African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5124

Full Length Research Paper

Development of a real-time PCR assay for the detection and quantification of Gluconacetobacter diazotrophicus in sugarcane grown under field conditions

Paulo Marcos Fernandes Boa Sorte
  • Paulo Marcos Fernandes Boa Sorte
  • 1. Soil Science department - UFRRJ, BR 465, km 7, Seropédica - RJ - CEP: 23891-000, Brazil; 5. Federal Institute of Alagoas - Campus Murici, Rua Astolfo Lopes, s/n, , Murici - AL - Cep: 57.820-000, Brazil.
  • Google Scholar
Jean Luiz Simões-Araújo
  • Jean Luiz Simões-Araújo
  • Embrapa Agrobiologia, BR 465, km 7, Seropédica - RJ - CEP: 23891-000, Brazil.
  • Google Scholar
Leona Henrique Varial de Melo
  • Leona Henrique Varial de Melo
  • 3. Plant Biotechnology department - UFRJ, Avenida Carlos Chagas Filho, 373, Rio de Janeiro - RJ - CEP: 21941-902, Brazil; 5. Federal Institute of Alagoas - Campus Murici, Rua Astolfo Lopes, s/n, , Murici - AL - Cep: 57.820-000, Brazil.
  • Google Scholar
Péricles de Souza Galisa
  • Péricles de Souza Galisa
  • Soil Science department - UFRRJ, BR 465, km 7, Seropédica - RJ - CEP: 23891-000, Brazil
  • Google Scholar
Lineu Leal
  • Lineu Leal
  • Ministry of Agrarian Development, Esplanada dos Ministérios, Bloco A/Ala Norte - Brasília-DF - CEP 70050-902, Brazil
  • Google Scholar
José Ivo Baldani*
  • José Ivo Baldani*
  • Embrapa Agrobiologia, BR 465, km 7, Seropédica - RJ - CEP: 23891-000, Brazil
  • Google Scholar
Vera Lúcia Divan Baldani
  • Vera Lúcia Divan Baldani
  • Embrapa Agrobiologia, BR 465, km 7, Seropédica - RJ - CEP: 23891-000, Brazil
  • Google Scholar


  •  Received: 14 March 2014
  •  Accepted: 27 June 2014
  •  Published: 30 July 2014

Abstract

The objective of this study was to evaluate the viability of real-time polymerase chain reaction (PCR) to detect Gluconacetobacter diazotrophicus in sugarcane inoculated and non-inoculated with diazotrophs which are grown under field conditions. The primer pair PAL5F and PAL5R yielded a specific band of 189 bp using real time PCR with SYBER Green I. This primer pair was the most sensitive one to detect endophytic bacteria in sugarcane plants grown under field conditions and inoculated or not with bacterium. The lower limit of detection was 5 fg of template DNA, which corresponds to 12 bacterial cells. In contrast, a cultivation-dependent approach was not capable of detecting the bacteria in the same sample. The quantification of G. diazotrophicus from field grown plants using real-time PCR and a set of specific primers can be used to determine the number of bacterial cells that colonize endophytically the plant after inoculation. A highly sensitive and specific assay was developed to quantify G. diazotrophicus in sugarcane plants grown under field conditionsThis assay can be used to evaluate the occurrence of the bacterium in different sample types.

Key words: Inoculant, diazotrophs, endophyte, colonization, quantitative polymerase chain reaction (qPCR).