{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T18:09:23Z","timestamp":1790100563048,"version":"4.0.1"},"reference-count":37,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100008798","name":"Ministry of Science Research and Technology","doi-asserted-by":"publisher","award":["4040260"],"award-info":[{"award-number":["4040260"]}],"id":[{"id":"10.13039\/501100008798","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"abstract":"ABSTRACT<\/jats:title>\n \n \n \n Clavibacter<\/jats:italic>\n species are the most important gram-positive bacterial plant pathogens in terms of both disease severity and economic importance. As a group of regulated and quarantined plant pathogens,\n Clavibacter<\/jats:italic>\n members are slow-growing organisms on general culture media, leading to difficulties in their isolation and identification. In this study, we developed an efficient, straightforward method for the isolation and identification of\n Clavibacter<\/jats:italic>\n members from plant materials. First, a semi-selective medium was developed from a modification of conventional YPGA and YDC media, namely ClaYPGA and ClaYDC, respectively. This semi-selective medium contained four antibiotics, that is\n ,<\/jats:italic>\n colistin (100.0 mg\/L), nalidixic acid (5.0 mg\/L), sulfamethoxazole (5.0 mg\/L), and trimethoprim (5.0 mg\/L) for the inhibition of non-target bacterial growth, as well as natamycin (500.0 mg\/L) for the inhibition of fungal growth. The ClaYPGA\/ClaYDC medium supported the growth of all\n Clavibacter<\/jats:italic>\n species while inhibiting the growth of almost all gram-negative bacteria, gram-positive\n Bacillus<\/jats:italic>\n and\n Streptomyces<\/jats:italic>\n , and fungi. Furthermore, ClaYPGA and ClaYDC did not affect the biological features, that is\n ,<\/jats:italic>\n cell density, and colony morphology of\n Clavibacter<\/jats:italic>\n species compared to the basal YPGA and YDC. To increase the specificity of semi-selective medium, the primer pair ClaFor\/ClaRev was designed based on the nucleotide sequence of a 259 bp DNA fragment found only in\n Clavibacter<\/jats:italic>\n members. These PCR primers were strictly specific to\n Clavibacter<\/jats:italic>\n species. The combination of semi-selective medium and ClaFor\/ClaRev PCR primers increased the precision of the isolation and identification of\n Clavibacter<\/jats:italic>\n species to such an extent that it has never been reported in the literature.\n <\/jats:p>\n \n IMPORTANCE<\/jats:title>\n \n Regulated plant pathogenic bacteria that are subjected to quarantine restrictions play a vital role in the global trade of agricultural products. Plant protection authorities and quarantine inspectors need sensitive, dependable, cost-effective, and easy-to-use, straightforward methods to monitor the status of regulated bacterial agents either in the field or in transportation ports.\n Clavibacter<\/jats:italic>\n species are the most destructive gram-positive bacterial plant pathogens. However, isolation and identification of these pathogens are affected by their slow-growing habit on general bacteriological culture media. Until 2015, only five\n Clavibacter<\/jats:italic>\n lineages had been identified infecting alfalfa, maize, potato, tomato, and wheat. Within the past decade, however, the range of plant-associated\n Clavibacter<\/jats:italic>\n members has been expanded to 13 standalone species. Selective or semi-selective culture media that have so far been developed for the isolation of\n Clavibacter<\/jats:italic>\n targeted only the five original pathogens. Thus, application of those culture media nowadays would leave the expanded range of\n Clavibacter<\/jats:italic>\n species untargeted. In this study, we aimed to develop a semi-selective medium to support the growth of all\n Clavibacter<\/jats:italic>\n lineages while combating the growth of gram-negative bacteria, non-target gram-positive bacteria, and fungi. Furthermore, we designed a PCR primer strictly specific for\n Clavibacter<\/jats:italic>\n members with no amplification in the other bacteria or any other living organism. Supplementing the results of semi-selective medium and specific PCR primers led to setting up a reliable, easy-to-use laboratory method for the isolation and identification of\n Clavibacter<\/jats:italic>\n species.\n <\/jats:p>\n <\/jats:sec>\n <\/jats:sec>","DOI":"10.1128\/aem.00871-26","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T17:42:33Z","timestamp":1790098953000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Isolation and identification of\n Clavibacter<\/i>\n made easy"],"prefix":"10.1128","author":[{"given":"Nastaran","family":"Zamani","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05vf56z40","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Plant Protection, College of Agriculture, University of Tehran","place":["Karaj, Iran"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zahra","family":"Sadeghi Mosavar","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05vf56z40","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Plant Protection, College of Agriculture, University of Tehran","place":["Karaj, Iran"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ardavan","family":"Soleimani","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05vf56z40","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Plant Protection, College of Agriculture, University of Tehran","place":["Karaj, Iran"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0359-0398","authenticated-orcid":true,"given":"Ebrahim","family":"Osdaghi","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05vf56z40","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Plant Protection, College of Agriculture, University of Tehran","place":["Karaj, Iran"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1094\/9780890546970"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1099\/00207713-34-2-107"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1080\/00288233.1977.10427375"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1099\/ijsem.0.002492"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02873-19"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1111\/mpp.70093"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1099\/ijsem.0.007154"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1111\/epp.12302"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02098-19"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1111\/mpp.13191"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1111\/mpp.13268"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1111\/ppa.13893"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02158-12"},{"key":"e_1_3_3_15_2","volume-title":"Laboratory guide for the identification of plant pathogenic bacteria","author":"Schaad NW","year":"2001","unstructured":"Schaad NW, Jones JB, Chun W. 2001. Laboratory guide for the identification of plant pathogenic bacteria. 3rd ed. American Phytopathological society (APS press).","edition":"3"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac803"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1094\/Phyto-69-82"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1099\/ijsem.0.006022"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1094\/PDIS-05-26-0881-RE"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1094\/PHYTO-02-11-0045"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/aem.63.7.2625-2630.1997"},{"key":"e_1_3_3_22_2","first-page":"104","volume-title":"Laboratory guide for identification of plant pathogenic bacteria","author":"Vidaver AK","year":"1988","unstructured":"Vidaver AK, Davis MJ. 1988. Coryneform plant pathogens, p 104\u2013113. 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Phytopathology 82:1072.","journal-title":"Phytopathology"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF01974227"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1094\/PD-76-0830"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1094\/PHYTO-07-24-0236-R"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.1128\/aem.63.2.498-505.1997"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10658-013-0329-x"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1007\/0-387-30743-5_43"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.1099\/ijsem.0.004786"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.1128\/aem.01245-25"},{"key":"e_1_3_3_34_2","volume-title":"Performance standards for antimicrobial susceptibility testing; informational supplement","author":"CLSI: Clinical and Laboratory Standards Institute","year":"2016","unstructured":"CLSI: Clinical and Laboratory Standards Institute. 2016. CLSI document M100-S. In Performance standards for antimicrobial susceptibility testing; informational supplement, 26th ed. 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