Development of a single-tube duplex real-time fluorescence method for the rapid quantitative detection of Fusarium oxysporum f. sp. cubense race 1 (FOC1) and race 4 (FOC4) using TaqMan probes

Banana fusarium wilt caused by Fusarium oxysporum f. sp. cubense race 1 (FOC1) and race 4 (FOC4) is a destructive disease that affects bananas in tropical and subtropical areas worldwide. A sensitive and specific detection method is the primary step to preventing spread of the disease. In this study, a real-time fluorescence PCR method was developed based on specific conserved primers from the markers of sequence characterized amplified region (SCAR) and TaqMan probes for detecting FOC1 and FOC4. The results showed that real-time fluorescence PCR could be used to detect FOC1 and FOC4 accurately and effectively within 90 min (not including DNA extraction). The developed method had high specificity and could therefore be used to distinguish F. oxysporum f. sp. cubense from other allied species and forma, such as Fusarium verticillioides, Fusarium oxysporum f. sp. melonis, Fusarium oxysporum f. sp. momodicae, Fusarium oxysporum f. sp. benincasae, and Fusarium oxysporum f. sp. opuntiarum, and other plant pathogens, such as Penicillium. This detection system also demonstrated high sensitivity, yielding a total copy number of 91,258, which was 100 times higher than that of endpoint PCR. We found that FOC1 had approximately 36 times higher abundance in banana ‘Guangfen #1’ pseudostem than FOC4 at 14 days after infection. In addition, banana ‘Guangfen #1’ root tissues showed an approximately 23 times higher abundance of FOC1 than corm tissues in field samples. In conclusion, the developed single-tube duplex real-time PCR method can sensitively distinguish FOC1 and FOC4 with high specificity. This method can be utilized to assist in the implementation of quarantine measures for the prevention and control of banana fusarium wilt caused by F. oxysporum f. sp. cubense.