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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
  • 14 Downloads

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.

Keywords

Green fluorescent protein Red fluorescent protein Molecular multigene identification Phylogeny Biological control 

Notes

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.

Supplementary material

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

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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

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