Agrobacterium-Mediated Transformation of Diaporthe schini Endophytes Associated with Vitis labrusca L. and Its Antagonistic Activity Against Grapevine Phytopathogens
- 14 Downloads
Fungus-caused diseases are among the greatest losses in grapevine culture. Biological control of pathogens by endophytes may be used to decrease fungicide application rates and environmental impacts. Previously, Diaporthe sp. B46-64 and C27-07 were highlighted as antagonists of grapevine phytopathogens. Herein, molecular multigene (ITS-TUB-TEF1) identification and phylogenetic analysis allowed the identification of these endophytes as belonging to Diaporthe schini species. Agrobacterium tumefaciens-mediated transformation was employed for obtaining 14 stable and traceable gfp- or DsRed-expressing transformants, with high transformation efficiency: 96% for the pFAT-GFP plasmid and 98% for pCAM-DsRed plasmid. Transformants were resistant to hygromycin B with gene hph confirmed by polymerase chain reaction and proved to be mitotically stable, expressing the fluorescent phenotype, with morphological differences in the colonies when compared with wild strains. In vitro antagonism tests revealed an increased antagonistic activity of some transformant strains. The current genetic transformation of D. schini mediated by A. tumefaciens proved to be an efficient technique within the randomized insertion of reporter genes for the monitoring of the strain in the environment.
KeywordsGreen fluorescent protein Red fluorescent protein Molecular multigene identification Phylogeny Biological control
The authors thank the SETI/UGF (TC n. 65/18), CNPq (307603/2017-2), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the Doctoral scholarship of A. C. Felber and CAPES/PNPD-UEM to postdoctoral scholarship of A. T. Costa. Authors also thank to EMBRAPA Uva e Vinho for providing the strains of the phytopathogenic fungi; Dr. Carlos Labate and Dr. Maria Carolina Quecine Verdi, for the donation of Agrobacterium tumefaciens strains with plasmids.
- 1.Food and Agriculture Organization of the United Nations. FAOSTAT. http://faostat3.fao.org/browse/Q/QC/E. Accessed 14 Oct 2018
- 3.Schulz B, Haas S, Junker C, Andrée N, Schobert M (2015) Fungal endophytes are involved in multiple balanced antagonisms. Curr Sci 109:39–45Google Scholar
- 5.Burruano S, Alfonzo A, Lo Piccolo S, Conigliaro G, Mondello V, Torta L, Moretti M, Assante G (2008) Interaction between Acremonium byssoides and Plasmopara viticola in Vitis vinifera. Phytopathol Mediterr 47:122–131. https://doi.org/10.14601/Phytopathol_Mediterr-2615 Google Scholar
- 7.Stepanenko OV, Verkhusha VV, Kuznetsova IM, Uversky VN, Turoverov KK (2008) Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes. Curr Protein Pept Sci 9:338–369. https://doi.org/10.2174/138920308785132668 CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Vu TX, Ngo TT, Mai LTD, Bui T, Le DH, Bui HTV, Nguyen HQ, Ngo BX, Tran V (2018) A highly efficient Agrobacterium tumefaciens-mediated transformation system for the postharvest pathogen Penicillium digitatum using DsRed and GFP to visualize citrus host colonization. J Microbiol Methods 144:134–144. https://doi.org/10.1016/j.mimet.2017.11.019 CrossRefPubMedGoogle Scholar
- 11.Pádua APSL, Freire KTLS, Oliveira TGL, Silva LF, Araújo-Magalhães GR, Agamez-Montalvo GS, Silva IR, Bezerra JDP, Souza-Motta CM (2018) Fungal endophyte diversity in the leaves of the medicinal plant Myracrodruon urundeuva in a Brazilian dry tropical forest and their capacity to produce l-asparaginase. Acta Bot Bras. https://doi.org/10.1590/0102-33062018abb0108 Google Scholar
- 12.Eckert M, Maguire K, Urban M, Foster S, Fitt B, Lucas J, Hammond-Kosack K (2005) Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp. FEMS Microbiol Lett 253:67–74. https://doi.org/10.1016/j.femsle.2005.09.041 CrossRefPubMedGoogle Scholar
- 13.Polonio JC, Ribeiro MA, Rhoden SA, Sarragiotto MH, Azevedo JL, Pamphile JA (2016) 3-Nitropropionic acid production by the endophytic Diaporthe citri: molecular taxonomy, chemical characterization, and quantification under pH variation. Fungal Biol 120:1600–1608. https://doi.org/10.1016/j.funbio.2016.08.006 CrossRefPubMedGoogle Scholar
- 20.Sebastianes FLS, Lacava PT, Fávaro LCL, Rodrigues MBC, Araújo WL, Azevedo JL, Pizzirani-Kleiner AA (2012) Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens. Curr Genet 58:21–33. https://doi.org/10.1007/s00294-011-0362-2 CrossRefPubMedGoogle Scholar
- 21.Santos PJC, Savi DC, Gomes RR, Goulin EH, Senkiv CC, Tanaka FAO, Almeida AMR, Galli-Terasawa L, Kava V, Glienke C (2016) Diaporthe endophytica and D. terebinthifolii from medicinal plants for biological control of Phyllosticta citricarpa. Microbiol Res 186–187:153–160. https://doi.org/10.1016/j.micres.2016.04.002 CrossRefPubMedGoogle Scholar
- 22.Pierron R, Gorfer M, Berger H, Jacques A, Sessitsch A, Strauss J, Compant S (2015) Deciphering the niches of colonisation of Vitis vinifera L. by the esca-associated fungus Phaeoacremonium aleophilum using a gfp marked strain and cutting systems. PLoS ONE. https://doi.org/10.1371/journal.pone.0126851 Google Scholar