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

Abstract

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.