ردیابی مولکولی زودهنگام Stilbocrea banihashemiana، بیمارگر نوظهور در درختان میوه

Stilbocrea banihashemiana is a recently described species of Ascomycota fungi (Bionectriaceae) and is the causative agent of Bionectria fig canker. This pathogen is spreading rapidly to other economically significant trees such as loquat, barberry, and walnut, and poses a substantial threat to the horticulture industry and urban trees. Early and accurate diagnosis of this pathogen is crucial for effective disease management. Our research focused on developing a polymerase chain reaction (PCR) assay using species-specific primers for S. banihashemiana. Ten primers were designed based on nucleotide sequence variations in the tef1, rpb2 genes, and the ITS region of rDNA. Four primer pairs, specifically targeting the tef1 gene, demonstrated exceptional specificity by accurately identifying the target species and avoiding detection of other non-target species associated with fig canker in both direct and nested PCR. Notably, the specific primers TEF-SbF1 and TEF-SbR2, which amplify a 577 base pair nucleotide fragment, efficiently detected DNA of the target species even at extremely low concentrations of one femtogram per microliter and 10 attogram per microliter in the direct and nested PCR assays, respectively. In addition, the selected primer pair demonstrated excellent performance in detecting the target pathogen in infected samples exhibiting trunk and branch canker during nested PCR. To detect the latent infection of S. banihashemiana, we collected 130 DNA samples from asymptomatic one-year-old branches of fig, loquat, and other important fruit trees in infected areas with this pathogen. Our molecular tool effectively identified latent infections in asymptomatic samples from fig and walnut trees across three different regions in Fars Province. This was a significant achievement, especially considering that traditional methods based on plant tissue culture could not detect these types of infections. The results of our study underscore the sensitivity, accuracy, and reliability of PCR-based evaluations using species-specific primers for the identification of S. banihashemiana from pure fungal cultures. Moreover, it proved to be an effective diagnostic tool for the pathogen in both symptomatic and asymptomatic trees. This approach paves the way for a rapid, cost-effective method for the monitoring, screening, and management of Bionectria canker caused by S. banihashemiana in both natural and potential host trees.