First specific detection and validation of tomato wilt caused by using a PCR assay.

Tomato wilt is a widespread soilborne disease of tomato that has caused significant yield losses in many tomato growing regions of the world. Previously, it was reported that tomato wilt can be caused by many pathogens, such as , , , , and . In addition, we have already reported that caused symptomatic disease of tomato wilt for the first time in China. The symptoms of tomato wilt caused by these pathogens are similar, making it difficult to distinguish them in the field. However, specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for to establish a more effective plan to control the disease. In this study, we designed -specific forward primers and reverse primers with a fragment size of 283bp located in the gene encoding carbamoyl phosphate synthase arginine-specific large chain by whole genome sequence comparison analysis of the genomes of eight spp.. We then tested different dNTP, Mg concentrations, and annealing temperatures to determine the optimal parameters for the PCR system. We evaluated the specificity, sensitivity and stability of the PCR system based on the optimized reaction system and conditions. The PCR system can specifically identify the target pathogens from different fungal pathogens, and the lower detection limit of the target pathogens is at concentrations of 10 pg/uL. In addition, we can accurately identify in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose .