Diversity and spatial distribution of fungal endophytes in a Eucalyptus grandis tree

Endophytic fungi of most woody trees are poorly studied due to their cryptic existence, the complexity of the community and the poor ability of traditional tools to characterize them. This study characterised the endophytic community present in commercially important Eucalyptus grandis trees in South Africa. Two different experimental approaches were used. Firstly environmental bar-coding, or metagenetics, was done using 454-pyrosequencing parallel sequencing of the barcode amplicons of all the fungal isolates present in the plants from the total DNA of the plant. Secondly, conventional DNA bar-coding of was done of fungal endophyte isolates. Isolates of the Botryosphaeriaceae family of latent endophytic pathogens were further characterised using a multi-gene phylogenetic approach from both E. grandis and related native S. cordatum that grew in close proximity. The endophytes within the three E. grandis trees were hyper diverse. A total of 1 281 Molecular Operational Taxonomic Units (MOTU) was identified based on 454- pyrosequencing of the E. grandis fungal endophyte infections. Only 85 fungal endophytic species were identified amongst isolates from one of these trees, using the conventional DNA bar-coding approach. Fifteen times more species/MOTU was thus recovered using a metagenetics compared to an isolation approach. Despite this high diversity the species accumulation curves indicate that more endophytic diversity is to be discovered. The multigene analysis of Botryosphaeriaceae isolates obtained from the E. grandis and S. cordatum trees show that three species co-infect both these hosts. Two novel host associations are also reported. This approach of verifying identities of cryptic species with appropriate multigene analyses is most likely needed for other diverse species complexes associated with these trees. A very thorough sampling strategy is required to adequately characterize the endophyte diversity in trees. The experimental approach, the 454-pyrosequening identification workflow and database described in this study will be useful to study these endophyte communities over time and space in future. Using these techniques and workflows described, questions related to host association, diversity and spatial distribution within hosts, and geographical delimitation of endophytes can be addressed.Dissertation (MSc)--University of Pretoria, 2012.GeneticsMScUnrestricte

Data from: Ion Torrent PGM as tool for fungal community analysis: a case study of endophytes in Eucalyptus grandis reveals high taxonomic diversity

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with insufficient taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths

seq.fna.zip

Sequences obtained from Ion Torrent Amplicon sequencing of the fungal community of Eucalyptus grandis in South Africa

Ion Torrent PGM as Tool for Fungal Community Analysis: A Case Study of Endophytes in<i>Eucalyptus grandis</i> Reveals High Taxonomic Diversity

<div><p>The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, <i>Eucalyptus grandis</i>, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with incomplete taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths.</p></div

Mean family richness.

<p>Mean family richness for the parameter settings (‘perfect match’, ‘fuzzy match’, ‘no primer’) and OTU sequence similarity values (90%, 95%, 97%).</p

OTU accumulation curve.

<p>Accumulation curve for OTUs of endophytic fungi of <i>Eucalyptus</i> for the parameter settings (black: ‘perfect match’; orange: ‘fuzzy match’; blue: ‘no primer’) and OTU sequence similarity values (solid: 90%; dashed: 95%; dotted: 97%). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081718#pone.0081718.s004" target="_blank">Figure S4</a> for 95% CIs.</p

Sequence quality and GC content for the different parameter settings.

<p>Black: average GC content using a sliding window approach (window size 10 bp); red: average sequence quality per base; blue: average sequence quality per base using a sliding window approach (window size 10 bp). a) ‘no primer’; b) ‘fuzzy match’; c) ‘perfect match’. Full sequence lengths before quality trimming is shown.</p

Overview of the results of the effects of the different quality and filtering approaches.

<p>*numbers in parentheses represent BLAST hits to database sequences with insufficient taxonomy.</p

Top fungal taxa recovered.

<p>The nine most abundant taxa for the three different parameter settings at a 95% OTU sequence similarity value. a) ‘no primer’; b) ‘fuzzy match’; c) ‘perfect match’.</p

Argasid and ixodid systematics: Implications for soft tick evolution and systematics, with a new argasid species list

International audienceThe systematics of the genera and subgenera within the soft tick family Argasidae is not adequately resolved. Different classification schemes, reflecting diverse schools of scientific thought that elevated or downgraded groups to genera or subgenera, have been proposed. In the most recent classification scheme, Argas and Ornithodoros are paraphyletic and the placement of various subgenera remains uncertain because molecular data are lacking. Thus, reclassification of the Argasidae is required. This will enable an understanding of soft tick systematics within an evolutionary context. This study addressed that knowledge gap using mitochondrial genome and nuclear (18S and 28S ribosomal RNA) sequence data for representatives of the subgenera Alectorobius, Argas, Chiropterargas, Ogadenus, Ornamentum, Ornithodoros, Navis (subgen. nov.), Pavlovskyella, Persicargas, Proknekalia, Reticulinasus and Secretargas, from the Afrotropical, Nearctic and Palearctic regions. Hard tick species (Ixodidae) and a new representative of Nuttalliella namaqua (Nuttalliellidae), were also se-quenced with a total of 83 whole mitochondrial genomes, 18S rRNA and 28S rRNA genes generated. The study confirmed the utility of next-generation sequencing to retrieve systematic markers. Paraphyly of Argas and Ornithodoros was resolved by systematic analysis and a new species list is proposed. This corresponds broadly with the morphological cladistic analysis of Klompen and Oliver (1993). Estimation of divergence times using molecular dating allowed dissection of phylogeographic patterns for argasid evolution. The discovery of cryptic species in the subgenera Chiropterargas, Ogadenus and Ornithodoros, suggests that cryptic speciation is common within the Argasidae. Cryptic speciation has implications for past biological studies of soft ticks. These are discussed in particular for the Ornithodoros (Ornithodoros) moubata and Ornithodoros (Ornithodoros) savignyi groups