Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31900
標題: Rhizoctonia solani AG 1-IC: 有效的一致性接種源製備和由原生質體所得之同核與異核菌株的確認與比較
Rhizoctonia solani AG 1-IC: production of uniform and effective inocula, and confirmation and comparison of homokaryotic and the heterokaryotic isolates derived from protoplasts.
作者: 蔡昀濃
Tsai, Yun-Nung
關鍵字: Rhizoctonia solani AG 1-IC
Rhizoctonia solani AG 1-IC
uniform
inoculum
disease suppressive soil
protoplast
tuft
homokaryon
heterokaryon
variant
一致性
接種源
抑病土
原生質體
絨毛菌絲叢
同核菌株
異核菌株
異常的菌株
出版社: 植物病理學系所
摘要: 第一章 將Rhizoctonia solani AG 1-IC纏據在滅過菌的油菜子上,開發出簡單的一致性接種源製備方法。最適合的製備條件為在光照下培養七天,而最適合的接種顆數為兩顆。將兩顆接種源,接種在五顆蘿蔔幼苗的中間,R. solani可使四棵以上的蘿蔔幼苗發生猝倒。一顆被纏據的油菜子,可以使兩棵以上的蘿蔔幼苗死亡。這個接種源不論是在相等數量上或相等重量上,其對蘿蔔幼苗的感染能力比菌核好。將R .solani AG 2-1、AG 4和AG 4 HG-I所纏據的油菜子接種蘿蔔幼苗,仍可以有效的使蘿蔔幼苗發病。利用R. solani AG 1-IC纏據其他十字花科的種子,包括芥菜、花椰菜、青花菜、青江菜、高麗菜和大白菜所製備出來的接種源,皆與纏據在油菜子上一樣有病原性。此技術不貴,製備方便、簡單而且材料容易取得。利用此方法製備出來的接種源,在形狀和大小上皆很一致而且有強感染潛力。結果也顯示,此方法適合用於偵測由R. solani及其他融合群所引起的病害的抑病土。 第二章 由於Rhizoctonia solani產生擔孢子的困難,因此利用製備原生質體的技術,來確定菌株的核型,並分析同核菌株與異核菌株及其從兩者配對後所產生的絨毛菌絲叢所分離的菌株。將從R. solani AG 1-IC所產生的原生質體菌株,利用絨毛菌絲叢的產判斷出擬同核菌株或擬異核菌株,再一次的分離原生質體菌株,並再用絨毛菌絲叢的產生再一次確認這些菌株的核型。三個擬異核菌株PR1-17、PR1-66和PR1-72經由此確認方法,皆可分離到兩種不同核型的菌株,因此確認為異核菌株。三個擬同核菌株PR1-9、PR1-35、PR1-68經由此確認方法,皆只分離到一種與自己相同的菌株,因此確認為同核菌株。另外,R. solani在這些實驗中,包括在經由不同的原生質體再生時間、不同營養、不同原生質體子代的分離、酵素處理過的菌絲片段、一至六代的原生質體子代經連續處理酵素2-4次後的原生質體菌株、製備原生質體前不同菌絲培養的溫度、單一菌絲尖端細胞分離,皆只有分離到單一個同核菌株和異核菌株,而沒有分離到兩種同核菌株。將同核菌株與異核菌株配對形成的絨毛菌絲叢後,分離出二種菌株,分別為異核菌株與異常菌株。異常菌株經由產生原生質體後,可分離出三種菌株。此三種菌株可以從形態和生長速度或利用絨毛菌絲叢形成的模式來判斷也可以分辨出來。異核菌株和同核菌株在PDA上生長比10% V-8培養基上快,而異常菌株在兩種培養上生長並無明顯差別。在PDA上的生長速度為異核菌株>同核菌株>異常菌株。在10% V-8培養基上的生長速度為異核菌株>異常菌株>同核菌株。異常菌株經由產生原生質體的方式確認後,確定為另一種異核菌株。
Chapter 1 A simple method for production of uniform inoculum of Rhizoctonia solani AG 1-IC was developed by colonization of autoclaved rape seeds with the fungus. When two colonized seeds were inoculated at the center of five radish seedlings in a pot, R. solani was able to kill more than four seedlings. The pathogen in one seed was able to cause death of more than two seedlings. Such colonized seeds have greater infection potential than sclerotia on both equal number and equal weight bases. Rape seeds colonized with AG 2-1, AG 4 or AG 4 HG-I were also effective in causing damping-off of radish seedlings. When other seeds of brassica including mustard cabbage, cauliflower, broccoli, spoon cabbage, head cabbage and Chinese cabbage were colonized with R. solani AG 1-IC, they were as pathogenic as colonized rape seeds. The technique is inexpensive and simple, and the material is readily available. Inoculum produced by this method is uniform in shape and sized, and is strong in infection potential. The result also shows that the method is suitable for use in the detection of soils suppressive to the disease caused by R. solani. Chapter 2 In Rhizcotonia solani, homokaryons and heterokaryons were usually confirmed by analysis of colony morphology of basidiospore progeny. However, production of basidiospores is frequently difficult and time assuming, and not all isolates of R. solani are capable of producing basidiospores. A simple method was, therefore, developed by analysis tuft formation pattern of protoplast regenerants. Three putative homokaryotic protoplast regenerants and three putative heterokaryotic protoplast regenerants were subjected to protoplast formation and analysis of tuft formation pattern of protoplasts regenerants. All the regenerants of putative homokaryons showed the same tuft formation pattern as their parents, thus comfirming the homokaryotic nature of these isolates. The regenerants of all putative heterokaryons show the same kind of homokaryotic tuft formation pattern and the original heterokaryotic tuft formation pattern, comfirming the heterokaryotic nature of these isolates. The protoplasts regenerants of the R. solani isolate used in this study gave rise to only one kind of homokaryon and the original heterokaryon. Repeated tests and trials using different methods to produce protoplasts failed to obtain another kind of homokaryon. The methods tested included: (i) isolation of late appearing protoplast regenerants, (ii) growing the fungus in different nutrient solutions before induction of protoplast formation, (iii) production of protoplasts from different generations of protoplast regenerants, (iv) isolation from mycelium fragments following release of protoplasts, (v) obtaining protoplasts from mycelia treated with lytic enzymes for two to four times, and (vi) growing the fungus at different temperatures before induction of protoplast formation (vii) single hyphal tip cell isolation. When single hyphal isolates were obtained from the tuft resulting from the pairing between homokaryon and heterokaryon, only original heterokaryon and a variant were obtained. The variant did not form tuft when paired with parental heterokaryon or homokaryon. Its protoplast regenerants gave rise to the original heterokaryon, the homokaryon and the variant, indicating that it is a new kind of heterokaryon. The growth of variant on PDA was shower than that of the original heterokaryon or homokaryon.
URI: http://hdl.handle.net/11455/31900
Appears in Collections:植物病理學系

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