{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T11:46:18Z","timestamp":1787312778703,"version":"build-2736575974"},"reference-count":53,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,5,19]],"date-time":"2021-05-19T00:00:00Z","timestamp":1621382400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Botrytis cinerea can attack over 500 genera of vascular plants and is considered the second phytopathogen in the \u2018top ten\u2019 for its economic importance. Traditional fungicides can be ineffective and with increasing fungicide resistance, new sustainable technologies are required. Lately, RNA interference-based fungicides are emerging for their potential uses in crop protection. Therefore, we assessed the potential of this innovative approach targeting the MAP kinase Bmp3 in B. cinerea, a gene involved in saprophytic growth, response to low osmolarity, conidiation, surface sensing, host penetration and lesion formation. After performing a prediction analysis of small interfering RNAs, a 427 nucleotides long dsRNA was selected as construct. We tested the effect of topical applications of dsRNA construct both in vitro by a fungal growth assay in microtiter plates and in vivo on detached lettuce leaves artificially inoculated. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination, an evident growth retardation and a strong reduction of necrotic lesions on leaves. These results correlated with a strongly reduced expression of Bmp3 gene. In accordance to these findings, the Bmp3 gene could be a promising target for the development of an RNAi-based fungicide against B. cinerea.<\/jats:p>","DOI":"10.3390\/ijms22105362","type":"journal-article","created":{"date-parts":[[2021,5,19]],"date-time":"2021-05-19T21:49:21Z","timestamp":1621460961000},"page":"5362","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":42,"title":["Silencing of the Slt2-Type MAP Kinase Bmp3 in Botrytis cinerea by Application of Exogenous dsRNA Affects Fungal Growth and Virulence on Lactuca sativa"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0151-2206","authenticated-orcid":false,"given":"Maria","family":"Spada","sequence":"first","affiliation":[{"name":"Department of Agriculture Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5846-7947","authenticated-orcid":false,"given":"Claudio","family":"Pugliesi","sequence":"additional","affiliation":[{"name":"Department of Agriculture Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Fambrini","sequence":"additional","affiliation":[{"name":"Department of Agriculture Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9017-4738","authenticated-orcid":false,"given":"Susanna","family":"Pecchia","sequence":"additional","affiliation":[{"name":"Department of Agriculture Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy"},{"name":"Interdepartmental Research Center Nutrafood \u201cNutraceuticals and Food for Health\u201d, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,5,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Fillinger, S., and Elad, Y. (2016). Plant Hosts of Botrytis Spp. Botrytis\u2014The Fungus, the Pathogen and Its Management in Agricultural Systems, Springer International Publishing.","DOI":"10.1007\/978-3-319-23371-0"},{"key":"ref_2","unstructured":"Watkinson, S.C., Money, N., and Boddy, L. (2016). Pathogens of autotrophs. The Fungi, Academic Press. [3rd ed.]."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"414","DOI":"10.1111\/j.1364-3703.2011.00783.x","article-title":"The top 10 fungal pathogens in molecular plant pathology","volume":"13","author":"Dean","year":"2012","journal-title":"Mol. Plant Pathol."},{"key":"ref_4","unstructured":"UIPP (2002). Annual Report, Union des Industries de la Protection des Plantes."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Fillinger, S., and Elad, Y. (2016). Chemical control and resistance management of Botrytis diseases. Botrytis\u2014The Fungus, the Pathogen and its Management in Agricultural Systems, Springer International Publishing.","DOI":"10.1007\/978-3-319-23371-0"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1016\/j.tim.2016.11.011","article-title":"Spray-induced gene silencing: A powerful innovative strategy for crop protection","volume":"25","author":"Wang","year":"2017","journal-title":"Trends Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Koch, A., Biedenkopf, D., Furch, A., Weber, L., Rossbach, O., Abdellatef, E., Linicus, L., Johannsmeier, J., Jelonek, L., and Goesmann, A. (2016). An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005901"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2543","DOI":"10.1111\/mpp.12728","article-title":"Secondary amplification of siRNA machinery limits the application of spray-induced gene silencing","volume":"19","author":"Song","year":"2018","journal-title":"Mol. Plant Pathol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"16151","DOI":"10.1038\/nplants.2016.151","article-title":"Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection","volume":"2","author":"Wang","year":"2016","journal-title":"Nat. Plants"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"7320","DOI":"10.1038\/s41598-018-25434-4","article-title":"Identification and application of exogenous dsRNA confers plant protection against Sclerotinia sclerotiorum and Botrytis cinerea","volume":"8","author":"McLoughlin","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Nerva, L., Sandrini, M., Gambino, G., and Chitarra, W. (2020). Double-stranded RNAs (dsRNAs) as a sustainable tool against gray mold (Botrytis cinerea) in grapevine: Effectiveness of different application methods in an open-air environment. Biomolecules, 10.","DOI":"10.3390\/biom10020200"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Kronstad, J.W. (2000). Infection strategies of Botrytis cinerea and related necrotrophic pathogens. Fungal Pathology, Kluwer Academic Publishers.","DOI":"10.1007\/978-94-015-9546-9"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1574-6968.2007.00930.x","article-title":"Botrytis cinerea virulence factors: New insights into a necrotrophic and polyphageous pathogen","volume":"277","author":"Choquer","year":"2007","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Islam, T.M., and Sherif, S. (2020). RNAi-based biofungicides as a promising next-generation strategy for controlling devastating gray mold diseases. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21062072"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1007\/978-3-642-22916-9_11","article-title":"Morphogenesis and infection in Botrytis cinerea","volume":"Volume 22","year":"2012","journal-title":"Morphogenesis and Pathogenicity in Fungi. Topics in Current Genetics"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Jiang, C., Zhang, X., Liu, H., and Xu, J.R. (2018). Mitogen-activated protein kinase signaling in plant pathogenic fungi. PLoS Pathog., 14.","DOI":"10.1371\/journal.ppat.1006875"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1111\/j.1364-3703.2007.00383.x","article-title":"The Slt2-type MAP kinase Bmp3 of Botrytis cinerea is required for normal saprotrophic growth, conidiation, plant surface sensing and host tissue colonization","volume":"8","author":"Rui","year":"2007","journal-title":"Mol. Plant Pathol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1111\/j.1364-3703.2007.00417.x","article-title":"Botrytis cinerea: The cause of grey mould disease","volume":"8","author":"Williamson","year":"2007","journal-title":"Mol. Plant Pathol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1146\/annurev-phyto-102313-050143","article-title":"Protein kinases in plant-pathogenic fungi: Conserved regulators of infection","volume":"52","author":"Turra","year":"2014","journal-title":"Annu. Rev. Phytopathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1327","DOI":"10.1105\/tpc.112.096156","article-title":"Mitogen-activated protein kinase signaling in plant-interacting fungi: Distinct messages from conserved messengers","volume":"24","author":"Hamel","year":"2012","journal-title":"Plant Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"51","DOI":"10.3389\/fpls.2020.00051","article-title":"A Perspective on RNAi-based biopesticides","volume":"11","author":"Fletcher","year":"2020","journal-title":"Front. Plant Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1111\/j.1574-6968.1990.tb04174.x","article-title":"An automated quantitative assay for fungal growth inhibition","volume":"69","author":"Broekaert","year":"1990","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1007\/s10658-018-1518-4","article-title":"RNA-based disease control as a complementary measure to fight Fusarium fungi through silencing of the azole target Cytochrome P450 Lanosterol C-14 \u03b1-Demethylase","volume":"152","author":"Koch","year":"2018","journal-title":"Eur. J. Plant Pathol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1155","DOI":"10.1002\/ps.3480","article-title":"In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f. sp. cubense and Mycosphaerella fjiensis","volume":"69","author":"Mumbanza","year":"2013","journal-title":"Pest Manag. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/j.fgb.2010.12.004","article-title":"The osmosensing signal transduction pathway from Botrytis cinerea regulates cell wall integrity and MAP kinase pathways control melanin biosynthesis with influence of light","volume":"48","author":"Liu","year":"2011","journal-title":"Fungal Genet. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Yin, Y., Wu, S., Chui, C., Ma, T., Jiang, H., Hahn, M., and Ma, Z. (2018). The MAPK kinase BcMkk1 suppresses oxalic acid biosynthesis via impeding phosphorylation of BcRim15 by BcSch9 in Botrytis cinerea. PLoS Pathog., 14.","DOI":"10.1371\/journal.ppat.1007285"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1111\/j.1364-3703.2010.00701.x","article-title":"The SLT2 mitogen-activated protein kinase-mediated signalling pathway governs conidiation, morphogenesis, fungal virulence and production of toxin and melanin in the tangerine pathotype of Alternaria alternata","volume":"12","author":"Yago","year":"2011","journal-title":"Mol. Plant Pathol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.fgb.2014.01.003","article-title":"PsMPK1, an SLT2-type mitogen-activated protein kinase, is required for hyphal growth, zoosporogenesis, cell wall integrity, and pathogenicity in Phytophthora sojae","volume":"65","author":"Li","year":"2014","journal-title":"Fungal Genet. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.1590\/S1517-83822013000400031","article-title":"Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioides","volume":"44","author":"Yong","year":"2013","journal-title":"Braz. J. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1119","DOI":"10.1094\/MPMI.2002.15.11.1119","article-title":"A Mitogen-Activated Protein Kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection","volume":"15","author":"Hou","year":"2002","journal-title":"Mol. Plant Microbe Interact."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"291","DOI":"10.3389\/fpls.2018.00291","article-title":"Silencing of the Mitogen-Activated Protein Kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis reduces growth and virulence on host plants","volume":"9","author":"Onyilo","year":"2018","journal-title":"Front. Plant Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1139\/cjm-2016-0091","article-title":"The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion","volume":"850","author":"Bashi","year":"2016","journal-title":"Can. J. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Giesbert, S., Siegmund, U., Schumacher, J., Kokkelink, L., and Tudzynski, P. (2014). Functional analysis of BcBem1 and its interaction partners in Botrytis cinerea: Impact on differentiation and virulence. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0095172"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"90","DOI":"10.5423\/PPJ.NT.07.2013.0064","article-title":"Evaluation of lettuce germplasm resistance to gray mold disease for organic cultivations","volume":"30","author":"Shim","year":"2014","journal-title":"Plant Pathol. J."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Caseys, C., Shi, G., Soltis, N., Gwinner, R., Corwin, J., Atwell, S., and Kliebenstein, D. (2020). Quantitative interactions drive Botrytis cinerea disease outcome across the plant kingdom. bioRxiv, 507491.","DOI":"10.1093\/g3journal\/jkab175"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1006\/fgbi.2000.1237","article-title":"MAP kinases in fungal pathogens","volume":"31","author":"Xu","year":"2000","journal-title":"Fungal Genet. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1094\/MPMI-19-0389","article-title":"MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth","volume":"19","author":"Mehrabi","year":"2006","journal-title":"Mol. Plant Microbe Interact."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"544","DOI":"10.1016\/j.fgb.2012.05.002","article-title":"The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana","volume":"49","author":"Luo","year":"2012","journal-title":"Fungal Genet. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1212","DOI":"10.3389\/fmicb.2016.01212","article-title":"Colletotrichum higginsianum mitogen-activated protein kinase ChMK1: Role in growth, cell wall integrity, colony melanization, and pathogenicity","volume":"7","author":"Wei","year":"2016","journal-title":"Front. Microbiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1239","DOI":"10.1002\/ps.4813","article-title":"RNAi technology in crop protection against arthropod pests, pathogens and nematodes","volume":"74","author":"Zotti","year":"2018","journal-title":"Pest Manag. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1002\/ps.5686","article-title":"RNA-based biocontrol compounds: Current status and perspectives to reach the market","volume":"76","author":"Taning","year":"2020","journal-title":"Pest Manag. Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1007\/BF00351784","article-title":"Variations in ploidy among isolates of Botrytis cinerea: Implications for genetic and molecular analyses","volume":"25","author":"Koch","year":"1994","journal-title":"Curr. Genet."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1006\/pmpp.1998.0142","article-title":"Detoxification of \u03b1-tomatine by Botrytis cinerea","volume":"52","author":"Quidde","year":"1998","journal-title":"Physiol. Mol. Plant Pathol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"e00647-15","DOI":"10.1128\/genomeA.00647-15","article-title":"Draft whole-genome sequence of the biocontrol agent Trichoderma harzianum T6776","volume":"3","author":"Baroncelli","year":"2015","journal-title":"Genome Announc."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1128\/EC.00295-12","article-title":"Transcriptome profiling of Botrytis cinerea conidial germination reveals upregulation of infection-related genes during the prepenetration stage","volume":"12","author":"Leroch","year":"2013","journal-title":"Eukaryot. Cell"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Pride, L., Vallard, G., and Agehara, S. (2021, February 22). How to Measure Leaf Disease Damage Using Image Analysis in ImageJ. Available online: https:\/\/edis.ifas.ufl.edu\/hs1382.","DOI":"10.32473\/edis-hs1382-2020"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1987","DOI":"10.1128\/AEM.67.5.1987-1994.2001","article-title":"Green fluorescent protein is lighting up fungal biology","volume":"67","author":"Lorang","year":"2001","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3389","DOI":"10.1093\/nar\/25.17.3389","article-title":"Gapped BLAST and PSI-BLAST: A new generation of protein database search programs","volume":"25","author":"Altschul","year":"1997","journal-title":"Nucleic Acids Res."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1093\/nar\/30.1.276","article-title":"The pfam protein families database","volume":"30","author":"Bateman","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/30.1.235","article-title":"The PROSITE database, its status in 2002","volume":"30","author":"Falquet","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Amselem, J., Cuomo, C.A., van Kan, J.A., Viaud, M., Benito, E.P., Couloux, A., Coutinho, P.M., de Vries, R.P., Dyer, P.S., and Fillinger, S. (2011). Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet., 7.","DOI":"10.1371\/journal.pgen.1002230"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1111\/mpp.12384","article-title":"A gapless genome sequence of the fungus Botrytis cinerea","volume":"18","author":"Stassen","year":"2017","journal-title":"Mol. Plant Pathol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using Real-Time quantitative PCR and the 2\u2212\u0394\u0394CT method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/10\/5362\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:04:21Z","timestamp":1760162661000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/10\/5362"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,5,19]]},"references-count":53,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["ijms22105362"],"URL":"https:\/\/doi.org\/10.3390\/ijms22105362","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,19]]}}}