*Corresponding author The genus Phytopythium is an oomycete plant pathogen that is morphologically similar to the genera Pythium and Phytophthora (Ghimire and Baysal-Gurel, 2023). Previously, Phytopythium was classified within Pythium clade K, but molecular phylogenetic analyses revealed it is more closely related to Phytophthora, leading to the designation of a new genus (Bala et al., 2010). While most Phytopythium species survive as saprophytes on host plants, some highly pathogenic species spread through aquatic and soil routes, causing root and stem rot in fruit and ornamental plants (Kahraman and Yıldız, 2024). Phytopythium vexans has been reported to cause root rot in kiwi, avocado, cherry, and apple (Baysal-Gurel et al., 2021; Jabiri et al., 2020; Polat et al., 2017; Rodriguez-Padron et al., 2018), while Phytopythium helicoides has been reported to cause root and stem rot in rose, citrus, and peach rootstocks (Browne et al., 2019; Chen et al., 2016; Kageyama et al., 2002).
In July 2022, symptoms of leaf and branch wilting, root rot, and tree death were observed on two grape vines (Vitis vinifera, cultivar ‘Stella’) at a vineyard in Pocheon, Gyeonggi Prov-ince (Fig. 1). White mycelium was present on the diseased root areas, which were then cut into 3-5 mm segments, disinfected with 70% ethanol and 1% sodium hypochlorite, and rinsed. The disinfected tissues were plated on potato dextrose agar (PDA) and incubated at 25°C for 3 days, after which the fungal isolate was purified by sub-culturing and designated as strain 22-141. The strain 22-141 was deposited into the Korean Agricultural Culture Collection (KACC) under accession no. KACC410774.
Fig. 1.
Symptoms of root rot disease caused by Phytopythium helicoides on grapevine. (A) Three-years-old grape plant showing severe wilting and yellowing. (B) Symptom of crown and root rot.
To identify strain 22-141, DNA was extracted and the sequences of the beta-tubulin and cytochrome C oxidase subunit II gene regions were analyzed using the primer pair, Bt2a (5’-GGT AAC CAA ATC GGT GCT GCT TTC-3’), Bt2b (5’-ACC CTC AGT GTA GTG ACC CTT GGC-3’), Cox2-F (5’-CCA CAA ATT TCA CTA CAT TGA-3’) and Cox2-R (5’-TAG GAT TTC AAG ATC CTG C-3’). The obtained sequences were aligned and compared with closely related species using Basic Local Alignment Search Tool (BLAST) in National Center for Biotechnology Information (NCBI), and a phylogenetic tree was constructed using the maximum-likelihood method (1,000 bootstrap replicates) in MEGA11 (Table 1). The analysis revealed that strain 22-141 was closely related to P. helicoides strains registered in GenBank (Fig. 2), and the sequence data of the isolate was deposited in the NCBI GenBank database (accession nos. PQ584366 and PQ584634).
Fig. 2.
Maximum-likelihood tree based on combined beta tubulin and cytochrome C oxidase subunit II sequences of Phytopythium helicoides 22-141 and related species. Numbers at nodes are bootstrap values for 1,000 replicates. The scale bar represents 0.02 nucleotide substitutions per site.
Table 1.
Genbank accession numbers of fungal strains used in phylogenetic analyses
To investigate the cultural characteristics of P. helicoides strain 22-141, mycelia were plated on PDA and V8 juice agar (V8A) and incubated in the dark at 25°C for 2 days. The colony diameter of P. helicoides 22-141 was 73-78 mm on PDA and 85-88 mm on V8A, with white, thin, cotton-like mycelia on both media (Fig. 3). For sporangia observation, we modified the method of Ghimire and Baysal-Gurel (2023) by incubating P. helicoides 22-141 on V8A at 25°C for 4 days, then adding sterile water and incubating under light conditions for 48 hr. The sporangia of P. helicoides 22-141 were globose or ovoid with papillate morphology, measuring 28.91-30.88×30.12-32.35 μm (Fig. 3). The oospores were globose, measuring 22.81-28.29 μm. These cultural and morphological characteristics were consistent with previously described features of P. helicoides (Ghimire and Baysal-Gurel, 2023).
Fig. 3.
Characteristics of Phytopythium helicoides 22-141 causing root rot disease on grape. (A) Colony after 3 days of incubation at 25°C on potato dextrose agar. (B) Colony on V8. (C) Globose sporangia. (D) Globose and aplerotic oospore. Scale bars=10 µm.
To test the pathogenicity of P. helicoides strain 22-141, 3-year-old ‘Stella’ grapevine cuttings were inoculated by immersing the roots in a spore suspension (105 spores/ml) for 2 hr, with control plants treated with distilled water. After 20 days in greenhouse conditions (25-30°C, 75% relative humidity), the roots of inoculated plants exhibited progressive blackening and rotting, and the pathogen was re-isolated from the diseased tissue (Fig. 4).
Fig. 4.
Pathogenicity test of Phytopythium helicoides 22-141 on root of grape. The inoculated plant (left) shows the root discoloration symptoms, while the un-inoculated plant (right) shows no visible symptoms in the pathogenicity test.
In South Africa, P. vexans has been reported as the causative agent of grapevine root rot, and Phytopythium species, including P. helicoides, are among the most commonly detected soilborne pathogens in South African grapevine nurseries, though the pathogenicity of P. helicoides has not been confirmed (Spies et al., 2011). In Korea, pathogenicity on grapevines by oomycetes has only been reported for Phytophthora drechsleri, which causes root rot (Choi et al., 2024).
Based on our results, P. helicoides was confirmed to cause root rot symptoms on grapevines, marking the first report of grapevine root rot caused by P. helicoides in Korea.
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