First Report of Bacterial Leaf Spot on Tobacco Caused by <i>Pseudomonas cichorii</i> in Fujian Province, China

Tobacco (Nicotiana tabacum), a major economic crop, is widely cultivated in China, serves as a vital source of income for the country's tobacco farmers. In May 2025, a severe bacterial leaf disease was observed on cigar tobacco (N. tabacum cv. Haiyan 101) in Xiamao Town (26°34′38.3″N, 117°40′33.8″E), Fujian Province, China. Across eight surveyed fields, disease incidence ranged from 7.5% to 36%. Symptoms primarily affected middle and lower leaves nearing maturity, beginning as small, water-soaked lesions. Under high humidity, these expanded into concentric rings with water-soaked margins and yellow halos, eventually merging into large necrotic areas, rendering leaves unmarketable. For pathogen identification, a single symptomatic leaf was collected from each of the eight fields. Lesions were excised and surface-sterilized by immersing sequentially in 75% ethanol for 10 s and 1% NaOCl for 30 s, rinsed three times with sterile water, homogenized and plated onto nutrient agar medium. Predominantly grayish-white, circular colonies with smooth margins were observed. All isolates were identified as Gram-negative with Gram staining (Moyes et al. 2009). Eight isolates (labeled FJSMCC4, 11, 15, 21, 28, 35, 37 and 42) were selected for further analysis. DNA was extracted from each isolate and used as the template for the 16S rRNA gene amplification by PCR with universal primers 27F/1492R (Frank et al., 2008). The 16S rRNA sequences were deposited in GenBank (PX148858 - PX148865) and showed 99.5-100% sequence identity to Pseudomonas cichorii strains (GenBank OR824302, AB724288 and MZ619032). Multilocus sequence analysis (MLSA) was performed on the housekeeping genes gyrB and rpoD for strains FJSMCC21 and FJSMCC37. The genes were amplified using the primer pairs gyrB-UP-1E/gyrB-AprU and rpoD-70F/rpoD-70R (Yamamoto et al., 2000), yielding amplicons of 861 bp (gyrB; GenBank PX207515 and PX207516) and 600 bp (rpoD; GenBank PX207513 and PX207514), respectively. The resulting sequences exhibited >99% nucleotide identity to those of known P. cichorii strains in the GenBank database. A phylogenetic tree was reconstructed using both the maximum likelihood (ML) and Bayesian inference (BI) methods based on two concatenated genes (gyrB and rpoD) (Ronquist et al. 2003; Stamatakis et al. 2008). Both topologies placed isolates FJSMCC21 and FJSMCC37 within the P. cichorii monophyletic clade with strong support (ML bootstrap = 100%; BI posterior probability = 1). To fulfill Koch's postulates, pathogenicity tests were performed on healthy tobacco (cv. Haiyan 101). Bacterial suspension (1×10⁸ CFU/mL) was aseptically needle-punctured into five leaves (four sites per leaf, three micropunctures per site) without full penetration. Controls were inoculated with sterile water. Plants were grown in a growth chamber with a 16-h light (30°C) /8-h dark (25°C) cycle and 80% relative humidity. Water-soaked spots appeared within one day, and all inoculated leaves developed field-like symptoms within five days, while controls remained asymptomatic. Bacteria were successfully reisolated from the lesions, these reisolates were morphologically identical and had 100% 16S rRNA sequence match with the original strain. Reports of P. cichorii damage on tobacco have been found exclusively on flue-cured tobacco in Guangxi Province in China (Lan et al., 2022). To our knowledge, this is the first report of P. cichorii causing bacterial leaf spot on tobacco in Fujian Province, China.