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研究生:蘇士閔
研究生(外文):Shih-Min Su
論文名稱:影響洋香瓜黑點根腐病菌子囊孢子發芽之因子暨田間土壤子囊孢子密度調查
論文名稱(外文):Factors Affecting Ascospore Germination of Monosporascus cannonballus and Investigation of Ascospore Densities in Field Soils
指導教授:林益昇
指導教授(外文):Yi-Sheng Lin
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
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:2005
畢業學年度:93
語文別:中文
論文頁數:91
中文關鍵詞:洋香瓜黑點根腐病菌子囊孢子發芽蔗糖溶液循環沈澱法子囊孢子密度土壤試管法根圈土壤放線菌包埋子囊孢子試管法
外文關鍵詞:Monosporascus cannonballusascospore germinationcircular sucrose and sedimentation tank methodascospore densitiessoil tube observation methodrhizosphere soilactinomycetesimmersed ascospore tube method
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本論文主要目的在探討影響Monosporascus cannonballus子囊孢子發芽的因子,和利用蔗糖溶液循環沈澱法進行田間土壤中子囊孢子密度的偵測。利用土壤試管法種植洋香瓜,12天後可觀察到子囊孢子在根圈土壤中發芽並侵入根表,在24℃的環境下發芽率可達54.8%,但若將土壤經高溫高壓滅菌後,則子囊孢子不發芽;添加多種藥劑或抗生素於土壤中進行試驗,發現多數能抑制細菌或放線菌生長者也能夠抑制或降低子囊孢子發芽。進一步自不同來源之土壤中分離得到真菌、細菌與放線菌等三群微生物來進行土壤試管法之試驗,結果發現在滅菌土中僅加入放線菌之處理能大量促進子囊孢子發芽。為了求得更精確的發芽率,並方便研究其影響因子,本實驗室研發包埋子囊孢子試管法,使得子囊孢子能夠在培養基上發芽。應用本方法在處理後第27天之觀察結果發現,子囊孢子在距離根表0-2mm範圍內才能大量發芽,且多集中在0-1mm內,超過3mm距離者則甚少發芽。而且無論添加耕作田土或非耕作田土,甚至是加入泥炭土與魚缸底砂的處理,子囊孢子均有發芽的表現,但在加入珍珠石取代覆蓋土壤之處理或利用珍珠石添加土壤抽出液替代覆蓋土壤的處理中,發現子囊孢子仍不發芽。另外,在不種植洋香瓜的狀況下,改以洋香瓜根的萃取液加入越瓜田土中進行試驗,子囊孢子同樣無法發芽。選取在土壤試管法中較能有效促進子囊孢子發芽的四個放線菌菌株,A17、A21、A22與A33,並改以包埋子囊孢子試管法進行試驗;結果發現將放線菌加入滅菌土中確實能觀察到子囊孢子大量發芽,但在珍珠石中或是沒有土壤的環境下放線菌則沒有促進子囊孢子發芽的效果。本論文亦以蔗糖溶液循環沈澱法進行田間土壤中病原菌密度的調查,發現除了子囊孢子之外,偶爾也可偵測得到子囊殼的存在。在東山鄉與芳苑鄉的試驗田表土(0-10cm)中,子囊孢子密度平均各為9.9與9.8個/克土壤;東山試驗田經一期的水稻輪作後,密度降至1.28個/克土壤。在垂直分佈上,於東山試驗田測得子囊孢子多分佈在0-30cm深的土壤中,並有愈往下子囊孢子密度愈低的趨勢;而在30-50cm深的土壤中則較少見到游離的子囊孢子,但可觀察到少數未釋放的子囊殼存在。除罹病田外,在清泉岡蘿蔔田、大里竹子田等非發病田仍能測得子囊孢子密度各為5.6與1.8個/克土壤;甚至於中興大學之中興湖、小禮堂旁與花蓮秀姑巒溪旁的非耕作田土也各有0.5、0.1及0.2個子囊孢子/克土壤的存在。
The main purpose of this thesis was to investigate the factors affecting the ascospore germination of Monosporascus cannonballus and to determine the ascospore densities in field soils by using circular sucrose and sedimentation tank method (CSSTM). Soil tube observation method (STOM) was employed to observe ascospore germination in rhizosphere soil and penetration into the root surface of muskmelon. Ascospore germination could reach 54.8% at 24℃, but ascospores did not germinate in the autoclaved soil. Several fungicides and antibiotics were used to treat the natural soil and the result showed that most of the tested chemicals could inhibit or reduce the ascospore germination. Several isolates of fungi, bacteria and actinomycetes isolated from different natural soils were added to the autoclaved soil and found that most tested actinomycete isolates could greatly increase ascospore germination. Using immersed ascospore tube method (IATM) to study the factors affecting the ascospore germination on agar medium, it was found that ascospores germinated at the distance of 0-2mm, especially greatly at 0-1mm, from the root surface, but rarely germinated beyond 3mm when the natural soils collected from crop fields were used as cover soil. Moreover, soils from non-crop fields, peat moss, and fish-jar sand also could stimulate ascospore germination but perlite (only 2.4%). By using IATM, when soil extract was added to perlite or muskmelon root extract was used instead of planting a muskmelon seedling, ascospores did not germinate, either. Four actinomycete isolates, A17, A21, A22 and A33, which could stimulate ascospore germination greatly by using STOM, were tested again for their abilities to stimulate the ascospore germination by operating IATM. They indeed greatly stimulated ascospore germination when autoclaved soil was used as cover soil. However, no ascospore germination was observed when their spore suspensions were added to the medium with no cover soil or with perlite as cover soil by using IATM. By using CSSTM, both ascospores and perithecia could be detected from the field soils and the ascospore densities of Monosporascus cannonballus in the top soils (depth of 0-10 cm) collected from Dongshan and Fangyuan muskmelon fields were 9.9 and 9.8 ascospores/g soil, respectively. The ascospore densities in Dongshan soil samples reduced to 1.28 from 10.4 ascospores/g soil after rotation with rice paddy once. Vertical distribution of the ascospores at Dongshan muskmelon field was also determined. It revealed that most wild ascospores existed within 30cm of soil depth and the deeper the lower ascospore numbers were detected. Few wild ascospores were detected within 30-50cm of soil depth, but sometimes intact perithecia could be observed. A carrot field at Chingchuankang and a bamboo field at Dalih were also subjected to determine their ascospore densities and yielded 5.6 and 1.8 ascospores/g soil, respectively. Furthermore, even in the soil samples collected from non-cropped fields, such as Chung Hsing Lake and Small Hall of National Chung Hsing University and Shougunuan River, the ascospore densities were 0.5, 0.1 and 0.2 ascospores/g soil, respectively.
目錄
誌謝 -----------------------------------------------------------------------------------------------I
中文摘要 -----------------------------------------------------------------------------------------II
英文摘要 -----------------------------------------------------------------------------------------IV
目錄 -----------------------------------------------------------------------------------------------VI
附表目錄 -----------------------------------------------------------------------------------------IX
附圖目錄 -----------------------------------------------------------------------------------------XI
前言 -----------------------------------------------------------------------------------------------1
前人研究 -----------------------------------------------------------------------------------------2
材料與方法 --------------------------------------------------------------------------------------10
一、洋香瓜黑點根腐病菌菌株之來源與培養 --------------------------------------10
1. 供試病原菌菌株之來源 -----------------------------------------------------------10
2. 病原菌之保存與更新 --------------------------------------------------------------10
二、供試植物、土壤來源與土壤質地測定 -----------------------------------------10
三、洋香瓜黑點根腐病菌生長測試 --------------------------------------------------11
1. 溫度對菌絲生長的影響-------------------------------------------------------------11
2. 培養基對子囊殼形成的影響 -----------------------------------------------------11
3. 溫度對子囊殼形成的影響 --------------------------------------------------------12
4. 光照對子囊殼形成的影響 --------------------------------------------------------12
四、影響洋香瓜黑點根腐病菌子囊孢子發芽之因子 -----------------------------13
1. 利用土壤試管法觀察子囊孢子發芽----------------------------------------------13
2. 溫度對子囊孢子發芽的影響 -----------------------------------------------------14
3. 滅菌土壤對子囊孢子發芽的影響 -----------------------------------------------14
a. 高溫高壓滅菌土對子囊孢子發芽的影響 -----------------------------------14
b. 藥劑滅菌土對子囊孢子發芽的影響 -----------------------------------------14
4. 土壤微生物群對子囊孢子發芽的影響-------------------------------------------15
5. 利用包埋子囊孢子試管法觀察子囊孢子之發芽 -----------------------------16
6. 耕作田土壤與非耕作田土壤對子囊孢子發芽的影響 -----------------------16
7. 非土壤介質對子囊孢子發芽的影響 --------------------------------------------17
8. 土壤抽出液對子囊孢子發芽的影響 --------------------------------------------17
9. 植物根的萃取液對子囊孢子發芽的影響 --------------------------------------17
10. 放線菌對子囊孢子發芽的影響 -------------------------------------------------18
五、田間洋香瓜黑點根腐病菌子囊孢子密度調查 --------------------------------18
1. 土壤採樣 -----------------------------------------------------------------------------18
2. 蔗糖溶液循環沉澱法 --------------------------------------------------------------19
結果 -----------------------------------------------------------------------------------------------20
一、洋香瓜黑點根腐病菌生長測試 --------------------------------------------------20
1. 溫度對菌絲生長的影響 -----------------------------------------------------------20
2. 培養基對子囊殼形成的影響 -----------------------------------------------------20
3. 溫度對子囊殼形成的影響 --------------------------------------------------------20
4. 光照對子囊殼形成的影響 --------------------------------------------------------21
二、影響黑點根腐病菌子囊孢子發芽之因子 --------------------------------------21
1. 利用土壤試管法觀察子囊孢子發芽 --------------------------------------------21
2. 溫度對子囊孢子發芽的影響 -----------------------------------------------------21
3. 滅菌土壤對子囊孢子發芽的影響 -----------------------------------------------22
a. 高溫高壓滅菌土對子囊孢子發芽的影響 ----------------------------------22
b. 藥劑滅菌土對子囊孢子發芽的影響 ----------------------------------------22
4. 土壤微生物群對子囊孢子發芽的影響 -----------------------------------------22
5. 利用包埋子囊孢子試管法觀察子囊孢子發芽 --------------------------------23
6. 耕作田土壤與非耕作田土壤對子囊孢子發芽的影響 -----------------------24
7. 非土壤介質對子囊孢子發芽的影響 --------------------------------------------24
8. 土壤抽出液對子囊孢子發芽的影響 --------------------------------------------24
9. 植物根的萃取液對子囊孢子發芽的影響 --------------------------------------25
10. 放線菌對子囊孢子發芽的影響 -------------------------------------------------25
三、田間洋香瓜黑點根腐病菌子囊孢子密度調查 ---------------------------------25
1. 田間子囊孢子密度與分布 -------------------------------------------------------25
2. 不同來源土壤中之子囊孢子密度 ----------------------------------------------26
討論 -----------------------------------------------------------------------------------------------27
引用文獻 -----------------------------------------------------------------------------------------33
附表 -----------------------------------------------------------------------------------------------42
附圖 -----------------------------------------------------------------------------------------------56
附錄 -----------------------------------------------------------------------------------------------74
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