臺灣博碩士論文加值系統

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

林慶元、呂文通。1991。利用蟲生真菌 (黑殭菌) 防治青蔥甜菜夜蛾。花蓮區農業改良場研究彙報 7:127-131。
萬鑫森。1991。基礎土壤物理學。國立編譯館。
蒲哲龍、李增智。1996。昆蟲真菌學。安徽科學技術出版社。
蔡三福、王順成。1997。微生物應用之管理及安全性評估。中華昆蟲學會。昆蟲生態及生物防治研討會專刊 161-168頁。
蔡三福、廖俊旺、洪文凱、王順成。1994。黑殭菌對白鼠肺急毒性、感染性及致病性之探討。植物保護學會會刊 36: 65-73。
蔡勇勝、高清文、侯豊男、高穗生。1993。黑殭菌抗殺菌劑菌株之篩選。中華昆蟲 13: 45-57。
蔡勇勝、高穗生、高清文、侯豐男。1992。數種殺蟲劑對黑殭菌感染甜菜夜蛾之影響。植物保護學會會刊 34: 216-226。
鄭清煥。1998。班飛蝨。植物疫情監測技術手冊 (I) 蟲害篇。台灣省政府農林廳編印。8~10頁。
關崇智。1987。昆蟲生理學。國立編譯館。
Ahrens, M. J., and Ingram, D. L. 1988. Heat tolerance of citrus leaves. Hort Sci. 23: 747-748.
Alexopoulos, C. J., Mims, C. W., and Blackwell, M. 1996. Introductory Mycology 4th ed. Wiley Press. New York.
Alves, R. T., Bateman, R. P., Prior, C., and Leather, R. 1998. Effects of simulated solar radiation on conidial germination of Metarhizium anisopliae in different formulations. Crop Prot. 17: 675-679.
Alves, S. B., Marchini, L. C., Pereira, R. M., and Baumgratz, L. L. 1996. Effects of some insect pathogens on the argicanized honey bee, Apis mellifera L. (Hym., Apidae). J. Appl. Ent. 120: 559-564.
Barnes, S. E., and Moore, D. 1997. The effect of fatty, organic or phenolic acids on the germination of conidia of Metarhizium flavoviride. Mycol. Res. 101: 662-666.
Barnett, H. L. and Hunter, B. B. 1987. Illustrated Genera of Imperfect. Fungi 4th ed. Macmillan Press. New York.
Barson, G., Renn, N., and Bywater, A. F. 1994. Laboratory evaluation of six species of entomopathogenic fungi for the control of the housefly (Musca domestica L.), a pest of intensive animal units. J. Invertebr. Pathol. 64: 107-113.
Bateman, R. P., Carey, M., Moore, D., and Prior, C. 1993. The enhanced infectivity of Metarhizium flavoviride in oil formulations to desert locusts at low humidities. Ann. Appl. Biot. 122, 145-152.
Baucias, D. G., and Pendland, J. C. 1991. Attachment of mycopathogens to cuticle: the initial event of mycoses in arthropod hosts. In The Fungal Spore and Disease Initiation in Plants and Animals (G. T. Cole and H. C. Hoch ed.), pp.101-127. Plenum Publishing Co., New York and London.
Blanford, S., Thomas, M. B., and Langewald, J. 1998. Behavioural fever in the Senegalese grasshopper, Oedaleus senegalensis, and its implications for biological control using pathogens. Ecol. Entomol. 23: 9-14.
Butt, T. M., Ibrahim, L., Ball., B. V., and Clark, S. J. 1994. Pahtogenicity of the entomogenous fungi Meatrhizium anisopliae and Beauveria bassiana against crucifer pests and the honey bee. Biocontrol Sci. and Technol. 4: 207-214.
Butt, T. M., Ibrahim, L., Clark, S. J., and Beckett, A. 1995. The germination behaviour of Metarhizium anisopliae on the surface of aphid and flea beetle cuticles. Mycol. Res. 99: 945-950.
Campbell, R. K., Barnes, B. L., Cartwright, B. O., and Eikenbary, R. D. 1983. Growth and sporulation of Beauveria bassiana and Metrhizium anisopliae in a basal medium containing various carbohydrate sources. J. Invertebr. Pathol. 41: 117-121.
Castillo, M. A., Pilar. M., Enrique, H., and Eduardo, P. Y. 2000. Susceptibility of Ceratitis capitata wiedemann (Diptera: Tephritidae) to entomopathogenic fungi and their extracts. Biocontrol 19: 274-282.
Cepero, D. G., Garcia, M. C., Arboleda, M. L., Barraquer, F., and Grose, E. 1997. Fungal keratitis caused by Metarhizium anisopliae var. anisopliae. J. Med. Vet. Mycol. 35: 361-363.
Chaisompongopan, N., Li, P. H., Davis, D. W., and Mackhart, A. H. 1990. Photosynthetic responses to heat stress in common bean genotypes differing in heat acclimation potential. Crop Sci. 30: 100-104.
Charnley, A. K., and St. Leger, R. J. 1991. The role of cuticle-degrading enzymes in fungal pathogenesis in insects. In “The fungal spore and disease initiation in plants and animals.” Cole, G. T., and Hoch, H. C. ed. Plenum, London. p.267-287
Crowe, J. H., Crowe, L. M., and Chapman, D. 1984. Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223: 701-703.
Daoust, R. A. and Roberts, D. W. 1986. Virulence of a natural isolate, auxotrophic mutants, and a diploid of Metarhizium anisopliae var. anisopliae to Rhodnius prolixus. J. Invertebr. Pathol. 47: 231-233.
Dara, S. K., and Semtner, P. J. 1998. Influence of substrate type and temperature on the developmental morphology of Pandora neoaphidis (Zygomycetes: Entomophthorales), a pathogen of the tobacco aphid (Homoptera: Aphididae). J. Invertebr. Pathol. 72: 112-118.
Doberski, J. W., and Tribe, H. T. 1980. Isolation of entomogenous fungi from elm bark and soil with reference to ecology of Beauveria bassiana and Metarhizium anisopliae. Trans. Br. Mycol. Soc. 74: 95-100.
Donovan-Peluso, M., Wasti, S. S., Hartmann, G. C., 1980. Safety of entomogenous fungi to vertebrate hosts. Appl. Entomol. Zoo. 15: 498-499.
Dwayne, D. H., and Khachatourians, G. G. 1996. The effects of temperature on the pathogenicity of heat-sensitive mutants of the entomopathogenic fungus, Beauveria bassiana, toward the migratory grasshopper, Melanoplus sanguinipes. J. J. Inverteber. Pathol. 68: 16165.
El-Sayed, G. N. 1993a. Insect cuticle and yease extract effects on germination, growth, and production of hydrolytic enzymes by Nomuraea rileyi. Mycopathologia 122: 143-1477.
El-Sayed, G. N., Ignoffo, C. M., Leathers, T. D., and Cupta, S. C. 1993b. Effect of cuticle source and concentration on expression of hydrolytic enzymes by an entomopathogenic fungus, Nomuraea rileyi. Mycopathologia 122: 149-152.
Elseth, G. D., and Baumgarduer, K. D. 1984. Genetics. Addisson-Wesley press. London.
Fargues, J., Goettel, M. S., Smits, N., Ouedraogo, A., Vidal, C., Lacey, L. A., Lomer, C. J., and Rougier, M. 1996. Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes. Mycopathologia 135: 171-181.
Gannarsson, S. G. S. 1988. Infection of Schistocerca gregaria by the fungus Metarhisium anisopliae: cellular reaction in the integument studied by scanning electon and light microscopy. J. Invertebr. Pathol. 52: 9-17.
Genthner, F. J., Chancy, C. A., Couch, J. A., Foss, S. S., Middaugh, D. P., George, S. E., Warren, M. A., and Bantle, J. A. 1998. Toxicity and pathogenicity testing of the insect pest control fungus Metarhizium anisopliae. Arch. Environ. Contam. Toxicol. 35: 317-324.
Genthner, F. J., Foss, S. S., Glas, P. S. 1997. Virulence of Metarhizium anisopliae to embryos of the grass shrimp Palaemonetes pugio. J. Invertebr. Pathol. 69:157-164.
Genthner, F. J., Middaugh, D. P., Foss, S. S. 1995. Validation of embryo tests for determining effects of fungal pest control agents on nontarget aquatic animals. Arch. Environ. Contam. Toxicol. 29: 540-544.
Gillespie, J. P., Bateman, R., and Charnley, A. K. 1998. Role of cuticle-degrading proteases in the virulence of Metarhizium spp. for the desert locust, Schistocerca gregaria. J. Invertebr. Pathol. 71: 128-137.
Goettel, M. S., Reger, R. J. S., Bhairi, S., Jung, M. K., Oakley, B. R., Roberts, D. W., and Staples, R. C. 1990. Pathogenicity and growth of Metarhizium anisopliae stably transformed to benomyl resistance. Curr. Genet. 17: 129-133.
Goettel, M. S., Stleger, R. J., Rizzo, N. W., Staples, R. C., and Roberts, D. W. 1989. Ultrastructural localization of a cuticle-degrading protease produced by the entomopathogenic fungus Metarhizium anisopliae during penetration of host (Manduca Sexta) cuticle. J. Gen. Microbiol. 135: 2233-2239.
Golkar, L., Lebrun, R. A., Ohayon, H., Gounon, P., Papierok, B., and Brey, P. T. 1993. Variation of larval susceptibility to Lagenidium giganteum in three mosquito species. J. Invertebr. Pathol. 62: 1-8.
Gupta, S. C., Leathers, T. D., El-Sayed, G., and Ignoffo, C. G. 1991. Production of degradative enzymes by Metarhizium anisopliae during growth on defined media and insect cuticle. Exp. Mycol. 15: 310-315.
Hajek, A. E. 1997. Ecology of terrestrial fungal entomopathogens. In “Advance in Microbial Ecology, Volume 15” (Jones ed.) p.193-249 Plenum Press, New York.
Hall, R. A., Peterkin, D. D., Ali, B., and Lopez, V. F. 1994. Inflence of culture age on rate of conidiospore germination in four deuteromycetous entomogenous fungi. Mycol. Res. 98: 763-768.
Hallsworth, J. E., and Magan, N. 1994. Improved biological control by changing polyols/trehalose in conidia of entomopathogens. “In Brighton Crop Protection Conference-Pests and Diseases.” British Crop Protection Council, Farnham. United Kingdom. p. 1091-1096.
Hallsworth, J. E., and Magan, N. 1995. Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability. Microbiology (UK) 141:1109-1115.
Hallsworth, J., and Magan, N. 1996. Culture age, temperature, and pH affect the polyol and trehalose contents of fungal propagules. Appl. Environ. Microbio. 62: 2345-2442.
Hankin, L., and Anagnostakis, S. L. 1975. The use of solid midia for detection of enzyme production by fungi. Mycologia 67:590-607.
Hashmi, S., Hashmi, G., and Gaugler, R. 1995. Genetic transformation an entomopathogenic nematode by microinjection. J. Invertebr. Pathol. 66: 293-296.
Hatting, J. L., Humber, R. A., Poprawski, T. J., Miller, R. M. 1999. A survey of fungal pathogens of aphids from South Africa, with special reference to cereal aphids. Biol. Control 16: 1-12.
Hedgecock, S., Moore, D., Higgins, P. M., and prior, C. 1995. Influence of moisture content on temperature tolerance and storage of Metarhizium flavoviride conidia in an oil formulation. Biocontrol Sci. and Technol. 5: 371-377.
Holdom, D. G., and Van Der Klashorst, G. 1986. Inexpensive culture methods and materials for Nomuraea rileyi. J. Invertebr. Pathol. 48: 159-176.
Hong, T. D., Ellis, R. H., and Morre, D. 1997. Development of a model to predict the effect of tempeature and moisture on fungal spore longevity. Ann. Bot. 79: 121-128.
Hong, T. D., Jenkins, N. E., Ellis, R. H., and Moore, D. 1998. Limits to the negative logarithmic relationship between moisture content and longevity in Metarhizium flavoviride. Ann. Bot. 81: 625-630.
Hywel-Jones, N. L., and Gillespie, A. T. 1990. Effect of temperature on spore germination in Metarhizium anisopliae and Beauveria bassiana. Mycol. Res. 94: 389-392.
Ignoffo, C. M., and Garcia, C. 1992. Influnce of conidial color on inactivation of several entomogenous fungi (Hyphomycetes) by simulated sunlight. Ann. Entomol. Soc. Am. 21: 913-917.
Inch, J. M., Humphreys, A. M., Trinci, A. P. J., and Gillespie, A. T. 1986. Growth and blastospore formation by Paceilomyces fumosoroseus, a pathogen of brown planthopper (Nilaaparvta lugens). Trans. Br. Mycol. Soc. 87: 215-222.
Inglis, G. D., Johnson, D. L., and Goettel, M. S. 1996. An oil-bait method used to test the efficacy of Beauveria baddiana against grasshoppers. J. Invertebr. Pathol. 67: 312-315.
James, R. R., Lighthart, B. 1994. Susceptibility of the convergent lady beetly to four entomopathogenic fungi. Environ. Entomol. 23: 188-190.
Jeanne, M., Inch, M., and Trinci, A. P. J. 1987. Effects of water activity on growth and sporulation of Paecilomyces farinosus in liquid and solid media. J. Gen. Microbiol. 133: 247-252.
Jensen, A. B., Gargas, A., Eilenberg, J., and Rosendahl, S. 1998. Relationships of the insect-pathogenic order Entomophthorales (Zygomycota, Fungi) based on phylogenetic analyses of nuclear small subunit ribosomal DNA sequences (SSU rDNA). Fungal Genet. Biol. 24: 325-334.
Kerwin, J. L. 1987. Fatty acids and fungal development: structure-ativity relationships. In Ecology and Metabolism of Plant Lipids (G. Fuller, and W. D. Nes ed.), pp.329-342. America Chemical Society, Symposium Series No. 325, Washington, DC.
Kintzios, S., Drossopoulos, J. B., and Lymperopoulos, C. 2000. Effect of vitamins and inorganic micronutrients on callus growth and somatic embryogenesis from young mature leaves of rose. J. Plant Nutr. 23: 1407-1420.
Krueger, S. R., Villani, M. G., Nyrop, J. P. and Roberts, D. W. 1991. Effect of soil environment on the efficacy of fungal pathogens against scarab grubs in laboratory bioassays. Biol. Control 1: 203-209.
Leopold, J., and Samsinakova, A. 1970. Quantitative estimation of chitinase and several other enzymes in the fungus Beauveria bassiana. J. Invertebr. Pathol. 15: 34-42.
Li, D. P., and Holdom, D. G. 1993. Effect of soil matric potential on sporulation and conidial survival of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). J. Invertebr. Pathol. 32: 273-277.
Li, D. P., and Holdom, D. G. 1994. Effects of pesticides on growth and sporulation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). J. Invertebr. Pathol. 63: 209-211.
Li, D. P., and Holdom, D. G. 1995. Effect of nutrients on colony formation, growth, and sporulation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). J. Invertebr. Pathol. 65: 253-260.
Lin, L., and Fu, T. 1988. Virulence of 14 Metarhizium anisopliae strains to mosquito larvae, flies, and cockroaches. “In Study and Application of Entomogenous Fungi in China, Volume II.” Application of entomogenous fungi in China ed. p.179. Academic Periodical Press. Beijing.
Lomer, C. J., Bateman, R. P., Godonou, I., Kpindou, D., Shah, P., Paraiso, A. and Prior, C. 1993. Field infection of Zonocerus fvariegatus following application of an oil based formulation of Metarhizium flavoviride conidia. Biocontrol Sci. and Technol.3: 337-346.
Lucarotti, C. J., and Shoulkamy, M. A. 2000. Coelomomyces stegomyiae infection in adult female Aedes aegypti following the first, second, and third host blood meals . J. Invertebr. Pathol. 75: 292-295
Luz, C., Silva, I. G., Cordeiro, C. M. T., and Tigano, M. S. 1999. Sporulation of Beauveria bassiana on cadavers of Triatoma infestans after infection at different temperatures and does of inoculum. J. Invertebr. Pathol. 73: 223-225.
Manocha, M. S. and Chen, Y. 1990. Specificity of attachment of fungal parasites to their hosts. Can. J. Microbiol. 36: 69-76.
Matewele, P., Trinci, A. P. J., and Gillespie, A. T. 1994. Mutants of entomopathogenic fungi that germinate and grow at reduced water activities and reduced relative humidities are more virulent to Nephotettix virescens (green leafhopper) than the parental strains. Mycol. Res. 98: 1329-1333.
McCammon, S. A., and Rath, A. C. 1994. Separation of Metarhizium anisopliae strains by temperature dependent germination rates. Mycol. Res. 98: 1253-1257.
Milner, R. J., Staples, J. A., and Lutton, G. G. 1997. The effect of humidity on germination and infection of termites by the Hyphomycete, Metarhizium anisopliae. J. Invertebr. Pathol. 69: 64-69.
Moorhose, E. R., and Gillespie, A. T., and Charnley, A. K. 1993. Application of Metarhizium anisopliae (Metsch.) Sor. Conidia to control Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae) larvae on glasshouse pot plants. Ann. Appl. Biol. 122: 523-636.
Morley-Daries, I., Moore, D., and Prior, C. 1995. Screening of Metarhizium and Beauveria spp. conidia with exposure to simulated sunlight and a range of temperatures. Mycol. Res. 100: 31-38.
Morre, D., Bridge, P. D., Higgins, P. M., Bateman, R. P., and Prior, C. 1993. Ultra-violet radiation damage to Metarhizium flavoviride conidia and the protection given by vegetable and mineral oil and chemical sunscreens. Ann. Appl. Biol. 122: 605-616.
Mulla, M. S. 1991. Biological control of mosquitoes with entomopathogenic bacteria. “In Proceedings of the IVth national vector control symposium Special Publ. No. 6. p.93-104. Taichung. Taiwan, R.O.C. Chinese Journal Entomology.
Paterson, I. C., Charnley, A. K., Cooper, R. M., and Clarkson, J. M. 1994. Specific induction of a cuticle-degrading protease of the insect pathogenic fungus Metarhizium anisopliae. Microbiology 140: 185-189.
Pelham, H. 1986. Speculations on the major heat shock and glucose regulated protein. Cell 46:959-961.
Pfyffer, G. E., and Rasts, D. M. 1988. The polyol pattern of fungi as influenced by the carbonhydrate source. New Phytol. 109: 121-126.
Quintela, E. D., and McCoy, C. W. 1998. Conidial attachment of Metarhizium anisopliae and Beauveria bassiana to the larval cuticle of Diaprepes abbreviatus (Coleoptera: Curculionidae) treated with imidacloprid. J. Invertebr. Pathol. 72: 220-230.
Roberts, D. W. 1966. Toxins from the entomogenous fungus Metarhizium anisopliae. J. Invertebr. Pathol. 8:22-227.
Roberts, D. W. 1981. Toxins of entomopathogenic fungi. “In Microbial Control of Pests and Plant Disease 1970-1980.” Burges, H. D. ed. p.441-464. Academic press. New York.
Rosato, Y. B., Messias, C. L., and Azevedo, J. L. 1981. Production of extracellular enzymes by isolates of Metarhizium anisopliae. J. Invertebr. Pathol. 38: 1-3.
Samuels, R. I., Charnley, A. K., Reynolds, S. E. 1988a. The role of destruxins in the pathogenicity of three strains of M. anisopliae for the tobacco hornworm Manduca sexta. Mycopathologia 104: 51-58.
Samuels, R. I., Reynolds, S. E., Charnley, A. K. 1988b. Calcium channel activation of insect muscle by destruxins, insecticidal compounds produced by the entomopathogenic fungus Metarhizium anisopliae. Comp. Biochem. Physiol. C 90: 403-413.
Senionitc, E., Manetos, Y., and Gavales, N. A. 1986. Co-operative effects of light and temperature on the activity of phosphoenolpyruvate carboxylase from Amaranthus paniculatus. Plnat Physiol. 82: 518-522.
Shadduck, J. A., Roberts, D. W., and Lause, S. 1982. Mammalian safety tests of Metarhizium anisopliae: preliminary results. Environ. Entomol. 11: 189-192.
Smith, N., Fargues, J., Rougier, M., Gouiet, R., and Itier, B. 1996. Effects of temperature and solar radiation interactions on the survival of quiescent conidia of the entomopathogenic hyphomycete Paecilomyces fumosoroseus (Wize) Brown and Smith. Mycopathologia 135: 163-170.
St. Leger, R. J. 1989a. Produfction in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae. Exp. Mycol. 13: 274-288.
St. Leger, R. J., But, T. M., Staples, R. C., and Roberts, D. W. 1989b. Synthesis of proteins including a cuticle-degrading protease during differentiation of the entomopathogenic fungus Metarhizium anisopliae. Exp. Mycol. 13: 253-262.
St. Leger, R. J., Butt, M. T., Goettel, M. S., Staples, R. C., and Roberts, D. W. 1989c. Production in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae. Exp. Mycol. 13: 274-288.
St. Leger, R. J., Charnley, A. K., and Cooper, R. M. 1986a. Cuticle-degrading enzymes of entomopathogenic fungi: mechanisms of interaction between pathogen enzymes and insect cuticle. J. Invertebr. Pathol. 47: 295-302.
St. Leger, R. J., Charnley, A. K., and Cooper, R. M. 1986b. Cuticle degrading enzymes of entomopathogenic fungi: synthesis in culture on cuticle. J. Invertebr. Pathol. 48: 85-95.
St. Leger, R. J., Durrands, P. K., Charnley, A. K., and Cooper, R. M. 1988. Role of extracellular chmoelastase in the virulence of Metarhizium anisopliae for Manduca sexta. J. Invertebr. Pathol. 52: 285-292.
St. Leger, R. J., Goettel, M., Roberts, D. E., and Staples, R. C. 1991. Prepenetration events during infection of host cuticle by Metarhizium anisopliae. J. Invrtebr. Pathol. 58:168-179.
St. Leger, R. J., Joshi, L., Bidochka, M. J., Rizzo, N. W., and Roberts, D. W. 1996. Characterization and ultrastructural localization of chitinases from Metarhizium anisopliae, M. flavoviride, and Beauveria bassiana during fungal invasion of host (Manduca: Sexta) cuticle. Appl. Environ. Microbiol. 62: 907-912.
St. Leger, R. J., Roberts, D. W., and Staples, R. C. 1989d. Calcium and calmodulin-mediated protein synthesis and protein phosphorylation during germination, growth, and protease production by Metarhizium anisopliae. J. Gen. Microbiol. 135: 2141-2154.
Valadares-Inglis, M. C., and Peberdy, J. F. 1997. Location of chitinolytic enzymes in protoplast and whole cells of the entomopathogenic fungus Metarhizium anisopliae. Mycol. Res. 101: 1393-1396.
Vanninen, I. 1995. Distribution and occurrence of four entomopathogenic fungi in Finland: effect of geographical location, habitat type and soil type. Mycol. Res. 100: 93-101.
Vestergaard, S., Butt, T. M., Bresciani, J., Gillespie, A. T., and Eilenbery, J. 1999. Light and electron microscopy studies of the infection of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae) by the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 73: 23-33.
Vilcinskas, A., Matha, V., and Gotz, P. 1997. Inhibition of phagocytic activity of plasmatocytes isolated from Galleria mellonella by entomogenous fungi and their secondary metabolites. J. Insect. Physiol. 43: 475-483.
Woods, S. P., and Grula, E. A. 1984. Utilisable surface nutrients on Heliothis zea available for growth of Beauveria bassiana. J. Invertebr. Pathol. 43:259-269.
Wu, J., Yang, L., and Wan, Y. 1990. Effect of lipids on infection of Metarhizium anisopliae to white grubs. In “Study and Application of Entomogenous Fungi in China, Volume I.” p.165-170. Academic Periodical Press. Beijing.
Zacharuk, R. Y. 1970. Fine structure of the fungus Metarhizium anisopliae infecting three species of larval elateridae (Coleoptera). J. Invertebr. Pathol. 15: 372-396
Zimmermann, G. 1982. Effect of high temperatures and artificial sunlight on the viability of conidia of Metarhizium anisopliae. J. Invertebr. Pathol. 40: 36-40.
Zimmermann, G. 1993. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pestic. Sci. 37: 375-379.
Zimmermann, G., Parierok, B., and Glare, T. 1995. Elias Metschnikoff, Elie metchnikoff or Ilya Ilich Mechnikov (1845-1916); A pioneer in insect pathology, the first describer of the entomopathogenic fungus Metarhizium anisopliae and how to translate a Russian name. Biocontrol Sci. and Technol. 5: 527-530.