NOVEL SPECIFIC PRIMERS FOR THE SPECIFIC DETECTION OF FUSARIUM OXYSPORUM F. SP. CUBENSE BASED ON SYBR GREEN REAL-TIME PCR

Abstract

Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Fusarium wilt of banana worldwide. Foc is transmitted to another banana plantation via an infected banana sucker. A specific and sensitive detection assay could be used for disease-free propagule screening to prevent disease dispersal effectively. In this study, SYBR green-based real-time PCR assay was developed based on novel specific primers targeting the large subunit of RNA polymerase II (RPB1) gene. The partial RPB1 gene was amplified and sequenced from Foc isolate FOC1708 and SK3-2. Regions specific for Foc were identified and used for designing real-time PCR primers. The specificity of the designed primers was evaluated on genomic DNA from Foc isolates and non-target microorganisms. The results showed that the designed primers were specific to only Foc isolates from race 1 and tropical race 4 (TR4). The detection efficiency of the designed primers was compared to other published primers through optimized SYBR green-based real-time PCR assay and nested PCR. The new primers could detect the Foc genomic DNA at a minimum of 5 pg and target pathogen in a symptomless banana sucker. The specificity and sensitivity of the new primers were comparable to the published real-time PCR primers and the nested PCR assay. This developed assay with these novel primers can aid the disease quarantine for effective prevention and control of the Fusarium wilt of banana.