臺灣博碩士論文加值系統

為篩選強致病力之蟲生真菌供作蚜蟲生物防治，本試驗以九個供試菌株，分別有三個菌株為黑殭菌(Metarhizium anisopliae)代號為MA126、3621、HO194，三個菌株為白殭菌(Beauveria bassiana)代號為BB-1、BBU、F121，二個菌株為蠟蚧輪枝菌(Verticillium lecanii)代號為VL-578、VL615，及一個曲霉菌(Aspergillus sp.)代號為M，測試對溫室內常見之偽菜蚜(Lipaphis erysimi)、桃蚜(Myzus persicae)及棉蚜(Aphis gossypii)之致病性。九個菌株培養在不同溫度下之菌絲生長及孢子發芽率則有顯著差異，但在高溫35℃時，HO194、BB-1、VL578及VL615之菌絲皆不生長；空氣中之相對溼度與九個菌株之孢子發芽率呈現正比之關係，且九個菌株之分生孢子發芽率是隨著培養基中水分活性下降而遞減；九個菌株均可產生蛋白質分解酵素，其中以黑殭菌MA126及輪枝菌VL615菌株，於測試培養基內產生之澄清透明環寬度直徑為最大，分別為2.86、2.98公分，同時九個菌株均檢測出lipase之酵素活性，此外利用API ZYM系統檢測出對蚜蟲有較高致病力之MA126、3621及VL615菌株，所產生之分解酵素如leucine aminopeptidase、valine aminopeptidase、trypsin、β-glucosidase及β-glucosaminidase的量較其它菌株為多。將蚜蟲以接種孢子濃度為1×108conidia/ml於28℃之室內試驗，發現MA126及VL615對三種蚜蟲之致死效果較其它菌株佳；另外於溫室試驗方面，在32℃以MA126對桃蚜之防治效果較好，危害指數(INFI)值為1.67，其次為3621及BBU，INFI值皆為2.0；在25.5℃則以VL615對桃蚜之防治效果較好，INFI值為1.0，其次為MA126，INFI值為1.5；因此在台灣秋冬低溫之季節，可施用輪枝菌VL615菌株來防治溫室內之蚜蟲，春夏較高溫之季節，可改用黑殭菌MA126菌株來噴施，藉由兩菌株於高低溫各有較強之致病性交替使用，在相對溼度高之溫室環境下，可有效控制溫室內之蚜蟲數量，同時應盡量避免和殺菌劑混合使用。

Selected isolates of entomopathogenic fungi were tested for their efficiency in controlling three major species of aphids, Lipaphis erysimi, Myzus persicae and Aphis gossypii commonly occurred in greenhouse. They were MA126, 3621 and HO194 of Metarhizium anisopliae, BB-1, BBU and F121 of Beauveria bassiana, VL578 and VL615 of Verticillium lecanii, M of Aspergillus sp. There were significantly difference in the effect of temperature on mycelial growth and germination rate of conidia of all nine tested isolates, but mycelial growth of HO194, BB-1, VL578, VL615 were inhibited at 35℃. The relative humidity was positively correlated with germination rate of conidia. In addition, the germination rate of conidia was low based on the decrease in water activity of medium. All nine isolates tested produced protease and lipase. But protease activities of MA126 and VL615 were better than those of the other isolates because the diameter of clearing ring from protease reaction for MA126 and VL615 were 2.86 and 2.98 cm, respectively. The three strong virulent isolates of MA126, 3621 and VL615 had higher enzymatic activities of leucine aminopeptidase, valine aminopeptidase, trypsin, β-glucosidase, and β-glucosaminidase, analyzed by the API ZYM system. Using conidial suspension concentration at 1×108conidia/ml as inoculum for pathogenicity bioassay of aphids, the results showed that the virulence of MA126 and VL615 against aphids was higher than that of other isolates at 28℃. The virulence of MA126 against Myzus persicae was better at 32℃, with an infestation index (INFI) value of 1.67, then for 3621 and BBU the INFI values were 2.0. However the virulence of VL615 against M. persicae was better at 25.5℃, the INFI value was 1.0, then MA126 the INFI value was 1.5. Therefore VL615 can be used against greenhouse aphids in the fall and winter seasons, but during spring and summer seasons MA126 can be used instead. Due to high relative humidity condition in the greenhouse these isolates can be used and reduce the over-usage of fungicides.

中文摘要-------------------------------------------------------------------Ⅰ
英文摘要-------------------------------------------------------------------Ⅲ
誌 謝-------------------------------------------------------------------Ⅴ
目 錄-------------------------------------------------------------------Ⅵ
圖次索引-------------------------------------------------------------------Ⅸ
表次索引-------------------------------------------------------------------Ⅺ
壹、前言--------------------------------------------------------------------1
貳、前人研究--------------------------------------------------------------4
一、 溫室內常見蚜蟲種類之簡介---------------------------------4
二、 蟲生真菌之定義及特性---------------------------------------6
(一)、蟲生真菌之定義----------------------------------------6
(二)、蟲生真菌之優缺點-------------------------------------6
(三)、蟲生真菌的分類----------------------------------------7
三、 感染蚜蟲之蟲生真菌種類及作用機制之探討---------- 10
(一)、感染蚜蟲之蟲生真菌種類-------------------------- 10
(二)、蟲生真菌感染蚜蟲之作用方式及機制-------------12
四、 影響蟲生真菌感染蚜蟲之因子---------------------------- 16
(一)、菌株間致病性之差異性-------------------------------16
(二)、溫度、溼度對蟲生真菌感染蚜蟲之影響----------17
(三)、農藥對蟲生真菌之影響------------------------------19
五、開發真菌性殺蟲劑防治蚜蟲之概況------------------------20
參、材料與方法-----------------------------------------------------------24
一、供試菌株來源與培養------------------------------------------25
二、供試蚜蟲之飼養------------------------------------------------27
三、接種源之配製---------------------------------------------------27
四、溫度對供試蟲生真菌菌絲生長之影響---------------------27
五、 供試蟲生真菌分生孢子發芽之最適溫度測試---------- 28
六、 水分活性對供試蟲生真菌分生孢子發芽之影響------- 30
七、 蟲生真菌對蚜蟲致病力之生物檢定(bioassay)----------32
(一)、室內試驗------------------------------------------------32
(二)、溫室試驗-- ---------------------------------------------35
八、 農藥對黑殭菌MA126、3621及輪枝菌VL615菌絲生長之影響- ---------------------------------------------------------37
(一)、殺菌劑對供試菌株生長影響之試驗----------------38
(二)、殺蟲劑對供試菌株生長影響之試驗----------------39
九、 蟲生真菌感染蚜蟲之致病過程觀察----------------------40
十、 各菌株產生之酵素檢測-------------------------------------41
(一)、固體培養基測試--------------------------------------41
(二)、API ZYM系統測試----------------------------------42
肆、結果------------------------------------------------------------------44
一、溫度對供試蟲生真菌菌絲生長之影響-------------------44
二、供試蟲生真菌分生孢子發芽之最適溫度測試----------44
三、水分活性對供試蟲生真菌分生孢子發芽之影響-------48
四、蟲生真菌對蚜蟲致病力之生物檢定----------------------55
(一)、室內試驗-----------------------------------------------55
(二)、溫室試驗-----------------------------------------------59
五、農藥對黑殭菌MA126、3621及輪枝菌VL615菌絲生長之影響----------------------------------------------------------66
六、蟲生真菌感染蚜蟲之致病過程----------------------------68
七、各菌株產生之酵素檢測-------------------------------------69
伍、討論------------------------------------------------------------------88
陸、參考文獻-----------------------------------------------------------108
柒、作者簡介-----------------------------------------------------------120
圖次索引
圖一、溫室內常見之蚜蟲種類A.偽菜蚜B.桃蚜C.棉蚜----------5
圖二、測試孢子發芽之V型玻棒高溼度培養皿裝置-------------29
圖三、測試孢子發芽可調控空氣相對濕度及培養基水分活性環境之雙層盒裝置-------------------------------------------------31
圖四、供試蟲生真菌測試蚜蟲致病力之生物檢定裝置--------- 33
圖五、噴藥塔裝置------------------------------------------------------ 34
圖六、高麗菜植株供蟲生真菌之生物檢定-------------------------36
圖七、九個供試蟲生真菌感染蚜蟲後於體表之產孢照片
----------------------------------------------------------------------56
圖八、桃蚜感染不同之蟲生真菌於高麗菜葉片死亡及產孢情形----------------------------------------------------------------------62
圖九、黑殭菌MA126於桃蚜體表菌絲發芽管前端形成膨大附著器之電子顯微鏡圖片-------------------------------------------77
圖十、輪枝菌VL615菌絲侵入桃蚜體表之電子顯微鏡圖片----------------------------------------------------------------------78
圖十一、黑殭菌HO194菌絲侵入偽菜蚜體表之電子顯微鏡圖片----------------------------------------------------------------------79
圖十二、輪枝菌VL578菌絲貫穿棉蚜腹管之電子顯微鏡圖片------------------------------------------------------------------80
圖十三、輪枝菌VL578菌絲佈滿於桃蚜體表之電子顯微鏡圖片------------------------------------------------------------------81
圖十四、黑殭菌3621感染桃蚜於體表產孢之電子顯微鏡圖片------------------------------------------------------------------82
圖十五、白殭菌BB-1感染偽菜蚜於體表產孢之電子顯微鏡圖片------------------------------------------------------------------82
圖十六、輪枝菌VL615感染桃蚜於體表產孢之電子顯微鏡圖片------------------------------------------------------------------83
圖十七、曲霉菌M感染偽菜蚜於體表產孢之電子顯微鏡圖片------------------------------------------------------------------83
圖十八、利用澄清環法測試供試蟲生真菌蛋白質分解酵素活性------------------------------------------------------------------84
圖十九、利用沉澱法測試供試蟲生真菌脂質分解酵素活性------------------------------------------------------------------85
圖二十、API ZYM系統檢測供試蟲生真菌產生分解酵素情形
-------------------------------------------------------------------86
表次索引
表一、九個供試蟲生真菌菌株之學名及來源---------------------26
表二、溫度對供試蟲生真菌培養七天之菌絲生長之影響---------------------------------------------------------------------45
表三、溫度對供試蟲生真菌經22小時培養之孢子發芽率之影
響------------------------------------------------------------------47
表四、空氣中97.5%R.H對供試菌株培養於不同水分活性培養基之孢子發芽率之影響------------------------------------------50
表五、空氣中91.5%R.H對供試菌株培養於不同水分活性培養基之孢子發芽率之影響------------------------------------------51
表六、空氣中84.5%R.H對供試菌株培養於不同水分活性培養基之孢子發芽率之影響------------------------------------------53
表七、供試蟲生真菌菌株於28℃100% R.H.下對偽菜蚜之致病
力------------------------------------------------------------------57
表八、供試蟲生真菌菌株於28℃100% R.H.下對桃蚜之致病力
---------------------------------------------------------------------61
表九、供試蟲生真菌菌株於28℃100% R.H.下對棉蚜之致病力
---------------------------------------------------------------------63
表十、利用蟲生真菌於25.5℃溫室中防治桃蚜經不同天數後桃蚜對高麗菜之危害指數----------------------------------------64
表十一、利用蟲生真菌於32℃溫室中防治桃蚜經不同天數後桃蚜對高麗菜之危害指數-------------------------------------65
表十二、殺菌劑對黑殭菌MA126菌株菌絲生長之抑制情形
-------------------------------------------------------------------71
表十三、殺菌劑對黑殭菌3621菌株菌絲生長之抑制情形
-------------------------------------------------------------------72
表十四、殺菌劑對蠟蚧輪枝菌VL615菌株菌絲生長之抑制情形
-------------------------------------------------------------------73
表十五、殺蟲劑對黑殭菌MA126菌株菌絲生長之抑制情形
-------------------------------------------------------------------74
表十六、殺蟲劑對黑殭菌3621菌株菌絲生長之抑制情形
-------------------------------------------------------------------75
表十七、殺蟲劑對蠟蚧輪枝菌VL615菌株菌絲生長之抑制情形
-------------------------------------------------------------------76
表十八、利用API ZYM系統測試供試蟲生真菌之酵素活性
-------------------------------------------------------------------87

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