The fate of a biocontrol agent released on pine phyllosphere in a greenhouse-confined trial was followed over 102 days. The microorganism used, a Pseudomonas sp., isolated from Pinus nigra, carries the cry9a toxin gene from Bacillus thuringiensis. In order to detect the GMM, specific primers were used, and a previously defined protocol for DNA isolation from bacteria colonizing pine needles was applied. The method, based on vortexing in a suspension of glass beads followed by microcolumn extractions, allowed sensitive PCR monitoring of the target transgenes. The presence of the released organism was recorded throughout the trial and compared with its entomocidal performance towards larvae of the pine processionary caterpillar Thaumetopoea pityocampa. At the same time the dynamics of the released Pseudomonas within the whole epiphytic bacterial community, was followed by amplifying 16S rDNA pools and comparing ARDRA profiles at seven sampling points. The resulting dendrogram allowed to follow the time-dependent progressive blending of the Pseudomonas profile into those of the resident biota. PCR-dominance of the released bacterium in the community was extended until 21 days from release while its activity against insect larvae lasted for over 3 months. The prokaryotic epiphytic population, irrespective of any particular impact from the released strain, showed no resilience but a general successional trend, which, remarkably, appeared synchronous on all trees tested, including non-inoculated controls. This observation suggests interesting patterns of concerted environmental shifts by phyllospheric microorganisms.

Monitoring a genetically modified Pseudomonas sp. released on pine leaves reveals concerted successional patterns of the bacterial phyllospheric community

ALBERGHINI, SARA;BATTISTI, ANDREA;SQUARTINI, ANDREA
2008

Abstract

The fate of a biocontrol agent released on pine phyllosphere in a greenhouse-confined trial was followed over 102 days. The microorganism used, a Pseudomonas sp., isolated from Pinus nigra, carries the cry9a toxin gene from Bacillus thuringiensis. In order to detect the GMM, specific primers were used, and a previously defined protocol for DNA isolation from bacteria colonizing pine needles was applied. The method, based on vortexing in a suspension of glass beads followed by microcolumn extractions, allowed sensitive PCR monitoring of the target transgenes. The presence of the released organism was recorded throughout the trial and compared with its entomocidal performance towards larvae of the pine processionary caterpillar Thaumetopoea pityocampa. At the same time the dynamics of the released Pseudomonas within the whole epiphytic bacterial community, was followed by amplifying 16S rDNA pools and comparing ARDRA profiles at seven sampling points. The resulting dendrogram allowed to follow the time-dependent progressive blending of the Pseudomonas profile into those of the resident biota. PCR-dominance of the released bacterium in the community was extended until 21 days from release while its activity against insect larvae lasted for over 3 months. The prokaryotic epiphytic population, irrespective of any particular impact from the released strain, showed no resilience but a general successional trend, which, remarkably, appeared synchronous on all trees tested, including non-inoculated controls. This observation suggests interesting patterns of concerted environmental shifts by phyllospheric microorganisms.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/2471216
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