Evaluation of methods to estimate production, biomass and turnover of ectomycorrhizal mycelium in forests soils : A review
TL;DR: The need to extend the application of current methods to focus on a greater range of habitats and mycorrhizal types enabling incorporation of mycor rhizal fungal biomass and turnover into biogeochemical cycling models is highlighted.
Abstract: Mycorrhizal fungi constitute a considerable sink for carbon in most ecosystems. This carbon is used for building extensive mycelial networks in the soil as well as for metabolic activity related to nutrient uptake. A number of methods have been developed recently to quantify production, standing biomass and turnover of extramatrical mycorrhizal mycelia (EMM) in the field. These methods include minirhizotrons, in-growth mesh bags and cores, and indirect measurements of EMM based on classification of ectomycorrhizal fungi into exploration types. Here we review the state of the art of this methodology and discuss how it can be developed and applied most effectively in the field. Furthermore, we also discuss different ways to quantify fungal biomass based on biomarkers such as chitin, ergosterol and PLFAs, as well as molecular methods, such as qPCR. The evidence thus far indicates that mycorrhizal fungi are key components of microbial biomass in many ecosystems. We highlight the need to extend the application of current methods to focus on a greater range of habitats and mycorrhizal types enabling incorporation of mycorrhizal fungal biomass and turnover into biogeochemical cycling models.
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Chinese Academy of Sciences1, Canterbury of New Zealand2, Pennsylvania State University3, Cornell University4, University of Minnesota5, University of Helsinki6, University of New Hampshire7, Oak Ridge National Laboratory8, Stanford University9, Indiana University10, University of Idaho11, College of Charleston12, University of Natural Resources and Life Sciences, Vienna13, Polish Academy of Sciences14
TL;DR: It is demonstrated how order-based and functional classification frameworks improve the understanding of dynamic root processes in ecosystems dominated by perennial plants.
Abstract: Fine roots acquire essential soil resources and mediate biogeochemical cycling in terrestrial ecosystems. Estimates of carbon and nutrient allocation to build and maintain these structures remain uncertain because of the challenges of consistently measuring and interpreting fine-root systems. Traditionally, fine roots have been defined as all roots 2mm in diameter, yet it is now recognized that this approach fails to capture the diversity of form and function observed among fine-root orders. Here, we demonstrate how order-based and functional classification frameworks improve our understanding of dynamic root processes in ecosystems dominated by perennial plants. In these frameworks, fine roots are either separated into individual root orders or functionally defined into a shorter-lived absorptive pool and a longer-lived transport fine-root pool. Using these frameworks, we estimate that fine-root production and turnover represent 22% of terrestrial net primary production globally - a c. 30% reduction from previous estimates assuming a single fine-root pool. Future work developing tools to rapidly differentiate functional fine-root classes, explicit incorporation of mycorrhizal fungi into fine-root studies, and wider adoption of a two-pool approach to model fine roots provide opportunities to better understand below-ground processes in the terrestrial biosphere.
834 citations
Cites background from "Evaluation of methods to estimate p..."
...Currently, several approaches exist to quantitatively estimate fungal biomass and activity in soils (Miller et al., 1995; Hobbie, 2006; Allen & Kitajima, 2013; Wallander et al., 2013)....
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TL;DR: An overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis are provided, and advice for leveraging next-generation technologies to explore mycobiome diversity in different habitats is provided.
Abstract: Fungi are major ecological players in both terrestrial and aquatic environments by cycling organic matter and channelling nutrients across trophic levels. High-throughput sequencing (HTS) studies of fungal communities are redrawing the map of the fungal kingdom by hinting at its enormous - and largely uncharted - taxonomic and functional diversity. However, HTS approaches come with a range of pitfalls and potential biases, cautioning against unwary application and interpretation of HTS technologies and results. In this Review, we provide an overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis. We also discuss upcoming trends and techniques in the field and summarize recent and noteworthy results from HTS studies targeting fungal communities and guilds. Our Review highlights the need for reproducibility and public data availability in the study of fungal communities. If the associated challenges and conceptual barriers are overcome, HTS offers immense possibilities in mycology and elsewhere.
461 citations
TL;DR: It is postulate that, by affecting turnover and decomposition of fungal tissues, mycorrhizal fungal identity and growth form are critical determinants of C and N sequestration in boreal forests.
Abstract: Summary
Boreal forest soils store a major proportion of the global terrestrial carbon (C) and below-ground inputs contribute as much as above-ground plant litter to the total C stored in the soil. A better understanding of the dynamics and drivers of root-associated fungal communities is essential to predict long-term soil C storage and climate feedbacks in northern ecosystems.
We used 454-pyrosequencing to identify fungal communities across fine-scaled soil profiles in a 5000 yr fire-driven boreal forest chronosequence, with the aim of pinpointing shifts in fungal community composition that may underlie variation in below-ground C sequestration.
In early successional-stage forests, higher abundance of cord-forming ectomycorrhizal fungi (such as Cortinarius and Suillus species) was linked to rapid turnover of mycelial biomass and necromass, efficient nitrogen (N) mobilization and low C sequestration. In late successional-stage forests, cord formers declined, while ericoid mycorrhizal ascomycetes continued to dominate, potentially facilitating long-term humus build-up through production of melanized hyphae that resist decomposition.
Our results suggest that cord-forming ectomycorrhizal fungi and ericoid mycorrhizal fungi play opposing roles in below-ground C storage. We postulate that, by affecting turnover and decomposition of fungal tissues, mycorrhizal fungal identity and growth form are critical determinants of C and N sequestration in boreal forests.
457 citations
TL;DR: It is called for studies that can functionally incorporate the root traits involved in resource uptake, the complex soil environment and the various soil resource uptake mechanisms - particularly the mycorrhizal pathway - in a multidimensional root trait framework.
Abstract: Contents 1159 I 1159 II 1161 III 1164 IV 1166 1167 References 1167 SUMMARY: The search for a root economics spectrum (RES) has been sparked by recent interest in trait-based plant ecology By analogy with the one-dimensional leaf economics spectrum (LES), fine-root traits are hypothesised to match leaf traits which are coordinated along one axis from resource acquisitive to conservative traits However, our literature review and meta-level analysis reveal no consistent evidence of an RES mirroring an LES Instead the RES appears to be multidimensional We discuss three fundamental differences contributing to the discrepancy between these spectra First, root traits are simultaneously constrained by various environmental drivers not necessarily related to resource uptake Second, above- and belowground traits cannot be considered analogues, because they function differently and might not be related to resource uptake in a similar manner Third, mycorrhizal interactions may offset selection for an RES Understanding and explaining the belowground mechanisms and trade-offs that drive variation in root traits, resource acquisition and plant performance across species, thus requires a fundamentally different approach than applied aboveground We therefore call for studies that can functionally incorporate the root traits involved in resource uptake, the complex soil environment and the various soil resource uptake mechanisms - particularly the mycorrhizal pathway - in a multidimensional root trait framework
374 citations
Cites background from "Evaluation of methods to estimate p..."
...…fungal exploration types based on their morphology (e.g. extraradical hyphae and rhizomorphs; Agerer, 2001), and quantifying mycelium abundance (Wallander et al., 2001, 2013; Ekblad et al., 2013)may further reveal the role of mycorrhizal fungi in soil exploration and exploitation capacities of…...
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TL;DR: 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.
Abstract: 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 similar to 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.
269 citations
Cites background or methods from "Evaluation of methods to estimate p..."
...A solution to this problemmay be to combine sequential harvesting of in-growth bags with a 13CO2 pulse labelling of the mycelium via the plant and analyses of 13C in structural components of the mycelium such as glucosamine (for further technical discussions, see Wallander et al. 2013)....
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...This large variation may derive from the factors regulating EMM production as well as from differences in the various methods used to assess mycelial biomass (ergosterol, phospholipid fatty acids, dry matter etc.; see Wallander et al. (2013))....
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...…and Wallander 1992; Ekblad and Näsholm 1996; Ekblad et al. 1998; Wallander et al. 2001; Dickie et al. 2002; Johnson et al. 2002; Leake et al. 2006; Högberg et al. 2010; Heinemeyer et al. 2007, 2011 and see Wallander et al. 2013 for a discussion of advantages and disadvantages of these methods)....
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References
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TL;DR: The cell content of 12 bacterial phospholipid fatty acids (PLFA) was determined in bacteria extracted from soil by homogenization/centrifugation and the soil content of the PLFA 18:2ω6 was correlated with the ergosterol content, which supports the use of this PLFA as an indicator of fungal biomass.
Abstract: The cell content of 12 bacterial phospholipid fatty acids (PLFA) was determined in bacteria extracted from soil by homogenization/centrifugation. The bacteria were enumerated using acridine orange direct counts. An average of 1.40×10-17 mol bacterial PLFA cell-1 was found in bacteria extracted from 15 soils covering a wide range of pH and organic matter contents. With this factor, the bacterial biomass based on PLFA analyses of whole soil samples was calculated as 1.0–4.8 mg bacterial C g-1 soil C. The corresponding range based on microscopical counts was 0.3–3.0 mg bacterial C g-1 soil C. The recovery of bacteria from the soils using homogenization/centrifugation was 2.6–16% (mean 8.7%) measured by PLFA analysis, and 12–61% (mean 26%) measured as microscopical counts. The soil content of the PLFA 18:2ω6 was correlated with the ergosterol content (r=0.92), which supports the use of this PLFA as an indicator of fungal biomass. The ratio 18:2ω6 to bacterial PLFA is therefore suggested as an index of the fungal:bacterial biomass ratio in soil. An advantage with the method based on PLFA analyses is that the same technique and even the same sample is used to determine both fungi and bacteria. The fungal:bacterial biomass ratio calculated in this way was positively correlated with the organic matter content of the soils (r=0.94).
2,256 citations
"Evaluation of methods to estimate p..." refers background in this paper
...A strong positive correlation was found between PLFA 18:2u6,9 and the fungal marker ergosterol in soils from cultivated fields, gardens, grasslands and forests (Frostegård and Bååth, 1996; Kaiser et al., 2010)....
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TL;DR: Results from principal component analysis showed that determining the levels of fatty acids present in both low and high concentrations is essential in order to correctly identify microorganisms and accurately classify them into taxonomically defined groups.
Abstract: This review discusses the analysis of whole-community phospholipid fatty acid (PLFA) profiles and the composition of lipopolysaccharides in order to assess the microbial biomass and the community structure in soils. For the determination of soil microbial biomass a good correlation was obtained between the total amount of PLFAs and the microbial biomass measured with methods commonly used for determinations such as total adenylate content and substrate-induced respiration. Generally, after the application of multivariate statistical analyses, whole-community fatty acid profiles indicate which communities are similar or different. However, in most cases, the organisms accounting for similarity or difference cannot be determined, and therefore artefacts could not be excluded. The fatty acids used to determine the biomass vary from those which determine the community structure. Specific attention has to be paid when choosing extraction methods in order to avoid the liberation of fatty acids from non-living organic material and deposits, and to exclude the non-target selection of lipids from living organisms, as well. By excluding the fatty acids which were presumed to be common and widespread prior to multivariate statistical analysis, estimates were improved considerably. Results from principal component analysis showed that determining the levels of fatty acids present in both low and high concentrations is essential in order to correctly identify microorganisms and accurately classify them into taxonomically defined groups. The PLFA technique has been used to elucidate different strategies employed by microorganisms to adapt to changed environmental conditions under wide ranges of soil types, management practices, climatic origins and different perturbations. It has been proposed that the classification of PLFAs into a number of chemically different subgroups should simplify the evaluating procedure and improve the assessment of soil microbial communities, since then only the subgroups assumed to be involved in key processes would be investigated.
1,895 citations
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...As eukaryotes and different groups of prokaryotes contain more or less specific ester-linked lipid fatty acids (Lechevalier and Lechevalier, 1988; Zelles, 1997, 1999), the analysis of PLFA composition and concentrations are useful as a tool for quantitative and qualitative examination of microbial…...
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TL;DR: How isotope measurements associated with the critical plant resources carbon, water, and nitrogen have helped deepen the understanding of plant-resource acquisition, plant interactions with other organisms, and the role of plants in ecosystem studies is reviewed.
Abstract: ▪ Abstract The use of stable isotope techniques in plant ecological research has grown steadily during the past two decades. This trend will continue as investigators realize that stable isotopes can serve as valuable nonradioactive tracers and nondestructive integrators of how plants today and in the past have interacted with and responded to their abiotic and biotic environments. At the center of nearly all plant ecological research which has made use of stable isotope methods are the notions of interactions and the resources that mediate or influence them. Our review, therefore, highlights recent advances in plant ecology that have embraced these notions, particularly at different spatial and temporal scales. Specifically, we review how isotope measurements associated with the critical plant resources carbon, water, and nitrogen have helped deepen our understanding of plant-resource acquisition, plant interactions with other organisms, and the role of plants in ecosystem studies. Where possible we also...
1,710 citations
"Evaluation of methods to estimate p..." refers background in this paper
...One of the problems with isotope techniques is the risk of differences in labelling of different chemical components of the fungal biomass, some of them having a high turnover rate (respiratory substrates), while the heavy isotopewill have longer residence time in structural cell materials (Dawson et al., 2002)....
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...…with isotope techniques is the risk of differences in labelling of different chemical components of the fungal biomass, some of them having a high turnover rate (respiratory substrates), while the heavy isotopewill have longer residence time in structural cell materials (Dawson et al., 2002)....
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TL;DR: The high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon, which would require anaer aerobic microhabitats in the aerated zones.
Abstract: The measurement of lipid phosphate is proposed as an indicator of microbial biomass in marine and estuarine sediments. This relatively simple assay can be performed on fresh, frozen or frozen-lyophilized sediment samples with chloroform methanol extraction and subsequent phosphate determination. The sedimentary lipid phosphate recovery correlates with the extractible ATP and the rate of DNA synthesis. Pulse-chase experiments show active metabolism of the sedimentary phospholipids. The recovery of added 14C-labeled bacterial lipids from sediments is quantitative. Replicate analyses from a single sediment sample gave a standard deviation of 11%. The lipid extract can be fractionated by relatively simple procedures and the plasmalogen, diacyl phospholipid, phosphonolipid and non-hydrolyzable phospholipid content determined. The relative fatty acid composition can be readily determined by gas-liquid chromatography. The lipid composition can be used to define the microbial community structure. For example, the absence of polyenoic fatty acids indicates minimal contamination with benthic micro-eukaryotes. Therefore the high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon. This would require anaerobic microhabitats in the aerated zones.
1,693 citations
"Evaluation of methods to estimate p..." refers background or methods in this paper
...The analytical procedure for PLFAs and NLFAs comprises four steps: (i) extraction of lipids, (ii) lipid fractionation, (iii) mild alkaline methanolysis, and (iv) GC analyses (White et al., 1979; Frostegård et al., 1991)....
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...PLFAs are essential components of cell membranes and they decompose quickly after cell death (White et al., 1979) and are commonly used as chemical markers of soil fungi....
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TL;DR: The results support the hypothesis that selection has favoured ericoid and ectomycorrhizal systems with well developed saprotrophic capabilities in those ecosystems characterized by retention of N and P as organic complexes in the soil.
Abstract: Progress towards understanding the extent to which mycorrhizal fungi are involved in the mobilization of nitrogen (N) and phosphorus (P) from natural substrates is reviewed here. While mycorrhiza research has emphasized the role of the symbiosis in facilitation of capture of these nutrients in ionic form, attention has shifted since the mid-1980s to analysing the mycorrhizal fungal abilities to release N and P from the detrital materials of microbial faunal and plant origins, which are the primary sources of these elements in terrestrial ecosystems. Ericoid, and some ectomycorrhizal fungi have the potential to be directly involved in attack both on structural polymers, which may render nutrients inaccessible, and in mobilization of N and P from the organic polymers in which they are sequestered. The advantages to the plant of achieving intervention in the microbial mobilization-immobilization cycles are stressed. While the new approaches may initially lack the precision achieved in studies of readily characterized ionic forms of N and P, they do provide insights of greater ecological relevance. The results support the hypothesis that selection has favoured ericoid and ectomycorrhizal systems with well developed saprotrophic capabilities in those ecosystems characterized by retention of N and P as organic complexes in the soil. The need for further investigation of the abilities of arbuscular mycorrhizal fungi to intervene in nutrient mobilization processes is stressed.
1,362 citations
"Evaluation of methods to estimate p..." refers background in this paper
...This is not the case because bacteria and precipitated SOM can be present in the mesh bags, but they probably contribute very little to the weight of putative fungal material extracted....
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...The majority of ergosterol from in-growth bags was found in the free form (90%), while the free ergosterol was below 20% in the mineral soil, supporting the view of increasing proportion of esterified ergosterol in older SOM (Wallander et al., 2010)....
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...The combination of ingrowth cores with a mesh allowing only hyphae or allowing both roots and hyphae filled with C4 dominated soils have been used to estimate the contribution of mycelia and roots to the formation of stable SOM (Godbold et al., 2006; Wallander et al., 2011)....
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...Much of this C is lost as respiration (Janssens et al., 2001) and a small but significant fraction enters the soil organic matter (SOM) pool....
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...Indeed, growth of arbuscular mycorrhizal fungi has been quantified in mesh bags amended with natural SOM using fatty acids (Labidi et al., 2007; Hammer et al., 2011), which are available for this mycorrhizal group (NLFA 16.1u5, Section 3.5)....
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