First Report of <i>Paracidovorax citrulli</i> as the Causal Agent of Bacterial Leaf Blight in Cucumber Seedlings in Mexico

In 2023, Mexico was the fourth largest producer of cucumber (Cucumis sativus L.), with 1,078,210 tons harvested. In September 2022, symptoms consisting of small water-soaked lesions surrounded by a yellow halo, later becoming dry, brown, and necrotic, were observed on seedlings of C. sativus cv. Azulan (slicer type cucumber) in a greenhouse in Culiacan, Sinaloa State, Mexico (Geographical coordinates 24°38'05.6"N 107°30'29.9" W). The disease incidence was 30 %, corresponding to three hectares with approximately 23,000 plants per ha, and the disease severity was 60 %. Bacterial streaming from the cut margin of leaf spots in distilled water was observed under a microscope at 100X magnification. Subsequently, a slice of cotyledon leaf was disinfected with 70 % ethanol for one minute and placed in sterile distilled water, and the suspension was plated onto King's B medium and incubated for three days at 27 °C. From five symptomatic seedlings, non-fluorescent bacterial colonies were obtained. After purification, colonies were cream white, circular in shape, with entire margins. The isolated bacteria were Gram-negative in the Gram staining process and caused a hypersensitive reaction on tobacco (Nicotiana tabacum) plants 24 h post-infiltration. Five isolates were tested in real-time PCR using SYBR green with the P. citrulli-specific primer pair AC158F (5’-CTTGGTGCTCCATGCTCGA-3’) and AC158R (5’-GGCTTGGTTGCGAATTCACT-3’) reported by Cho et al. (2015). To confirm the PCR results (partial) 16S rRNA gene sequencing was performed on a representative isolate using the FD2 (5’-AGAGTTTGATCATGGCTCAG-3’) and RP1 (5’-ACGGTTACCTTGTTACGACTT-3’) primers (Weisburg et al., 1991). Comparison of sequence data of strain CIAD_66 (GenBank Accession Number OR233591.1) with P. citrulli strains ICMP 7500 and BC525 (Accession Numbers NR_041758 and NR_118397, respectively) showed a 100 % homology. A discriminative PCR according to Lee et al. (2024) was performed from five P. citrulli isolates based on gltA gene analysis, and it was determined that our strains belong to clonal complex group I. Koch postulates were fulfilled by inoculating both cucumber seeds and plants using a bacterial suspension containing 3 x 108 cfu mL-1. For seeds, immersion in the bacterial suspension was carried out for two minutes and then two seeds were placed per pot containing sterile substrate, 6 pots in total. Five 14-day-old cucumber plants were inoculated by spraying and placed in a humid chamber at 27 °C. Control treatments were inoculated using sterile water. A water-soaked lesion was visible mainly on the cotyledons. Inoculated plants showed symptoms 7-10 days post-inoculation. Control plants (originating from seeds and seedlings did not show symptoms). Cream white bacterial colonies were re-isolated on King's B medium only from inoculated seedlings and plants, and their identity was checked by real-time PCR, confirming the pathogenicity test. Paracidovorax citrulli (formerly known as Acidovorax citrulli) (Du et al., 2023) is a seed-borne pathogen reported to affect mainly Cucurbitaceae with watermelon and melon as primary hosts; however, pumpkin, squash, and cucumber may also be infected. This bacterium is distributed worldwide, especially in Asia, North America, and South America, where two genetically different groups have been reported: Group I (moderately aggressive across most cucurbit hosts), and Group II (more aggressive on watermelon) (EPPO, 2025). In México, Hernández-Anguiano et al. (2023) reported infestations of P. citrulli in watermelon production fields. For cucumbers, there were reports from Australia and South Korea (Martin et al., 1999; Song et al., 2015). To our knowledge, this is the first report of P. citrulli clonal complex group I causing bacterial leaf blight in cucumber seedlings in Mexico. Currently, strategies for managing this disease are limited, and resistant commercial cultivars are not known; therefore, the presence of this pathogen poses a latent risk to cucurbit production in Mexico, and the development and establishment of control measures are essential to minimize the production losses and pathogen distribution.