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標題: 辣椒炭疽病菌(Colletotrichum acutatum)入侵過程及其可能毒力因子探討
Infection process and potential virulence factors of Colletotrichum acutatum on chili pepper
作者: 廖建堯
Liao, Chein-Yao
關鍵字: pepper anthracnose;辣椒炭疽病;Colletotrichum acutatum;internal infection structure;virulence factor;內生型侵入構造;毒力因子
出版社: 植物病理學系所
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第一章 辣椒炭疽病菌毒力因子分析
在台灣辣椒在栽培過程中及採收後遭受炭疽病菌Colletotrichum acutatum危害相當嚴重,其造成的果腐嚴重降低辣椒採收後的經濟價值,然而針對C. acutatum對辣椒的致病及毒力因子研究相當有限。因此本實驗利用在辣椒上表現不同毒力的三菌株C. acutatum Coll-153、Coll-365及Coll-524做為研究對象,藉由比較三菌株差異以期尋找出C .acutatum在感染辣椒的毒力因子。在生長及分子特性研究中,低毒力Coll-365 在固態培養基PSA中有生長緩慢,產孢量也較低;然而三菌株在液態PDB生長並沒有明顯差異。經由雙股RNA純化,Coll-365純化出2.5kb及1.3kb雙股RNA,Coll-524則純化出1.7 kb及1.4 kb雙股RNA。比較三菌株的孢子附著能力、發芽能力、附著器形成能力及其附著器膨壓累積大小,低毒力Coll-365附著器膨壓累積較低,但其cutinase活性較另兩株中高毒力菌株高。在辣椒果實致病能力分析中,以孢子懸浮液無傷口接種PBC-81、PBC-932、9955-15、群香、朝天椒朱雀及雞心椒果實,結果顯示PBC-932及PBC-81綠色果實為抗病性,其餘品種之果實為感病性。三菌株在感病品種群香綠色果實上發病時間上沒有差異,主要差異在病勢進展,當病徵出現後高毒力Coll-524引起之病斑擴展速度較為中低毒力菌株快,低毒力Coll-365發展至一定程度即停止,且在辣椒果實上沒有產孢情形,三株病菌毒力表現差異主要在於侵入寄主之後的病勢進展,後期發展關係到病菌細胞壁分解能力及對抗寄主防禦反應的能力。分析各菌株之細胞壁分解能力,纖維素及果膠分解能力沒有差異;而低毒力Coll-365在蛋白質分解能力比中高毒力菌株高。此外也針對常被報導能分解眾多酚化合物laccase活性進行分析,結果顯示高毒力Coll-524 的laccase活性為中低毒力菌株的3~6倍,因此推測laccase可能為C. acutatum在辣椒感染過程中的毒力相關因子。
第二章 Colletotrichum acutatum在辣椒的侵入過程
由Colletotrichum acutatum所引起的炭疽病危害許多辣椒栽培地區,然而對其在辣椒侵入過程研究卻相當稀少,因此本研究目的是藉由觀察三株不同毒力的C. acutatum Coll-153、Coll-365、Coll-524菌株在抗病及感病辣椒侵入過程,其中以高毒力Coll-524作為主要觀察對象,為了方便觀察侵入過程,成功以Agrobacterium轉殖GFP基因於此三菌株中,共計獲得133株GFP轉殖菌株。於顯微鏡下觀察,C. acutatum於感病群香綠色果實侵入過程,首先最早於接種後四小時孢子在果表發芽產生發芽管形成附著器,並在周圍分泌孢外物質幫助其附著,於孢子及附著器下方的角質層形成多分叉結構,此結構未被報導過,取名為內生型侵入構造 (internal infection structure),以GFP菌株觀察此結構發出綠色螢光,於接種72小時,三菌株菌絲均已在果實表皮細胞生長,於接種後第五天,病徵處果實表皮出現許多裂縫,中毒力Coll-153及Coll-524由裂縫叢生出分生孢子梗,產生大量孢子,而低毒力Coll-365 卻沒有從裂縫中產生分生孢子梗或產生大量孢子。於抗病PBC-81綠色果實觀察,三菌株在內生型侵入構造形成後,菌絲沒有在表皮細胞生長,於第13天觀察內生型侵入構造已部分褐化。針對內生型侵入構造觀察。此外也發現內生型侵入構造為C. acutatum在番椒普遍的侵入結構,在番茄及芒果葉均無產生,探討內生型侵入構造形成原因,推測可能為辣椒角質成分及致密結構所造成。此外C. acutatum在石蠟膜也出現疑似內生型侵入構造,但以GFP菌株接種觀察,此類似結構不會發出螢光,其特性與辣椒上的內生型侵入構造有所不同。在本章研究中發現C. acutatum 於辣椒感染形成一個特殊的入侵結構 (內生型侵入構造),其可能在辣椒侵入過程中扮演重要角色。

Anthracnose of chili pepper (Capsicum spp.) caused by Colletotrichum acutatum results in fruit decay and severe losses in Taiwan, but only a few studies focusing on the pathogenicity of C. acutatum on chili pepper were published. In this research, the pathogenicity of C. acutatum on chili pepper was investigated by comparing the pathogenesis of three C. acutatum isolates which showed different virulence on chili pepper Capsicum chinense ABRDC accession PBC-932 by wound inoculation. The three isolates (Coll-153, Coll-365 and Coll-524) were characterized at physiological and molecular level. On agar medium, Coll-365 grew much slower and produced less conidia than the other two isolates. However, there was no significant difference on growth among the three isolates when cultured in liquid medium. Moreover no difference was found on pigment formation, conidial shape and size and optimal temperature for growth. ITS sequence analysis showed that they were all closely related to other C. acutatum isolates. Two dsRNA fragments were isolated from Coll-365 (2.5 kb and 1.3 kb) and Coll-524 (1.7 kb and 1.4 kb) but none from Coll-153. Factors related to pre-penetration and penetration were analyzed in vitro, including spore attachment and germination, appressorium formation and turgor pressure accumulation. No significant difference was found among the three isolates on these factors except that Coll-365 appressoria accumulated less turgor pressure on plastic surface. However, microscopic examination showed that appressorium might not be required for the penetration of the three isolates on chili pepper. Results showed that Coll-365 has stronger protease and cutinase activity than the other two isolates, while Coll-524 has highest laccase activity. In pathogenicity assay, seven chili pepper cultivars or accessions, one bell pepper cultivar and other potential host plants including mango fruits and leaf and tomato fruit were inoculated without wound. On susceptible Capsicum fruits, Coll-524 caused largest lesion, while Coll-365 caused smallest lesion. No sporulation was found on all plant materials infected by Coll-365 except on tomato fruit. No difference was observed on the timing and ability of the three isolates on spore germination, appressorium formation, infection and symptom initiation on resistant chili pepper Ca. baccatum AVRDC accession PBC-81or on susceptible Ca. annuum cv. “GroupZest”. It indicates that the factors contributing to the difference of the three isolates on virulence are involved in fungal colonization inside infected host tissue. Thus, compound detoxification (e.g., laccase) and cell-wall degrading enzyme production might be important factors causing high virulence of Coll-524.
Anthracnose of Capsicum spp. caused by Colletotrichum acutatum is a severe disease in chili pepper in Taiwan. To understand how the pathogen infects the plant, the infection process of C. acutatum on resistant and susceptible fruits were studied using three isolates with various virulences on chili pepper. The three isolates formed young appressoria at 4 hours post-inoculation (hpi) and formed penetration hyphae in the epidermal cells of susceptible hosts at 72 hpi. Interestingly, this fungus forms a novel internal infection structure on resistant and susceptible chili pepper fruits before penetrating the epidermal cell. The internal infection structure was located in the cuticle layer and was demonstrated by light and fluorescent microscopy with GFP-tagged transformants. The internal infection structure formed within 24 hpi and kept growing in the cuticle layer and subsequently penetrated into the epidermal cell at 72 hpi. Formation of this internal structure by C. acutatum appears to be a response to the thick cuticle layer of pepper fruits. This fungus also formed similar internal structures on resistant hosts. However, the structure turned dark 13 days post-inoculation and no further development was observed. This structure was not found on infected tomato fruit and mango leaf, but was found on infected sweet pepper. Moreover, this fungus can form this structure in isolated chili pepper cuticle layer and Parafilm membrane. Therefore, factors involved in the induction and regulation of this structure can be easily studied in the future. Based on many examinations in this study, this internal infection structure seems to be required for C.
acutatum penetration on chili pepper.
其他識別: U0005-2308201022155700
Appears in Collections:植物病理學系

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