Cloning and sequence analysis of the plasmid DNA found inRhizoctonia solani AG-2-2 LP isolate and its potential use for fungal detection |
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Authors: | Susumu Takamatsu Manami Nakano Kaewalin Kunasakdakul Hideyuki Yokota Hitoshi Kunoh |
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Institution: | (1) Laboratory of Plant Pathology, Faculty of Bioresources, Mie University, 1515, Kamihama, 514-8507 Tsu, Japan;(2) Department of Plant Pathology, Faculty of Agriculture, Chiangmai University, 50200 Chiangmai, Thailand |
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Abstract: | A plasmid DNA (PE-42 plasmid) obtained fromRhizoctonia solani AG-2-2 LP isolate PE-42, the causal agent of large patch disease of zoysiagrass (Zoysia spp.), was partially cloned. Sequence analyses of the 1.2-kb and 0.2-kb cloned fragments revealed that the nucleotide sequence
of the 0.2-kb fragment was similar to that of the 5′ region of the 1.2-kb fragment (pSH4). Southern hybridization analysis
of total DNA of a large patch isolate using the 1.2-kb fragment as a probe showed two bands differing slightly in size. These
results indicated that the PE-42 plasmid consisted of at least two components having similar nucleotide sequences with different
sizes. The nucleotide sequence of the pSH4 fragment showed no significant homology with known DNA sequences. The pSH4 fragment
hybridized to all of the 22 large patch isolates tested, but not to other subgroup isolates in AG-2-2, other anastomosis groups
ofR. solani, or other pathogens of zoysiagrass. These results indicated that the pSH4 fragment can be used as a specific probe to detect
the large patch fungus. The detection limit for the large patch fungus using the pSH4 fragment as a probe was 0.1 μg of the
total DNA of the fungus, which was significantly higher than those for other fungi. However, with improvement of the detection
sensitivity and simplification of the detection procedure, the pSH4 fragment has potential for use in molecular diagnosis
of the large patch disease of zoysiagrass.
Contribution No. 140 from the Laboratory of Plant Pathology, Mie University. |
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Keywords: | large patch disease molecular diagnosis plasmid Rhizoctonia solani zoysiagrass |
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