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研究生:邱安隆
研究生(外文):CHIOU, AN-LONG
論文名稱:防治百合灰黴病生物製劑之研發及作用機制探討
論文名稱(外文):Developement and mechanism of bio-pesticide to control gray mold of lily
指導教授:吳文希吳文希引用關係
指導教授(外文):WU, WEN-SHI
學位類別:博士
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:2002
畢業學年度:90
語文別:中文
論文頁數:135
中文關鍵詞:百合灰黴病菌Bacillus amyloliquefaciens生物防治添加物轉型作用存活
外文關鍵詞:lilyBotrytis ellipticaBacillus amyloliquefaciensbiocontroladjuvanttransformationsurvive
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自罹患百合灰黴病植株上分離並篩選最具病原性之Botrytis elliptica BE009為供試菌株,當添加1 %麥芽抽出物,可顯著地(p = 0.05)促進灰黴病菌對百合的感染。在東方型百合品種中,以Casa Blanca最感病。從百合植株上所分離到之700株微生物,在四種不同低營養成份之培養基中,與百合灰黴病菌進行對峙及共同培養,菌株B99、B111、B128、B131、B171、B190、B196、B203、B501及BS對灰黴病菌具有明顯的拮抗能力,以百合葉片於培養皿中,或以整株百合於溫室中進行生物檢定各拮抗菌種之效果時,也有相同效果。應用百合切葉篩選防治灰黴病所得之拮抗菌,與溫室中之生物檢測結果呈正相關。經Biolog系統之初步鑑定,B99、B111為Burkholderia gladioli,B128及B190為Bacillus amyloliquefaciens,B131及B501為Actinomycetes,而B171及B196為Bacillus megaterium,B203為Corynebacterium jeikeium及BS為Bacillus subtilis。以載玻片發芽法(slide germination method)檢測八種殺菌劑抑制病原發芽的能力, 1 ppm 之flusilazole及prochloraz對灰黴病菌之發芽管伸長之抑制較其他殺菌劑顯著,而benomyl、iprodione、prochloraz及prochloraz+MgCl2,其濃度需達100 ppm,亦比其他處理具顯著性地(p = 0.05)抑制灰黴病菌分生孢子發芽之效果;1 ppm 之flusilazole、iprodione或prochloraz,即可抑制百合灰黴病菌在馬鈴薯瓊脂平板培養基上之生長。當殺菌劑存在於液體培養基中,1 ppm 之flusilazole或prochloraz+MgCl2,對百合灰黴病菌之抑制作用比其它六種殺菌劑的效果顯著(p = 0.05)。本研究中,在兩次的田間試驗中,證實百合經噴灑B. gladioli B111、B. amyloliquefaciens B128及B190或100 ppm flusilazole可顯著有效地(p = 0.05)防治百合灰黴病。
應用1或5 %米糠、黃豆粉或酵母粉培養基,可促進B. gladioli B111及B. amyloliquefaciens B190之生長, Bacillus amyloliquefacies B128則在1 % 2-deoxyl-D-glucose、黃豆粉或酵母粉及5 %麥芽萃取物、米糠、黃豆粉或甘蔗渣粉培養基中生長顯著地較佳。0.1 %氧化鈣及氫氧化鈣可促進B. amyloliquefaciens B190的生長,並對百合灰黴病具顯著性地(p = 0.05)抑制效果;當0.1 %氫氧化鈣或氧化鈣溶液中,添加B. amyloliquefaciens B190,具顯著性地(p = 0.05)降低百合灰黴病之罹病嚴重度效果。另外,0.1 %磷酸氫二鉀、碳酸鈉或硝酸銨亦具顯著性地(p = 0.05)抑制百合灰黴病的效果,上列三種鹽類物質或0.1 %氫氧化鈣中添加B. amyloliquefacien B190,皆具有顯著性地(p = 0.05)抑制灰黴病的效果。在0.025 %氫氧化鈣、0.05 %碳酸鈉、0.025 %硝酸銨或0.025 %磷酸氫鉀中添加B. amyloliquefaciens B190,也具抑病效果。上列四種配方,單獨或分別組成不同試劑配方,以0.05 %碳酸鈉、0.025 %氫氧化鈣添加0.05 %碳酸鈉、0.025 %氫氧化鈣混合0.025 %硝酸銨,或0.025 %氫氧化鈣添加0.05 %碳酸鈉、0.025 %硝酸銨及0.025 %磷酸氫鉀之四種組合,並添加B. amyloliquefaciens B190可顯著性地(p = 0.05)抑制灰黴病的效果至少有10天。以0.025 %氫氧化鈣、0.05 %碳酸鈉與0.1 % Tween 80之混合亦具抑病效果。上列三種物質混合液中,添加0.05 % 4-methoxycinamate、礦物油(light或heavy)及salicylic acid 2-ethylhexyl ester,抑病效果則較佳。在溫室中的生物檢定, 0.025 %氫氧化鈣混合0.05 %碳酸鈉、0.1 % Tween 80及0.05 %礦物油,有或未添加B. amyloliquefacien B190,以及0.025 %氫氧化鈣混合0.05 %碳酸鈉、0.025 %硝酸銨、0.025 %磷酸氫鉀、0.1 % Tween 80及0.05 %礦物油,並添加B. amyloliquefaciesn B190,和flusilazole之防治效果最顯著。在田間的防治效果測定中,單獨施用B. amyloliquefaciens B190,或施用含B. amyloliquefaciens B190之乳劑,以及施用flusilazole,均具顯著性地(p = 0.05)抑制百合灰黴病的功效。Bacillus amyloliquefaciens B190與十九種植物病原菌進行對峙及共同培養,可顯著性地(p = 0.05)抑制其中十四種病原菌。而B. amyloliquefaciens B190對百合灰黴病菌防治作用之機制,應包含抗生及寄生作用。Bacillus amyloliquefaciens B190在乳劑配方中,於第九個月時,族群仍與最初的菌量相若。
噴佈帶有Tn 917之B. amyloliquefaciens B190轉型株於百合花器及葉片上,15天後,花器上轉型株之族群數呈顯著性地(p = 0.05)增加,在上位葉及中位葉上,5天後,及下位葉上10天後,轉型株之菌落數均呈顯著性地(p = 0.05)增加。當轉型株有或未添加製劑配方,分別噴於葉表並置於20、28或36℃下,10及15天後,轉型株在葉片上之族群數量顯著性地(p = 0.05)較在5天者多。Bacillus amyloliquefaciens B190轉型株在溫度為12、20或28℃及相對濕度為98或100 %時,有或未製成製劑均可顯著性地(p = 0.05)提高於葉表的存活能力。Bacillus amyloliquefaciens B190轉型株在相對濕度為98 %以上時,於葉部正面及背面,比相對濕度為55 %者,具顯著性地(p = 0.05)促進於葉表上之遷移能力。轉型株有或未添加製劑配方,於百合葉正面及葉背,當溫度為28℃,相對濕度為90、95、98或100 %時,比相對濕度為55 %者,能顯著性地(p = 0.05)提高其遷移能力。於黑暗或經254 nm光線波長照射10天後,轉型株存活能力顯著性地(p = 0.05)比315 nm光線波長處理者提高;其中,又以黑暗處理之轉型株菌落數,比光線波長為254 nm之處理組高,且呈顯著性地(p = 0.05)差異。單獨應用B. amyloliquefaciens B190並無誘導百合葉片抗灰黴病菌感染的能力。
Botrytis elliptica BE009 was isolated from diseased lily plants and selected and used in this study due to its consistent virulence on 3 tested cultivars. Adding 1 % malt extract in spore suspension promoted B. elliptica to infect lily. Oriental hybrid cultivar Casa Blanca was the most susceptible lily. Among 700 microorganisms isolated from lily plants and screened by dual and concomitant cultures against Botrytis elliptica, ten isolates ( B99、B111、B128、B131、B171、B190、B196、B203、B501 and BS ) had antagonistic effects against B. elliptica on detached leaves of three lily cultivars in petri dish and on whole plant in greenhouse trials. Based on Biolog system, B99 and B111 were identified as Burkholderia gladioli, and B128 and B190 as Bacillus amyloliquefaciens, B131 and B501 as actinomycetes, B171 and B196 as Bacillus megaterium, B203 as Corynebacterium jeikeium, BS as Bacillus subtilis. 1 ppm of flusilazole、iprodione or prochloraz inhibited significantly ( p = 0.05 ) the growth of B. elliptica on potato dextrose agar plate. 1 ppm flusilazole or prochloraz+MgCl2 was able to inhibit significantly ( p = 0.05 ) the growth of B. elliptica in PDA broth. 1 ppm flusilazole or prochloraz and 100 ppm benomyl, iprodione, prochloraz or prochloraz+ MgCl2 was able to inhibit significantly ( p = 0.05 ) the germination of B. elliptica on slide culture. In the field, B. gladioli B111, B. amyloliquefaciens B128, B190 or 100 ppm flusilazole controlled effectively ( p = 0.05 ) the occurrence of lily gray mold.
1 or 5 % (w/v) of rice chaff, soybean meal or yeast powder promoted the growth of B. gladioli B111 and B. amyloliquefaciens B190, 1 % 2-deoxyl-D-glucose, soybean meal or yeast powder and 5 % malt extract, rice chaff, soybean meal or sugarcan dreg powder promoted the growth of B. amyloliquefaciens B128(w/v)significantly ( p = 0.05 ). 0.1 % calcium hydroxide or calcium oxide increased significantly ( p = 0.05 ) the growth of Bacillus amyloliquefaciens B190 and decreased the disease severity caused by B. elliptica on lily. Bacillus amyloliquefaciens B190 cultured in 1 % rice chaff and mixed with 0.1 % calcium hydroxide or calcium oxide controlled lily gray mold effectively. Spraying 0.1 % di-potassium hydrogenphosphate, sodium carbonate, ammonium nitrate or calcium hydroxide with or without B. amyloliquefaciens B190 on lily leaves decreased significantry ( p = 0.05 ) the gray mold. Spraying B. amyloliquefaciens B190 which was mixed either with 0.025 % calcium hydroxide, 0.05 % sodium carbonate, 0.025 % ammonium nitrate or 0.025 % di-potassium hydrogenphosphate on lily leaves decreased significantry ( p = 0.05 ) the gray mold. Bacillus amyloliquefaciens B190 was mixed with 0.025 % sodium carbonate, 0.025 % calcium hydroxide added 0.05 % sodium carbonate, or with 0.025 % ammonium nitrate, or with the mixed solution of 0.025 % calcium hydroxide, 0.05 % sodium carbonate, 0.025 % ammonium nitrate and 0.025 % di-potassium hydrogenphosphate, and with the mixed solution of 0.025 % calcium hydroxide, 0.05 % sodium carbonate, 0.025 % ammonium nitrate and 0.025 % di-potassium hydrogenphosphate. These mixtures controlled lily gray mold, significantly ( p = 0.05 ) on detached leaves for 10 days. 0.1 % Tween 80 mixed with 0.025 % calcium hydroxide and 0.05 % sodium carbonate decreased the severity of gray mold on lily leaves. 0.05 % 4-methoxycinamate, mineral oil(heavy or light)or salicylic acid 2-ethylhexyl ester mixed with 0.025 % calcium hydroxide and 0.05 % sodium carbonate increased the effeciency of controlling lily gray mold. 0.025 % calcium hydroxide, 0.025 % sodium carbonate, 0.1 % Tween 80 and 0.05 % mineral oil mixed with or without B. amyloliquefaciens B190 controlled lily gray mold effectively(p = 0.05)as flusilazole in greenhouse. Pure culture or formulated B. amyloliquefaciens B190 and flusilazole controlled(p = 0.05)significantly gray mold of lily at four different fields. Twenty transformants at dually and concomitantly with B. amyloliquefaciens B190 inhibited significantly ( p = 0.05 ) the growth of 14 species of plant pathogens. The effect of B. amyloliquefaciens B190 to inhibit B. elliptica was due to antibiosis and mycoparasitism. Formulated B. amyloliquefaciens B190 was able to survive under room temperature(25℃± 5℃)for 9 months at least without decling, while maintain 14 months with consistent population in steriled pure water.
Bacillus amyloliquefaciens B190 transformant could exist on lily flower for 15 days. The population of transformant sprayed on upper and central lily leaves for 5 day or on lower lily leaves for 10 days, increased significantly (p = 0.05)compared to the initial population. Transformant could exist well under 20, 28 or 36℃on lily leaves for 10 and 15 days. Bacillus amyloliquefaciens B190 transformant could migrate on lily leaves when the relative humidity(RH)was 98 or 100 %. Intact or formulated B. amyloliquefaciens B190 could migrate well under 28℃ with 90, 95, 98 or 100 % RH compared to 55 % RH on lily leaves. Transformant grew significantly(p = 0.05)more under dark than under 315 nm light on lily leaves. Bacillus amyloliquefaciens B190 could not induce lily resistant to gray mold.
壹、前言------------------------------------------------------ 6貳、前人研究-------------------------------------------------- 9
一、灰黴病菌之分類及生態-------------------------------------- 9
二、灰黴病菌感染寄主作物之作用機制--------------------------- 10
三、應用化學防治法防治灰黴病及其引發之問題------------------- 10
四、應用拮抗微生物防治灰黴病--------------------------------- 11
五、拮抗微生物防治灰黴病之作用機制--------------------------- 12
六、運用非生物與非化學防治法防治灰黴病----------------------- 15
七、生物製劑之製備條件、應用實例及防治病害之作用原理--------- 16
八、拮抗微生物於作物體上之存活表現--------------------------- 18
九、葉表細菌之存活及纏據------------------------------------- 20
參、 材料與方法---------------------------------------------- 22
一、百合灰黴病菌及其拮抗菌之分離、篩選及鑑定----------------- 22
(一)、百合灰黴病菌之分離、收集及鑑定----------------------- 22
(二)、拮抗菌之篩選----------------------------------------- 22
(三)、在百合葉片上營養基質影響百合灰黴病菌之致病性----------23
(四)、葉片上生物防治之生物檢定----------------------------- 24
(五)、溫室中生物防治之生物檢定----------------------------- 24
(六)、殺菌劑的篩選 ---------------------------------------- 25
1、殺菌劑對灰黴病菌分生孢子發芽之抑制測試------------------ 25
2、殺菌劑對灰黴病菌菌絲生長之抑制測試---------------------- 25
3、殺菌劑對灰黴病菌生物量之抑制測試------------------------ 25
(七)、拮抗菌之鑑定----------------------------------------- 26
(八)、田間生物防治效果之測定------------------------------- 26
二、應用拮抗細菌及添加物製備生物製劑防治百合灰黴病----------- 26
(一)、篩選培養拮抗菌之基本基質----------------------------- 26
(二)、含鈣物質對百合灰黴病菌孢子發芽的影響----------------- 27
(三)、含鈣物質對B. amyloliquefaciens B190生長之影響-------- 27
(四)、含鈣物質對百合灰黴病菌致病性之影響------------------- 27
(五)、氫氧化鈣及氧化鈣對B. amyloliquefaciens B190防治百合灰黴病之影響----------------------------------------------------- 28
(六)、營養培養基質及含鈣物質對B. amyloliquefaciens B190防治百合灰黴病之影響----------------------------------------------- 28
(七)、鹽類物質對B. amyloliquefaciens B190防治百合灰黴病之影響---------- 29
(八)、不同濃度氫氧化鈣或鹽類物質對B. amyloliquefaciens B190防治百合灰黴病之影響------------------------------------------- 29
(九)、不同濃度氫氧化鈣或鹽類物質之組合對B. amyloliquefaciens B190防治百合灰黴病之影響------------------------------------- 29
(十)、不同輔助劑添加至B. amyloliquefaciens B190之組合中對防治百合灰黴病之影響--------------------------------------------- 30
(十一)、不同乳化劑及油類物質添加至B. amyloliquefaciens B190之半製劑配方中對防治百合灰黴病之影響--------------------------- 31
(十二)、含B. amyloliquefaciens B190之各種組合處理於溫室中防治百合灰黴病之效果--------------------------------------------- 31
(十三)、含B. amyloliquefaciens B190之各種組合處理於田間防治百合灰黴病之效果----------------------------------------------- 32
(十四)、Bacillus amyloliquefaciens B190於各種製劑配方中之存活表現--------------------------------------------------------- 32
(十五)、Bacillus amyloliquefaciens B190與各種植物病原菌在稀釋馬鈴薯瓊脂平板上對峙與共同培養時之抑制效果測定--------------- 32
三、Bacillus amyloliquefaciens B190防治百合灰黴病之原理探討-- 33
(一)、抗生及營養競爭作用----------------------------------- 33
(二)、寄生作用--------------------------------------------- 34
(三)、增殖、分佈及佔據能力--------------------------------- 34
1、拮抗菌分子標記製備及轉型作用------------------------------ 34
(1)、Tn 917之質體製備-------------------------------------- 34
(2)、轉型作用---------------------------------------------- 35
2、Bacillus amyloliquefaciens B190轉型株於百合葉片及花器上的增殖現象--------------------------------------------------------- 36
3、溫度、濕度及光線波長對B. amyloliquefaciens B190轉型株於百合葉片上的存活及遷移影響----------------------------------------- 36
(1)、溫度對B. amyloliquefaciens B190轉型株於百合葉片上的存活影響----------------------------------------------------------- 36
(2)、溫度及相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的存活影響------------------------------------------------- 37
(3)、相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的遷移影響------------------------------------------------------- 37
(4)、溫度及相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的遷移影響------------------------------------------------- 38
(5)、不同波長之光線及相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的存活影響--------------------------------------- 38
(四)、誘導抗病性之測定------------------------------------- 38
肆、結果----------------------------------------------------- 40
一、百合灰黴病菌及其拮抗菌之分離、篩選及鑑定----------------- 40
(一)、百合灰黴病菌之分離、收集及鑑定----------------------- 40(二)、拮抗菌之篩選----------------------------------------- 40
(三)、在百合葉片上營養基質影響百合灰黴病菌之致病性--------- 41
(四)、葉片上生物防治之生物檢定----------------------------- 41
(五)、溫室中生物防治之生物檢定----------------------------- 41
(六)、殺菌劑的篩選 ---------------------------------------- 41
1、殺菌劑對灰黴病菌分生孢子發芽之抑制測試------------------ 41
2、殺菌劑對灰黴病菌菌絲生長之抑制測試---------------------- 42
3、殺菌劑對灰黴病菌生物量之抑制測試------------------------ 42
(七)、拮抗菌之鑑定----------------------------------------- 42
(八)、田間生物防治效果之測定------------------------------- 43
二、應用拮抗細菌及添加物製備生物製劑防治百合灰黴病----------- 43
(一)、篩選培養拮抗菌之基本基質----------------------------- 43
(二)、含鈣物質對百合灰黴病菌孢子發芽的影響----------------- 43
(三)、含鈣物質對B. amyloliquefaciens B190生長之影響-------- 44
(四)、含鈣物質對百合灰黴病菌致病性之影響------------------- 44
(五)、氫氧化鈣及氧化鈣對B. amyloliquefaciens B190防治百合灰黴病之影響----------------------------------------------------- 44
(六)、營養培養基質及含鈣物質對B. amyloliquefaciens B190防治百合灰黴病之影響----------------------------------------------- 44
(七)、鹽類物質對B. amyloliquefaciens B190防治百合灰黴病之影響-------------------------------------------------------------- 45
(八)、不同濃度氫氧化鈣或鹽類物質對B. amyloliquefaciens B190防治百合灰黴病之影響------------------------------------------- 45
(九)、不同濃度氫氧化鈣或鹽類物質之組合對B. amyloliquefaciens B190防治百合灰黴病之影響------------------------------------- 45
(十)、不同輔助劑添加至B. amyloliquefaciens B190之組合配方中對防治百合灰黴病之影響----------------------------------------- 46
(十一)、不同乳化劑及油類物質添加至B. amyloliquefaciens B190之半製劑配方中對防治百合灰黴病之影響--------------------------- 46
(十二)、含B. amyloliquefaciens B190之各種組合處理於溫室中防治百合灰黴病之效果--------------------------------------------- 47
(十三)、含B. amyloliquefaciens B190之各種組合處理於田間防治百合灰黴病之效果----------------------------------------------- 47
(十四)、Bacillus amyloliquefaciens B190於各種製劑配方中之存活表現--------------------------------------------------------- 48
(十五)、Bacillus amyloliquefaciens B190與各種植物病原菌在稀釋馬鈴薯瓊脂平板上對峙與共同培養時之抑制效果測定--------------- 48
三、Bacillus amyloliquefaciens B190防治百合灰黴病之原理探討-- 49
(一)、抗生及營養競爭作用----------------------------------- 49
(二)、寄生作用--------------------------------------------- 49
(三)、增殖、分佈及佔據能力--------------------------------- 49
1、拮抗菌分子標記製備及轉型作用------------------------------ 49
2、Bacillus amyloliquefaciens B190轉型株於百合葉片及花器上的增殖現象--------------------------------------------------------- 50
3、溫度、濕度及光線波長對B. amyloliquefaciens B190轉型株於百合葉片上的存活及遷移影響----------------------------------------- 50
(1)、溫度對B. amyloliquefaciens B190轉型株於百合葉片上的存活影響----------------------------------------------------------- 50
(2)、溫度及相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的存活影響-------------------------------------------------------------------- 50
(3)、相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的遷移影響----------------------------------------------------------------------------- 51
(4)、溫度及相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的遷移影響-------------------------------------------------------------------- 51
(5)、不同波長之光線及相對濕度對B. amyloliquefaciens B190轉型株於百合葉片上的存活影響----------------------------------------------------- 51
(四)、誘導抗病性之測定----------------------------------------------------------------- 52
伍、表-------------------------------------------------------------------------------- 53
陸、圖-------------------------------------------------------------------------------------------- 91
柒、討論---------------------------------------------------------------------------------------- 98
捌、中文摘要---------------------------------------------------------------------------------- 109
玖、英文摘要--------------------------------------------------------------------------------- 112
拾、參考文獻--------------------------------------------------------------------------------- 115
拾壹、附錄一--------------------------------------------------------------------------------- 135
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