Four Methylobacterium extorquens strains were isolated from strawberry (Fragaria x ananassa cv. Elsanta) leaves, and one strain, called ME4, was tested for its ability to promote the growth of various plant seedlings. Seedling weight and shoot length of Nicotiana tabacum, Lycopersicon esculentum, Sinapis alba, and Fragaria vesca increased significantly in the presence of the pink-pigmented facultative methylotroph (PPFM), but the germination behaviour of seeds from six other plants was not affected. The cell-free supernatant of the bacterial culture stimulated germination, suggesting the production of a growth-promoting agent by the methylotroph. Methanol emitted from N. tabacum seedlings, as determined by proton-transfer-reaction mass spectrometry (PTR-MS), ranged from 0.4 to 0.7 ppbv (parts per billion by volume), while significantly lower levels (0.005 to 0.01 ppbv) of the volatile alcohol were measured when the seedlings were co-cultivated with M. extorquens ME4, demonstrating the consumption of the gaseous methanol by the bacteria. Additionally, by using cells of the methylotrophic yeast Pichia pastoris transformed with the pPICHS/GFP vector harbouring a methanol-sensitive promoter in combination with the green fluorescence protein (GFP) reporter gene, stomata were identified as the main source of the methanol emission on tobacco cotyledons. Methylobacterium extorquens strains can nourish themselves using the methanol released by the stomata and release an agent promoting the growth of the seedlings of some crop plants.

Epiphytic bacteria were isolated from strawberry plants cultivated in the field or in the greenhouse in order to investigate their interaction with leaf-surface transport properties. Colonization of lower leaf sides was higher on field-grown plants, whereas upper leaf sides were more densely colonized on plants cultivated in the greenhouse. Fungal isolates significantly contributed to total microbial biomass on leaf surfaces of greenhouse-grown strawberry plants, whereas these organisms were rarely abundant on field-grown plants. Microscopic investigations of bacteria in the phyllosphere revealed that the highest densities of bacteria were observed on living trichomes, which obviously provide a source of nutrients. Isolated strains were characterized by colony morphology, microscopy and histochemistry. About 324 isolated bacterial strains were grouped into 38 morphotypes. Of the morphotypes, 12 were identified by 16S rRNA gene sequencing. Dominating bacteria belonged to the genus Pseudomonas, Stenotrophomonas, Bacillus and Arthrobacter. Cuticular water permeability of isolated cuticular membranes and intact leaf disks was measured before and after treatment with one of the most prominent epiphytic bacteria, Pseudomonas rhizosphaerae. Results showed that cuticular transpiration was significantly increased by P. rhizosphaerae. This shows that leaf-surface properties, such as cuticular water permeability, can be influenced by bacteria, leading to improved habitable conditions in the phyllosphere.

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Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.

The biotransformation of a series of aliphatic aldehydes (C(8)-C(12)) by Bacillus megaterium isolated from strawberry leaf surfaces was investigated. Products were isolated by liquid/liquid extraction and analyzed by gas chromatography (GC) combined with mass spectrometry (MS). In addition to aliphatic alcohols and the remaining aldehydes, major transformation products included the corresponding acids as well as 2,3-dialkylacroleins, dehydrated aldol addition products, which were detected for the first time as biotransformation products. To verify the structures, 2,3-dialkylacroleins were chemically synthesized from the appropriate aldehydes by base-catalyzed aldol condensation reactions and characterized by (1)H and (13)C NMR spectroscopy. Time-course studies showed that the maximum yield of the acrolein derivatives was obtained after 6 days of incubation.