番石榴果實黑腐病病原鑑定、生理特性及防治之探討

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論文名稱: 番石榴果實黑腐病病原鑑定、生理特性及防治之探討
研究生姓名: 蔡篤鑫
指導教授姓名: 王惠亮;陳瑞祥
出版年: 2003
學校名稱: 國立高雄師範大學
系所名稱: 生物科學研究所
關鍵字: 番石榴;果實黑腐病;ITS核酸序列;生物殺菌劑;木黴菌;化學藥劑;Botryodiplodia theobromae Pat.;rDN Botryodiplodia fruit rot A ITS;Botryodiplodia fruit rot;commercial fungicides;biological control agents
摘要: 番石榴果實黑腐病為台灣夏季高溫時嚴重病害之一,果實初期產生淡褐色圓形小病斑,之後迅速擴大,病斑中間並有許多黑色點狀的柄子殼產生,外圍則為水浸狀。依照柯霍氏法則 (Koch’s postulates)系列測試,病原菌經鑑定為Botryodiplodia theobromae Pat.,病原菌培養於PDA上,菌落初期為白色,有許多氣生菌絲,而後轉為暗灰色,而柄孢子形狀初期為圓形至橢圓形,無色單室,成熟後轉為橢圓形,暗褐色,雙室,壁變厚,其大小為20~29 μm × 10~14 μm。菌絲生長以30 ℃時生長速率最快,柄子殼的產生範圍為15~25 ℃,而以20 ℃為最多。病原菌利用碳素源以果糖、蔗糖、與葡萄糖為最好,而氮素源則以硝酸銨及硫酸銨較佳,水份潛勢以 -4.6 ~ -9.1 bars時生長最快速,病原菌在pH值 4~10的範圍內菌絲均能良好生長,但以酸性較佳,光照會促進菌絲生長,而要產孢更需要在有光照的條件。測試培養基對菌絲生長以培養基maltose extract agar、 potato dextrose agar(PDA)、potato sucrose agar(PSA)為最適生長,孢子發芽的數目以25 ℃~30 ℃為最高,經6小時後病原菌的孢子發芽率可達90 ﹪以上。而探討菌株之間的遺傳歧異程度,由ITS核酸序列與GenBank資料庫進行比對分析,結果顯示ITS核酸序列在同屬菌株間有很高的相同度可達97 ﹪以上,顯示將來可利用rDNA ITS區間序列作為檢測鑑定病原菌的輔助依據。室內測試化學藥劑對B. theobromae菌絲生長之影響,結果發現62.5 ﹪(Fludioxonil + Cyprodinil)賽普護汰寧混合水份散性粒劑、37 ﹪(Flusilazol)護矽得乳劑、25.9 ﹪(Tebuconazole)得克利乳劑、23.7 ﹪(Iprodione)依普同水懸劑、56 ﹪(Dithianon-MBC)貝芬硫酉昆可濕性粉劑、25 ﹪(Prochloraz)撲克拉乳劑對病原菌的菌絲生長表現較佳的抑制效果。至於23 ﹪(Azoxystrobin)亞托敏水懸劑,對病原菌的菌絲生長抑菌效果不佳,而在生物防治試驗中,以玻璃紙抗生法測定拮抗菌對引起番石榴果實黑腐病的B. theobromae之拮抗能力,可發現木黴菌菌株FT-010、FT-023及放線菌SS31對菌絲生長的抑制皆有良好的效果,而市售之兩種生物殺菌劑枯草桿菌 Bacillus subtilis Y1336及放線菌 Streptomyces candidus Y21007-2,試驗結果顯示Y1336在稀釋倍數100倍及1000倍時,對病原菌的菌絲生長抑制可分別達到90 ﹪及80 ﹪以上,而Y21007-2於稀釋100倍時,對病原菌的菌絲生長抑制也可達80 ﹪以上。將藥劑依其建議施用濃度稀釋23.7 %依普同水懸劑1000倍、56 ﹪貝芬硫酉昆可濕性粉劑1000倍、62.5 %賽普護汰寧混合水份散性粒劑1500倍、37 ﹪護矽得乳劑10000倍、安心寶600倍、台灣寶800倍直接噴灑於病果,顯示病果經藥劑處理後,均有抑制病斑及產孢的效果,其中化學藥劑以依普同及護矽得的防治效果較佳,而在市售生物殺菌劑中,則以安心寶的效果較佳。 The Botryodiplodia fruit rot of guava fruit is one of the most serious diseases in summer season in Taiwan. The symptoms begin with a small brown pit, and then enlarge to a circle-like spot with pycnidia within the fruit wounds, and circular water-soaked spots on fruit. According to the morphological characteristics of the pathogen, the causal agent was identified as Botryodiplodia theobromae Pat.. The colonies of B. theobromae cultured on PDA plates were white and gradually turned to gray, fluffily with abundant aerial mycelium. Pycnidiospores were initially unicellular hyaline, oblong or elliptical and maturely pycnidiospores were brown with two-cell, and 20-29 μm × 10-14μm size. The optimal temperature for mycelial growth was 30 ℃. The temperature for germination of pycnidia was 15-25 ℃ with the optimum at 20 ℃. Fructose, sucrose and dextrose of carbon sources and ammonium nitrate and ammonium sulfate of nitrogen sources were suitable for mycelial growth. The optimal water potential was between -4.6 to -9.1 bars. B. theobromae could grow at a pH value ranging from 4 to 10. However, they grew better under an acidic condition. The mycelial and sporulation growths of B. theobromae were stimulated when cultured under light. Of the various media tested, the best for mycelial growth were potato dextrose agar, maltose extract agar and potato sucrose agar. The optimal temperature for pycnidiospores germination was 25-30 ℃. Comparison of the identity of rDNA ITS-full gene sequence between B. theobromae isolate names and genus of Botryodiplodia in NCBI reached 97 %. Nine commercial fungicides were evaluated on PDA for their abilities to inhibit mycelial growth of the pathogen. Except 23 % Azoxystrobin SC, 62.5 % Fludioxonil + Cyprodinil WG, 37 % Flusilazol EC, 25.9 % Tebuconazole EC, 23.7 % Iprodione SC, 56 % Dithianon-MBC WP and 25 % Prochloraz EC were highly effective against this fungus. Among biological control agents, two isolates of Trichoderma (FT-010, FT-023) and one isolate of Streptomyces (SS31) showed strongly antagonistic to the mycelial growth of B. theobromae. The result of two biological agents already available in market Bacillus subtilis Y1336 (Bio-aid) and Streptomyces candidus Y21007-2 (Bio-bac) showed that when Y1336 were diluted to 100, 1000 times, the percentage of inhibition were over 90 % and 80 %, respectively, and Y21007-2 was diluted to 100 times, the percentage of inhibition were 80 %. After spraying the diluted chemical and biological agents on the infected fruits, 23.7 % Iprodione SC at 1:1000 dilution, 56 % Dithianon-MBC WP at 1:1000 dilution, 62.5 % Fludioxonil + Cyprodinil WG at 1:1500 dilution, 37 % Flusilazol EC at 1:10000 dilution, S. candidus Y21007-2 at 1:600 dilution, and B. subtilis Y1336 at 1:800 dilution proved to be effective. In chemicals fungicides, both Flusilazol and Iprodione showed the best efficacy, and Y21007-2 were better in biological control agents.
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