Species-specific PCR primers for the detection of Phyllosticta minima, the causal agents of leaf spot diseases on Acer pycnanthum
2016
Yano, A. (Tokyo University of Agriculture (Japan). Graduate School of Agriculture, Department of Forest Science) | Motohashi, K.
Acer pycnanthum is a maple tree indigenous to Japan, which grows in limited areas of the Aichi, Gifu, and Nagano Prefectures. A. pycnanthum is currently an endangered species, with a declining population. This tree is considered to be a vulnerable species according to the Red List of the Ministry of the Environment. Leaf spot disease caused by Phyllosticta minima, a phytopathogenic fungus, inflict severe damage on A. pycnanthum. These fungi secrete inhibitory factors, which have been suggested to repress natural regeneration. In the present study, we designed species-specific primers with the aim of specifically detecting pathogens in tissue samples of A. pycnanthum, thereby identifying infection routes crucial for disease control. The species-specific primers PmiF and PmiR, which were designed based on the sequence of the P. minima rDNA Internal Transcribed Spacer region, were verified for their specificity in PCR tests using the total DNA of isolated strains. In A. pycnanthum tissue samples from Hachioji, Tokyo and Nagoya, Aichi Prefecture, pathogenic fungi were isolated not only from affected but also asymptomatic leaves. Furthermore, in trees planted at Hachioji, Tokyo, pathogens were observed in tissue samples of leaves, petioles, twigs, winter buds, and axillary buds. These results suggest that P. minima infects axillary (winter) buds from June to July via previously infected leaves, thus becoming latent and finally triggering primary infections in the following year.
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