First Report of Soft Rot of <i>Ranunculus ternatus</i> Caused by <i>Pseudomonas fluorescens</i> in China

Ranunculi ternati Radix is a perennial herb of Ranunculaceae Ranunculus plant Ranunculus ternatus Thunb.. It is the only medicinal plant of the buttercup genus included in the 1977 edition of the Chinese Pharmacopoeia. As an important traditional medicinal plant,Ranunculus ternatus Thunb. is rich in organic acids, sugars, esters, sterols, volatile oils,alkaloids, flavonoids, triterpenoids, total saponins and other components. It is mainly distributed in Guangxi, Taiwan, Jiangsu, Zhejiang, Jiangxi, Hunan, Anhui, Hubei, Henan another provinces in China. In November of 2024, a severe outbreak of Ranunculus ternatus Thunb. soft rot disease occurred in Lichun Town, Pengzhou City, Sichuan Province of China (103°10′-103°40′ E, 30°54′-31°26′ N), causing symptoms such as water-soaked lesions on stems and leaves that turn brown and rot inward. Affected plants gradually wilt, defoliate, emit a foul odor, and eventually die (Figure 1). In a field of 0.8 ha,the disease incidence was 30% . Symptomatic samples were surface sterilized in 75% ethanol for 30 seconds and then in 2% NaClO for 60 seconds. After sterilization, the samples were rinsed three times with sterile water and dried on sterilized filter paper. The samples were then cut into pieces and suspended in sterile water. Finally, the suspension was spread onto culture plates containing nutrient agar medium (NA) and incubated for 2 days at 28 °C in the dark. Representative strain (fy02-1) was convex, circular with smooth margins on NA medium. The bacteria were gram-negative, short straight rods, 0.5 to 0.9 × 1.5 to 3.0 μm, single polar flagellum. The bacterium grew at temperatures up to 42 °C. The purified bacterial strain was inoculated into NA liquid medium and cultured at 28°C for 18-24 h. The bacterial concentration was adjusted to 108 CFU/mL with sterile water, and 20 μL was injected into the stem base of Ranunculus ternatus Thunb., with sterile water as the control. The plants were grown under conditions of 28°C and 70% humidity, and disease symptoms were observed, with the color, size, location, and severity of lesions recorded at different stages of disease development. Symptoms appeared on the second day after inoculation, with initial lesions appearing brown. By the third day, the entire plant had rotted and withered, while the control group showed no symptoms (Figure 2). Pathogenic bacteria was reisolated from the infected plants, confirming Koch’s postulates and the pathogenicity on Ranunculus ternatus Thunb. For bacterial species identification, a single-colony culture was subjected to genomic DNA extraction, gene amplification, and sequencing of 16S rRNA, rpoD, and gyrB. The universal primers 27F/1492R (Lane 1991) were used to amplify the 16S rRNA gene, and the specific primers 70F/70R and UP-1E/APrU (Hwang et al. 2005) were used to amplify the rpoD and gyrB genes, respectively. In the BLAST analysis, the 16S rRNA sequence (GenBank PV926360.1) of the isolate shared 99% similarity (1428/1527 bp) with the Pseudomonas fluorescens strain MFAF76a (KT350501.1), and the rpoD ( PV963828.1) and gyrB (PV963827.1) sequences showed >98% identity to rpoD (695/1260 bp; KT350504.1) and gyrB (657/940 bp; KT350502.1) of the P. fluorescens strain MFAF76a. Based on the multigene phylogeny, the isolated bacterium was identified as P. fluorescens. P. fluorescens has also been shown to cause certain plant diseases, such as stem rot disease on melon (Zhang et al. 2016), bacterial head rot on broccoli (Li et al. 2009), and head rot on Brassica oleracea convar. botrytis var. italica (Catore et al. 2007). As far as we know, this is the first report of P. fluorescens causing Ranunculus ternatus Thunb. soft rot disease in China. Soft rot caused by P. fluorescens may be a threat to Ranunculus ternatus Thunb. cultivation,and this report of its occurrence is the first step in determining potential spread and effective control measures.