1. |
- Bengtsson-Palme, Johan, 1985, et al.
(författare)
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Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data
- 2013
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Ingår i: Methods in Ecology and Evolution. - 2041-210X. ; 4:10, s. 914-919
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and blast searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 – as well as full-length ITS sequences – from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. ITSx paves the way for more sensitive blast searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.
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2. |
- Bengtsson-Palme, Johan, 1985, et al.
(författare)
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Megraft: A software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes
- 2012
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Ingår i: Research in Microbiology. - : Elsevier BV. - 0923-2508. ; 163:6-7, s. 407-412
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Tidskriftsartikel (refereegranskat)abstract
- Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of a community sequencing effort, rarefaction analysis of the ribosomal small subunit (SSU/16S/18S) gene in the metagenome is usually performed. The fragmentary, non-overlapping nature of SSU sequences in metagenomic libraries poses a problem for this analysis, however. We introduce a software package – Megraft – that grafts SSU fragments onto full-length SSU sequences, accounting for observed and unobserved variability, for accurate assessment of species richness and sequencing depth in metagenomics endeavors.
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3. |
- Eusemann, Pascal, et al.
(författare)
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Habitat conditions and phenological tree traits overrule the influence of tree genotype in the needle mycobiome–Picea glauca system at an arctic treeline ecotone
- 2016
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Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 211:4, s. 1221-1231
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Tidskriftsartikel (refereegranskat)abstract
- Plant-associated mycobiomes in extreme habitats are understudied and poorly understood. We analysed Illumina-generated ITS1 sequences from the needle mycobiome of white spruce (Picea glauca) at the northern treeline in Alaska (USA). Sequences were obtained from the same DNA that was used for tree genotyping. In the present study, fungal metabarcoding and tree microsatellite data were compared for the first time. In general, neighbouring trees shared more fungal taxa with each other than trees growing in further distance. Mycobiomes correlated strongly with phenological host traits and local habitat characteristics contrasting a dense forest stand with an open treeline site. Genetic similarity between trees did not influence fungal composition and no significant correlation existed between needle mycobiome and tree genotype. Our results suggest the pronounced influence of local habitat conditions and phenotypic tree traits on needle-inhabiting fungi. By contrast, the tree genetic identity cannot be benchmarked as a dominant driver for needle-inhabiting mycobiomes, at least not for white spruce in this extreme environment.
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4. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
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A comprehensive, automatically updated fungal ITS sequence dataset for reference-based chimera control in environmental sequencing efforts
- 2015
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Ingår i: Microbes and Environments. - 1342-6311 .- 1347-4405. ; 30:2, s. 145-150
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric—artificially joined—DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.
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5. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
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A comprehensive, automatically updated fungal ITS sequence dataset for reference-based chimera control in environmental sequencing efforts
- 2015
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Ingår i: Microbes and Environments. - 1342-6311 .- 1347-4405. ; 30:2, s. 145-150
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric—artificially joined—DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.
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6. |
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7. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
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Improving ITS sequence data for identification of plant pathogenic fungi
- 2014
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Ingår i: Fungal Diversity. - : Springer Science and Business Media LLC. - 1560-2745 .- 1878-9129. ; 67:1, s. 11-19
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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8. |
- Abarenkov, Kessy, et al.
(författare)
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Annotating public fungal ITS sequences from the built environment according to the MIxS-Built Environment standard – a report from a May 23-24, 2016 workshop (Gothenburg, Sweden)
- 2016
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Ingår i: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; 16, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Recent molecular studies have identified substantial fungal diversity in indoor environments. Fungi and fungal particles have been linked to a range of potentially unwanted effects in the built environment, including asthma, decay of building materials, and food spoilage. The study of the built mycobiome is hampered by a number of constraints, one of which is the poor state of the metadata annotation of fungal DNA sequences from the built environment in public databases. In order to enable precise interrogation of such data – for example, “retrieve all fungal sequences recovered from bathrooms” – a workshop was organized at the University of Gothenburg (May 23-24, 2016) to annotate public fungal barcode (ITS) sequences according to the MIxS-Built Environment annotation standard (http://gensc.org/mixs/). The 36 participants assembled a total of 45,488 data points from the published literature, including the addition of 8,430 instances of countries of collection from a total of 83 countries, 5,801 instances of building types, and 3,876 instances of surface-air contaminants. The results were implemented in the UNITE database for molecular identification of fungi (http://unite.ut.ee) and were shared with other online resources. Data obtained from human/animal pathogenic fungi will furthermore be verified on culture based metadata for subsequent inclusion in the ISHAM-ITS database (http://its.mycologylab.org).
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9. |
- Albrectsen, Benedicte Riber, 1960-, et al.
(författare)
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Both plant genotype and herbivory shape aspen endophyte communities
- 2018
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Ingår i: Oecologia. - : Springer. - 0029-8549 .- 1432-1939. ; 187:2, s. 535-545
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Tidskriftsartikel (refereegranskat)abstract
- Salicinoid phenolic glycosides are common defence substances in salicaceous trees and specialist leaf beetles use these compounds for their own defence against predators. Salicinoids vary qualitatively and qualitatively in aspen (Populus tremula) and this variation has a genetic basis. The foliar endophyte mycobiome is plentiful and we hypothesised that it is related to plant genotype, potentially mediated by salicinoid composition, and that interactions with the leaf beetle Chrysomela tremula may alter this relationship. We studied these three-way interactions in controlled greenhouse experiments. Endophytic fungi were isolated from sterilised leaf tissues with and without beetle damage, and from beetles. We confirmed that endophyte composition was influenced by host genotype. Beetle activity added generalist morphs to the mycobiome that overrode the initial host association. Yeast-like genera (Cryptococcus and Rhodotorula) were isolated only from beetle-damaged tissues and from beetles, whereas fast-growing filamentous fungi dominated beetle-free control plants. Competition experiments between filamentous fungi of plant origin and beetle-related yeasts suggested interaction of both stimulating and inhibiting modes of action amongst the fungi. As a result, we detected examples of amensalism, commensalism, parasitism and competition between the morphs tested, but we found no evidence of mutualism, and consequently no co-evolutionary relationship could be demonstrated, between yeasts carried by beetles, host genotype and associated filamentous morphs. Endophyte studies are method-dependent and high-throughput sequencing technology best define the fungal mycobiome, culturing however continues to be a cheap way to provide fundamental ecological insights and it is also required for experimental studies.
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10. |
- Begerow, D., et al.
(författare)
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Current state and perspectives of fungal DNA barcoding and rapid identification procedures
- 2010
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 87:1, s. 99-108
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Forskningsöversikt (refereegranskat)abstract
- Fungal research is experiencing a new wave of methodological improvements that most probably will boost mycology as profoundly as molecular phylogeny has done during the last 15 years. Especially the next generation sequencing technologies can be expected to have a tremendous effect on fungal biodiversity and ecology research. In order to realise the full potential of these exciting techniques by accelerating biodiversity assessments, identification procedures of fungi need to be adapted to the emerging demands of modern large-scale ecological studies. But how should fungal species be identified in the near future? While the answer might seem trivial to most microbiologists, taxonomists working with fungi may have other views. In the present review, we will analyse the state of the art of the so-called barcoding initiatives in the light of fungi, and we will seek to evaluate emerging trends in the field. We will furthermore demonstrate that the usability of DNA barcoding as a major tool for identification of fungi largely depends on the development of high-quality sequence databases that are thoroughly curated by taxonomists and systematists.
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11. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
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Taxonomic annotation of public fungal ITS sequences from the built environment - a report from an April 10-11, 2017 workshop (Aberdeen, UK)
- 2018
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Ingår i: Mycokeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; :28, s. 65-82
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Tidskriftsartikel (refereegranskat)abstract
- Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi - whether transient visitors or more persistent residents - may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxonomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions - such as country and host/substrate of collection - are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10-11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS bar-code sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes - including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences - were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.
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12. |
- Siddique, Abu Bakar, et al.
(författare)
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Mycobiomes of Young Beech Trees Are Distinguished by Organ Rather Than by Habitat, and Community Analyses Suggest Competitive Interactions Among Twig Fungi
- 2021
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Beech trees (Fagus sylvatica) are prominent keystone species of great economic and environmental value for central Europe, hosting a diverse mycobiome. The composition of endophyte communities may depend on tree health, plant organ or tissue, and growth habitat. To evaluate mycobiome communalities at local scales, buds, and twigs were sampled from two young healthy mountain beech stands in Bavaria, Germany, four kilometers apart. With Illumina high-throughput sequencing, we found 113 fungal taxa from 0.7 million high-quality reads that mainly consisted of Ascomycota (52%) and Basidiomycota (26%) taxa. Significant correlations between richness and diversity indices were observed (p < 0.05), and mycobiomes did not differ between habitats in the current study. Species richness and diversity were higher in twigs compared to spring buds, and the assemblages in twigs shared most similarities. Interaction network analyses revealed that twig-bound fungi shared similar numbers of (interaction) links with others, dominated by negative co-occurrences, suggesting that competitive exclusion may be the predominant ecological interaction in the highly connected twig mycobiome. Combining community and network analyses strengthened the evidence that plant organs may filter endophytic communities directly through colonization access and indirectly by facilitating competitive interactions between the fungi.
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13. |
- Siddique, Abu Bakar, First research engineer, et al.
(författare)
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What do we learn from cultures in the omics age? High-throughput sequencing and cultivation of leaf-inhabiting endophytes from beech (Fagus sylvatica L.) revealed complementary community composition but similar correlations with local habitat conditions
- 2017
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Ingår i: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; 20, s. 1-16
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Tidskriftsartikel (refereegranskat)abstract
- Comparative simultaneous studies of environmental high-throughput sequencing (HTS) and cultivation of plant-associated fungi have rarely been conducted in the past years. For the present contribution, HTS and extinction culturing were applied for the same leaf samples of European beech (Fagus sylvatica) in order to trace both “real” environmental drivers as well as method-dependent signals of the observed mycobiomes. Both approaches resulted in non-overlapping community composition and pronounced differences in taxonomic classification and trophic stages. However, both methods revealed similar correlations of the fungal communities with local environmental conditions. Our results indicate undeniable advantages of HTS over cultivation in terms of revealing a good representation of the major functional guilds, rare taxa and biodiversity signals of leaf-inhabiting fungi. On the other hand our results demonstrate that the immense body of literature about cultivable endophytic fungi can and should be used for the interpretation of community signals and environmental correlations obtained from HTS studies and that cultivation studies should be continued at the highest standards, e.g. when sequencing facilities are not available or if such surveys are expanded into functional aspects with experiments on living isolates.
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14. |
- Unterseher, Martin, et al.
(författare)
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Diversity and Composition of the Leaf Mycobiome of Beech (Fagus sylvatica) Are Affected by Local Habitat Conditions and Leaf Biochemistry
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- Comparative investigations of plant-associated fungal communities (mycobiomes) in distinct habitats and under distinct climate regimes have been rarely conducted in the past. Nowadays, high-throughput sequencing allows routine examination of mycobiome responses to environmental changes and results at an unprecedented level of detail. In the present study, we analysed Illumina-generated fungal ITS1 sequences from European beech (Fagus sylvatica) originating from natural habitats at two different altitudes in the German Alps and from a managed tree nursery in northern Germany. In general, leaf-inhabiting mycobiome diversity and composition correlated significantly with the origin of the trees. Under natural condition the mycobiome was more diverse at lower than at higher elevation, whereas fungal diversity was lowest in the artificial habitat of the tree nursery. We further identified significant correlation of leaf chlorophylls and flavonoids with both habitat parameters and mycobiome biodiversity. The present results clearly point towards a pronounced importance of local stand conditions for the structure of beech leaf mycobiomes and for a close interrelation of phyllosphere fungi and leaf physiology.
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15. |
- Unterseher, Martin, et al.
(författare)
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Mycobiomes of sympatric Amorphophallus albispathus (Araceae) and Camellia sinensis (Theaceae) – a case study reveals clear tissue preferences and differences in diversity and composition
- 2018
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Ingår i: Mycological Progress. - : Springer Science and Business Media LLC. - 1617-416X .- 1861-8952. ; 17:4, s. 489-500
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Tidskriftsartikel (refereegranskat)abstract
- Multiple biotic and abiotic parameters influence the dynamics of individual fungal species and entire communities. Major drivers for tropical plant endophytes are undoubtedly seasonality, local habitat conditions and biogeography. However, host specialization and tissue preferences also contribute to the structuring of endophytic mycobiomes. To elucidate such specializations and preferences, we sampled two commercially important, unrelated plant species, Amorphophallus albispathus and Camellia sinensis (tea plant) simultaneously at close proximity. The mycobiomes of different tissue types were assessed with high-throughput amplicon sequencing of the internal transcribed spacer DNA region. Both plants hosted different fungal communities and varied in α- and β-diversity, despite their neighboring occurrence. However, the fungal assemblages of Amorphophallus leaflets shared taxa with the mycobiomes of tea leaves, thereby suggesting common driving forces for leaf-inhabiting fungi irrespective of host plant identity. The mycobiome composition and diversity of tea leaves was clearly driven by leaf age. We suggest that the very youngest tea leaves are colonized by stochastic processes, while mycobiomes of old leaves are rather similar as the result of progressive succession. The biodiversity of fungi associated with A. albispathus was characterized by a large number of unclassified OTUs (at genus and species level) and by tissue-specific composition.This study is the first cultivation-independent high-throughput assessment of fungal biodiversity of an Amorphophallus species, and additionally expands the knowledge base on fungi associated with tea plants.
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