An entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata subsp temperata (Ptt), suppresses insect immune responses and facilitates its symbiotic nematode development in target insect. Benzylideneacetone (BZA), PY, cPY, Ac-FGV, indole, 2-oxindole and 3-(4-hydroxyphenylpropionic) acid (PHPP) were compounds derived from the bacterial. Their immunosuppressive activities have been induced by inhibitory activity against eicosanoid biosynthesis and used to develop an additive to enhance control efficacy of other commercial microbial insecticides. This study investigated any cytotoxicity of their culture broth and bacterial metabolites on Spodoptera exigua hemocyte. When Xn or Ptt (<100 cells per larva) were injected to larval of S. exigua, the bacteria increased in density with incubation time, while the insent hemocyte numbers significantly and the resulting culture broths were sampled for analysis of their cytotoxicity against S. exigua hemocytes. In addition, the sequential culture broth samples were analyzed in active component chemicals using a reverse phase HPLC. Finally, seven bacterial metabolites were analyzed in relative cytotoxicity against S. exigua. These results suggest that BZA is a major cytotoxic compound.

Bacillus thuringiensis (Bt) is a bacterial biopesticide against insect pests, mainly lepidopterans. Spodoptera exigua and Plutella xylostella exhibit significant decreases in Bt susceptibility in late larval instars. To enhance Bt pathogenicity, we used a mixture treatment of Bt and other bacterial metabolites which possessed significant immunosuppressive activities. Mixtures of Bt with culture broths of Xenorhabdus nematophila (Xn) or Photorhabdus temperata ssp. temperata (Ptt) significantly enhanced the Bt pathogenicity against late larval instars. Different ratios of Bt to bacterial culture broth had significant pathogenicities against last instar P. xylostella and S. exigua. Five compounds identified from the bacterial culture broth also enhanced Bt pathogenicity. After determining the optimal ratios, the mixture was applied to cabbage infested by late ins tar P. xylostella or S. exigua in greenhouse conditions. A mixture of Bt and Xn culture broth killed 100% of both insect pests when it was sprayed twice, while Bt alone killed less than 80% or 60% of P. xylostella and S. exigua, respectively. Other Bt mixtures, including Ptt culture broth or bacterial metabolites, also significantly increased pathogenicity in the semi-field assays. These results demonstrated that the Bt mixtures collectively names 'Bt-Plus' can be developed into potent biopesticides to increase the efficacy of Bt.

An entomopathogenic bacterium, Photorhabdus temperata ssp. temperata (Ptt), suppresses insect immune responses and facilitates its symbiotic nematode development in target insects. The immunosuppressive activity of Ptt enhances pathogenicity of various microbial pesticides including Bacillus thuringiensis (Bt). This study was performed to select a cheap and efficient bacterial culture medium for large scale culturing of the bacteria. Relatively cheap industrial bacterial culture media (MY and M2) were compared to two research media, Luria-Bertani (LB) and tryptic soy broth (TSB). In all tested media, a constant initial population of Ptt multiplied and reached a stationary phase at 48 h. However, more bacterial colony densities were detected in LB and TSB at the stationary phase compared to two industrial media. All bacterial culture broth gave significant synergism to Bt pathogenicity against third instars of the diamondback moth, Plutella xylostella. Production of bacterial metabolites extracted by either hexane or ethyl acetate did not show any significant difference in total mass among four culture media. Reverse phase HPLC separated the four bacterial metabolites, which were not much different in quantities among four bacterial culture broths. This study suggests that two industrial bacterial culture media can be used to economically culture Ptt in a large scale.

Oral toxicities of 5 Photorhabdus temperata ssp. temperata (Ptt) strains collected in different regions of Korea were determined against the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, Culex pipiens pallens and Paratlanticus ussuriensis. When a diet or water containing culture media of 5 different Ptt strains were ingested to immature insects, mortalities of the first instar larvae of G. mellonella, L. caesar, P. ussuriensis and young nymphs of C. pipiens pallens were rapidly increased and 100% within 3-5 days after treatments. However, mortality of P. interpunctella neonate larvae was slightly slower and 94.4-100% within 7 days after treatments. As controls, a diet containing either water, the medium without culturing bacteria, or E. coli culture medium did not effective on their mortalities. As another control group, the culture medium of P. temperata ssp. laumondii (KACC) were variously effective to mortalities of 4 species, namely, 100, 45.3, 2.8 and 0% to Galleria, Lucilia, Plodia and Culex, respectively. Culture media of Ptt strains inhibited developmental late of late larvae of P. interpunctella. Our results suggest that the oral administration of the culture medium of Ptt symbiotic bacteria was highly effective to control various immature insects.

Two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata, are known to be potent against the diamondback moth, Plutella xylostella, when the bacteria are injected into the hemocoel. This study investigated any pathogenic effect of their culture broth on P. xylostella by oral administration. Only culture broth of both bacterial species did not give enough pathogenic effects by the oral administration. However, when the culture broth was orally treated together with Bacillus thuringiensis (Bt), both cell-free culture broth significantly enhanced Bt pathogenicity against the 3rd instar larvae of P. xylostella. The culture broth was then fractionated into hexane, ethyl acetate, and aqueous extracts. Most synergistic effect on Bt pathogenicity was found in ethyl acetate extracts of both bacterial species. Thin layer chromatography of these extracts clearly showed that ethyl acetate extracts of both bacterial culture broths possessed metabolites that were different to those of hexane and aqueous extracts. These results suggest that the both entomopathogenic bacteria produce and secrete different factors to give significant synergistic effect on Bt pathogenicity.

Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata temperata (Ptt), are symbionts of nematodes, Steinernema carpocapsae and Heterorhabditis megidis, respectively. When the nematodes enter host insect hemocoel, the bacteria are released from the nematode intestine to insect hemocoel and cause immunosuppression, which results in septicemia. Culture broth of both bacteria had insecticidal effects when injected into hemocoel of Plutella xylostella larvae, but did not when orally administered. However, either mixture of Xn or Ptt with Bacillus thuringiensis (Bt) significantly enhanced the Bt pathogenicity against P. xylostella. The culture broth was fractionated with hexane and diethylacetate extracts. Diethylacetate extract had potent factor (s) to increase Bt pathogenicity. A compound, benzylideneacetone, identified from the diethylacetate fraction had oral toxicity against P. xylostella. This compound also showed high acaricidal effect on the two spotted spider mite, Tetranychus urticae

Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata temperata (Ptt), maintain monoxenic condition within host insect cadaver by synthesizing and releasing various antibiotics. These two bacteria were cultured in tryptic soy broth during different times, which were screened in their antibacterial activities. Both bacterial culture broth had high antibacterial activities against Escherichia coli at their stationary growth phase. The potent culture broth was used to screen target plant bacterial pathogens using both inhibition zone assay and liquid culture assay. Ralstonia sp. was most susceptible, while Xanthomonas sp. was highly resistant. Pseudomonas sp. and Bacillus sp. showed hemi-susceptible. The culture broth was further fractionated into hexane and diethylether extracts. Significant antibacterial effect was found in the diethylether extract

Two entomopathogenic bacteria, Xenorhadus nematophila and Photorhabdus temperata temperata, are known to suppress immune responses of target insects by inhibiting eicosanoid biosynthesis. This study analyzed these bacterial metabolites in their effects on hemocyte-spreading behavior of the beet armyworm, Spodoptera exigua. Both bacterial culture broth significantly inhibited the hemocyte-spreading behavior, at which the culture broth derived from the stationary growth phase had the most potent effect. Three identified eicosanoid synthesis inhibitors (benzylideneacetone, PY and Ac-FGV) impaired the hemocyte-spreading behavior of S. xigua, at which benzylideneacetone was the most potent. These three compounds share a common chemical structure: a pentenebenzene ring. Alternation of this common structure resulted in significant loss of their inhibitory activity to the hemocyte-spreading behavior.