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  • Blackwell Publishing Ltd  (2)
  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 48 (2004), S. 0 
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Population levels of total and ampicillin-resistant culturable bacteria and the putative horizontal bla gene acquirement from Bt-corn were studied in commercial fields of transgenic corn in Spain during the years 2000–2003. Commercial fields consisting of conventional corn (Dracma) and Bt176 transgenic corn (Compa CB) were located in three climatic regions. The effect of corn type, plant material, field location, stage of sampling and year of study were studied on total and ampicillin resistant bacterial population levels, on median effective dose and on the slope of the dose–response curve to ampicillin. None of the parameters measured were significantly different (P〈0.01) between transgenic and non-transgenic cornfields under the diverse conditions studied. However, in population levels of ampicillin resistant bacteria, the minimum difference between sample means to be significant with a likelihood of 80% was 8.9%. Specific detection of putative bacteria harbouring bla TEM-1 ampicillin resistance genes acquired from Bt176 corn was performed with a method based on the extraction of DNA from the culturable bacterial fraction and with PCR. Primers for PCR were targeted to the bla gene and the corresponding flanking regions present in the pUC18 cloning vector or the Bt176 construct. The culturable bacterial fraction of 144 field samples (up to 864 analysis, including ampicillin enrichments) was analysed by PCR. The estimated total number of bacteria analysed was 108. The level of detection of a transfer event according to the sensitivity of the methods used was 10−6. Four samples of transgenic and five of non-transgenic corn gave positive signals. However, the amplification products did not correspond to the ones expected from Bt176 or pUC18. The limitations of the sampling design and of the methods used are discussed.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU (g f.w.)−1, which in turn decreased progressively to 4–5 log CFU (g f.w.)−1 after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU (g f.w.)−1, thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.
    Type of Medium: Electronic Resource
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