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Identification, Infection Process and Telemorph Formation of the Pathogen of Chinese Amaranth Leaf Spot in Taiwan
|關鍵字:||Chinese amaranth;莧菜;Chinese amaranth leaf spot disease;Rhizoctonia solani AG 2-2 IIIB;Thanatephorus cucumeris;Teleomorph;Infection process;Control;莧菜葉斑病;Rhizoctonia solani AG 2-2 IIIB;Thanatephorus cucumeris;有性世代;侵染過程;防治||出版社:||植物病理學系||摘要:||
台灣地區有機農場生產莧菜 (Amaranthus mangostanus L.) 的栽培田，最近出現莧菜葉斑病，按照柯霍氏法則 (Koch’s postulates)系列測試，證實它的病原菌是Rhizoctonia solani Kuhn [有性世代Thanatephorus cucumeris (Frank) Donk]。將莧菜葉斑病菌R. solani RSA-03和RSA-09與R. solani標準菌株行菌絲融合群測定，發現兩菌株與R. solani AG 2-2 IIIB (ATCC 76124) 具有高菌絲融合率 (> 70%); 惟與標準菌株R. solani AG 2-1 (ATCC 76168)、AG 2-2IV (ATCC 76125)、AG 2-3 (R6) 及AG BI (ATCC 76132) 的菌絲融合率則小於5%。探討兩菌株對於硫胺素 (thiamine-HCl) 的需求，發現莧菜葉斑病菌與標準菌株R. solani AG 2-2 IIIB (ATCC 76124) 皆屬於營養缺陷菌株。另外莧菜葉斑病菌與標準菌株ATCC 76124的菌絲生長溫度及生長速度相近，且標準菌株ATCC 76124的菌絲塊對莧菜亦具有病原性，因此將莧菜葉斑病菌鑑定為R. solani AG 2-2 IIIB。利用土壤覆蓋法誘使莧菜葉斑病菌RSA-03和RSA-09大量產生擔孢子後，配製每毫升105個擔孢子懸浮液，接種於株齡四星期的莧菜植株，在28℃的濕室中保濕，六天後葉片開始出現水浸狀病徵。受害莧菜葉片初期病斑呈圓形，水浸狀的透化小斑，直徑大小約1mm左右，接著病斑擴展為二級不規則的爪狀斑; 若病勢發展嚴重，病斑間會相互癒合，造成葉片枯萎死亡。利用光學與螢光顯微鏡，觀察擔孢子侵染莧菜葉片的過程，發現接種擔孢子9小時後，發芽管會侵入葉片，18小時後團狀菌絲形成，直到第21小時成團狀絲體會逐漸形成子座般的構造 (stroma-like structure)，並存在於病斑中央。
測試7種土壤覆蓋資材對莧菜葉斑病菌形成有性世代的影響，結果發現BVB No. 4栽培介質可穩定地誘使莧菜葉斑病菌RSA-03和RSA-09兩菌株大量形成子實層。因此將Naito氏土壤覆蓋法 (soil-over-culture method) 生產擔孢子的流程修正如下: 即將R. solani接種於含1% (w/v) 酵母抽取物之Potato-yeast extract-dextrose agar (PYDA) 培養基平板 (內徑9公分)，在28℃下培養四天，隨後覆蓋90ml土壤 [含有40% (v/v) BVB No.4 栽培介質及40% ~ 50% (v/v) 水分]，在濕室中靜置四天後，即可在覆蓋的土壤表面穩定產生大量絨毛狀灰白色的子實層，修正後的栽培介質土壤覆蓋法 (peat moss- soil-over-culture method) 所產生的子實層表面積約為Naito氏土壤覆蓋法所生產的三至四倍。本研究證明溫度、濕度、通氣性及培養基營養成分等因子均會影響R. solani形成有性世代。莧菜葉斑病菌RSA-03和RSA-09兩菌株形成有性世代的最適溫度與酸鹼值分別為24 ~ 28℃及pH 5 ~ 7。在土壤中加入有機及無機添加物，拮抗微生物及農藥等均會干擾本菌子實層的形成，試驗結果指出土壤中添加1% (v/v) 魚粉、苦茶粕及苦楝可顯著抑制本菌產生子實層與擔孢子。若於覆蓋土壤中加入放線菌 Stretomyces padanus PMS-702、S. sioyaensis PMS-502、S. saraceticus SS-31和S. misionensis PMS 101亦會抑制本菌子實層與擔孢子的形成，惟加入枯草桿菌 Bacillus pumilus PMB-102、B. thermoglucosidasius PMB-101和B. subtilis BS-001卻不具有抑菌的功效。此外，本研究發現鋅錳乃浦、 免賴得、 貝芬替、 福多寧、 五氯硝苯、 依普同和賓克隆等農藥，也皆會抑制本菌子實層的形成。
A new leaf spot disease of Chinese amaranth (Amaranth mangotanus L.) caused by Rhizoctonia solani was frequently observed at the so-called organic farms in Taiwan during the summer season. The hyphae of RSA-03 and RSA-09 isolates obtained from Chinese amaranth leaf spot were able to anastomose in high frequency (> 70%) with R. solani AG 2-2 IIIB (ATCC 76124), but in low frequency (< 5%) with R. solani AG 2-1 (ATCC 76168), AG 2-2 IV (ATCC 76125), AG 2-3 (R6) and AG BI (ATCC 76132). When five isolates from Chinese amaranth leaf spot were respectively cultured in liquid glucose asparagine (GA) medium with or without thiamine-HCl, they were auxtrophic for thiamine-HCl and more closely resembled the ATCC 76124 isolate of R. solani AG 2-2 IIIB. The optimum temperature for mycelial growth of RSA-09 isolate from Chinese amaranth leaf spot was similar to one of R. solani AG 2-2 IIIB (ATCC 76124). Inoculation tests revealed that both RSA-03 and RSA-09 from Chinese amaranth leaf spot and R. solani AG 2-2 IIIB (ATCC 76124) were pathogenic to Chinese amaranth. Based on the anastomosis, thiamine-HCl requirement, growth temperature, and pathogenicity tests, the isolates from Chinese amaranth leaf spot were recommended as R. solani AG 2-2 IIIB. According to Koch's postulates tests, it was proved that the Chinese amaranth leaf spot was caused by the basidiospores of T. cucumeris, the telemorph of R. solani AG 2-2 IIIB. The basidiospores (105 spore/ml) of the pathogen were sprayed to leaf surface of Chinese amaranth. The inoculated plants were put in the moist chamber at 28℃. Primary lesions appeared as small, circular water-soaked spots on leaves of Chinese amaranth six days after inoculation. After additional incubation, claw-like lesions growing out from the primary lesions expanded into the leaves tissues and caused secondary lesions with large-sized irregular necrotic spots. When Chinese amaranth leaves were inoculated, basidiospores of the pathogen germinated and penetrated into the epidermal cell walls nine hours after inoculation. Mass mycelia were formed 18hrs after inoculation, then development of mass mycelia into stroma-like structure 21hrs after inoculation. In the study, Naito's soil-over-culture method for production of hymenia was modified. It was found that the peat moss and soil-over culture method (PSC method) was much more effective in producing hymenia of T. cucumeris RSA-03 and RSA-09. The procedures of PSC method were as follows: (1) to inoculate the fungus onto potato- yeast extract-dextrose agar plate in a 9-cm petri dish, (2) to incubate at 28℃for 4 days until the fungal colony covered the agar plate surface, (3) the agar plate surface was covered with 90ml soil [included 40% (v/v) BVB No. 4 peat moss and maintained the soil moisture at 40 ~ 50% (v/v)], (4) experiments were kept in moist chamber. After 4-day-incubation hymenial formation was observed. The PSC method was suitable for hymenial formation of the pathogen and able to markedly produce 3-4 fold hymenial amount compared to Naito's soil-over-culture method. The factors affecting hymenial formation of the pathogen included temperature, humidity, light, aeration, and culture substrate. The temperatures were favorable for R. solani RSA-03 and RSA-09 hymenial formation at 24 ~ 28℃and the covered soil was at pH 5 ~ 7. The amendments of covered soil with various organic and inorganic materials, antagonists, and fungicides did significantly influence the hymenia formation of T. cucumeris RSA-03 and RSA-09. The hymenia of the fungus were completely inhibited in the covered soils amended with 1﹪(v/v) fish meal, tea seed pomace and chinaberry meal. Amendments of covered soil with Stretomyces padanus PMS-702, S. sioyaensis PMS-502, S. saraceticus SS-31 and S. misionensis PMS 101 also inhibited hymenial formation, but Bacillus pumilus PMB-102, B. thermoglucosidasius PMB-101 and B. subtilis BS-001 did not. The fungicides, mancozeb, benomyl, carbendazim, flutolanil, PCNB, iprodione and pencycuron were significantly effective in inhibiting the hymenial formation.
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