Molecular Mechanism Associated With Insecticidal Activity Of Xenorhabdus Nematophila Strain CB6

Xenorhabdus and Photorhabdus are two genera of insect pathogenic bacteria that are symbiotically associated with entomopathogenic nematode belonging to two genera Steinernema and Heterorhabditis. Xenorhabdus and Photorhabdus bacteria were detected to have oral insecticidal activity, which is associated with the insecticidal toxin protein-a new group of toxins protein. The insecticidal activities of metabolite from Xenorhabdus and Photorhabdus were also investigated for their potential application as biocontrol agent. Exploring the molecular mechanism of insecticidal activity from Xenorhabdus and Photorhabdus bacteria is not only helpful for seeking new insecticidal protein genes, but also offer some clues for exploiting new types micro-insecticidal agent. In this paper, the insecticidal activity of 30 strains from Xenorhabdus and Photorhabdus were tested. An insecticidal toxin gene fragment from X. nematophila CB6 was cloned and expressed in Escherichia coli, and the insecticidal activity of the lysates from E.coli cells was determined, Eight gene fragments was detected based on the AFLP fingerprint analysis of phase I and phase II cells from X. nematophila CB6, which was the symbiotic bacterium of the entomopathogenic nematode, Steinernema carpocapsae that isolated from a soil sample in the west of Beijing. The primary results are as follows:1 ) The oral toxicity of the culture broths of 30 strains from Xenorhabdus and Photorhabdus to Helicoverpa armigera and Pieris rapae was tested. The most of bacteria strains showed to have insecticidal activity. There are differences of insecticidal activity between two genera, or species, even different strains of the same species. The X. nematophila bacteria displayed highly oral toxicity that average corrected mortality to neonatal H. armigera larvae was 89.3%. The oral toxicity of X. nematophila CB6 to different instars of H. armigera was tested. The corrected mortality of neonatal larvae, the rate of growth inhibition of 3rd instar larvae and the rate of anti-feeding of 5th instar larvae were 88.7%, 95.1% and 83.6%, respectively.2) The insecticidal toxin gene fragments cb6Al, cb33Al, cb43A\ and cb54A1 were gained and showed strong homology from Xenorhabdus strains CB6, CB33, CB43 and CB54 that showed different insecticidal activity. A point mutation at 1038 site of cb43A1 and cb54A\ was detected. The frame-shift mutation that the guanine was deletion at 1038 site was likely to reduce the insecticidal activity of strains CB43 and CB54.3 ) Bacterial non-fusion and fusion expression vectors of cb6A\ gene were constructed and successfully expressed in E. coli. The oral toxicity of non-fusion expressionproduct in E. coli was detected, and the corrected mortality to neonatal larvae of H.arrnigera reached 91.67 % when the larvae were fed on the artificial diet that mixed with non-fusion expression product (20u.g recombine protein/g diet) for 3 days.4) X. nematophila CB6 exhibit a phase variation that the phase I cell can change to phase II cells that involved in insecticidal activity at low-level in vitro culture. The homology of 16SrRNA and insecticidal toxin gene fragments between phase I and phase II cells were 99.85% and 100%, respectively.5) Eight different DNA fragments from phase I and phase II cells of X. nematophila CB6 were identified by amplified fragment length polymorphism (AFLP) analysis with 400 primers combination. The homology search (Blast at GenBank) indicated that the seven gene fragments were first detected in Xenorhabdus and photorhabdus The comparative analysis of deduced amino acid sequence indicated that eight genes fragment involved in transcription, metabolize control and biosynthesis of membrane protein. Eight GenBank accession numbers were obtained (AY317150, AY317151, AY317152, AY317153, AY321438, AY321439, AY321440, AY321441). The results first demonstrated that the phase variation had implications of genetic instability between phase I and phase II.