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First Report of Bacterial Leaf Spot Caused by Pseudomonas viridiflava on Chinese Cabbage in China

Liu, Y., Guan, X. Y., Kong, X. D., Wu, X. L., Liu, S. N., Ren, J. H., Guo, M., Zhang, Y. W.
Plant disease 2019 v.103 no.7 pp. 1764
Brassica oleracea, Brassica rapa, Chinese cabbage, Gram-negative bacteria, Pseudomonas viridiflava, acetone, arginine deiminase, carbon, citrates, cultivars, downy mildew, essential genes, financial economics, flagellum, gelatin, greenhouses, growing season, hydrogen peroxide, inbred lines, leaf spot, leaves, levan, liquefaction, nitrate reduction, nucleotide sequences, pathogenicity, phenotype, rain, relative humidity, ribosomal DNA, spraying, tobacco, viruses, China
Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important and widely cultivated vegetable in China and is consumed worldwide. However, the yield and quality of this vegetable can be affected by soft rot, downy mildew, and virus diseases during the growing season. In October 2015 and 2017, outbreaks of a disease of Chinese cabbage caused significant economic losses in many varieties (yield loss between 60 and 100%) after rain events in Harbin, Heilongjiang province, China. Water-soaked lesions were first observed on lower leaves and later developed into necrotic spots surrounded by yellow haloes and water-soaked rings on the underside of leaves, which were consistent with leaf spots symptoms described by Kudela (1986). The lesions were along the veins in some varieties, causing the leaves to dry or rot and eventually killing the plant. Three gram-negative bacterial isolates (X1, X2, and X3) were obtained with an average size of 1.05 ± 0.04 × 0.5 ± 0.02 μm and clear signs of polar flagella. LOPAT tests indicated that X1 to X3 isolates tested negative for levan, pectolysis, arginine dihydrolase, and oxidase and positive for tobacco hypersensitivity. Furthermore, the three isolates were also positive for gelatin liquefaction, hydrogen peroxide, and acetone production (V-P) and were negative for nitrate reduction and methyl red. Moreover, the isolates fluoresced on King’s medium B and utilized malonate and citrate as carbon sources. 16S rDNA was amplified by polymerase chain reaction using universal primers 27 F and 1492 R (Ying et al. 2012). The 16S rDNA sequences of X1, X2, and X3 (GenBank accession nos. MG686550.1, MG706127.1, and MG708224.1) shared 100, 99, and 100% identity to Pseudomonas viridiflava strain CFBP 1590 (LT855380.1), P. viridiflava strain +Y15 (JX 077099.1), and P. viridiflava strain 45 (GQ398129.1), respectively. The identities of the three isolates were confirmed by sequencing the housekeeping gene gyrB (Yamamoto et al. 2000) and 16S-23S intergenic transcribed spacer region (Tanti et al. 2012) (GenBank accession nos. MH389073.1, MH389074.1, MH389075.1, MH348157.1, MH348158.1, and MH348159.1). Pathogenicity tests were performed to complete Koch’s postulates. Chinese cabbage cultivar ‘Xixing Qiuguan’ and inbred line A95-1-1 were cultivated in a greenhouse. After 5 weeks, five completely expanded leaves from each of 10 plants were inoculated by spraying with about 2 ml of bacterial suspension (10⁵ CFU/ml). The inoculated plants were then placed in a greenhouse under 80 to 90% relative humidity at 20°C daytime and 10°C nighttime. After 14 days, lesions similar to the initial symptoms were observed on plants inoculated by the three P. viridiflava strains; however, the control plants treated with sterile water did not show symptoms. Pathogens were reisolated from inoculated plants and identified as P. viridiflava according to the phenotypic, physiological, and biochemical characteristics and the molecular methods described above. This work is the first report of bacterial leaf spot on Chinese cabbage caused by P. viridiflava in China.