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標題: 萵苣黑斑病菌之鑑定及其微生物防治試驗
Identification for the Causal Agent of Lettuce Black Leaf Spot and Its Microbial Control Experiments
作者: 高郁琇
Kao, Yu-Hsiu
Contributors: 黃振文
植物病理學系所
關鍵字: 萵苣黑斑病;形態鑑定;ITS序列分析;寄主範圍測試;A. lactucae;B. amyloliquefaciens;黃豆粉-糖蜜培養基;花生油;乳化劑;Iturin A;Surfactin
lettuce black leaf spot;morphological characteristics;ITS sequencing analysis;host range test;A. lactucae;B. amyloliquefaciens;Soybean meal-Molasses medium;peanut oil;emulsified agent;Iturin A;Surfactin
日期: 2013
Issue Date: 2013-11-19 12:38:35 (UTC+8)
Publisher: 植物病理學系所
摘要: 西元2011年3月於雲林縣二崙鄉萵苣栽培區,首次發現結球萵苣植株之葉片出現許多大小不一之黑褐色病斑,罹病嚴重時病斑相互融合致使葉片黃化乾枯死亡,進而造成缺株之現象。自栽培區取得之結球萵苣苗盤其罹病率高達98%且缺株率達16%。由罹病株分離獲得之菌株皆歸屬於鏈格孢菌屬 (Genus Alternaria) 真菌,隨後依柯霍氏法則測定病原性,證實分離獲得之26株Alternaria sp.菌株皆可引起萵苣黑斑病,且病徵與田間原罹病株相仿。將Alternaria sp. AL1-2及AL3-11兩菌株分別接種於不同栽培種之萵苣、番茄、馬鈴薯、甜椒、甘藍、白菜、茼蒿、紅莧菜、菠菜、蕹菜、北蔥、胡蘿蔔、胡瓜以及西瓜等作物測試其寄主範圍,結果發現該病原菌僅危害萵苣,於接種7天後即會導致萵苣葉片黃化乾枯進而造成植株死亡;此外,其對於番茄及甘藍則引起疑似過敏性反應。分析Alternaria sp. AL1-2及AL3-11菌株之核醣體去氧核醣核酸之轉錄內區間 (internal transcribed spacer, ITS) 序列,發現兩菌株之序列與A. solani較為相近,將其與財團法人食品工業研究所生物資源保存及研究中心提供之A. solani (BCRC No. 32123) 序列比較,兩者之相似度為99%。該病原菌於蔬菜瓊脂 (V8 juice agar) 平板上之菌落呈黑褐色,具輪紋,產孢梗大多具單一產孢點,少數則具2-4個產孢點,每一產孢點僅產生單一分生孢子,分生孢子大小平均為82-90 × 13 μm,呈長橢圓形或長卵形,具6-10個橫隔膜及0-3個縱隔膜,大部分分生孢子具單一個喙,少部分則具兩個喙,喙 (beak) 屬於基部較寬而尖端纖細 (narrow-taper) 之形式且長達163-176 μm。病原菌菌絲生長、分生孢子發芽及感染萵苣之最適溫度皆為24℃。綜合上述病原菌之形態、生長特徵、ITS序列分析及其對萵苣之致病性,進而與國內外相關文獻比較,該萵苣黑斑病之病原菌係屬於一新發現種,因此命名為Alternaria lactucae Kao & Huang sp. nov.。測試15株拮抗菌株對萵苣黑斑病菌之拮抗效果,結果發現本研究分離獲得之Bacillus amyloliquefaciens BA01菌株對萵苣黑斑病菌之拮抗效果最佳,其於馬鈴薯牛肉煎汁瓊脂 (PNA) 平板上對A. lactucae AL1-2及AL3-11菌株之菌絲生長抑制率分別達57%及56%;將BA01菌株培養於牛肉煎汁培養液 (NB) 中四天,其醱酵液會誘使病原菌之發芽管膨大,進而抑制病原菌分生孢子發芽率達80%以上。由結球萵苣切離葉生物分析法評估B. amyloliquefaciens BA01菌株之防病能力,結果發現BA01菌株可抑制病斑擴展率達89%。本研究利用結球萵苣切離葉評選BA01菌株合適之培養基配方,發現以黃豆粉-糖蜜培養基 (SYM) 培養BA01菌株時,其100倍稀釋醱酵液可抑制病原菌AL1-2及AL3-11菌株危害萵苣之百分率分別為36%與41%;進一步測試SYM培養基添加植物油對BA01菌株防病能力之影響,結果發現將BA01菌株培養於添加2.5% (w/v) 花生油之SYM培養基,其醱酵液之防治成效最佳,200倍稀釋醱酵液對病害之抑制率可達46%以上;此外,於SYM培養基中添加0.125% (w/v) 乳化劑亦可顯著提升BA01菌株之防病能力,其200倍稀釋醱酵液對病害之抑制率可達56%以上。在溫室,將上述兩種BA01菌株之250倍稀釋醱酵液施用於結球萵苣植株,結果兩者皆可顯著降低病害之發生,其中尤以於接種病原菌前一天施用者之防治效果最佳。利用高壓液相層析儀 (HPLC) 分析BA01菌株之代謝產物,發現BA01菌株具有產生Iturin A及Surfactin等抑菌物質之能力。本研究所研製之兩種Bacillus amyloliquefaciens BA01菌株醱酵液證實具有進一步研發成為微生物源植物保護製劑產品之潛力。
During spring season in 2011, a disease previously unreported in Taiwan was observed on commercial plantings of lettuce (Lactuca sativa L.) at Erlun in Yunlin County. A lot of black-brown spots showed on leaves of diseaed head lettuce plants. When spots are so numerous, they coalesce to form areas extending whole leaf and resulting in poor standing of head lettuce with leaf yellow, drying and death. Disease incidence and the rate of poor standing of head lettuce seedlings occurred in cultivation trays at Erlun were 98% and 16%, respectively. A species of Alternaria was consistently isolated from diseased portions of the infected leaves on 2% (w/v) water agar. Spore suspensions (105 conidia/ml) of the pathogen were used to inoculate fourteen crops to confirm its host range. The results showed that leaf spots were similar to the original symptoms developed only on inoculated plants of ten lettuce cultivars. However, inoculated tomato and cabbage plants did show hypersensitive-like reaction. Other inoculated plants remained symptomless. Based on ribosomal DNA internal transcribed spacer (ITS) sequencing analysis, the ITS sequence of the pathogen was similar to A. solani. The ITS sequence identity between the pathogen and A. solani (BCRC No. 32123) obtained from Food Industry Research and Development Institute was 99%. On V8 juice agar, colonies of the pathogen are dark-brown with concentric rings. Most of conidiophores with single conidiogenous site and a few with 2-4 conidiogenous sites. Conidia borne singly from each conidiogenous site on the conidiophores, long-ellipsoid or long-ovoid, 6-10 transverse septa and 0-3 longitudinal septa, 1-2 beaks, 82-90 × 13 μm. Beaks are narrow-tapered and become up to 163-176 μm long. The optimum temperature for mycelial growth, conidial germination and infection on lettuce of Alternaria sp. isolates AL1-2 and AL3-11 was 24℃. The results indicated that the fungus may be a new species of Alternaria on Lactuca sativa in the world and we named it Alternaria lactucae Kao & Huang sp. nov.. Fifteen antagonistic bacteria were evaluated for their effects on mycelial growth and conidial germination of A. lactucae isolates AL1-2 and AL3-11. Among tested bacteria, Bacillus amyloliquefaciens isolate BA01 was an effective biocontrol agent which was able to inhibit 55% of mycelial growth and 80% of conidial germination and did also cause germ tubes swelling of A. lactucae. Application of detached head lettuce leaf for evaluating the efficacy of BA01 on controlling lettuce black leaf spot showed that BA01 was able to reduce 89% of development of black spot on detached head lettuce leaf. The fermented broth of BA01 cultured in Soybean meal-Molasses (SYM) medium was analyzed by bioassay method of detached head lettuce leaf for disease control. 100-fold dilution of fermented broth of BA01 cultured in SYM media could inhibit 36% and 41% of development of black spot caused by A. lactucae isolates AL1-2 and AL3-11, respectively. Adding different plant oils into SYM media to evaluate their effects on the control efficacy of BA01, the results indicated that fermented broth of BA01 cultured in SYM media amended with 2.5% (w/v) peanut oil did show to be the most effective in controlling lettuce black leaf spot. Inhibition rate of 200-fold dilution of the fermented broth was more than 46%. In addition, we also found that SYM media amended with 0.125% (w/v) emulsified agent could enhance the ability of BA01 for controlling lettuce black leaf spot. Inhibition rate of 200-fold dilution of the fermented broth reached more than 56%. In greenhouse test, spraying two fermented broths of BA01 at 250-fold dilution to head lettuce plants one day before inoculating with the pathogen (105 conidia/ml) did show to be the most effective in controlling the disease. Analysis of antifungal compounds produced from cultural filtrate of BA01 by high pressure liquid chromatography (HPLC) showed that BA01 could not only produce surfactin but also produce iturin A. This study demonstrates that the two fermented broths of B. amyloliquefaciens BA01 have the potential to be developed into microbial plant protectants for controlling lettuce black leaf spot.
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