Persistence of Pseudomonas aureofaciens strains and DNA in soil

The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10 2 cells g (fresh weight)؊1 and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10 7 to 10 8 cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.

Section: Discussionmentioning

confidence: 99%

Assessment of the Environmental Fate of the Biological Control Agent of Fire Blight, Pseudomonas fluorescens EPS62e, on Apple by Culture and Real-Time PCR Methods

Pujol

Badosa

Manceau³

et al. 2006

“…Fragments of chromosomal bacterial DNA have been shown to persist in soil for weeks (9,43). However, this physical stability has not been reflected in similar data demonstrating the long-term biological activity (e.g., transforming activity) of chromosomal DNA in soil.…”

Section: Discussionmentioning

confidence: 99%

Natural Transformation of Acinetobacter sp. Strain BD413 with Cell Lysates of Acinetobacter sp., Pseudomonas fluorescens , and Burkholderia cepacia in Soil Microcosms

Nielsen

Smalla²,

Elsas

2000

To elucidate the biological significance of dead bacterial cells in soil to the intra-and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80°C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.

“…Romanowski et al (35) demonstrated the persistence of plasmid DNA in soil for weeks and even for months after its release from the cell. England et al (8) detected a Pseudomonas aureofaciens gene in soil 4 weeks after inoculation of the cell lysates. However, in contrast to these reports, Young et al (48) showed that PCR amplification of a specific gene is directly correlated to the presence of viable cells.…”

Section: Discussionmentioning

confidence: 99%

Culture- and Quantitative IS 900 Real-Time PCR-Based Analysis of the Persistence of Mycobacterium avium subsp. paratuberculosis in a Controlled Dairy Cow Farm Environment

Morávková

Babák

Králová

et al. 2012

ABSTRACTThe aim of this study was to monitor the persistence ofMycobacterium aviumsubsp.paratuberculosisin environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive forM. aviumsubsp.paratuberculosisby cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average meanM. aviumsubsp.paratuberculosiscell number of 3.09 × 103were positive forM. aviumsubsp.paratuberculosisbefore destocking compared to 43% with an average meanM. aviumsubsp.paratuberculosiscell number of 5.86 × 102after 24 months.M. aviumsubsp.paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas.M. aviumsubsp.paratuberculosiswas detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs.M. aviumsubsp.paratuberculosisDNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers ofM. aviumsubsp.paratuberculosiscells were comparable.