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

Abstract

‘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 rapidly increased in incidence and spread, resulting in significant qualitative and quantitative yield reductions. Disease management strategies remain insufficient, without reliable 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. Primers 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 reaction efficiencies from 97% to 100%, and high reproducibility across operators and sessions. 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.