Genetic diversity and pathogenicity of Botryosphaeriaceae and Diaporthaceae causing defects of hazelnut nuts from Italy.

Italy is the second-largest hazelnut producing country (110,000 t/year) after Turkey, on a surface of 81,000 ha. Major constraints for hazelnut production are members of Botryosphaeriaceae family and genus Diaporthe which are responsible for several defects (internal discoloration, necrosis, blemishes) in hazelnut kernel, and can reduce the hazelnut quality and yield by altering its kernel. In order to investigate the phenomenon of rotten hazelnuts and to identify the responsible agents, a survey was initiated during 2020. A total of 383 samples having the symptoms of black rot (incidence: 32%), mouldy rot (incidence: 41%) and necrosis (incidence: 27%) were collected from Piedmont, northern Italy. Fungal genomic DNA was extracted, and multi-locus phylogeny was performed based on combined partial genomic region ITS and the partial gene tef-1α. ITS and tef-1α sequences were obtained after PCR amplification using the primers ITS1/ITS4 and EF1-728F/EF1-986R, sequencing and phylogenetic analysis for species identification. Pathogenicity tests were performed on ripening hazelnuts ‘Tonda Gentile del Piemonte’. Three nuts per isolate, and per three replicates were surface disinfected with 1% NaClO. A piece of shell (5 mm diameter) from nuts was removed with a sterile cork borer and inoculated with mycelium plug cut #Fungal22 | 169 from 7 days old PDA colony. Research results revealed that isolates represent 6 species of Diaporthe (D. eres, D. rudis, D. novem, D. oncostoma, D. ravennica, D. foeniculina) and 3 species of Botryosphaeriaceae (Botryosphaeria dothidea, Diplodia seriata and Neofusicoccum parvum). Overall incidence of Diaporthe spp., B. dothidea, D. seriata and N. parvum were 39%, 20%, 15% and 5%, respectively. Pathogenicity results revealed that all these species are pathogenic to the tested cultivar. All the hazelnut kernels showed abundant development of pycnidia with different disease index. Additionally, isolates from B. dothidea, D. seriata and N. parvum produced black lesions with softening pulp. D. eres was more virulent compared with the other Diaporthe spp. N. parvum was the most pathogenic species among Botryosphaeriaceae. The present study improves our understanding of the species associated with hazelnut defects and provides useful information for effective management of the nut disease.