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

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 conditions. This assay can be used to evaluate the occurrence of the bacterium in different sample types.

Current Methods, Common Practices, and Perspectives in Tracking and Monitoring Bioinoculants in Soil

“…Several examples in the literature reported that using Real-Time PCR coupled with trait-based PCR permit the detection of the introduced species, simultaneously evaluating their relative abundance within the microbial community ( Clesceri et al, 1999 ; Babić et al, 2008 ; Couillerot et al, 2010 ; Paulo et al, 2014 ; Canfora et al, 2016 , 2017 ; Mandakovic et al, 2018 ; Tartanus et al, 2021 ). However, the suitability of a polyphasic approach based on DNA-based methods emerged clearly from a 3-year long study on the persistence of two entomopathogenic fungi ( Beauveria brongniartii and B. bassiana ) on two sites ( Canfora et al, 2016 ; Tartanus et al, 2021 ).…”

Effectiveness of Plant Beneficial Microbes: Overview of the Methodological Approaches for the Assessment of Root Colonization and Persistence

“…Among these techniques, qPCR is a sensitive and suitable approach for determining the abundance of functional genes from soil-derived DNA and RNA (Fiorentino et al, 2016), and it has therefore been extensively used to track and quantify inoculated strains in soil systems (Providenti et al, 2009;Timmusk et al, 2009). For instance, Sorte et al (2014) used this method to design specific PCR primers targeting a 16S rRNA variable region to specifically measure the abundance of Gluconacetobacter diazotrophicus following coinoculation with other diazotrophic strains in sugarcane plants grown under field conditions ( Table 3). The validation of employed species-specific primers allow the use of this method to evaluate the occurrence of endophytic diazotrophic G. diazotrophicus species in any soil type and plant tissue.…”

Long-term detection of Hartmannibacter diazotrophicus on winter wheat and spring barley roots under field conditions revealed positive correlations on yield parameters with the bacterium abundance

“…qPCR has been used to monitor the populations of various types of legume-nodulating rhizobia in the field (Maluk et al 2022 , 2023 ). Only a few long-term studies under field conditions have been performed to monitor the dynamics of PGPR in the rhizosphere soil (Zhang et al 2020 ), roots (Soares et al 2021 , Urrea-Valencia et al 2021 ), or stalks (Fernandes et al 2014 ) of different plants, including maize, Brachiaria grasses, soybean, and sugarcane.…”