First Report of Curtobacterium flaccumfaciens Causing Black Shank on Tobacco cultivar K326 in China

Tobacco is one of the important cash crops, which is widely planted in China. In July 2023, black stem plants in some tobacco cultivar K326 fields were observed in Xinning County, Shaoyang City, Hunan Province of China (87.113° N, 49.3° E). The incidence in the 0.67 hectares of area surveyed was approximately 15.3%. The growth of field symptomatic plants showed signs of yellow leaves and black stems. To identify the causal agent of this disease, symptomatic tobacco black stems were washed in tap water, surface sterilized in 1.5% sodium hypochlorite for 1 min and then 75% ethanol for 30 s, rinsed with sterilized distilled water three times, and then cultured on Nutrient agar (NA) plates at 30℃ for 48 h. Three colonies with the same morphology, named H6J-2-1, H6J-2-2, and H6J-2-3, were obtained and transferred to NA plates using the conventional repetitive streaking technique to obtain pure cultures. The colonies were light yellow, round and smooth. The bacterial isolates were Gram positive. DNA was extracted from three isolates. Sequence analysis (Richert et al. 2005) of 16S rRNA gene sequences (GenBank accession nos. PP065621, PP065622, PP065623) showed 100%similarity with Curtobacterium flaccumfaciensc qsV11 (16SrRNA, MN826580.1), while gyrB gene sequences (GenBank accession: PP153626, PP153627, PP153628) showed 99.73%, 99.71%, 99.72% similarity with C. flaccumfaciens Cw101 (gyrB, KX591724.1), respectively. Whole genome sequencing of strain H6J-2-1 was performed using PacBio Sequel IIe sequencer & Illumina NovaSeq6000 system (Majorbio BioPharm Technology Co., Ltd., Shanghai, China) (accession ID: CRA040750) (CNCB-NGDC Members and Partners 2025). The strain H6J-2-1 was identified as C. flaccumfaciens via MLSA analysis of 31 house-keeping genes. For pathogenicity test, 50 mL of the pathogen suspension (108 CFU/mL) was poured around the rhizosphere of 35-day-old tobacco (cv. Yunyan 87), and the control seedlings were inoculated with sterilized water. All treatments were kept in a growth chamber at 28±0.5℃. After 15 days of inoculation, the stems of tobacco turned brown and wilted, while the control plants showed no symptoms. Strains with the same morphological characteristics and gyrB gene sequence as the original culture were re-isolated from the inoculated plants. It has previously been reported that Soybeans (Tarakanov et al. 2022), Bean Seeds (Tegli et al. 2017; Maringoniet al. 2006) and Sunflowers (Pilik et al. 2023) are hosts of C. flaccumfaciens. To our knowledge, this is the first report of C. flaccumfaciens associated with black shank on tobacco cultivar K326 in China. Identifying C. flaccumfaciens as the causative agent of the observed tobacco black shank is significant for future prevention and control of this disease.