A novel TaqMan qPCR system for the detection of Diaporthe citri, the causative agent of citrus melanose

Abstract

Citrus melanose, caused by Diaporthe citri, is one of the most economically significant fungal diseases in China. Given that the application of fungicides is the main technique used for the control of citrus melanose, many studies have focused on enhancing the effectiveness and efficiency of the use of fungicides for the control of this economically significant disease. Here, we developed a TaqMan quantitative polymerase chain reaction (qPCR) protocol for monitoring citrus melanose via detection of suspected infected leaves and airborne conidia of D. citri. A primer pair and TaqMan probe targeting D. citri were designed to amplify a region of the fungal β-tubulin gene. The limits of detection and quantification of our TaqMan qPCR method were 159.57 and 683 copies/μL, respectively. The method detected the DNA equivalent to 47 conidia when fungal conidia were cultured in vitro. To evaluate the feasibility of our TaqMan qPCR method, symptomatic and asymptomatic leaf samples, airborne conidia samples were collected from a citrus orchard and tea garden, respectively. D. citri was detected in leaves with citrus melanose symptoms in an orchard; airborne fungal conidia of D. citri were also detected. These findings suggested that this novel TaqMan qPCR method could be used to detect D. citri both in vivo and in vitro. Our methodology could be applied as an alternative method for monitoring the changes in the incidence of D. citri in orchards.

Keywords:

• Citrus melanose
• Diaporthe citri
• TaqMan quantitative PCR
• fungal conidia

Acknowledgements

The authors would like to thank Sangong Bioengineering (Shanghai Co., Ltd.) for provision of sequencing of the recombinant plasmid.

Author’s contributions

Conceived, designed the experiments, writing-original draft preparation, writing-review and editing, Z.X.P., F.H., Z.D.H.; Investigation, performed the experiments, sampling, Z.X.P., L.M.L., D.C.D., L.Z., X.R.H, Z.D.H.; Analysed the data, Z.X.P., Z.D.H. and L.M.L. Funding acquisition, Z.X.P., Z.D.H.; All authors have read and approved the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data that support the findings reported in this study are available from the corresponding author(Z.D.H.)upon reasonable request.

Additional information

Funding

This study was supported by the Key Research and Development Program of Zhejiang Province (2019C02022), the China Agriculture Research System (CARS-27), and the Science and Technology Plan Project of Taizhou (1901ny13).