First Report of Walnut Anthracnose Caused by Colletotrichum fioriniae on English (Persian) Walnut Fruits in Hungary

In September 2017 brown or black huge circular sunken necrotic lesions were observed on walnut fruits (Juglans regia L.) in the fruit variety collection of NARIC Research Institute in Érd, Hungary. Similar symptoms were visible on walnut fruits in three other locations of the country in 2018; the disease symptoms occurred diffusely (in orchards of Lengyeltóti, Nagyoroszi) or widely (in orchards of Sarród, Érd). Early symptom stages were observed in June 2018, with small dry spots affecting the surface of the husk. Acervuli were formed on collected fruit samples, and orange conidial masses appeared under wet conditions. Conidia were hyaline, unicellular, and fusiform, pointed at one or both ends, and measured 11.0 to 17.6 × 3.8 to 5.0 µm (n = 100). The fungus was isolated from necrotic tissues and conidial masses on potato dextrose agar (PDA) medium amended with chloramphenicol (25 mg/liter). The colonies were white to pink on the upper side and pink with black spots on the reverse. Acervuli formed and produced conidial masses on PDA on 23°C after 6 days; later conidia formations were observed partly on hyphae. The isolates were morphologically identified as Colletotrichum acutatum sensu lato. The C. acutatum isolates were identified first based on the internal transcribed spacer (ITS) region. The actin (ACT) and calmodulin (CAL) gene sequences were determined to identify the Colletotrichum strains. ITS, ACT, and CAL genes were amplified by ITS1F/ITS4R, ACT512F/ACT783R, and CAL1/CAL2 primers (Carbone and Kohn 1999; O’Donnell et al. 2000; White at al. 1990). The sequences of the ITS region (accession nos. MK367397, MK367400, and MK367403) showed 99% similarity (query cover: 100%) with several Colletotrichum fioriniae sequences from walnut in GenBank (e.g., MF554910 to MF554914) and based on ACT gene (nos. MK415990, MK415993, and MK415996) showed 99% identity to C. fioriniae (e.g., MF554940) from walnut. The obtained sequences of CAL gene (nos. MK415997, MK416000, and MK416003) were the same and showed 99% similarity (query cover: 98%) with other C. fioriniae sequences from other host plants (e.g., Fagus sylvatica, KY828143) (Pszczolkowska et al. 2018). The fungus was identified as C. fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan. Its teleomorph, Glomerella fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan, was not found. Pathogenicity tests were accomplished in the field and under laboratory conditions (23°C on thermostat) on 10 green walnut fruits each. Tests were conducted both on living trees and collected fruits by inserting mycelial agar plugs onto wounded pericarp tissues, which were then wrapped with wet cotton and Parafilm. Wounded tissues on five fruits were treated with noncolonized PDA plugs as noninoculated controls. Necrotic lesions 10 to 19 mm in diameter developed after 14 days of incubation both on field and on laboratory-inoculated walnut fruits, whereas no necrosis developed around control wounds. The morphology of the reisolated fungus was consistent with the inoculated one, fulfilling Koch’s postulates. Leaf spot disease of walnut caused by C. fioriniae was reported from China (Zhu et al. 2015), and an epidemic event of walnut anthracnose caused by C. fioriniae and other Colletotrichum species was reported in France (Da Lio et al. 2018). To our knowledge, this is the first report of anthracnose of walnut fruits caused by C. fioriniae in Hungary and the second in the world. This disease may become a major problem on Persian walnut trees.