First Report of Twig Dieback Caused by Neopestalotiopsis clavispora on Blueberry in Korea

Abstract

Blueberry (Vaccinium corymbosum) is an economically important fruit with increasing cultivation in Korea, expanded to about 4,000 ha in 2017. In January 2018, dieback symptoms were observed on young twigs and stems of blueberry plants at an orchard located in Goheung, Jeonnam Province, Korea. Initial red spots developed into larger lesions with brown margins, which gradually enlarged and developed into gray cankers. Affected twigs showed blight symptoms that gradually expanded along the stems, culminating in dieback. No acervuli and pycnidia were observed on diseased twigs and/or stems. Ten twigs displaying typical blight lesions were collected from each of 10 individual V. corymbosum ‘Duke’ plants. Small pieces of tissue were cut from the edge of each lesion, surface sterilized with 70% ethanol for 1 min, rinsed three times in sterilized water, and cultured on potato dextrose agar at 25°C. A total of 19 isolates were identified by morphological examination and molecular analysis. Colonies were white with cottony aerial mycelium, irregular margins, and light yellow on the reverse side of the plate, with black, globular acervuli (120 to 400 μm, average 230 μm) distributed in concentric circles. Pycnidial conidiomata were swollen and globose to oval. Conidia were straight to fusiform, measuring 19.5 to 30.1 × 5.0 to 11.0 μm (n = 50) and consisted of five cells with the three median cells being much darker than the end cells. The three median cells were doliform, rugose, and versicolored and had septa darker than the rest of the cell. The apical cell was hyaline, conic, and subcylindric, with two to three apical hyaline appendages (average 26.5 μm). The sexual stage was observed. These morphological characteristics are consistent with those of Neopestalotiopsis clavispora (Borrero et al. 2018). The ITS (MH423964-82), TUB2 (MH423945-63) and tef1 (MH423926-44) sequences matched with N. clavispora strains LB14 (99%; KY319134), FC-DB1 (99%; KY568918), and TOR-802-803-804 (99%; KU096881), respectively. Using MEGA X program (Kumar et al. 2018), maximum likelihood analysis based on the concatenated ITS, TUB2, and tef1 sequences placed the isolates within a clade comprising N. clavispora (G.F. Atk.) (Jayawardena et al. 2016; Song et al. 2014). To confirm pathogenicity of the isolates, three twigs of three 1-year-old V. corymbosum ‘Duke’ were inoculated by spraying conidial suspension (1 × 10⁵ conidia/ml) with and without wounding using a sterilized needle. Inoculated plants were maintained in a plastic bag for 16 h in a greenhouse with 16-h photoperiod, temperature between 18 and 30°C, and humidity maintained at 75%. Red-brown specks were observed on both the wounded and nonwounded twigs at 3 days postinoculation (dpi) and 2 months postinoculation, respectively. In the wound-inoculated twigs, the developed specks gradually grew larger at 7 to 14 dpi. After 14 dpi, the twigs showed gray canker symptoms typically observed on naturally infected plants. Control plants showed no symptoms on both the wounded and nonwounded plants. The pathogen was successfully reisolated using the methods described above and identified via analysis of the three genes and morphological characteristics. This pathogen has been previously reported to cause twig blight on blueberry in Chile, Uruguay, China, and Spain (Borrero et al. 2018; Chen et al. 2016; Espinoza et al. 2008; Gonzalez et al. 2012). To our knowledge, this is the first report of N. clavispora causing twig dieback on V. corymbosum ‘Duke’ in Korea, and this information will aid development of disease control strategies.