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Resistance to the quinone outside inhibitor (Qol) fungicides in the tea gray blight-causing fungus Pestalotiopsis longiseta is a serious problem in Japanese tea cultivation. We conducted a population genetic analysis of Qol-resistant P. longiseta isolates on the Makinohara Plateau, Shizuoka Prefecture, Japan's largest tea-growing area, to elucidate the disease's epidemiology and the spread of Qol resistance. Inter simple sequence repeat (ISSR) analysis of 1,083 isolates from 395 fields collected from 2009 to 2012 detected 42 ISSR types, designated as PL01 to PL42. A total of 18, seven, and 38 ISSR types were detected in highly resistant, moderately resistant, and sensitive isolates, respectively. No distinct phylogenetic relationship corresponding to Qol sensitivity or sampling location was observed. No annual changes in the population genetic structure of highly resistant isolates were observed during the study period. A different ISSR type was predominant among Qol-resistant isolates in each region. Analysis of molecular variance revealed significant genetic differentiation in populations of highly resistant isolates among regions (FCT= 0.213) and farmers (FCT = 0.071). Consequently, we speculate that Qol-resistant P. longiseta strains occurred in a number of clonal lineages and spread by both natural and artificial transmission, such as rain splash and plucking machines, throughout each region on the Makinohara Plateau. [ABSTRACT FROM AUTHOR]