Liliane Ruess

Fatty acids of fungi and nematodes: possible biomarkers in the soil food chain?

Abstract The fatty acid composition of 16 different soil fungi (ascomycetes, basidiomycetes, mitosporic fungi) and a fungal-feeding nematode Aphelenchoides sp. reared on seven fungal species was investigated. Additionally, fatty acid profiles of Aphelenchoides sp. and A. saprophilus grown on the same fungal food source,Agrocybe gibberosa, were compared. Thirteen predominant fatty acids were detected in the fungi. Most of them occurred in each of the 16 species, but relative quantities of individual fatty acids differed, in particular those of unsaturated ones. Most fungal species could be differentiated from each other on this basis. Our study revealed convergence, but also, differences, in the fatty acid composition of systematically related fungi, i.e. a taxonomic or phylogenetic relationship was not necessarily accompanied by similarity in fatty acid profiles. Nematodes comprised a wider fatty acid spectrum than fungi, with 17 predominant fatty acids, and a higher amount of long-chain, polyunsaturated acids than their fungal diet. Fungal host tissue may have supplied palmitic, oleic and linoleic acid present, whereas most of the long-chain unsaturated fatty acids were synthesized by the nematodes. Unsaturated fatty acids mainly belonged to the ω6 and 9 family, indicating a carboxyl-directed desaturation as a major metabolic pathway. The fungal host significantly affected the fatty acid profile of the nematodes. However, we could not assign individual fatty acids as biomarkers reflecting the dietary source, likely due to the considerable convergence within tested fungal species. Of the basidiomycetes analyzed Laccaria laccata, was distinctly separated from the others and this difference could also be detected in the nematodes showing the influence of food type. We conclude that the lipid composition of nematodes is controlled by both the nematode and its diet and that monitoring fatty acid patterns of soil animals may therefore provide a way to detect trophic interactions in belowground food webs.

Simulated climate change affecting microorganisms, nematode density and biodiversity in subarctic soils

Arctic terrestrial ecosystems are strongly dominated by temperature, and global warming is expected to have a particularly strong impact in high latitudes. The Arctic will therefore be an important region for early detection of global change. In the present study the effects of environmental manipulations simulating climate change on soil microorganisms and nematode populations were investigated. Study sites were a dwarf shrub dominated tree-line heath (450 m a.s.l.) and a high altitude fellfield (1150 m a.s.l.) at Abisko, Swedish Lapland. Soil temperature was enhanced by using passive greenhouses and the impact on soil organisms with and without NPK fertilizer addition was assessed. The nematode community was strongly affected by warming and nutrient application. Population density was twice as high for all treatments at the fellfield as compared to controls. At the heath temperature enhancement with or without fertilizer application also led to a doubling of the population density, whereas fertilization alone caused an increase of about one third. The environmental manipulations resulted in a greater microbial biomass C and active fungal biomass in the heath soil. Increased density was also recorded for bacterial and fungal feeding nematodes at both sites. The results suggest that nematodes have an important impact on microbial biomass and turnover rates in the two subarctic systems. Elevated soil temperature apparently will lead to increased grazing on microorganisms, contributing to enhanced net N and P mineralization rates and plant nutrient availability. However, biodiversity was generally affected negatively by the environmental manipulations. The effects were more severe at the high altitude fellfield indicating that the influence of elevated temperature will be more pronounced in systems already stressed by extreme climatic conditions.

Nitrogen isotope ratios and fatty acid composition as indicators of animal diets in belowground systems

This study analyses trophic interactions between soil fungi, micro- and mesofauna in microcosm experiments. The trophic shift of 15N and fatty acids (FAs) was investigated in different food chains, which comprised either two (fungi and grazers) or three (fungi, nematodes and Collembola) levels. Contrary to the widely accepted assumption of 15N enrichment in trophic cascades the experiments revealed enrichment, depletion or no change in 15N of consumers compared to their diet. Factors responsible for this pattern were suggested to be: (1) the main metabolic pathway used for N excretion in ammonotelic nematodes to be similar or depleted in the heavier isotope, and uricotelic Collembola mostly enriched in the heavier isotope; (2) a higher shift in 15N with a high-protein diet (e.g. for predators); (3) compensation due to low-quality food altering the fractionation of 15N. Analysis of the lipid composition showed phospholipids to be generally unaffected and neutral lipids closely related to the FA pattern of the food source. Dietary routing of FAs into neutral lipids occurred, as evidenced by corresponding frequencies of FAs in host and consumer profiles. Additionally, several FAs were only detected in the grazer when present in the food source. Oleic acid showed a shift over three trophic levels, from fungi to nematodes to Collembola. The assimilation of dietary FAs resulted in a more diverse neutral lipid profile, i.e. animals higher in the food chain contained more individual FAs compared to animals lower in the food chain. The results indicate that monoenoic C18 and monoenoic C20 FAs have the potential to act as tools for the bioindication of feeding strategies in belowground systems. We suggest that primary consumers will have no or only trace amounts of monoenoic C20 acids in their neutral lipid profile, whereas consumers feeding on a eukaryote diet will show a considerably higher frequency.

Long-term manipulation of the microbes and microfauna of two subarctic heaths by addition of fungicide, bactericide, carbon and fertilizer

Nutrient availability is a major constraint to plant production and carbon storage in arctic ecosystems, but there are few studies coupling processes in the decomposer and microbial food web and the implications these processes have on the control of nutrient mineralization. We studied the relationship between microbial biomass and the abundance of microbivore and the role of grazing on nutrient turnover after annual addition of carbon (sucrose), fertilizer (NPK), fungicide (benomyl) and bactericides (streptomycin and penicillin) to two dwarf shrub communities, a low and a high altitude heath. After four years of repeated additions, we measured microbial biomass by fumigation-extraction and phospholipid fatty acid (PLFA) analysis, the fungal to bacterial biomass ratio by PLFA analysis and estimated the numbers of protozoa and nematodes, assigned into feeding groups. The fungal to bacterial ratio of nematode feeding groups was around 0.2, indicating a bacterial-based food web in both communities. The size of the microbial biomass did not change after the additions, except when the amount of available carbon was increased (low altitude heath) or when addition of carbon was combined with fertilizer (high altitude heath). In contrast, fertilizer but not carbon increased the number of microbivores. This suggests that the amount of available carbon and not grazing pressure controls the size of the microbial biomass. Furthermore, it suggests that the food quality, e.g. nutrient content of the micro-organisms, had a larger effect on the microbivore than the size of the microbial biomass. The addition of bactericides and fungicide did not significantly change the fungal to bacterial biomass ratio of the micro-organisms. We could not detect any effects of the bactericides. In contrast, the fungicide strongly decreased nematode density, least in the fungal feeders, probably due to increased abundance of the insensitive Aphelenchoides ssp.

APPLICATION OF LIPID ANALYSIS TO UNDERSTAND TROPHIC INTERACTIONS IN SOIL

Trophic interactions in cryptic belowground systems are difficult to assess, either experimentally or by direct observation. We used lipid analysis to determine feeding strategies in Collembola raised on various bacterial, fungal, plant, or nematode diets. Dietary fatty acids (FAs) were conserved and transferred through the trophic cascade into the neutral lipids of consumers. The presence of vaccenic type FAs (v7 family) was indicative of a bacterial diet in general. More specifically, methyl-branched (iso, anteiso) and cyclic forms of FAs were markers for consumption of gram-positive and gram-negative bacteria, re- spectively. Fungal-feeding Collembola comprised a higher proportion of linoleic acid, whereas the profile of plant feeders showed an increased amount of oleic acid. The FA 20:1 v9 was only present in Collembola with nematodes as prey. Based on the assigned marker FAs, field populations of Collembola in three deciduous forest stands were ascribed to feeding guilds (i.e., fungivores, bacterivores, herbivores, predators). In conclusion, FA biomarkers provide a high-resolution method to define feeding strategies of decomposer invertebrates and to determine their diets in situ. Lipid analysis has considerable potential as a new tool in soil food web studies.

Chapter Three – Empirically Characterising Trophic Networks: What Emerging DNA-Based Methods, Stable Isotope and Fatty Acid Analyses Can Offer

Abstract Food webs in agricultural systems are complex and trophic linkages are difficult to track using conventional methodologies. Here, we review three alternative approaches that allow empirical assessment of feeding interactions: DNA-based techniques, and stable isotope and fatty acid analyses. DNA-based methods, namely multiplex PCR and next-generation sequencing, allow identification of food types and host–parasitoid linkages, resulting in taxonomically highly resolved feeding networks. Stable isotopes and fatty acids reflect the assimilation of broader categories of resources, as metabolised into the consumers’ tissue, together with the associated energy and nutrient fluxes in the food web. We discuss the strengths of the approaches but also highlight their limitations, providing practical advice on which technique is best suited to answer specific questions in examining food web interactions in agroecosystems. Future refinements of these techniques, especially when used in combination, could herald a new era in agricultural food web ecology, enabling management and environmental impact to be placed in the mechanistic context of trophic networks.

Studies On the Nematode Fauna of an Acid Forest Soil: Spatial Distribution and Extraction

The distribution of free living soil nematodes was examined by collecting samples consisting of 16 cores in a small plot. The k of the negative binomial was used to describe patterns in the spatial distribution of total numbers and of several species. Both the horizontal and the vertical distributions were aggregated. The efficiency of the Baermann extraction method was compared with direct microscopic examination. Also, the effect of the extraction time on the recovery was tested. For quantitative extraction the Baermann funnel method was a useful technique, but it was less reliable in providing results of community compositions when compared with direct microscopic examination of field material.

Food preferences of a fungal-feeding Aphelenchoides species.

The growth of Aphelenchoides sp. populations was investigated in vitro with 17 different fungal species as food source. Nematode mass cultures were obtained with saprophytic ( Agrocybe , Chaetomium ) and especially with mycorrhizal fungi ( Cenococcum , Hymenoscyphus , Laccaria ). Mitosporic species, like Alternaria , Monocillium or Penicillium , were generally meagre or non-hosts. This poor host suitability is likely due to the release of toxic metabolites (e.g. antibiotics) and/or to morphological differences (e.g., forming of conidiophores) by the fungi. Frequent grazing of nematodes on mycorrhizal mycelia may be of major significance for the establishment and maintenance of mycorrhizal associations in the field. Food preference of Aphelenchoides sp. was tested in choice chamber experiments. Nematodes showed a marked preference for particular fungal species. They changed food source with time, indicating a “mixed diet” selection, probably a strategy to avoid the concentration of toxic metabolites. The attractiveness of a fungus was not necessarily correlated with its suitability as a host. That a poor fungal host can be a strong nematode attractant and influence their spatial distribution in the soil has implications for nematode populations in the field. In Laborexperimenten wurde die Vermehrung des Nematoden Aphelenchoides sp. mit 17 verschiedenen Pilzspezies als Nahrungsgrundlage untersucht. Neben saprophytischen Arten ( Agrocybe , Chaetomium ) eigneten sich insbesondere Mykorrhizapilze ( Cenococcum , Hymenoscyphus , Laccaria ) fur eine Massenvermehrung. Eine schlechte Nahrungsquelle stellten mitosporische Arten, wie Alternaria , Monocillium oder Penicillium , dar. Dies durfte auf toxische Stoffwechselprodukte (z.B., Antibiotika) und/oder auf morphologische Unterschiede (z.B., Sporenbildung) zuruckzufuhren sein. Die gute Vermehrung der Nematoden an Mykorrhizapilzen ist von weitreichender Bedeutung fur das Freiland. Negative Auswirkungen auf die Ausbildung und Funktion von Mykorrhiza im Boden sind zu erwarten. In Nahrungswahlexperimenten zeigte Aphelenchoides sp. eine ausgepragte Praferenz fur bestimmte Pilzarten. Das Wechseln zwischen den einzelnen Pilzspezies weist auf die Bevorzugung von “Mischnahrung” hin. Dies durfte eine Strategie zur Vermeidung von hohen Konzentrationen toxischer Nahrungsbestandteile sein. Praferenz und Nahrungsqualitat standen nur in geringem Zusammenhang. Somit konnen auch Pilze, die eine schlechte Nahrungsquelle darstellen, attraktiv auf Nematoden wirken und deren Verbreitung in Boden und Rhizosphare beeinflussen.

OakContigDF159.1, a reference library for studying differential gene expression in Quercus robur during controlled biotic interactions: use for quantitative transcriptomic profiling of oak roots in ectomycorrhizal symbiosis

Oaks (Quercus spp.), which are major forest trees in the northern hemisphere, host many biotic interactions, but molecular investigation of these interactions is limited by fragmentary genome data. To date, only 75 oak expressed sequence tags (ESTs) have been characterized in ectomycorrhizal (EM) symbioses. We synthesized seven beneficial and detrimental biotic interactions between microorganisms and animals and a clone (DF159) of Quercus robur. Sixteen 454 and eight Illumina cDNA libraries from leaves and roots were prepared and merged to establish a reference for RNA-Seq transcriptomic analysis of oak EMs with Piloderma croceum. Using the Mimicking Intelligent Read Assembly (MIRA) and Trinity assembler, the OakContigDF159.1 hybrid assembly, containing 65 712 contigs with a mean length of 1003 bp, was constructed, giving broad coverage of metabolic pathways. This allowed us to identify 3018 oak contigs that were differentially expressed in EMs, with genes encoding proline-rich cell wall proteins and ethylene signalling-related transcription factors showing up-regulation while auxin and defence-related genes were down-regulated. In addition to the first report of remorin expression in EMs, the extensive coverage provided by the study permitted detection of differential regulation within large gene families (nitrogen, phosphorus and sugar transporters, aquaporins). This might indicate specific mechanisms of genome regulation in oak EMs compared with other trees.

Nematode soil faunal analysis of decomposition pathways in different ecosystems

Carbon flow via decomposition of organic matter proceeds through different energy channels depending on the input and turnover rate of plant material. Generally, energy channels in grasslands are considered to be bacteria-dominated, whereas in forests the importance of fungal decomposition increases. Agroecosystems are regarded as intermediate. This hypothesis was tested using indices based on nematode faunal analysis that have been proposed as indicators of decomposition pathways. The ratio of fungal- to bacterial-feeding nematodes and the channel index (Ferris et al., 2001) were calculated from a range of studies, taking into account 131 different sites or sampling times. No distinct differences in the fungal- to bacterial-feeder ratio between grassland, field and forest were observed. However, the channel index indicated a fungal-based energy channel in coniferous forest sites. Generally, the results suggest that soil and climate affect nematode faunal indices more strongly than ecosystem type.