Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31336
標題: 產氣細菌防治柳橙綠黴病的效果與其抑菌氣體成分的分析
Control of Orange Green Mold with Gas-Producing Bacteria and Analysis of Their Major Gas Components Suppressing the Pathogen
作者: 陳泊菘
Chen, Po-Sung
關鍵字: biocontrol;生物防治;dimethyl disulfide;gas-producing bacteria;orange;orange green mold;perlite;柳橙綠黴病;柳橙;產氣細菌;珍珠石;二甲基二硫醚
出版社: 植物病理學系所
引用: 丁姵分. 2006. 番茄萎凋病之生物防治菌的鑑定與防病潛力評估. 國立中興大學植物病理學系碩士論文. 51 pp. 石信德. 2003. 鏈黴菌PMS-702防治作物病害的功效與其抑菌主要代謝物治黴色基素之鑑定. 國立中興大學植物病理學系博士論文. 151 pp. 行政院農業委員會. 2007. 民國96年農業統計年報. 行政院農委會出版. 339pp. 台北. 呂明雄. 2002. 第二章:品種及其特性. p.11-42. 柑桔整合管理. 楊秀珠彙編. 行政院農委會農業藥物毒物試驗所編印. 台中. 178 pp. 呂明雄. 2007. 優質柑桔果園作業管理. p.11-28. 安全高品質柑桔生產手冊. 國立中興大學農業暨目然資源學院農業推廣中心編印. 台中. 113 pp. 柯勇. 2008. 臺灣經濟果樹病害彩色圖鑑. 藝軒圖書出版社. 台北. 431 pp. 孫守恭. 2000. 臺灣果樹病害. 世維出版社. 台中. 429 pp. 麥富德、江妮蓁、楊筱蕙、鍾文梅. 2006.質子轉移反應質譜儀的原理與應用. 科儀新知 27: 69-77. 費雯綺、王玉美. 2002. 植物保護手冊(果樹篇). 行政院農業委員會農業藥物毒物試驗所. 台中. 255 pp. 黃靜淑. 2008. Bacillus mycoides防治甘藍幼苗病害之效果評估. 國立中興大學植物病理學系碩士論文. 58 pp. 楊秀珠. 2002. 第十五章:貯藏病害之發生與防治. p.155-168. 柑桔整合管理. 楊秀珠彙編. 行政院農委會農業藥物毒物試驗所. 台中. 178 pp. 楊維哲. 2007. 利用產氣細菌防治萵苣猝倒病. 國立中興大學植物病理學系碩士論文. 76 pp. 廖龍盛. 2005. 實用農藥 (全新修訂第八版). 得力興業股份有限公司. 1311 pp. 謝慶昌. 2007. 柑橘採收後處理及檢疫. p.96-100. 安全高品質柑桔生產手冊. 國立中興大學農業暨自然資源學院農業推廣中心. 台中. 113 pp. 羅幹成、安寶貞、蔣慕琰. 2002. 壹、前言. p.2-8. 柑橘保護(上冊). 行政院農業委員會動植物防疫檢疫局出版. 台中. 174 pp. Agnioni, A., Cabras, P., Dhallewin, G., Pirisi, F. M., Reniero, F., and Schirra, M. 1998. Synthesis and inhibitory activity of 7-geranoxy coumarin against Penicillium species in citrus fruits. Phytochemistry 47: 1521-1525. Agrios, G. N. 2005. Plant Pathology. 5th ed. Academic Press. U.S.A. 922 pp. Archbold, D. D., Hamilton-Kemp, T. R., Clements, A. M., and Collins, R. W. 1999. Fumigating “Crimson Seedless” table grapes with (E)-2-hexenal reduces mold during long-term postharvest storage. HortScience. 34: 705-707. Bower, J. P., and Dennison, M.T. 2003. An integrated approach to postharvest disease management in citrus. Acta Hort. 628: 715-720. D’Aquino, S., Schirra, M., Palma, A., Angioni, A., Cabras, P., and Migheli, Q. 2006. Residue level and effectiveness of pyrimethanil vs imazalil when using heated postharvest treatments for control of Penicillium decay on citrus fruit. J. Agric. Food Chem. 54: 4721-4726. D’hallewin, G., Arras, G., Castia, T., and Piga, A. 1993. Reducing decay of “Avena” mandarin fruit by the use of UV, heat and thiabendazole treatments. Acta Hort. 368: 387-394. Droby, S., Porat, R., Cohen, L., Weiss, B., Shapira, B., Philosoph-Hadas, S., and Meir, S. 1999. Suppressing green mold decay in grape fruit with postharvest jasmonates application. J. Am. Soc. Hort. Sci. 124: 184-188. Eckert, J. W., and Eaks, I. L. 1989. Postharvest disorders and diseases of citrus fruits. Pages 179-260 in: The Citrus Industry, vol. 4. Reuther, W., Calavan, E. C., and Carman, G. E. eds. University of California Press, Berkeley, U.S.A. Ezra, D., Japer, J., Rogers, T., Knihton, B., Grimsrud, E., and Strobel, G. 2004. Proton transfer-mass spectrometry as a technique to measure volatile emissions of Muscodor albus. Plant Sci. 166: 1471-1477. Federal Register. 2004. Muscodor albus strain QST 20799: notice of filing a pesticide petition to establish an exemption from tolerance for a certain pesticide in or on food. Fed. Reg. 69: 1-16. Fernando, W. G. D., Ramarathnam, R., Krishnamoorthy, A. S., and Savchuk, S. C. 2005. Identification and use of potential bacterial organic antifungal volatiles in biocontrol. Soil Biol. Biochem. 37: 955-964. Hamilton-Kemp, K. J., Diepenhorst, P., Bakker, W., and Gorris, L. G. M. 1995. Effect of some natural volatile compounds on the pathogenic fungi Alternaria alternata and Botrytis cinerea. J. Chem. Ecol. 18: 1083-1091. Holmes, G. J., and Eckert, J. W. 1999.Sensitivity of Penicillium digitatum and P. italicum to postharvest citrus fungicides in California. Phytopathology 89: 716-721. Janisiewicz, W. J., and Korsten, L. 2002. Biological control of postharvest diseases of fruits. Annu. Rev. Phytopathol. 40: 411-441. Kanetis, L., Förster, H., and Adaskaveg, J. E. 2008. Optimizing efficacy of new postharvest fungicides and evaluation of sanitizing agents for managing citrius green mold. Plant Dis. 92: 261-269. Leelasuphakul, W., Hemmanee, P., and Chuenchitt, S. 2008. Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen (Penicilium digitatum Sacc.) of citrus fruit. Postharv. Biol. Tech. 48: 113-121. Liu, W. T., Chu, C. L., and Zhou, T. 2002. Thymol and acetic acid vapors reduce post harvest brown rot of apricot and plums. HortScience. 37: 151-156. Mari, M., Bertoii, P., and Prateiia, G. C. 2003. Non-conventional methods for the control of post harvest pear diseases. J. Appl. Microbiol. 94: 761-766. Mari, M., Leoni, O., Bernardi, R., Neri, F. and Palmieri, S. 2008. Control of brown rot on stonefruit by synthetic and glucosinolate-derived isothiocyanates. Postharv. Biol. Tech. 47:61-67. Mari, M., Leoni, O., Lori, R., and Cembali, T. 2002. Antifungal vapour-phase activity of allyl isothiocyanate against Penicillium expansum on pears. Plant Pathol. 51: 231-236. Mercier, J., and Jiménez, J. I. 2004. Control of fungal decay of apples and peaches by the biofumigant fungus Muscodor albus. Postharv. Biol. Tech. 42: 121-123. Mercier, J., and Smilanick, J.L. 2005. Control of green mold and sour rot of stored lemon by biofumigation with Muscodor albus. Biol. Control 32:401-407. Mollet, C., Drancourt, M., and Raoult, D. 1997. rpoB sequence analysis as a novel basis for bacterial identification. Mol. Microbiol. 26(5):1005-1011. Munoz, A., Lopez-Garcia, B., and Marcos, J. F. 2007. Comparative study of antimicrobial peptides to control citrus postharvest decay caused by Penicillium digitatum. J. Agric. Food Chem. 55: 8170-8176. Neri, F., Mari, M., Brigati, S., and Paolo, B. 2007. Fungicidal activity of plant volatile compounds for controlling Monilinia laxa in stone fruit. Plant Dis. 91: 30-35. Porat, R., Pavoncello, D., Peretz, J., Weiss, B., Daus, A., Cohen, L., Ben-Yehoshua, S., Fallik, E., Droby, S., and Lurie, S. 2000. Induction of resistance to Penicillium digitatum and chilling injury in “Star Ruby” grapefruit by a short hot water rinse and brushing. J. Hort. Sci. Biotechnol. 75:428-432. Ragsdale, N. N., and Sisler, H. D. 1994. Social and political implications of managing plant diseases with decreased availability of fungicides in the United States. Annu. Rev. Phytopahol. 32: 545-557. Ramin, A. A., Braun, P. G., Prange, R. K., and Delong, J. M. 2005. In vitro effects of Muscodor albus and three volatile components on growth of selected postharvest microorganisms. HortScience 40: 2109-2114. Ryan, F. J., Leesch, J. G., Palmquist, D. E., and Aung, L. H. 2007. Glutathione concentration and phytotoxicity after fumigation of lemons with methyl iodide. Postharv. Biol. Tech. 45: 141-146. Schneider, S. M., Rosskpf, E. N., Leesch, J. G., Chellemi, D. O., Bull, C. T., and Mazzola, M. 2003. United States Department of Agriculture- Agricultural Research Service research on alternatives to methyl bromide: pre-plant and post-harvest. Pest Manag. Sci. 59: 814-826. Sholberg, P. L., and Gaunce, A. P. 1995. Fumigation of fruit with acetic acid to prevent post harvest decay. HortScience 30: 1271-1275. Sholberg, P. L., Reynolds, A. G., and Gaunce, A. P. 1996. Fumigation of table grapes with acetic acid to prevent post harvest decay. Plant Dis. 80: 1425-1428. Smilanick, J. L., Mackey, B. E., Reese, R., Usall, J., and Margosa, D. A. 1997. Influence of soda ash, temperature, and immersion period on the control of postharvest green mold of oranges. Plant Dis. 81: 379-382. Smilanick, J. L., Mansour, M. F., and Sorenson, D. 2005a. Pre- and postharvest treatments to control green mold of citrus during ethylene degreening. Plant Dis. 90: 89-96. Smilanick, J. L., Mansour, M. F., Margosan, D. A., Mlikota Gabler, F., and Goodwine, W. R. 2005b. Influence of pH and NaHCO3 on effectiveness of Penicillium digitatum and to control postharvest green mold on citrus fruit. Plant Dis. 89: 640-648. Smilanick, J. L., Margosan, D. A., Mlikota, F., Usall, J., and Michale, I. F. 1999. Control of citrus green mold by carbonate and bicarbonate salts and the influence of commercial postharvest practices on their efficacy. Plant Dis. 83: 139-145. Spotts, R. A., and Cervantes, L. A. 1992. Effect of ozonated water on postharvest pathogens of pear in laboratory and packing house tests. Plant Dis. 76: 256-259. Spotts, R. A., and Peters, B. B. 1980. Chlorine and chlorine dioxide for control of d’Anjou pear decay. Plant Dis. 64: 1095-1097. Strobel, G. 2006. Muscodor albus and its biological promise. J. Ind. Microbiol. Biotechnol. 33: 514-522. Strobel, G. A., Dirkse, E., Sears, J., and Markworth, C. 2001. Volatile antimicrobials from Muscodor albus, a novel endophytic fungus. Microbiology 147: 2943-2950. Tripathi, P., and Dubey, N. K. 2004. Exploitation of natural products as an alternative strategy to control postharvest fungal rotting of fruit and vegetables. Postharv. Biol. Tech. 32: 235-245. Tsao, R., and Zhou, T. 2000. Antifungal activity of monoterpenoids against postharvest pathogens Botrytis cinerea and Monilinia fructicola. J. Essential Oil Res. 12: 113-121. Utama, I. M. S., Wills, R. B. H., Ben-Ye-Hoshua, S., and Kuek, C. 2002. In vitro efficacy of plant volatiles for inhibiting the growth of fruit and vegetable decay microorganisms. J. Agric. Food Chem. 50: 6371-6377. Whangchai, K., Saengnil, K., and Uthaibutra, J. 2005. Control of postharvest diseases in longan fruit by ozone. Acta Hort. 682: 2121-2126. White, T.J., Bruns, T.D., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Innis, M. A., and Gelfand, D. H. eds. Academic Press, London. Wilson, C. L., and Wisniewski, M. E. 1989. Biological control of postharvest diseases of fruits and vegetables: an emerging technology. Annu. Rev. Phytopathol. 27: 425-441. Woropong, J., Strobel, G. A., Ford, E. J., Li, J. Y., Baird G., and Hess, W. M. 2001. Muscodor albus anam. nov. an endophyte from Cinnamomum zeylanicum. Mycotaxon 79: 67-79.
摘要: 
Penicillium digitatum引起的柳橙綠黴病,是柳橙於貯藏期間損失的主要原因之一。過去常用於防治貯藏病害的化學藥劑已出現多重抗藥性菌株導致逐漸失去效用;至於常用於燻蒸消毒的溴化甲烷 (Methyl bromide) 也由於聯合國蒙特婁議定書之決議而將遭禁用,是故尋找替代的防治策略成為刻不容緩的課題。本研究主要的目的在於探討Enterobacter cloacae E006、E010與Bacillus mycoides CHT2401、CHT2402菌株所產生之氣體的抑菌效果,進而評估它們於密閉貯藏環境下產氣防治柳橙綠黴病害的效果。首先將E. cloacae E006、E010與B. mycoides CHT2401、CHT2402菌株分別培養於King’s medium B (KB)與 Tryptic soy agar (TSA) 平板上,利用兩培養皿對扣法測試其所產生之氣體對Penicillium digitatum P51與DOB-2孢子發芽及菌絲生長的影響,結果發現E010培養於KB與CHT2401培養於TSA上所產生的氣體對於病原菌之分生孢子發芽及菌絲生長皆具有較優異的抑制效果。探討溫度與酸鹼值對E010與CHT2401產氣抑制分生孢子發芽的能力的影響,結果顯示在24℃,P51與DOB-2的分生孢子發芽率最佳,而E010與CHT2401於該溫度產氣抑制P51與DOB-2分生孢子發芽之效果也是最佳; 至於E010培養於酸鹼值6的KB及CHT2401培養於酸鹼值為7-8的TSA時,產生氣體抑制P51與DOB-2的孢子發芽的效果最好。將E. cloacae E010與B. mycoides CHT2401菌株分別培養於混拌有KB或Tryptic soy broth (TSB) 的基質上,置於盛有接種過P. digitatum P51之柳橙果實的密封塑膠盒內進行小型燻蒸室模擬試驗法,結果顯示珍珠石混拌KB與E010或TSB與CHT2401所形成之製劑 (E010-KB-Perlite formula, EKP; CHT2401-TSB-Perlite formula, CTP) 皆可以產氣防治柳橙綠黴病的發生,其罹病度分別為0%及18.9%。使用150mL之調整培養基成份濃度至2.5倍的EKP或CPT之產氣防治柳橙綠黴病的效果最佳;結果發現EKP的培養基成份濃度提高到5倍,才能有效延長E010產氣抑制病害的時間,至於CHT2401則不需提高CPT內培養基成份濃度即可長時間產氣抑制柳橙綠黴病害的發生。利用產氣細菌製劑所產生之氣體與腐絕及免賴得等化學藥劑比較防治柳橙綠黴病的效果,發現E010與CHT2401產氣防治病害發生的效果遠優於化學藥劑。模擬不同貯藏溫度利用產氣細菌製劑所產生之氣體防治柳橙綠黴病害的效果中,只有E. cloacae E010能夠在低溫之下仍然保持良好的產氣防治病害發生的能力,罹病度皆在15%以下。進一步以氣相層析質譜儀分析E. cloacae E006與E010培養於KB及混拌有KB的珍珠石製劑所釋放之氣體,分別是苯乙醇 (phenylethyl alcohol)、乙酸丁酯 (butyl actate)及4,5-二甲基-1-己烯 (4,5-dimethyl-1-hexene),其中4,5-二甲基-1-己烯可能是抑菌的主要氣體,惟目前無法取得4,5-二甲基-1-己烯之標準品;B. mycoides CHT2401與CHT2402可釋放二甲基二硫醚 (dimethyl disulfide)。因此取標準品測試,發現二甲基二硫醚具有燻蒸抑制P. digitatum P51與DOB-2分生孢子發芽及菌絲生長的功效;進一步使用二甲基二硫醚標準品燻蒸接種過P51分生孢子之柳橙,證實其具有良好防治病害發生的效果,並可使柳橙果實罹病度近於0%。

Orange green mold incited by Penicillium digitatum causes a great part of postharvest losses on orange fruits. Fungicides are the primary means of controlling postharvest diseases. However, multiple drug resistant strains of postharvest pathogens have occurred due to frequently use of traditional chemical treatments and limited their effectiveness. In addition, methyl bromide, a fumigant that commonly used in post-harvest management of pests and pathogens on fresh products and durable commodities, has been suggested to be banned by the Montreal Protocol. Thus, finding an alternative way in safe means to humans to displace methyl bromide in postharvest management might be needed. The main purpose of this research is to evaluate a useful biocontrol formulation for producing gas by four strains of gas-producing bacteria, Enterobacter cloacae isolates E006、E010 and Bacillus mycoides isolates CHT2401、CHT2401, to control orange green mold. In vitro tests, E. cloacae E006 and E010 grown on plates of King’s medium B (KB) and B. mycoides CHT2401 and CHT2401 grown on plates of Tryptic soy agar (TSA) were conducted for suppressing conidial germination and mycelial growth of Penicillium digitatum P51 and DOB-2. E. cloacae E010 and B. mycoides CHT2401 expressed better effectiveness in inhibiting the two pathogens. Temperature at 24℃ was optimal for E010 and CHT2401 releasing gas to inhibit conidial germination of P51 and DOB-2 compared to the control. KB at pH6 for culturing E. cloacae E010 and TSA at pH7-8 for culturing B. mycoides CHT2401 were the most optimal for two bacteria to release gas for inhibiting conidial germination of P51 and DOB-2. Gas released from perlite mixed with KB or TSB ingredients culturing E. cloacae E010 or B. mycoides CHT2401 (E010-KB-Perlite formula, EKP; CHT2401-TSB-Perlite formula, CTP) was effective in reducing disease severity of orange green mold caused by P. digitatum P51 from 80% to 0% or from 90.0% to 18.9%, respectively. The amount of 150mL EKP or CTP which added 2.5 times amount of original KB or TSB ingredients was the best substrate for culturing E010 and CHT2401 to produce gas for controlling orange green mold. After the fruits were treated with the EKP or CTP for 7 days, development of disease lesions on P. digitatum P51-inoculated orange fruits was much slower compared to the other treatment. Adding 5 times amount of KB ingredients to EKP could prolong the gas-releasing period of the bacterium to control orange green mold for one month. Comparison of gas-producing bacteria incubated in EKP or CTP and chemical treatments (thiabendazole and benomyl) showed that gas-producing bacteria incubated in EKP or CTP was much more effective in controlling orange green mold than chemical treatments. Only E. cloacae E010 incubated in EKP could successfully inhibit disease development under low temperatures (8-16℃). The volatile organic compounds (VOCs) produced by E. cloacae E006 and E010 on KB plate or KB-mixed perlite formula were identified by Gas chromatography- mass spectrometry. The major VOCs produced by E006 and E010 were identified as phenylethyl alcohol、butyl actate and 4,5-dimethyl-1-hexene. In our studies, 4,5-dimethyl-1-hexene was supposed to be a major compound for inhibiting conidial germination of P. digitatum P51 by E006 and E010. The major VOCs produced by B. mycoides CHT2401 and CHT2401 was identified as dimethyl disulfide. Dimethyl disulfide was able to significantly suppress P. digitatum P51 conidial germination and mycelial growth in vitro and completely control orange green mold caused by P. digitatum P51 in vivo tests.
URI: http://hdl.handle.net/11455/31336
其他識別: U0005-2008200921060600
Appears in Collections:植物病理學系

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