First Report of Polygonatum kingianum Leaf Spot Caused by Pantoea ananatis in Yunnan Province, China

Polygonatum kingianum Coll. et Hemsl. is a medicinal herb widely cultivated in Southwestern China (Tang et al. 2024). In September 2024, we observed a leaf spot disease on P. kingianum with 45% incidence in commercial plantings in Wenshan, Yunnan Province, China (23.3680°N, 104.2462°E). Initial small brown spots on lower leaves enlarge into tan-brown lesions that may become thin and perforated; lesions coalesce into large necrotic patches, spreading upward and causing blighting of above-ground plant parts. For pathogen isolation, small pieces from lesion margins were surface sterilized, macerated in sterile water, and the suspension was streaked onto Luria Bertani agar. A representative yellow colony was obtained. Following incubation at 28°C for 48 h,representative yellow, smooth colonies growing on the plates from the 10⁻⁴ dilution were selected from the samples and purified by streaking onto fresh LB. The purified isolate was designated as strain Pa-A8. To confirm the species identity of Pa-A8, genomic DNA was extracted and several conserved genes were amplified and sequenced, including the 16S rDNA gene (primers 27F/1492R) (Song et al. 2024), the gyrB gene (primers gyrB-F/gyrB-R), and the rpoD gene (primers rpoD-F/rpoD-R), respectively. The PCR products were sequenced in Kunming by Tsingke Biotech (Beijing). BLAST analysis of the 16S rDNA (accession no. SRX31354997, 1415 bp), rpoD (accession no. SRX31354998, 1096 bp), and gyrB sequences (accession no. SRX31354999, 1039 bp) showed 99.54 to 99.81% homology (100% query coverage) to Pantoea ananatis strain OC5a (CP059084.1). Phylogenetic tree analysis of 16S rDNA, gyrB, and rpoD genes further verified that the isolate Pa-A8 was in close proximity to P. ananatis. Based on the morphology, physiological and biochemical characteristics, which were determined according to standard tests (Ashmawy et al. 2020), and sequence analyses, the isolate Pa-A8 was identified as P. ananatis. To fulfill Koch’s postulates, pathogenicity tests were conducted on healthy (lesion-free and disease-free) 3-year-old P. kingianum Coll. et Hemsl. plants. Nine healthy plants were inoculated by injecting a bacterial suspension (1 × 108 CFU/ml) of the isolate Pa-A8 and an equal volume of control LB. The plants were then incubated at 28/25°C (day/night) in an artificial climate room. Within 5 days post-inoculation (dpi), water-soaked lesions gradually developed on the leaves of the inoculated plants, and the inoculation sites had completely necrosed by 30 dpi. Throughout the experimental period, the control plants remained asymptomatic. Bacterial colonies re-isolated from the necrotic lesions of inoculated leaves matched the original strain Pa-A8 in morphological characteristics, and were molecularly identical to the original isolate Pa-A8. To our knowledge, this is the first report of P. kingianum leaf spot disease caused by P. ananatis, in China. This finding has the potential to inform effective management strategies for this plant pathogenic bacterium.