Korean journal of applied entomology (한국응용곤충학회지)

Korean Society of Applied Entomology (한국응용곤충학회)
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Identification of Metarhizium sp. Isolated from Protaetia brevitarsis seulensis (Kolbe) Using Ribosomal DNA Sequence

흰점박이꽃무지로부터 Metarhizium속 사상균의 분리 및 ribosomal DNA 염기서열에 의한 동정

  • 최지영 (농촌진흥청 농업과학기술원 잠사곤충부) ;
  • 김철학 (강원대학교 농업생명과학대학) ;
  • 제연호 (서울대학교 농생명공학부) ;
  • 최영철 (농촌진흥청 농업과학기술원 잠사곤충부) ;
  • 김종길 (농촌진흥청 농업과학기술원 잠사곤충부) ;
  • 박규택 (강원대학교 농업생명과학대학) ;
  • 김근영 (농촌진흥청 농업과학기술원 잠사곤충부)
  • Published : 2003.03.01
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Abstract

For the purpose of the protection of beneficial insects from pathogens and the development of control agent against pests, a strain of Metarhizium sp. was isolated from the infected Protaetia brevitarsis seulensis larvae in Korea. Under the scanning electron microscope, the isolate, Metarhizium sp. KMA-1, showed distinct formation of conidia on the palisade-like masse which were comprised of elongate chains and this shape is a typical feature of Metarhizium species. PCR techniques were used to identify the isolate and the primers used were designed on the basis of two kinds of rRNAs sequences, 28S rRNA and internal transcribed spacer(ITS). The specific PCR products from each primer set were amplified and the DNA sequences were determined for the similarity comparison. Sequence alignment of these fragments using GenBank database resulted in the highest homology similarity between the isolate Metarhizium sp. KMA-1 and M. anisopliae. From these results, the isolate Metarhizium sp. KMA-1 in this study was identified as M. anisopliae.

곤충자원의 대량사육을 위한 병 발생 예방과 해충의 효과적인 방제를 위하여 흰점박이꽃무지 이병충으로부터 곤충병원 사상균을 분리하였다. 전자현미경 관찰 결과 분리균주 KMA-1은 Metharizium속의 전형적인 쇠사슬형의 분생자를 paliside-like masse에 형성하였다. 따라서, 정확한 동정을 위하여 28S rRNA와 ITS염기 서열을 바탕으로 제작한 특이 프라이머쌍을 사용하여 PCR 반응을 수행하였다. 각각의 프라이머쌍을 사용한 PCR반응으로부터 특이 밴드가 검출되었으며 이 증폭 산물들의 염기 서열을 결정, 비교하였다. 분리 균주 KMA-1의 PCR산물인 28S rRNA와 ITS DNA염기서열을 GenBank데이터베이스에 등록된 염기서열 정보와의 상동성을 검색한 결과, 모두 Metarhizium anisopliae와 가장 높은 서열 상동성을 보였다. 이상의 결과로서 본 실험에서 분리 명명된 KMA-1는 M. anisopliae로 동정되었다.

Keywords

References

  1. Anderson, J.B., S.S. Bailey and P.J. Pukkila. 1989. Variation in ribosomal DNA among biological species of Armillasria, a genus of root-infecting fungi. Evolution. 43: 1652-1662 https://doi.org/10.2307/2409381
  2. Boucias, D.G. and J.C. Pendland. 1998. Principle of insect pathol-ogy. 537 pp. Kluwer academic publishers, Boston
  3. Cerenius, L., P.O. Thornqvist, A. Vey, M.W. Johansson and K. Soderhall. 1990. The effect of the fungal toxin destruxin E on isolated crayfish haemocytes. J. Insect Physiol. 36: 785-789 https://doi.org/10.1016/0022-1910(90)90052-H
  4. Curran, J., F. Driver, J.W.O. Ballard and R.J. Milner. 1994. Phy-logeny of Metarhizium : sequence analysis of the internally transcribed and 5.8s region of the ribosomal DNA repeat. My-cological Research 9: 547-552
  5. Driver, F., R.J. Milner and J.W.H. Trueman. 2000. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rRNA sequence data. Mycol. Res. 104: 134-150 https://doi.org/10.1017/S0953756299001756
  6. Dumas, C., V. Matha, J.M. Quiot and A. Vey. 1996. Effects of dextruxins, cyclic depsipeptide mycotoxins, on calcium balance and phosphorylation on intracellular proteins in lepidopteran cell lines. Comp. Biochem. Physiol. 114C: 213-219
  7. Glare, T.R., R.J. Milner and C.D. Beaton. 1996. Variation in Metarhizium, a genus of fungal pathogen attacking Orthoptera: Is phialide morphology a useful criterion? J. Orthopteran Res. 5: 19-27
  8. Hanel, H. 1982. The life cycle of the insect pathogenic fungus Metarhizium anisopliae in the termite Nasutitermes exitiosus. Mycopathologia. 80: 137-145 https://doi.org/10.1007/BF00437576
  9. Kang, L.J., H.K. Kim, C.K. Chung, S.I. Kim and D.W. Oh. 2000. Effects of Protaetia orientalis (Gory et Perchlon) larva on the lipid metabolism in ethanol administered rats. J. Kor. Soc. Food Sci. Nutr. 29: 479-484
  10. King, E.J. and M.F. Brown. 1983. A technique for preserving aerial fungal structures for scanning electron microscopy. Can. J. Microbiol. 29: 653-658 https://doi.org/10.1139/m83-106
  11. Knudsen, G.R.M., D.J. Eschen, L.M. Dandurand and Z.G. Wang. 1991. Method to enhance growth and sporulation of pelletized biological fungi. Appl. Environ. Microbiol. 57: 2864-2867
  12. Leal, S.C.M., D.J. Bertioli, B.V. Ball and T.M. Butt. 1994. Pre-sence of double-stranded RNAs and virus-like particles in the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Science and Technology 4: 89-94 https://doi.org/10.1080/09583159409355316
  13. Nunn, G.B., B.F. Theisen, B. Christensen and P. Arctander. 1996. Simplicity corrected size growth of the nuclear 28S ribosomal RNA D3 expansion segment in the crustacean order Isopoda. J. Mol. Evol. 42: 211-223 https://doi.org/10.1007/BF02198847
  14. Park, H.Y., S.S. Park, H.W. Oh and J.I. Kim. 1994. General char-acteristics of the white-spotted flower chafer, Protaetia brevi-tarsis reared in the laboratory. Kor. J. Entomol. 24: 1-5
  15. Pipe, N.D., D. Chandler, B.W. Bainbridge and J.B. Heale. 1995. Restriction fragment length polymorphisms in the ribosomal RNA gene complex of isolates of the entomopathogenic fungus Metarhizium anisopliae. Mycol. Res. 99: 485-491 https://doi.org/10.1016/S0953-7562(09)80651-5
  16. Rakotonirainy, M.S., M. Dutertre, Y. Brygoo and G. Riba. 1994. Phylogenetic relationships within the genus Metarhizium based on 28S rRNA sequences and isozyme comparisons. Mycol. Res. 98: 225-230 https://doi.org/10.1016/S0953-7562(09)80190-1
  17. Schabel, H.G. 1976. Oral infection of Hylodius pales by Metarhi-zium anisopliae. J. Invertebr. Pathol. 27: 377-383 https://doi.org/10.1016/0022-2011(76)90100-2
  18. Sosa-Gomez, D.R., D.G. Boucias and J.L. Nation. 1997. Attach-ment of Metarhizium anisopliae to the southern green stink bug Nezara viridula cuticle and fungistatic effect of cuticular lipids and aldehydes. J. Invertebr. Pathol. 69: 31-39 https://doi.org/10.1006/jipa.1996.4619
  19. Suh, Y., L.B. Thien, H.E. Reeve and E.A. Zimmer. 1993. Molecu-lar evolution and phylogenetic implications of internal trans-cribed spacer sequences of ribosomal DNA in witeraceae. American J. Botany. 80: 1042-1055 https://doi.org/10.2307/2445752
  20. Tulloch, M. 1976. The genus Metarhizium. Transactions of the British Mycological Society 66: 407-411 https://doi.org/10.1016/S0007-1536(76)80209-4
  21. White, T.J., T. Bruns, S. Lee and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phy-logenies. pp. 315-322. In PCR Protocols: A Guide to Method and Applications. ed. by M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White. 482 pp. Academic Press. New York