Integration and holistic analysis of multiple multidimensional soil data sets.

Complex matrices such as soil have a range of measurable characteristics, and thus data to describe them can be considered multidimensional. These characteristics can be strongly influenced by factors that introduce confounding effects that hinder analyses. Traditional statistical approaches lack the flexibility and granularity required to adequately evaluate such matrices, particularly those with large dataset of varying data types (i.e. quantitative non-compositional, quantitative compositional). We present a statistical workflow designed to effectively analyse complex, multidimensional systems, even in the presence of confounding variables. The developed methodology involves exploratory analysis to identify the presence of confounding variables, followed by data decomposition (including strategies for both compositional and non-compositional quantitative data) to minimise the influence of these confounding factors such as sampling site/location. These data processing methods then allow for common patterns to be highlighted in the data, including the identification of biomarkers and determination of non-trivial associations between variables. We demonstrate the utility of this statistical workflow by jointly analysing the chemical composition and fungal biodiversity of New Zealand vineyard soils that have been managed with either organic low-input or conventional input approaches. By applying this pipeline, we were able to identify biomarkers that distinguish viticultural soil from both approaches and also unearth links and associations between the chemical and metagenomic profiles. While soil is an example of a system that can require this type of statistical methodology, there are a range of biological and ecological systems that are challenging to analyse due to the complex interplay of global and local effects. Utilising our developed pipeline will greatly enhance the way that these systems can be studied and the quality and impact of insight gained from their analysis.

Dose-Dependent Genetic Resistance to Azole Fungicides Found in the Apple Scab Pathogen.

The evolution of azole resistance in fungal pathogens presents a major challenge in both crop production and human health. Apple orchards across the world are faced with the emergence of azole fungicide resistance in the apple scab pathogen Venturia inaequalis. Target site point mutations observed in this fungus to date cannot fully explain the reduction in sensitivity to azole fungicides. Here, polygenic resistance to tebuconazole was studied across a population of V. inaequalis. Genotyping by sequencing allowed Quantitative Trait Loci (QTLs) mapping to identify the genetic components controlling this fungicide resistance. Dose-dependent genetic resistance was identified, with distinct genetic components contributing to fungicide resistance at different exposure levels. A QTL within linkage group seven explained 65% of the variation in the effective dose required to reduce growth by 50% (ED50). This locus was also involved in resistance at lower fungicide doses (ED10). A second QTL in linkage group one was associated with dose-dependent resistance, explaining 34% of variation at low fungicide doses (ED10), but did not contribute to resistance at higher doses (ED50 and ED90). Within QTL regions, non-synonymous mutations were observed in several ATP-Binding Cassette and Major Facilitator SuperFamily transporter genes. These findings provide insight into the mechanisms of fungicide resistance that have evolved in horticultural pathogens. Identification of resistance gene candidates supports the development of molecular diagnostics to inform management practices.

Inferring bacterial transmission dynamics using deep sequencing genomic surveillance data.

Identifying and interrupting transmission chains is important for controlling infectious diseases. One way to identify transmission pairs - two hosts in which infection was transmitted from one to the other - is using the variation of the pathogen within each single host (within-host variation). However, the role of such variation in transmission is understudied due to a lack of experimental and clinical datasets that capture pathogen diversity in both donor and recipient hosts. In this work, we assess the utility of deep-sequenced genomic surveillance (where genomic regions are sequenced hundreds to thousands of times) using a mouse transmission model involving controlled spread of the pathogenic bacterium Citrobacter rodentium from infected to naïve female animals. We observe that within-host single nucleotide variants (iSNVs) are maintained over multiple transmission steps and present a model for inferring the likelihood that a given pair of sequenced samples are linked by transmission. In this work we show that, beyond the presence and absence of within-host variants, differences arising in the relative abundance of iSNVs (allelic frequency) can infer transmission pairs more precisely. Our approach further highlights the critical role bottlenecks play in reserving the within-host diversity during transmission.

Soil and bark biodiversity forms discrete islands between vineyards that are not affected by distance or management regime.

Within geographic regions, the existing data suggest that physical habitat (bark, soil, etc.) is the strongest factor determining agroecosystem microbial community assemblage, followed by geographic location (site), and then management regime (organic, conventional, etc.). The data also suggest community similarities decay with increasing geographic distance. However, integrated hypotheses for these observations have not been developed. We formalized and tested such hypotheses by sequencing 3.8 million bacterial 16S, fungal ITS2 and non-fungal eukaryotic COI barcodes deriving from 108 samples across two habitats (soil and bark) from six vineyards sites under conventional or conservation management. We found both habitat and site significantly affected community assemblage, with habitat the stronger for bacteria only, but there was no effect of management. There was no evidence for community similarity distance-decay within sites within each habitat. While communities significantly differed between vineyard sites, there was no evidence for between site community similarity distance-decay apart from bark bacterial communities, and no correlations with soil and bark pH apart from soil bacterial communities. Thus, within habitats, vineyard sites represent discrete biodiversity islands, and while bacterial, fungal and non-fungal eukaryotic biodiversity mostly differs between sites, the distance by which they are separated does not define how different they are.

Mixed yeast communities contribute to regionally distinct wine attributes.

There is evidence that vineyard yeast communities are regionally differentiated, but the extent to which this contributes to wine regional distinctiveness is not yet clear. This study represents the first experimental test of the hypothesis that mixed yeast communities-comprising multiple, region-specific, isolates, and species-contribute to regional wine attributes. Yeast isolates were sourced from uninoculated Pinot Noir fermentations from 17 vineyards across Martinborough, Marlborough, and Central Otago in New Zealand. New methodologies for preparing representative, mixed species inoculum from these significantly differentiated regional yeast communities in a controlled, replicable manner were developed and used to inoculate Pinot Noir ferments. A total of 28 yeast-derived aroma compounds were measured in the resulting wines via headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. Yeast community region of origin had a significant impact on wine aroma, explaining ∼10% of the observed variation, which is in line with previous reports of the effects of region-specific Saccharomyces cerevisiae isolates on Sauvignon Blanc ferments. This study shows that regionally distinct, mixed yeast communities can modulate wine aroma compounds in a regionally distinct manner and are in line with the hypothesis that there is a microbial component to regional distinctiveness, or terroir, for New Zealand Pinot Noir.

Meta-analysis of global soil data identifies robust indicators for short-term changes in soil organic carbon stock following land use change.

The restoration of degraded lands and minimizing the degradation of productive lands are at the forefront of many environmental land management schemes around the world. A key indicator of soil productivity is soil organic carbon (SOC), which influences the provision of most soil ecosystem services. A major challenge in direct measurement of changes in SOC stock is that it is difficult to detect within a short timeframe relevant to land managers. In this study, we sought to identify suitable early indicators of changes in SOC stock and their drivers. A meta-analytical approach was used to synthesize global data on the impacts of arable land conversion to other uses on total SOC stock, 12 different SOC fractions and three soil structural properties. The conversion of arable lands to forests and grasslands accounted for 91 % of the available land use change datasets used for the meta-analysis and were mostly from Asia and Europe. Land use change from arable lands led to 50 % (32-68 %) mean increase in both labile (microbial biomass C and particulate organic C - POC) and passive (microaggregate, 53-250 µm diameter; and small macroaggregate, 250-2000 µm diameter) SOC fractions as well as soil structural stability. There was also 37 % (24-50 %) mean increase in total SOC stock in the experimental fields where the various SOC fractions were measured. Only the POC and the organic carbon stored in small macroaggregates had strong correlation with total SOC: our findings reveal these two SOC fractions were predominantly controlled by biomass input to the soil rather than climatic factors and are thus suitable candidate indicators of short-term changes in total SOC stock. Further field studies are recommended to validate the predictive power of the equations we developed in this study and the use of the SOC metrics under different land use change scenarios.

The Efficacy of Yeast Phagostimulant Baits in Attract-and-Kill Strategies Varies between Summer- and Winter-Morphs of Drosophila suzukii.

Drosophila suzukii (Matsumura), is a globally invasive pest of soft and stone fruit. To survive winter in temperate zones it enters a reproductive diapause in a morphologically distinct phenotype. Phagostimulant baits can be combined with insecticides in attract-and-kill strategies for control. We investigated the effectiveness of single yeast species and combinations of co-fermented yeast phagostimulant baits when combined with insecticides in laboratory assays against both summer- and winter-morph D. suzukii. Candida zemplininia or Hanseniaspora uvarum + C. zemplininia combined with lambda-cyhalothrin or cyantraniliprole, and H. uvarum combined with cyantraniliprole caused significantly higher mortality in winter- compared to summer-morph D. suzukii. Additionally, lambda-cyhalothrin combined with M. pulcherrima + H. uvarum resulted in greater mortality compared to single yeasts, H. uvarum for both summer- and winter-morphs and C. zemplininia for summer-morphs. M. pulcherrima + H. uvarum with spinosad significantly reduced the time-to-kill (50%) of summer-morphs compared to insecticide alone. Most yeast-based baits were comparable in terms of attract-and-kill efficacy to Combi-protec, a commercially available bait, although M. pulcherrima or H. uvarum + C. zemplininia in with cyantraniliprole were less effective. Our study suggests that yeast phagostimulants in attract-and-kill strategies should be adjusted for summer- and winter-morph D. suzukii for more effective control.

A restatement of the natural science evidence base regarding the source, spread and control of Campylobacter species causing human disease.

Food poisoning caused by Campylobacter (campylobacteriosis) is the most prevalent bacterial disease associated with the consumption of poultry, beef, lamb and pork meat and unpasteurized dairy products. A variety of livestock industry, food chain and public health interventions have been implemented or proposed to reduce disease prevalence, some of which entail costs for producers and retailers. This paper describes a project that set out to summarize the natural science evidence base relevant to campylobacteriosis control in as policy-neutral terms as possible. A series of evidence statements are listed and categorized according to the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

The relative abundances of yeasts attractive to Drosophila suzukii differ between fruit types and are greatest on raspberries.

Fungal metabolic volatiles attract Drosophila suzukii which oviposits in ripening fruits, but there are few data describing the fungal microbiomes of commercial fruits susceptible to this insect pest. We tested the hypothesis that fruit type and ripening stage have a significant effect on fruit surface fungal communities using DNA metabarcoding approaches and found strong support for differences in all three fungal community biodiversity metrics analysed (numbers, types, and abundances of taxa). There was an average fivefold greater difference in fungal communities between sites with different fruit types (strawberry, cherry, raspberry, and blueberry) than across fruit developmental stages, demonstrating site and/or fruit type is the greater factor defining fungal community assemblage. The addition of a fungal internal standard (Plectosphaerella cucumerina) showed cherry had relatively static fungal populations across ripening. Raspberry had a greater prevalence of Saccharomycetales yeasts attractive to D. suzukii, including Hanseniaspora uvarum, which aligns with reports that raspberry is among the fruits with greatest susceptibility and attraction to D. suzukii. Greater knowledge of how yeast communities change during fruit maturation and between species or sites may be valuable for developing methods to manipulate fruit microbiomes for use in integrated pest management strategies to control D. suzukii.

A small effect of conservation agriculture on soil biodiversity that differs between biological kingdoms and geographic locations.

Larger easily visible animals and plants are negatively affected by agrochemicals used for intensive food production, but we do not understand the general spatial and temporal effects of agrochemicals on the multitudes of bacteria, fungi, and small invertebrate animals that underpin ecosystem productivity. We sequenced the 16S, ITS2, and COI DNA barcode regions from 648 New Zealand vineyard soil samples managed under either conventional or low-agrochemical-input conservation approaches across two regions and three seasons in 1 year and discovered at least 170,000 phylotypes (taxa) with >97% genetic identity. Management approach correlated with a significant 2%-10% difference in the abundances of phylotypes that differed over regions and seasons. Although the data show that agrochemicals do not have a large effect on soil biodiversity on average, the important finding is that the magnitude of impact differs between taxa types and locations, and some taxa most affected also influence the quality of agricultural produce.

Whole-genome sequencing from the New Zealand Saccharomyces cerevisiae population reveals the genomic impacts of novel microbial range expansion.

Saccharomyces cerevisiae is extensively utilized for commercial fermentation, and is also an important biological model; however, its ecology has only recently begun to be understood. Through the use of whole-genome sequencing, the species has been characterized into a number of distinct subpopulations, defined by geographical ranges and industrial uses. Here, the whole-genome sequences of 104 New Zealand (NZ) S. cerevisiae strains, including 52 novel genomes, are analyzed alongside 450 published sequences derived from various global locations. The impact of S. cerevisiae novel range expansion into NZ was investigated and these analyses reveal the positioning of NZ strains as a subgroup to the predominantly European/wine clade. A number of genomic differences with the European group correlate with range expansion into NZ, including 18 highly enriched single-nucleotide polymorphism (SNPs) and novel Ty1/2 insertions. While it is not possible to categorically determine if any genetic differences are due to stochastic process or the operations of natural selection, we suggest that the observation of NZ-specific copy number increases of four sugar transporter genes in the HXT family may reasonably represent an adaptation in the NZ S. cerevisiae subpopulation, and this correlates with the observations of copy number changes during adaptation in small-scale experimental evolution studies.

De Novo Resistance to Arg10-Teixobactin Occurs Slowly and Is Costly.

Bacterial pathogens are rapidly evolving resistance to all clinically available antibiotics. One part of the solution to this complex issue is to better understand the resistance mechanisms to new and existing antibiotics. Here, we focus on two antibiotics. Teixobactin is a recently discovered promising antibiotic that is claimed to "kill pathogens without detectable resistance" (L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al., Nature 517:455-459, 2015, https://doi.org/10.1038/nature14098). Moenomycin A has been extensively used in animal husbandry for over 50 years with no meaningful antibiotic resistance arising. However, the nature, mechanisms, and consequences of the evolution of resistance to these "resistance-proof" compounds have not been investigated. Through a fusion of experimental evolution, whole-genome sequencing, and structural biology, we show that Staphylococcus aureus can develop significant resistance to both antibiotics in clinically meaningful timescales. The magnitude of evolved resistance to Arg10-teixobactin is 300-fold less than to moenomycin A over 45 days, and these are 2,500-fold and 8-fold less than evolved resistance to rifampicin (control), respectively. We have identified a core suite of key mutations, which correlate with the evolution of resistance, that are in genes involved in cell wall modulation, lipid synthesis, and energy metabolism. We show the evolution of resistance to these antimicrobials translates into significant cross-resistance against other clinically relevant antibiotics for moenomycin A but not Arg10-teixobactin. Lastly, we show that resistance is rapidly lost in the absence of antibiotic selection, especially for Arg10-teixobactin. These findings indicate that teixobactin is worth pursuing for clinical applications and provide evidence to inform strategies for future compound development and clinical management.

Small scale fungal community differentiation in a vineyard system.

Microbes influence the quality of agricultural commodities and contribute to their distinctive sensorial attributes. Increasingly studies have demonstrated not only differential geographic patterns in microbial communities and populations, but that these contribute to valuable regionally distinct agricultural product identities, the most well-known example being wine. However, little is understood about microbial geographic patterns at scales of less than 100 km. For wine, single vineyards are the smallest (and most valuable) scale at which wine is asserted to differ; however, it is unknown whether microbes play any role in agricultural produce differentiation at this scale. Here we investigate whether vineyard fungal communities and yeast populations driving the spontaneous fermentation of fruit from these same vineyards are differentiated using metagenomics and population genetics. Significant differentiation of fungal communities was revealed between four Central Otago (New Zealand) Pinot Noir vineyard sites. However, there was no vineyard demarcation between fermenting populations of S. cerevisiae. Overall, this provides evidence that vineyard microbiomes potentially contribute to vineyard specific attributes in wine. Understanding the scale at which microbial communities are differentiated, and how these communities influence food product attributes has direct economic implications for industry and could inform sustainable management practices that maintain and enhance microbial diversity.

Are Drosophila preferences for yeasts stable or contextual?

Whether there are general mechanisms, driving interspecific chemical communication is uncertain. Saccharomycetaceae yeast and Drosophila fruit flies, both extensively studied research models, share the same fruit habitat, and it has been suggested their interaction comprises a facultative mutualism that is instigated and maintained by yeast volatiles. Using choice tests, experimental evolution, and volatile analyses, we investigate the maintenance of this relationship and reveal little consistency between behavioral responses of two isolates of sympatric Drosophila species. While D. melanogaster was attracted to a range of different Saccharomycetaceae yeasts and this was independent of fruit type, D. simulans preference appeared specific to a particular S. cerevisiae genotype isolated from a vineyard fly population. This response, however, was not consistent across fruit types and is therefore context-dependent. In addition, D. simulans attraction to an individual S. cerevisiae isolate was pliable over ecological timescales. Volatile candidates were analyzed to identify a common signal for yeast attraction, and while D. melanogaster generally responded to fermentation profiles, D. simulans preference was more discerning and likely threshold-dependent. Overall, there is no strong evidence to support the idea of bespoke interactions with specific yeasts for either of these Drosophila genotypes. Rather the data support the idea Drosophila are generally adapted to sense and locate fruits infested by a range of fungal microbes and/or that yeast-Drosophila interactions may evolve rapidly.

Co-evolution as Tool for Diversifying Flavor and Aroma Profiles of Wines.

The products of microbial metabolism form an integral part of human industry and have been shaped by evolutionary processes, accidentally and deliberately, for thousands of years. In the production of wine, a great many flavor and aroma compounds are produced by yeast species and are the targets of research for commercial breeding programs. Here we demonstrate how co-evolution with multiple species can generate novel interactions through serial co-culture in grape juice. We find that after ~65 generations, co-evolved strains and strains evolved independently show significantly different growth aspects and exhibit significantly different metabolite profiles. We show significant impact of co-evolution of Candida glabrata and Pichia kudriavzevii on the production of metabolites that affect the flavor and aroma of experimental wines. While co-evolved strains do exhibit novel interactions that affect the reproductive success of interacting species, we found no evidence of cross-feeding behavior. Our findings yield promising avenues for developing commercial yeast strains by using co-evolution to diversify the metabolic output of target species without relying on genetic modification or breeding technologies. Such approaches open up exciting new possibilities for harnessing microbial co-evolution in areas of agriculture and food related research generally.

From vineyard to winery: a source map of microbial diversity driving wine fermentation.

Humans have been making wine for thousands of years and microorganisms play an integral part in this process as they not only drive fermentation, but also significantly influence the flavour, aroma and quality of finished wines. Since fruits are ephemeral, they cannot comprise a permanent microbial habitat; thus, an age-old unanswered question concerns the origin of fruit and ferment associated microbes. Here we use next-generation sequencing approaches to examine and quantify the roles of native forest, vineyard soil, bark and fruit habitats as sources of fungal diversity in ferments. We show that microbial communities in harvested juice and ferments vary significantly across regions, and that while vineyard fungi account for ∼40% of the source of this diversity, uncultivated ecosystems outside of vineyards also prove a significant source. We also show that while communities in harvested juice resemble those found on grapes, these increasingly resemble fungi present on vine bark as the ferment proceeds.

European derived Saccharomyces cerevisiae colonisation of New Zealand vineyards aided by humans.

Humans have acted as vectors for species and expanded their ranges since at least the dawn of agriculture. While relatively well characterised for macrofauna and macroflora, the extent and dynamics of human-aided microbial dispersal is poorly described. We studied the role which humans have played in manipulating the distribution of Saccharomyces cerevisiae, one of the world's most important microbes, using whole genome sequencing. We include 52 strains representative of the diversity in New Zealand to the global set of genomes for this species. Phylogenomic approaches show an exclusively European origin of the New Zealand population, with a minimum of 10 founder events mostly taking place over the last 1000 years. Our results show that humans have expanded the range of S. cerevisiae and transported it to New Zealand where it was not previously present, where it has now become established in vineyards, but radiation to native forests appears limited.

Microbiology: Mixing Wine, Chocolate, and Coffee.

Yeasts associated with cocoa and coffee beans are genetically distinct. These populations have been created through the migration and mixing of populations associated with vineyards, trees in America, and the ancestral seat of this species in Far East Asia.