Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31757
標題: 萊豆苗莖枯病菌的生物特性及其病害防治
Studies on the Biological Characteristics of Botryodiplodia theobromae and its Disease Control
作者: 郭章信
Kuo, Chang-Hsin
關鍵字: lima bean;萊豆;seedling stem blight;Botryodiplodia theobromae;pathogenicity tests;hydrolases;control;苗莖枯病;Botryodiplodia theobromae;病原性測定;水解酵素;病害防治
出版社: 植物病理學系
摘要: 
在台南縣麻豆鎮種植之萊豆發生之苗莖枯病,病原菌經鑑定為Botryodiplodia theobromae,接種證明萊豆為病原菌的新寄主。病原菌之分生孢子器(pycnidia)單生或聚生,埋生而後突出植物表皮組織,具孔口,分生孢子為全出芽型(holoblastic),未成熟器孢子(pycnidiospores)無色單室,橢圓形至長方形,成熟器孢子厚壁深褐色雙室,橢圓形,基部呈楔形(truncate),孢子壁上有條狀直紋(striate)。比較田間採集之單室無色孢子與澤田氏之模式標本Macrophoma phaseoli-lunati,顯示兩者應為同物異名。病原菌菌絲生長與孢子發芽的最適溫度範圍為25~35℃,30℃為最適溫度。病原菌在10∼35℃間,皆可形成分生孢子器,以20℃為最適產孢溫度。網室內經兩年測試結果顯示溫度與萊豆苗莖枯病之發生有密切關係,植株發病率隨溫度之升高而增高,日均溫高於25℃,每日最低溫在20℃以上,植株發病率可達90%以上,其病徵在接種後第12~15天後表現;若日均溫在18~22℃間,每日最低溫在18℃以下,植株發病率明顯降低,病徵延至接種第21~24天後方出現;日均溫低於18℃,萊豆未表現病徵。測試不同豆類蔬菜對病原菌的抗感性,發現大粒萊豆白仁種最為感病。病原菌也會感染大粒萊豆花仁種、Luna、UC-92及小粒萊豆W.U.N品種,但是對小粒萊豆MercLa等8種供試豆類蔬菜不具病原性。試驗證實病原菌可經由萊豆種子傳播。分離自夾竹桃、龍血樹及珊瑚刺桐之B. theobromae 菌株均可感染大粒萊豆白仁種;相反的,白仁種經接種分離自蕃荔枝之菌株,則未表現病徵。
試驗證明苗莖枯病菌侵入萊豆幼苗必須經由子葉方能夠感染幼苗莖部表現病徵。評估影響子葉接種法的因子,包括相對濕度、乾燥、水分潛勢對病原菌器孢子發芽的影響,接種源濃度、萊豆苗齡及子葉水分潛勢。結果顯示,器孢子在自由水及100%相對濕度中,其發芽率均可達到87%以上,相對濕度在93%以下,單室孢子的發芽率已經下降至30%,而雙室孢子則仍無發芽。器孢子經自然乾燥後,環境若未能維持前述的相對濕度,孢子發芽率隨乾燥時間的增加而顯著下降。器孢子在水分潛勢0∼-10 bars,經過6小時,單室孢子發芽達98%,雙室孢子達80%以上;單室孢子在水分潛勢-40 bars以下,雙室孢子在水分潛勢-60 bars以下均無法發芽。萊豆幼苗經接種每毫升103孢子濃度時,植株的罹病率就可達60%以上。病原菌接種於不同株齡的萊豆幼苗,以出土5天幼苗的罹病率最高。
孢外分泌物(spore matrix)不影響病原菌孢子的發芽,並且孢子不論是否具有孢外分泌物,對萊豆皆有致病力,接種後的幼苗發病率均可達47%以上,但是孢外分泌物經過加熱處理後,幼苗的發病率由64%降低為34%,則有顯著差異。以API ZYM試劑 (bioMérieux, Inc. France)測定孢外分泌物、孢子發芽分泌物、罹病組織液及健康組織液內的酵素活性,孢外分泌物含有13種、孢子發芽分泌物含有9種、罹病組織液含有11種及健康組織液含有11種供測試的酵素,其中僅esterase及β-glucosidase在孢外分泌物、孢子發芽分泌物及罹病組織液中呈現,健康植物組織中則未發現。進一步利用聚丙醯銨膠體電泳確定esterase、β-glucosidase、cellulase及catalase的存在,esterase在孢外分泌物有1條及罹病組織液有4條條帶 (band)而健康組織液內沒有;β-glucosidase在孢外分泌物有2條及罹病組織液有5條條帶而健康組織液內沒有;四種供試材料中均未發現cellulase及catalase的活性。利用DNS定量不同齡期罹病幼苗內β-glucosidase,結果顯示植株雖然在接種後第9天表現病徵,但是β-glucosidase的活性在接種後第6天,已經開始提高0.4單位;在接種12天後,酵素活性達到最高為1.8單位。比較罹病組織與健康組織內還原醣的含量,兩者均隨著植株的成長而增加,但是罹病組織內還原醣的含量增加更快,在接種後12天,組織內還原醣的含量已經升到544.8 µMole/mg,相較於健康組織內337.9 µMole/mg的還原醣的含量,高出許多。
室內測試12種化學藥劑對萊豆苗莖枯病菌Botryodiplodia theobromae菌絲生長及孢子發芽的影響,結果發現依普同、撲滅寧、保粒黴素、免賴得、得克利及貝芬替等藥劑對菌絲生長及孢子發芽均具抑制效果。溫室盆栽測試結果顯示50%免賴得可溼性粉劑1500倍、50%撲滅寧可溼性粉劑1500倍、23.8%得克利乳劑2000倍及23.7%依普同水懸劑1000倍等處理,均具防治萊豆苗莖枯病的功效,其中以撲滅寧及依普同的效果最佳,保粒黴素不具顯著的防治效果。兩次田間藥劑防治試驗顯示,萊豆幼苗出土後約一週內施藥一次,無論處理50%撲滅寧可溼性粉劑 1500倍或2000倍,23.7%依普同水懸劑1000倍或1500倍,均可有效防治萊豆苗莖枯病的發生,減少植株罹病率達6.6%~19.4%。試驗期間各藥劑對萊豆均無藥害產生。

Seedling stem blight of lima bean severely (Phaseolus limensis Macf.) occurred at Matou, Tainan. The causal organism was identified as Botryodiplodia theobromae. This is the first report of lima bean as a new host of B. theobromae. Pycnidia simple, or compound, often aggregated, stromatic, and ostiolate. Conidiogenous cells are holoblastic. Conidia are initially unicellular, hyaline, ellipsoide-oblong, and mature conidia are uniseptate, cinnamon to fawn, ellipsoide, base truncate, often longitudinally striate. On the basis of comparison between unicellular, hyaline conidia of B. theobromae and holotype of Macrophoma phaseoli-lunati collected by Sawada, these two species were suggested to be a synonym. The most suitable temperatures for both mycelial growth and spore germination ranged between 25℃and 35℃ with optimum being 30℃. However, the optimum temperature for pycnidia formation was around 20℃. Testing under net-house conditions for a period of 2 years, the disease was most severe during the season when daily average temperatures reached 25℃ and the minimum average temperature was above 20℃, the highest disease severity was recorded. Percentage of disease incidence was reduced and symptom development was delayed when daily average temperatures ranged between 18℃ and 22℃, and minimum average temperature was below 18℃. There was no disease development during the winter periods when temperatures were below 18℃. Most of cultivars of lima bean including Pai-Jen, Hwa-Jen, Luna, UC-92, and sieva W.U.N were susceptible to B. theobromae, the Pai-Jen being the most susceptible cultivar. The fungus was not able to infect sieva MercLa and other leguminous crops. B. theobromae was transmitted by seeds. About 2% of lima bean seeds obtained from farmers developed stem blight symptoms after sowing. In cross inoculations, B. theobromae isolated from Nerium indicum, Dracaena fragrans, Erythrina corallodendron, Carica papaya and Psidium guajava were pathogenic on lima bean, whereas that isolate from Annona squamosa was not.
Factors affecting cotyledon-inoculation on stem blight of lima bean caused by Botryodiplodia theobromae were tested. Percentage of spore germination of B. theobromae was more than 87% both in distilled water and under 100% relative humidity at 25℃, but no spores germinated when the relative humidity was below 89%. Both unicellular and uniseptate conidia failed to germinate after being air dried for 48 and 72 hours, respectively. Spore germination was more than 98% for unicellular conidia and 80% for uniseptate conidia on water agar with water potential ranging from 0 to -10 bars at 25℃, and no germination at or lower -40 bars. The cotyledon-inoculation technique was developed for disease assessment. The stem blight symptoms developed when lima bean (cv. Pai-Jen) was inoculated with unicellular and uniseptate conidia separately. The disease severity was more than 60% when spore suspensions containing 103 to 105 conidia per ml were used for inoculation on lima bean. When lima bean seedlings of different ages were inoculated with B. theobromae, the 5-day-old seedlings showed the most susceptible. The fungus was unable to cause infection on the seedlings whose cotyledons have dropped naturally or were removed artificially. The results suggest that the cotyledons of lima bean provide an entry for successful infection of B. theobromae.
The spore matrix did not affect conidial germination but was able to stimulate stem blight disease development on the seedling of lima bean. The washed spores + spore matrix caused 64% infection as compared to 47% only caused by washed spores + sterilized deionized water or sterilized water. The stimulating effect of matrix was heat unstable, because washed spores + heated spore matrix caused 34% of infection which is significantly lower than that caused by washed spores + spore matrix. Enzyme activities of spore matrix (SM), extracellular conidial matrix (ECM), extract of infected stem tissue (IST), and healthy stem tissue (HST) of lima bean were assayed by using API ZYM (bioMérieux, Inc. France) and non-denaturing polyacryamide gel electrophoresis. In API ZYM, 13 enzymes of SM, 9 enzymes of ECM, 11 enzymes of IST, and 11 enzymes of HST of lima bean were detected. Among them esterase and β-glucosidase were consistently detected in the SM, ECM, and IST, but not in HST. In the further study by using non-denaturing gel electrophoresis to assay esterase and β-glucosidase, the results were about the same as found in API ZYM tests except these two enzymes were not found in ECM and the esterase was found in HST. Esterase zymogram of polyacryamide gel electrophoresis under non-denaturing conditions indicated the presence of 1 band in SM, 4 bands in IST with esterase activity which were absent in HST. β-Glucosidase zymogram under the same conditions indicated the presence of 2 bands in SM, 5 bands in IST with β-glucosidase activity which were absent in HST. No cellulase and catalase activities were detected in all tested material by using gel electrophoresis. To analyse the enzyme activities in seedling of lima bean inoculated with B. theobromae, by using the DNS (3,5-dinitrosalicyclic acid) method, maximum β-glucosidase activity, 1.8 units, was detected on the 12th day after inoculation. In the meantime, the reducing sugar contents in the infected tissue was increased to the maximal level 544.8 µMole/mg, as compared to 337.9 µMole/mg only, in the healthy tissue of lima bean.
Among a total of twelve commercial fungicides evaluated in vitro for their ability to inhibit mycelial growth and spore germination of the pathogen, iprodione, procymidone, polyoxin, benomyl, tebuconazole and carbendazim, were found to be effective. In a potted plant trial, 4 kinds of fungicides, i.e., 50% benomyl WP at 1:1500 dilution, 50% procymidone WP at 1:1500 dilution, 23.8% tebuconazole EC at 1:2000 dilution and 23.7% iprodione F at 1:1000 dilution, were found effective for controlling seedling stem blight of lima bean. Both procymidone and iprodione showed the best efficacy in the field trial. Application of these two fungicides, 50% procymidone WP at 1:1500 or 1:2000 dilution, 23.7% iprodione F at 1:1000 or 1:1500 dilution, markedly reduced the disease severity in further two separate field trials. Application of both fungicides caused no phytotoxic damages to lima bean seedling both in pot and in field test.
URI: http://hdl.handle.net/11455/31757
Appears in Collections:植物病理學系

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