First Report of Root Rot Caused by Pythium dissotocum on Tobacco in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop that is widely grown around the world. Its annual production in China is estimated at 2.2 million tons (Berbeć and Matyka 2020). Since 2022, a root rot disease was sporadically observed on tobacco seedlings on cultivar Yunyan 87 in cultivated tobacco fields in the Hunan province of China. A disease incidence of about 10% occurred across 48 ha of tobacco fields. The affected tobacco plants had slow and stunted growth with yellowing leaves. The roots turned grayish brown, decayed, and died. Diseased roots were collected from six fields and cut into small pieces (5 mm ×5 mm) from the edge of the rotted portions, and then sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 1 min, and washed in sterilized water three times. All the sterilized tissue were placed on potato dextrose agar (PDA) medium and cultured at 26 ℃ in the dark. About 3 days later, colonies with similar morphology were removed and sub-cultured on fresh PDA. A total of six strains were obtained from six tobacco samples. Strains were white and had radial growth on PDA. Hyphae were aseptate and the sporangia were filamentous. The oogonia were subglobose, smooth, 16.04 ± 0.25 µm (n=50) in diameter, and developed on unbranched stalks. The antheridia were barrel shaped and clavate. Oospores were globose, aplerotic or nearly plerotic, measuring 6.62 ± 0.33 µm (n=50). These morphological characteristics were consistent with the description of Pythium spp. (van der Plaats-Niterink 1981). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and cytochrome c oxidase subunit I (Cox I) of a representative isolate, GF-3, were amplified and sequenced (GenBank accession nos. OR228424 for ITS and OR237556 for Cox I) using universal primers ITS1/ITS4 (White et al. 1990) and FM58/FM66, respectively (Villa et al. 2006). BLASTn analysis revealed that the ITS and Cox I sequences were 99.76 % (838/840 bp) and 99.85% (671/672 bp) identical to the corresponding sequences of P. dissotocum strain CBS 166.68 (AY598634.2) and UM982 (MT981147.1), respectively. A neighbor-joining phylogenetic tree based on the Cox I sequence showed that GF-3 grouped in the P. dissotocum branch. Based on morphological and molecular characteristics, GF-3 was identified to be P. dissotocum. For pathogenicity testing, four- to five-leaf-old healthy potted tobacco seedlings of the Yunyan 87 cultivar were inoculated with a zoospore suspension (1 × 105 zoospores/ml), which was induced on V8-juice medium. The zoospore suspension was introduced into the soil around plant roots and 10 mL of inoculum was used for each plant. In the control group, plants were inoculated with sterilized water. All of the treated plants were kept in humid chambers at 26°C under a 12 h/12 h photoperiod. The pathogenicity assays were performed twice, with each treatment having three replicated plants. After 5 days, tobacco seedlings inoculated with P. dissotocum showed symptoms resembling that observed in the field. However, the control plants remained healthy. Pythium dissotocum was re-isolated from the infected plants and identified by morphological and molecular methods, thus confirming Koch's postulates. Pythium dissotocum has been reported causing root rot in other plants, including hydroponic lettuce (McGehee et al. 2018) and spinach (Huo et al. 2020). Also, many Pythium species have recently been recovered from float-bed tobacco transplant production greenhouses (Zhang et al. 2022). However, to our knowledge, this is the first report of root rot on tobacco caused by P. dissotocum in China. Since this disease could greatly affect tobacco seedling establishment in the field, appropriate management strategies need to be developed to reduce further losses in tobacco planting fields.