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

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Abstract

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.

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References

  1. Food and Agriculture Organization of the United Nations. FAOSTAT. http://faostat3.fao.org/browse/Q/QC/E. Accessed 14 Oct 2018

  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 407–428

    Chapter  Google Scholar 

  3. Schulz B, Haas S, Junker C, Andrée N, Schobert M (2015) Fungal endophytes are involved in multiple balanced antagonisms. Curr Sci 109:39–45

    Google Scholar 

  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. https://doi.org/10.4238/2012.December.6.2

    Article  CAS  PubMed  Google 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

    Article  Google Scholar 

  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. https://doi.org/10.1007/s13213-015-1162-6

    Article  CAS  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

    Article  CAS  PubMed  PubMed Central  Google 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

    Article  PubMed  Google Scholar 

  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. https://doi.org/10.1017/S0953756203008086

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1007/s00284-017-1421-9

    Article  CAS  Google 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

    Article  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

    Article  CAS  PubMed  Google 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

    Article  CAS  PubMed  Google Scholar 

  14. Bernardi-Wenzel J, Quecine MC, Azevedo JL, Pamphile JA (2016) Agrobacterium-mediated transformation of Fusarium proliferatum. Genet Mol Res. https://doi.org/10.4238/gmr.15027944

    Article  PubMed  Google Scholar 

  15. Knight CJ, Bailey AM, Foster GD (2010) Investigating Agrobacterium-mediated transformation of Verticillium albo-atrum on plant surfaces. PLoS ONE. https://doi.org/10.1371/journal.pone.0013684

    Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1590/S1517-838246220131042

    Article  Google Scholar 

  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. https://doi.org/10.4238/2013.April.2.10

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1111/jam.13769

    Article  CAS  PubMed  Google Scholar 

  19. Anco DJ, Kim S, Mitchell TK, Madden LV, Ellis MA (2009) Transformation of Phomopsis viticola with the green fluorescent protein. Mycologia 101:8538–8558

    Article  Google 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

    Article  CAS  PubMed  Google 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

    Article  PubMed  Google 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

    Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1094/PHYTO-09-15-0236-R

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1111/ppa.12302

    Article  CAS  Google Scholar 

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Biotechnology, Genetics and Cell Biology, State University of Maringá, Maringá, São Paulo, 87020-900, Brazil

    Aretusa Cristina Felber, Julio Cesar Polonio, Ravely Casarotti Orlandelli, Alessandra Tenório Costa, Eliane Papa Ambrosio-Albuquerque & João Alencar Pamphile

  2. Department of Genetics, College of Agriculture Luiz de Queiroz, University of São Paulo, Piracicaba, São Paulo, 13418-900, Brazil

    Renata Assis Castro, Maria Carolina Quecine-Verdi & João Lúcio de Azevedo

  3. Department of Biology, State University of Maringá, Avenida Colombo, 5.790, Jd. Universitário, Maringá, Paraná, 87020-900, Brazil

    Káthia Socorro Mathias Mourão

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  1. Aretusa Cristina Felber
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  2. Julio Cesar Polonio
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  3. Ravely Casarotti Orlandelli
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  4. Alessandra Tenório Costa
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Correspondence to João Alencar Pamphile.

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Felber, A.C., Polonio, J.C., Orlandelli, R.C. et al. Agrobacterium-Mediated Transformation of Diaporthe schini Endophytes Associated with Vitis labrusca L. and Its Antagonistic Activity Against Grapevine Phytopathogens. Indian J Microbiol 59, 217–224 (2019). https://doi.org/10.1007/s12088-019-00787-0

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