Molecular mechanism of virulence in the bacterium, Photorhabdus luminescens (Thomas and Poinar) against Tetranychus truncatus Ehara (Prostigmata: Tetranychidae)

Abstract

Spider mites (Tetranychidae) are a major group of sucking pests of many agricultural and horticultural crops worldwide. Tetranychus truncatus Ehara is the predominant mite species infesting economically important crops of Kerala. Novel acaricides are being extensively used for managing mite infestation, which has led to development of significant level of resistance in mite populations to these acaricides. This, along with other adverse effects of synthetic acaricides to the environment, calls for alternative strategies in mite pest management. The entomopathogenic bacteria Photorhabdus luminescens (Thomas and Poinar), a natural symbiont of the entomopathogenic nematode, Heterorhabditis spp. has potential virulent properties and the ability to infect a wide range of insect pests and a few mite pests. An array of toxins produced by the bacteria are responsible for this appreciable insecticidal activity. Most of the orally active toxins are encoded by tca and tcd loci of TC genes. These toxins might also be responsible for the acaricidal activity of the bacterium. In this context, the present study was undertaken to identify the virulent strain of P. luminescens against T. truncatus and to elucidate the molecular mechanism of virulence against T. truncatus. Eleven bacterial isolates associated with the entomopathogenic nematodes, Heterorhabditis spp. were isolated in the study. Cultural characterization of the bacterial isolates showed that the bacterial colonies were circular to irregular, entire, opaque, smooth colonies on NBTA medium. All the isolates were found to be Gram negative and rod shaped. For molecular characterization, the 16S rRNA gene of the isolates was amplified, sequenced and subjected to BLASTn for homology search. One species of EPN symbiotic bacteria, P. luminescens and eight nonsymbiotic/ associated bacteria namely, two isolates of Pseudomonas aeruginosa, five isolates of Ochrobactrum and one isolate of Stenotrophomonas maltophila were identified. Phylogenetic tree was constructed based on the gene sequences of 16S rRNA to validate the bacterial identification.Laboratory bioassays were carried out to evaluate the efficacy of the both cell suspension and cell free supernatant (CFS) of five bacterial isolates (1 isolate of P. luminescens, 2 isolates of P. aeruginosa, and 2 isolates of Ochrobactrum sp.) separately against the eggs and adults of T. truncatus. Though the isolates did not show any significant ovicidal action, they recorded significant adulticidal action against the mite. The P. luminescens isolate was superior over other isolates recording 64.67 and 60.67 per cent mortality for CFS and cell suspension, respectively at 96 h of treatment The virulent P. luminescens isolate was subjected to characterization of the toxin complex (TcdA gene). The full length gene (7.5 kb) was split into eight regions and the primers were designed to amplify each regions separately. Among the eight regions, only six got amplified and were sequenced. Toxin gene sequence of the isolated P. luminescens bacteria involved in the virulence against T. truncatus was partially sequenced and the variations in the sequence were analyzed. The study identified a potential isolate of P. luminescens with significant adulticidal action against T. truncatus and the major gene TcdA encoding the toxin complexes which possess appreciable insecticidal activity was sequenced and characterized. The P. luminescens isolate obtained in the study can be evaluated for efficacy against major insect pests in the region in order to identify the potential in pest management.