All-Food-Seq (AFS): a quantifiable screen for species in biological samples by deep DNA sequencing

Background

DNA-based methods like PCR efficiently identify and quantify the taxon composition of complex biological materials, but are limited to detecting species targeted by the choice of the primer assay. We show here how untargeted deep sequencing of foodstuff total genomic DNA, followed by bioinformatic analysis of sequence reads, facilitates highly accurate identification of species from all kingdoms of life, at the same time enabling quantitative measurement of the main ingredients and detection of unanticipated food components.

Results

Sequence data simulation and real-case Illumina sequencing of DNA from reference sausages composed of mammalian (pig, cow, horse, sheep) and avian (chicken, turkey) species are able to quantify material correctly at the 1% discrimination level via a read counting approach. An additional metagenomic step facilitates identification of traces from animal, plant and microbial DNA including unexpected species, which is prospectively important for the detection of allergens and pathogens.

Conclusions

Our data suggest that deep sequencing of total genomic DNA from samples of heterogeneous taxon composition promises to be a valuable screening tool for reference species identification and quantification in biosurveillance applications like food testing, potentially alleviating some of the problems in taxon representation and quantification associated with targeted PCR-based approaches.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-639) contains supplementary material, which is available to authorized users.     

Soil-plant compartments affect fungal microbiome diversity and composition in grapevine

Plant compartments provide unique niches that lead to specific microbial associations. The microbiota colonizing the endophytic compartment (endorhizosphere) and the rhizosphere contribute to productivity, plant growth, phytoremediation and carbon sequestration. The main objective of this study was to investigate how fungal communities are enriched in different habitats outside and inside of grapevine roots. For this purpose, the spatial dynamics of the fungal communities associated with three soil-plant compartments (bulk soil, rhizosphere and endorhizosphere) were characterized by ITS high-throughput amplicon sequencing (HTAS). Fungal communities were largely affected in their diversity and composition by soil-plant compartments, whereas the spatial variation (i.e. across five vineyards) was low. The endorhizosphere compartment differed most from the other two, suggesting that the root tissues entail a barrier for fungal colonization. The results of functional prediction via FUNGuild suggested an increase in the relative abundances of potential plant pathogens, endophytes and arbuscular mycorrhiza, and a decrease in wood, dung and undefined saprotrophs from bulk soil towards the endorhizosphere. Roots of asymptomatic vines were a microbial niche that is inhabited by soilborne fungi associated with grapevine trunk diseases, which opens up new perspectives in the study of the endophytic role of these pathogens on grapevines. Results obtained in this study provide helpful information to better know how the host shapes its microbiome and the implications for vineyard productivity and management.     

Rhizosphere fungal community structure succession of Xinjiang continuously cropped cotton

The large-scale long-term plantation of cotton in the Xinjiang region has been accompanied by a regular and wide outbreak of soil-borne fungal diseases such as verticillium wilt, which significantly damaged the local cotton industry. High-throughput sequencing data showed that the cotton field cultivation management measures pose a significant influence upon the original ecological soil fungal community structure. During long-term continuous cropping of cotton, a new soil fungal community structure emerges after several repeated adjustments over five years. The number of verticillium wilt pathogens in the soil increased rapidly with prolonged continuous cropping time, reaching a maximum at around the 10th y; moreover, the abundance of the verticillium wilt pathogen only serves as one of numerous essential factors for disease occurrence. The fungal community structure and the abundance of verticillium wilt pathogens in local cotton fields are gradually formed under joint effects of year-long continuous cropping and supporting cultivation management measures.     

纳帕海高原湿地真菌群落多样性和组成的分布

【背景】位于滇西北的纳帕海高原湿地,是我国唯一的低纬度、高海拔、季节性半封闭型高原湿地。真菌在湿地生态系统的维持和稳定中发挥着特殊作用,然而关于纳帕海高原湿地真菌群落多样性和组成的研究目前仍无报道。【目的】对纳帕海高原湿地不同季节和土壤类型真菌群落多样性和组成及与环境因子的关系开展系统研究分析,促进对高原湿地微生物多样性的深入认识。【方法】采用荧光定量PCR和高通量测序技术,分析了纳帕海高原湿地不同季节和土壤类型中真菌的数量、群落多样性和组成及其与环境因子的关系。【结果】真菌数量级的变化对于人为干扰下的湿地土壤退化是敏感的响应指标。在真菌群落组成中,约有60%以上未确定的分类信息,40%有确定分类信息的包括6个门17个纲37个目53个科63个属,大部分分类信息集中在Ascomycota门,相对优势属为Gibberella。通过分类水平、OTU水平和β多样性分析比较,在纳帕海高原湿地整体真菌群落多样性和组成受季节变化影响不显著,但不同土壤类型的变化呈显著差异,推测是由于不同采样区植物根际效应和种类的影响。CCA (Canonical correlation analysis)分析表明,在不同采样区受不同土壤理化因子的影响。【结论】揭示了纳帕海高原湿地土壤真菌群落多样性和组成的区域特征,从微生物学角度进一步提出了对纳帕海高原湿地环境保护和恢复的重要性。     

Changes in fungal diversity and composition along a chronosequence of Eucalyptus grandis plantations in Ethiopia

Eucalyptus tree species are widely used in Ethiopian plantations, but the impact of these plantations on the soil fungal communities is still unknown. We assessed the changes in diversity, species composition and ecological guilds of the soil fungal communities across tree ages of Eucalyptus grandis plantations by DNA metabarcoding of ITS2 amplicons. Changes in soil fungal species composition, diversity and ecological guilds were related to stand age but also to fertility changes. The relative abundance of saprotrophs and pathogens were negatively correlated with stand age, and positively with soil fertility. In contrast, the relative abundance and diversity of ectomycorrhizal species were higher in older, less fertile stands, including well-known cosmopolitan species but also species associated with Eucalyptus, such as Scleroderma albidum and Descomyces albellus. We show that soil fungal community changes are linked to progressive soil colonization by tree roots but are also related to soil fertility changes.     

Effect of tree host species on fungal community composition in a tropical rain forest in Panama

Abstract.  To determine whether host species influence the composition of fungal communities, the ascomycetes and basidiomycetes present on three tree species ( Prioria copaifera (Fabaceae), Quararibea asterolepis (Bombacaceae), and Trichilia tuberculata (Meliaceae)) were sampled on the 50-ha Forest Dynamics Project plot in lowland moist tropical forest on Barro Colorado Island, Panama. The most abundant fungal morphotypes of both ascomycetes and basidiomycetes were generalists found on all three hosts, but detrended correspondence analysis revealed distinct differences in fungal community composition among host trees. These differences among hosts were constant across census years. Randomization tests revealed that there were significantly fewer host-generalist fungi than expected for ascomycetes but not for basidiomycetes. These results indicate that host composition plays a role in structuring both ascomycete and basidiomycete fungal communities, but that the most successful fungal morphotypes are capable of colonizing multiple host species.     

A common garden experiment with Porphyra umbilicalis (Rhodophyta) evaluates methods to study spatial differences in the macroalgal microbiome

While macroalgal microbiomes are the focus of many recent studies, there is little information about microbial spatial diversity across the thallus. Reliance on field material makes it difficult to discern whether recovered microbiomes belong to the host or its epiphytes, and technical comparisons of macroalgal samples for microbial studies are needed. Here, we use a common garden approach that avoids the problem of epiphytes, particularly at holdfasts, to examine the microbiome of Porphyra umbilicalis (strain Pum1). We used the V6 hypervariable region of the 16S rDNA with Illumina HiSeq sequencing and developed PNA clamps to block recovery of organelle V6 sequences. The common garden approach allowed us to determine differences in the microbiome at the holdfast versus blade margin. We found a notable increase in the relative abundance of Planctomycetes and Alphaproteobacteria at the holdfast, particularly of the possible symbiont Sulfitobacter sp. Nonadjacent 1.5 cm² samples of blade margin had microbiomes that were not statistically different. The most abundant phylum in the overall microbiome was Proteobacteria, followed by Bacteroidetes. Because phycologists often work in remote sites, we compared three stabilization and preparation techniques and found silica gel desiccation/bead‐beating and flash‐freezing/lyophilization/bead‐beating to be interchangeable. Core taxa (≥0.1% of sequences) across treatments were similar and accounted for ≥95% of all sequences. Finally, statistical conclusions for all comparisons were the same, regardless of which microbial community analysis tool was used: mothur or minimum entropy decomposition.     

Limited dispersal prevents Quercus rubra invasion in a 14‐species common garden experiment

Aim

Information about the importance of propagule pressure and habitat invasibility in invasion success of dispersal‐limited species is scarce. We aimed to assess invasiveness of Quercus rubra within stands of 14 tree species, and the effects of distance from propagule source on invasion success, to highlight limiting factors for further application in nature conservation.

Location

Siemianice Experimental Forest—a common garden forest experiment with 14 tree species, western Poland.

Methods

We investigated aboveground biomass, leaf area index and density of Q. rubra natural regeneration within 53 experimental plots, as well as distance from the seed source. We also analysed light availability changes between 2005 and 2015 on plots of each tree species. We used multiple linear regression and variable importance to quantify the effect of each factor.

Results

All factors tested influenced ecological success of Q. rubra. Invasion success decreased with increasing distance from the seed source and decreasing light availability and was higher within stands of pioneer tree species. Leaf area index depended mostly on tree stand species, density depended on distance from the propagule source and biomass depended on both. Light availability explained 7.2%–30.2% of the variance; tree species—from 36.1% to 57.4%; and distance from the propagule source—from 12.4% to 56.7%.

Main conclusions

Tree stand species, light availability and distance from the propagule source influence ecological success of invasive Q. rubra, displaying their importance for spread of this species. These factors are controllable in forest/conservation management and may be used to prevent Q. rubra invasion. Planting late‐successional tree species that cast dense shade, maintaining canopy closure and removing fruiting trees from surrounding more invasible stands may prevent Q. rubra invasion.

     

代表活性污泥中苯酚降解菌群的ERIC-PCR产物片段的多态性

对可能代表活性污泥中两个时期的主要苯酚降解菌群的：ERIC-PCR指纹图谱中的两条2．1kb的条带(P1和P8)中的DNA片段进行克隆、转化,得到193个转化子。．HinfⅠ物理图谱分析得到35种酶切类型,将两端各进行1次测序后,同源性大于90％的克隆归为一类,共得到10种序列类型。对各类型的代表克隆进行了全长测序,5种类型为P1条带所有,3种类型为P8所有,二者共享的类型为2种,丰度最高的片段为S3,P1条带中76．3％的转化子和P8条带中66．7％转化子属该类型。对所得序列进行检索分析,它们与GenBank中已知基因序列均无显著同源性。用S3特异性引物对活性污泥样品及其它在LB、dCGY、MP和FWM培养基上的回收菌进：行扩增,除LB上的回收菌没有显示目的条带外,活性污泥和其回收菌中均检测到带有该基因组片段的菌种的存在。研究为专一性分离降酚活性污泥中的优势菌提供了分子探针。     

Do foliar fungal communities of Norway spruce shift along a tree species diversity gradient in mature European forests?

Foliar fungal species are diverse and colonize all plants, though whether forest tree species composition influences the distribution of these fungal communities remains unclear. Fungal communities include quiescent taxa and the functionally important and metabolically active taxa that respond to changes in the environment. To determine fungal community shifts along a tree species diversity gradient, needles of Norway spruce were sampled from trees from four mature European forests. We hypothesized that the fungal communities and specific fungal taxa would correlate with tree species diversity. Furthermore, the active fungal community, and not the total community, would shift along the tree diversity gradient. High-throughput sequencing showed significant differences in the fungal communities in the different forests, and in one forest, tree diversity effects were observed, though this was not a general phenomenon. Our study also suggests that studying the metabolically active community may not provide additional information about community composition or diversity.     

长期不同磷肥施用量对砂姜黑土真菌多样性、群落组成和种间关系的影响

以安徽蒙城砂姜黑土氮磷钾肥肥效长期定位试验为平台,选取P0(不施磷肥)、P1(P_2O_5 45 kg/hm~2)和P2(P_2O_5 90 kg/hm~2)3个磷肥施用梯度,采用现代高通量测序技术,探索长期施用磷肥对真菌多样性、群落组成和种间关系的影响。结果发现真菌多样性随磷肥施入量增加而降低,且与全磷和有效磷均成显著负相关。磷肥施用导致真菌群落组成发生明显改变,冗余分析结果表明这与全磷和可溶性有机碳密切相关。P1和P2中子囊菌门和子囊菌纲显著升高,而被孢霉菌门和被孢霉菌纲则显著降低。在属水平上,施磷处理中腐生营养型真菌显著升高,而病原型和共生型真菌显著降低:其中P0中Sagenomella、Simplicillium、Magnaporthiopsis、Schizothecium和Nigrospora等5个属相对丰度最高;P1中Plenodomus、Penicillium和Arthrobotrys相对丰度最高;P2中Cyphellophora、Zopfiella、Guehomyces、Mortierella和Mucor等5个属相对丰度最高。网络分析发现施磷后真菌网络复杂度和稳定性升高,物种间正相关关系增加,负相关关系减少。因此在砂姜黑土区长期施用磷肥可降低真菌多样性,增加土壤中腐生营养型真菌、减少共生或病原菌数量,同时提高物种间网络复杂度和稳定性。以上结果表明磷肥施用对促进土壤养分循环,控制植物病害、维持地下生态系统稳定性具有重要作用。