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標題: 台灣產芒果與草莓炭疽病菌對Strobilurin (QoI) 類 及 Benzimidazole 類殺菌劑之抗感性
Sensitivity and resistance of mango and strawberry anthracnose fungi to Strobilurins (QoIs) and Benzimidazoles in Taiwan
作者: 彭孟慈
Peng, Meng-Tzu
關鍵字: Colletotrichum gloeosporioides;炭疽病菌;QoIs;benzimidazoles;resistance;QoIs;benzimidazoles;抗藥性
出版社: 植物病理學系所
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American Phytopathological Society, St. Paul, MN. Sutton, B. C. 1992. The genus Glomerella and its anamorph Colletotrichum. In:Bailey, J. A. and Jeger, M. J. (eds.). Colletotrichum biology, pathology andcontrol (11-15). CAB international, UK. Albertini, C., Gredt, M. and Leroux, P. 1999. Mutationsof the -tubulin geneassociated with different phenotypes of benzimidazole resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis. Pesticide Biochemistry and Physiology 64:17-31. Anonymous 1998. Strobilurin-resistant mildew found in Germany. AGROW 318:11. Araki, Y., Sugihara, M., Sawada, H., Fujimoto, H. and Masuko, M. 2005. Monitoring of the sensitivity of Magnaporthe grisea to Metominostrobin 2001-2003:No emergence of resistant strains and no mutations at codon 143 or 129 of the cytochrome b gene. Pesticide Science 3:203-208. Avila-Adame, C., Olaya, G. and Köller, W. 2003. 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A new species of Curvularia from Japan. Mycotaxon 91:49-54. Chung, W. H., Ishii, H., Nishimura, K. and Fukaya, Y. 2006. Fungicide sensitivity and phylogenetic relationship of anthracnose fungi isolated from various fruit crop in Japan. Plant Disease 90:506-512. Davidse, L. C. 1986. Benzimidazole fungicides:Mechanism of action and biological impact. Annual Review of Phytopathology 24:43-65. Erichsen, E. 1999. Problems in mildew control in northern Germany. Getreide 1:44-46. Farman, M. L. 2001. The molecular basis of field resistannce to QoI fungicides in Pyricularia grisea. Phytopathology 91:110. (Abstract) Farungsang, U. and Farungsang, N. 1992. Benomyl resistance of Colletotrichum spp. associated with rambutan and mango fruit rot in Tailand. Acta Horticulturae 2:891-897. Fitzell, R. D. 1981. Effect of regular applications of benomyl on the population of Colletotrichum in mango leaves. Transactions of the British Mycological Society 77:529-533. Grasso, V., Sierotzki, H., Garibaldi, A., Gisi, U. 2006. Characterization of cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides. Pesticide Biochemistry and Physiology 84:72-82. Hnatova, M., Gbelska, Y., Obernauerova, M., Subikova, V. and Subik, J. 2003.Cross-Resistance to strobilurin fungicides in mitochondrial and nuclear mutants of Saccharomyces cerevisiae. Folia Microbiologica 48:496-500. Inada, M., Ishii, H., Chung, W. H., Yamada, T., Yamaguchi, J. and Furuda, A. 2008.Occurrence of strobilurin-resistant strains of Colletotrichum gloeosporioides (Glomerella cingulata) the causal fungus of strawberry anthracnose. Japan Journal of Phytopathology 74:114-117. Ishii, H., B. A., Fraaije, T., Sugiyama, K., Noguchi, K., Nishimura, T., Takeda, T., Amano and Hollomon, D. W. 2001. Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. Phytopathology 91:1166-1171. Koenraadt, H., Somerville, S. C. and Jones, A. L. 1992. Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalisand other plant pathogenic fungi. Phytopathology 82:1348-1354. Kraiczy, P., Haase, U., Gencic, S., Flindt, S., Anke, T., Brandt, U. and Von jagow, G.1996. The molecular basis for the natural resistance of the cytochrome bc,complex from strobilurin-producing basidiomycetes to center QP inhibitors.European Journal of Biochemistry 235:54-63. Kuo, K. C. 2001. Sensitivity of mango anthracnose pathogen, Colletotrichum gloeosporioides, to the fungicide Prochloraz in Taiwan. Proceedings of the National Science Council 25:174-179. McKay, G. J., Egan, D., Morris, E. and Brown A. E. 1998. Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR based method. Mycological Research 102:671-676. Mills, P. R., Sreenivasaprasad, S. and Brown, A. E. 1992. Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR. FEMS Microbiology Letters 98:137-144. Nakaune, R. and Nakano, M. 2007. Benomyl resistance of Colletotrichum acutatum is caused by enhanced expression of -tubulin1 gene regulated by putative leucine 30 zipper protein CaBEN1. Fungal Genetics and Biology 44:1324-1335. Olaya, G.., Cleere, S., Stanger, C., Burbidge, J., Hall, A. and Windass, J. 2003. A novel potential target site QoI fungicide resistance mechanism in Pythium aphanidermatum. Phytopathology 93:67. (Abstract) Pasche, J. S., Wharam, C. M. and Gudmestad, N. C. 2004. Shift in sensitivity of Alternaria solani in response to QoI fungicides. Plant Disease 88:181-187. Peres, N. A. R., Souza, N. L., Peever, T. L. and Timmer, L. W. 2004. Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from citrus. Plant Disease 88:125-130. Russell, P. E. 2005. Acentury of fungicide evolution. Journal of Agricultural Science 143:11-25. Sreenivasaprasad, S., Sharda, S. K., Brown, A. E. and Mills, P. R. 1996. PCR-based detection of Colletotrichum acutatum on strawberry. Plant Pathology 45:650-655. Tamura, H., Mizutani, A., Yukioka, H., Miki, N., Ohba, K. and Masuko, M. 1999.Effect of the methoxyiminoacetamide fungicide, SSF129, on respiratory activity in Botrytis cinerea. Pesticide Science 55:681-686. Vanlerberghe, G. C. and McIntosh, L.1997. Alternative oxidase: from gene to function. Annual Review of Plant Physiology and Plant Molecular Biology 48:703-734. Yarden, O. and Katan, T. 1993. Mutations leading to substitutions at amino acids 198 and 200 of beta-tubulin that correlate with benomyl-resistance phenotypes of field strains of Botrytis cinerea. Phytopathology 83:1478-1483. Zheng, D., Olaya, G. and Köller, W. 2000. Characterization of laboratory mutants of Venturia inaequalis resistant to the strobilurin-related fungicide kresoximmethyl.Current Genetics 38:148-155.
Colletotrichum 屬真菌可引起多種作物之炭疽病,其中亦包含草莓及芒果,除影響水果商品價值外,亦是縮短櫥架壽命的主要原因。本研究自台灣中南部草莓及芒果的主要產區,由罹病葉片及果實上分離到炭疽病菌共 105 株,經形態學與分子鑑定後得知,其中 3 株菌株屬於 C. acutautm,其餘 102 株均屬於 C. gloeosporioides。另以 Strobilurin (QoI) 類殺菌劑中的克收欣 (Kresoxim- methyl)、亞托敏 (Azoxystrobin)、三氟敏 (Trifloxystrobin) 及百克敏 (Pyraclostrobin) 對所有菌株進行感受性測試,結果顯示百克敏於 10 ppm 時即能有效抑制芒果及草莓炭疽病菌之菌絲生長,而在 500 ppm 有效濃度下,克收欣對供試菌株之平均抑制率為 45.0%,亞托敏與三氟敏則分別為 44.6% 與 46.1%。前人研究指出,cytochrome b 基因中第 143 密碼子處發生點突變為主要控制 QoIs 抗藥性產生的機制,然本研究對台灣芒果及草莓炭疽病菌菌株之 cyt b 基因片段進行分析後發現第 143 密碼子位置並無突變現象產生,顯示 143 位置並非影響台灣芒果及草莓炭疽病菌對 QoIs 產生抗性的主要原因。此外,於含有 100 ppm 亞托敏原體之 PDA 培養基中添加 SHAM (Salicyhydroxamic acid) 抑制 alternative oxidase 之活性,亦無法有效提升亞托敏的殺菌效果,顯示 alternative pathway respiration 並非引起抗藥性產生的機制。另分析 cyt b 基因結果中顯示,分離自芒果、草莓、柑橘、山藥及蕃薯等作物之 C. gloeosporioides 菌株,皆在第 144 與 145 密碼子位置間出現了核苷酸缺失 (deletion) 的現象,造成第 144 密碼子後的胺基酸發生改變,推測此現象可能為影響菌株抗性之原因。本研究選取來自不同地區的 30 株芒果及草莓炭疽病菌進行苯丙咪唑系 (Benzimidazoles) 藥劑感受性測試,並觀察 QoIs 與 Benzimidazoles 藥劑間是否具有交互抗性 (cross-resistance) 之現象。結果顯示,有 12 株供試菌株對免賴得 (benomyl)、腐絕 (Thiabendazole) 及貝芬替 (Carbendazim) 具有抗藥性,其餘 18 株菌株則在 1ppm 有效濃度處理下即明顯受藥劑所抑制,得知目前台灣芒果與草莓炭疽病菌之抗藥性菌株與感受性菌株同時存在於田間。此外,觀察供試菌株對 QoIs 與 Benzimidazoles 藥劑之抗感性反應,得知台灣芒果與草莓炭疽病菌對此兩類殺菌劑已有交互抗性現象產生。而在菌株抗 Benzimidazole 類藥劑之機制方面,於分析 β-tubulin 基因片段後確定抗藥性菌株在第 198 密碼子位置發生由GAG (Gly) 置換成 GCG (Ala) 的點突變,然未發現菌株在第200密碼子的位置發生改變,顯示台灣芒果炭疽病菌對苯丙咪唑類藥劑之抗藥性產生導因於β-tubulin 基因上第 198 密碼子的改變。為能快速診斷出抗 Benzimidazole 類藥劑的炭疽病菌,本研究針對β-tubulin 基因設計專一性引子對 TubGF1 與 TubGR,並利用 PCR-RFLP 方法對 50 株供試菌株進行分子檢測,並配合觀察其對 Benzimidazoles 藥劑的抗感性反應,結果顯示所設計的專一性引子對配合 PCR-RFLP 可正確診斷田間芒果及草莓的炭疽病菌株對Benzimidazoles 藥劑是否具抗藥性。

Anthracnose caused by Colletotrichum spp., including mango and strawberry, is an important crops disease throughout the world. Anthracnose is one of factor to reduce economic worth and time of post-harvest of fruits. A total of 105 Colletotrichum isolates obtained from infected fruits and leaves of strawberry and mango in main production area. Based on morphological and molecular characteristics, three C. acutatum and 102 C. gloeosporioides isolates were identified. For compaŠnng sensitivities to strobilurins (QoIs), all C. gloeosporioides isolates were examined sensitivities to four QoIs, kresoxim- methyl, azoxystrobin, trifloxystrobin and pyraclostrobin at 1, 10, 100 and 500 ppm (a.i.) by mycelial growth. Pyraclostrobin was highly efficacy to inhibit mycelial growth of all C. gloeosporioides isolates at 10 ppm (a.i.). Contrary to pyraclostrobin, kresoxim- methyl, azoxystrobin and trifloxystrobin only showed 45.0%, 44.6% and 46.1% mycelial growth inhibition rate at 500 ppm (a.i.), respectively. Previous studies indicated that the mutation at codon 143 in cytochrome b gene (cyt b) is the principal action of QoIs-resistant fungal pathogen. However, the QoIs-resistant isolates of C. gloeosporioides obtained from mango and strawberry did not be found the mutation at codon 143 of cyt b. The result indicated that the mutation at codon 143 of cyt b is not available character to increase the resistance of C. gloeosporioides isolates from mango and strawberry to QoIs in Taiwan. Moreover, the azoxystrobin did not raise the inhibiting efficacy of C. gloeosporioides isolates at salicylhydroxamic acid (SHAM) added PDA medium. Thus, alternative pathway respiration did not play an important role for C. gloeosporioides isolates enhanced resistance to QoIs. Interestingly, the nucleotide deletion of cyt b between codon 144 and 145 was detected among C. gloeosporioides isolates from mango, strawberry, orange, yam and sweet potato. The result suggests that the nucleotide deletion may change the protein structure of cyt b and enhance the resistance of C. gloeosporioides isolates to QoIs in Taiwan. In this study, thirty C. gloeosporioides isolates obtained from mango and strawberry were tested the sensitivities to benzimidazole fungicides, benomyl, thiabendazole and carbendazim. Results showed that mycelial growth of 18 C. gloeosporioides isolates were significantly inhibited at 1 ppm (a.i.) and 12 C. gloeosporioides isolates were resistant to those three benzimidazoles. Thus, the benzimidazoles-sensitive and -resistant C. gloeosporioides isolates were simultaneously existed in the field at Taiwan. Moreover, the positive cross-resistance among benzimidazoles on C. gloeosporioides isolates was existence, and the negative cross-resistance between benzimidazoles and QoIs were detected on some C. gloeosporioides isolates. For the benzimidazoles-resistant mechanism analysis, the resistant C. gloeosporioides isolates showed point mutation at codon 198 in β-tubulin gene and the amino acid was substituted glycine (Gly) to alanine (Ala). While, the codon 200, caused the moderately resistant to benzimidazoles, did not be detected among all C. gloeosporioides isolates. The results suggest that the mutation at codon 198 in β-tubulin gene is major mechanism of benzimidazoles-resistant C. gloeosporioides isolates in Taiwan. Consequently, the specific primers of TubGF1 and TubGR were designed to amplify partial β-tubulin gene, including codon 198, and used PCR-RFLP method to detect the benzimidazoles -resistant C. gloeosporioides isolates in field. According to the result, this method could be rapidly and precisely to diagnose benzimidazoles-resistant C. gloeosporioides isolates obtained from mango and strawberry in Taiwan.
其他識別: U0005-0407200817303200
Appears in Collections:植物病理學系

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