First report of bacterial leaf spot on Hami melon caused by Pseudomonas aeruginosa in China.

Abstract

Hami melon (Cucumis melon var. saccharinus) is an economically important crop all over the world. It is being extensively planted in greenhouse in the southwest part of Hainan province, China. A new bacterial leaf spot was observed in a 20 hm2 Hami melon plantation in Huangliu town, Ledong county, Hainan province, in January 2022. The incidence of the disease was approximately 5%. Symptoms were irregularly shaped, brown lesions with yellow haloes on mature leaves, and first appeared as small, dark-green, water-soaking spots. Specimens from the lesion margin were disinfected by submersion in 0.1% mercuric chloride for 1 min, then soaked with 75% alcohol for 30 s, and rinsed with sterilized distilled water. The tissues were then ground in 2 ml of sterile water and allowed to stand for 5min. The supernatant was streaked onto nutrient agar (NA) and incubated for 48h at 30°C. Colonies were round, smooth, colorless, nearly transparent, bead-shaped at first, and then became lightly blue. After being cultured for days on NA at 30℃, the bacteria can turn the media brown. Yellow green pigments (pyoverdin) that fluoresce under ultraviolet light could be produced by the isolates in the Luria Broth. The bacteria were gram-negative, rod shaped with a single polar flagellum, 0.4 to 1.1 × 1.4 to 3.4 µm. Its physiological and biochemical characteristics were as follows: positive for the oxidase, aerobic, arginine dihydrolase, gelatin liquefaction, denitrification, lipase, growth at 41℃, utilization of mannitol, and production of pyocyanin tests; negative for the hydrolysis of starch, levan formation, lecithinase, growth at 4℃, growth in media supplemented with 8.5% NaCl, and utilization of maltose, xylose, and ethylene glycol tests. The 16S rRNA (1,437 bp), gyrB (1,181 bp), and rpoB genes (1,510 bp) were amplified with 27F/1492R (Zhang et al. 2016）, UP-1s/UP-2sr（Hannula M，2007）, and rpoB-F/rpoB-R (Ogier, JC. et al., 2019) primer sets respectively. One of the 5 isolates collected was sequenced. A BLASTn search of GenBank revealed that the sequence of 16S rRNA gene (OQ918303) had 99.7% identity and 98% coverage comparing with the best hit Pseudomonas aeruginosa strain DSM 50071(NR_117678.1), and both gyrB (OR261077) and rpoB (OR261078) had 99.9% identity and over 98% coverage comparing with P. aeruginosa E90 (CP044006.1). A pathogenicity test was conducted by spraying a suspension of the bacteria (108 CFU/mL) onto 10 Hami melon seedlings with two true leaves. Controls were inoculated with sterile water. All inoculated plants were maintained at 28℃ with 80 to 85% relative humidity in a greenhouse. Dark-green, water soaking spots appeared on the cotyledon and stems of treated seedlings 3-5 days after inoculation, and dark green lesions with halos were observed on the true leaves at the same time. Symptoms did not occur on the control plants. The bacteria which were re-isolated from the inoculated plants were confirmed as P. aeruginosa based on the 16S rRNA gene sequence. The bacterium was not isolated from control plants. P. aeruginosa has been reported to cause disease on a variety of plants including tomato (Zhang et al., 2021), poplar (Liu, et al., 2019), ginseng (Gao et al., 2014), tinda (Mondal et al., 2012), onion (Abd-Alla et al., 2011), tobacco (Yu et al., 2008) and sweet basil (Walker et al., 2004). As far as we know, this is the first report of P. aeruginosa causing leaf spot on Hami melon in China.. This report will contribute to the recognition and diagnosis of the new disease for the Hami melon growers.