Abstract:

Citrus melanose is a worldwide fungal disease which seriously harms citrus production. Rapid detection of Diaporthe citri, the pathogen of citrus melanose, is of great practical significance for early diagnosis and scientific prevention and control of citrus melanose. In this study, a specific primer pair for D.citri was designed based on β-tubulin gene sequence, and a rapid molecular detection system was established based on conventional PCR and SYBR Green I Real-time PCR(qRT-PCR) to detect and verify the mycelia and typical infected leaves of D.citri. The results showed that in conventional PCR, the primers could obtain a 244 bp specific amplification fragment from D.citri, and the detection sensitivity was up to 0.78 ng·μL^-1. In qRT-PCR, the primers could only obtained a unique product absorpation peak from D.citri, and the detection sensitivity was up to 0.35 ng·μL^-1.The typical infected leaves were detected by PCR detection system. The results showed that the positive detection rate of conventional PCR was 30%, and that of qRT-PCR was 60%, indicating that qRT-PCR was more sensitive than conventional PCR. Therefore, the specific primers designed in this study and the established conventional and qRT-PCR detection method have the advantages of fast speed, strong specificity and high sensitivity, which can be used for molecular identification of D. citri and also have reference value for disease diagnosis.

Key words: Diaporthe citri, β-tubulin sequence, SYBR Green I Real-time PCR, Conventional PCR detection