A TaqMan Real-Time PCR Assay for Early and Accurate Detection of Hypomontagnella monticulosa, an Emerging Pathogen Causing White Leaf Spot on Pachira glabra Pasq.

Hypomontagnella monticulosa is an emerging pathogen of Pachira glabra Pasq. causing white leaf spot, a damaging fungal disease of P. glabra in southern China. The early and proper detection and qualification is of fundamental for understanding epidemiology and developing preventive measures of this fungus. Using the second largest subunit of the RNA polymerase II gene as target, a quantitative TaqMan real-time polymerase chain reaction assay was developed for the detection and quantification of H. monticulosa in P. glabra leaves. This method could specifically recognize all tested H. monticulosa strains, while no cross-reaction was observed in closely related Hypoxylon species. Sensitivity of the assays was determined to be as low as 0.05 fg/μL (2, 300 copies/μL) of plasmid DNA, 0.5 pg/μL of mycelia genomic DNA, and 0.001% of target DNA mixed with leaf tissue DNA. The two-stage induction of H. monticulosa DNA was observed during the infection process, suggesting that this assay could be used to monitor the growth dynamics of this fungus in the whole disease process. Additionally, the assay could not only effectively detected H. monticulosa in naturally infected P. glabra trees in fields, but also accurately evaluate the differences in resistance among varieties of P. glabra. Therefore, the current study provides a rapid and accurate technology for monitoring and qualification of H. monticulosa infection in P. glabra, and will be applicable for prediction and control of the disease but also for the study of plant-H. monticulosa interaction.