Rasmus Kjøller

Fungal community analysis by high-throughput sequencing of amplified markers – a user's guide

Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.

Towards global patterns in the diversity and community structure of ectomycorrhizal fungi

Global species richness patterns of soil micro-organisms remain poorly understood compared to macro-organisms. We use a global analysis to disentangle the global determinants of diversity and community composition for ectomycorrhizal (EcM) fungi-microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro-organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next-generation sequencing techniques and relevant soil metadata.

The production and turnover of extramatrical mycelium of ectomycorrhizal fungi in forest soils: role in carbon cycling

There is growing evidence of the importance of extramatrical mycelium (EMM) of mycorrhizal fungi in carbon (C) cycling in ecosystems. However, our understanding has until recently been mainly based on laboratory experiments, and knowledge of such basic parameters as variations in mycelial production, standing biomass and turnover as well as the regulatory mechanisms behind such variations in forest soils is limited. Presently, the production of EMM by ectomycorrhizal (EM) fungi has been estimated at ~140 different forest sites to be up to several hundreds of kg per ha per year, but the published data are biased towards Picea abies in Scandinavia. Little is known about the standing biomass and turnover of EMM in other systems, and its influence on the C stored or lost from soils. Here, focussing on ectomycorrhizas, we discuss the factors that regulate the production and turnover of EMM and its role in soil C dynamics, identifying important gaps in this knowledge. C availability seems to be the key factor determining EMM production and possibly its standing biomass in forests but direct effects of mineral nutrient availability on the EMM can be important. There is great uncertainty about the rate of turnover of EMM. There is increasing evidence that residues of EM fungi play a major role in the formation of stable N and C in SOM, which highlights the need to include mycorrhizal effects in models of global soil C stores.

Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (Single Stranded Conformation Polymorphism)

PCR amplification of a region of the large subunit ribosomal DNA sequence with Glomus specific primers was used to detect arbuscular mycorrhizal fungi in root tissue of four plant species. The primers were specific to Glomus mosseae, Glomus caledonium, Glomus geosporum, Glomus coronatum, Glomus fragilistratum and Glomus constrictum, and did not recognise sequences from Glomus claroideum. Sequence differences between isolates were detected by Single Stranded Conformation Polymorphisms (SSCPs) in polyacrylamide gels under non-denaturing conditions. Isolates of G. mosseae, G. caledonium and G. coronatum could be separated by their SSCP patterns, while three isolates of G. geosporumshowed no variation. Specific SSCP patterns from isolates of G. mosseae and G. caledonium allowed detection of both fungi in the same root segment. Sequence differences leading to variations in SSCP patterns were confirmed by direct sequencing.

Identification of mycorrhizal fungi from single pelotons of Dactylorhiza majalis (Orchidaceae) using single‐strand conformation polymorphism and mitochondrial ribosomal large subunit DNA sequences

The mitochondrial ribosomal large subunit (Ls) DNA was used to identify the orchid mycorrhizal fungi found in roots of Dactylorhiza majalis. The gene was amplified using DNA extracted from single pelotons obtained from fresh and silica gel dried roots. Furthermore, sequencing a variety of well‐characterized orchid isolates expanded the fungal database of the mitochondrial ribosomal LsDNA. Polymerase chain reaction product length variants present in D. majalis were sequenced and identified using the expanded database. These analyses revealed two different peloton‐forming fungi in samples from D. majalis, which sometimes occurred together as a single two‐taxa peloton within the same cortex cell. The first taxon belonged to the genus Tulasnella and the second taxon was distantly related to Laccaria.

Dramatic changes in ectomycorrhizal community composition, root tip abundance and mycelial production along a stand‐scale nitrogen deposition gradient

• Nitrogen (N) availability is known to influence ectomycorrhizal fungal components, such as fungal community composition, biomass of root tips and production of mycelia, but effects have never been demonstrated within the same forest. • We measured concurrently the abundance of ectomycorrhizal root tips and the production of external mycelia, and explored the changes in the ectomycorrhizal community composition, across a stand-scale N deposition gradient (from 27 to 43 kg N ha⁻¹ yr⁻¹) at the edge of a spruce forest. The N status was affected along the gradient as shown by a range of N availability indices. • Ectomycorrhizal root tip abundance and mycelial production decreased five and 10-fold, respectively, with increasing N deposition. In addition, the ectomycorrhizal fungal community changed and the species richness decreased. The changes were correlated with the measured indices of N status, in particular N deposition and N leaching. • The relationship between the altered ectomycorrhizal community, root tip abundance and mycelial production is discussed in the context of the N parameters. We suggest that increased N deposition to forests will cause large changes in ectomycorrhizal fungal community structure and functioning, which, in turn, may result in reduced N uptake by roots and fungi, and increased losses of N by leaching.

Effect of phosphate and the arbuscular mycorrhizal fungus Glomus intraradices on disease severity of root rot of peas (Pisum sativum) caused by Aphanomyces euteiches

Abstract The effects of inorganic phosphate levels and the presence of arbuscular mycorrhiza on disease severity of Aphanomyces euteiches in pea roots were studied. Disease severity on roots and epicotyl as well as the oospore number within infected root tissue were correlated with the phosphorus (P) level in the growth medium. The arbuscular mycorrhizal fungus Glomus intraradices increased P uptake and the P concentration in the plant but reduced disease development in peas. Polyacrylamide gel electrophoresis followed by densitometry of glucose-6-phosphate dehydrogenase specific to A.euteiches was used to measure the activity of the pathogen in roots. The enzyme activity increased with disease severity and disease incidence, except in plants supplemented with P at the highest level, where a peak in activity was seen 12 days after inoculation with the pathogen, followed by a decrease in activity. The epicotyl of mycorrhizal plants showed a reduction in disease severity although this part of the plants was not mycorrhizal. Thus, an induced systemic factor may be responsible for increased resistance in mycorrhizal plants.

Towards standardization of the description and publication of next‐generation sequencing datasets of fungal communities

Bonfante P, Genre A. 2008. Plants and arbuscular mycorrhizal fungi: an evolutionary-developmental perspective. Trends in Plant Science 13: 492–498. Bonfante P, Selosse MA. 2010. A glimpse into the past of land plants and of their mycorrhizal affairs: from fossils to evo-devo. New Phytologist 186: 267–270. Boullard B. 1979. Considerations sur la symbiose fongique chez les pteridophytes. Syllogeus 19: 1–58. Brachmann A, Parniske M. 2006. The most widespread symbiosis on Earth. PLoS Biology 4: e239. Brundrett MC. 2002. Coevolution of roots and mycorrhizas of land plants. New Phytologist 154: 275–304. Brundrett MC. 2004. Diversity and classification of mycorrhizal associations. Biological Reviews 78: 473–495. Koltai H, Kapulnik Y, Eds. 2010. Arbuscular mycorrhizas: physiology and function, 2nd edn. Dordrecht, the Netherlands: Springer. Parniske M. 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Reviews: Microbiology 6: 763–775. Phillips TL, DiMichele WA. 1992. Comparative ecology and life-history biology of arborescent lycopsids in Late Carboniferous swamps of Euramerica. Annals of the Missouri Botanical Garden 79: 560–588. Pressel S, Bidartondo MI, Ligrone R, Duckett JG. 2010. Fungal symbioses in bryophytes: new insights in the twenty first century. Phytotaxa 9: 238–253. Rothwell GW, Erwin DM. 1985. The rhizomorphic apex of Paurodendron; implications for homologies among the rooting organs of Lycopsida. American Journal of Botany 72: 86–98. Stewart WN. 1947. A comparative study of stigmarian appendages and Isoetes roots. American Journal of Botany 34: 315–324. Strullu-Derrien C, Rioult JP, Strullu DG. 2009. Mycorrhizas in Upper Carboniferous Radiculites-type cordaitalean rootlets. New Phytologist 182: 561–564. Strullu-Derrien C, Strullu DG. 2007. Mycorrhization of fossil and living plants. Comptes Rendus Palevol 6: 483–494. Stubblefield SP, Rothwell GW. 1981. Embryology and reproductive biology of Bothrodendrostrobus mundus (Lycopsida). American Journal of Botany 68: 625–634. Sudová R, Rydlová J, Ctvrtlı́ková M, Havránek P, Adamec L 2011. The incidence of arbuscular mycorrhiza in two submerged Isoëtes species. Aquatic Botany 94: 183–187. Taber RA, Trappe JM. 1982. Vesicular-arbuscular mycorrhiza in rhizomes, scale-like leaves, roots, and xylem of ginger. Mycologia 74: 156–161. Taylor TN, Taylor EL, Krings M. 2009. Paleobotany. The biology and evolution of fossil plants, 2nd edn. New York, NY, USA: Elsevier ⁄ Academic Press. Wagner CA, Taylor TN. 1981. Evidence for endomycorrhizae in Pennsylvanian age plant fossils. Science 212: 562–563. Winther JL, Friedman WE. 2008. Arbuscular mycorrhizal associations in Lycopodiaceae. New Phytologist 177: 790–801.

Rhizopogon spore bank communities within and among California pine forests

In this study we examine the distribution of Rhizopogon species in spore banks from five California pine forests. Four of the forest sites were discontinuous populations of Pinus muricata and a fifth was a Pinus ponderosa stand in Sierra National Forest. Rhizopogon species were retrieved by bioassaying the soils with pine seedlings followed by isolation of axenic cultures from individual root tips with typical Rhizopogon ectomycorrhizal morphology. The cultures were screened by ITS-RFLP and all unique patterns were sequenced. These sequences then were compared with those derived from identified sporocarp material. Bioassaying proved to be an efficient way to bring Rhizopogon species into culture. Approximately 50% of the pots contained ectomycorrhizal tips with Rhizopogon-like morphology, and axenic Rhizopogon cultures were obtained from half these pots. Our results showed that Rhizopogon spores usually are well distributed within local forest areas, while there is significant structuring of species at the regional scale. Spore longevity and homogenization by soil and water movement might explain their distribution within local forest areas, while the regional pattern might be explained by limited long distance dispersal or climatic and edaphic differences.

The distance decay of similarity in communities of ectomycorrhizal fungi in different ecosystems and scales

Summary 1. Despite recent advances in understanding community ecology of ectomycorrhizal fungi, little is known about their spatial patterning and the underlying mechanisms driving these patterns across different ecosystems. 2. This meta-study aimed to elucidate the scale, rate and causes of spatial structure of ectomycorrhizal fungal communities in different ecosystems by analysing 16 and 55 sites at the local and global scales, respectively. We examined the distance decay of similarity relationship in species- and phylogenetic lineage-based communities in relation to sampling and environmental variables. 3. Tropical ectomycorrhizal fungal communities exhibited stronger distance-decay patterns compared to non-tropical communities. Distance from the equator and sampling area were the main determinants of the extent of distance decay in fungal communities. The rate of distance decay was negatively related to host density at the local scale. At the global scale, lineage-level community similarity decayed faster with latitude than with longitude. 4. Synthesis. Spatial processes play a stronger role and over a greater scale in structuring local communities of ectomycorrhizal fungi than previously anticipated, particularly in ecosystems with greater vegetation age and closer to the equator. Greater rate of distance decay occurs in ecosystems with lower host density that may stem from increasing dispersal and establishment limitation. The relatively strong latitude effect on distance decay of lineage-level community similarity suggests that climate affects large-scale spatial processes and may cause phylogenetic clustering of ectomycorrhizal fungi at the global scale.