial toxicity testing was done against three species of mosquito larvae(Culex quinquefasciatus, Anopheles aconitus and Aedes aegypti) and isolates that showed more than 50% larvae killing will be assayed to obtain LC_(50) and LC_(90)values within 48 h.PCR technique were conducted to obtain 16 s r DNA amplicon for sequencing and to detect toxin-expressing genes(using multiplex PCR).Results: Twenty isolates of B. sphaericus have been collected from 20 determined locations and their characteristics were in agreement with standard B. sphaericus characteristics. Bioassay testing showed that four isolates(namely isolate MNT, SLG, TJL2 and PLG) were mildly toxic against all larvae. The rests were either low toxic or non-toxic at all.Phylogenetic analysis showed that all four isolates were clustered with other known mildly and highly toxic strains. The multiplex PCR result showed four toxic isolates owned 1–2bands from Bin toxin genes and three bands from Mtx toxin genes, whereas 16 isolates with low to non-toxic characteristics showed only three bands from Mtx toxin genes.Conclusions: Four toxic isolates of B. sphaericus were isolated from beach area of Lombok Island. They showed mild toxicity against larvae of three mosquito species.