Agrobacterium-Mediated Transformation of Diaporthe schini Endophytes Associated with Vitis labrusca L. and Its Antagonistic Activity Against Grapevine Phytopathogens

  • Aretusa Cristina Felber
  • Julio Cesar Polonio
  • Ravely Casarotti Orlandelli
  • Alessandra Tenório Costa
  • Eliane Papa Ambrosio-Albuquerque
  • Renata Assis Castro
  • Maria Carolina Quecine-Verdi
  • João Lúcio de Azevedo
  • João Alencar PamphileEmail author
  • Káthia Socorro Mathias Mourão
Original Research Article


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.


Green 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.

Supplementary material

12088_2019_787_MOESM1_ESM.docx (2.1 mb)
Supplementary material 1 (DOCX 2171 kb)


  1. 1.
    Food and Agriculture Organization of the United Nations. FAOSTAT. Accessed 14 Oct 2018
  2. 2.
    Gomès E, Coutos-Thévenot P (2009) Molecular aspects of grapevine pathogenic fungi interactions. In: Roubelakis-Angelakis KA (ed) Grapevine molecular physiology and biotechnology, 2nd edn. Springer, Dordrecht, pp 407–428CrossRefGoogle Scholar
  3. 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
  4. 4.
    Brum MCP, Araújo WL, Maki CS, Azevedo JL (2012) Endophytic fungi from Vitis labrusca L. (‘Niagara Rosada’) and its potential for the biological control of Fusarium oxysporum. Genet Mol Res 11:4187–4197. CrossRefPubMedGoogle Scholar
  5. 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. Google Scholar
  6. 6.
    Felber AC, Orlandelli RC, Rhoden SA, Garcia A, Costa AT, Azevedo JL, Pamphile JA (2016) Bioprospecting foliar endophytic fungi of Vitis labrusca Linnaeus, Bordô and Concord cv. Ann Microbiol 66:765–775. CrossRefGoogle Scholar
  7. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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. CrossRefPubMedGoogle Scholar
  9. 9.
    Fitzgerald AM, Mudge AM, Gleave AP, Plummer KM (2003) Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis. Mycol Res 107:803–810. CrossRefPubMedGoogle Scholar
  10. 10.
    Ribeiro AS, Polonio JC, Costa AT, Santos CM, Rhoden SA, Azevedo JL, Pamphile JA (2018) Bioprospection of culturable endophytic fungi associated with the ornamental plant Pachystachys lutea. Curr Microbiol 75:588–596. CrossRefGoogle Scholar
  11. 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. Google Scholar
  12. 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. CrossRefPubMedGoogle Scholar
  13. 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. CrossRefPubMedGoogle Scholar
  14. 14.
    Bernardi-Wenzel J, Quecine MC, Azevedo JL, Pamphile JA (2016) Agrobacterium-mediated transformation of Fusarium proliferatum. Genet Mol Res. PubMedGoogle Scholar
  15. 15.
    Knight CJ, Bailey AM, Foster GD (2010) Investigating Agrobacterium-mediated transformation of Verticillium albo-atrum on plant surfaces. PLoS ONE. Google Scholar
  16. 16.
    Orlandelli RC, Almeida TT, Alberto RN, Polonio JC, Azevedo JL, Pamphile JA (2015) Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw. Braz J Microbiol 2015:359–366. CrossRefGoogle Scholar
  17. 17.
    Rhoden SA, Garcia A, Azevedo JL, Pamphile JA (2013) In silico analysis of diverse endophytic fungi by using ITS1-5,8S-ITS2 sequences with isolates from various plant families in Brazil. Genet Mol Res 12:935–950. CrossRefPubMedGoogle Scholar
  18. 18.
    Soares DA, Oliveira DP, Santos TT, Marson PG, Pimenta RS (2018) Multiloci identification of Diaporthe fungi isolated from the medicinal plant Costus spiralis (Jacq.) Roscoe (Costaceae). J Appl Microbiol 125:172–180. CrossRefPubMedGoogle Scholar
  19. 19.
    Anco DJ, Kim S, Mitchell TK, Madden LV, Ellis MA (2009) Transformation of Phomopsis viticola with the green fluorescent protein. Mycologia 101:8538–8558CrossRefGoogle Scholar
  20. 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. CrossRefPubMedGoogle Scholar
  21. 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. CrossRefPubMedGoogle Scholar
  22. 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. Google Scholar
  23. 23.
    Wu L, Conner LR, Wang X, Xu R, Li H (2016) Variation in growth, colonization of maize, and metabolic parameters of gfp- and DsRed-labeled Fusarium verticillioides strains. Phytopathology 106:890–899. CrossRefPubMedGoogle Scholar
  24. 24.
    Bitsadze N, Siebold M, Koopmann B, von Tiedemann A (2015) Single and combined colonization of Sclerotinia sclerotiorum sclerotia by the fungal mycoparasites Coniothyrium minitans and Microsphaeropsis ochracea. Plant Pathol 64:690–710. CrossRefGoogle Scholar

Copyright information

© Association of Microbiologists of India 2019

Authors and Affiliations

  1. 1.Department of Biotechnology, Genetics and Cell BiologyState University of MaringáMaringáBrazil
  2. 2.Department of Genetics, College of Agriculture Luiz de QueirozUniversity of São PauloPiracicabaBrazil
  3. 3.Department of BiologyState University of MaringáMaringáBrazil

Personalised recommendations