Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers for characterizing complex microbial communities in explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias toward Alphaproteobacteria. In silico, primer pair 341f/785r showed the highest coverage of the domain Bacteria (96.1%) with no obvious bias toward the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies.

Metal contaminated soils are increasing worldwide. Metal-tolerant plants growing on metalliferous soils are fascinating genetic and microbial resources. Seeds can vertically transmit endophytic microorganisms that can assist next generations to cope with environmental stresses, through yet poorly understood mechanisms. The aims of this study were to identify the core seed endophyte microbiome of the pioneer metallophyte Crotalaria pumila throughout three generations, and to better understand the plant colonisation of the seed endophyte Methylobacterium sp. Cp3. Strain Cp3 was detected in C. pumila seeds across three successive generations and showed the most dominant community member. When inoculated in the soil at the time of flowering, strain Cp3 migrated from soil to seeds. Using confocal microscopy, Cp3-mCherry was demonstrated to colonise the root cortex cells and xylem vessels of the stem under metal stress. Moreover, strain Cp3 showed genetic and in planta potential to promote seed germination and seedling development. We revealed, for the first time, that the seed microbiome of a pioneer plant growing in its natural environment, and the colonisation behaviour of an important plant growth promoting systemic seed endophyte. Future characterization of seed microbiota will lead to a better understanding of their functional contribution and the potential use for seed-fortification applications.

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Background A diverse community of microbes naturally exists on the phylloplane, the surface of leaves. It is one of the most prevalent microbial habitats on earth and bacteria are the most abundant members, living in communities that are highly dynamic. Today, one of the key challenges for microbiologists is to develop strategies to culture the vast diversity of microorganisms that have been detected in metagenomic surveys. Results We isolated bacteria from the phylloplane of Hedera helix (common ivy), a widespread evergreen, using five growth media: Luria–Bertani (LB), LB01, yeast extract–mannitol (YMA), yeast extract–flour (YFlour), and YEx. We also included a comparison with the uncultured phylloplane, which we showed to be dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Inter-sample (beta) diversity shifted from LB and LB01 containing the highest amount of resources to YEx, YMA, and YFlour which are more selective. All growth media equally favoured Actinobacteria and Gammaproteobacteria, whereas Bacteroidetes could only be found on LB01, YEx, and YMA. LB and LB01 favoured Firmicutes and YFlour was most selective for Betaproteobacteria. At the genus level, LB favoured the growth of Bacillus and Stenotrophomonas, while YFlour was most selective for Burkholderia and Curtobacterium. The in vitro plant growth promotion (PGP) profile of 200 isolates obtained in this study indicates that previously uncultured bacteria from the phylloplane may have potential applications in phytoremediation and other plant-based biotechnologies. Conclusions This study gives first insights into the total bacterial community of the H. helix phylloplane, including an evaluation of its culturability using five different growth media. We further provide a collection of 200 bacterial isolates underrepresented in current databases, including the characterization of PGP profiles. Here we highlight the potential of simple strategies to obtain higher microbial diversity from environmental samples and the use of high-throughput sequencing to guide isolate selection from a variety of growth media.

Ambient air pollution exerts deleterious effects on our environment. Continuously exposed to the atmosphere, diverse communities of microorganisms thrive on leaf surfaces, the phylloplane. The composition of these communities is dynamic, responding to many environmental factors including ambient air pollution. In this field study, over a 2 year period, we sampled Hedera helix (ivy) leaves at six locations exposed to different ambient air pollution conditions. Daily, we monitored ambient black carbon (BC), PM2.5, PM10, nitrogen dioxide, and ozone concentrations and found that ambient air pollution led to a 2–7-fold BC increase on leaves, the phylloplane BC load. Our results further indicated that the phylloplane BC load correlates with the diversity of bacterial and fungal leaf communities, impacting diversity more than seasonal effects. The bacterial genera Novosphingobium, Hymenobacter, and Methylorubrum, and the fungal genus Ampelomyces were indicators for communities exposed to the highest phylloplane BC load. Parallel to this, we present one fungal and two bacterial phylloplane strains isolated from an air-polluted environment able to degrade benzene, toluene, and/or xylene, including a genomics-based description of the degradation pathways involved. The findings of this study suggest that ambient air pollution shapes microbial leaf communities, by affecting diversity and supporting members able to degrade airborne pollutants.