First Report of Leaf Spot on Great Burnet ( <i>Sanguisorba officinalis</i> L.) Caused by <i>Pantoea agglomerans</i> in China

Great burnet (Sanguisorba officinalis L.) is a perennial herb of the family Rosaceae. Its dried roots are commonly used as a traditional Chinese medicine to treat burns, scalds, and internal hemorrhage (Committee of China Pharmacopoeia, 2020). In September 2019, bacterial leaf spot was observed in a 120 m² great burnet cultivation garden planted in Min County, Gansu Province, China (35°17’N, 104°02’E). Initial symptoms appeared as water-soaked lesions on the leaves, which subsequently developed into yellow to brown lesions. Yellow bacterial exudate appeared on the abaxial surface of the leaves, leading to the leaves turning black, withering, and dying. Disease incidence was approximately 75%. Symptomatic leaf tissues were cut into 5 × 5 mm sections and surface sterilized for 1 min with 0.1% HgCl2 and rinsed three times in sterile distilled water before grinding with a glass rod. A loopful of bacterial suspension was streaked onto nutrient agar (NA) and cultured at 28°C for 48h. Colonies were yellow, round, opaque, smooth, and sticky, with a raised center. Three isolates DY1-DY3 were selected for further analysis. The isolated bacteria were Gram-negative rods. They caused a hypersensitivity reaction on tobacco, tested negative for methyl red reaction, tyrosinase, starch hydrolase, and tested positive for catalase, gelatin liquefaction, and casein hydrolysis. Additionally, they can decompose phenylalanine and utilize xylose and citrate (Gavini et al. 1989). These isolates exhibited morphological and biochemical characteristics consistent with the genus Pantoea. To test the pathogenicity of the strain (DY1), 30 healthy leaves of one-year-old great burnet were selected. They were sprayed with a 1 × 10 ⁸ CFU/mL bacterial suspension. The treatment included both leaves wounded with a sterile needle and an unwounded group. The control group was sprayed with water. The plants were bagged and maintained at 80% RH, 25 ± 1°C, in a light incubator with a 12h light/12h dark photoperiod. Twenty-four hours post-inoculation, wounded leaves demonstrated black necrotic spots, and unwounded leaves exhibited water-soaked lesions and these symptoms were consistent with field observations. The control group showed no symptoms. Tests were repeated 2 times. Re-isolated pathogen from inoculated leaves were consistent with those of the original isolate both morphological examination and molecular identification, fulfilling Koch's postulates. The 16S rDNA, gyrB, fusA, and leuS genes of the isolate DY1 were amplified and sequenced with previously reported primers (Zang et al. 2021). The sequences were submitted in GenBank with accession Nos. OR225701, OR827065, PP537634, and PP537635, respectively. The gene sequences exhibited 99, 99, 100 and 99% similarity with the corresponding sequences of the Pantoea agglomerans type strain LMG 1286, respectively. Aligned concatenated sequences of the strains DY1 were used to construct a maximum likelihood phylogenetic tree in MEGA11.0, revealing clustering with P. agglomerans LMG 1286, with 98.7 to 99.2% nucleotide similarity. To our knowledge, this is the first report of bacteria leaf spot on S. officinalis caused by P. agglomerans in China. The results of this study will help prevent and control this disease.