First Report of Xanthomonas campestris Causing Leaf Blight on Buttercup (Ranunculus asiaticus) in South Carolina, USA.

Buttercup (Ranunculus asiaticus L.) is a popular and high value ornamental species grown in landscapes and gardens and as cut flowers. It is mostly cultivated in Europe, the Mediterranean, and the Americas (Beruto and Debergh, 2004). In January 2022, leaf blight was observed on approximately 24 of forty 4-month-old R. asiaticus plants grown in a high tunnel at a cut flower farm located in Anderson County, South Carolina, USA. Symptoms included irregular, vein-limited, and necrotic leaf lesions and yellowing. Some lesions had a chlorotic halo. Two diseased plants were submitted to the Clemson University Plant and Pest Diagnostic Clinic. Symptomatic leaves were surface sterilized with 10% bleach for 1 min and rinsed in sterile water. Small leaf portions (1 × 1 cm2) were excised from the margin of lesions. They were macerated in 500 µl of sterile water and incubated at room temperature for 10 min. A loopful of suspension was streaked on nutrient agar (NA). Slightly convex, yellowish-mucoid colonies appeared after incubation at 28°C for 48 h. Two isolates, 23A and 23B, from two plants were obtained by transferring single colonies to new NA plates. Both isolates were identified as X. campestris (probability values > 0.8) using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of these two isolates were performed for housekeeping genes gyrB, rpoD, and dnaK (Young et al. 2008). The amplicon sequences (GenBank accession nos.: OR101193 and OR101194 [dnaK]; OR101195 and OR101196 [gyrB]; OR101197 and OR101198 [rpoD]) were identical between the two isolates based on sequence alignment in MEGA11 (Tamura et al. 2021). Nucleotide BLAST of these three genes showed 94.6 to 98.9% identity (dnaK: 912 of 922 bp; gyrB: 827 of 839 bp; rpoD: 803 of 849 bp) with 100% coverage with the Xanthomonas campestris pv. campestris type strain (AE008922). A neighbor joining phylogenetic tree with the concatenated sequences of these three genes showed that 23A and 23B had the closest match with X. campestris pv. campestris. However, these two isolates tested negative in the probe-based qPCR assay specific for X. campestris pv. campestris with only the positive control amplified (Köhl et al. 2011), suggesting that they may belong to a new pathovar of X. campestris. To confirm the pathogenicity of these isolates, three healthy R. asiaticus plants each were spray inoculated with suspensions of 23A and 23B in sterile tap water until runoff (OD600 = 0.1, approx. 108 CFU/ml). The non-inoculated control plants received a sterile tap water spray. The experiment was conducted twice. All plants were maintained in a growth chamber at 24°C with 10-h photoperiod. Seven to 15 days after inoculation, necrotic lesions with chlorotic halo and leaf yellowing, similar to those observed in the field, were observed on inoculated plants, while the non-inoculated control plants remained symptomless. Koch's postulates were fulfilled by reisolating the bacteria from the symptomatic plants and confirming the bacterial identity with the sequence analysis described above. The disease was first reported in California in 1996 (Azad et al. 1996) but to the best of our knowledge has not been reported again in the United States. This is the first report of X. campestris causing bacterial leaf blight in R. asiaticus in South Carolina. Since more than 50% of the flower producers/farmers grow Ranunculus in South Carolina, further work is necessary to determine how widespread the disease is and its economic impact.