The Korean Journal of Pesticide Science (농약과학회지)

The Korean Society of Pesticide Science (한국농약과학회)
권호 표지

Protoplast Formation of Collectotrichum acutum and the Assessment of Antifungal Activity of Several by using its Protoplasts

고추 탄저병군(Colletotrichum acutatum)의 원형질체 형성과 원형질체를 이용한 살균제 효과 검정

  • 김명기 (충북대학교 농업생명환경대학 응용생명환경학부 식물의학) ;
  • 김흥태 (충북대학교 농업생명환경대학 응용생명환경학부 식물의학)
  • Published : 2008.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

To obtain protoplasts of Colletotrichum acutatum JC24, conidia were inoculated onto cellophane membrane placed on PDA and incubated at $25^{\circ}C$ for 20 hrs under the dark condition. Cellophane membranes, where mycelia were incubated, were soaked into 2% lysing enzyme solution prepared with 0.02 M phosphate buffer (pH 7.0) including 1.2 M sorbitiol. After treatment in 2% enzyme solution for 2 - 3 hrs, it could be possible to harvest $2-3\;{\times}\;10^6$ protoplasts/mL. The effect of several fungicides on reversion ratio was determined by using the protoplasts obtained from C. acutatum JC24. Any protoplasts could not be reversed to mycelia on reversion PDA amended with $10\;{\mu}\;g\;mL^{-1}$ of propineb. With tebuconazole, inhibition ratio of protoplast reversion was 100 and 0.9% at 0.5 and $0.1\;{\mu}\;g\;mL^{-1}$, respectively, while inhibitory effect on mycelial growth was 85.1 and 75.7%. The inhibitory tendency of carbendazim on protoplast reversion was as same as mycelial growth. In the case of strobilurins, trifloxystrobin and kresoxim-methyl, they only could inhibit protoplast reversion of C. acutatum JC24, when salicylhydroxamic acid (SHAM) was amended into reversion PDA with strobilurins.

고추 탄저병균인 Colletotrichum acutatum JC24는 PDA 배지에 치상한 cellophane막에 포자 접종하여 $25^{\circ}C$의 암상태에서 20시간 배양한 균사체를 2%의 lysing enzyme에 2-3시간 처리하여 $2-3\;{\times}\;10^6$ 개/mL의 원형질체를 수확할 수 있었다. 이때에는 0.02 M phosphate buffer(pH 7.0)와, 삼투압 안정제로는 1.2 M sorbitol용액을 사용하였을 때가 원형질체의 생성량이 가장 높았다. C. acutatum JC24의 균사체에서 얻은 원형질체에 propineb를 $10\;{\mu}\;g\;mL^{-1}$를 처리하였을 경우에, 균사생장 억제효과는 미미하였지만 원형질체의 재생은 100% 억제하였다. Tebuconazole의 재생 억제효과는 균사생장 억제효과와는 큰 차이를 보였는데, $0.5\;{\mu}\;g\;mL^{-1}$ 처리구에서는 원형질체의 재생이 100% 억제되었고, $0.1\;{\mu}\;g\;mL^{-1}$ 처리구에서는 0.9%밖에 억제하지 못하였다. 하지만 동일한 농도에서 균사생장에 대한 억제효과는 85.1과 75.7%로 억제의 경향이 매우 달랐다. Carbendazim의 경우에는 원형질체 재생과 균사생장에 미치는 효과가 비슷하였다. Trifloxystrobin과 kresoxim-methyl을 SHAm과 동시에 처리하였을 원형질체의 재생에 대한 억제 효과가 크게 상승하였다.

Keywords

References

  1. Bachmann, B. J. and D. M. Bener (1981) Protoplasts from Neurospora crassa. J. Bacteriol. 78:550-556
  2. Dahmen, H., H. C. Hoch and T. Staub (1988) Differential effects of sterol inhibitors on growth, cell membrane permeability, and ultrastructure of two target fungi. Phytopathology 78:1033-1042 https://doi.org/10.1094/Phyto-78-1033
  3. Davidse, L. C. (1986) Benzimidazole fungicides: mechanism of action and biological impact. Ann. Rev. Phytopathol. 24:43-65 https://doi.org/10.1146/annurev.py.24.090186.000355
  4. Davis, B. (1985) Factors influencing protoplast isolation. p 45-71. ed. by J. F. Peberdy and L. Ferenczy in Fungal protoplasts; Application in biochemistry and genetics. pp. 354
  5. Harris, G. M. (1982) Protoplasts from Gibberella fujikuroi. Phytopathology 72:1403-1407 https://doi.org/10.1094/Phyto-72-1403
  6. He, Z., M. S. Price, G. R. Brian, D. R. Georgianna and G. A. Payne (2007) Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus. BMC Microbiology 7:104-115 https://doi.org/10.1186/1471-2180-7-104
  7. Kim, C. H. and K. S. Park (1988) A predictive model of disease progression of red-pepper anthracnose. Kor. J. Plant Pathol. 4:325-331
  8. Kim, J. T., S. Park, W. Choi, Y. Lee and H. T. Kim (2008) Characterization of Colletotrichum isolates causing anthracnose of pepper in Korea. Plant Pathol. J. 24:17-23 https://doi.org/10.5423/PPJ.2008.24.1.017
  9. Kreger, D. R. and M. Kopecka (1975) On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: An electromicroscopic, X-ray diffraction and chemical study. J. Gen. Microbiol. 92:207-220
  10. Moore, P. M. and J. F. Peberdy (1976) Release and regeneration of protoplast of the conidia of Aspergillus flavus. Trans. Br. Mycol. Soc. 66:421-425 https://doi.org/10.1016/S0007-1536(76)80211-2
  11. Ogawa, K., N. Yoshida, W. Gesnara, C. A. Omumasaba and C. Chamuswarng (2000) Hybridization and breeding of the benomyl resistant mutant, Trichoderma harzianum antagonized to phytopathogenic fungi by protoplast fusion. Biosci. Biotechnol. Biochem. 64:833-836 https://doi.org/10.1271/bbb.64.833
  12. Olaya, G., D. Zheng and W. Koller (1999) Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl. Pesticide Sci. 54:230-236
  13. Park, K. S. and C. H. Kim (1992) Identification, distribution and etiological characteristics of anthracnose fungi of red pepper in Korea. Kor. J. Plant Pathol. 8:61-69
  14. Peberdy, J. F., C. E. Buckley, D. C. Daltry and P. M. Moore (1976) Factors affecting protoplast release in some filamentous fungi. Trans. Br. Mycol. Soc. 67:23-26 https://doi.org/10.1016/S0007-1536(76)80003-4
  15. Sagara, Y. (1969) Studies on protoplasts of Geotrichum candidum: Mechanism of formation of protoplasts and their physical and morphological properties. Tokushima J. Exp. Med. 16:57-69
  16. Sancholle, M., J. D. Weete and C. Montant (1983) Effects of triazoles on fungi: I. Growth and cellular permeability. Pesticide Biochem. Physiol. 21:31-44
  17. Shin, H. J., Z. J. Chen, J. M. Hwang and S. G. Lee (1999) Comparison of pepper anthracnose pathogen from Korea and China. Plant Pathol. J. 15:323-329
  18. Stephen, E. R. and A. Nasim (1981) Production of protoplasts indifferent yeasts by mutanase. Can. J. Microbiol. 27:550-553 https://doi.org/10.1139/m81-082
  19. Talhinhas, P., S. Muthumeenakshi, J. Neves-Martins, H. Oliveira and S. Sreenivasaprasad (2008) Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles. Mol. Biotechnol. (in press)
  20. Teraoka, T., Y. Shimura, D. Hosokawa and M. Watanabe (1992) Giant protoplast of Pyricularia oryzae Cavara. Ann. Phytopath. Soc. Japan 58:726-733 https://doi.org/10.3186/jjphytopath.58.726
  21. Wood, P. M. and D. W. Hollomon (2003) A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Qo site of complex III. Pest Manag. Sci. 59:499-511 https://doi.org/10.1002/ps.655
  22. Ziogas, B. N., G. Oesterhelt, P. Masner, C. C. Steel and R. Furter (1991) Fenpropomorph: A three site inhibitor of ergosterol biosynthesis in Nectria haematococca var. cucurbitae. Pesticide Biochem. Physiol. 39:74-83 https://doi.org/10.1016/0048-3575(91)90215-8
  23. 김재정, 김준태, 박성우, 박은숙, 김흥태 (2003) 고추 탄저병균의 포자 발아와 부착, 균사 생장에 미치는 화합물의 활성 검정법 확립 및 살균제의 효과. 농약과학회지 7:159-168