臺灣博碩士論文加值系統

西瓜蔓割病係由土媒病原菌Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hansen所引起，防治頗為不易。目前台灣主要以化學藥劑或嫁接扁蒲(Lagenaria siceraria Ser.) 防治本病害。本研究為了尋找其它替代的防治方法，因此嘗試利用介質添加物與微生物，用以防治西瓜幼苗蔓割病的發生。首先以香菇太空包堆肥 (spent forest mushroom compost, SFMC) 按不同體積比例與荷蘭泥炭苔 (Bas Van Burren No. 4, BVB No.4) 混拌，結果發現兩者體積混拌比例在50：50 (SFMC50) 時，除可降低西瓜蔓割病的發生，西瓜幼苗尚可正常生長。在SFMC50栽培介質添加0.75%蝦蟹殼粉 (SC75) 或0.25%牛血粉 (SB25) 後，兩者栽培介質水萃取液皆可降低西瓜蔓割病菌分生孢子的發芽百分率；其中SC75栽培介質可顯著降低蔓割病的發生達30.48%。將不同組成的栽培介質以熱蒸氣 (100℃，30分鐘) 滅菌處理後，即喪失對於蔓割病菌分生孢子發芽的抑制作用。由SFMC50與蝦蟹殼粉中分離而得之18株細菌菌株分別於馬鈴薯牛肉煎汁瓊脂 (PNA) 平板上與蔓割病菌進行對峙培養，發現Bacillus subtilis Ju020菌株對病原菌FNH0103拮抗效果最佳，可抑制菌絲生長達51.3%。取SFMC50添加0.75%蝦蟹殼粉與Ju020 (106 cfu/mL medium) 製成SCJ20栽培介質，可顯著降低蔓割病發病率達40.3%。利用玻片洋菜薄膜培養法觀察SFMC50、SC75以及SCJ20栽培介質對於病原菌分生孢子發芽的影響，結果發現SCJ20栽培介質可使西瓜蔓割病菌FNH0103與FNP0401菌株分生孢子發芽率分別下降37.33%與36.67%，發芽管長度減少68.99±5.61 μm與104.65±1.85 μm，發芽管不正常膨大比例增加30.33%與26.00%，且分生孢子於第24小時會出現瓦解的情形，FNH0103及FNP0401菌株分生孢子瓦解率分別達45.67%與44.00%。以高壓液相層析儀 (HPLC) 分析栽培介質中可能之抑菌物質，發現有類似iturin A之波峰產生，但是不具有surfactin相似波峰。綜言之，SFMC50搭配0.75%蝦蟹殼粉與B. subtilis Ju020所製成的SCJ20栽培介質，具有防治西瓜蔓割病發生的功效。至於其防病的部分原理可歸因於SCJ20介質中存在有iturin A抑菌物質及擁有較豐富的微生物相與活性，致使西瓜蔓割病菌分生孢子發芽率下降，發芽管出現不正常膨大及瓦解等的現象，因而喪失感染植株的能力。

Fusarial wilt of watermlon is caused by soil-borne fungal Fusarium oxysporum (Schl.) f. sp. niveum (E. F. Smith) Snyder & Hansen, and it is hard to be controlled. Chemical fungicides and grafting bottle gourd (Lagenaria siceraria Ser.) are mainly used to control the disease in Taiwan. In order to search alternative methods, the purpose of this study is to use amendments and microorganism to develop a bioformulated cultural medium for controlling watermelon fusarial wilt. To compare different composition of spent forest mushroom compost (SFMC) mixed with Bas Van Burren No. 4 (BVB No.4) peat moss, it was found that the cultural medium consisted of SFMC and BVB No.4 at the ratio of 50：50 (v/v) was effective in reducing watermelon fusarial wilt, and made watermelon seedlings grown normally. The water-soluble extract of cultural medium SFMC50 amended with 0.75% shrimp and crab shell powder (SC75) or 0.25% blood meal (SB25) could reduce the conidial germination of F. oxysporum f. sp. niveum. The disease incidence of watermelon seedlings grown in cultural medium SC75 was reduced by 30.48%. Inhibition of conidial germination of the pathogen was nullified when cultural media SC75 and SB25 were steamed at 100℃ for 30 min. Eighteen bacterial isolates obtained from SFMC50 and SC75 were dually cultured with F. oxysporum f. sp. niveum on the potato nutrient agar plates. The results indicated that Bacillus subtilis Ju020 was able to inhibit the mycelial growth of F. oxysporum f. sp. niveum isolate FNH0103 up to 51.3%. The cultural medium SFMC50 amended with 0.75% (w/v) SCSP and antagonistic bacterium Ju020 (106 cfu/mL medium) (SCJ20) could reduce 40.3% of fusarial wilt on watermelon seedlings. The effect of cultural media SFMC50, SC75 and SCJ20 on the conidial germination of F. oxysporum f. sp. niveum was analyzed by slide agar-film culture method. The results showed that the conidial germination of F. oxysporum f. sp. niveum isolates FNH0103 and FNP0401 in the cultural medium SCJ20 were decreased by 37.33% and 36.67%, the length of conidial germ tube were reduced by 68.99±5.61 μm and104.65±1.85 μm, abnormal germ tubes were incited by 30.33% and 26.00%. The germ tube lysis percentage of F. oxysporum f. sp. niveum isolates FNH0103 and FNP0401 were observed by 45.67% and 44.00% 24hrs after treatment. Analyzed antifungal compounds in the cultural media by High pressure liquid chromatography (HPLC), it was found that the water-soluble extract of cultural medium SCJ20 had peaks similar to iturin A, but it didn’t have peaks similar to surfactin. The partial mechanisms for reducing fusarial wilt of watermelon seedlings with the cultural medium SCJ20 may attribute plenty of microbial activity and the antifungal activity of iturin A existed in the medium.

中文摘要................................................................................................i
英文摘要................................................................................................iii
目次.......................................................................................................v
表次索引................................................................................................ix
圖次索引................................................................................................x
前言.......................................................................................................1
材料與方法..............................................................................................5
一、供試植株..........................................................................................5
二、供試菌株來源、培養與保存................................................................5
三、病原性之測定....................................................................................5
四、接種病土之製作................................................................................6
五、土壤病原菌密度測定..........................................................................6
六、病害調查方法....................................................................................6
七、病原菌之形態與生理..........................................................................7
(一)病原菌之形態.....................................................................................7
(二)時間對分生孢子發芽的影響..................................................................7
八、感染率測試........................................................................................7
九、西瓜栽培介質之基質的篩選.................................................................8
(一)不同處理香菇太空包堆肥水萃取液對西瓜蔓割病菌分生孢子發芽的影響.....8
(二)香菇太空包堆肥添加荷蘭泥炭苔對西瓜幼苗生長的影響...........................8
(三)香菇太空包堆肥添加荷蘭泥炭苔對西瓜幼苗蔓割病發生的影響.................9
十、抑病栽培介質的調製與評選.................................................................9
(一)不同組成之栽培介質水萃取液製備.........................................................9
(二)玻片洋菜薄膜培養法...........................................................................10
(三)不同組成之栽培介質水萃取液對西瓜蔓割病菌分生孢子發芽的影響..........10
(四)以玻片洋菜薄膜培養法評選不同組成之栽培介質對西瓜蔓割病菌分生孢子發芽的影響....................................................................................................10
(五)不同組成之栽培介質對西瓜幼苗蔓割病發生的影響.................................11
十一、拮抗微生物之篩選..........................................................................11
(一)拮抗菌株來源.....................................................................................11
(二)對峙培養...........................................................................................12
(三)拮抗菌株之鑑定與生理生化特性...........................................................12
1.DNA萃取..............................................................................................12
2.聚合酵素鏈鎖反應.................................................................................12
3.增幅片段之定序比對..............................................................................13
4.Biolog分析............................................................................................14
十二、栽培介質添加蝦蟹殼粉與拮抗細菌對西瓜幼苗蔓割病發生之影響.........14
十三、栽培介質添加蝦蟹殼粉與拮抗細菌防治西瓜幼苗蔓割病之機制初探......14
(一)蒸氣滅菌法.........................................................................................15
(二)蒸氣滅菌處理過之栽培介質對於西瓜蔓割病菌分生孢子發芽的影
響...........................................................................................................15
(三)不同組成之荷蘭泥炭苔與香菇太空包堆肥對西瓜幼苗蔓割病發生的影響...........................................................................................................16
(四)抑病栽培介質對西瓜蔓割病菌分生孢子瓦解的影響..................................16
(五)抑病栽培介質萃取液之HPLC分析..........................................................16
1.硫酸銨萃取法........................................................................................16
2.利用HPLC分析Surfactin以及Iturin............................................................17
結果........................................................................................................18
一、病原菌之形態與生理...........................................................................18
(一)西瓜蔓割病菌之形態............................................................................18
(二)時間對西瓜蔓割病菌分生孢子發芽的影響...............................................18
二、感染率測試........................................................................................18
三、西瓜栽培介質之基質的篩選.................................................................19
(一)不同處理香菇太空包堆肥水萃取液對西瓜蔓割病菌分生孢子發芽的影響....19
(二)香菇太空包堆肥添加荷蘭泥炭苔對西瓜幼苗生長的影響...........................19
(三)香菇太空包堆肥添加荷蘭泥炭苔對西瓜幼苗蔓割病發生的影響.................19
四、抑病栽培介質的調製與評選.................................................................20
(一)不同組成之抑病栽培介質水萃取液對西瓜蔓割病菌分生孢子發芽的影響....20
(二)以玻片洋菜薄膜培養法評選不同組成之栽培介質對西瓜蔓割病菌分生孢子發芽的影響.....................................................................................................20
(三)不同組成之栽培介質對西瓜幼苗蔓割病發生的影響..................................20
五、拮抗微生物之篩選..............................................................................21
(一)對峙培養............................................................................................21
(二)拮抗細菌Ju020之16S rDNA鑑定...........................................................21
(三)Biolog鑑定分析...................................................................................21
六、栽培介質添加蝦蟹殼粉與拮抗細菌對西瓜幼苗蔓割病發生之影響.............22
七、栽培介質添加蝦蟹殼粉與拮抗細菌防治西瓜幼苗蔓割病之機制初探..........22
(一)以蒸氣滅菌處理不同栽培介質對於西瓜蔓割病菌分生孢子發芽的影
響...........................................................................................................22
(二)不同組成之荷蘭泥炭苔與香菇太空包堆肥對西瓜幼苗蔓割病發生的影響...........................................................................................................22
(三)抑病栽培介質對西瓜蔓割病菌分生孢子瓦解的影響..................................23
(四)抑病栽培介質萃取液之HPLC分析..........................................................23
1.HPLC分析Surfactin................................................................................23
2.HPLC分析Iturin A..................................................................................23
討論........................................................................................................25
參考文獻.................................................................................................29
圖表........................................................................................................34

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