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

Abstract

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 Fusarium oxysporum, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Fusarium acuminatum, and Plectosphaerella cucumerina. In addition, we have already reported that Fusarium brachygibbosum 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, F. brachygibbosum specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for Fusarium brachygibbosum to establish a more effective plan to control the disease. In this study, we designed F. brachygibbosum-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 Fusarium spp.. We then tested different dNTP, Mg2+ 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 F. brachygibbosum in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose F. brachygibbosum.

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

“…PCR systems with dNTPs at 0.2–0.4 mM are usually the most favorable for amplification, and amplification is rapidly inhibited above this value, while lower dNTP concentration (dNTPs at 0.1 mM) allows PCR amplification with reduced products ( Markoulatos et al, 1999 ; Markoulatos, Siafakas & Moncany, 2002 ). In addition, optimization of Mg 2+ is crucial as excessive Mg 2+ concentration stabilizes DNA double strand and prevents complete denaturation of DNA, thus reducing amplification yield, while insufficient Mg 2+ concentration would also reduce PCR product ( Markoulatos, Siafakas & Moncany, 2002 ; Deng et al, 2023 ). In this study, we optimized our PCR system with 0.8 μmol/L Fu-4F, 0.2 μmol/L Fgram-R, 0.2 μmol/L Fpseu-R, 0.2 μmol/L Fprol-R, 0.2 μmol/L Fvert-R, 2 mM MgCl 2, 0.2 mM dNTPs with the annealing temperature of 53 °C in a 50 μl reaction.…”