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研究生:高宏遠
研究生(外文):Hung-Yuan Kao
論文名稱:除草劑促進草莓炭疽病發生的副效應
論文名稱(外文):Side-effect of Herbicides on Enhancing Occurrence of Strawberry Anthracnose
指導教授:黃振文黃振文引用關係
指導教授(外文):Jenn-Wen Huang
口試委員:謝廷芳鍾文全
口試委員(外文):Ting-Fang HsiehWen-Chuan Chung
口試日期:2017-07-17
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:2017
畢業學年度:105
語文別:中文
論文頁數:69
中文關鍵詞:除草劑草莓炭疽病菌Colletotrichum gloeosporioides副作用草莓巴拉刈嘉磷塞固殺草
外文關鍵詞:herbicidesstrawberry anthracnose fungus Colletotrichum gloeosporioidesside-effectstrawberryparaquatglyphosateglufosinate-ammonium
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本研究主要目的在於:(1)研發一種可以偵測與分離田間草莓炭疽病菌的選擇性培養基,及(2)探討巴拉刈、嘉磷塞及固殺草等三種除草劑(a)對於草莓炭疽病菌(Colletotrichum gloeosporioides (Penz.) Sacc.) MD-02與ME-03菌株在基質及植物體上生長繁殖的影響;(b)對土壤微生物相的影響;(c)對草莓植株生長與病害發展的影響,期有助於瞭解田間施用除草劑是否會影響草莓炭疽病的發生。首先以查氏培養基配方作為基礎培養基,評估十三種碳素源及十六種氮素源對草莓炭疽病菌菌絲生長的影響,發現果糖、麥芽糖及蔗糖等三種碳素源及硝酸鈉及硝酸鉀等兩種氮素源可顯著促進炭疽病菌菌絲的生長。比較七種化學藥劑對草莓炭疽病菌菌絲生長的影響,發現100 ppm的白克列、滅達樂及四環黴素均不具抑制菌絲生長的功效。評估化學藥劑對於土壤中真菌的抑制作用,發現25 ppm的免賴得和腐絕,50 ppm的巴拉刈和滅達樂及100 ppm的白克列可顯著抑制土壤中真菌的生長。進一步,比較前述不同濃度的化學藥劑對草莓炭疽病菌發芽管生長的影響,結果顯示這些藥劑亦有不同程度抑制發芽管生長的功效。將蔗糖30 g、硝酸鈉3 g、磷酸氫二鉀1 g、硫酸鎂0.5 g、氯化鉀0.5 g、硫酸鐵 0.01 g、洋菜粉15 g及蒸餾水1L均勻混合,並經高溫高壓(121°C, 15 lb)滅菌後,逐一加入免賴得25 mg、腐絕25 mg、白克列100 mg、滅達樂50 mg、巴拉刈50 mg及四環黴素50 mg,製成SSBP選擇性培養基(Sucrose-sodium nitrate-boscalid-paraquat selective medium),可有效偵測存活於土壤中的草莓炭疽病菌。此外,本試驗研究發現病原菌處理過巴拉刈5 ppm與嘉磷塞50 ppm四小時後,分別可促進394.4及178.9%草莓炭疽病菌的分生孢子發芽;固殺草則未觀察到有促進之效果。在查氏培養基(Czapek solution agar)分別添加不同濃度的除草劑後製成平板,接種炭疽病菌菌絲塊,五天後,發現巴拉刈2 ppm、嘉磷塞25 ppm及固殺草1.5 ppm皆可促進菌絲生長約9%;在平板上以分生孢子懸浮液塗佈的方式,三天後,巴拉刈5 ppm與嘉磷塞250 ppm則分別可促進炭疽病菌產孢達198.6與419.5%,固殺草則無促進之效果。將感染炭疽病菌的草莓葉柄段片,以不同濃度的除草劑處理後,結果顯示巴拉刈500 ppm與嘉磷塞10 ppm分別可促進炭疽病菌產孢達351.5與273.7%。將經過除草劑處理過的草莓葉柄段片,埋入混有炭疽病菌的病土中,兩天後發現巴拉刈500 ppm與嘉磷塞250 ppm可提升病原菌纏據草莓葉柄的百分比。在含有炭疽病菌分生孢子的土壤中分別添加巴拉刈、嘉磷塞或固殺草,五天後分析炭疽病菌的菌量變化,發現土壤中添加巴拉刈2500 ppm或嘉磷塞12500 ppm,炭疽病菌族群量分別可提升832.3或1223.5%;至於添加固殺草的處理組則與對照組相仿,結果呈現逐日遞減的趨勢。土壤添加巴拉刈、嘉磷塞或固殺草後,分析土壤中細菌、放線菌、真菌及酵母郡的族群量變化,結果顯示嘉磷塞2500 ppm可提升真菌與酵母菌的族群量約1205.3及363.1%;至於添加巴拉刈500 ppm及固殺草675 ppm的處理組與對照組間無顯著差異。進一步,以巴拉刈10 ppm與嘉磷塞50 ppm噴施草莓植株後,翌日接種草莓炭疽病菌,發現處理過巴拉刈及嘉磷塞可顯著提高植株的罹病度達305及117%。此外,先將巴拉刈或嘉磷塞添加於土壤中再種植草莓植株,結果發現巴拉刈濃度超過320 ppm或嘉磷塞濃度超過40 ppm時,草莓的新葉會出現黃化的症狀;若處理巴拉刈濃度在240 ppm或嘉磷塞20 ppm時,草莓植株外觀雖無異樣,然而接種炭疽病菌後,植株炭疽病罹病度卻可提高61.6〜64.1%左右。
The purposes of this study were to: (1) develop the selective medium for detecting anthracnose fungus Colletotrichum gloeosporioides of strawberry from soil; (2) evaluate the effect of herbicides paraquat, glyphosate and glufosinate-ammonium on the growth and sporulation of strawberry anthracnose fungus C. gloeosporioides isolates MD-02 and ME-03; (3) understand microbial flora in soils treated with and without herbicides.; and (4) analyze the effect of herbicides on the disease severity of strawberry inoculated with and without C. gloeosporioides. In the study, thirteen carbohydrates and sixteen nitrogenous compounds were evaluated for their effects on mycelial growth of both isolates MD-02 and ME-03. Fructose, maltose and sucrose as the carbohydrates, sodium nitrate and potassium nitrate as the nitrogenous compounds were more effective than the others to enhance growth of the pathogen. Seven pesticides were evaluated for the effect on mycelial growth of both isolates MD-02 and ME-03. The results showed that 100 ppm boscalid, metalaxyl and tetracycline didn’t inhibit mycelial growth of both isolates. Benomyl, thiabendazole, paraquat, boscalid, metalaxyl and tetracycline were respectively used to evaluate for their

inhibiting ability in population proliferation of the fungi in soil. The results showed that 25 ppm benomyl, 25 ppm thiabendazole, 50 ppm paraquat, 50 ppm metalaxyl, and 100 ppm boscalid could significantly inhibit fungal populations in soils. To evaluate the effect of above pesticides at the same concentration on germ tube growth of both isolates MD-02 and ME-03, the results indicated those could also inhibit two isolates in vary degree. The sucrose-sodium nitrate-boscalid-paraquat selective medium (SSBP selective medium) consisting of 30 g sucrose, 3 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.01 g FeSO4·7H2O, 15 g agar, 25 mg benomyl, 25 mg thiabendazole, 50 mg paraquat, 50 mg metalaxyl, 50 mg tetracycline, 100 mg boscalid and 1 L distilled water was hence formulated for isolating C. gloeosporioides from soil. In addition, Conidia of both isolates MD-02 and ME-03 of C. gloeosporioides treated with paraquat at 5 ppm and glyphosate at 50 ppm resulted in 394.4% and 178.9% promotion of germination rates, respectively. Glufosinate-ammonium had no promotive effect on conidial germination. Mycelial growth of both isolates was increased by 9% on Czapek solution agar amended with 2 ppm of paraquat, 25 ppm of glyphosate or 1.5 ppm of glufosinate-ammonium. Czapek solution agar amended with 5 ppm paraquat or 25 ppm glyphosate significantly promoted the sporulation of isolates MD-02 and ME-03 of C. gloeosporioides. Furthermore, 351.5% and 273.7% promotion of sporulation on strawberry petiole segments treated with 500 ppm paraquat and 10 ppm glyphosate were also respectively observed. Strawberry petiole segments treated with paraquat at 500 ppm and glyphosate at 250 ppm were markedly increased colonization by C. gloeosporioides. To evaluate the effect of herbicides on survival ability of C. gloeosporioides in soil, the results showed that population density of C. gloeosporioides was significantly higher in the soil treated with 2500 ppm paraquat

and 12500 ppm glyphosate compared to untreated soil as the control. Furthermore, population of fungi and yeasts was also markedly enhanced by 1205.3% and 363.1% in the glyphosate-treated soil. Spraying 10 ppm paraquat and 50 ppm glyphosate solution to strawberry plants prior to inoculating C. gloeosporioides, the results indicated that paraquat and glyphosate could increase the anthracnose disease severity by 305% and 117%. Planting strawberry seedlings in soils treated with paraquat and glyphosate, yellowing of young leaves had been observed in the treatment of 320 ppm paraquat and 40 ppm of glyphosate. However, no symptom could be observed in the treatment of 240 ppm paraquat and 20 ppm of glyphosate, but anthracnose disease severity of strawberry plants could be enhanced by 61.6 to 64.1%.
中文摘要 i
Abstract iv
目錄 vii
表次索引 x
圖次索引 xi
前言 1
材料與方法 5
一、供試植株來源 5
二、供試菌株來源、培養及保存 5
(一)菌株來源 5
(二)病原性測試 5
(三)菌株培養與保存 6
三、供試菌株之鑑定 6
(一)形態鑑定 6
(二)分子鑑定 6
1.DNA萃取 6
2.聚合酶連鎖反應(Polymerase Chain Reaction) 7
3.增幅片段之定序和比對 8
四、偵測草莓炭疽病菌的選擇性培養基之研發 8
(一)不同碳素源對草莓炭疽病菌菌絲生長之影響 8
(二)不同氮素源對草莓炭疽病菌菌絲生長之影響 9
(三)不同化學藥劑對草莓炭疽病菌菌絲生長的影響 9
(四)不同藥劑濃度對草莓炭疽病菌發芽管生長與土壤中雜菌的影響 10
1.對草莓炭疽病菌發芽管生長的效應分析 10
2.對土壤中雜菌抑制的效應分析 10
(五)SSBP選擇性培養基偵測土壤中草莓炭疽病菌的回收效率 11
(六)利用SSBP選擇性培養基偵測田間土壤中草莓炭疽病菌的存活情形 11
五、除草劑對草莓炭疽病菌之影響 11
(一)除草劑對病原菌分生孢子發芽之影響 12
(二)除草劑對病原菌菌絲生長之影響 12
(三)除草劑對病原菌產孢之影響 12
(四)除草劑對土壤中病原菌纏據之影響 13
六、除草劑對土壤中的草莓炭疽病菌及微生物之影響 13
(一)除草劑對土壤中病原菌族群量之影響 13
(二)除草劑對土壤中微生物相之影響 14
七、除草劑對植株生長與病害發展之影響 14
(一)葉面噴施除草劑對草莓植株生長之影響 14
(二)草莓葉面噴施除草劑對其炭疽病罹病度之影響 14
(三)土壤中的除草劑對植株生長及草莓炭疽病罹病度之影響 15
八、統計分析 15
結果 16
一、病原菌形態與分子序列鑑定 16
二、偵測草莓炭疽病菌的選擇性培養基之調製與應用 16
(一)不同碳素源對病原菌菌落生長之影響 16
(二)不同氮素源對病原菌菌落生長之影響 16
(三)不同化學藥劑對病原菌菌落生長之影響 17
(四)不同藥劑濃度對草莓炭疽病菌發芽管生長與土壤中雜菌的影響 17
1.對草莓炭疽病菌發芽管生長的效應分析 17
2.對土壤中雜菌抑制的效應分析 17
(五)SSBP選擇性培養基偵測土壤中草莓炭疽病菌的回收效率 18
(六)利用SSBP選擇性培養基偵測田間土壤中草莓炭疽病菌的存活情形 18
三、除草劑對草莓炭疽病菌之影響 18
(一)除草劑對病原菌分生孢子發芽之影響 18
(二)除草劑對病原菌菌絲生長之影響 19
(三)除草劑對病原菌產孢之影響 19
(四)除草劑對主壤中病原菌纏據之影響 19
四、除草劑對土壤中草莓炭疽病菌及微生物之影響 20
(一)除草劑對土壤中草莓炭疽病菌族群量之影響 20
(二)除草劑對土壤中微生物相之影響 20
五、除草劑對植株生長與病害發展之影響 21
(一)葉面噴施除草劑對草莓植株生長之影響 21
(二)葉面噴施除草劑對草莓炭疽病害發展之影響 21
(三)土壤中的除草劑對草莓植株及草莓炭疽病罹病度之影響 21
討論 22
參考文獻 27
圖表 34
附錄 66

表次索引

表一、不同碳素源對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03菌絲生長之影響............................34
表二、不同氮素源對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03菌絲生長之影響............................35
表三、不同化學藥劑對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03菌絲生長之影響..............36
表四、巴拉刈對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03於查氏培養基上產孢之影響.............37
表五、嘉磷塞對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03於查氏培養基上產孢之影響........................38
表六、固殺草對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03於查氏培養基上產孢之影響........................39
表七、巴拉刈對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03於草莓葉柄上產孢之影響..........................40
表八、嘉磷塞對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03於草莓葉柄上產孢之影響..........................41
表九、巴拉刈對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03於草莓葉柄上纏據之影響..........................42
表十、嘉磷塞對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03於草莓葉柄上纏據之影響..........................43

圖次索引

圖一、不同化學藥劑對草莓炭疽病菌Colletotrichum gloeosporioides發芽管生長之影響..........................44
圖二、查氏培養基中添加不同化學藥劑的偵測土壤中雜菌的出現率...45
圖三、利用SSBP選擇性培養基由土壤中偵測草莓炭疽病菌Colletotrichum gloeosporioides的效率.....................46
圖四、利用SSBP選擇性培養基偵測田間土壤中草莓炭疽病菌Colletotrichum gloeosporioides的族群量..................47
圖五、巴拉刈對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03分生孢子發芽之影響..............................48
圖六、嘉磷塞對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03分生孢子發芽之影響..............................49
圖七、固殺草對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03分生孢子發芽之影響.............................50
圖八、巴拉刈對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03菌絲生長之影響................................51
圖九、嘉磷塞對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03菌絲生長之影響..................................52
圖十、固殺草對草莓炭疽病菌Colletotrichum gloeosporioides MD-02與ME-03菌絲生長之影響.................................53
圖十一、巴拉刈對土壤中的草莓炭疽病菌Colletotrichum gloeosporioides MD-02族群量的影響.......................54
圖十二、嘉磷塞對土壤中的草莓炭疽病菌Colletotrichum gloeosporioides ME-03族群量的影響.......................55
圖十三、固殺草對土壤中的草莓炭疽病菌Colletotrichum gloeosporioides MD-02族群量的影響........................56
圖十四、除草劑對土壤中的微生物相的影響.....................57
圖十五、植株葉表噴施10 ppm巴拉刈對草莓炭疽病病勢發展的影響...58
圖十六、草莓葉表噴施10 ppm巴拉刈對草莓炭疽病病勢發展的影響...59
圖十七、植株葉表噴施50 ppm嘉磷塞對草莓炭疽病病勢發展的影響...60
圖十八、草莓葉表噴施50 ppm嘉磷塞對草莓炭疽病病勢發展的影響...61
圖十九、土壤中的巴拉刈對草莓植株生長及炭疽病病勢發展的影響...62
圖二十、草莓植株種植在含有不同濃度巴拉刈的土壤中14天,然後接種病原菌後第6天之植株炭疽病之罹病度比較........................63
圖二十一、土壤中的嘉磷塞對草莓植株生長及炭疽病病勢發展的影響.64
圖二十二、草莓植株種植在含有不同濃度嘉磷塞的土壤中14天,然後接種病原菌後第6天之植株炭疽病之罹病度比較......................65
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