First Report of Bacterial Canker Caused by Clavibacter michiganensis subsp. michiganensis on Pepino ( Solanum muricatum

Pepino (Solanum muricatum), also known as melon pear, is an emerging solanaceous fruit crop cultivated in several regions of China. In August 2025, a severe bacterial canker-like disease was observed on pepino seedlings (cv. Yuanzi) propagated by cuttings in six commercial greenhouse nurseries in Wuwei City, Gansu Province, China (37.94° N, 102.61° E). The nurseries covered approximately 0.5 ha, and disease incidence reached 40% to 50% in heavily affected seedbeds. Symptomatic plants showed unilateral chlorosis and wilting of leaves, curling of young petioles, leaf distortion, brown cracking lesions on stems, and vascular browning in longitudinally cut stems and roots. Small symptomatic tissue pieces (5 × 5 mm) excised from the advancing margins of stem and leaf lesions of 13 plants (two pieces per plant, 26 pieces total) were surface-disinfested in 75% ethanol for 30 s and 1% sodium hypochlorite for 60 s, rinsed twice in sterile distilled water, macerated in sterile water, and streaked onto nutrient agar. After incubation at 28°C for 96 h, six bacterial isolates exhibiting identical colony morphology were obtained from symptomatic tissues, while no other consistent bacterial isolates were recovered. Two representative isolates, CMM-2 and CMM-3, obtained from stem and leaf tissues, respectively, were selected for further study. Their colonies were creamy yellow, circular, smooth, and convex, and cells were Gram-positive, rod-shaped. For molecular identification, the nearly full-length 16S rRNA gene of the two selected isolates was amplified with primer pair 27F/1492R (Weisburg et al. 1991). BLASTn analysis of the 16S rRNA gene showed 100% identity with Clavibacter michiganensis subsp. michiganensis (Cmm) type strain NCPPB 382 (GenBank accession no. AM711867), supporting their placement in the genus Clavibacter. To accurately identify the isolates at the subspecies level, as 16S rRNA alone is often insufficient for closely related Clavibacter taxa, partial sequences of five housekeeping genes were amplified and sequenced from both strains as described by Jacques et al. (2012). BLASTn results for both isolates showed 100% query coverage with Cmm type strain NCPPB 382 for all loci; the sequence lengths and percent identities were: atpD (JX889820, 561 bp, 99.29%), dnaK (JX889998, 576 bp, 100%), gyrB (JX890087, 744 bp, 99.73%), ppk (JX890176, 564 bp, 97.80%), and recA (JX890265, 594 bp, 100%). The sequences were deposited in GenBank for strains CMM-2 and CMM-3 under accession numbers OR752409 and OR752410 (atpD), OR752412 and OR752413 (dnaK), OR752415 and OR752416 (gyrB), OR752418 and OR752419 (ppk), and OR752421 and OR752422 (recA), respectively. A maximum-likelihood phylogenetic tree based on concatenated sequences of the five loci provided the primary evidence for subspecies identification, as it grouped both isolates with reference strains of Cmm and separated them from other Clavibacter subspecies. Pathogenicity of both strains was tested on healthy pepino seedlings (cv. Yuanzi) at the 6-leaf stage by clipping the two youngest apical leaves (one cut per leaf) with scissors dipped in a bacterial suspension (107 CFU/ml). Healthy pepino seedlings inoculated with sterile distilled water served as controls (Zaluga et al. 2013). Three seedlings were inoculated per isolate per experiment, and the experiment was repeated three times. All inoculated plants developed symptoms, and both isolates (CMM-2 and CMM-3) induced identical symptoms. Plants were maintained at 25 to 28°C and about 90% relative humidity. Five days after inoculation, pale green to chlorotic lesions developed on leaves, followed by stem browning. After 14 days, stem cracking, vascular discoloration, and wilting developed, consistent with symptoms observed in the field. Control plants remained symptomless. Bacteria reisolated from inoculated plants showed the same colony morphology as the original isolates, and their identities were confirmed by 16S rRNA gene sequencing (100% identity with the inoculated strains), thus fulfilling Koch’s postulates. Cmm is an important pathogen of economic solanaceous crops, including potato (Ignatov et al. 2019), and has occasionally been reported on non-solanaceous hosts such as sugar beet (Ignatov et al. 2018). To our knowledge, this is the first report of bacterial canker caused by Cmm on pepino in China. Although currently a localized outbreak, the emergence of this disease raises concern for pepino seedling production in greenhouse propagation systems, highlighting the need for continued disease monitoring and sanitary management measures.