First Report of <i>Pectobacterium versatile</i> Causing Soft Rot in <i>Aconitum carmichaelii</i> in China

Aconitum carmichaelii Debeaux is a perennial herbaceous plant within the Ranunculaceae family. The processed lateral roots of A. carmichaelii, known as "Fuzi", are widely used as traditional Chinese medicinal materials for the treatment of various human diseases such as rheumatoid arthritis, cardiovascular diseases, and tumors in Asian countries (He et al., 2023). A. carmichaelii has been cultivated for over 1,000 years (Yu et al., 2016). Jiangyou, in Sichuan, China, has been considered the geo-authentic cultivating area of A. carmichaelii (Zhou et al., 2015). An outbreak of soft rot in A. carmichaelii caused up to 60%-80% of plant death in Jiangyou in April, 2024. Soft rot blackened lesions may develop on infected stems, roots, or both, leading to the collapse and wilting of the leaves, and death of the plant. To isolate the causal agent, tissue fragments (5 mm × 5 mm) from lesion margins of 5 symptomatic plants were surface-sterilized by soaking in 75% ethanol for 30 s, and 2% NaOCl for 1 min, followed by rinsing with sterilized water for 5 times. The materials were then macerated in sterile water, and streaked onto Luria-Bertani (LB) agar plates. After incubation at 28°C for 48 h, four bacterial isolates (SR-1, SR-2, SR-3, and SR-5) were picked and purified on LB plates. Single colonies of these isolates were milky white, round, opaque, glossy, and smooth-edged, with 0.2-0.3 cm diameter. All the strains were Gram-negative. Genomic DNA was extracted from each strain using Bacterial DNA Kit (Sangon, Shanghai) and was used as template for the amplification of 16S rRNA, atpD, gyrA and recA genes (Menna et al., 2009; Wang et al., 2017). Sequences of strains SR-1, SR-2, SR-3, and SR-5 have been deposited in the GenBank under accession numbers PV776673-PV776676 (16S rRNA), PV799309-PV799312 (atpD), PV799313-PV799316 (gyrA), and PV799317-PV799320 (recA). Phylogenetic tress based on 16S rRNA gene and multilocus sequence analysis (MLSA) of concatenated 16S rRNA, atpD, gyrA, and recA sequences were constructed using the Neighbor-Joining method in MEGA X with 1,000 bootstrap replicates. SR-1, SR-2, SR-3, and SR-5 clustered together with Pectobacterium versatile CFBP 6051T (MK226529) with similarities ranging from 97.66% to 98.00% in the phylogenetic tree based on 16S rRNA gene. Consistently, MLSA-based phylogeny revealed that SR-1, SR-2, SR-3, and SR-5 clustered with P. versatile CFBP 6051T (RMBU01000000), sharing 98.73%, 98.70%, 98.70%, and 98.70% identities, respectively. To verify the pathogenicity of the strains, bacterial suspension of each isolate (1×10⁸ CFU/mL, 1 mL) was injected to the roots of A. carmichaelii plants using a sterilized syringe (5 plants per treatment, 3 biological replicates on cultivar MianFu No. 1). Sterilized water was used as a negative control. The control plants were healthy throughout the experiment. SR-1, SR-2, and SR-5 induced wilting of the plants at 3 days post inoculation (dpi), while SR-3 did at 5 dpi. Plants inoculated with test strains all died at 10 dpi. Strains were successfully reisolated from the symptomatic plants and confirmed as P. versatile by the methods mentioned above. P. versatile has been reported as causing pathogen of potato, cabbage, tobacco, and Hydrangea macrophylla (Chen et al., 2022; Handique et al., 2023; Lu et al., 2023; Su et al., 2023). To our knowledge, this is the first report of P. versatile causing soft rot in A. carmichaelii in China, expanding its known host range. Soft rot posed remarkable threat to the yield and quality of A. carmichaelii. Further researches on epidemiology and management are warranted.