臺灣博碩士論文加值系統

西元1928至1966年間，許多科學家對於十字花科蔬菜黃葉病菌的分類地位
及生理小種有不一致的意見，直到1986年Bosland氏等人根據病原菌的病原性、營養體親和群 (vegetative compatibility group, VCG)、地理分布、生理、生化及基因序列等特性，才又將十字花科蔬菜黃葉病菌之分化種及生理小種重新歸納成Fusarium oxysporum f. sp. conglutinans (FOC) race 1及2 , F. oxysporum f. sp. raphani (FOR), F. oxysporum f. sp. matthioli (FOM) race 1及2；其中conglutinans分化種名係於西元1935年，由Wollenweber氏依據病原菌菌落產生黏性分生孢子堆 (Sporodochia) 的特徵命名。十字花科蔬菜種類繁多，彼此間遺傳基因互有重疊，導致尖鐮孢菌對十字花科蔬菜之致病毒性亦有明顯的差異，因此，本研究的目的在於針對不同來源的十字花科蔬菜黃葉病菌，分別探討它們對寄主範圍之病原性反應、營養體親和性及親緣關係，藉以釐清十字花科蔬菜黃葉病菌之分化種。首先赴台灣各地採集黃葉病菌的供試菌株，它們的代碼分別是由甘藍分離的FOC-JR01、FOCN15、FOCN19、FOCN20、FOCN22；由芥藍分離的FOCN06、FOCN07、FOCN18；由芥菜分離的FOCN05、FOCN36、FOCN38；由蘿蔔分離的F19、F21、F24、F26菌株及由白菜分離的FOCN33、FOCN34、FOCN43。此外亦由美國菌種保存中心 (American Type Culture Collection, ATCC) 購得FOC標準菌株 (ATCC 52557、ATCC 16600)、FOR標準菌株 (ATCC 16601) 及FOM標準菌株 (ATCC 16602、ATCC 16603)。供試的寄主植物有Brassica oleracea變種群之甘藍及芥藍、Raphanus sativus變種群之蘿蔔、Brassica juncea變種群之芥菜及Brassica rapa變種群之小松菜、塔菇菜、不結球白菜及油菜等，試驗結果發現由甘藍與芥藍分離的菌株可感染除芥菜以外的其他十字花科蔬菜；至於由蘿蔔、芥菜、白菜分離的菌株則無法感染甘藍與芥藍，顯示上述諸菌株可區分成兩大類群。Bosland氏 (1986) 報導甘藍和芥菜黃葉病菌均屬於同一conglutinans分化種，然而台灣的甘藍與芥菜分離的菌株間卻有不一致的病原性反應；其中來自甘藍與芥藍的黃葉病菌的寄主病原性顯然異於來自芥菜、蘿蔔及白菜黃葉病的菌株。至於分析各菌株之VCG，發現目前所分離的台灣供試菌株與ATCC之標準菌株間均不具親和性，其中台灣的甘藍及芥藍黃葉病菌之變異菌株間則可彼此相互親和，顯示來自該兩寄主的菌株可獨立成為一個新的營養體親和群。另外，以FIGS11/FIGS12引子對增幅菌株之IGS片段後，再將解序的序列進行親緣樹的構築，亦可將來自甘藍及芥藍的菌株與來自芥菜、蘿蔔及白菜的菌株區分開。顯然，以甘藍 (高峰品種)、芥藍 (蕙津品種)、芥菜 (青和品種)及蘿蔔 (明和品種) 等四者作為尖鐮孢菌感染十字花科蔬菜的指示植物，再搭配各病原菌菌株間的VCG分析與IGS片段之序列分析，筆者認為甘藍與芥藍黃葉病菌可由F. oxysporum f. sp. conglutinans之複合分化種中獨立出來，重新命名為F. oxysporum f. sp. oleraceae nov. f. sp.。

The pathogen of Fusarium yellows and wilt of cruciferae producing colonies with slime sporodochia was first named as Fusarium conglutinans by Wollenweber (1913). From 1928 to 1996, the classification of the causal agent of Fusarium yellows and wilt of cruciferae had been changed several times based on pathogenicity assays. In 1940, Snyder and Hansen revised the scientific name of the pathogen in the genus Fusarium causing yellows and wilt of cruciferae as Fusarium oxysporum. Afterwards, many formae speciales of F. oxysporum have been defined based on their host preference, such as radish- f. sp. raphani, tomato- f. sp. lycopersici and watermelon- f. sp. niveum. Based on pathogenicity, isozyme polymorphism, vegetative compatibility, and geographic origin, Bosland et al. (1986) further classified the isolates from Fusarium yellows and wilt of cruciferae into F. oxysporum f. sp. conglutinans (race 1 and race 2), F. oxysporum f. sp. raphani, F. oxysporum f. sp. matthioli (race 1 and race 2). Recently, we have found that F. oxysporum caused severe damage to cabbage, radish, and mustard in Taiwan. In total, 18 isolates of F. oxysporum were collected from diseased cruciferous plants. Five isolates, designated FOC-JR01, FOCN15, FOCN19, FOCN20, and FOCN22, were obtained from diseased cabbage. Isolates FOCN06, FOCN07, and FOCN18 were obtained from diseased Chinese kale. Isolates FOCN05, FOCN36, and FOCN38 were obtained from diseased mustard. Isolates F19, F21, F24 and F26 were obtained from diseased radish. Isolates FOCN 33, FOCN34, and FOCN43 were obtained from diseased pakchoi. However, the true identity of these F. oxysporum isolates remains largely unknown. Pathogenicity assays revealed that the causal agents isolated from each of the cruciferous crops exhibited different pathogenicity tested on cabbage, Chinese kale, radish, pakchoi, rape, and mustard. For example: isolates FOC-JR01, FOCN06 and FOCN07 were capable of infecting almost all tested cruciferous crops except mustard. The isolates obtained from diseased radish and mustard were pathogenic to their original host plants, but failed to infect cabbage and Chinese kale. Quantitative analyses revealed that the isolates of F. oxysporum obtained from mustard, radish and pakchoi did not show pathogenicity on cabbage and Chinese kale. The isolates obtained from pakchoi were able to infect radish, pakchoi and mustard varieties, but not cabbage and Chinese kale. Therefore, it was recommended that four indicator plants, cabbage (cv. Summer Summit), Chinese kale (cv. Huei-Jin), mustard (cv. Ching-He) and radish (cv. Ming-Ho) were used to evaluate the pathogenicity of F. oxysporum isolated from diseased cruciferous plants. Based on our pathogenicity analyses, the causal agent of Fusarium yellows and wilt of cruciferae in Taiwan could be divided into at least two groups, each containing multiple physiological races. Bosland et al. (1986) grouped the causal agent of cabbage and mustard yellows and wilt into the same forma specialis- conglutinans, inconsistent with our results. To resolve these discrepancies and to precisely define the formae speciales of F. oxysporum isolated from crucifers in Taiwan, further analyses based on VCGs (vegetative compatibility groups) and molecular characteristics were carried out in this study. The results of VCG and phylogenetic tree of isolates based on sequences of about 580 bp of the rDNA intergenic spacer region also showed high varience between cabbage and mustard isolates. Our research exhibited that the causal agent of cabbage and Chinese kale yellows was independent from F. oxysporum f. sp. conglutinans complex based on the pathogenicity test of four indicator plants, VCG testing and IGS region sequencing. We suggested that the causal agent of cabbage and Chinese kale yellows should be renamed as F. oxysporum f. sp. oleraceae nov. f. sp.

目次
中文摘要 i
英文摘要 iii
目次 v
表次索引 vii
圖次索引 viii
前言 1
材料與方法 6
一、供試菌株來源、分離、鑑定、培養與保存 6
(一) 供試菌株來源 6
(二) 供試菌株之分離及鑑定 6
(三) 供試菌株之培養與保存 7
二、溫度對於十字花科黃葉病菌菌絲生長的影響 7
三、病菌土製作及菌量密度測定 8
(一) 病菌土製作 8
(二) 土壤中病原菌密度的測定 8
四、病原性測定 8
(一) 不同接種源濃度的黃葉病菌對於甘藍植株罹病度的影響 8
(二) 病原菌對不同十字花科作物之病原性 9
(三) 病害調查方法 10
(四) 統計分析 11
五、營養體親和性試驗 11
(一) 供試培養基 11
(二) 硝酸營養突變缺陷株之篩選 11
(三) 硝酸的營養突變缺陷株之表現型測定 12
(四) 供試菌株之異核自體親和性試驗 12
(五) 親和性試驗 13
六、分子特性分析 13
(一)菌株的培養與菌絲的收集 13
(二) 總量DNA的萃取 14
(三) IGS區域DNA片段之增幅 14
(四) 增幅片段之定序和比對 15
(五) 分子親緣性分析 15
結果 17
一、十字花科黃葉病菌之形態鑑定 17
二、溫度對於十字花科黃葉病菌菌絲生長的影響 17
三、病原性測定 17
(一) 不同接種源濃度的黃葉病菌對於甘藍植株罹病度的影響 17
(二) 病原菌對不同十字花科作物之病原性 18
四、營養體親和性試驗 19
(一) 供試菌株之異核自體親和性試驗 19
(二) 親和性試驗 20
五、分子親緣性分析 20
(一) 增幅片段之定序和比對 20
(二) 分子親緣性分析 20
討論 22
參考文獻 27
附錄一、供試培養基 (Medium) 之製備 57


表次索引
表一、本試驗之供試菌株來源 33
表二、本試驗供試植物之學名與品種名 34
表三、分子親緣性分析中所使用的菌株資料 35
表四、供試十字花科黃葉病菌菌株之孢子大小 37
表五、十字花科黃葉病菌菌株對不同十字花科作物之罹病度 38
表六、十字花科黃葉病菌菌株對不同十字花科作物之病原性發病率測試結果 40
表七、不同十字花科作物對各十字花科黃葉病菌菌株之抗感性反應 42
表八、供試菌株營養突變缺陷株之表現型測定 44
表九、供試菌株之營養突變缺陷株 (nit 3 或Nit M mutants) 與配對標準菌株之營養突變缺陷株 (nit 1 mutants) 之配對結果 47
表十、不同供試菌株的不同表現型營養突變缺陷株間之互相配對結果 49


圖次索引
圖一、十字花科蔬菜黃葉病菌 (FOCN36) 的不同表現型之nit突變缺陷株在五種不同氮源培養基上之生長情形。 50
圖二、十字花科黃葉病菌之形態。 51
圖三、溫度對甘藍及芥藍黃葉病菌菌絲生長之影響 52
圖四、溫度對芥菜、蘿蔔及白菜黃葉病菌菌絲生長之影響 53
圖五、不同黃葉病菌接種源濃度對於甘藍植株在42天內罹病度的影響 54
圖六、十字花科黃葉病菌菌株對不同十字花科作物之病原性測試接種結果 55
圖七、以十字花科黃葉病菌之IGS片段以近鄰結合法 (Neighbor-joining method, NJ) 所建構之親緣樹 56

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