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

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.

Emerging Perspectives on the Natural Microbiome of Fresh Produce Vegetables

“…Similarly, the diversity and structure of the bacterial community in the phyllosphere of a number of tree species has been described [19][20][21][22]. More than basic descriptions of community structure, 16S rRNA techniques have also been used to examine how the phyllosphere community is influenced by environmental factors, such as ultraviolet light [15] and rainfall [23], as well as pesticide [24,25] or biological control agent [16,26] application. At an ecological level, both spatial [20] and temporal [21] patterns in the composition of the phyllosphere have been examined.…”

Effects of conventional and transgenicBacillus thuringiensisgalleriaetoxin onExorista larvarum(Diptera: Tachinidae), a parasitoid of forest defoliating Lepidoptera

“…These results suggested the potential of epiphytic bacteria modified with Bt genes for more efficient and persistent toxin delivery to the target species. We further assessed the biosafety of the transgenic Pseudomonas by first devising a procedure to optimally rescue bacteria from pine needles (Alberghini, Battisti, and Squartini 2008a) which was then tested to assess the GM strain persistence and its impact on resident phyllospheric microbiota on Pinus mugo under greenhouse conditions (Alberghini, Battisti, and Squartini 2008b). The results were reassuring in terms of biosafety as they showed that, in spite of an extended plant protection, lasting over 100 days from release, microbial communities continue to follow their natural succession and are not shifted by the effect of the biocontrol inoculant.…”