Flower pollen is a plant microhabitat which was overseen for microbial analyses compared to the intensively studied leaf and root habitats. Pollen is important for plant reproduction and provides nutrients for insects e.g. for honeybees and humans such as honey and propolis. Pollen of wind-pollinated plants is a major airborne allergen all over the world, causing severe allergic rhinitis. In this work the abundance, structure and diversity of the microbiota associated with the pollen of nine different plants, including four wind-pollinated, high allergenic species (birch, winter rye, common hazel and common mugwort), four insect-pollinated, low allergenic species (autumn crocus, winter rapeseed, blackthorn and cherry plum) and one wind-pollinated but low allergic species (hemp) were compared. The microbiota was analysed by high-throughput sequencing approach based on bacterial 16S rRNA gene and fungal ITS2 region. In parallel, culture-dependent methods were used to estimate the extent of the cultivable bacterial fraction, and microscopic methods were used to visualise the colonization of bacteria on pollen grains. Furthermore, bacterial endotoxin levels (lipopolysaccharides and lipoteichoic acids) of pollen were compared with those of the bacterial isolates, by using enzyme-linked immunosorbent assay.Proteobacteria (bacteria) and Ascomycota (fungi) were the most abundant phyla, while Pseudomonas (bacteria) and Cladosporium (fungi) were the most abundant genera found in the pollen microhabitat. Archaea sequences were not detected. Furthermore, the bacterial and fungal alpha diversity indices were significantly lower in the low allergenic pollen and in hemp, compared to the high allergenic pollen. The most significant influencing factors in bacterial and fungal microbiotas were allergenic potential followed by plant species and pollination type (wind- and insect-pollinating) of the pollen. Notably, the hemp clustered closer to the other low allergenic pollen species. A core microbiome consisting of 12 bacterial and 33 fungal genera was found in the pollen of the nine plant species investigated. The most abundant core genera found were Pseudomonas and Rosenbergiella (bacteria), and Cladosporium and Aureobasidium (fungi). Co-occurrence analysis highlighted significant inter- and intra-kingdom interactions, and the interaction network was shaped by four bacterial hub taxa: Methylobacterium (two OTUs), Friedmanniella and Rosenbergiella. Methylobacterium prevailed in wind-pollinated high allergenic pollen and Rosenbergiella in the insect-pollinated low allergenic pollen; the latter was negatively correlated with the other three hubs, indicating habitat preference. For evaluation of the allergic potential of the bacterial isolates and pollen, the bacterial endotoxins level were assessed. In high allergenic pollens endotoxin concentrations were higher than low allergenic ones. Interestingly, the lipopolysaccharide concentrations of Gram-negative bacteria isolated from high allergenic pollen were also significantly higher than those of low allergic pollen isolates. The levels of endotoxins in the pollen and in the corresponding bacterial isolates were highly correlated which supports our hypothesis that pollen microorganisms may play a role in pollen allergy. In total 157 morphologically different bacterial strains, belonging to 27 bacterial families, were isolated from the nine different pollens. Among them, a new species from the genus Spirosoma was isolated from common hazel, characterized by phenotypic, phylogenetic and genotypic (draft genome sequence) variations and described as the new species Spirosoma pollinicola HA7T.This study enhances our basic knowledge of the pollen microbiome, provide insights on the role of pollen-associated microbes in pollen allergy, and poses the basis for further inter- and intra-kingdom interaction studies of the plant reproductive organs.
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