請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/31116
標題: 發展人工孢子以研究立枯絲核菌在土壤中的生物學
Development of artificial conidia for the study of biology of Rhizoctonia solani in soil
作者: 劉東憲
Liu, Tung-Hsen
關鍵字: Protoplast
原生質體
Artificial conidia
Rhizoctonia solani
人工孢子
Rhizoctonia solani
出版社: 植物病理學系所
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摘要: 摘 要 真菌的原生質體能夠作為真菌遺傳學及分子生物學的研究工具。本試驗是發展立枯絲核菌 (Rhizoctonia solani Kühn) 產生原生質體的方法,取代過去使用已停產的酵素Novozym 234。將培養48小時R. solani AG-1 IC的菌絲,以Driselase (20 mg/ml) 結合Lysing enzyme (10 mg/ml) 反應在37℃ 15分鐘然後在34℃ 105分鐘,原生質體能夠達到最高的產量。原生質體產生濃度大約為6 × 105 /ml,而大小為8到12 μm。而其他R. solani菌絲融合群的分離株以這個方法亦可產生原生質體。原生質體培養在24℃ 4小時以Ca-sucrose溶液形成細胞壁效果最好。光照不會影響原生質體形成細胞壁。原生質體形成細胞壁所用的蔗糖以購自林純藥理 (Hayashi Co.) 的效果最好。產生的原生質體有0 ~ 7個細胞核,而人工孢子有1 ~ 8個細胞核。原生質體的大小與細胞核的數目沒有顯著相關性,而人工孢子的大小與細胞核的數目相關性低。不像菌核或菌絲,R. solani的人工孢子敏感於土壤靜菌作用。人工孢子的發芽管在土上生長也同樣會受土壤靜菌作用抑制。以人工孢子發芽測定殺菌劑福多寧的劑量反應,發芽情形在土上和在培養基上相似。從台灣收集52種的土壤樣本中,有三種土能抑制R. solani之人工孢子的發芽,而人工孢子在不同土中發芽率與土壤酸鹼值無關。有些土可能不抑制人工孢子發芽,但是會抑制菌絲生長。在抑菌土上人工孢子的發芽管分解速度比導菌土快。實驗結果顯示以R. solani之人工孢子能夠定量研究此病原菌在土中之生物學。
Abstract The fungal protoplasts can be used as a tool for the studies of fungal genetics and molecular biology. A new protocol of protoplast formation by Rhizoctonia solani was developed to replace the old protocol that used Novozym 234 which is no longer available. The greatest number of protoplasts was produced by treating 48-h-old mycelium of R. solani AG-1 IC with Driselase (20 mg/ml) and Lysing enzyme (10 mg/ml) for 15 min at 37℃ and then 105 min at 34℃. The yield of protoplasts was about 6 × 105 /ml, and the size of the protoplasts ranged from 8 to 12 μm. Other anastomosis groups of R. solani also released protoplasts by using this protocol. The best conditions for cell wall synthesis of protoplasts were incubation of protoplasts in Ca-sucrose solution for 4 h at 24℃. The light did not affect the cell wall synthesis of protoplasts. The sucrose purchased from Hayashi Co. is the best source for cell wall synthesis of protoplasts. Each protoplast contained 0 ~ 7 nuclei while each artificial conidium contained 1 ~ 8 nuclei. No significant correlation or small correlation was found between nuclear number and size of protoplasts or artificial conidia. Artificial conidia were bigger than protoplasts. Unlike sclerotia or mycelia, artificial conidia of R. solani were sensitive to soil fungistasis. Growth of germ tubes of R. solani on nature soil was also inhibited. Dosage response of artificial conidia of R. solani to flutolanil on soil was similar to that on water agarose. Three of 52 soil samples tested suppressed to germination of artificial conidia of R. solani, and germination rates of artificial conidia on soils were not correlated with soil pH. Some soils were conducive to germination of artificial conidia, but suppressive to hyphal growth. Lysis of germ tube of artificial conidia was faster on suppressive soil than conducive soil. Results showed that artificial conidia of R. solani are suitable for the quantitative studies of the biological activities of this pathogen in soil.
URI: http://hdl.handle.net/11455/31116
其他識別: U0005-0102200914491500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0102200914491500
顯示於類別:植物病理學系

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