한국응용곤충학회지 (Korean journal of applied entomology)

  • 제58권2호
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  • Pages.101-109
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  • 2019
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  • 1225-0171(pISSN)
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  • 2287-545X(eISSN)
한국응용곤충학회 (Korean Society of Applied Entomology)
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곤충병원세균(Xenorhabdus ehlersii KSY)의 곤충면역 억제 능력과 이를 이용한 Bacillus thuringiensis 의 살충력 증가 효과

Immunosuppressive Activity of an Entomopathogenic Bacteria, Xenorhabdus ehlersii KSY, and Its Application to Enhance Insecticidal Activity of Bacillus thuringiensis

  • 김효일 (안동대학교 생명과학대학 식물의학과) ;
  • 김용균 (안동대학교 생명과학대학 식물의학과)
  • 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)
  • 투고 : 2019.03.27
  • 심사 : 2019.04.08
  • 발행 : 2019.06.01
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초록

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.

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.

키워드

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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).

OOGCBV_2019_v58n2_101_f0005.png 이미지

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.

OOGCBV_2019_v58n2_101_f0006.png 이미지

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.

참고문헌

  1. Ahmed, S., Kim, Y., 2019. An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua. Sci. Rep. In Press.
  2. Ahmed, S., Stanley, D., Kim, Y., 2018. An insect prostaglandin $E_2$ synthase acts in immunity and reproduction. Front. Physiol. 9, 1231. https://doi.org/10.3389/fphys.2018.01231
  3. Akhurst, R.J., 1980. Morphological and functional dimorphism in Xenorhabdus spp. bacteria symbiotically associated with the insect pathogenic nematodes Neoplectana and Heterorhabditis. J. Gen. Microbiol. 121, 303-309.
  4. Baines, D., Downer, R.G., 1994. Octopamine enhances phagocytosis in cockroach hemocytes: involvement of inositol trisphosphate. Arch. Insect Biochem. Physiol. 26, 249-261. https://doi.org/10.1002/arch.940260402
  5. Berry, C., Crickmore, N., 2017. Structural classification of insecticidal proteins - towards an in silico characterisation of novel toxins. J. Invertebr. Pathol. 142, 16-22. https://doi.org/10.1016/j.jip.2016.07.015
  6. Bode, H.B., 2009. Entomopathogenic bacteria as a source of secondary metabolites. Curr. Opin. Chem. Biol. 13, 224-230. https://doi.org/10.1016/j.cbpa.2009.02.037
  7. Boemare, N.E., Akhurst, R.J., Mourant, R.G., 1993. DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. Int. J. Syst. Bacteriol. 43, 249-255. https://doi.org/10.1099/00207713-43-2-249
  8. Bravo, A., Likitvivatanavong, S., Gill, S.S., Soberon, M., 2011. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 41, 423-431. https://doi.org/10.1016/j.ibmb.2011.02.006
  9. Broderick, N.A., Raffa, K.F., Handelsman, J., 2006. Midgut bacteria required for Bacillus thuringiensis insecticidal activity. Proc. Natl. Acad. Sci. USA 103, 15196-15199. https://doi.org/10.1073/pnas.0604865103
  10. Campos-Herrera, R., Barbercheck, M., Hoy, C.W., Stock, S.S., 2012. Entomopathogenic nematodes as a model system for advancing the frontiers of ecology. J. Nematol. 42, 162-176.
  11. Clark, K.D., Pech, L.L., Strand, M.R., 1997. Isolation and identification of a plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens. J. Biol. Chem. 272, 23440-23447 https://doi.org/10.1074/jbc.272.37.23440
  12. Corey, E.J., Albright, J.O., Barton, A.E., Hashimoto, S., 1980. Chemical and enzymic syntheses of 5-HPETE, a key biological precursor of slow-reacting substance of anaphylaxis (SRS) and 5-HETE. J. Am. Chem. Soc. 102, 1435-1436. https://doi.org/10.1021/ja00524a044
  13. Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J., Dean, D.H., 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 807-813. https://doi.org/10.1128/MMBR.62.3.807-813.1998
  14. Dowds, B.C.A., Peters, A., 2002. Entomopathogenic nematology, In: Gaugler, R. (Ed.), Virulence Mechanisms. CABI, New York, pp. 79-98.
  15. Eom, S., Park, Y., Kim, H., Kim, Y., 2014b. Development of a high efficient "Dual Bt-Plus" insecticide using a primary form of an entomopathogenic bacterium, Xenorhabdus nematophila. J. Microbiol. Biotechnol. 24, 507-521. https://doi.org/10.4014/jmb.1310.10116
  16. Eom, S., Park, Y., Kim, Y., 2014a. Sequential immunosuppressive activities of bacterial secondary metabolites from the entomopathogenic bacterium Xenorhabdus nematophila. J. Microbiol. 52, 161-168. https://doi.org/10.1007/s12275-014-3251-9
  17. ffrench-Constant, R., Waterfield, N., Daborn, P., Joyce, S., Bennett, H., Au, C., Dowling, A., Boundy, S., Reynolds, S., Clarke, D., 2003. Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiol. Rev. 26, 433-456. https://doi.org/10.1111/j.1574-6976.2003.tb00625.x
  18. Forst, S., Clarke, D., 2002. Bacteria-nematode symbiosis, In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New Brunswick, New Jersey, pp. 57-78.
  19. Furlong, M.J., Wright, D.J., Dosdall, L.M., 2013. Diamondback moth ecology and management: problems, progress, and prospects. Annu. Rev. Entomol. 58, 517-541. https://doi.org/10.1146/annurev-ento-120811-153605
  20. Gatsogiannis, C., Lang, A.E., Meusch, D., Pfaumann, V., Hofnagel, O., Benz, R., Aktories, K., Raunser, S., 2013. A syringe-like injection mechanism in Photorhabdus luminescens toxins. Nature 495, 520-523. https://doi.org/10.1038/nature11987
  21. Gaugler, R., 2002. Entomopathogenic Nematology. CABI Publishing, Wallingford, UK.
  22. Gillespie, J.P., Kanost, M.R., Trenczek, T., 1997. Biological mediators of insect immunity. Annu. Rev. Entomol. 42, 611-643. https://doi.org/10.1146/annurev.ento.42.1.611
  23. Godjo, A., Afouda, L., Baimey, H., Decraemer, W., Willems, A., 2018. Molecular diversity of Photorhabdus and Xenorhabdus bacteria, symbionts of Heterorhabditis and Steinernema nematodes retrieved from soil in Benin. Arch. Microbiol. 200, 589-601. https://doi.org/10.1007/s00203-017-1470-2
  24. Grizanova, E.V., Dubovskiy, I.M., Whitten, M.M., Glupov, V.V., 2014. Contributions of cellular and humoral immunity of Galleria mellonella larvae in defence against oral infection by Bacillus thuringiensis. J. Invertebr. Pathol. 119, 40-46. https://doi.org/10.1016/j.jip.2014.04.003
  25. Ishii, K., Adachi, T., Hamamoto, H., Oonishi, T., Kamimura, M., Imamura, K., Sekimizu, K., 2013. Insect cytokine paralytic peptide activates innate immunity via nitric oxide production in the silkworm Bombyx mori. Dev. Comp. Immunol. 39, 147-153. https://doi.org/10.1016/j.dci.2012.10.014
  26. Ji, D., Yi, Y., Kim, G.H., Choi, Y.H., Kim, P., Baek, N.I., Kim, Y., 2004. Identification of an antibacterial compound, benzylideneacetone, from Xenorhabdus nematophila against major plant-pathogenic bacteria. FEMS Microbiol. Lett. 239, 241-248. https://doi.org/10.1016/j.femsle.2004.08.041
  27. Jung, J.K., Seo, B.-Y., Park, J.H., Moon, J.-K., Choi, B.-S., Lee, Y.-H., 2007. Developmental characteristics of soybean podworm, Matsumuraeses phaseoli (Lepidoptera: Tortricidae) and legume pod borer, Maruca vitrata (Lepidoptera: Pyralidae) on semi-synthetic artificial diets. Korean J. Appl. Entomol. 46, 393-399. https://doi.org/10.5656/KSAE.2007.46.3.393
  28. Kim, H., Keum, S., Hasan, A., Kim, H., Jung, Y., Lee, D., Kim, Y., 2018b. Identification of an entomopathogenic bacterium, Xenorhabdus ehlersii KSY, from Steinernema longicaudum GNUS101 and its immunosuppressive activity against insect host by inhibiting eicosanoid biosynthesis. J. Invertebr. Pathol. 159, 6-17. https://doi.org/10.1016/j.jip.2018.10.014
  29. Kim, Y., Ahmed, S., Stanley, D., An, C., 2018a. Eicosanoid-mediated immunity in insects. Dev. Comp. Immunol. 83, 130-143. https://doi.org/10.1016/j.dci.2017.12.005
  30. Kim, Y., Ji, D., Cho, S., Park, Y., 2005. Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase $A_2$ to induce host immunodepression. J. Invertebr. Pathol. 89, 258-264. https://doi.org/10.1016/j.jip.2005.05.001
  31. Kim, Y., Kim, K., Kim, H., Park, Y., Kim, G.H., 2013. An integrated biological control using an endoparasitoid wasp (Cotesia plutellae) and a microbial insecticide (Bacillus thuringiensis) against the diamondback moth, Plutella xylostella. Korean J. Appl. Entomol. 52, 35-43. https://doi.org/10.5656/KSAE.2013.01.1.080
  32. Kim, Y., Sadekuzzaman, M., Kim, M., Kim, K., Park, Y., Jung, J.K., 2016. Genetic character and insecticide susceptibility on a Korean population of a subtropical species, Maruca vitrata. Korean J. Appl. Entomol. 55, 257-266. https://doi.org/10.5656/KSAE.2016.06.0.024
  33. Lavine, M.D., Strand, M.D., 2002. Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol. 32, 1295-1309. https://doi.org/10.1016/S0965-1748(02)00092-9
  34. Nalini, M., Kim, Y., 2007. A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior. J. Insect Physiol. 53, 1283-1292. https://doi.org/10.1016/j.jinsphys.2007.07.004
  35. Park, J., Stanley, D., Kim, Y., 2013. Rac1 mediates cytokinestimulated hemocyte spreading via prostaglandin biosynthesis in the beet armyworm, Spodoptera exigua. J. Insect Physiol. 59, 682-689. https://doi.org/10.1016/j.jinsphys.2013.04.012
  36. Park, Y., Kim, Y., 2000. Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the entomopathogenic nematode Steinernema carpocapsae. J. Insect Physiol. 46, 1469-1476. https://doi.org/10.1016/S0022-1910(00)00071-8
  37. Sadekuzzaman, M., Stanley, D., Kim, Y., 2018. Nitric oxide mediates insect cellular immunity via phospholipase $A_2$ activation. J. Innate Immun. 10, 70-81. https://doi.org/10.1159/000481524
  38. Seo, S., Lee, S., Hong, Y., Kim, Y., 2012. Phospholipase $A_2$ inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata. Appl. Environ. Microbiol. 78, 3816-3823. https://doi.org/10.1128/AEM.00301-12
  39. Shi, Y.M., Bode, H.B., 2018. Chemical language and warfare of bacterial natural products in bacteria-nematode-insect interactions. Nat. Prod. Rep. 35, 309-335. https://doi.org/10.1039/C7NP00054E
  40. Shrestha, S., Kim, Y., 2009. Biochemical characteristics of immune-associated phospholipase $A_2$ and its inhibition by an entomopathogenic bacterium, Xenorhabdus nematophila. J. Microbiol. 47, 774-782. https://doi.org/10.1007/s12275-009-0145-3
  41. Stanley, D.W., 2000. Eicosanoids in invertebrate signal transduction systems. Princeton, New Jersey, NY.
  42. Stanley, D.W., Kim, Y., 2014. Eicosanoid signaling in insects: from discovery to plant protection. Crit. Rev. Plant Sci. 33, 20-63. https://doi.org/10.1080/07352689.2014.847631
  43. Stock, S.P., Goodrich-Blair, H., 2008. Entomopathogenic nematodes and their bacterial symbionts: the inside out of a mutualistic association. Symbiosis 46, 65-76.
  44. Sung, E.J., Ryuda, M., Matsumoto, H., Uryu, O., Ochiai, M., Cook, M.E., Yi, N.Y., Wang, H., Putney, J.W., Bird, G.S., Shears, S.B., Hayakawa, Y., 2017. Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress. Proc. Natl. Acad. Sci. USA 114, 13786-13791. https://doi.org/10.1073/pnas.1712453115
  45. Tabashnik, B.E., Liu, Y.B., Malvar, T., Heckel, D.G., Masson, L., Ballester, V., Granero, F., Mensua, J.L., Ferre, J., 1997. Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis. Proc. Natl. Acad. Sci. USA 94, 12780-12785. https://doi.org/10.1073/pnas.94.24.12780
  46. Talekar, N.S., Shelton, A.M., 1993. Biology, ecology and management of the diamondback moth. Annu. Rev. Entomol. 38, 275-301. https://doi.org/10.1146/annurev.en.38.010193.001423
  47. Waterfield, N.R., Ciche, T., Clarke, D., 2009. Photorhabdus and a host of hosts. Annu. Rev. Microbiol. 63, 557-574. https://doi.org/10.1146/annurev.micro.091208.073507
  48. Wu, G., Yi, Y., 2018. Transcriptome analysis of differentially expressed genes involved in innate immunity following Bacillus thuringiensis challenge in Bombyx mori larvae. Mol. Immunol. 103, 220-228. https://doi.org/10.1016/j.molimm.2018.10.006
  49. Xu, J., Morisseau, C., Yang, J., Lee, K.S., Kamita, S.G., Hammock, B.D., 2016. Ingestion of the epoxide hydrolase inhibitor AUDA modulates immune responses of the mosquito, Culex quinquefasciatus during blood feeding. Insect Biochem. Mol. Biol. 76, 62-69. https://doi.org/10.1016/j.ibmb.2016.06.011
  50. Yooyangket, T., Muangpat, P., Polseela, R., Tandhavanant, S., Thanwisai, A., Vitta, A., 2018. Identification of entomopathogenic nematodes and symbiotic bacteria from Nam Nao National Park in Thailand and larvicidal activity of symbiotic bacteria against Aedes aegypti and Aedes albopictus. PLoS One. 13, e0195681. https://doi.org/10.1371/journal.pone.0195681