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Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil

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In this study, we show that noncoding sequences from amplified fragment length polymorphisms (AFLPs) can provide robust and sensitive genetic markers suitable for PCR-based discrimination of closely related strains of Bacillus and Paenibacillus, and quantitative PCR (qPCR)-based tracking of the strains in complex natural systems like soil. Quantitative PCR was accurate in the ~1 × 109 to ~1 × 104 colony forming units (CFU)/g soil range. The detection limit was improved to ~1 × 102 CFU/g when amplicons were analyzed by gel electrophoresis. Studies with laboratory-contained intact soil-core microcosms indicated that environmental persistence trends vary among different strains. For example, Bacillus circulans ATCC 9500, Bacillus amyloliquefaciens DSL 13563-0, Bacillus licheniformis ATCC 12713, Paenibacillus polymyxa NRRL B-4317, and 3 Bacillus subtilisstrains (ATCC 6051A, ATCC 55405, and NRRL B-941) died down to below the 1 × 102 CFU/g detection limit by days 28-105. In contrast, over a 105-day period, B. licheniformis ATCC 55406, Bacillus megaterium NRRL B-14308, and P. polymyxa strains ATCC 55407 and DSL 13540-4 died down but persisted at levels just above the detection limit, whereas Bacillus thuringiensis ATCC 13367 experienced a less than 10-fold decrease in cell numbers.

Dans cette étude, nous démontrons que les séquences non codantes obtenues par AFLPs (amplified fragment length polymorphisms) peuvent constituer des marqueurs forts et sensibles, appropriés à la discrimination par PCR de souches fortement apparentées de Bacillus et Paenibacillus, ainsi qu’au dépistage par qPCR de souches présentes dans des systèmes naturels complexes comme le sol. La quantification par qPCR était exacte à l’intérieur d’un intervalle de ~1 × 109 à ~1 × 104 UFC/g de sol. La limite de détection a été améliorée jusqu’à ~1 × 102 UFC/g lorsque les amplicons étaient analysés par électrophorèse sur gel. Des études réalisées en microcosmes expérimentaux contrôlés constitués de sols intacts ont indiqué que la tendance à la persistance environnementale variait en fonction des souches. Par exemple, les populations de Bacillus circulans ATCC 9500, Bacillus amyloliquefaciens DSL 13563-0, Bacillus licheniformis ATCC 12713, Paenibacillus polymyxa NRRL B-4317, et 3 souches de Bacillus subtilis (ATCC 6051A, ATCC 55405 et NRRL B-941) décroissaient pour passer sous la limite de détection de 1 × 102 UFC/g entre le jour 28 et le jour 105. Par contre, pendant une période de 105 jours, les populations de B. licheniformis ATCC 55406, Bacillus megaterium NRRL B-14308 et les souches ATCC 55407 et DSL 13540-4 de P. polymyxa décroissaient mais persistaient à des niveaux tout juste au-dessus de la limite de détection, alors que la population de Bacillus thuringiensis ATCC 13367 diminuait de moins de 10 fois en terme de nombre de cellules.

Document Type: Research Article

Publication date: October 1, 2009

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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