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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
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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.
其他識別: U0005-2008200921060600
Appears in Collections:植物病理學系

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