The influence of genetics,defensive chemistry and the fungal microbiome on disease outcome in whitebark pine trees

The invasive fungal pathogen Cronartium ribicola infects and kills whitebark pine (Pinus albicaulis) throughout western North America. Whitebark pine has been proposed for listing under the Endangered Species Act in the USA, and the loss of this species is predicted to have severe impacts on ecosystem composition and function in high‐elevation forests. Numerous fungal endophytes live inside whitebark pine tissues and may influence the severity of C. ribicola infection, either directly by inhibition of pathogen growth or indirectly by the induction of chemical defensive pathways in the tree. Terpenes, a form of chemical defence in pine trees, can also influence disease. In this study, we characterized fungal endophyte communities in whitebark pine seedlings before and after experimental inoculation with C. ribicola, monitored disease progression and compared fungal community composition in susceptible vs. resistant seedlings in a common garden. We analysed the terpene composition of these same seedlings. Seed family identity or maternal genetics influenced both terpenes and endophyte communities. Terpene and endophyte composition correlated with disease severity, and terpene concentrations differed in resistant vs. susceptible seedlings. These results suggest that the resistance to C. ribicola observed in natural whitebark pine populations is caused by the combined effects of genetics, endophytes and terpenes within needle tissue, in which initial interactions between microbes and hosts take place. Tree genotype, terpene and microbiome combinations associated with healthy trees could help to predict or reduce disease severity and improve outcomes of future tree breeding programmes.     

Foliar fungal endophyte community structure is independent of phylogenetic relatedness in an Asteraceae common garden

Phylogenetic distance among host species represents a proxy for host traits that act as biotic filters to shape host‐associated microbiome community structure. However, teasing apart potential biotic assembly mechanisms, such as host specificity or local species interactions, from abiotic factors, such as environmental specificity or dispersal barriers, in hyperdiverse, horizontally transmitted microbiomes remains a challenge. In this study, we tested whether host phylogenetic relatedness among 18 native Asteraceae plant species and spatial distance between replicated plots in a common garden affects foliar fungal endophyte (FFE) community structure. We found that FFE community structure varied significantly among host species, as well as host tribes, but not among host subfamilies. However, FFE community dissimilarity between host individuals was not significantly correlated with phylogenetic distance between host species. There was a significant effect of spatial distance among host individuals on FFE community dissimilarity within the common garden. The significant differences in FFE community structure among host species, but lack of a significant host phylogenetic effect, suggest functional differences among host species not accounted for by host phylogenetic distance, such as metabolic traits or phenology, may drive FFE community dissimilarity. Overall, our results indicate that host species identity and the spatial distance between plants can determine the similarity of their microbiomes, even across a single experimental field, but that host phylogeny is not closely tied to FFE community divergence in native Asteraceae.     

Community composition of root‐associated fungi in a Quercus‐dominated temperate forest: “codominance” of mycorrhizal and root‐endophytic fungi

In terrestrial ecosystems, plant roots are colonized by various clades of mycorrhizal and endophytic fungi. Focused on the root systems of an oak‐dominated temperate forest in Japan, we used 454 pyrosequencing to explore how phylogenetically diverse fungi constitute an ecological community of multiple ecotypes. In total, 345 operational taxonomic units (OTUs) of fungi were found from 159 terminal‐root samples from 12 plant species occurring in the forest. Due to the dominance of an oak species (Quercus serrata), diverse ectomycorrhizal clades such as Russula, Lactarius, Cortinarius, Tomentella, Amanita, Boletus, and Cenococcum were observed. Unexpectedly, the root‐associated fungal community was dominated by root‐endophytic ascomycetes in Helotiales, Chaetothyriales, and Rhytismatales. Overall, 55.3% of root samples were colonized by both the commonly observed ascomycetes and ectomycorrhizal fungi; 75.0% of the root samples of the dominant Q. serrata were so cocolonized. Overall, this study revealed that root‐associated fungal communities of oak‐dominated temperate forests were dominated not only by ectomycorrhizal fungi but also by diverse root endophytes and that potential ecological interactions between the two ecotypes may be important to understand the complex assembly processes of belowground fungal communities.     

Species abundance distributions and richness estimations in fungal metagenomics--lessons learned from community ecology

Results of diversity and community ecology studies strongly depend on sampling depth. Completely surveyed communities follow log-normal distribution, whereas power law functions best describe incompletely censused communities. It is arguable whether the statistics behind those theories can be applied to voluminous next generation sequencing data in microbiology by treating individual DNA sequences as counts of molecular taxonomic units (MOTUs). This study addresses the suitability of species abundance models in three groups of plant-associated fungal communities - phyllosphere, ectomycorrhizal and arbuscular mycorrhizal fungi. We tested the impact of differential treatment of molecular singletons on observed and estimated species richness and species abundance distribution models. The arbuscular mycorrhizal community of 48 MOTUs was exhaustively sampled and followed log-normal distribution. The ectomycorrhizal (153 MOTUs) and phyllosphere (327 MOTUs) communities significantly differed from log-normal distribution. The fungal phyllosphere community in particular was clearly undersampled. This undersampling bias resulted in strong sensitivity to the exclusion of molecular singletons and other rare MOTUs that may represent technical artefacts. The analysis of abundant (core) and rare (satellite) MOTUs clearly identified two species abundance distributions in the phyllosphere data - a log-normal model for the core group and a log-series model for the satellite group. The prominent log-series distribution of satellite phyllosphere fungi highlighted the ecological significance of an infrequent fungal component in the phyllosphere community.     

城乡不同绿地中毛白杨土壤及根系真菌群落结构研究

土壤微生物在土壤养分循环和能量流动等生物过程中起着重要的作用,并且能敏感地响应环境条件的变化。本研究以天津市宝坻区毛白杨Populus tomentosa为研究对象,探究3种不同环境条件(社区绿地CG、公园绿地PG和高速公路旁绿地HG)对毛白杨土壤和根系真菌群落结构的影响。研究结果表明,3个绿地间土壤真菌Shannon多样性指数存在显著差异(HG>CG>PG);但根系真菌Shannon多样性指数无显著差异;此外,不同环境条件对真菌群落的组成起着重要作用。毛白杨土壤和根系真菌群落组成主要为丝盖伞属Inocybe、地孔块菌属Geopora、被孢霉属Mortierella、棉革菌属Tomentella、蓝状菌属Talaromyces、块菌属Tuber、层腹菌属Hymenogaster、链格孢属Alternaria、革菌属Thelephora、克努夫菌属Knufia和桩菇菌属Paxillus等,且差异分析表明丝盖伞属Inocybe、棉革菌属Tomentella和地孔块菌属Geopora的相对多度在3个样地中差异显著。比较分析土壤和根系样品发现土壤真菌群落α多样性显著高于根系真菌群落,两部位的真菌群落结构存在显著差异,块菌属Tuber的相对多度在根系样品中显著高于土壤样品。真菌群落功能预测表明腐生功能类群在不同环境条件下和不同部位间均存在显著差异。研究结果阐述了环境变化对土壤微生物群落中真菌组的影响,为森林生态系统的保护和林地可持续发展提供重要理论依据。     

黄山典型植被类型土壤真菌群落特征及其影响因素

黄山地势高差明显,植被类型多样,生态系统保存完整,是研究森林生态系统土壤真菌群落的天然实验室。本研究采集黄山典型植被下土壤样本,利用Illumina NovaSeq高通量测序技术分析土壤真菌群落结构,结合土壤理化性质探讨不同植被类型影响真菌群落组成的潜在因素。结果共检测到13个真菌门,优势真菌门依次为：担子菌门Basidiomycota,获得38目,202属,相对丰度介于7.30%-90.71%,在常绿落叶阔叶混交林、山地矮林及落叶阔叶林中出现高值,局部呈现先增后减的单峰变化格局;子囊菌门Ascomycota有56目,393属,相对丰度介于4.69%-53.07%,随着典型植被类型变化无明显变化规律;被孢霉门Mortierellomycota获得1目和2属,相对丰度介于2.88%-29.92%,随着典型植被类型变化呈现U型变化模式;5种植被类型土壤中共检测到34个不同分类单元的真菌指示类群,落叶阔叶林土壤真菌指示类群最为丰富,占67%;pH显著影响土壤真菌α多样性（Pearson,P<0.001）,是黄山土壤真菌群落变异的主控因子（Monte Corlo 检验,P<0.01）。     

Specificity of fungal associations of Pyroleae and Monotropa hypopitys during germination and seedling development

Mycoheterotrophic plants obtain organic carbon from associated mycorrhizal fungi, fully or partially. Angiosperms with this form of nutrition possess exceptionally small ‘dust seeds’ which after germination develop ‘seedlings’ that remain subterranean for several years, fully dependent on fungi for supply of carbon. Mycoheterotrophs which as adults have photosynthesis thus develop from full to partial mycoheterotrophy, or autotrophy, during ontogeny. Mycoheterotrophic plants may represent a gradient of variation in a parasitism–mutualism continuum, both among and within species. Previous studies on plant–fungal associations in mycoheterotrophs have focused on either germination or the adult life stages of the plant. Much less is known about the fungal associations during development of the subterranean seedlings. We investigated germination and seedling development and the diversity of fungi associated with germinating seeds and subterranean seedlings (juveniles) in five Monotropoideae (Ericaceae) species, the full mycoheterotroph Monotropa hypopitys and the putatively partial mycoheterotrophs Pyrola chlorantha, P. rotundifolia, Moneses uniflora and Chimaphila umbellata. Seedlings retrieved from seed sowing experiments in the field were used to examine diversity of fungal associates, using pyrosequencing analysis of ITS2 region for fungal identification. The investigated species varied with regard to germination, seedling development and diversity of associated fungi during juvenile ontogeny. Results suggest that fungal host specificity increases during juvenile ontogeny, most pronounced in the fully mycoheterotrophic species, but a narrowing of fungal associates was found also in two partially mycoheterotrophic species. We suggest that variation in specificity of associated fungi during seedling ontogeny in mycoheterotrophs represents ongoing evolution along a parasitism–mutualism continuum.     

Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐rDNA in Environmental Microbial Eukaryotic Diversity Surveys

High‐throughput sequencing platforms are continuing to increase resulting read lengths, which is allowing for a deeper and more accurate depiction of environmental microbial diversity. With the nascent Reagent Kit v3, Illumina MiSeq now has the ability to sequence the eukaryotic hyper‐variable V4 region of the SSU‐rDNA locus with paired‐end reads. Using DNA collected from soils with analyses of strictly‐ and nearly identical amplicons, here we ask how the new Illumina MiSeq data compares with what we can obtain with Roche/454 GS FLX with regard to quantity and quality, presence and absence, and abundance perspectives. We show that there is an easy qualitative transition from the Roche/454 to the Illumina MiSeq platforms. The ease of this transition is more nuanced quantitatively for low‐abundant amplicons, although estimates of abundances are known to also vary within platforms.     

神农架国家公园林线过渡带土壤真菌多样性

林线过渡带是陆地生态系统对气候变化响应的敏感区域,研究林线过渡带土壤真菌的群落结构和形成机制,对于预测气候变化对土壤养分循环和维持陆地生态系统功能的影响具有重要意义。利用Illumina高通量测序技术分析了神农架国家公园林线上下的灌木林和针叶林的土壤真菌群落结构和多样性。结果表明,在真菌物种组成上,两种植被类型的土壤优势菌门、属和种类不同,针叶林和灌木林的优势菌门分别是担子菌门(Basidiomycota)和接合菌门(Zygomycota)。除趋势对应分析(DCA)和不相似性检验(Dissimilarity test)表明两种林型的土壤真菌群落结构组成存在显著差异,且针叶林土壤真菌Shannon指数、Chao值和Richness指数均显著(P0.05)高于灌木林。典范对应分析(CCA)和Mantel检验显示土壤真菌群落结构与土壤p H、植物多样性、土壤温度和土壤湿度存在显著相关性。因此,林线过渡带上下的土壤真菌群落结构和多样性存在显著差异,土壤p H、植物多样性、土壤湿度和土壤温度可能是影响土壤真菌群落结构的重要因素。     

比较形态学、聚丙酰胺凝胶电泳与二代测序在AM真菌群落研究中的应用

采集内蒙古荒漠草原沙化梁地羊柴Hedysarum laeve根围土样,采用形态学方法、聚丙酰胺凝胶电泳（denatured gradient gel electrophoresis,DGGE）技术和二代测序（next generation sequencing,NGS）技术分析羊柴根围AM真菌群落组成和多样性。结果表明,应用NGS共鉴定AM真菌3纲4目10科18属,DGGE鉴定2纲2目2科5属,形态学鉴定1纲2目2科3属。AM真菌分类等级越低,NGS、DGGE和形态学鉴定结果差异越大,尤其在属水平上差异显著。与NGS技术相比,形态学和DGGE方法所反映AM真菌种类主要为优势菌种,显著低估了AM真菌物种组成并高估其丰度,分子测序是对形态学分类的补充和完善。     

Vertical and Horizontal Distribution of Bacterial Communities in Alluvial Groundwater of the Nakdong River Bank

As a collaborative effort to characterize a pilot test site for managed aquifer recharge (MAR), vertical and horizontal distributions of microbial communities in the river bank subsurface were investigated to assess the ecological effects after the operation of the MAR using the river water adjacent to the site. Along with a geochemical analysis, barcoded pyrosequencing was performed using the genomic DNAs extracted from the subsurface groundwater/sediment samples retrieved from three multilevel wells among the installed cluster of 14 boreholes. A total of 9 samples from 3 depths (10, 15–25, and 33 m below the ground surface) of each borehole showed higher bacterial abundance and diversity in the shallow (10 m) depths than in the deep (33 m) groundwater. In addition, there was a slight separation of the microbial communities between the depths based on the nonmetric multidimensional scaling analysis of the Yue and Clayton distance and the distance-weighted UniFrac analysis. The phylum Proteobacteria was dominant in all the samples at the sequence abundance of 64.0–97.8% with the total operational taxonomic units of 3375 at the species level, while among the total 288 genera, the genus Pseudomonas and an unclassified genus from Betaproteobacteria were the most abundant across the samples. The community separation between the shallow and the deep groundwater seemed to be correlated with depth differences, supported by differences in the dissolved oxygen (DO) concentration and oxidation-reduction potential (ORP). In the study site, unusually high values of electrical conductivity (EC) were found in the deep groundwater, but those values were unlikely to contribute to the community separation between the shallow and deep groundwater, unlike the DO and ORP values, which were found to influence the community differences.     

SNP genotyping for detecting the ‘rare allele phenomenon’ in hybrid zones

Hybrid zones are regions where genetically distinct populations meet, mate and produce offspring. In such zones, genetically less compatible gene combinations are usually generated, resulting in reduced fitness, and hybrid zones are often maintained because of continuous removal of unfit genotypes, balanced by gene flow into the zone from the parental populations (and are then referred to as ‘tension zones’). Tension zones often display unexpectedly high frequencies of gene variants that are rare outside the zone. Previous work has shown that this ‘rare allele phenomenon’ is not the result of intragenic recombination or increased mutation rates. Further understanding of the population genetics of the phenomenon requires an approach in which both the numbers of individuals and the numbers of loci is increased. Here, we report an approach using a combination of Illumina next‐generation sequencing and mass spectrophotometer genotyping to identify markers that may be used for genome‐wide investigations of the rare allele phenomenon. We test this approach on a hybrid zone in the land snail Albinaria hippolyti from Greece.     

Temporal variation of Bistorta vivipara‐associated ectomycorrhizal fungal communities in the High Arctic

Ectomycorrhizal (ECM) fungi are important for efficient nutrient uptake of several widespread arctic plant species. Knowledge of temporal variation of ECM fungi, and the relationship of these patterns to environmental variables, is essential to understand energy and nutrient cycling in Arctic ecosystems. We sampled roots of Bistorta vivipara ten times over two years; three times during the growing‐season (June, July and September) and twice during winter (November and April) of both years. We found 668 ECM OTUs belonging to 25 different ECM lineages, whereof 157 OTUs persisted throughout all sampling time‐points. Overall, ECM fungal richness peaked in winter and species belonging to Cortinarius, Serendipita and Sebacina were more frequent in winter than during summer. Structure of ECM fungal communities was primarily affected by spatial factors. However, after accounting for spatial effects, significant seasonal variation was evident revealing correspondence with seasonal changes in environmental conditions. We demonstrate that arctic ECM richness and community structure differ between summer (growing‐season) and winter, possibly due to reduced activity of the core community, and addition of fungi adapted for winter conditions forming a winter‐active fungal community. Significant month × year interactions were observed both for fungal richness and community composition, indicating unpredictable between‐year variation. Our study indicates that addressing seasonal changes requires replication over several years.     

Exploring the accuracy of amplicon‐based internal transcribed spacer markers for a fungal community

With the continual improvement in high‐throughput sequencing technology and constant updates to fungal reference databases, the use of amplicon‐based DNA markers as a tool to reveal fungal diversity and composition in various ecosystems has become feasible. However, both primer selection and the experimental procedure require meticulous verification. Here, we computationally and experimentally evaluated the accuracy and specificity of three widely used or newly designed internal transcribed spacer (ITS) primer sets (ITS1F/ITS2, gITS7/ITS4 and 5.8S‐Fun/ITS4‐Fun). In silico evaluation revealed that primer coverage varied at different taxonomic levels due to differences in degeneracy and the location of primer sets. Using even and staggered mock community standards, we identified different proportions of chimeric and mismatch reads generated by different primer sets, as well as great variation in species abundances, suggesting that primer selection would affect the results of amplicon‐based metabarcoding studies. Choosing proofreading and high‐fidelity polymerase (KAPA HiFi) could significantly reduce the percentage of chimeric and mismatch sequences, further reducing inflation of operational taxonomic units. Moreover, for two types of environmental fungal communities, plant endophytic and soil fungi, it was demonstrated that the three primer sets could not reach a consensus on fungal community composition or diversity, and that primer selection, not experimental treatment, determines observed soil fungal community diversity and composition. Future DNA marker surveys should pay greater attention to potential primer effects and improve the experimental scheme to increase credibility and accuracy.     

Decrease in diversity and changes in community composition of arbuscular mycorrhizal fungi in roots of apple trees with increasing orchard management intensity across a regional scale

Understanding which factors drive the diversity and community composition of arbuscular mycorrhizal fungi (AMF) is important due to the role of these soil micro‐organisms in ecosystem functioning and current environmental threats to AMF biodiversity. Additionally, in agro‐ecosystems, this knowledge may help to evaluate their use in making agriculture more sustainable. Here, we used 454‐pyrosequencing of small subunit rRNA gene amplicons to quantify AMF diversity and community composition in the roots of cultivated apple trees across 24 orchards in central Belgium. We aimed at identifying the factors (soil chemical variables, organic vs. conventional farming, and geographical location) that affect AMF diversity and community composition. In total, 110 AMF OTUs were detected, of which the majority belonged to the Glomeraceae (73%) and the Claroideoglomeraceae (19%). We show that soil characteristics and farming system, rather than the geographical location of the orchards, shape AMF communities on apple trees. Particularly, plant‐available P content of the soil was associated with lower AMF diversity. In orchards with a lower plant‐available P content of the soil (P < 100 mg/kg soil), we also found a significantly higher AMF diversity in organically managed orchards as compared to conventionally managed orchards. Finally, the degree of nestedness of the AMF communities was related to plant‐available P and N content of the soil, pointing at a progressive loss of AMF taxa with increasing fertilization. Overall, we conclude that a combination of organic orchard management and moderate fertilization may preserve diverse AMF communities on apple trees and that AMF in the roots of apple trees appear not to be dispersal limited at the scale of central Belgium.     

Summer season and long-term drought increase the richness of bacteria and fungi in the foliar phyllosphere of Quercus ilex in a mixed Mediterranean forest

We explored the changes in richness, diversity and evenness of epiphytic (on the leaf surface) and endophytic (within leaf tissues) bacteria and fungi in the foliar phyllosphere of Quercus ilex, the dominant tree species of Mediterranean forests. Bacteria and fungi were assessed during ontogenic development of the leaves, from the wet spring to the dry summer season in control plots and in plots subjected to drought conditions mimicking those projected for future decades. Our aim was to monitor succession in microbiota during the colonisation of plant leaves and its response to climate change. Ontogeny and seasonality exerted a strong influence on richness and diversity of the microbial phyllosphere community, which decreased in summer in the whole leaf and increased in summer in the epiphytic phyllosphere. Drought precluded the decrease in whole leaf phyllosphere diversity and increased the rise in the epiphytic phyllosphere. Both whole leaf bacterial and fungal richness decreased with the decrease in physiological activity and productivity of the summer season in control trees. As expected, the richness of epiphytic bacteria and fungi increased in summer after increasing time of colonisation. Under summer dry conditions, there was a positive relationship between TRF (terminal restriction fragments) richness and drought, both for whole leaf and epiphytic phyllosphere, and especially for fungal communities. These results demonstrate that changes in climate are likely to significantly alter microbial abundance and composition of the phyllosphere. Given the diverse functions and large number of phyllospheric microbes, the potential functional implications of such community shifts warrant exploration.     

南亚热带乡土树种与桉树人工林土壤真菌群落多样性和功能类群的比较

营造乡土树种人工林和桉树人工林是我国南亚热带森林经营的常见模式。为探究土壤真菌群落多样性及功能对乡土树种和桉树人工林的响应特征与机制,该研究以南亚热带4个乡土树种人工林[马尾松(Pinus massoniana)、火力楠(Michelia macclurei)、米老排(Mytilaria laosensis)、红锥(Castanopsis hystrix)]和外来树种尾巨桉(Eucalyptus urophylla × E. grandis)人工林为对象,基于各林分土壤(0～20 cm)真菌18S rRNA高通量测序数据,利用FUNGuild数据库,比较分析乡土树种与尾巨桉人工林土壤真菌群落多样性和功能类群的差异特性及影响的主导土壤环境因子。结果表明:(1)5个研究林分的土壤真菌优势门均为子囊菌门和担子菌门,但不同乡土树种林分与尾巨桉林的土壤真菌优势目存在差异。(2)尾巨桉林土壤真菌群落α多样性高于乡土树种人工林,其群落组成结构也与乡土树种人工林存在显著差异(P<0.05)。(3)4个乡土树种人工林土壤的腐生营养型的相对丰度高于尾巨桉林,并且火力楠林和米老排林土壤丛枝菌根真菌的相对丰度明显高于尾巨桉林,尾巨桉林土壤共生营养型以及外生菌根真菌和木材腐生菌的相对丰度明显高于乡土树种人工林。(4)pH是导致尾巨桉林与乡土树种人工林土壤真菌群落多样性和功能类群差异的主要土壤环境因子。综上认为,在南亚热带地区将尾巨桉林改建成火力楠林或米老排林可提高土壤养分水平,提升土壤生态功能。