LAUSR

Grapevine trunk diseases (GTDs) are a complex of diseases that strongly affect grape productivity, wine quality, and vineyard longevity worldwide. GTDs occur in diverse regions, and in some areas disease agents are present in up to 100% of vines (Pintos et al. 2018). Recently, species belonging to Botryosphaeria and Diaporthe were identified as pathogens associated with GTDs in China (Dissanayake et al. 2015; Yan et al. 2013). During 2017 to 2018, interveinal leaf necrosis and wood white decay surrounded by brown necrosis in longitudinal section and black spots in cross section were observed in several grapevines (Cabernet) in Huailai County, Hebei Province, China. Disease incidence was up to 2 to 3% of the vineyard. Diseased samples (whole trunk and roots of 19-year-old vines) were collected and taken to the laboratory. Woody samples were cut into small chips, surface sterilized in 1.5% NaOCl for 3 min followed by 70% ethanol for 30 s, and rinsed three times with sterile distilled water (SDW). Once the samples were dried, they were placed onto PDA plates amended with ampicillin (0.1 g/liter). Plates were incubated at 25°C under dark conditions. After 14 days of incubation, hyphal tips of fungi growing from wood pieces were transferred onto new PDA plates and incubated until they produced conidia. One type of colony was consistently isolated from the discolored tissue, with honey brown mycelium, and producing a yellow pigment on PDA. Conidia were ellipsoid to allantoid, 3.8 to 6.2 μm long, and 1.6 to 3.2 μm wide (n = 50). Morphologically these isolates resembled species belonging to Phaeoacremonium (Mostert et al. 2006). For species confirmation, genomic DNA of three representative isolates (JZB3190001, JZB3190003, and JZB3190005) was extracted. PCR amplification was performed using two phylogenetic markers (actin and β-tubulin) amplified with primers ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1/Bt2b (Glass and Donaldson 1995). The sequences obtained were deposited in GenBank under the accession numbers MK994188 to MK994193. Phylogenetic analysis was conducted using maximum likelihood in RAxML, which was accomplished using RAxML-HPC2 on XSEDE in the CIPRES Science Gateway platform (http://www.phylo.org/). In the phylogenetic tree, the isolates from the present study clustered together with Phaeoacremonium minimum (CBS 246.91), with 100 bootstrap values. Based on morphological characters and phylogenetic results, the species isolated in this study was identified as P. minimum (Tul. & C. Tul.) D. Gramaje, L. Mostert & Crous (Gramaje et al. 2015). The pathogenicity test was conducted on healthy, 2-month-old, rooted ‘Furcal’ grapevines that were grown in an inoculation chamber. Roots were washed using flow water and rinsed with SDW. The ends of SDW-treated roots (roughly 1 cm) were cut with sterilized scissors, and the roots and trunk base of Furcal vines were dipped in a 10⁶/ml P. minimum spore suspension for 30 min. Inoculated plants were immediately planted in individual pots, and 10 ml of spore suspension was added to the soil per pot. SDW was used as a control. Two P. minimum isolates (JZB3190001 and JZB3190003) were used to do the pathogenicity test, and 10 plants were inoculated with each isolate and the control water. Temperature of the inoculation chamber was controlled between 24 and 25°C, and humidity was maintained at 60%. After 114 days of inoculation, the inoculated plants developed black necrosis at the base of wood but did not show leaf necrosis, whereas the control plants showed no symptoms both in wood and leaves. Koch’s postulates were confirmed by reisolating and identification based on cultural and morphological characters of the inoculated isolates. To our knowledge, this is the first report of P. minimum associated with GTDs in China. The results of this study will enhance the capability of controlling GTDs in China by correct identification of the causal organism.