Molecular diagnostics of Paecilomyces formosus in Zagros forests using species-specific primers

Accurate identification of the host species is critically important for disease detection and informing appropriate disease management decisions. Paecilomyces formosus, a causal agent of dieback and decline of oak, is an emerging threat that may cause severe risk to the Zagros forests of Iran in the future. In this study, a nested PCR assay for the identification of P. formosus was developed with the species-specific primer pairs PaMF and PaMR designed from the comparisons of nucleotide sequences of the nuclear ribosomal DNA internal transcribed space regions (ITS) from P. formosus isolates and other closely related taxa. To accomplish this, we sampled forest trees with dieback symptoms in Kermanshah and Ilam provinces. The Paecilomyces isolates were identified based on morphological characteristics, acid production on keratin sucrose agar medium, and sequencing of the ITS-rDNA region and part of the beta tubulin gene. Nested PCR was successfully amplified a 441 bp product exclusively from P. formosus genomic DNA, and no cross-reactions were observed with any other species, and also P. variotii. The nested PCR method can detect 100 fg of P. formosus genomic DNA. Sixty of symptomatic forest trees from seven locations in Zagros foreste were assayed, resulting in the discovery of Amygdalus lycioides, Cerasus avium, Cerasus microcarpa, Quercus libani, Acer spp., Acer monspessulanum, Ficus carica, Ziziphus spina-christi, Tamarix ramosissima and Ziziphus spina-christi as new host species, and all seven infested areas. To fulfill Koch’s postulates, the experiments were carried out on detached branches and attached healthy branches of trees at the forests in Kermanshah and Ilam provinces, Iran. The PCR-based method developed here can be used for a fast and reliable diagnosis of P. formosus, monitoring the epidemics, and assessing management strategies in Zagros forests.