First Report of Leaf Spot on Cucumber Caused by Pantoea ananatis in Hainan, China

HomePlant DiseaseVol. 107, No. 4First Report of Leaf Spot on Cucumber Caused by Pantoea ananatis in Hainan, China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Leaf Spot on Cucumber Caused by Pantoea ananatis in Hainan, ChinaG. S. Lao, P. F. Jin, W. G. Miao, and W. B. LiuG. S. LaoSchool of Plant Protection, Hainan University, Haikou 570228, Hainan, ChinaSearch for more papers by this author, P. F. Jin†Corresponding author: P. F. Jin; E-mail Address: [email protected]https://orcid.org/0000-0003-0730-1263School of Plant Protection, Hainan University, Haikou 570228, Hainan, ChinaSearch for more papers by this author, W. G. MiaoSchool of Plant Protection, Hainan University, Haikou 570228, Hainan, ChinaSearch for more papers by this author, and W. B. LiuSchool of Plant Protection, Hainan University, Haikou 570228, Hainan, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations G. S. Lao P. F. Jin † W. G. Miao W. B. Liu School of Plant Protection, Hainan University, Haikou 570228, Hainan, China Published Online:5 Apr 2023https://doi.org/10.1094/PDIS-04-22-0819-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleCucumber (Cucumis sativus L.) is one of the most important vegetables in the world. It is widely cultivated and mostly grown in greenhouses (Sallam et al. 2021). It has a long growth cycle and is particularly susceptible to bacterial diseases. In May 2021, bacterial leaf spot was found on cucumbers of the variety Lyuyou No.3 in Hainan Province, China. In the early stage of the disease, leaves showed small yellow-brown spots in the shape of water stains. When exposed to light, a yellow halo around the disease spot could be seen. In later stages, the lesions gradually become larger and more yellow. The leaf veins around the disease site also gradually turned yellow. In serious cases, the whole leaf turned yellow and died. We collected plants with the same symptoms from 25 different farms in Hainan Province. Five plants were selected from each farm by the classic five-point sampling method and three leaves were selected from each plant, for a total of 15 leaves per farm. Three leaves were randomly selected from the 15 leaves on each farm for pathogen isolation, and a total of 75 leaves were isolated. The incidence of the disease was 20 to 30% based on a diagnostic test of these 75 leaves. Using microscopy, bacterial streaming was observed to tentatively identify the causal agent as a bacteria. Tissue isolation was used to isolate the responsible pathogens. A 5 × 5 mm sample of tissue at the junction of diseased and healthy sections was collected. The surface of the tissue was disinfected in a 75% ethanol solution for 30 s, soaked in 2% NaOCl for 5 to 7 min, and washed thrice in sterile distilled water. The tissues were inoculated onto lysogen broth culture media (LB) and cultured in a 28°C incubator for 2 days. Bacterial colonies that emerged from the tissues were cultured in LB. Four isolated colonies were selected for verification. The colonies isolated from the diseased leaves of cucumber were round, egg yellow, and slightly sticky. The isolate PA-1 was identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F/1492R (Lane 1991) and gyrB gene (Li et al. 2019). Sequences were stored in GenBank with accession numbers OK576932.1 (16S rRNA, PA-1) and OL978577 (gyrB); BLASTn was used to compare these with other GenBank sequences. Sequencing of the 16S rRNA gene showed that PA-1 had a sequence length of 1403 bp, with 99.78% genetic similarity to Pantoea ananatis strain MZ007857.1. Sequencing of the gyrB gene showed that the sequence length of PA-1 was 1136bp, with 99.29% genetic similarity to P. ananatis strain MW981331.1. Then, a pathogenicity test was conducted to verify Koch’s postulates by first inoculating P. ananatis into LB liquid medium (shake culture at 28°C, 180 r/min). The log phase cell was collected by centrifugation (5,000 r/min for 2 min at 4°C), and inoculated strains were resuspended in sterile water at OD600 = 0.5. The bacterial suspension was inoculated on healthy cucumber leaves with a syringe. The control was sterile water, which was injected onto healthy cucumber leaves using the same methods. The plants were placed in a greenhouse with a diurnal temperature difference of 21 to 27°C and were observed daily. After 2 weeks, all bacterial inoculated plants developed symptoms of shriveling and necrosis, while the control group showed no symptoms. From the symptomatic plants, the pathogen was reisolated and identified by morphological and molecular characterization. The sequences of the isolates recovered from the inoculated experiment matched 100% the sequences of isolate PA-1. Koch’s postulates were completed by following the previously described method. To our knowledge, this is the first report of P. ananatis causing leaf spot of cucumber.The author(s) declare no conflict of interest.References:Lane, D. J. 1991. Page 115 in: Nucleic Acid Techniques in Bacterial Systematics. Wiley, New York, NY. Google ScholarLi, F., et al. 2019. Aquac. Res. 50:1515. Crossref, ISI, Google ScholarSallam, B. N., et al. 2021. Horticulturae 7:256. https://doi.org/10.3390/horticulturae7080256 Crossref, ISI, Google ScholarFunding: Funding was provided by Scientific Research Foundation for Advanced Talents, Hainan University (grant/award no. KYQD(ZR)1842), Hainan Provincial Natural Science Foundation-the Scientific Research Foundation for Advanced Talents (grant/award no. 2019RC163), Hainan Association for Science and Technology Plans to Youth R & D Innovation (grant/award no. QCXM201903), and National Natural Science Foundation of China (grant/award no. 31960552).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 107, No. 4 April 2023SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Article History Issue Date: 27 Apr 2023Published: 5 Apr 2023First Look: 15 Sep 2022Accepted: 11 Sep 2022 Page: 1214 Information© 2023 The American Phytopathological SocietyFundingScientific Research Foundation for Advanced Talents, Hainan UniversityGrant/Award Number: KYQD(ZR)1842Hainan Provincial Natural Science Foundation-the Scientific Research Foundation for Advanced TalentsGrant/Award Number: 2019RC163Hainan Association for Science and Technology Plans to Youth R & D InnovationGrant/Award Number: QCXM201903National Natural Science Foundation of ChinaGrant/Award Number: 31960552Keywordsdisease managementpathogen diversityprokaryotesvegetablesThe author(s) declare no conflict of interest.PDF downloadCited byPest categorisation of Pantoea ananatisEFSA Journal, Vol. 21, No. 3