First Report of Ralstonia pseudosolanacearum Causing Bacterial Wilt of Artemisia selengensis in China

Lihao (Artemisia selengensis), a perennial herbaceous plant belonging to Asteraceae family, has been widely used as Chinese traditional medicine and vegetable (Shao et al. 2010). In September 2025, typical wilting symptoms were observed in Lihao plants in the field of Yangxin County (29.83°N 115.21°E), Huangshi City, Hubei Province, China. The initial symptom of the disease was progressive wilting of leaves from the top downward, usually wilted at noon and recovered in the morning and evening. This cycle repeated for 3-5 days until the entire plant withered irreversibly and died eventually, although the plants remained green with a lighter hue before the leaves eventually dried up and fell off. The stems of wilted plants gradually turned brown, and in later stages, white bacterial ooze exuded from the transversely cut ends when the stems were soaked in clean water or squeezed by hand. Under high-humidity conditions, the infected parts of plants emitted a sour, foul odor. Disease incidence in the fields ranged from 30% to 70%. Twelve symptomatic plants were randomly collected from fields for pathogen isolation. The stems of infected plants were washed under running tap water for 3-5 min, and the stem segments approximately 5 cm above the base of infected plants were cut using a sterilized scalpel. The stem samples were sterilized with 70% ethanol for 1 min and then rinsed three times with sterile distilled water. Each sample was ground separately into a slurry, and serially diluted 10-fold four times with sterile distilled water to obtain a final dilution of 10 -4 . Then, 100 μL of each diluted suspension was spread on the triphenyltetrazolium chloride (TTC) medium and incubated at 28°C for 48 h. Typical colonies on TTC medium were fluid and nearly circular or oval with pink centers and cream peripheries (Fig. 1a1, a2). All colonies were confirmed as Gram-negative after standard Gram staining (crystal violet primary staining, followed by iodine mordanting, ethanol decolorization, and safranin counterstaining), presenting a red color. The cells were short rods measuring 0.6-0.9 μm × 1.4-1.8 μm (n = 30) under light microscopy. These morphological characteristics are similar to those of Ralstonia solanacearum (Hayward 1991) and Ralstonia pseudosolanacearum (Safni et al. 2014). For further molecular identification, genomic DNA of the two representative strains (A1 and A2) was extracted using the TINAamp Bacteria DNA Kit. PCR was performed with six primer pairs: universal primers 27F/1496R (Weisburg et al. 1991), specific primers 759f/760r (Opina et al. 1997), hrpB-F/hrpB-R (Shi 2023), and RS-F-759/RS-R-760 (Umesha and Avinash 2015), as well as RalIs-1/ RalIs-2 (Guo et al. 2012) and RS-1/2 (Yang et al. 2012). Amplicons of the expected sizes were observed on 1.2% agarose gels, and the PCR products were subsequently Sanger sequenced bidirectionally by Sangon Biotech (Wuhan China) using an ABI 3500 Genetic Analyzer. Sequence assembly and chromatogram inspection were performed using Unipro UGENE (version 53.1) with the integrated CAP3 assembler, and base calls were manually verified based on chromatograms. BLAST analysis against the NCBI GenBank database revealed that sequences amplified with the first four primer pairs showed >99% identity to the Ralstonia solanacearum species complex (RSSC) (Table 1), whereas those amplified with RalIs-1/ RalIs-2 and RS-1/2 showed 99% identity, as the top BLAST hits, to R. pseudosolanacearum (Table 1). The complete genome of strain A1 was sequenced using a combined second- and third-generation approach and deposited in GenBank (accession number JBXOVA000000000). Whole-genome average nucleotide identity (ANI) analysis showed 99.92% identity to R. pseudosolanacearum (CP104494) and 91.55% to R. solanacearum (CP115946). Phylotype I was confirmed by phylotype-specific multiplex PCR (Pmx-PCR) with the Nmult primer series (Fegan and Prior 2005), together with the RSSC-specific primers 759f/760r (Opina et al. 1997), with banding patterns matching those of the reference phylotype I strain (Fig. 1c). A ~750-bp egl fragment amplified with Endo-F/Endo-R (Poussier et al. 2000) was sequenced bidirectionally, and trimmed at the 3′ end according to the AGCAGTGG pattern (terminal motif GATGCCGACCGAGCAGTGG, confirmed by Sanger sequencing) to the 698-nt alignment window (Cellier et al. 2025); the corrected 698-nt sequence was deposited in GenBank (accession PZ753157). A maximum-likelihood (ML) phylogenetic tree based on the 110 curated reference sequences of the egl database (Cellier et al. 2025) placed the isolate in a clade with Sequevar 15 reference strains RUN2140 and CFBP765 with 100% ML bootstrap support (Fig. 1d), identifying it as R. pseudosolanacearum Phylotype I, Sequevar 15. To determine the pathogenicity, both strains were cultured in SPA broth ((sucrose-peptone), and cultured at 28°C with shaking for 2 days until the OD 600 reached 1.3. Twenty plants per treatment were inoculated by drenching the roots with the 20-fold diluted bacterial suspension (OD 600 = 0.25), with sterile water as the control (CK). The inoculated plants were maintained in a greenhouse at 25°C under a 12-h light/dark cycle. Disease symptoms were observed 3 days post-inoculation (Fig. 1b), with treated plants exhibiting bacterial wilt symptoms consistent with those observed in the field. To fulfill Koch's postulates, strain A1 was re-isolated from symptomatic seedlings on TTC medium, yielding fluid, pink-centered, with cream margins colonies identical in morphology to those of the original isolate. The re-isolated strain was further confirmed by Sanger sequencing of the 759f/760r amplicon to be 100% identical to the original strain. Based on the combined results, the pathogen causing wilt of A. selengensis was identified as R. pseudosolanacearum. This is the first global report of R. pseudosolanacearum Phylotype I, Sequevar 15 infecting A. selengensis, broadening the documented host range and sequevar diversity of the RSSC.