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

  • Kim, Myoung-Ki (Dept. of Plant Medicine, Chungbuk National University) ;
  • Kim, Heung-Tae (Dept. of Plant Medicine, Chungbuk National University)
  • 김명기 (충북대학교 농업생명환경대학 응용생명환경학부 식물의학) ;
  • 김흥태 (충북대학교 농업생명환경대학 응용생명환경학부 식물의학)
  • Published : 2008.03.31


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.


  1. 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
  2. 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
  3. Stephen, E. R. and A. Nasim (1981) Production of protoplasts indifferent yeasts by mutanase. Can. J. Microbiol. 27:550-553
  4. 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
  5. Teraoka, T., Y. Shimura, D. Hosokawa and M. Watanabe (1992) Giant protoplast of Pyricularia oryzae Cavara. Ann. Phytopath. Soc. Japan 58:726-733
  6. Olaya, G., D. Zheng and W. Koller (1999) Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl. Pesticide Sci. 54:230-236
  7. 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
  8. 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
  9. 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)
  10. 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
  11. 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
  12. 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
  13. 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
  14. 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
  15. 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
  16. Bachmann, B. J. and D. M. Bener (1981) Protoplasts from Neurospora crassa. J. Bacteriol. 78:550-556
  17. Davidse, L. C. (1986) Benzimidazole fungicides: mechanism of action and biological impact. Ann. Rev. Phytopathol. 24:43-65
  18. 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
  19. 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
  20. Harris, G. M. (1982) Protoplasts from Gibberella fujikuroi. Phytopathology 72:1403-1407
  21. 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
  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
  23. 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