High Sensitive Method for the Simultaneous Detection of Pathogens in Chrysanthemum morifolium: A Nested Multiplex PCR Assay

Abstract

Chrysanthemum morifolium, a kind of medicine and food homology product, is threatened by plant pathogens. It is crucial to establish a sensitive and accurate method for diagnosing its pathogens. Therefore, the internal transcribed spacer regions of the ribosomal DNA (rDNA) sequences of three common pathogens in C. morifolium (Chaetomium globosum, Fusarium incarnatum, and Alternaria alternata) were compared, and the three pairs of specific primers were achieved. A two-step nested multiplex PCR system which consists of two sequential amplification steps was successfully developed to detect those three pathogens in C. morifolium. This method was sensitive due to its low detection limits, which were 10 pg/μL, 1 pg/μL, and 10 fg/μL for C. globosum, F. incarnatum, and A. alternata, respectively. The entire detection process of this method only took 5–6 h, demonstrating its high efficiency. The nested multiplex PCR system could be successfully applied to detect actual samples, and a 90% detection rate for those three pathogens in selected C. morifolium was achieved. This suggests that our assay could be an operator-friendly tool for diagnosing C. morifolium diseases.