First Report of pv. as the Causal Agent of Bacterial Leaf Spot on Lettuce in China.

Lettuce (Lactuca sativa) is a leafy vegetable that belongs to the family Asteraceae. This cool-season crop grows well during Spring and Fall in China (Qi et al., 2021). In October 2023, a leaf disease with dark brown to black lesions was observed on lettuce (var. iceberg) in Yongning Town, Yanqing District, Beijing, China (40°53'N, 116°16'E). The disease incidence ranged from 10 to 40% in the surveyed greenhouse and field. Translucent and water-soaked brown spots were observed on the margins of leaf, then coalesced into large necrotic lesions surrounded by a chlorotic halo. Infected sections were soaked with 75% ethyl alcohol for 7 second, rinsed with sterile water for 15 second twice, and cut into pieces in the sterile water. The sample extracts were streaked on Nutrient agar medium. After incubation for 48 hours, 12 colonies were obtained and all the colonies were Gram-negative and aerobic with yellow, round-shape, smooth and glistening appearance. Four isolates SCZX1-SCZX4 with typical characteristics were selected for further identification tests. Pathogenicity test of SCZX1-SCZX4 was performed on the three-week-old lettuce (var. butterhead) by spraying with the bacterial suspension (108 CFU·ml-1). Inoculated lettuce were incubated at 26℃ and 70% relative humidity in the growth chamber. Another set of lettuce plants were mock inoculated with sterile distilled water. Three trials were carried out per isolate, and each treatment included fifteen lettuce plants. Symptoms appeared within 7-10 days after inoculation and are identical to those naturally infected lettuces. Negative control plants had no symptoms. The 16S rRNA region and two housekeeping genes (gyrB and atpD) of each isolate were amplified with universal primers F27/R1492 (Monciardini et al., 2006) and specific primers (Roach et al., 2018), respectively. According to BLAST analysis of each isolate (Genbank accession number PP027925, PP140779, PP140781 to PP140783, PP137422 to PP137428), BLAST searches of the obtained sequences revealed 100.0% of 16S rRNA region(1400/1400 nucleotides), 100.0% of gyrB(774/774 nucleotides), and 100.0% of atpD(768/768 nucleotides) identity and query coverage to Xhv CFBP 498 (Genbank accession number LR828257.1). Phylogenetic analysis revealed that SCZX1-SCZX4 clustered with the neopathotype strain Xhv LMG 938PT, which was isolated from a diseased L. sativa in Zimbabwe (Timilsina, S et al., 2015). Physiological and biochemical tests of SCZX1-SCZX4 were conducted by the BIOLOG GENIII microplate system (Biolog, Hayward, CA, USA), and the test results are consistent with the Xhv LMG 938PT (Morinière, L et al., 2020) and L1-L7 (Pernezny, K et al., 1995). Strains were re-isolated from re-inoculated lettuce, then re-identified as Xhv by the same method, fulfilling Koch's postulates. Bacterial leaf spot (BLS) is a worldwide-spread lettuce disease caused by numerous bacterial pathogens, including Xhv (Dia, N.C et al., 2022), Pseudomonas cichorii (Patel, N et al., 2021) and P. viridiflava (Alippi, A. M et al., 1999) etc. Xhv had been widely reported as the causal pathogen of BLS in Turkey (Sahin, F et al., 2000), United States (Bull, C.T et al., 2007), Saudi Arabia (Al-Saleh, M et al., 2008), and France (Morinière et al., 2020). However, the first report of Xhv causing BLS on lettuce in China is significant to provide pathogen information for growers in making effective measures to manage this disease.