First report of Pythium myriotylum causing root rot in Nicotiana tabacum (L.) in China

Tobacco (Nicotiana tabacum L.) is an important economic crop in China. In June 2025, tobacco plants exhibiting root rot were observed in Liutu Town (26.20 °N, 112.23 °E) and XiaoSongbai Town (26.20 °N, 112.23 °E) in Changning city, Hunan province, China. Symptomatic and healthy plants were uprooted, and symptomatic plants had reduced root mass, reduced root hair density, and necrosis. Roots and growth substrate samples were taken from symptomatic (n=10) and healthy plants for further analysis. Roots with brown lesions and healthy roots were surface sterilized by soaking in 0.1% sodium hypochlorite for five minutes, rinsed in sterile deionized water, and two-centimeter segments were plated on potato dextrose agar (PDA) medium and cultured at 25°C in the dark. About 3 days later, morphologically similar colonies were recovered from 80% of the symptomatic roots and subcultured on fresh PDA. Strains were white with radial growth, hyphae were aseptate and hyaline, and sporangias were filamentous. Oogonia were subglobose, smooth, and 18.04 ± 0.25 µm (n=50) in diameter and developed on unbranched stalks. Antheridia were barrel shaped and clavate. Oospores were globose, aplerotic, or nearly plerotic, measuring 12.62 ± 0.33 µm (n=50). Morphological characteristics were consistent with the description of Pythium spp. (van der Plaats-Niterink 1981). The internal transcribed spacer (ITS) region of rDNA and cytochrome oxidase Ⅱ (COX Ⅱ) of four representative isolates were amplified and sequenced using the universal primers ITS1/ITS4 (White et al. 1990) and FM58/FM66 (Villa et al. 2006), respectively, and sequences were submitted to NCBI (GenBank accession nos. PV805134 and PV805014 for ITS and CoxⅡ, respectively). The four isolates had identical DNA sequences, and BLASTn analysis revealed that the ITS and COX Ⅱ sequences were 99.76% (825/840 bp) and 100% (515/595 bp) identical to the corresponding sequences of the P. myriotylum isolate WYJPM0310 for ITS (MT229364.1) and P. myriotylum isolate ND3-3 for Cox II (PP212859.1). Furthermore, had 99.51% homology with the ITS sequence for P. myriotylum type isolate CBS 254.70.. For pathogenicity testing, four-to five-leaf stage healthy potted tobacco seedlings of the cultivar Yunyan 87 were inoculated with a zoospore suspension (1×105 zoospores/ml), which was induced on V8 juice agar 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 blank V8 juice agar medium. 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. myriotylum showed symptoms resembling those observed in the field. However, the control plants remained healthy. P. myriotylum was reisolated from the infected plants and identified by morphological and molecular methods, thus fulfilling Koch’s postulates. P. myriotylum has been reported causing root rot in other plants, including ginger (Stirling et al. 2009) and peanut (Yu et al. 2019). However, to our knowledge, this is the first report of root rot on tobacco caused by P. myriotylum 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.