Fig. 5.

Validation of virulence activities of 12 virulent candidate secondary metabolites produced by two highly potent bacterial isolates of Photorhabdus temperata Subsp. temperata (‘Ptt’) and Xenorhabdus hominickii (‘Xh’). A reference metabolite was selected from compounds produced by a low virulent isolate of X. ehlersii (‘Xe’). For virulence tests, suppression of hemocyte nodulation, inhibition of sPLA₂, and inhibition of cPLA₂ were determined. To induce hemocyte nodulation, 4 × 10⁴ cfu/larva of E. coli was injected to L5 larvae of S. exigua. Bacterial metabolites (10 μg/larvae) were injected to inhibit nodule formation. For each treatment, 15 larvae were used. To measure sPLA₂ and cPLA2 enzyme activities, a commercial assay kit was used with PLA₂-specific substrate as described in Materials and methods. As control (‘CON’), DMSO was used. Each treatment was independently replicated three times. Different letters above standard error bars indicates significant differences among means at Type I Error = 0.05 (LSD test). Bacterial metabolites used in this assay included benzyl alcohol (‘BA’), benzeneethanol-4-hydroxy (‘BH’), o-cyanobenzoic acid (‘CBA’), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (‘DHF’), 2-ethyl-1-hexanol (‘EH’), 3-ethoxy-4-methoxyphenol (‘EMP’), indole-3-aceticacid hydrazide (‘IAAH’), indole (‘IND’), 2-mercaptophenol (‘MP’), 2-mercaptobenzothiazole (‘MT’), N-(2-phenylethyl) acetamide (‘NPA’), 1-phenyl-1,2-ethanediol (‘PE’), and tryptophol (‘TPL’). These compounds are classified as bacterial metabolites synthesized by both Ptt and Xh (‘Ptt + Xh’), only Xh (‘Xh’), only Ptt (‘Ptt’), and common to Ptt, Xh, and Xe (‘Xe’)