Quantitative real-time PCR assay for detection and quantification of Anthostoma decipiens, the cause of ‘mal dello stacco’ in hazelnut

‘Mal dello stacco’, a destructive fungal trunk disease of hazelnut, is caused by Anthostoma decipiens. This disease manifests as wedge-shaped cankers, vascular necroses, branch cracking, and reddish masses on bark surfaces. The disease has rap-idly increased in incidence and spread, resulting in significant qualitative and quantita-tive yield reductions. Disease management strategies remain insufficient, without reli-able tools for early pathogen detection in hazelnut nurseries or field plantations. This study developed and validated an SYBR Green quantitative real-time PCR (qPCR) assay for the detection and quantification of A . decipiens in hazelnut wood. Prim-ers were designed on the nuclear ribosomal internal transcribed spacer (ITS) region. The assay was optimized and validated according to EPPO standard PM 7/98 (6), for analytical specificity and sensitivity, selectivity, repeatability, and reproducibility. The primer pair AD8_F/AD16_R successfully amplified a 154 bp fragment in all of 32 A . decipiens isolates, without amplification for 42 off-target species up to 35 cycles. The assay detected up to 100 fg of pathogen DNA in water and hazelnut DNA, with reac-tion efficiencies from 97% to 100%, and high reproducibility across operators and ses-sions. The assay was validated on artificially inoculated and naturally infected hazelnut plants from Italy, Spain, and Serbia, and from ten hazelnut cultivars. The qPCR assay developed is a reliable, rapid, and cost-effective diagnostic tool for early detection of A . decipiens, with potential applications in nursery certification, orchard monitoring and epidemiological studies