Development of duplex PCR molecular rapid detection for fusarium wilt of Dajiao

【Objective】The objective of this study was to establish duplex PCR molecular detection for fusarium wilt strain of Dajiao based on gene sequences specific to 2 different differentiation lineages of Dajiao fusarium wilt strain, which could provide theoretical basis for prevention and control of banana fusarium wilt.【Method】Multiple pairs of PCR primers were designed based on rDNA intergene region(IGS) unique to pathogen of fusariun wilt pathogen of Dajiao, DNAs of 8 fusariun wilt strains of Dajiao, 3 fusarium wilt strains of Fenjiao from Guangdong, 2 fusarium wilt strains of Fenjiao from Guangxi and Hainan respectively, 5 F. oxysporum strains and 5 outgrouped strains were amplified by PCR as templates to screen the specific primers for the fusarium wilt strain of Dajiao. The specificity and sensitivity of the primers were tested, and the lower limit of strain content detection of duplex PCR system was determined.【Result】Several pairs of PCR primers were designed based on the specific gene sequence of fusarium wilt strain of Dajiao. After screening and verification, 2 pairs of specific primers were obtained that could be used to detect 2 different lineages of fusarium wilt strains(Lineage Ⅰ and Lineage Ⅱ), which could be directly applied to the detection of Dajiao fusarium wilt strains in Dajiao planting soil and Dajiao plants. The amplified fragments were 755 and 590 bp, respectively. When the amplified band was 755 bp, it indicated that Dajiao fusarium wilt strain was contained in the sample. When the amplified band was 590 bp, it indicated that the sample contained the strains of Fenjiao or Dajiao fusarium wilt. The sensitivity of the duplex PCR detection system for Dajiao fusarium wilt strain hypha was up to 0.4 μg, and the lower limit of soil strain content was 40 spores per gram of soil.【Conclusion】The established duplex PCR detection technique can simultaneously detect and distinguish two lineages of fusariun wilt strains of Dajiao and the dominant group of fusarium wilt of Fenjiao in Guangdong area in one PCR reaction, which can be used for the detection and screening of disease-free Fenjiao and Dajiao seedlings and planting soil.