First Report of Bacterial Leaf Spot on <i>Physalis pubescens</i> Caused by <i>Xanthomonas euvesicatoria</i> pv. <i>euvesicatoria</i> in Inner Mongolia, China

Ground cherry (Physalis pubescens L.) is a perennial herb of the Solanaceae family, native to China and distributed across Eurasia. Its fruit is edible and contains a variety of beneficial substances (Li et al. 2018). In July 2022, an unknown leaf spot disease was observed on ground cherry in Nierji Town, Morin Dawa Daur Autonomous Banner, Hulun Buir City, Inner Mongolia, China (48.658837°N, 123.770639°E), affecting approximately 2,000 ha. The disease incidence exceeded 85%, with observed plant mortality rates ranging from 5% to 10%. Initial symptoms appeared as water-soaked lesions between the veins of leaves, which subsequently developed into brown necrotic spots, surrounded by chlorotic margins, and then coalesced, leading to leaf perforation. For pathogen isolation, the diseased leaf tissues were surface sterilized with 75% ethanol for 3 min, followed by three consecutive rinses in distilled water. The leaves were macerated, and the resulting homogenate was streaked onto Luria-Bertani (LB) agar plates. Plates were incubated at 28℃ for 48 h. Dominant colony types were purified by subculturing onto fresh LB agar. Pale yellow, round, slightly raised, smooth, and mucoid colonies were consistently obtained. Further analysis using the BIOLOG microbial identification system (BUG agar medium for culturing isolates, IF-A inoculant solution and GEN III microplates for metabolic tests, and GEN III MicroStation System for reading data) confirmed that all ten isolates (designated nm-1, nm-3, nm-5, nm-6, nm-7, nm-8, nm-9, nm-G1, nm-Y1, and nm-Y2) were Xanthomonas euvesicatoria. In addition, genomic DNA was extracted from bacterial isolates using the AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen, U.S.A.) and quantified using a NanoVue Plus spectrophotometer (GE Healthcare, U.S.A.). Four housekeeping genes (lepA, gyrB, gapA, and gltA) were amplified and sequenced from the ten isolates, using primers described by Young et al. (2008) and Almeida et al. (2010). The GenBank accession numbers are as follows: lepA (PQ489597-PQ489606), gyrB (PQ539452-PQ539461), gapA (PQ489577-PQ489586), and gltA (PQ489587-PQ489596). A phylogenetic tree was constructed using the neighbor-joining method based on multilocus sequence analysis (MLSA) in MEGA 7 software (Kumar et al. 2016), which confirmed the isolates as X. euvesicatoria pv. euvesicatoria. Biochemical characterization revealed that all ten strains were Gram negative, catalase positive, oxidase and nitrate reductase negative. None exhibited amylolytic or pectolytic activity, consistent with the phenotypic profile of X. euvesicatoria pv. euvesicatoria strain ICMP 98PT. X. euvesicatoria pv. euvesicatoria-specific PCR assays using primers BS-XeF/BS-XeR (Koenraadt et al. 2009) yielded the expected 173-bp amplicon from all ten isolates, as with the control strain X. euvesicatoria pv. euvesicatoria ICMP 98PT. Leaves of 6-week-old ground cherry grown in climatic incubators were spray-inoculated with a bacterial suspension (108 CFU/ml). The inoculated plants were enclosed in clear plastic bags for 24 h to maintain high humidity and subsequently maintained at 60% relative humidity, with a 16-h light period at 28°C and an 8-h dark period at 22°C. Characteristic symptoms, identical to those observed in the field, developed on inoculated leaves within 7 days after inoculation: initial water-soaked lesions expanded into necrotic areas, accompanied by leaf thinning and chlorosis. Koch’s postulates were fulfilled by reisolating bacteria from typical lesions on inoculated plants, and the reisolated bacteria were identical to the inoculated ones. While X. euvesicatoria pv. euvesicatoria has been previously reported to infect ground cherry in Heilongjiang, China and eggplant in Liaoning, China (Song et al. 2019; Liu et al. 2024), this is the first report of its occurrence on ground cherry in Inner Mongolia, China. This finding lays a foundation for further research on disease management and the biological characteristics of the pathogen. This first report of X. euvesicatoria pv. euvesicatoria infecting ground cherry in Inner Mongolia undercores the expanding geographic range of this pathogen and its potential threat to local production. Given the high disease incidence observed in the field, there is an urgent need to develop effective detection methods and sustainable control strategies to manage this emerging disease and mitigate its impact on crop yield and quality.