Real-time PCR for detection and quantification of C. gloeosporioides s.l. growth in Stylosanthes and Arabidopsis

Abstract

Colletotrichum gloeosporioides sensu lato (s.l.) is one of the most economically and scientifically important fungal pathogens, causing serious diseases in tropical and subtropical region. The detection of C. gloeosporioides s.l. is of importance for disease control. In this study, an ACT-based real-time PCR assay was developed for quantification of C. gloeosporioides s.l., which reliably detected as little as 100 fg genomic DNA, 100 copies of target DNA and 20 conidia. This method could recognize all four tested C. gloeosporioides s.l. isolates, while no amplification was observed in other Colletotrichum species and Botrytis cinerea, indicating the specificity of this assay. Detection and quantification of C. gloeosporioides s.l. was demonstrated in artificially and naturally infected host leaves. First, the real-time PCR analysis was performed using leaf samples collected at different time points post inoculation to monitor the growth of C. gloeosporioides s.l. over time. Secondly, the method was used to compare the resistance in two Stylosanthes cultivars and two Arabidopsis cultivars. Finally, naturally infected and symptomless leaves of Stylosanthes in the fields were tested by the real-time PCR method. Overall, the real-time PCR assay could allow the detection at early symptomless phase of infection; and the results was well correlated with microscopic observation and later disease symptoms. Therefore, our studies have provided a rapid and effective detection method for studying the plant-Colletotrichum interaction as well as disease prediction and control.