First Report of Nut Rot Caused by Neofusicoccum parvum on Hazelnut (Corylus avellana) in Italy

Hazelnut (Corylus avellana L.), which is native to Europe and Western Asia, is a widely distributed and economically important crop in Italy, cultivated on 82,104 ha and its production is 110,618 t (ISTAT 2021). A total of one hundred and eleven black rotted nuts (incidence: 41%) with sunken lesions from Lu and Cuccaro (45°00'21.8"N/8°28'59.6"E), north-western Italy, were collected during the ripening stage of hazelnuts during October-December 2020. Symptomatic half cut kernels were sterilized in 1% NaClO for 1 min, washed in sterile water twice, and dried on sterile filter paper. The fragments were placed onto potato dextrose agar (PDA) containing streptomycin. After 48 to 72 h of incubation at 25°C, fast-growing white colonies with abundant aerial mycelia were observed. On the opposite side of the plates, colonies were initially white, then turned to dark-grayish olive after one week of incubation. Dark colonies produced globose, hyaline, ellipsoidal, unicellular conidia ranging from 12.23-15 μm x 5-6.71 μm. Morphologically, the causal agent was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (Crous et al. 2006; Zhang et al. 2021). The DNA from the isolates HMa-19-2 and Hwb-4b-2 was extracted with the E.Z.N.A. Fungal DNA mini kit (Omega Bio-tek) according to manufacturer instructions. Molecular identification was confirmed by sequencing of rDNA internal transcribed spacer (ITS) using primers ITS1/ITS4 (White et al. 1990) and translation elongation factor 1-alpha (tef-1α) gene by using the primers EF1-728F/EF1-986R (Carbone & Kohn, 1999). The sequences of both isolates were deposited in GenBank for ITS (accession numbers MZ848132 and MZ848133) and for tef-1α gene (accession numbers MZ913266 and MZ913267). The BLAST analysis showed 99% identity with ex-type strain of N. parvum (CMW9081) for ITS and tef-1α. Maximum likelihood method based on combined sequences of ITS and tef-1α genes was performed and the isolates of N. parvum clustered with ex-type strain of N. parvum (CMW9081; eXtra file). Pathogenicity of both isolates were tested on ripening hazelnuts (BBCH: 85) to evaluate Koch's postulates. 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, then nuts were inoculated with PDA mycelium plugs of the same diameter cut from 7 days old PDA colony (Seddaiu et al. 2021). The control nuts were treated with sterilized PDA plugs. All inoculated nuts produced black lesions with softening pulp (eXtra file). Additionally, abundant white-gray mycelium developed on the inoculation sites. Control nuts showed no symptoms. Neofusicoccum parvuwas recently reported in Italy on chestnut (Seddaiu et al. 2021) and blueberry (Guarnaccia et al. 2021). The fungal pathogen was also reported as agent of grey necrosis of hazelnut in Chile (Duran et al. 2020). However, this is the first report of N. parvum on hazelnut nut in Italy. The findings suggest that N. parvum could severely affect hazelnut production in Italy. Accurate identification of the pathogen will support the growers to manage the disease.