台灣枇杷枝枯病菌的鑑定與其感染過程及防治方法

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論文名稱: 台灣枇杷枝枯病菌的鑑定與其感染過程及防治方法
研究生姓名: 蔡宜儒
指導教授姓名: 黃振文
出版年: 2010
學校名稱: 國立中興大學
系所名稱: 植物病理學系所
關鍵字: Cylindrocladiella peruviana;Cylindrocladium reteaudii;枇杷;枇杷枝枯病;入侵感染;酯解酵素;非農藥防治;傷口防護劑;loquat;Cylindrocladiella peruviana;Cylindrocladium reteaudii;infection process;esterase;Chinese medicines;disease control;non-chemical control;a wound protectant ointment
摘要: 西元2008與2009年間,台灣中部地區枇杷園發現枇杷植株葉片呈現失水、萎凋下垂,及黃化之病徵。由罹病株分離獲得的菌株分別歸屬於 Cylindrocladium 屬與 Cylindrocladiella 屬等兩群的真菌,隨後按柯霍氏法則逐一證實 Lsc-01、02 及 Lscy-01三菌株皆可引起枇杷枝枯病,且病徵與田間原罹病株相仿。依據它們的形態特徵,並與 ITS 與 D1/D2 區域核醣體去氧核醣核酸序列分析,結果,將 Lsc-01 與 Lsc-02 的學名鑑定為 Cylindrocladiella peruviana;而 Lscy-01 菌株則鑑定為 Cylindrocladium reteaudii。Lsc-01 與 Lsc-02菌株最適菌絲生長與孢子發芽溫度均介於 24~28 ℃,最適產孢溫度為 28 ℃;而 Lscy-01 菌株最適菌絲生長與孢子發芽溫度介於 24~28 ℃,最適產孢溫度為 16 ℃。將 Ce. peruviana Lsc-01、Lsc-02 及 Cy. reteaudii Lscy-01 之孢子懸浮液濃度,調整至每毫升含有 104 個孢子,分別以噴霧方式接種於枇杷、番石榴、桃子、李子、蓮霧及葡萄等 6 種作物植株,結果發現 Lscy-01可感染枇杷、番石榴、李子及蓮霧等4種作物植株。另外,將 Lsc-01、Lsc-02 及 Lscy-01 之孢子懸浮液(104 spores/ml)接種於枇杷、番石榴、桃子、梅子、李子及蓮霧等6種作物的果實,只有 Lscy-01 不經由傷口可以直接感染果實,造成果實腐爛,且病斑擴展速度會隨接種濃度的提高而加快。探討病原菌感染枇杷的過程,發現 Lscy-01 會由發芽管產生類似附著器結構,經由氣孔處進入或是直接經由表皮細胞侵入寄主的細胞間。測試供試三菌株產生酯解酵素的能力,結果顯示只有 Lscy-01 菌株之孢子表面與周圍有酯解酵素的活性反應。測試 12 種化學藥劑對枇杷枝枯病菌菌絲生長之抑制效果,發現 1 ppm 有效濃度之 33.5 %快得寧水懸劑可完全抑制枇杷枝枯病菌的菌絲生長。此外,篩選 31 種中草藥對枇杷枝枯病菌菌絲生長之影響,結果發現 2 %(W/W)木香、黃芩及五倍子對枇杷枝枯病菌菌絲的生長最具良好抑制效果。隨後利用 2 %(W/W)木香與凡士林製成傷口防護劑,證實可有效對防治枇杷枝枯病的發生與擴展。A new disease, loquat twig blight has occurred in central Taiwan since 2008. Cylindrocladiella isolates Lsc-01 and Lsc-02 and Cylindrocladium isolate Lscy-01 were isolated from diseased loquat twigs. The pathogenicity was confirmed by inoculating loquat seedlings, which showed the same symptoms as diseased plants in the fields. Based on morphological characteristics, ITS and D1/D2 sequences analyses, three isolates were respectively identified as Cylindrocladiella peruviana isolates Lsc-01 and Lsc-02 and Cylindrocladium reteaudii isolate Lscy-01. The optimal temperatures for mycelial growth and conidial germination of Ce. peruviana isolates Lsc-01 and Lsc-02 was at 24-28 ℃, and Cy. reteaudii isolate Lscy-01 also was at 24-28 ℃. The optimal temperature for sporulations of Lsc-01 and Lsc-02 was at 28 ℃, and isolate Lscy-01 was at 16 ℃. Furthermore, conidial suspensions of Ce. peruviana Lsc-01 & Lsc-02 and Cy. reteaudii Lscy-01were respectively sprayed on six fruit crops. The results showed that isolate Lscy-01 could infect seedlings of loquat, guava, plum and wax apple, but Lsc-01 & Lsc-02 could not. Lscy-01 was also able to directly infect fruit without wounds. The fruit rot became more severe when inoculum density of Lscy-01 was increased. After Lscy-01 was inoculated on fruit surface of loquat for 16 hrs, it could form appressorium-like structures around stomata and penetrated directly into the intercellular epidermis. Based upon indoxyl acetate reaction under microscopy observation, it was proved that Lscy-01 was able to produce esterase, but were not Lsc-01 and Lsc-02. Twelve synthetic chemical fungicides were evaluated their effect on suppression of mycelial growth of Lsc-01, Lsc-02 and Lscy-01 on MEA. The results showed that 33.5 % Oxine Copper was very effective in inhibiting compeletly mycelial growth of three isolates at one ppm. Thirty one Chinese medicines were evaluated their effect on suppression mycelial growth of Lsc-01, Lsc-02 and Lscy-01 on MEA. The results showed that 2 % Aucklandiae Radix, Scutellariac Radix and Chinensis Galla were effective in inhibiting mycelial growth of three isolates. A wound protectant ointment was formulated with petrolatum and 2 % (w/w) Aucklandiae Radix powder.It was significantly effective in controlling twig blight of loquat when wounded petiole amd twig were treated.
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