First Report of Coniella vitis Causing White Rot on Virginia Creeper (Parthenocissus quinquefolia) in China

Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) belongs to the genus of Parthenocissus and Vitaceae family, which is very common in vineyards and where wild grape occurs (Bergh et al., 2011). In September of 2021, a severe white rot disease was observed on Virginia creeper around the vineyard of wine grapevine (Cabernet Sauvignon) located in Penglai city (37º 75'38" N, 120º 84'28" E), Shandong province of China. The disease incidence was about 75%, and infected leaf of Virginia creeper exhibited irregular necrotic lesion with brown center, and most lesion occurred on leaf margin, black pycnidia were also observed on the infected leaf at the late stage of infection. To determine the causal agent, symptomatic leaves with typical lesions were cut into small pieces (5 mm × 3 mm), surface sterilized with 75% ethanol for 1 min, followed by three times rinsed in sterile water. Leaf sections were plated onto potato dextrose agar (PDA) medium and incubated at 28°C for 3 days. Totally, five isolates (referred to as JD01, JD07, JD09, JD12 and JD16) were collected and transferred on to fresh PDA medium for incubation at 28°C. Seven days later, colonies on PDA plates had crenulated edges with concentric rings, the upper surface of colonies was mostly flat and white with many pycnidia. The conidia were hyaline at immature and became brown later, spherical or ellipsoid, aseptate, and 7.92 ± 1.20 μm × 5.18 ± 0.61 μm (n=50), length : width ratio is nearly 2. Morphologically, the isolates were identified as Coniella vitis (Chethana et al., 2017). Further to confirm the fungal species, the internal transcribed spacer region (ITS) of the ribosomal RNA gene, large subunit rRNA gene (LSU) and the translation elongation factor 1-alpaha gene (TEF1-α) were amplified using primers ITS1/ ITS4, LR7/ LROR, and TEF1- 728F/ TEF1- 986R (Chethana et al., 2017; Raudabaugh et al., 2018). The amplification products were sequenced and deposited in GenBank database. The sequences were compared to type sequences in GenBank. The results showed that ITS (GenBank accession numbers ON329769, ON329770, ON329771, ON329772 and ON329773), LSU (ON358423，ON358424, ON358425, ON358426 and ON358427) and TEF (ON297671, ON229071, ON229072, ON229073 and ON297672) sequences of the five isolates were 99.66%, 96.90% and 98.79% identical with the sequences data from C. vitis isolates in GeneBank (MFLUCC 18-0093, JZB3700020 and MFLUCC 18-0093, respectively). Furthermore, concatenated sequences of the three genes (ITS, LSU and TEF) were used to conduct a phylogenetic tree using maximum likehood MEGA-X (Raudabaugh et al., 2018). The phylogenetic analysis showed that the five isolates (JD01, JD07, JD09, JD12 and JD16) belong to C. vitis clade among the 41strains of Coniella spp. In the pathogenicity tests, detached leaves of Virginia creeper (1-year-old) were inoculated with mycelia plugs (5 mm diameter) (form 3-day-old of isolate JD07 culture), and control were inoculated with PDA plugs (5 mm diameter). Virginia creeper live plants (1-year-old) were inoculated with conidial suspension (2.5×106 spores/ml) of the isolate JD07 of one week old, and control plants were inoculated with sterile water. All treated Virginia creeper plants (detached leaves) were placed in a greenhouse maintained at 28°C and 95% relative humidity. Virginia creeper plants (detached leaves) inoculated with the conidial suspension (fungal mycelia) had brown lesion on leaves, the disease symptoms were similar to those observed in field. No such symptoms were observed on control plants (detached leaves). The pathogen was reisolated from inoculated Virginia creeper plants and re-identified, thus fulfilling Koch's postulates. C. vitis had been reported to cause grape white rot in China (Chethana et al., 2017). Virginia creeper, as an excellent host of C. vitis, will increase the transmission risk of the pathogens. To our knowledge, this is the first report of C. vitis causing white rot on Virginia creeper, and this finding will provide useful information for developing effective control strategies for white rot disease.