Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31761
標題: 杏鮑菇褐斑病菌之鑑定、生物特性、偵測與防治
Identification, Biological Characteristics, Detection, and Control of Gliocladium roseum, the Causal Agent of Brown Spot of the King Oyster Mushroom Pleurotus eryngii
作者: 陳錦桐
Chen, Jin-Tong
關鍵字: 杏鮑菇
Pleurotus eryngii
褐斑病菌
生物特性
核糖體轉錄外區間
28S 核糖體DNA
半選擇性培養基
偵測
防治
Gliocladium roseum
biological characteristics
inter transcribed spacer (ITS)
28S ribosome DNA (28S rDNA)
semiselective medium
detection
control
出版社: 植物病理學系
摘要: 西元1998年,在台中縣霧峰鄉與行政院農委會農業試驗所之杏鮑菇栽培場中,發現菇體出現褐斑、畸形、龜裂與停滯生長的病徵。將罹病菇體分離到的GR-6、GR-7、GR-12、GR-13、GR-16、GR-18、GR-23、GR-28、GR-31及GR- 37等10個菌株,重新接種於杏鮑菇體上,3-4天後菇體均出現如同栽培場相似的病徵。本病原菌菌株培養在20℃,麥芽萃取蛋白洋菜培養基平板生長10天,菌落大小為32.5-35 mm,並呈現橘紅至鮭紅色,其菌絲有隔膜;分生孢子柄直立、有兩種型態,一種頗似Verticillium狀的分生孢子柄,具有2-6個瓶狀枝,分生孢子柄長為110-170µm,瓶狀枝長為13-28µm;另一種則類似Penicillium狀的分生孢子柄,分生孢子柄密緻,瓶狀枝長為10-16µm;分生孢子卵圓形,由瓶狀枝產生,基部微凸,透明、單胞,大小為4.6-6.5 × 2.0-3.5µm,有黏液泌出黏聚成球形。參照Domsch等氏(1980)、Schroer等氏(1999)及Smalley與Hansen兩氏(1957)等分類文獻,將本病原菌鑑定為Gliocladium roseum Bainier。進一步利用核糖體轉錄外區間(Inter transcribed spacer, ITS)及28S rDNA等分子生物技術佐證病原菌的鑑定,證實本病原菌確為G. roseum。本病原菌GR-12與GR-28菌株孢子發芽和菌絲生長最適溫度為24-28℃。利用孢子懸浮液接種法接種於去皮後12天之杏鮑菇,發現杏鮑菇的罹病度會隨著接種菌量濃度之增加,愈趨嚴重。本病害罹病度會隨溫度提高(由12℃至28℃),愈趨嚴重。G. roseum GR-12與GR-28菌株可感染洋菇[Agaricus bisporus (Lange) Imbach]、香菇[Lentinus edodes (Berk.) Sing.]、金針菇[Flammulina velutipes (Curt.: Fr.) Sing.]、秀珍菇[Pleurotus sajor (Fr.) Sing.]、夏季鮑魚菇[Pleurotus cystidosus Miller]、柳松菇[Agrocybe cylindracea (DC: Fr) Mre.]、鴻喜菇[Hypsizigus marmoreus Bunashimeji]、雞腿菇[Coprinus comatus (Müll.: Fr. ) Pers.]等食用菇類。評估12種碳素源與12種氮素源對G. roseum GR-12與GR-28菌株菌絲生長的影響,發現澱粉、蔗糖、麥芽糖及己六醇等四種碳素源;硝酸鉀、硝酸鈉及亞硝酸鈉等三種氮素源均可顯著促進菌絲生長。利用麥芽糖與亞硝酸鈉作為碳氮素源時,除適合本菌生長外,尚可以菌落與其他菌類加以區別。比較七種化學藥劑對本菌菌絲生長的影響,發現200 ppm福多寧(flutolanil)與4 ppm腐絕(mertect)等兩者不具有抑制本菌生長之效果;至於在抗生素方面,鏈黴素、青黴素與氯黴素在500 ppm以下,均不會抑制本菌菌絲生長。將麥芽糖30公克、亞硝酸鈉2公克、磷酸氫二鉀1公克、硫酸鎂0.5公克、氯化鉀0.5公克、硫酸鐵0.01公克、洋菜20公克及蒸餾水1公升均勻混合,高壓滅菌(121℃,15 lb,15min.)後,添加福多寧200 ppm、腐絕4 ppm、鏈黴素200 ppm、青黴素200 ppm及氯黴素200 ppm後,製成之MNa半選擇性培養基,可以有效偵測出木屑基質中的杏鮑菇褐斑病菌,並可有效鑑別本菌感染的罹病菇。本病原菌的主要感染源為栽培室內的空氣濾網,此外,在山黃麻木屑與菇場鄰近土壤,也可偵測到本菌的存在。 至於防治方面,以Streptomyces padanus PMS-702在菌量濃度104cfu./ml、Bacillus subtillis BS 6-14菌株在105 cfu./ml菌量濃度或植物健素中興一百2000倍稀釋液,與杏鮑菇褐斑病菌孢子懸浮液106 spores/ml同時接種或先噴佈拮抗微生物、植物健素一天後,再接種杏鮑菇褐斑病菌,兩種處理皆可顯著降低菇體之罹病度達30%以上。以清水或2%次氯酸鈉清洗菇類栽培室之空氣濾網,具有顯著降低菇舍內褐斑病菌菌量的功效。
In 1998, commercial cultivation of Pleurotus eryngii (Dc. : Fr.)Quēl. at Taiwan Agricultural Research Institute (TARI) and Wufeng areas around central Taiwan were severely affected by an unknown disease. Symptoms consisted of brown spot, curling of the tissues, sometimes even shrinking and cracking of the infected fruit bodies. Ten isolates, GR-6、GR-7、GR-12、GR-13、GR-16、GR-18、GR-23、GR-28、GR-31 and GR-37 were isolated from diseased fruiting bodies. Pathogenicity was confirmed by inoculating the fungus onto fruit-bodies of P. eryngii for 3 - 4 days under high humidity (RH>93%) at 16℃. Discoloration of brown spot similar to the original symptoms developed only on inoculated king oyster mushrooms. Noninoculated king oyster mushrooms P. eryngii remained symptomless. On malt extract peptone agar, colony diameter reached 32.5-35.0 mm 10 days after inoculation at 20℃, appeared to be orange to salmon red. Mycelium with septa, two kinds of conidiophores, one consists of verticillate conidiophores 110-170 µm, bearing 2-6 whorls of phialides 13-28 µm; the other bearing the “densely penicillate” phialides 10-16 µm ; Conidia hyaline, one-celled, ovoid , accumulating in a single, slimy, base protruding, smooth-walled, 4.0-6.8 x 3.0-4.5 µm. The fungus was identified as Gliocladium roseum Bainier according to the references of Domsch, et al (1980)、Schroer, et al (1999) and Smalley & Hansen (1957). In advance, the causal agent was also confirmed by comparing the ITS and 28S rDNA sequences of ten isolates of the pathogen with NCBI database. The optimum temperature for conidial germination and mycelial growth of G. roseum isolates GR-12 and GR-28 was at 24-28℃. The best temperature for infection of G. roseum isolates GR-12 and GR-28 onto fruit-bodies of oyster mushroom P. eryngii was at 24-28℃. Pathogenicity of G. roseum isolates GR-12 and GR-28 was confirmed by inoculating onto fruit-bodies of Agaricus bisporus (Lange) Imbach、Lentinus edodes (Berk.) Sing.、Flammulina velutipes (Curt.: Fr.) Sing.、Pleurotus sajor (Fr.) Sing.、Pleurotus cystidosus Miller、Agrocybe cylindracea (DC: Fr) Mre.、Hypsizigus marmoreus Bunashimeji、Coprinus comatus (Müll.: Fr. ) Pers. Twelve carbohydrates and twelve nitrogenous compounds were evaluated for their effects on mycelial growth of two isolates, GR-12 and GR-28 of the pathogen. Among those compounds, sucrose, sorbitol, starch and maltose were more effective than other carbohydrates to enhance the growth of G. roseum. As to nitrogenous compounds, NaNO3, NaNO2 and KNO3 were more effective to enhance growth of the pathogen. Seven pesticides were respectively added into the basal medium (a modified Czapek’s medium containing 3% (w/v) maltose and 0.2% (w/v) NaNO2 , MNa medium) for indexing their suppressive effectiveness. Flutolanil at 200 ppm, mertect at 4 ppm, streptomycin sulfate at 500 ppm, chloramphenicol at 500 ppm and penicillin G at 500 ppm were obviously not effective in inhibiting growth of the pathogen. Finally, maltose-NaNO2 semiselective medium (MNa semiselective medium) consisting of 30 g maltose, 2 g NaNO2, 1 g K2HPO4, 0.5 g MgSO4 7H2O, 0.5 g KCl, 0.01 g FeSO4 7H2O, 20 g agar, 200 ppm flutolanil, 4 ppm mertect, 200 ppm streptomycin sulfate, 200 ppm chloramphenicol, 200 ppm penicillin G and 1L distilled water was hence formulated for the enumeration and isolation of G. roseum from the infested sawdust. The results showed that G. roseum could be accurately detected from artificially and naturally infested sawdust by use of MNa semiselective medium. Population density of the pathogen in naturally infested sawdust was 0 - 1.0×102cfu/g sawdust and in naturally infested soil was 0 — 3.0×102cfu/g soil. High population density of the fungus was also detected from air filter of the mushroom cultivation room by MNa semiselective medium. The disease severity could be reduced more than 30% by spraying Streptomyces padanus PMS-702 at 104cfu/ml, Bacillus subtilis BS 6-14 at 105cfu/ml or CH100 at 2000-fold dilution at the same time or one day before inoculation with the pathogen onto the fruit-bodies of P. eryngii. Environmental hygiene of the mushroom cultivation room is the determinant factor for the disease control. The study indicated that washing air filter of the mushroom cultivation room with water or 2%(v/v) sodium hypochlorite was effective in reducing population density of the pathogen for the disease control.
URI: http://hdl.handle.net/11455/31761
Appears in Collections:植物病理學系

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.