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Sphingomonas is one of the most abundant bacterial genera in the phyllosphere of wild Arabidopsis thaliana, but relative to Pseudomonas, the ecology of Sphingomonas and its interaction with plants is poorly described. We analyzed the genomic features of over 400 Sphingomonas isolates collected from local A. thaliana populations, which revealed much higher intergenomic diversity than for the considerably more uniform Pseudomonas isolates found in the same host populations. Variation in Sphingomonas plasmid complements and additional genomic features suggest high adaptability of this genus, and the widespread presence of protein secretion systems hints at frequent biotic interactions. While some of the isolates showed plant-protective phenotypes in lab tests, this was a rare trait. To begin to understand the extent of strain sharing across alternate hosts, we employed amplicon sequencing and a bulk-culturing metagenomics approach on both A. thaliana and neighboring plants. Our data reveal that both Sphingomonas and Pseudomonas thrive on other diverse plant hosts, but that Sphingomonas is a poor in or dead leaves., Proceedings of the National Academy of Sciences of the United States of America, 119 (52), ISSN:0027-8424, ISSN:1091-6490

Plant-associated fungi are incredibly diverse, comprising over a million species of mycorrhiza, endophytes, saprophytes and pathogens worldwide. This diverse fungal community is highly important for plant health. Many fungi are effective biocontrol agents that can kill or suppress fungal pathogens, with pathogen biocontrol found for both individual microorganisms and plant-associated fungal consortia. Meanwhile, increased plant community diversity aboveground corresponds to an increase in below-ground fungal community diversity, which contributes in turn to improved rhizosphere soil health and pathogen suppression. In this review, we discuss the role of fungal diversity in soil health and plant disease suppression and the various mechanisms by which mycorrhizal and endophytic fungi combat plant pathogenic fungi. We also discuss the array of diagnostic tools, both well-established and newly developed, which are revolutionising fungal pathogen detection and rhizosphere community analysis.

Relative contributions of pre-existing vs de novo genomic variation to adaptation are poorly understood, especially in polyploid organisms. We assess this in high resolution using autotetraploid Arabidopsis arenosa, which repeatedly adapted to toxic serpentine soils that exhibit skewed elemental profiles. Leveraging a fivefold replicated serpentine invasion, we assess selection on SNPs and structural variants (TEs) in 78 resequenced individuals and discover significant parallelism in candidate genes involved in ion homeostasis. We further model parallel selection and infer repeated sweeps on a shared pool of variants in nearly all these loci, supporting theoretical expectations. A single striking exception is represented by TWO PORE CHANNEL 1, which exhibits convergent evolution from independent de novo mutations at an identical, otherwise conserved site at the calcium channel selectivity gate. Taken together, this suggests that polyploid populations can rapidly adapt to environmental extremes, calling on both pre-existing variation and novel polymorphisms.

Sphingomonas is one of the most abundant bacterial genera in the phyllosphere of wild Arabidopsis thaliana, but relative to Pseudomonas, the ecology of Sphingomonas and its interaction with plants remains elusive. We analyzed the genomic features of over 400 Sphingomonas isolates collected from local A. thaliana populations, which revealed high intergenomic diversity, in contrast to genetically much more uniform Pseudomonas isolates found in the same host populations. Variation in Sphingomonas plasmid complements and additional genomic features suggest high adaptability of this genus, and the widespread presence of protein secretion systems hints at frequent biotic interactions. While some of the isolates showed plant-protective properties in lab tests, this was a rare trait. To begin to understand the extent of strain sharing across alternate hosts, we employed amplicon sequencing and a novel bulk-culturing metagenomics approach on both A. thaliana and neighboring plants. Our data reveal that Sphingomonas and Pseudomonas both thrive on other diverse plant hosts, but that Sphingomonas is a poor competitor in dying or dead leaves.

Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low‐cost genomic DNA extraction method for soil samples. We also present an Illumina‐compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non‐commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad‐leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column‐based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low‐cost genomic DNA extraction method for soil samples that can be coupled to an Illumina‐compatible simple two‐step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high‐quality genomic DNA at a fraction of the cost of commercial kits and enables cost‐effective, large‐scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics., We present an inexpensive and high‐throughput magnetic particle‐based soil genomic DNA (gDNA) extraction method "SDE" ($0.36) for next‐generation sequencing quality gDNA extraction, scalable to thousands of samples with little hands‐on time. SDE can also be used in combination with our low‐cost amplicon library construction workflow (16S, ITS, $2.5), enabling multiplexing of up to 2304 libraries at a time. Combining SDE and our custom library construction workflow allows utilization of state‐of‐the‐art Illumina sequencing platforms (e.g., NovaSeq) for large‐scale microbiome studies.

BackgroundCommon bottlenecks in environmental microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in PCR inhibitors. To address this, we have established a low-cost genomic DNA extraction method for inhibitor rich samples alongside an Illumina-compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non-commercial extraction method by Zou et al. (2017). Our benchmarking experiment used four different soil types (coniferous, broad leafed, and mixed forest plus a standardised cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analysing sequence variants of 16S V4 and ITS rRNA amplicons.ResultsWe found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column-based DNA extraction with extensive washing delivered the cleanest genomic DNA e.g. best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits.ConclusionHere we present a low-cost genomic DNA extraction method for inhibitor rich sample types such as soil that can be coupled to an Illumina-compatible simple two step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high quality genomic DNA at a fraction of the cost of commercial kits and enables cost-effective, large scale amplicon sequencing projects. Notably our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, Dovetail genomics etc.

Relative contributions of pre-existing vs de novo genomic variation to adaptation are poorly understood, especially in polyploid organisms. We assess this in high resolution using autotetraploid Arabidopsis arenosa, which repeatedly adapted to toxic serpentine soils that exhibit skewed elemental profiles. Leveraging a fivefold replicated serpentine invasion, we assess selection on SNPs and structural variants (TEs) in 78 resequenced individuals and discover significant parallelism in candidate genes involved in ion homeostasis. We further model parallel selection and infer repeated sweeps on a shared pool of variants in nearly all these loci, supporting theoretical expectations. A single striking exception is represented by TWO PORE CHANNEL 1, which exhibits convergent evolution from independent de novo mutations at an identical, otherwise conserved site at the calcium channel selectivity gate. Taken together, this suggests that polyploid populations can rapidly adapt to environmental extremes, calling on both pre-existing variation and novel polymorphisms.