Korean journal of applied entomology (한국응용곤충학회지)

Korean Society of Applied Entomology (한국응용곤충학회)
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Immunosuppressive Activity of an Entomopathogenic Bacteria, Xenorhabdus ehlersii KSY, and Its Application to Enhance Insecticidal Activity of Bacillus thuringiensis

곤충병원세균(Xenorhabdus ehlersii KSY)의 곤충면역 억제 능력과 이를 이용한 Bacillus thuringiensis 의 살충력 증가 효과

  • Kim, Hyoil (Department of Plant Medicals, College of Life Sciences, Andong National University) ;
  • Kim, Yonggyun (Department of Plant Medicals, College of Life Sciences, Andong National University)
  • 김효일 (안동대학교 생명과학대학 식물의학과) ;
  • 김용균 (안동대학교 생명과학대학 식물의학과)
  • Received : 2019.03.27
  • Accepted : 2019.04.08
  • Published : 2019.06.01
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Abstract

An entomopathogenic bacterium, Xenorhabdus ehlersii KSY, is symbiotic to a nematode, Steinernema longicaudum, and exhibits high entomopathogenic virulence against lepidopteran insects. This study showed that the bacterial pathogenicity is induced by its inhibitory activity against eicosanoid biosynthesis of target insects, resulting in immunosuppression. To be applied for insect pest control, the bacteria should be infected to insect hemocoel. To deliver X. ehlersii to inset hemocoel, Bacillus thuringiensis (Bt) was mixed with the bacteria to breakdown the physical barrier (= midgut epithelium) from midgut lumen to hemocoel. The bacterial mixture significantly enhanced insecticidal activity of Bt only against larvae of Plutella xylostella and Maruca vitrata. For formulation, X. ehlersii cells were freeze-dried and mixed with sporulated Bt cells. The formulated bacterial mixture was applied to semi-field cultivating cabbage crop infested by P. xylostella. The bacterial mixture treatment showed over 95% control efficacy, while Bt alone gave 80% control efficacy. These results suggest that X. ehlersii can be applied to develop a novel insect control agent.

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 수화제와 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.

Keywords

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Fig. 1. Primary form of X. ehlersii KSY.

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Fig. 2. Immunosuppression activity of X. ehlersii against hemocyte-spreading behavior of P. xylostella larvae. Hemocytes were collected from fourth intar larvae and incubated with X. ehlersii -cultured broth (Xe). For control, hemocytes were incubated in 10 μL TC-100. For treatments hemocyte in 9 μL TC-100 were incubated with 1 μL of Xe or TSB. For rescue experiment, 8 μL hemocyte suspension are incubated with 1 μL of Xe and 1 μL of arachidonic acid (AA, 100mM). Hemocyte-spreading assay followed the method described in materials and method. Each treatment are replicated three times.

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Fig. 3. Enhanced insecticidal activity of B. thuringiensis kurstaki (Btk) against P. xylostella larvae by addition of X. ehlersii-cultured broth (Xe). Forth instar larvae were treated with Btk (30 ppm) by a leaf-dipping method. Xe was obtained by 48 h culture and the bacterial suspension are used to prepare Btk (30 ppm) suspension (Btk+Xe). Each treatment used 10 larvae and are replicated three times. Asterisks indicate complete difference between Btk and Btk+Xe treatment at Type 1 error = 0.05 (LSD test).

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Fig. 4. Effect of bacterial mixture treatment on control efficacy of M. vitrata larvae. X. ehlersii was cultured in TSB for 48 h and used for preparation of bacterial mixtures with B. thuringiensis kurstaki (BtK) or B. thuringiensis aizawaii (BtA). (A) Mixture effect of X. ehlersii and BtK (B) Mixture effect of X. ehlersii and BtA. The bacterial mixtures were treated to fourth instar larvae at M. vitrata by a dipping method. For reference, BtK or BtA alone was treated. Each treatment used 10 larvae and replicated three times.

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Fig. 5. Semi-field test of a bacterial mixture (XeBt) of B. thuringiensis kurstaki (Btk) and X. ehlersii (Xe) against P. xylostella larvae infesting cabbages cultivated in pots. Btk (30 ppm) was mixed in Xe-cultured broth to prepare XeBt. XeBt was sprayed against the cabbages after counting initial numbers per experimental unit (= a cabbage plant). Each experiment unit had more than 30 individuals (mostly 3rd to 4th instar larvae). Each treatment are replicated three times. (A) Survival rates at different time points after bacterial treatment. (B) Host damage analysis induced by P. xylostella larval feeding behavior. Damage intensities (%) are calculated by scoring damage rate (0: less than 5% feeding damage, 1: 5~10% feeding damage, 2: 10~15% feeding damage, 3: over 15% feeding damage) using a following formular: Damage Intensity (%) ={Σ (Number of damage leave × damage rate)}/(Number of total test leaves) × 100.

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