{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T10:54:55Z","timestamp":1769597695988,"version":"3.49.0"},"reference-count":48,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2019,12,11]],"date-time":"2019-12-11T00:00:00Z","timestamp":1576022400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100008530","name":"European Regional Development Fund","doi-asserted-by":"publisher","award":["AGL2014-55287-C2-1-R, BFU2015-66806-R, RTI2018-094263-B-I00, and AGL2017-84389-C2-2-R"],"award-info":[{"award-number":["AGL2014-55287-C2-1-R, BFU2015-66806-R, RTI2018-094263-B-I00, and AGL2017-84389-C2-2-R"]}],"id":[{"id":"10.13039\/501100008530","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011033","name":"Agencia Estatal de Investigaci\u00f3n","doi-asserted-by":"publisher","award":["RTI2018-094263-B-I00"],"award-info":[{"award-number":["RTI2018-094263-B-I00"]}],"id":[{"id":"10.13039\/501100011033","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["AGL2014-55287-C2-1-R, BFU2015-66806-R, RTI2018-094263-B-I00, and AGL2017-84389-C2-2-R"],"award-info":[{"award-number":["AGL2014-55287-C2-1-R, BFU2015-66806-R, RTI2018-094263-B-I00, and AGL2017-84389-C2-2-R"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples.<\/jats:p>","DOI":"10.3390\/genes10121033","type":"journal-article","created":{"date-parts":[[2019,12,12]],"date-time":"2019-12-12T03:20:16Z","timestamp":1576120816000},"page":"1033","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7482-1715","authenticated-orcid":false,"given":"Silvia","family":"Rodr\u00edguez-Pires","sequence":"first","affiliation":[{"name":"Department of Plant Protection, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de La Coru\u00f1a Km. 7, 28040 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5873-6059","authenticated-orcid":false,"given":"Eduardo Antonio","family":"Espeso","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Biology, Centro de Investigaciones Biol\u00f3gicas, Consejo Superior de Investigaciones Cient\u00edficas, Ramiro de Maeztu 9, 28040 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2561-9304","authenticated-orcid":false,"given":"Nuria","family":"Bar\u00f3-Montel","sequence":"additional","affiliation":[{"name":"IRTA, XaRTA-Postharvest, Edifici Fruitcentre, Parc Cient\u00edfic i Tecnologic Agroalimentari de Lleida, 25003 Lleida, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1806-9626","authenticated-orcid":false,"given":"Rosario","family":"Torres","sequence":"additional","affiliation":[{"name":"IRTA, XaRTA-Postharvest, Edifici Fruitcentre, Parc Cient\u00edfic i Tecnologic Agroalimentari de Lleida, 25003 Lleida, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paloma","family":"Melgarejo","sequence":"additional","affiliation":[{"name":"Department of Plant Protection, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de La Coru\u00f1a Km. 7, 28040 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7725-7782","authenticated-orcid":false,"given":"Antonieta","family":"De Cal","sequence":"additional","affiliation":[{"name":"Department of Plant Protection, Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de La Coru\u00f1a Km. 7, 28040 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,12,11]]},"reference":[{"key":"ref_1","unstructured":"Byrde, R.J.W., and Willetts, H.J. (1977). The Brown Rot Fungi of Fruit: Their Biology and Control, Pergamon Press Ltd."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1094\/PDIS-06-17-0867-RE","article-title":"Distribution and characterization of Monilinia spp. causing apple fruit decay in Serbia","volume":"102","author":"Vico","year":"2018","journal-title":"Plant Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"763","DOI":"10.1094\/PDIS-93-7-0763A","article-title":"First report of brown rot caused by Monilinia fructicola in peach orchards in Ebro Valley, Spain","volume":"93","author":"Gell","year":"2009","journal-title":"Plant Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1007\/s10658-013-0292-6","article-title":"Occurrence of Monilinia laxa and M. fructigena after introduction of M. fructicola in peach orchards in Spain","volume":"137","author":"Villarino","year":"2013","journal-title":"Eur. J. Plant Pathol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.ijfoodmicro.2016.01.023","article-title":"Growth and aggressiveness factors affecting Monilinia spp. survival peaches","volume":"227","author":"Villarino","year":"2016","journal-title":"Inter. J. Food Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Garcia-Benitez, C., Melgarejo, P., De Cal, A., and Fontaniella, B. (2016). Microscopic analyses of latent and visible Monilinia fructicola infections in nectarines. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0160675"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1071\/AP00013","article-title":"First record of Monilinia fructicola on strawberry fruit in Victoria, Australia","volume":"29","author":"Washington","year":"2000","journal-title":"Australas. Plant Pathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1823","DOI":"10.1094\/PDIS-04-17-0507-PDN","article-title":"First report of Monilinia fructicola causing postharvest decay of strawberries (Fragaria \u00d7 ananassa) in the United States","volume":"101","author":"Janisiewicz","year":"2017","journal-title":"Plant Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1094\/PHYTO-07-17-0250-R","article-title":"A model for temporal dynamics of brown rot spreading in fruit orchards","volume":"108","author":"Bevacqua","year":"2018","journal-title":"Phytopathology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1094\/PDIS-91-7-0907B","article-title":"First report of brown rot caused by Monilinia fructicola on various stone and pome fruits in the Czech Republic","volume":"91","year":"2007","journal-title":"Plant Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1094\/PDIS-02-11-0113","article-title":"First report of brown rot of apple caused by Monilinia fructicola in Germany","volume":"95","author":"Grabke","year":"2011","journal-title":"Plant Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1094\/PDIS-06-11-0531","article-title":"First report of brown rot caused by Monilinia fructicola on stored apple in Serbia","volume":"96","author":"Duduk","year":"2012","journal-title":"Plant Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"556","DOI":"10.1094\/PDIS-10-14-1006-PDN","article-title":"First report of asiatic brown rot (Monilinia polystroma) and brown rot (Monilinia fructicola) on pears in Italy","volume":"99","author":"Martini","year":"2015","journal-title":"Plant Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1094\/PDIS-11-14-1099-PDN","article-title":"First report of brown rot on apple fruit caused by Monilinia fructicola in Pennsylvania","volume":"99","author":"Peter","year":"2015","journal-title":"Plant Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2240","DOI":"10.1094\/PDIS-03-16-0325-RE","article-title":"Monilinia species associated with brown rot of cultivated apple and pear fruit in China","volume":"100","author":"Zhu","year":"2016","journal-title":"Plant Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2657","DOI":"10.1094\/PDIS-05-18-0755-PDN","article-title":"First report of brown rot caused by Monilinia fructicola on apple in Brazil","volume":"102","author":"Pereira","year":"2018","journal-title":"Plant Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1094\/PHYTO.2002.92.7.743","article-title":"Use of green fluorescent protein-transgenic strains to study pathogenic and nonpathogenic lifestyles in Colletotrichum acutatum","volume":"92","author":"Horowitz","year":"2002","journal-title":"Phytopathology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1961","DOI":"10.1111\/nph.14297","article-title":"Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens","volume":"213","author":"Romero","year":"2017","journal-title":"New Phytol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"768","DOI":"10.1094\/PHYTO.2003.93.7.768","article-title":"Green fluorescent detection of fungal colonization and endopolygalacturonase gene expression in the interaction of Alternaria citri with citrus","volume":"93","author":"Isshiki","year":"2003","journal-title":"Phytopathology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"134","DOI":"10.3923\/ppj.2007.134.140","article-title":"Transformation of Botrytis cinerea with a Green Fluorescent Protein (GFP) Gene for the Study of Host-pathogen Interactions","volume":"6","author":"Li","year":"2006","journal-title":"Plant Pathol. J."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1111\/j.1365-3059.2009.02022.x","article-title":"Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts","volume":"58","author":"Bolton","year":"2009","journal-title":"Plant Pathol."},{"key":"ref_22","first-page":"152","article-title":"Exploring the Monilinia vaccinii-corymbosi-blueberry pathosystem for the discovery of genes governing host-specificity","volume":"106","author":"Bansal","year":"2016","journal-title":"Phytopathology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1007\/s10658-014-0579-2","article-title":"Sensitivity of Monilinia fructicola from Spanish peach orchards to thiophanate-methyl, iprodione, and cyproconazole: Fitness analysis and competitiveness","volume":"141","author":"Melgarejo","year":"2015","journal-title":"Eur. J. Plant Pathol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"R4","DOI":"10.1186\/gb-2009-10-1-r4","article-title":"Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis","volume":"10","author":"Michielse","year":"2009","journal-title":"Genome Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1671","DOI":"10.1038\/nprot.2008.154","article-title":"Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori","volume":"3","author":"Michielse","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"532","DOI":"10.1111\/mmi.12294","article-title":"Phospho-regulation and nucleocytoplasmic trafficking of CrzA in response to calcium and alkaline-pH stress in Aspergillus nidulans","volume":"89","author":"Espeso","year":"2013","journal-title":"Mol. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1590\/S1516-89132010000300003","article-title":"Isolation and characterization by asymmetric PCR of the ENDO1 gene for glucan endo-1,3-\u03b2-D-glucosidase in Phytophthora cinnamomi associated with the ink disease of Castanea sativa Mill","volume":"53","author":"Meirinho","year":"2010","journal-title":"Braz. Arch. Biol. Technol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"W123","DOI":"10.1093\/nar\/gkt418","article-title":"WebAUGUSTUS\u2014A web service for training AUGUSTUS and predicting genes in eukaryotes","volume":"41","author":"Hoff","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"745","DOI":"10.1094\/Phyto-76-745","article-title":"Disinfection of seed surfaces with sodium hypochlorite","volume":"76","author":"Sauer","year":"1986","journal-title":"Phytopathology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3177","DOI":"10.1105\/tpc.110.078048","article-title":"Tissue-adapted invasion strategies of the rice blast fungus Magnaporthe oryzae","volume":"22","author":"Marcel","year":"2010","journal-title":"Plant Cell"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Jones, K., Kim, D.W., Park, J.S., and Khang, C.H. (2016). Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae. BMC Plant. Biol., 16.","DOI":"10.1186\/s12870-016-0756-x"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Bolton, M.T.B. (2012). Confocal Microscopy in Plant\u2013Pathogen Interactions. Plant Fungal Pathogens. Methods in Molecular Biology (Methods and Protocols), Humana Press.","DOI":"10.1007\/978-1-61779-501-5"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.ijfoodmicro.2012.10.005","article-title":"Use of GFP-tagged strains of Penicillium digitatum and Penicillium expansum to study host-pathogen interactions in oranges and apples","volume":"160","author":"Usall","year":"2012","journal-title":"Inter. J. Food Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1094\/MPMI.2002.15.2.172","article-title":"Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker","volume":"15","author":"Lagopodi","year":"2002","journal-title":"Mol. Plant Microbe Interact."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/j.mimet.2017.11.019","article-title":"A highly efficient Agrobacterium tumefaciens-mediated transformation system for the postharvest pathogen Penicillium digitatum using DsRed and GFP to visualize citrus host colonization","volume":"144","author":"Vu","year":"2018","journal-title":"J. Microbiol. Methods."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1616","DOI":"10.1094\/PDIS-92-12-1616","article-title":"AFLP analysis of intraspecific variation between Monilinia laxa isolates from different hosts","volume":"92","author":"Gril","year":"2008","journal-title":"Plant Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1111\/mpp.12387","article-title":"Function of the genetic element \u201cMona\u201d associated with fungicide resistance in Monilinia fructicola","volume":"18","author":"Chen","year":"2017","journal-title":"Mol. Plant Pathol."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Chou, C.M., Yu, F.Y., Yu, P.L., Ho, J.F., Bostock, R.M., Chung, K.R., Huang, J.-W., and Lee, M.H. (2015). Expression of five endopolygalacturonase genes and demonstration that MfPG1 overexpression diminishes virulence in the brown rot pathogen Monilinia fructicola. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0132012"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1007\/s00294-006-0059-0","article-title":"Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola","volume":"49","author":"Lee","year":"2006","journal-title":"Curr. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1094\/MPMI-23-2-0176","article-title":"Overexpression of a redox-regulated cutinase gene, MfCUT1, increases virulence of the brown rot pathogen Monilinia fructicola on Prunus spp","volume":"23","author":"Lee","year":"2010","journal-title":"Mol. Plant Microbe Interact."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1354","DOI":"10.1094\/PHYTO.2003.93.11.1354","article-title":"Development of spontaneous hygromycin B resistance in Monilinia fructicola and Its impact on growth rate, morphology, susceptibility to demethylation Inhibitor fungicides, and sporulation","volume":"93","author":"Dai","year":"2003","journal-title":"Phytopathology"},{"key":"ref_42","first-page":"1","article-title":"YAP1 homologue-mediated redox sensing is crucial for a successful infection by Monilinia fructicola","volume":"1","author":"Yu","year":"2016","journal-title":"Mol. Plant Pathol."},{"key":"ref_43","unstructured":"Marek, S.M., Pan, Z., Ciuffetti, L.M., and Bostock, R.M. (2002, January 27\u201331). Agrobacterium tumefaciens-mediated transformation of Monilinia fructicola with green fluorescent protein. Proceedings of the APS Annual Meeting, Milkwaukee, WI, USA. P2002-0673-AMA."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1007\/s00294-007-0122-5","article-title":"Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae","volume":"51","author":"Li","year":"2007","journal-title":"Curr. Genet."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1111\/j.1365-2958.2007.05918.x","article-title":"Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae","volume":"66","author":"Choi","year":"2007","journal-title":"Mol. Microbiol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1050","DOI":"10.1016\/j.fgb.2007.04.002","article-title":"A systematic analysis of T-DNA insertion events in Magnaporthe oryzae","volume":"44","author":"Meng","year":"2007","journal-title":"Fungal Genet. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1534\/g3.112.002048","article-title":"Incidence of genome structure, DNA asymmetry, and cell physiology on T-DNA integration in chromosomes of the phytopathogenic fungus Leptosphaeria maculans","volume":"2","author":"Bourras","year":"2012","journal-title":"G3 Genes Genomes Genet."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Villarino, M., Etxebeste, O., Mendizabal, G., Garzia, A., Ugalde, U., and Espeso, E.A. (2017). Boron tolerance in Aspergillus nidulans is sustained by the SltA pathway through the SLC-family transporters SbtA and SbtB. Genes, 8.","DOI":"10.3390\/genes8070188"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/10\/12\/1033\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:41:23Z","timestamp":1760190083000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/10\/12\/1033"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,12,11]]},"references-count":48,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2019,12]]}},"alternative-id":["genes10121033"],"URL":"https:\/\/doi.org\/10.3390\/genes10121033","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,12,11]]}}}