First Report of Branch Dieback of Walnut Caused by Neofusicoccum parvum in Korea

Abstract

Walnut (Juglans sinensis Dode) is an economically important tree in the world, both for its wood and its fruit. Walnut fruits, as rich sources of omega-3 essential fatty acid, are valuable nutritionally. Consumer interest in Korea for walnuts has increased in recent years, and production has increased to 1,042 ha with the Kyoungbuk region consisting of 402 ha (2). In May 2012, lethal dieback disease of walnut tree was detected in two orchards in Andong, Kyoungbuk region, Korea, each with an incidence of 25 to 30%. Disease symptoms included blight and dieback of the stems, flowing resin, dark decay inside the bark of dead twigs, and defoliation. The bark of dead twigs was removed and sliced thinly using a razor blade, and water-mounted, without staining, for observation of fungal structures, if present. Pycnidia were found embedded within the bark of dead twigs and conidia were mostly characterized by fusoid, hyaline, smooth, thin-walled, unicellular and 16.25 to 21.25 μm long and 4.37 to 6.87 μm wide. These characteristics are consistent with those reported previously for Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (1). Diseased branch tissues collected from the two locations were surface sterilized with 1% NaOCl, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA). The fungal isolates, recovered from the two different orchards, produced white, aerial mycelium and became light gray within a week after incubating plates at 25°C. To confirm the identities of the isolates, the complete internal transcribed spacer (ITS) rDNA of the fungi was amplified and sequenced using PCR. The sequences were compared with other DNA sequences in the GenBank database, using a BLAST search. BLAST analysis of the PCR product showed that the sequence had 99% identity with the nucleotide sequences for N. parvum (JQ411396.1 and GU997688.1). Additionally, the chitin synthase 1 gene was sequenced and analyzed using the BLAST server. The sequence of PCR product had 100% identity with the nucleotide sequences of N. parvum strain CMW9080 chitin synthase 1 gene (EU339501). Thus, both morphological and molecular characters confirmed this species as N. parvum. Pathogenecity tests were performed by inoculating 2-year-old J. sinensis trees. Inoculations consisted of inserting 5-mm-diamter agar plug bearing fresh mycelium of the fungal isolates into the wounds. Within 2 weeks, black lesions appeared on all inoculated plants accompanied by defoliation, whereas no symptoms were observed in the control plants. N. parvum has been reported a member of Botryosphaeriaceae, commonly associated with dieback and cankers of woody plants (1). To our knowledge, this study is the first report of N. parvum as a pathogen of Juglans sinensis in Korea. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) Statistics Korea. Forest Households by Growing Area of Walnut/Total Area. 2010.