Soil mycobiomes in native European aspen forests and hybrid aspen plantations have a similar fungal richness but different compositions, mainly driven by edaphic and floristic factors

BackgroundThe cultivation of short-rotation tree species on non-forest land is increasing due to the growing demand for woody biomass for the future bioeconomy and to mitigate climate change impacts. However, forest plantations are often seen as a trade-off between climate benefits and low biodiversity. The diversity and composition of soil fungal biota in plantations of hybrid aspen, one of the most planted tree species for short-rotation forestry in Northern Europe, are poorly studied.MethodsThe goal of this study was to obtain baseline knowledge about the soil fungal biota and the edaphic, floristic and management factors that drive fungal richness and communities in 18-year-old hybrid aspen plantations on former agricultural soils and compare the fungal biota with those of European aspen stands on native forest land in a 130-year chronosequence. Sites were categorized as hybrid aspen (17–18-year-old plantations) and native aspen stands of three age classes (8–29, 30–55, and 65-131-year-old stands). High-throughput sequencing was applied to soil samples to investigate fungal diversity and assemblages.ResultsNative aspen forests showed a higher ectomycorrhizal (EcM) fungal OTU richness than plantations, regardless of forest age. Short-distance type EcM genera dominated in both plantations and forests. The richness of saprotrophic fungi was similar between native forest and plantation sites and was highest in the middle-aged class (30–55-year-old stands) in the native aspen stands. The fungal communities of native forests and plantations were significantly different. Community composition varied more, and the natural forest sites were more diverse than the relatively homogeneous plantations. Soil pH was the best explanatory variable to describe soil fungal communities in hybrid aspen stands. Soil fungal community composition did not show any clear patterns between the age classes of native aspen stands.ConclusionWe conclude that edaphic factors are more important in describing fungal communities in both native aspen forest sites and hybrid aspen plantation sites than forest thinning, age, or former land use for plantations. Although first-generation hybrid aspen plantations and native forests are similar in overall fungal diversity, their taxonomic and functional composition is strikingly different. Therefore, hybrid aspen plantations can be used to reduce felling pressure on native forests; however, our knowledge is still insufficient to conclude that plantations could replace native aspen forests from the soil biodiversity perspective

Corrigendum for: “Oomycete-specific ITS primers for identification and metabarcoding” published in MycoKeys, doi: 10.3897/mycokeys.14.9244

The oomycete-specific ITS primers published by Riit et al. (2016) have been put to use in the scientific community working with oomycetes.  Recently, however, it has been brought to our attention that the sequences of the primers ITS1oo and ITS3oo shown in the first Figure of the published manuscript are incomplete, when compared to the sequences of the same primers as listed on the UNITE website. This discrepancy is derived from re-checking primer sequences from tube labels that are restricted to the first 18 bases.Closer examination revealed that the sequence of primer ITS1oo in Figure 1 is missing one nucleotide from the 3’ end and the primer ITS3oo is missing two nucleotides from the 3’ end. These errors are expected to reduce relative primer specificity to Oomycetes, which probably results in lower proportion of this group in metabarcoding studies. We hereby provide the updated figure (Figure 1) with correct information. We apologize to all users of these erroneous primers for their suboptimal performance. We are grateful to Dr. Diana Marčiulynienė and Dr. Sannakajsa Velmala for pointing out these problems

Oomycete-specific ITS primers for identification and metabarcoding

Microbial metabarcoding studies using high throughput sequencing technologies generate unprecedented amounts of DNA sequence data and make it possible to determine not only the composition of the communities but also the underlying factors powering the evolution of these communities. Despite the potential of community level studies in helping to better understand the ecology of pathogens and to manage the losses caused by them, very few oomycete addressing metabarcoding studies have been carried out and with highly variable results. The aim of this study was to develop new oomycete-specific ITS region PCR primers with improved specificity for metabarcoding and identification of oomycetes. The modified ITS1oo and the newly developed ITS3oo primers show improved in silico specificity for oomycetes and when paired with the universal ITS4 successfully amplified the DNA from all eleven tested oomycete species from six genera. High throughput sequencing of 20 soil samples from forest nurseries and bordering areas, using the primer pair ITS1oo/ITS4, recovered more than 400 oomycete OTUs, which is a significant increase over previous studies, and indicates the ability of the new method to detect various oomycete groups from complex substrates. The average fraction of oomycete reads per soil samples was 32–36%, with a maximum of 69%. The recovered oomycete OTUs represented the groups Lagenidiales, Peronosporales, Pythiales and Saprolegniales, with Pythiales dominating in all samples. In addition, the new primers were successfully used in identifying pathogens directly from infected plant tissues with Sanger sequencing. The pathogen was identified to the species or genus level in four samples out of six. In conclusion, the developed oomycete-specific primers provide a reliable method for the identification and metabarcoding of oomycetes

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain.publishedVersio

Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi

Rapid development of high-throughput (HTS) molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA) regions often used in metabarcoding analyses. The internal transcribed spacer (ITS) barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs) at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU) and large subunit (LSU) genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6-38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups

The effect of stump harvesting on tree growth and the infection of root rot in young Norway spruce stands in hemiboreal Estonia

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Oomycete Soil Diversity Associated with <i>Betula</i> and <i>Alnus</i> in Forests and Urban Settings in the Nordic–Baltic Region

This study aimed to determine the differences and drivers of oomycete diversity and community composition in alder- and birch-dominated park and natural forest soils of the Fennoscandian and Baltic countries of Estonia, Finland, Lithuania, Norway, and Sweden. For this, we sequenced libraries of PCR products generated from the DNA of 111 soil samples collected across a climate gradient using oomycete-specific primers on a PacBio high-throughput sequencing platform. We found that oomycete communities are most affected by temperature seasonality, annual mean temperature, and mean temperature of the warmest quarter. Differences in composition were partly explained by the higher diversity of Saprolegniales in Sweden and Norway, as both total oomycete and Saprolegniales richness decreased significantly at higher longitudes, potentially indicating the preference of this group of oomycetes for a more temperate maritime climate. None of the evaluated climatic variables significantly affected the richness of Pythiales or Peronosporales. Interestingly, the relative abundance and richness of Pythiales was higher at urban sites compared to forest sites, whereas the opposite was true for Saprolegniales. Additionally, this is the first report of Phytophthora gallica and P. plurivora in Estonia. Our results indicate that the composition of oomycetes in soils is strongly influenced by climatic factors, and, therefore, changes in climate conditions associated with global warming may have the potential to significantly alter the distribution range of these microbes, which comprise many important pathogens of plants

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain

Improving ITS sequence data for identification of plant pathogenic fungi

Plant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort