Development and application of multiplex PCR for the rapid identification of four Fusarium spp. associated with Fusarium crown rot in wheat

Fusarium crown rot (FCR), caused by Fusarium spp., is a devastating disease in wheat growing areas. Previous studies have shown that FCR is caused by co-infection of F. graminearum, F. pseudograminearum, F. proliferatum and F. verticillioides in Hubei Province, China. In this study, a method was developed to simultaneously detected DNAs of F. graminearum, F. pseudograminearum, F. proliferatum and F. verticillioides that can efficiently differentiate them. Whole genome sequence comparison of these four Fusarium spp. was performed and a 20 bp sequence was designed as an universal upstream primer. Specific downstream primers of each pathogen was also designed, which resulted in a 206, 482, 680, and 963 bp amplicon for each pathogen, respectively. Multiplex PCR specifically identified F. graminearum, F. pseudograminearum, F. proliferatum and F. verticillioides but not from other 46 pathogens, and the detection limit of target pathogens is about 100 pg/μl. Moreover, we accurately determined the FCR pathogen species in wheat samples using the optimized multiplex PCR method. These results demonstrate that the multiplex PCR method established in this study can efficiently and rapidly identify F. graminearum, F. pseudograminearum, F. proliferatum, and F. verticillioides, which should provide technical support for timely and targeted prevention and control of FCR.