Corresponding author: Abu Bakar Siddique ( firstname.lastname@example.org )
Academic editor: Imke Schmitt
© 2017 Abu Bakar Siddique, Anis Mahmud Khokon, Martin Unterseher.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Siddique AB, Khokon AM, Unterseher M (2017) 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. MycoKeys 20: 1-16. https://doi.org/10.3897/mycokeys.20.11265
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.
Cultivation, high-throughput sequencing, metabarcoding, fungal endophytes, biodiversity
Fungal endophytes reside in the living tissues of plants without causing visible disease symptoms (e.g.
It is well recognized that interpretation of diversity of endophytes and other fungi depends on the applied methods (
To date, published studies about the comparative assessment of fungal biodiversity are rare.
In this study, we investigated endophytic phyllosphere fungi with dilution-to-extinction cultivation (and ITS barcoding) and Illumina sequencing of the same DNA region and from the same material. In accordance with existing knowledge, we expected lower OTU (operational taxonomic unit) richness in the cultivation data and a preferential isolation of ubiquitous, primary saprobic taxa. Consequently, we hypothesized that the cultivable mycobiome exhibits different ecological signals compared with the mycobiome obtained by HTS.
The samples were obtained from an experimental site established in 2013 (
Isolation of endophytes followed the dilution-to-extinction cultivation (
Instead of classifying the fungal cultures according to macroscopic and microscopic characters (
Total genomic DNA was extracted with the Charge Switch® gDNA Plant Kit (Invitrogen, Germany) from the same fresh leaf particle mass that was used for cultivation. Library preparation consisted of two consecutive amplification steps in order to add sample-specific tag combination for multiplexing. Please refer to
Demultiplexing and quality filtering of Illumina reads relied on QIIME (
The analysis of fungal biodiversity comprised the assessment of OTU richness and further indicators of diversity (Fisher’s Alpha, Shannon index and three numbers of Hill’s series of diversity, the latter considering different levels of rarity) (
Community composition was assessed with PCoA (principal coordinate analysis) and NMDS (non-metric multidimensional scaling) and tested with PERMANOVA (permutational multivariate analysis of variance). Functional guild analysis was performed according to
Data volumes differed strongly between Illumina sequencing and cultivation. Illumina sequencing resulted in 597 OTUs from 170480 curated ITS1 reads and cultivation revealed 70 OTUs from 438 culture-based Sanger sequences with the same settings for OTU clustering. The combined data set comprised 630 OTUs (+ 33 OTUs compared with Illumina data). Thirty-seven OTUs were detected with both methods (see Table S1 on Suppl. material
An insignificant trend of lower fungal diversity at the mountain site across all indexes was observed for HTS data (Fig.
Diversity indexes and accumulation curves for a Illumina and b cultivation data of fungal leaf-inhabiting endophytes of beech. Except of the accumulation curves of cultivation data, both methods revealed a clear and partly significant trend of higher fungal diversity at the valley site.
The analysis of community composition with non-metric multidimensional scaling (NMDS), principal coordinate analysis (PCoA) and PERMANOVA discovered a significant influence of the isolation method (df = 1, F = 7.58, R2 = 0.30, p= 0.001) as well as of locality (df = 1, F = 2.87, R2 = 0.14, p= 0.014) (Fig.
. Principal coordinate analysis (PCoA) of fungal leaf-inhabiting endophytes of beech display strongly differing assemblages obtained with Illumina sequencing and cultivation. Both methods revealed differing mycobiomes from valley and from mountain leaves, although these differences were less pronounced for cultivation data. Abbreviations: IM = Illumina data from mountain samples, IV = Illumina data from valley samples, CM = cultivation data from mountain samples, CV = cultivation data from valley samples
Three of the five most abundant orders from Illumina data were also most abundant in cultivation data (Capnodiales – both methods, Helotiales – both methods, Saccharomycetales – Illumina only, Pleosporales – both methods; all Ascomycota) (Fig.
The five main guilds (pathotrophs, patho-saprotrophs, patho-symbiotrophs, saprotrophs and symbiotrophs) were all detected by HTS. Cultivation largely failed to detect pathotrophs (including patho-saprotrophs and -symbiotrophs). The relative abundance of saprotrophs was clearly higher in cultivation than in HTS data (Fig.
Relative abundance of fungal leaf-inhabiting endophytes of beech among the five main trophic guilds as revealed by analysis with FUNGuild (
When analysing the influence of locality for the occurrence of different ecological guilds, it turned out that the abundance of pathotrophs was significantly higher in leaves of mountain trees than of valley trees (Fig.
Results from HTS and cultivation data were congruent in as much as saprotrophs and symbiotrophs revealed similar abundance patterns for both methods.
Relative abundance distribution of fungal leaf-inhabiting endophytes of beech among the five main trophic guilds as revealed by analysis with FUNGuild (
The most abundant orders were the same for both cultivation and HTS, namely Capnodiales, Helotiales and Pleosporales (Fig.
The compositional difference in the two mycobiomes also corresponded to the presence of parasitic taxa (Taphrinales, Erysiphales) and yeast-like fungi (Saccharomycetales and Tremellales) in the HTS data, whereas the cultivation data were devoid of fungi with obligate parasitic, biotrophic or pathogenic lifestyle. The latter guilds usually cannot be cultivated, and yeasts are often detected only during cultivation studies when growth of filamentous fungi is slowed down with low-temperature incubation. In this study, HTS retained a wide range of taxa (compare
A poor comparability of cultivation and HTS data, as it is presented here, was recently reported for a microbiome study (
On the one hand side our results clearly demonstrate the limitations and biases of cultivation approaches for comprehensive biodiversity assessments. On the other hand, the results did not meet our expectations (see above), because significant correlations to environmental parameters (here, it was the difference between valley and mountain samples) were still recognized. The present cultivation data are in concordance with similar studies (
Our results clearly justify the co-existence of cultivation and high-throughput approaches. Despite the fast improvement and diversification of HTS technologies with many undeniable advantages in microbial biodiversity assessments (
Our results suggest that the immense body of literature about cultivable endophytic fungi can and should be consulted for the interpretation of community signals obtained from HTS studies.
This study was funded by the Deutsche Forschungsgemeinschaft (DFG) as a part of the project grant to MU (UN262/9-1). The authors thank Andreas Brachmann (Munich) and Derek Peršoh (Bochum) for profound help with Illumina sequencing.