First Report of Bacterial Leaf Spot of Cucumber Caused by Pseudomonas cichorii in China

Abstract

Cucumber (Cucumis sativus L.) is a globally important vegetable. From September to May every year, more than 600 groups from 29 provinces of China conduct experiments in crop breeding and seed reproduction in Hainan Province. A new cucumber bacterial leaf spot disease was discovered in 2015, during a survey in a cucumber breeding facility in Sanya, Hainan province. The disease incidence reached 90% in severely infected fields since 2015. Initial symptoms on cucumber leaves are water soaking and light green lesions. Leaves are thinner. Lesion margins gradually turn yellow, and the lesion centers turn brown. The range of irregular lesions is 1.5 to 2.0 cm long and 1 to 1.5 cm wide. Severely affected leaves show chlorotic lesions, which coalesce to produce broad necrotic areas. More than 120 bacterial isolates with consistent appearance were isolated from symptomatic cucumber leaf samples using nutrient agar (NA), and three isolates were selected for further characterization. Physiological and morphological characteristics of the isolated bacteria were determined using standard microbiological techniques (Schaad et al. 2001). Colonies are round, pale yellow, smooth, with undulate margins. Bacterial cells were gram negative, with multiple polar flagella, positive for catalase and oxidase, produced fluorescent blue color under ultraviolet light on King’s medium B agar, and metabolized glucose and fructose. Negative results were produced for growth at 41°C, gelatin hydrolysis, fructosan production, arginine dihydrolase presence, Tween 80 hydrolysis, use of trehalose, and sorbitol. Based on these morphological and biochemical characteristics, the pathogen was identified as Pseudomonas cichorii Stapp (Bergey et al. 1994). The bacterial identity was further confirmed by amplifying the 16S rRNA gene using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) and the rpoB gene using primers LAPS/LAPS27 (Ait Tayeb et al. 2005). The sequences were deposited in GenBank (accession nos. MF979525.1 [16S rRNA] and MF979524.1 [rpoB]) and exhibited a 99% identity with P. cichorii (accession nos. KU923374.1 and JQ994485.1). A pathogenicity assay was carried out using potted, greenhouse-grown cucumber plants. The inoculum was grown in NA for 24 h at 28°C and then was suspended in distilled water. Leaves (three replications, cv. Yansi) were sanitized in 0.1% mercuric chloride for 30 s, washed and rinsed with sterile distilled water, and then sprayed with a bacterial suspension (5.0 × 10⁸ CFU/ml). Distilled water was used as negative control. Inoculated and control plants were incubated at 90 to 100% relative humidity at 25°C. Water-soaked, thin, and light-green lesions appeared after 24 h, and enlarged lesions with yellow halos were observed after 48 h. The lesion margins gradually turned yellow, and the lesion centers turned brown after 5 days. These symptoms were not observed on control plants. The pathogenicity test was repeated three times. The same bacteria were consistently reisolated. Thus, the Koch’s postulates were fulfilled. To our knowledge, this is the first isolation of cucumber leaf spot caused by P. cichorii in China.