Detection of Diaporthe cinerascens from latent and symptomatic fig tree cankers using species-specific primers

Abstract

Fig canker is a multi-agent and complex disease, and since its identification, Diaporthe canker, primarily caused by Diaporthe cinerascens, has emerged as one of the most persistent and destructive diseases affecting fig trees in regions such as Iran, California, Canada, and Italy. The economic implications of this disease are considerable, further aggravated by the lack of effective therapeutic interventions. This study presents the development and validation of novel species-specific PCR primers designed to enhance the early and accurate detection of Diaporthe canker in artificially and naturally infected samples with mixed and latent infections. We utilized pure cultures of well-characterized D. cinerascens isolates, whose proven pathogenicity and genetic homogeneity were validated in earlier studies. Among the 82 developed primers, the TUB-Dc1 and HIS-Dc1 pairs were chosen after a thorough analysis of the β-tubulin (tub) and histone 3 (his3) genes, along with the internal transcribed spacer of rDNA (ITS) region. These selected primers successfully amplified target gene sequences in pure cultures and infected tissue samples using direct and nested PCR. The diagnostic sensitivity thresholds achieved were 100 fg μL⁻¹ for direct PCR and 1 ag μL⁻¹ for nested PCR. Notably, nested PCR significantly enhanced detection capabilities by up to 100,000-fold compared to direct PCR. The TUB-Dc1 primer pair effectively identified D. cinerascens in 80 % of samples with Type A canker, 22.7 % with Type C canker, and 8.8 % showing dieback symptoms. In comparison, conventional cultural methods detected the pathogen in only 30 % of Type A canker samples, and 4.4 % of Type C canker samples, with no detections in samples showing dieback. Furthermore, our results underscore the potential utility of our molecular tools for precise identification and detecting latent infections in asymptomatic samples via nested PCR. Our findings indicate that the species-specific assays are highly sensitive, specific, and reliable for detecting D. cinerascens in symptomatic and asymptomatic environmental samples. Additionally, this tool paves the way for future applications aimed at certifying pathogen-free fig saplings, an essential component for sustainable fig cultivation practices.