Assessment of a qPCR technique for the detection and quantification of Azospirillum brasilense in roots

CABRERA A.; VAZQUEZ A.; ZAWOZNIK M.S.; BENAVIDES M.P.; GROPPA M.D.

Congreso; LV annual SAIB meeting and XIV PABMB Congress, Salta, Argentina; 2019

ASSESSMENT OF A qPCR TECHNIQUE FOR THE DETECTION AND QUANTIFICATION OF AZOSPIRILLUM BRASILENSE IN ROOTSCabrera, A1; Vazquez, A; Zawoznik,MS1; Benavides, MP2; Groppa, MD21Universidad de Buenos Aires, Fac.de Farmacia y Bioquímica, Departamento de Química Biológica, Cátedra de Química Biológica Vegetal, Buenos Aires, Argentina2Universidad de Buenos Aires. Consejo Nacional de lnvestigaciones Cientificas y Tecnicas. Instituto de Química y Fisicoquímica Biológicas (IQUIFIB). Fac.de Farmacia y Bioquímica, Buenos Aires, Argentina.Email:andreacab86@gmail.com Because culture-dependent counting techniques tend to produce erroneous results mainly if the count is based on the morphology of the colony, our objective was to evaluate a molecular technique (qPCR) to perform a specific and more accurate count of Azospirillum brasilense Az39, a plant-growth promoting rizobacterium used in commercial inoculants. For this purpose, the sequence of the NifA transcription factor gene was searched in the Az39 genome by alignments of the same gene from other strains of the same species, and specific primers for Az39 were designed. With these primers, specificity curves were obtained using DNA extracted from Az39 and other bacteria. The expected amplicon had a Tm of 85.15 and a Ct value of 19.06 using 6.8 ng of the template of Az39. Samples from other bacteria showed non-specific amplifications with different Tm and higher Ct values, except for bacteria belonging to the Azospirillum brasilense species, whose Tm and Ct were close to that of Az39. Once the technique was optimized, it was used to quantify A. brasilense present in the roots of wheat plants inoculated with Az39 and in non-inoculated plants, and the results were compared with the data obtained using a plate counting technique.We can conclude that the primers designed were specific for Azospirillum brasilense species but not for Az39 strain. When comparing this method with a culture technique, we verified that the plate counting method underestimated the number of Azospirillum in the exorizosphere and overestimated its number in the endorizosphere.The molecular counting technique was useful to prove the endophytic nature of this rhizobacterium as well as the preferential colonization of the exorizosphere by the inoculated microorganisms.