The increasing use of biological control agents (BCAs) against Botrytis cinerea in strawberry raises the question of whether there are any undesirable impacts of foliar applications of BCAs on nontarget microorganisms in the phyllosphere. Therefore, our objective was to investigate this issue within a field study. Strawberry plants were repeatedly sprayed with three BCAs—namely, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum-P (Trichoderma harzianum T22), and Naturalis (Beauveria bassiana ATCC 74040)—to suppress Botrytis cinerea infections. Microbial communities of differentially treated leaves were analyzed using plate counts and pyrosequencing and compared with the microbial community of nontreated leaves. Plate count results indicate that the applied Bacillus and Trichoderma spp. survived in the strawberry phyllosphere throughout the strawberry season. However, no significant impacts on the leaf microbiota could be detected by this culture-dependent technique. Pyrosequencing of internal transcribed spacer ribosomal RNA and 16S RNA sequences revealed a change in fungal composition and diversity at class level after the introduction of T. harzianum T22 to the phyllosphere, whereas the bacterial composition and diversity was not affected by either this Trichoderma preparation or the other two BCAs. Our results suggest that pyrosequencing represents a useful method for studying microbial interactions in the phyllosphere.

Currently, there is little knowledge of the establishment of repeatedly applied biological control agents (BCAs) in the phyllosphere of plants and, in particular, their interactions with the resident microbiome. Under field conditions, the BCA Aureobasidium pullulans was applied as a model organism to organically grown strawberries during two subsequent years (2011, 2012), either as single strain treatment or with the co-application of the entomopathogenic fungus Beauveria bassiana. Fungal and bacterial communities of strawberry leaves were investigated by means of plate counts and 454 pyrosequencing. The establishment of the introduced A. pullulans strains considerably differed between the two years, presumably due to distinct environmental conditions. Short-term and long-term effects of BCA applications on the composition and diversity of fungal communities could be observed as a result of successful establishment of A. pullulans, in 2011, showing, for instance, reduced diversity of fungal communities by competitive displacement shortly after BCA introduction. Due to considerable dynamics in untreated resident microbial communities in the phyllosphere in general, however, we suggest that even the effects caused by the applied BCA preparations in 2011 are negligible under practical conditions.