Mycobiomes of Young Beech Trees Are Distinguished by Organ Rather Than by Habitat, and Community Analyses Suggest Competitive Interactions Among Twig Fungi

Siddique, Abu Bakar; Biella, Paolo; Unterseher, Martin; Albrectsen, Benedicte Riber

doi:10.3389/fmicb.2021.646302

Cited by 9 publications

(14 citation statements)

References 95 publications

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“…As a cultivation-independent method it is frequently used to study the diversity and potential function of microbial communities [1][2][3]. Mycobiomes (or microbial fungi) in the phyllosphere of trees have been studied to investigate their association with tree performance related to plant internal and external factors such as plant genotype [4,5], organ specificity [6], and geographic and climatic parameters [7][8][9][10][11][12]. Sequencing protocols and bioinformatic pipelines that are used to dissect metagenome data are also under rapid development [1,3].…”

Section: Introductionmentioning

confidence: 99%

Optimization of Protocol for Construction of Fungal ITS Amplicon Library for High-Throughput Illumina Sequencing to Study the Mycobiome of Aspen Leaves

et al. 2022

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High-Throughput Illumina Sequencing (HTS) can be used to study metagenomes, for example, those of importance for plant health. However, protocols must be optimized according to the plant system in question, the focal microorganisms in the samples, the marker genes selected, and the number of environmental samples. We optimized the protocol for metagenomic studies of aspen leaves, originating from varied genotypes sampled across the growing season, and consequently varying in phenolic composition and in the abundance of endo- and epiphytic fungal species. We optimized the DNA extraction protocol by comparing commercial kits and evaluating five fungal ribosomal specific primers (Ps) alone, and with extended primers that allow binding to sample-specific index primers, and we then optimized the amplification with these composite Ps for 380 samples. The fungal DNA concentration in the samples varied from 561 ng/µL to 1526 ng/µL depending on the DNA extraction kit used. However, binding to phenolic compounds affected DNA quality as assessed by Nanodrop measurements (0.63–2.04 and 0.26–2.00 absorbance ratios for 260/280 and 260/230, respectively), and this was judged to be more important in making our choice of DNA extraction kit. We initially modified the PCR conditions after determining the concentration of DNA extract in a few subsamples and then evaluated and optimized the annealing temperature, duration, and number of cycles to obtain the required amplification and PCR product bands. For three specific Ps, the extended Ps produced dimers and unexpected amplicon fragments due to nonspecific binding. However, we found that the specific Ps that targeted the ITS2 region of fungal rDNA successfully amplified this region for every sample (with and without the extension PP) resulting in the desired PCR bands, and also allowing the addition of sample-specific index primers, findings which were successfully verified in a second PCR. The optimized protocol allowed us to successfully prepare an amplicon library in order to subject the intended 380 environmental samples to HTS.

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Section: Introductionmentioning

confidence: 99%

Optimization of Protocol for Construction of Fungal ITS Amplicon Library for High-Throughput Illumina Sequencing to Study the Mycobiome of Aspen Leaves

et al. 2022

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“…Fungal communities in tree foliage are not uniformly distributed or stationary ( Toju et al., 2019 ); their composition is influenced by several factors including host genotype ( Albrectsen et al., 2010b ), growth site ( Siddique et al., 2021 ), seasonal factors ( Albrectsen et al., 2010b ), ontogeny ( Bourassa et al., 2005 ; Boutigny et al., 2013a ), tissue type ( Siddique et al., 2021 ), and host-associated consumers ( Arnold et al., 2003 ; Albrectsen et al., 2010b ; Siddique et al., 2021 ). The mechanisms underpinning these relationships are not fully understood but it has been suggested that both the host’s chemical defense responses ( Ullah et al., 2017 ; Witzell et al., 2022 ) and competition between members of the mycobiome may help shape the microbial community ( Wemheuer et al., 2019 ; Siddique et al., 2021 ). In the present study, we also obtained results suggesting the occurrence of a transition in the composition of the fungal community.…”

Section: Discussionmentioning

confidence: 99%

Molecular studies of rust on European aspen suggest an autochthonous relationship shaped by genotype

Siddique

Menke²,

Dinedurga³

et al. 2023

Front. Plant Sci.

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Forests are at increasing risk from pathogen outbreak. Climate change for example enhance the risk of local disease outbreaks, and naturalization of exotic pathogens may follow human activities, warranting robust pest surveillance routines to support forest management. Melampsora pinitorqua (pine twisting rust) is of concern in Swedish forestry, and here we evaluate the use of visible rust scores (VRS) on its obligate summer host, European aspen (Populus tremula) as a tool for quantification of the pathogen. With use of species-specific primers, we could detect the native rust, but we failed to detect two exotic rusts (M. medusae and M. larici-populina). We found that aspen genotype determined the presence of fungal genetic markers (amplifying the ITS2 region of the fungal rDNA sequence) as well as DNA sequences specific to M. pinitorqua. We correlated VRS with the amount of fungal DNA in the same leaf, and we related the findings to aspen genotype-specific parameters such as the ability to synthesize and store leaf condensed tannins (CT). At the genotype level both positive and negative relationships were observed between CTs, fungal markers, and rust infestations. However, at the population level, foliar CT concentrations correlated negatively with general fungal- and rust-specific marker abundances. Our results, therefore, do not support the use of VRS to assess Melampsora infestation in Aspen. They do, however, suggest that the relationship between European aspen and rust infestation may be characterized as autochthonous in northern Sweden.

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“…Even if this approach has rarely been applied, to date, in the pollen DNA metabarcoding literature (Table 1), it was specifically developed to distinguish "true signals" from "noise" [102] in molecular biology and could constitute a promising avenue for processing data of pollen DNA metabarcoding. For instance, it has been used in other DNA-based research fields [103,104], such as for eDNA where it is proved to increase the reliability of data [105]. However, it would be promising to investigate how different parametrizations of the ROC approach would impact the estimations of cutting thresholds.…”

Section: Discussionmentioning

confidence: 99%

Harnessing the Power of Metabarcoding in the Ecological Interpretation of Plant-Pollinator DNA Data: Strategies and Consequences of Filtering Approaches

et al. 2021

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Although DNA metabarcoding of pollen mixtures has been increasingly used in the field of pollination biology, methodological and interpretation issues arise due to its high sensitivity. Filtering or maintaining false positives, contaminants, and rare taxa or molecular features could lead to different ecological results. Here, we reviewed how this choice has been addressed in 43 studies featuring pollen DNA metabarcoding, which highlighted a very high heterogeneity of filtering methods. We assessed how these strategies shaped pollen assemblage composition, species richness, and interaction networks. To do so, we compared four processing methods: unfiltering, filtering with a proportional 1% of sample reads, a fixed threshold of 100 reads, and the ROC approach (Receiver Operator Characteristic). The results indicated that filtering impacted species composition and reduced species richness, with ROC emerging as a conservative approach. Moreover, in contrast to unfiltered networks, filtering decreased network Connectance and Entropy, and it increased Modularity and Connectivity, indicating that using cut-off thresholds better describes interactions. Overall, unfiltering might compromise reliable ecological interpretations, unless a study targets rare species. We discuss the suitability of each filtering type,plead for justifying filtering strategies on biological or methodological bases and for developing shared approaches to make future studies more comparable.

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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

Cited by 9 publications

References 95 publications

Optimization of Protocol for Construction of Fungal ITS Amplicon Library for High-Throughput Illumina Sequencing to Study the Mycobiome of Aspen Leaves

Optimization of Protocol for Construction of Fungal ITS Amplicon Library for High-Throughput Illumina Sequencing to Study the Mycobiome of Aspen Leaves

Molecular studies of rust on European aspen suggest an autochthonous relationship shaped by genotype

Harnessing the Power of Metabarcoding in the Ecological Interpretation of Plant-Pollinator DNA Data: Strategies and Consequences of Filtering Approaches

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