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

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.