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研究生:徐偉城
研究生(外文):Wei-Cheng Hsu
論文名稱:廢棄木屑堆肥添加康氏木黴菌防治樹木褐根病之探討
論文名稱(外文):Application of waste mushroom sawdust amended with Trichoderma koningii to control tree brown root rot caused by Phellinus noxius
指導教授:梁文進梁文進引用關係
指導教授(外文):Wen-Jinn Liang
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:植物醫學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2012
畢業學年度:101
語文別:中文
論文頁數:71
中文關鍵詞:褐根病生物防治康氏木黴菌廢棄木屑堆肥
外文關鍵詞:Phellinus noxiusBiological controlTrichoderma koningiiMushroom waste sawdust
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本研究以木黴菌添加至再堆肥化廢棄木屑堆肥中,探討於溫室防治褐根病之效果。自屏東地區土壤及植物組織中分離獲得35株木黴菌菌株,利用對峙培養法、玻璃紙抗生法測定其對褐根病菌(Phellinus noxius(Corner))之拮抗作用,篩選出TAS、TDS、TFS三株木黴菌菌株; 可抑制由四個不同地區,分離之褐根病菌PCCR、PGSR、PCFR、PMZR菌株菌絲的生長。利用顯微鏡觀察載玻片上三種菌之對峙培養,發現木黴菌TAS、TDS、TFS菌株均可造成褐根病原菌菌絲之超寄生現象。藉由核醣體DNA比對鑑定,證明TAS、TDS、TFS菌株皆為Hypocera koningii (anamorph: Trichoderma koningii) 。將化學藥劑添加於PDA培養基中,測定不同濃度之藥劑對褐根病菌之抑制效果,在供試之6種藥劑中,以25%普克利乳劑及45.5%待普克利乳劑之抑菌效果最好。於廢棄木屑二次堆肥化過程中,經檢測堆肥化過程堆肥之溫度、pH值,電導度EC值變化比,得知堆積發酵4天後,就可進行木黴菌之接種培養。測試不同含水率之廢棄木屑堆肥對木黴菌生長之影響,70%含水率試驗第十三天時,TAS、TDS、TFS於試管中菌絲之生長可達9cm為最好之生長;測試不同含量之CaCO3對菌絲生長之影響時,添加5%及7%CaCO3之廢棄木屑堆肥試驗第十天時,TAS、TDS、TFS菌絲於試管中可達9cm最好之生長。溫室測試廢棄木屑對枇杷植株生長之影響,土中添加不同比例之TAS、TDS、TFS木黴菌菌株之再堆肥化廢棄木屑堆肥,結果發現添加2:1(w/w)、3:1(w/w)之比例,枇杷幼苗植株死亡率為0%;而1:1(w/w)者則有30%死亡率,完全木屑堆肥之死亡率為100%。將病土與木黴菌廢棄木屑堆肥按2:1(w/w)比例混合,結果顯示,接種第35天時病土處理之枇杷幼苗植株死亡率為100%;含木黴菌廢木屑棄堆肥處理及待普克利化學藥劑處理防治枇杷幼苗植株死亡率為0%,顯示上述二處理具有抑制樹木褐根病之發生效果。


關鍵字:褐根病、生物防治、康氏木黴菌、廢棄木屑堆肥

Phellinus noxius is major wood decay fungi causing decline of many species tree in Taiwan. This research focuses on utilization of waste mushroom sawdust amended with Trichoderma antagonists to experiment the control efficiency of tree brown root rot caused by Phellinus noxius. 35 isolates of Trichoderma were isolated from soils and dead materials of plants at different areas in Pingtung. With cellophane antagonism test and dual culture, 3 isolates of Trichoderma. were selected based on the inhibitory effect of antagonism on mycelial growth of Phellinus noxius. Dual culture on slide between TAS、TDS and TFS isolates of Trichoderma and Phellinus noxius showed that these three isolates could degrade the mycelium of Phellinus noxius. By coling and penetzation all of three isolates were identified as species of Hypocera koningii (amamorph: Trichoderma koningii) by comparison of the sequence of ribosomal DNA. Potato dextrose ager (PDA) amended with individual fungicide was used to evaluate effective fungicides on suppression of Phellinus noxius. Among the 6 fungicides tested against Phellinus noxius, 45% difenoconazole propiconazde EC and 25% propiconazde EC were most effective in completely inhibiting the mycelial growth at the active ingredient (ai) dosage of 10 ppm.
In composting process of waste sawdust, through the monitory of temperature, pH value and electronic conductivity of composted sawedust and Trichoderma cultivate test, the available sawdust was harvested and ultilized at 4 days after composting fermentation for Trichoderma culture. Effect of pH value and water content of sawdust culture substrate on mycelia growth of TAS、TDS、and TFS were experimented in large glass tube culture.The Trichoderma mycelia grow well at 5 and 7% of CaCO3 in sawdust, and was better tham than that of other CaCO3 content at 1and 3% Trichoderma grow its mycelium better at 70% water content of sawdust substrate than that of any other water content at 50, 60 and 80% rspectively. The study was conducted to determine survival of loquat in soil amended with different amount of waste mushroom sawdust. Results showed that soil amended with waste mushroom sawdust in 2:1(w/w) or 3:1(w/w) was effective growth of loguat in all the plants after treating for 1 month. When waste mushroom sawdust which had been grown T. koningii TAS, TDS, and TFS isolate was amended to diseased soil, the results showed that soil supplemented with the waste mushroom sawdust amended with Trichoderma koningii and drenched synthetic fungicide,were more excellent in disease control with no infected loquat occurred in all the plants after treating for 35 days.

目錄

摘要-------------------------------------------------------------------------- I
Abstract---------------------------------------------------------------------- III

謝誌-------------------------------------------------------------------------- V

目錄-------------------------------------------------------------------------- Ⅶ
圖表目錄-------------------------------------------------------------------- XI
壹、前言-------------------------------------------------------------------- 1
貳、前人研究-------------------------------------------------------------- 2
一、褐根病(Brown root rot)之介紹------------------------------- 2
二、褐根病目前的防治方法---------------------------------------- 4
三、木黴菌(Trichoderma spp.)之介紹----------------------------- 6
四、木黴菌在生物防治上之應用---------------------------------- 8
五、利用堆肥防治植物病害--------------------------------------- 10
參、材料方法-------------------------------------------------------------- 13
ㄧ、供試材料來源---------------------------------------------------- 13
(一)褐根病菌(P. noxius)菌株及其採集、分離及保存 13
(二)供試木黴菌菌株之採集、分離及保存---------------- 13
(三)供試作物---------------------------------------------------- 14
(四)木黴菌及P. noxius穀粒菌種之製備------------------- 14
(五)供試土壤之製備------------------------------------------- 14
(六)廢棄木屑堆肥與木黴菌混合物之製備---------------- 14
二、篩選具拮抗能力之木黴菌(Trichoderma spp.)菌株------ 15
(一)以對峙培養法(Dual culture method)測試不同木黴菌菌株對P. noxius BCRC菌株菌落生長之影響- 15
(二)以玻璃紙抗生法(Cellophane paper method)測試不同木黴菌菌株對P. noxius BCRC菌株菌落生長之影響------------------------------------------------------ 15
三、木黴菌TAS、TDS、TFS菌株對不同P. noxius PCCR、PGSR、PCFR、PNZR菌株之拮抗能力------------------- 16
(一)以對峙培養法測試木黴菌TAS、TDS、TFS菌株對不同P. noxius菌株生長之影響---------------------
16
(二)以玻璃紙抗生法測試木黴菌TAS、TDS、TFS菌株對不同P. noxius菌株生長之影響------------------ 16
(三) 木黴菌TAS、TDS、TFS菌株對P. noxius菌絲纏繞及超寄生拮抗作用之觀察--------------------------- 16
四、利用分子技術鑑定木黴菌TAS、TDS、TFS菌株------- 17
(一)萃取Genomic DNA -------------------------------------- 17
(二)擴增核醣體基因------------------------------------------- 17
(三)純化回收PCR產物--------------------------------------- 18
(四)核醣體基因選殖至pGEM-T easy載體---------------- 18
(五)yT&;A cloning plasmid 之小量萃取------------------- 19
(六)限制酶確認------------------------------------------------- 20
五、化學藥劑對P. noxius (BCRC)菌絲生長之影響---------- 20
六、化學藥劑待普克利、普克利對不同P. noxius PCCR、PGSR、PCFR、PNZR菌株菌絲生長之影響--------------- 20
七、菇蕈栽培後廢棄木屑再利用--------------------------------- 21
(一)廢棄木屑再堆肥化---------------------------------------- 21
(二)廢棄木屑再堆肥化之堆肥性狀調查------------------- 21
八、廢棄木屑堆肥化堆肥之含水率對木黴菌菌絲生長之影響------------------------------------------------------------------ 21
九、廢棄木屑堆肥化堆肥添加不同含量之CaCO3對木黴菌菌絲生長之影響------------------------------------------------ 22
十、含木黴菌堆肥化廢棄木屑與化學藥劑防治褐根病之溫室試驗------------------------------------------------------------ 22
十一、不同含量再堆肥化之廢棄木屑堆肥對枇杷植株生長之影響------------------------------------------------------------ 23
十二、含木黴菌之堆肥化廢棄木屑堆肥與化學藥劑對防治褐根病之溫室試驗------------------------------------------ 23
肆、結果--------------------------------------------------------------------- 24
一、篩選優良拮抗能力之木黴菌菌株---------------------------- 24
(一)以對峙培養法測試不同木黴菌菌株對P. noxius BCRC菌株菌落生長之影響 --------------------------

24
(二)以玻璃紙抗生法測試不同木黴菌菌株對P. noxius BCRC菌株菌落生長之影響--------------------------- 24
二、木黴菌TAS、TDS、TFS菌株對不同P. noxius PCCR、PGSR、PCFR、PNZR菌株之拮抗能力------------------- 24
(一)以對峙培養法測試木黴菌TAS、TDS、TFS菌株對不同P. noxius菌株生長之影響--------------------- 24
(二)以玻璃紙抗生法測試木黴菌TAS、TDS、TFS菌株對不同P. noxius菌株生長之影響------------------ 25
(三)木黴菌TAS、TDS、TFS菌株對P. noxius菌絲纏繞及超寄生拮抗作用之觀察--------------------------- 25
三、利用分子技術鑑定木黴菌TAS、TDS、TFS菌株------- 25
四、化學藥劑對P. noxius (BCRC)菌絲生長之影響--------- 26
五、化學藥劑待普克利、普克利對不同P. noxius PCCR、PGSR、PCFR、PNZR菌株菌絲生長之影響---------------- 27
六、菇蕈栽培後廢棄木屑再利用--------------------------------- 27
(一)廢棄木屑再堆肥化之堆肥性狀調查------------------- 27
七、廢棄木屑堆肥化堆肥之含水率對木黴菌菌絲生長之影響------------------------------------------------------------------ 28
八、廢棄木屑堆肥化堆肥添加不同含量之CaCO3對木黴菌菌絲生長之影響------------------------------------------------ 28
九、含木黴菌堆肥化廢棄木屑與化學藥劑防治褐根病之溫室試驗--------------------------------------------------------- 28
十、不同含量再堆肥化之廢棄木屑堆肥對枇杷植株生長之影響------------------------------------------------------------ 29
十一、含木黴菌之堆肥化廢棄木屑堆肥與化學藥劑對防治褐根病之溫室試驗------------------------------------------
29
伍、討論-------------------------------------------------------------------- 30
陸、結論-------------------------------------------------------------------- 34
參考文獻-------------------------------------------------------------------- 35
圖表-------------------------------------------------------------------------- 41
附錄-------------------------------------------------------------------------- 67
作者簡介-------------------------------------------------------------------- 71

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