Rapid detection and identification of the free-living nitrogen fixing genus Azospirillum by 16S rRNA-gene-targeted genus-specific primers

Abstract

The modern agricultural practice utilizing plant growth promoting rhizobacteria (PGPR) has brought great benefits in the promotion of crop growth. Among PGPR, Azospirillum is considered as an important genus which is not only closely-associated with plants but also shows potential in the degradation of organic contaminants. However, lack of media for selective isolation or techniques for specific detection or identification limit the exploration of these rhizobacteria. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Azospirillum by means of PCR-specific amplification. The sensitivity and specificity of the newly designed primer pair Azo494-F/Azo756-R were tested against 12 Azospirillum type strains and other closely-related genera. The Azospirillum-specific 16S rRNA gene fragment (263 bp) was successfully amplified for all the reference Azospirillum species with the designed primer pair. No amplification was noted for closely-related species from other genera. The genus specificity was validated with 18 strains including environmental isolates. Interestingly, two strains assigned earlier as Azospirillum amazonense (DSM 2787(T)) and Azospirillum irakense (DSM 11586(T)) failed to produce an Azospirillum-specific fragment with this primer pair. Further phylogenetic analysis of these two isolates based on 16S rRNA gene sequences shows that these two strains might belong to other genera rather than Azospirillum. Preliminary screening of isolates and soil samples with the Azospirillum-specific primers was successful in terms of the rapid detection of Azospirillum isolates. By using real-time PCR analysis the minimum limit of Azospirillum detection was 10(2) CFU g(-1) in the seeded soil sample. The newly designed primers can be used to study the diversity of Azospirillum in ecosystems and aid in the exploration of novel species.

Diversity and functional characterization of bacterial endophytes dwelling in various rice (Oryza sativa L.) tissues, and their seed-borne dissemination into rhizosphere under gnotobiotic P-stress

“…PCR conditions were maintained as follows: 95°C for 4 min followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min and finally 1 cycle at 72°C for 5 min. The purification of PCR amplicons and DGGE run condition were according to Lin et al (2011). After the run, gels were stained with ethidium bromide; DGGE patterns were visualized by UV-illumination and documented using a digital camera.…”

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

“…Although Couillerot et al (2010) could quantify the presence of Azospirillum lipoferum in the rhizosphere of younger maize plants using qPCR with primers specific for this specie, most species-specific primers used to monitor nitrogen-fixing bacteria asso-ciated with Poaceae family plants were designed for use with conventional PCR (Lin et al, 2011;Muthukumarasamy et al, 2005;Sevilla et al, 2001). As such, there are few studies that evaluate the establish-ment of inoculated nitrogen-fixing bacteria in plants.…”

Azospirillum agricola sp. nov., a nitrogen-fixing species isolated from cultivated soil

“…T (Lavrinenko et al, 2010), A. rugosum DSM 19657 T (Young et al, 2008) and Azospirillum zeae LMG 23989 T (Mehnaz et al, 2007b) were analysed under the conditions described by Lin et al (2011Lin et al ( , 2012Lin et al ( , 2013Lin et al ( , 2014Lin et al ( , 2015 and Young et al (2015). For direct comparative analysis, all these strains were grown on NA at 30 8C for 2 days, unless specified otherwise.…”

“…A genus-specific PCR primer pair was used for rapid identification of the genus Azospirillum. The forward primer Azo494-F and reverse primer Azo756-R were used to amplify a genus-specific fragment (Lin et al, 2011). For the analysis of DNA G+C content, DNA samples were prepared and degraded enzymically into nucleosides as described by Mesbah et al (1989).…”