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研究生:曾敏南
研究生(外文):89ntu00364005
論文名稱:黑殭菌(Metarhiziumanisopliaevar.anisopliae)耐高溫突變菌株之篩選及其生理、病原性之探討
論文名稱(外文):Metarhizium anisopliae var. anisopliae : high temperature tolerant mutant selection, physiology and infectivity.
指導教授:曾顯雄曾顯雄引用關係
指導教授(外文):Shean-Shong Tzeng
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理學研究所
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:2001
畢業學年度:89
語文別:中文
中文關鍵詞:黑殭菌高溫誘變生理
外文關鍵詞:Metarhizium anisopliaeheat tolerantmutantphysiology
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黑殭菌 (Metarhizium anisopliae) 為昆蟲寄生真菌 (entomopathogenic fungi) ,其寄主範圍廣泛,可多達200餘種 (species) 昆蟲。自1878年發現以來,曾被使用於許多有害昆蟲做為生物防治藥劑。然而許多不適合之田間環境條件,如高溫、乾燥及紫外線之傷害,可大幅降低昆蟲寄生真菌之防治效果。如果能改善黑殭菌對不良環境條件之耐受性,提昇其田間存活率,應可有效提高黑殭菌田間之應用效果。
本實驗利用紫外線照射的誘變方法,篩選耐高溫 (36oC) 之黑殭菌突變菌株。經誘變後,選出可於36oC中存活之菌株,並進一步檢測各突變株產生protease、lipase、chitinase及amylase之酵素活性,及其生長速率,最後獲得M. anisopliae 3615耐高溫且生長較快速之突變菌株。經形態特徵之觀察,突變株M. anisopliae 3615與野生株M. anisopliae 3621在菌落外觀上具有明顯之差異,前者在菌落培養之早期,即可發現由菌落中央向外呈現許多凹痕且菌落色澤在培養後期呈現灰褐色;後者菌落平滑並且呈現墨綠色澤;而光學顯微鏡之觀察,兩者之產孢構造並無差異。
在生長條件之評估上,於所有溫度條件,突變株之生長速率均比野生株來得快。突變株與野生株在32oC以下之生長條件時,孢子發芽率並無差異,但突變株之分生孢子在34及36oC高溫條件下,其發芽快,且發芽率明顯比野生株高。更有意義的是,突變株分生孢子培養於36oC中,第7天可形成直徑約0.3公分之菌落,而野生株之孢子雖可發芽,卻無菌落形成。在36oC中突變株雖然菌落生長緩慢,卻顯示可於此逆境下存活。諸多氮源中,以soy peptone對突變株生長及產孢之促進效果最為良好,其次為KNO3。此外,8種碳源中,以dextrose及mannose對突變株生長及產孢最佳,L-sorbose則明顯抑制生長並完全抑制產孢;在產孢方面,除L-sorbose外,所有碳素源均有助於產孢。而維生素中只有niacin對突變株生長有不良影響,其餘均無顯著差異;而且除了niacin之外,所有維生素均有促進產孢之效果。
試驗5種蔬菜油 (包括橄欖油、花生油、芝麻油、葵花子油及麥胚芽油) 及1種礦物油對突變株孢子發芽影響。所有油質對孢子第6小時之發芽率均具明顯之促進效果,比懸浮於0.5% Tween 80水溶液中高出6~12倍不等,其中又以麥胚芽油最佳,花生油次之。此外,懸浮於水中分生孢子受光波長280~320nm 紫外線照射8分鐘後,發芽完全受抑制;但添加麥胚芽油後,照射2小時後,培養24小時發芽率仍達100%。針對班飛蝨 (Laodelphax striatellus)、亞洲棕梠象鼻蟲 (Rhabdoscelus lineatocollis)、斜紋夜蛾 (Spodoptera litura)、條背土蝗 (Patanga succincta) 及紅斑獵椿象 (Eocanthecona furcellate) 檢測致病性之結果,突變株M. anisopliae 3615對亞洲棕梠象鼻蟲及紅斑獵椿象完全不具致病能力,對班飛蝨之成蟲致病力最佳,接種後第七天之死亡率為78 %。對條背土蝗之致死速率雖緩慢,但接種後明顯影響其取食行為。
The entomopathogenic fungus, Metarhizium anisopliae has broad host range covering more than 200 species of insects. It has been long recognized since 1878 as a promising agent for biological control of insect pests. However, a number of suppressive environmental factors, such as, high temperature, low relative humidity and ultraviolet damage always reduce the efficacy of entomopathogenic fungi in field trial. In order to improve the tolerance of unfavorable abiotic factors, experiments attempt on improving the efficacy of M. anisopliae in pest control were conducted.
In this study, several high temperature (36oC) tolerance mutants of M. anisopliae have been obtained by ultraviolet mutation. The mutants were further subjected to screening by the activities of protease, lipase, chitinase, and amylase and also by the growth rates. As a result, a heat tolerant and more rapid growth mutant M. anisopliae 3615 was gained. Under light microscopy, morphologically there are no obvious different in sporulating structures and conidia characteristic between the mutant and the wild type. However the colonies of the mutant showed distinct furrow in the early stages, and the color turned brownish gray in the late stage; in contrast colonies of the wild type were smooth, never sulcate, dark green.
Under a range of temperature regimes, the mutant 3615 exhibited faster growth rate than that of wild strain. Although germination rate of the both isolates was similar between 20~32oC, the mutant showed up a higher and faster germination rate than the wild type at 34oC and 36oC. Significantly, the mutant formed a colony around 0.3 cm at 7 day when incubated at 36 oC, whereas wild type, though conidia rarely germinated but no colony accessible under the same condition. The result showed even though M. anisopliae 3615 growth rate was somewhat retarded at 36 oC, it still can survive under such adversarial condition. To optimize inoculum source for field application, an array of nutritional factors were tested. Of various hydrogen sources, soy peptone was the most optimal for growth and sporulation of M. anisopliae 3615, follow by KNO3. Among the 8 carbon sources, dextrose and mannose were the most conducive for growth and sporulation, but L-sorbose considerably suppressed the growth and totally inhibited sporulation. All carbon sources except L-sorbose favored sporulation. All vitamins except niacin increased sporulation, but had no noticeable effect on the growth of M. anisopliae 3615, compared to control.
Of five vegetable oils, olive oil, peanut oil, sesame oil, sunflower oil and wheat germ oil, and one mineral oil tested to evaluate its’ effect on germination and UV radiation protection towards M. anisopliae 3615. The results indicated that all six oils increased the germination rate of spore 6~12 times than water containing 0.5 % Tween 80 surfactant. Wheat germ oil appeared to be the best for germination, follow by peanut oil. Moreover, the germination was nil when conidia suspended in water exposed to 280~320nm ultraviolet light for 8 minutes. While wheat germ oil added, even after exposure to ultraviolet light for 2 hours, the germination rate can still reach 100 % after 24 hours incubation. The pathogenicity of the mutant, M. anisopliae 3615 was assessed toward various insects: brown planthopper (Laodelphax striatellus), yellow palm beetle (Rhabdoscelus lineatocollis), tobacco cutworm (Spodoptera litura), locust (Patanga succincta), and hunter sting bug (Eocanthecona furcellate). Of them, the brown planthopper was the most susceptible, with a 78% mortality in 7 days after inoculation. Conversely, yellow palm beetle and hunter sting bug were immune, no infection taking place.
中文摘要-------------------------------------------------------1
英文摘要-------------------------------------------------------3
壹、前言-------------------------------------------------------5
貳、前人研究---------------------------------------------------7
一、昆蟲寄生菌之分類概況-----------------------------------7
二、黑殭菌之歷史-------------------------------------------8
三、黑殭菌形態特徵與分類體系-------------------------------9
四、昆蟲寄生性真菌之侵染模式-------------------------------9
(一) 昆蟲之抗真菌體系---------------------------------10
(二) 黑殭菌對寄主昆蟲的侵染過程-----------------------11
(三) 侵染過程酵素之參與-------------------------------16
五、黑殭菌之生態特性--------------------------------------18
(一) 溫度對黑殭菌之影響-------------------------------19
(二) 濕度對黑殭菌之影響-------------------------------20
(三) 紫外線對黑殭菌之影響-----------------------------21
六、對動物及非目標昆蟲之安全性以及對生態系之衝擊----------22
七、改善黑殭菌抗逆境能力之研究現況------------------------24
參、材料方法--------------------------------------------------27
一、致突變方法--------------------------------------------27
(一)野生菌株來源--------------------------------------27
(二)野生株之紫外線誘變--------------------------------27
二、突變株Metarhizium anisopliae 3615之形態特徵-----------30
(一)分生孢子之形態特徵--------------------------------30
(二)不同生長時期之分生孢子發芽率----------------------31
(三)產孢枝之形態特徵----------------------------------31
(四)菌落形態特徵及生長率------------------------------31
三、M. anisopliae 3615 生理特性探討-----------------------32
(一)溫度對M. anisopliae 3615 菌落生長之影響-----------32
(二)不同培養溫度對M. anisopliae 3615 產孢之影響-------32
(三)不同培養溫度對M. anisopliae 3615所產生之孢子發芽之
影響----------------------------------------------33
(四)溫度對M. anisopliae 3615 孢子發芽之影響-----------33
(五)水分活性 (water activity) 對M. anisopliae 3615生長
之影響--------------------------------------------34
(六)紫外線對M. anisopliae 3615孢子發芽之影響----------34
(七)不同碳、氮素源及維生素 (vitamin) 對M. anisopliae
3615生長、菌落形成及產孢之影響--------------------34
(八)農業藥劑對M. anisopliae 3615之影響----------------37
(九) 不同油質對M. anisopliae 3615孢子發芽之影響-------38
(十) 不同油質對紫外線之保護效果-----------------------38
(十一) 脂肪酸對M. anisopliae 3615孢子發芽之影響-------38
四、M. anisopliae 3615之寄主範圍與致病力------------------
(一) M. anisopliae 3615對斜紋夜蛾 (Spodoptera litura)
之致病性-----------------------------------------39
(二) M. anisopliae 3615對班飛蝨 (Laodelphax
striatellus) 之致病性----------------------------40
(三) M. anisopliae 3615對亞洲棕梠象鼻蟲 (Rhadoscelus
lineatocollis) 之致病性--------------------------40
(四) M. anisopliae 3615條背土蝗 (Patanga succincta) 之
致病性-------------------------------------------41
(五) M. anisopliae 3615對紅班獵椿象 (Eocanthecona
furcellate) 之致病性-----------------------------41
肆、結果------------------------------------------------------43
一、耐高溫突變菌株之誘變----------------------------------43
(一) 致死率測定---------------------------------------43
(二) 耐高溫突變菌株篩選-------------------------------43
二、突變株Metarhizium anisopliae 3615之形態特徵-----------44
(一) 產孢枝及分生孢子之形態特徵-----------------------44
(二) 不同培養時期之孢子發芽率-------------------------44
(三) 菌落形態特徵及生長速率---------------------------45
三、Metarhizium anisopliae 3615生理特性探討---------------46
(一)溫度對M. anisopliae 3615生長之影響----------------46
(二)溫度對M. anisopliae 3615產孢之影響----------------47
(三)不同培養溫度對M. anisopliae 3615所產生之孢子發芽率
的影響--------------------------------------------47
(四)溫度對M. anisopliae 3621及M. anisopliae 3615孢子發47
芽率之影響----------------------------------------48
(五)水分活性 (water activity) 對M. anisopliae 3615之影
響------------------------------------------------48
(六)紫外線對M. anisopliae 3615孢子發芽之影響----------48
(七)不同氮素源對M. anisopliae 3615生長、菌落形成及產孢
之影響--------------------------------------------49
(八)不同碳素源對M. anisopliae 3615生長、菌落形成及產孢
之影響--------------------------------------------49
(九)不同維生素對M. anisopliae 3615生長、菌落形成及產孢
之影響--------------------------------------------49
(十) 農對藥劑對M. anisopliae 3615之影響---------------50
(十一) 不同油質對M. anisopliae 3615孢子發芽之影響-----50
(十二) 麥胚芽油對紫外線輻射傷害之保護效果-------------51
(十三) 脂肪酸對M. anisopliae 3615孢子發芽之影響-------51
四、M. anisopliae 3615之寄主範圍與致病力------------------52
(一) M. anisopliae 3615對斜紋夜蛾 (Spodoptera litura)
之致病性------------------------------------------52
(二) M. anisopliae 3615對班飛蝨 (Laodelphax
striatellus) 之致病性-----------------------------52
(三) M. anisopliae 3615對亞洲棕梠象鼻蟲 (Rhadoscelus
lineatocollis) 之致病性---------------------------52
(四) M. anisopliae 3615對條背土蝗 (Patanga succincta)
之致病性性----------------------------------------52
(五) M. anisopliae 3615對紅班獵椿象 (Eocanthecona
furcellate) 之致病性------------------------------52
伍、討論------------------------------------------------------54
一、與侵染有關之酵素檢測----------------------------------54
二、突變株之形態特徵--------------------------------------55
三、M. anisopliae 3615生理探討----------------------------56
四、M. anisopliae 3615之寄主範圍與致病------------------------66
圖------------------------------------------------------------69
表------------------------------------------------------------80
陸、參考文獻--------------------------------------------------92
附錄---------------------------------------------------------104
圖序
圖一、M. anisopliae 3621(A)及M. anisopliae 3615(B)產生蛋白質分解酵素 (protease) 活性之檢測------------------------------------69
圖二、光學顯微鏡觀察M. anisopliae 3615及M. anisopliae 3621之分生孢子梗,瓶形小梗,以及分生孢子--------------------------------70
圖三、於黑殭菌突變菌株 (M. anisopliae 3615) 不同培養時期之菌落形態特徵--------------------------------------------------------71
圖四、突變株M. anisopliae 3615及野生株M. anisopliae 3621培養第25天之菌落特徵比較----------------------------------------------72
圖五、突變株M. anisopliae 3615及野生株M. anisopliae 3621於36oC (A) 及25oC (B) 溫度條件中之菌落特徵比較-----------------------73
圖六、M. anisopliae 3615感染之條背土蝗 (Patanga succincta) (A,B)及班飛蝨 (Laodelphax striatellus) (C,D)-----------------------74
圖七、溫度對突變株 (M. anisopliae 3615)及野生株 (M. anisopliae 3615) 生長之影響----------------------------------------------75
圖八、 (A)溫度對Metarhizium anisopliae 3615 (突變株) 及Metarhizium anisopliae 3621 (野生株) 培養12小時後孢子發芽之影響---------------------------------------------------------------76
圖八、(B)溫度對Metarhizium anisopliae 3615 (突變株) 及Metarhizium anisopliae 3621 (野生株) 培養24小時後孢子發芽之影響---------------------------------------------------------------76
圖九、不同polyethylene glycol (PEG) 莫耳濃度對M. anisopliae 3615 生長之影響-----------------------------------------------77
圖十、(A)黑殭菌突變菌株 (M. anisopliae 3615) 不同濃度孢子懸浮液對2~3齡班飛蝨若蟲之致死率-------------------------------------78
圖十、(B)黑殭菌突變菌株 (M. anisopliae 3615) 不同濃度孢子懸浮液對班飛蝨成蟲之致死率------------------------------------------78
圖十一、(A)黑殭菌突變菌株 (M. anisopliae 3615) 不同濃度孢子懸浮液對2~3齡斜紋夜蛾之致死率-------------------------------------79
圖十一、(B)黑殭菌突變菌株 (M. anisopliae 3615) 不同濃度孢子懸浮液對條背土蝗之致死率------------------------------------------79
表序
表一、16株耐高溫黑殭菌(Metarhizium anisopliae) 突變菌株及野生株M.anisopliae 3621生長速率及酵素活性之比較---------------------80
表二、於黑殭菌突變株 (Metarhizium anisopliae 3615)不同培養時期之孢子發芽率----------------------------------------------------81
表三、不同溫度範圍對M. anisopliae 3615 (突變株) 產孢量及所產生孢子發芽率之影響------------------------------------------------82
表四、不同polyethylene glycol (PEG) 莫耳濃度對M. anisopliae 3615 生長及孢子發芽之影響-------------------------------------83
表五、紫外線輻射對M. anisopliae 3615分生孢子之傷害------------84
表六、氮素源對M. anisopliae 3615菌落形成,菌絲生長及產孢量之影響------------------------------------------------------------85
表七、碳素源對M. anisopliae 3615菌落形成,菌絲生長及產孢量之影響------------------------------------------------------------86
表八、維生素對M. anisopliae 3615 菌落形成,菌絲生長及產孢量之影響------------------------------------------------------------87
表九、農業藥劑對M. anisopliae 3615 生長及孢子發芽率之影響-----88
表十、不同油質對M. anisopliae 3615分生孢子發芽之影響----------89
表十一、麥胚芽油保護M. anisopliae 3615分生孢子受紫外線輻射傷害之效果----------------------------------------------------------90
表十二、不同脂肪酸對M. anisopliae 3615之分生孢子發芽影響------91
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