Characterization Of Xenorhabdus Spp. Xptb1 And Xptc1 Toxin Genes And Effect Of The Bacteria On Chilo Partellus And Busseola Fusca.

Abstract

Xenorhabdus spp. (Enterobacteriaceae) are endosymbionts of entomopathogenic nematodes from the Steinernema genus. They cause insect mortality by producing potent insecticidal toxin complexes composed of XptA1, XptA2 (class A), XptB1 (class C) and XptC1 (Class B) proteins. However, the use of only the bacterium as a biopesticide is limited as they depend on their nematode hosts for survival in the environment. The XptAs exhibit different spectra of activity, requiring XptB1 and XptC1 for full activity. Studies of their tccC homologues from the related Photorhabdus sp. revealed that class C proteins have enzymatic activity. The objective of this study was to characterize XptB1 and XptC1 novel toxin genes and proteins and investigate the bacteria’s potential as alternative pest control agents against two lepidopteran pests; Chilo partellus (Spotted stem borer) and Busseola fusca (African stem borer). These pests are a constraint to maize production in Kenya, causing estimated annual yield losses of about 15%. Pure bacterial cultures were isolated by sampling infected Galleria mellonella (Wax moth) haemolymph. The bacteria were identified morphologically by observing colony characteristics on NBTA (Nutrient-Bromothymol Blue-Triphenyltetrazolium chloride Agar) plates and by Gram staining. The 16S rRNA gene sequence was used to confirm the identity of the bacteria, by similarity searching in public databases. Primer design, PCR amplification and sequencing that targeted the XptB1 and XptC1 regions was done using one pair of gene specific and three pairs of degenerate primers. Proteins were partially purified from bacterial crude cell lysate using a Sepharose CL6B size exclusion chromatography column. Xenorhabdus sp. resuspended in PBS (Phosphate buffered saline) was assayed for efficacy against the target pests through diet (ground maize leaves, bean powder, nutrient supplements, and agar) incorporation and direct injection. The three Xenorhabdus isolates studied were closely related (99% identity) to X. griffiniae using Phylogenetic analysis. Xenorhabdus sp. (OD600 = 2.1847A) was found to be effective against the target pests causing mortality in injected larvae of C. partellus (72%) and B. fusca (78%), within 6 days of exposure. Xenorhabdus sp. incorporated in diet (OD600 = 0.015A) caused mortality (33%) in C. partellus after 24 days of exposure as well as sub-lethal effects such as stunted larval development. Sequences amplified by gene specific primers were found to be similar to two conserved genes of Xenorhabdus doucetiae; a bacterial surface antigen D15 related to the ShlB (VB-type) membrane protein and a poly (A) polymerase. The partially purified bacterial cell lysate also yielded proteins within the size range of 110 – 160 kDa, suggesting the presence of the two target proteins. The study has demonstrated that Xenorhabdus sp. has a potent bioactive agent against C. partellus and B. fusca larvae when delivered through injection