| Summary: | Melanose disease caused by <i>Diaporthe citri</i> is considered as one of the most important and destructive diseases of citrus worldwide. In this study, isolates from melanose samples were obtained and analyzed. Firstly, the internal transcribed spacer (ITS) sequences were used to measure <i>Diaporthe</i>-like boundary species. Then, a subset of thirty-eight representatives were selected to perform the phylogenetic analysis with combined sequences of ITS, beta-tubulin gene (<i>TUB</i>), translation elongation factor 1-α gene (<i>TEF</i>), calmodulin gene (<i>CAL</i>), and histone-3 gene (<i>HIS</i>). As a result, these representative isolates were identified belonging to <i>D. citri</i>, <i>D. citriasiana</i>, <i>D. discoidispora</i>, <i>D. eres</i>, <i>D. sojae</i>, and <i>D. unshiuensis</i>. Among these species, the <i>D. citri</i> was the predominant species that could be isolated at highest rate from different melanose diseased tissues. The morphological characteristics of representative isolates of <i>D. citri</i> were investigated on different media. Finally, a molecular tool based on the novel species-specific primer pair TUBDcitri-F1/TUBD-R1, which was designed from <i>TUB</i> gene, was developed to detect <i>D. citri</i> efficiently. A polymerase chain reaction (PCR) amplicon of 217 bp could be specifically amplified with the developed molecular tool. The sensitivity of the novel species-specific detection was upon to 10 pg of <i>D. citri</i> genomic DNA in a reaction. Therefore, the <i>D. citri</i> could be unequivocally identified from closely related <i>Diaporthe</i> species by using this simple PCR approach.
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