Development of a strain-specific genomic marker for monitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato

Abstract

A strain-specific molecular marker enabling the detection and tracking of the biological control agent Bacillus subtilis 101, when released into the environment, was developed. Random amplified polymorphic DNA (RAPD) technique was used to differentiate this from other B. subtilis strains. A differentially amplified fragment obtained from RAPD profiles was sequenced and characterized as sequence-characterized amplified region (SCAR) marker, and four primer pairs were designed and evaluated for their specificity towards this strain. The sensibility of the selected SCAR primer pair was evaluated by qualitative PCR and Southern blotting, and the detection limit was assessed around 10(2) CFU (g dry wt soil)(-1), thus providing a reliable tool for the traceability of this B. subtilis strain in greenhouse or field trials. A plating assay coupled to PCR with the SCAR primer pair was then used as a detection method in microcosm experiments for monitoring the population of B. subtilis 101 in the rhizosphere of tomato, grown under two different soil conditions, i.e. nonsterile peat-based substrate and sandy-loam agricultural soil, respectively. The data of rhizosphere colonization indicated that the soil conditions significantly affected the rhizosphere establishment of strain 101.

Specific detection ofLysobacter enzymogenes(Christensen and Cook 1978) strain 3.1T8 with TaqMan®PCR

“…2009), Pseudomonas fluorescens EPS62e (Pujol et al. 2005) and Bacillus subtilus 101 (Felici et al. 2008).…”

“…Strain-specific Q-PCR methods have recently been developed for other bacteria using random amplified polymorphic DNA (RAPD), i.e. Lactobacillus rhamnosus GG (Ahlroos and Tynkkynen 2009), Pseudomonas brassicacearum MA250 (Holmberg et al 2009), Pseudomonas fluorescens EPS62e (Pujol et al 2005) and Bacillus subtilus 101 (Felici et al 2008). Strain-specific markers for bacterial as well as fungal strains have mostly been developed based on discriminatory fragments from RAPD profiles (Pujol et al 2005;Rubio et al 2005).…”

Persistence ofPseudomonas fluorescensLBUM677 in the rhizosphere of corn gromwell (Buglossoides arvensis) under field conditions and its impact on seed oil and stearidonic acid bioaccumulation

“…; Felici et al . ). Extrapolations of these results to field conditions have been hampered by biological, chemical and physical complexities found under field conditions (De Weger et al .…”

Development of a Sequence-Characterized Amplified Region Marker-Targeted Quantitative PCR Assay for Strain-Specific Detection ofOenococcus oeniduring Wine Malolactic Fermentation

“…Due to increased specificity, SCAR amplifications are less sensitive to changes in reaction conditions and are more reproducible than traditional typing methods. Strain-specific SCAR markers have been widely and successfully used to monitor bacteria (14,39,61) and yeasts (9,30) in environmental samples. Recently, PCR-based methods with strain-specific primer sets have been set up for quantification of Lactobacillus probiotics in feces (12,15,29).…”