The Fungal Community

Its Organization and Role in the Ecosystem, Fourth Edition

John Dighton, James F. White, John Dighton, James F. White

• Publisher: CRC Press
• Year: 2017
• eBook ISBN: 9781351645805
• Edition: 4
• Topic: Biological Sciences
• Subtopic: Ecology
• Index: Biological Sciences

"…a number of chapters provide excellent summaries of the modern methods available for studying fungal ecology, along with those more traditional methods that are still extremely valuable…overall it is a hugely valuable compendium of fungal ecology research. It is a must for the library shelf."

-Lynne Boddy, Cardiff University, UK, Mycological Research, 2006"These 44 chapters are an excellent starting point for anyone interested in fungal communities, in the broadest sense of the term. It is a book for dipping into…may be the last comprehensive treatment of fungal communities before the molecular revolution."
-Meriel Jones, University of Liverpool, UK, Microbiology Today

"… the scope of the work is tremendous. … Excellent chapters providing overviews of methods … provide a snap shot of the current approaches used to understand fungal communities at several levels of organization. This book should probably be on the shelf of every student of mycology, and many ecologists too. For all students, this book should be a valuable resource and source of inspiration."

-Daniel Henk, Imperial College Faculty of Medicine, London, in Inoculum, Vol. 59, No. 3, May 2008

"Thorough taxonomic and subject indices further aid the reader in navigating through multiple authors' treatments of subjects of interest."

- Anthony Amend, Department of Botany, University of Hawaii at Manoa in Economic Botany, V. 61

In all subjects in science, new findings and the use of new technologies allow us to develop an ever-greater understanding of our world. Expanded and updated coverage in the fourth edition includes:

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• Adds new sections on Integrating Genomics and Metagenomics into Community Analysis, Recent Advances in Fungal Endophyte Research, Fungi in the Built Environment, and Fungal Signaling and Communication

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• Includes a broader treatment of fungal communities in natural ecosystems with in-depth coverage of fungal adaptations to stress and conservation

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• Expands coverage of the influence of climate change on fungi and the role of fungi in organically polluted ecosystems

Includes contributions from scientists from 20 nations to illustrate a true global approach for bridging gaps between ecological concepts and mycology

PART I
Integrating Genomics and Metagenomics into Community Analysis

CHAPTER 1

Molecular Community Ecology of Arbuscular Mycorrhizal Fungi

Joe D. Taylor, Thorunn Helgason, and Maarja Öpik

CONTENTS

1.1 Introduction

1.2 Contribution of Molecular Methods to Understanding AM Fungal Diversity

1.2.1 From Morphology to Molecules

1.2.2 From Community Fingerprinting to Deep Sequencing

1.2.3 From Taxonomic Expert Knowledge to Sequence Databases

1.2.4 Molecular Quantification of AM Fungi

1.2.5 Stable Isotope Probing for AM Research

1.2.6 Analysis of Physiologically Active AM Fungal Communities

1.3 Sampling AM Fungi to Study Taxonomic and Functional Diversity

1.3.1 Sampling Design

1.3.2 Sample Preservation

1.3.3 DNA and RNA Extraction from AM Fungal Samples

1.4 High-Throughput Sequencing for AM Fungal Research

1.4.1 Marker Choice

1.4.2 HTS Platform Choice

1.4.3 Bioinformatics and Databases

1.5 Taxonomy, Phylogeny, and Genomics of AM Fungi

1.5.1 Historical and Current Taxonomy

1.5.2 Genomic and Multigene Data

1.6 Metagenomics and Metatranscriptomics for AM Fungal Research

1.6.1 Metagenomics

1.6.2 Metatranscriptomics

1.7 Outlook

References

1.1 INTRODUCTION

The arbuscular mycorrhiza (AM) is a symbiosis between fungi of the phylum Glomeromycota and roots or belowground organs of plants (Smith and Read 2008). Approximately, two-thirds of plant species form AM symbiosis (Wang and Qiu 2006; Smith and Read 2008). Arbuscular mycorrhizal fungi are obligate symbionts and rely on carbon sources obtained from the photosynthetic partner (Fitter et al. 1998). Host plants receive phosphorus (Javot et al. 2007; Smith et al. 2015a) and nitrogen (Hodge and Storer 2015) via AM fungal partners and frequently show positive growth responses to AM fungi (Artursson et al. 2006). In addition, AM plants can show increased resistance to biotic stress, such as pathogens (Jung et al. 2012; Pozo et al. 2015) and herbivores (Vannette and Rasmann 2012), and abiotic stress, such as salinity, drought, and increased heavy metal concentrations (Smith and Read 2008; Porcel et al. 2012). At the community and ecosystem levels, AM fungal diversity is positively related to plant diversity and productivity (van der Heijden et al. 1998, 2008; Hiiesalu et al. 2014).

The benefits that host plants gain from the AM interaction depend on the identities of both plants and AM fungi involved. There is evidence that AM fungal species and isolates can differ in terms of benefits provided to the host (Munkvold et al. 2004). Arbuscular mycorrhizal fungi potentially have a large impact on the competitive interactions between plant species (Facelli et al. 2010; Moora and Zobel 2010). However, meta-analysis of various studies has shown that analysis of such benefits is incredibly complex and involves a multitude of biotic and abiotic factors (Hoeksema et al. 2010). Thus, it has been proposed that diversity of AM fungal communities may be a major driver of the dynamics of terrestrial plant communities (van der Heijden and Cornelissen 2002; Bever et al. 2010; Klironomos et al. 2011; Zobel and Öpik 2014).

Arbuscular mycorrhizal fungal communities are now being studied both to gather empirical information about their patterns of composition and abundance and to understand the underlying factors generating the patterns (Öpik et al. 2006, 2010; Dumbrell et al. 2010; Kivlin et al. 2011; Davison et al. 2015). Our knowledge of AM fungal field ecology has increased markedly in the past two decades with the development of molecular biology techniques and their increasing accessibility to mycorrhizal ecologists. Due to the microscopic size, shortage of morphological traits suitable for identification of field material, and limited knowledge about the natural history of AM fungi, studying their large-scale dynamics on the basis of micromorphological methods has made slow progress. Molecular techniques revolutionized the AM fungal community ecology research by making rapid community-level analysis possible. Further development of molecular techniques and molecular data analysis approaches, specifically high-throughput sequencing (HTS), is allowing field-based community ecology of AM fungi increasingly more feasible, reliable, and reproducible.

This chapter summarizes the analysis of AM fungal communities by using modern molecular techniques, describing where molecular techniques have provided new knowledge and enabled major discoveries, providing a short guide to functional analysis of AM fungal communities by using molecular techniques, and presenting an outlook for the future. In particular, we aim to highlight how molecular techniques can move the field of AM fungal community ecology from focusing on taxonomic diversity to functioning and enabling research questions such as “Who is there?” to be followed by “ … and what are they doing?”

1.2 CONTRIBUTION OF MOLECULAR METHODS TO UNDERSTANDING AM FUNGAL DIVERSITY

1.2.1 From Morphology to Molecules

Assessment of AM fungal diversity and dynamics has been one of the major foci of research into the ecology of AM fungi. Microscopy-based studies paved the way for AM fungal research, raising many questions that molecular techniques have since been able to answer. Early work was based solely on microscopical identification of AM fungal spores sampled from field-collected soil or multiplied in trap cultures (Mosse 1973). This is a slow process, relying heavily on expert knowledge. Furthermore, the spore-based detection of AM fungi has its known limitations (Sanders 2004). Importantly, spores of AM fungi are resting and dispersal organs, and factors driving sporulation are not well understood. Thus, the presence of AM fungal spores is evidence of the species present, but the absence of spores is not evidence of the absence of species (e.g., Clapp et al. 1995; Varela-Cervero et al. 2015). Instead, this indicates the absence of sporulation.

The importance of spore-based studies to AM fungal community ecology for gathering observational evidence and developing and answering essential questions cannot be underestimated. Such studies showed that AM fungal diversity can have seasonal and spatial patterns (Merryweather and Fitter 1998; Zangaro et al. 2013), including successional dynamics (Johnson et al. 1991). The first field evidence to which AM fungal diversity and plant diversity were related was based on soil spore identification (Landis et al. 2004). Sporulation dynamics provided data for developing the concept of plant-AM fungal feedback (Bever 1994; Bever et al. 1997) and differential host-AM fungal relationships (Bever et al. 1996). Not only have spore-based studies provided us with important field-based observations, but sporulating and culturable AM fungi are an important source of clean material for conducting experiments in controlled conditions (van der Heijden et al. 1998) and for genomics, genetics, and physiology of AM fungi (e.g., Tisserant et al. 2012, 2013; Lin et al. 2014).

Molecular techniques are currently the prevailing approach for studying AM fungal communities. Compared to studying AM fungal spores, the deoxyribonucleic acid (DNA)- and particularly ribonucleic acid (RNA)-based methods allow active components of the community to be analyzed. Data generated from root samples are currently the norm in molecular AM fungal community ecology. This reflects the interest in the plant–fungus association and also the difficulty in extracting AM fungal DNA directly from soil (Gamper et al. 2004). The major advances brought about by DNA-based studies increased understanding of the global biodiversity of AM fungi (Öpik et al. 2013; Davison et al. 2015), and improved knowledge of their taxonomy (Schüßler et al. 2001; Oehl et al. 2011; Redecker et al. 2013). These are the prerequisites to studying community dynamics of AM fungi.

1.2.2 From Community Fingerprinting to Deep Sequencing

The first eukaryotic nuclear ribosomal RNA (rRNA) gene sequences (Medlin et al. 1988) and subsequent design of universal polymerase chain reaction (PCR) primers for fungi (White et al. 1990) led to the first eukaryotic 18S rRNA gene sequences from Glomeromycotan fungi (Simon et al. 1992). The development of PCR-based techniques started molecular taxonomy of AM fungi and enabled linking phenotypic data (mostly spore morphology) with genotypic data (DNA sequences) and yielding better understanding about phylogenetic relationships of Glomeromycota. Early studies of AM fungal community diversity were performed using cloning and sequencing (Clapp et al. 1995; Sanders et al. 1995). The next big development was the design of fungal primers that exclude plant sequences (Helgason et al. 1998). The paradigm shift driven by these studies was the unambiguous evidence that multiple colonizations, that is, several AM fungi cocolonizing a root space, even in short root lengths (Helgason et al. 1999), were widespread and that AM fungi are nonrandomly distributed among their host plants (Helgason et al. 2002). The shift from spore identification to study AM fungal DNA and RNA in roots meant that active components of the fungal community could be analyzed. Furthermore, cloning and Sanger sequencing permitted detection and identification of multiple co-occurring AM fungi in situ, without the need for recognizable morphological features.

It is important to highlight the fact that AM fungi are a monophyletic fungal group (Phylum Glomeromycota), unlike fungi forming other mycorrhizal types; the design of group-specific primers is easier. Such primers helped identify AM fungi in planta, excluding plant sequences and sequences of non-AM fungi colonizing roots and rhizoplane. Several other primer sets specific for AM fungi as a group or smaller subsets of them (e.g., families) have been designed, further improving the detection of AM fungal diversity (Redecker 2000; Lee et al. 2008; Krüger et al. 2009; summarized by Hart et al. 2015). Improvements in primer systems have made the large-scale field studies possible by enabling capture of almost all of the diversity of AM fungi in studied ecosystems.

Co-occurrence of multiple AM fungal species in a (root or soil) sample is common. Quantifying community composition necessitates the separation of PCR products of individual fungi by molecular community techniques, by using either fingerprinting or DNA sequencing methods. A range of fingerprinting techniques have been applied to the study of AM fungi: polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP; Helgason et al. 1999; Husband et al. 2002; Vandenkoornhuyse et al. 2002); single-stranded conformation polymorphism (SSC; Kjøller and Rosendahl 2000; Nielsen et al. 2004); terminal (t)-RFLP (Vandenkoornhuyse et al. 2003); denaturing gradient gel electrophoresis (DGGE; Kowalchuk et al. 2002; Öpik et al. 2003); and temperature gradient gel electrophoresis (TGGE; Cornejo et al. 2004). The advantage of these techniques was rapid and relatively inexpensive profiling of AM fungal communities; however, without sequence data, the comparison and re-evaluation of individual studies are usually not possible.

In the early molecular AM fungal community studies, there was a trade-off between the high sample throughput but low study-to-study comparability offered by fingerprinting techniques and the costly choice of cloning and Sanger sequencing to identify individual fungal taxa, providing easily comparable and reanalyzable sequence data. High-throughput sequencing (HTS) or next-generation sequencing (although this term is almost outdated as there is now a newer generation of sequencers present, Kircher and Kelso 2010; Venkatesan and Bashir 2011) was initially costly and had low sample throughput. This has now developed to enable both high sample throughput and sufficient sequencing depth per sample at affordable costs to mycorrhizal ecologists. High-throughput sequencing techniques, as they sequence by synthesis, have also removed the need for separation of individual PCR products either via fingerprinting or via cloning techniques. HTS yields more accurate data about rarer members of AM fungal communities via increased sequence numbers per sample (sequencing depth), thus typically reporting higher richness values (Öpik et al. 2009). It is noteworthy that typical root or soil samples used in AM fungal community surveys contain an average of 5–40 species (operational taxonomic unit [OTUs], molecular taxa; Hart et al. 2015), which is lower than the reported richness values of general fungal (Toju et al. 2013) or bacterial communities (Mantar et al. 2010). Therefore, the sufficient sample-based sequencing depth is lower in the case of AM fungi than that in some other soil microbes (Hart et al. 2015).

The shift from cloning and Sanger sequencing to HTS approaches has been both disruptive, completely changing the scale and design of field-based experiments, and transformative, revealing a new understanding of AM fungal diversity and dynamics. High-throughput sequencing can now be used to relatively rapidly profile dynamics of AM fungal communities in large-scale field studies to describe temporal (Dumbrell et al. 2011; Cotton et al. 2015) and spatial (Öpik et al. 2013; Davison et al. 2015) variations in these communities. One of the transformative results stemming from HTS data has been the change in understanding about associations between AM fungi and host plant species. Early evidence on AM fungal-host plant species level specificity or preference (Vandenkoornhuyse et al. 2002, 2003) may be better explained by preference among ecological groups of AM fungi and host plants (Öpik et al. 2009).

The swift accumulation of DNA-based AM fungal community data sets has revealed diversity patterns from local to global scales. The first AM fungal biogeographical meta-analyses described diversity patterns related to biome, spatial (continents), and environmental (edaphic and climatic) factors (Öpik et al. 2006, 2010; Kivlin et al. 2011). These were followed by HTS-based large-scale case studies, revealing lower global endemism of AM fungi than what was thought earlier (Öpik et al. 2013; Davison et al. 2015). Observations made in early sequencing studies, such as the dramatic decrease in AM...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Contents
• Introduction
• Editors
• Contributors
• Part I Integrating Genomics and Metagenomics into Community Analysis
• Part II Recent Advances in Fungal Endophyte Research
• Part III Fungal Communities in Terrestrial Ecosystems
• Part IV Fungal Communities in Marine and Aquatic Ecosystems
• Part V Fungal Adaptations to Stress and Conservation
• Part VI Fungal–Faunal Interactions
• Part VII Fungal Communities, Climate Change, and Pollution
• Part VIII Fungi in the Built Environment
• Part IX Fungal Signaling and Communication
• Index