First Report of Xanthomonas campestris Causing Leaf Blight on Ranunculus asiaticus in Shanghai, China

Ranunculus asiaticus L., a species widely cultivated for its ornamental value, has emerged as an economically significant crop for Shanghai floriculture industry. In February 2025, severe leaf blight symptoms were observed affecting approximately 50% (15,000/30,000) of two-month-old R. asiaticus seedlings within a commercial nursery in Pudong New District, Shanghai. Initial symptoms included irregular necrotic lesions with distinct chlorotic halos (2-5 mm diameter), followed by rapid yellowing and leaf collapse. To isolate the causal agent, leaf tissues from the margins of lesions from symptomatic plants were surface-sterilized (70% ethanol, 30 s), rinsed with sterile distilled water, and macerated in 0.1 M phosphate-buffered saline using sterile mortar and pestle. Then, the resultant homogenate was streaked onto nutrient agar plates. After 48h incubation at 28°C, predominant yellow-pigmented, mucoid colonies exhibiting morphological consistency were selectively isolated and purified. To confirm species identity, genomic DNA was extracted from three independent representative isolates (SHPD01 to SHPD03). Four conserved loci were amplified for sequencing using specific primer pairs: 16S rRNA gene (27F/1492R), gyrB gene (XgyrB1F/XgyrB1R), dnaK gene (XdnaK1F/ XdnaK1R), and rpoD gene (XrpoD1F/XrpoD1R) (Lelliot et al., 1966; Panth et al., 2023; Young et al., 2008). The results demonstrated that all three representative isolates exhibited identical genetic profiles. Through sequence analysis, the 16S rRNA gene sequences (accession no. PV362695) showed 99.72% (1436/1440 nt) identity with Xanthomonas campestris pv. campestris ATCC 33913 (AE008922). Subsequent analysis of the conserved housekeeping genes revealed that gyrB (accession no. PV404776), dnaK (accession no. PV404778) and rpoD (accession no. PV404777) sequences displayed 96.93 to 98.52% identity (gyrB, 801/813 nt; dnaK, 888/900 nt; rpoD, 790/815 nt) with 100% coverage with the X. campestris pv. campestris ATCC 33913 (AE008922). Additionally, specific primers for X. campestris pv. campestris were used for polymerase chain reactions (PCR), with specific primers HrcCF2/HrcCR2 (Zaccardelli et al., 2007). All isolates could be detected by specific primers. Phylogenetic tree analysis of 16S rDNA, gyrB, dnaK and rpoD genes further verified that the bacteria isolate was in close proximity to X. campestris. To confirm pathogenicity, a bacterial suspension of SHPD01 (10⁸ CFU/mL) was applied via spray inoculation to five healthy two-month-old R. asiaticus seedlings. Five seedlings were treated with sterile distilled water as a negative control. Inoculated plants were covered with plastic bags for 24 h to maintain humidity and incubated at 28°C with 80% relative humidity. The plants inoculated with the bacteria showed water-soaked lesions developed within 3-4 days post-inoculation (dpi), progressing to characteristic leaf blight symptoms by 10 dpi. Control plants remained asymptomatic. Koch's postulates were fulfilled through bacterial re-isolation and confirmed by specific primers. This outbreak represents the first documentation of X. campestris-induced leaf blight in Chinese Ranunculus cultivation systems. The high infection rate observed within this nursery suggests a potential epidemiological threat within the humid subtropical climate of East China. Given Shanghai status as a leading national center for floricultural production, the implementation of urgent phytosanitary measures and the development of resistance breeding programs are strongly recommended.