Xenorhabdus와 Photorhabdus 속은 각각 곤충병원성 선충인 Steinernema와 Heterorhabditis에 공생하는 공생세 균이다. 감염성 선충의 유충은 공생세균을 표적 곤충의 혈강에 전달하고, 여기서 세균이 증식하여 숙주 선충의 발달을 돕는다. 이러한 선충과 세균 복합체의 성공적 공생관계는 세균의 이차대사산물을 통한 숙주의 면역억제 에 달려져 있다고 알려져 있다. 본 연구에서는 서로 다른 살충력을 보이는 6종의 Xenorhabdus를 확보하고 이러한 차이가 세균의 성장속도와 NRPS (Non ribosomal peptide synthease)에 의해 생성되는 세균의 이차대사산물 발현에 서 기원한다는 것을 확인하였다. 서로 다른 균주들은 콩명나방 (Tenebrio molitor)에 대한 살충력에 차이를 가지고 있었다. 이러한 세균들은 TSB 배지에서는 세균 성장 속도에 차이가 존재하지 않았지만 콩명나방 혈강 내에서는 세균의 성장 속도에 차이가 존재하는 것으로 나타났다. 또한 각 세균의 이차대사산물 추출물을 통한 곤충의 면역 억제 실험 결과 PLA2 활성 억제, 세포독성 능력들이 살충력과 상관관계가 있는 것으로 나타났다. 이러한 이차대 사산물의 경우 많은 물질이NRPS (Non ribosomal peptide synthease)에 의해 생성되므로 각 세균 별 NRPS의 유전자 발현을 보았을 때 흥미롭게도 살충력이 더 높은 스트레인의 세균이 일부 NRPS 유전자의 발현이 더 높은 것으로 나타났다. NRPS에 의해 합성되는 물질을 포함한 세균의 이차대사산물의 차이를 서로 비교하기 위하여 이차대사 산물 추출액을 GC-MS/MS를 이용하여 분석하였다. 본 연구를 통해 곤충병원세균에 살충력의 기원이 NRPS를 통해 합성되는 이차대사산물에 있다는 것을 확인하였으며 이를 이용한 다양한 NRPS 유래 물질 연구는 신규 살충 물질 개발에 들어가는 비용과 시간을 획기적으로 줄일 수 있을 것으로 기대된다.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. Success parasitism of the nematode-bacterial complex depends on the host immunosuppression by the bacteria via their secondary metabolites. Lrp (Leucine-responsive regulatory protein) is a global transcriptional factor of the bacteria and play a crucial role in the parasitism. However, its regulatory targets to suppress the insect immunity were not clearly determined. This study investigated the regulatory target genes and subsequent secondary metabolites by Lrp in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage in a target insect, Spodoptera exigua. Among eight non-ribosomal peptide synthetase (NRPS1-NRPS8) genes, six gene (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites along with alteration of the NRPS expression levels. The immunosuppressive activities of X. hominickii depended on the Lrp expression level. The metabolites produced by Lrp expression possessed the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (= cPF) was produced under Lrp control and identified to inhibit phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays crucial role in insect immunosuppression by modulating NRPS expressions.

곤충병원선충인 Steinernema longicaudum에 공생하는 Xenorhabdus ehlersii KSY 세균은 나방류에 대한 높은 병원력을 발휘한다. 본 연구에서 이 세균의 병원력이 아이코사노이드 생합성을 억제하여 기주 곤충의 면역 저하를 유발한다는 것을 확인하였다. 그러나 이 세균의 병원력은 혈강 주입에 의해 야기된다. 섭식을 통해 이 세균을 혈강으로 전달하기 위해 곤충의 중장벽을 파괴하여 병원력을 발휘하는 Bacillus thuringiensis (Bt)와 혼합하여 처리하였다. 배추좀나방(Plutella xylostella) 유충에 대해서 X. ehlersii 세균 배양액의 혼합 처리는 Bt 살충력을 현격하게 증가시켰다. 이러한 살충효과는 또 다른 나비목 해충인 콩명나방에 대해서도 확인되었다. 제형화를 위해 X. ehlersii 세균 배양액을 동결건조하여 Bt 수 화제와 혼합하였다. 이를 기반으로 간이 포장실험을 수행하였다. Bt 단독으로 처리한 결과 약 80%의 방제 효과를 보인 반면 X. ehlersii 혼합제는 95% 이상의 방제효과를 나타냈다. 본 연구는 곤충병원세균 X. ehlersii가 새로운 해충 방제제로 개발될 가능성을 제시하고 있다.

The entomopathogenic bacteria Xenorhabdus/Photorhabdus inhibit insect immune responses by inhibiting eicosanoid biosynthesis. Especially, the bacterial secondary metabolites inhibit PLA2 that release eicosanoid biosynthesis precursors. Some organic extracts of the bacteria-cultured broth possessed PLA2 inhibitory activity. This study used butanol to extract the metabolites and showed that the extract possessed potent inhibitory activity against insect immunity. The inhibition was reversed by the addition of eicosanoid biosynthesis precursor, suggesting PLA2 inhibition. Furthermore, sub-fractionation of the butanol extract separated the secondary metabolites and the fractions exhibited differential PLA2 inhibition. The active fractions appeared to contain novel compounds that are not known in PLA2 inhibition of these bacteria.

Entomopathogenic nematodes (EPNs) of the genus Steinernema are pathogenic to the insects and well known as ideal models for understanding parasite-host interaction. EPNs harbor a number of bacterial symbionts in their gut belonging to the noble genus Xenorhabdus which are capable of killing insects by themselves or by combination with nematodes by suppressing insect immune defense. Here, we report host range of Steinernema monticolum and its symbiont Xenorhabdus hominickii. S. monticolum has a diverse host range including lepidopteran and coleopteran insects although they showed higher pathogenicity to the lepidopteran insects. Especially, X. hominickii suppressed insect immune responses. A target insect, Spodoptera exigua, exhibited both cellular and humoral immune responses by expressing antimicrobial peptides and forming nodules in response to heat-killed X. hominickii. However, live bacteria significantly suppressed the immune responses. An addition of arachidonic acid to the bacterial infection significantly rescued the immune responses, suggesting eicosanoid biosynthetic pathway as a pathogenic target of X. hominickii.

A phase variation has been reported in an entomopathogenic bacterium, Xenorhabdus nematophila. Compared to a wild type primary form, a secondary form usually lose several physiological and biochemical characters. This study showed that the phase variation of X. nematophila caused a significant alteration in its immunosuppressive activity and subsequent entomopathogenicity. A secondary form of X. nematophila was detected in laboratory colonies and exhibited significant differences in dye absorption and entomopathogenicity. In addition, the secondary form was different in production of eicosanoid-biosynthesis inhibitors (EBIs) compared to the primary form of X. nematophila. Production of oxindole and p-hydroxypropionic acid was significantly reduced in the culture broth of the secondary form of X. nematophila. The reduced EBI production resulted in significant suppression in the inhibitory effects on a cellular nodule formation and phenoloxidase activity. Culture broth of the primary form of X. nematophila significantly more enhanced the pathogenicity of Bacillus thuringiensis (Bt) than the culture broth of the secondary form. Furthermore, this study developed a high efficient ‘Dual Bt-Plus’ to control both lepidopteran insect pests of Plutella xylostella and Spodoptera exigua by mixing two effective Bt strains along with the addition of potent bacterial metabolites or 100-fold concentrated X. nematophila culture broth.

An entomopathogenic bacterium, Xenorhabdus nematophila, secretes at least eight bacterial metabolites, which have suppressive effects on insect immunity. This study quantified their sequential production during bacterial growth and analyzed their individual immunosuppressive activities against an insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth in both insect host and culture medium, in which eight metabolites were secreted in different time points. At early growth phase (6 to 12 h), Ac-FGV, Cis-cPY, PHPP and indole metabolites were detected in the culture broth. During early growth phase, PHPP was highly potent to inhibit phenoloxidase activity as well as nodule formation. At late growth phase (24 to 48 h), BZA, HPA, PY were detected at 10 – 140 ppm in the culture broth, their metabolites were highly potent to inhibit phospholipase A2 and to induce cytotoxicity to hemocytes. These results suggest that X. nematophila sequentially produces the immune suppressive metabolites, which cooperatively inhibit different steps of insect immune responses.

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.

 ,  , The entomopathogenic bacterium, Xenorhabdus sp., was isolated from an entomopathogenic nematode, Steinernema monticolum. When these bacteria were injected into the hemocoel of the diamondback moth, Plutella xylostella, they caused significant mortality. However, the bacterium was not pathogenic when it was administered orally. This study showed that Xenorhabdus sp. significantly enhanced oral pathogenicity of Bacillus thuringiensis (Bt) against the last instar larvae of P. xylostella. Different ratios of culture broth of Xenorhabdus sp. and Bt showed significantly different pathogenicities against P. xylostella. In field tests, the optimal bacterial mixture significantly enhanced control efficacy against P.xylostella compared to Bt treatment alone. These results demonstrated that Xenorhabdus sp. culture broth can be developed as a potent biopesticide by enhancing the insecticidal efficacy of Bt.

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, Xenorhabdus sp., symbiotic to Steinernema monticolum was investigated in its insecticidal activity. The bacteria induced septicemia of two lepidopteran insects (Plutella xylostella, Spodoptera exigua), a coleopteran insect (Tribolium castaneum), and a hemipteran insect (Riptortus clavatus) when they were injected into hemocoel. The bacterial culture broth contained immunosuppressive factor(s) that inhibited hemocyte nodulation in response to heat-killed bacteria. The immunosuppressive activity appeared to be caused by inhibition of two main immune-associated enzymes, phospholipase A2 (PLA2) and phenoloxidase (PO). HPLC analysis of the bacterial culture broth contained several PLA2 inhibitors. The bacterial culture broth significantly enhanced Bt pathogenicity. There results support a novel insect pest control strategy using eicosanoid-biosynthesis inhibitors.

A bacterial colony was isolated from the gut of the bean bug, Riptortus clavatus. From morphological and biochemical tests, the bacterial isolate showed the highest similarity to Staphylococcus succinus. DNA sequence of 16S rRNA gene of the bacterium supported the identification. Oral administration of penicillin G to adults of R. clavatus gave a dose-dependent mortality of adults of R. clavatus to adults along with significant decrease of the bacterial population in the gut. Similarly, three metabolites (benzylideneacetone, proline-tyrosine, and acetylated phenylalanine-glycine-valine) derived from an entomopathogenic bacterium, Xenorhabdus nematophila, also inhibited growth of the gut bacterial population and gave significant mortalities to R. clavatus. These results suggest that a gut bacterial population classified as Staphylococcus sp. is required for survival of R. clavatus and that the three bacterial metabolites had toxic effects on the bugs due to their antibacterial properties.

Two groups of entomopathogenic bacteria, Xenorhabdus and Photorhabdus, are known to suppress insect immune responses by inhibiting eicosanoid biosynthesis. This study used these bacterial culture broths to develop novel biochemical insecticides against the diamondback moth, Plutella xylostella. Though the bacterial culture broths alone showed little insecticidal activity, they significantly enhanced pathogenicity of Bacillus thuringiensis against the fourth instar larvae of P. xylostella. Sterilization of the bacterial culture broth by autoclaving or 0.2 ㎛ membrane filtering did not influence the synergistic effect on the pathogenicity of B. thuringiensis. Three metablites identified in the culture broth of X. nematophila also showed similar synergistic effects. In field test, both entomopathogenic bacterial culture broth also enhanced the control efficacy of B. thuringiensis against P. xylostella.