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標題: 調製栽培介質防治甘藍黃葉病
Formulation of Cultural Media for Control of Cabbage Yellows
作者: 莊茗凱
Chuang, Min-Kai
Contributors: 黃振文
植物病理學系所
關鍵字: 香菇太空包堆肥;抑病栽培介質;枯草桿菌;蝦蟹殼粉;牛血粉;甘藍;甘藍黃葉病
Spent mushroom compost;disease suppressive cultural medium;Bacillus;Shrimp and crab shell powder;Blood meal;Cabbage;Cabbage yellows
日期: 2012
Issue Date: 2013-11-19 12:38:46 (UTC+8)
Publisher: 植物病理學系所
摘要: 甘藍黃葉病是由Fusarium oxysporum f. sp. conglutinans引起,為重要的土壤傳播性病害,可造成苗期或本田期甘藍植株黃化、萎凋及死亡。目前防治甘藍黃葉病的防治方法中,最有效的兩種方法為抗病育種及土壤燻蒸。土壤燻蒸劑對環境安全有疑慮,故不建議使用;此外發展抗病育種需要較長的時間,且育成不易,因此亟須發展其他有效防治甘藍黃葉病的策略。本研究主要目的在於利用香菇太空包堆肥調配適於甘藍幼苗發育,且兼具有抑制甘藍黃葉病發生的栽培介質;此外也嘗試配合有益微生物及有機添加物增強栽培介質對於甘藍黃葉病的抑病功效。
自雲林、苗栗等地採集甘藍罹病植株分離獲得15菌株後,依病原性測定、形態特徵及其ITS序列比對,將FOC-JR01及 FOC-YL08菌株學名鑑定為Fusarium oxysporum f. sp. conglutinans (Sch. ex Fr.) Snyd. & Hans.。本病原菌菌絲生長的最適溫度為28℃。將甘藍(農友,高峰品種)種子播種於菌量濃度為103、104及105 ( cfu/ g soil ) 的人工病菌土中,7天後甘藍幼苗被感染率分別達60%、78%及100%。香菇太空包堆肥的水萃取液對於病原菌FOC-JR01及FOC-YL08的分生孢子發芽抑制率分別為26%及11%。在荷蘭BVB No.4泥炭苔栽培介質中加入香菇太空包堆肥的濃度超過50 % (v/v)時,不利於甘藍幼苗的生長;惟若以香菇太空包堆肥與泥炭苔以1:1 (v/v)比例組合成的SFMC50栽培介質,則可使甘藍幼苗的鮮重與乾重表現最為優異;此外將甘藍種植於含有病原菌菌量104 cfu/ ml cultural medium的SFMC50栽培介質中,可使甘藍幼苗的發病率較對照組低17%,也可以使甘藍黃葉病菌的存活率明顯較對照組減少20%。在SFMC50混拌入牛血粉或蝦蟹殼粉2% (w/v),在28℃,7天後可以降低病原菌在栽培介質內的存活率達100%或39%。自發病甘藍田土、甘藍健株根部、SFMC50栽培介質及添加牛血粉(2% w/v)或蝦蟹殼粉之栽培介質中分離獲得183菌株的細菌分別與F. oxysporum f. sp. conglutinans FOC-YL08 在馬鈴薯葡萄糖牛肉煎汁培養基平板進行對峙培養,結果顯示有128株菌株具有抑制 F. oxysporum f. sp. conglutinans FOC-YL08 菌絲生長的效果,其中有56株菌株對於FOC-YL08菌絲生長抑制率可達50%以上。在SFMC50中以106 cfu/ ml 的生物防治菌株AU-001, JU-019, JU-020, 2402 或 NP01分別均勻混拌,結果發現它們和未處理之對照組相比可以使甘藍黃葉病的發生率降低60%以上。將拮抗微生物之16s rDNA序列與NCBI基因資料庫作比對,發現具有防治潛力的菌株AU-001屬於Bacillus licheniformis,JU-019及JU-020皆屬於Bacillus subtilis,而2402及NP-01則是屬於Bacillus mycoides。
綜合前述成果,本研究成功研發出兩種抑制甘藍黃葉病的栽培介質,其一為 SFMC50 栽培介質和0.75% (w/v) 蝦蟹殼粉均勻混合後接種 JU-020 菌株,製成SFMC50-S20栽培介質,和對照組相比可以有效抑制甘藍幼苗發病率達76%;其二則是SFMC50 栽培介質和0.25% (w/v) 牛血粉均勻混合並接種JU-019,製成SFMC50-B19栽培介質,和對照組相比可以有效抑制甘藍幼苗發病率達85%。SFMC50-S20栽培介質抑制甘藍黃葉病發生的機制,可能與其有較高的pH值、微生物活性及Bacillus屬細菌在栽培介質中產生的幾丁質分解酵素與Iturin A抑菌物質等因素存在有密切的關係。
Cabbage yellows caused by Fusarium oxysporum f. sp. conglutinans (Wollenweb.) W. C. Snyder and H. N. Hansen, is one of the most common soilborne diseases of cabbage in the world. It cause yield loss of cabbage seedlings and plants. The best ways to control cabbage yellows are breeding resistant cultivars and using soil fumigation. Soil fumigation was banned due to concerns of human health and environmental safety. Furthermore, there is no resistant cultivar reported in Taiwan. So it is in an urgent need for us to develop new methods to control cabbage yellows. The objective of the research is to develop a bio-formulated cultural medium from spent forest mushroom compost (SFMC) amended with beneficial bacteria and organic materials for control of cabbage yellows.
In the study, fifteen isolates FOC-JR01~06 and FOC-YL01~09 were obtained from diseased cabbage plants from Yun-lin and Meau-li, and identified as F. oxysporum f. sp. conglutinans according to their pathogenicity test, morphological and molecular characteristics. Isolate FOC-JR01 and FOC-YL08 grew well at 24-30℃, but optimal at 28℃.The infection rates of cabbage seedlings by the pathogen in 103, 104, 105 (cfu/ g soil) infested soil were 60%, 78% and 100% respectively at the 7th day after planting. Water extract of SFMC could inhibit 11%-26% of conidial germination of the pathogen. Nine cultural media prepared from the spent forest mushroom compost (SFMC) mixed with different ratio of BVB No.4 peat moss were tested for their efficacy on supporting the growth of cabbage seedlings. SFMC50 cultural medium consisted of spent forest mushroom compost and BVB No.4 peat moss at the ratio of 1:1 (v/v) was much more suitable for the growth of cabbage seedlings, and it could also reduce 17% disease incidence of cabbage yellows compared to BVB No.4 peat moss as a control. Amendment of SFMC50 with 2% (w/v) blood meal or shrimp and crab shell powder did respectively reduce 100% or 39% population densities of the pathogen for seven days at 28℃ in the growth chamber. Fifty six bacterial strains isolated from healthy cabbage stem, root, infested soil and SFMC50 with amendment of 2% (w/v) blood meal or shrimp and crab shell powder were significantly effective in inhibiting more than 50% of mycelial growth on potato nutrient agar plates. SFMC50 mixed respectively with bacterial suspension of strain AU-001, JU-019, JU-020, 2402 or NP01 at a concentration of 106 (cfu/ ml) was more effective in reducing more than 60% disease incidence of cabbage seedlings caused by F. oxysporum f. sp. conglutinans compared to non treatment SFMC50 as a control. These antagonistic bacteria were analyzed by 16S rDNA and comparing the gene sequence of ITS region of these antagonistic bacterial strains with the NCBI data base, the results indicated that AU-001 is identified as Bacillus licheniformis, JU-019 and JU-020 are Bacillus subtilis, 2402 and NP-01 are Bacillus mycoides. A SFMC50-S20 cultural medium was formulated with SFMC50, 0.75% (w/v) SCSP and cell suspension of B. subtilis JU-020. The SFMC-S20 was very effective in reducing 76% disease incidence of cabbage seedlings compared to SFMC50 as a control. In addition, SFMC50-B19 cultural medium was also formulated with using SFMC50, 0.25% (w/v) BLM and cell suspension of JU-019. The SFMC50-B19 was very effective in reducing 85% disease incidence of cabbage seedlings compared to SFMC50 as a control. In our studies, the results indicated that higher pH value, microbial activity and the production of chitinase and iturin A from Bacillus spp. in the SFMC50-S20 cultural medium might be related with its mechanism for control of cabbage yellows.
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