Calibration of quantitative real-time TaqMan PCR by correlation with hyphal biomass and ITS copies in mycelia of Piloderma croceum

DNA-based quantification methods such as real-time TaqMan PCR allow a rapid and highly sensitive species-specific quantification of isolated fungal DNA material, but most quantification systems are only able to measure relative amounts of biomass or biomass changes during different treatments. In this experiment, an already established DNA quantification system for the ectomycorrhizal fungus Piloderma croceum, based on the ITS region of ribosomal DNA, was calibrated to absolute biomass to obtain a direct correlation between mycelial biomass and isolated ITS copies. Thin layers of sterile mycelia were cultured on slides. The mycelial biomass was calculated from measurements of the total hyphal length using image analysis, followed by determination of the mycelial volume, and multiplied by the specific weight of hyphae obtained from literature data. Using the very same mycelium, the number of ITS copies was quantified by TaqMan PCR. The mean value of 1047 (+/- 185) copies per mm hypha results in possible data for a direct conversion: one billion (10 (9)) ITS copies corresponded to 0.79 mg hyphal dry weight. For the ribosomal ITS multi-copy genes, the number of ITS copies could be calculated to approx. 152 (+/- 26) copies per dikaryotic cell. These conversion data now allow determination of the mycelial biomass of Piloderma croceum using real-time TaqMan PCR, a prerequisite for competition experiments with Piloderma croceum.     

Solubilisation and colonisation of wood ash by ectomycorrhizal fungi isolated from a wood ash fertilised spruce forest

In Sweden application of granulated wood ash has been suggested as a method to supplement nutrient loss resulting from harvesting of forest residues for bioenergy production. Mycelia of two ectomycorrhizal fungi Piloderma sp. 1 and Ha-96-3, were commonly found to colonise ash granules in a wood ash fertilised spruce forest. Thirty-eight fungal isolates were selected from 10 taxa to investigate the possible role of different ectomycorrhizal fungi in nutrient mobilisation from ash. The taxa were Cenococcum geophilum Fr., Piloderma croceum Erikss. and Hjortst., Piloderma sp. 1, Thelephora terrestris (Ehrenb.) Fr., Tylospora fibrillosa Donk, and five unidentified species, all originating from a wood ash fertilised spruce forest. The isolates were tested for their ability to solubilise tricalcium phosphate (TCP) or hardened wood ash (HWA) in vitro. Ha-96-3, P. croceum and Piloderma sp. 1 were the only taxa which solubilised TCP. Abundant calcium oxalate crystals were formed in TCP and HWA plates with Piloderma sp. 1. Ha-96-3 and two isolates of P. croceum produced intermediate amounts of crystals. Ha-96-1 and T. fibrillosa produced low amounts of crystal but no crystal formation was observed by any of the other isolates. Piloderma sp. 1 from HWA plates had significantly higher concentrations of P, compared to P. croceum or Ha-96-3. Piloderma sp. 1 and P. croceum were further tested for their ability to colonise wood ash in microcosms containing intact mycorrhizal associations. After 7 months Piloderma sp. 1 colonised ash amended patches with a dense, mat like mycelium, whereas P. croceum mycelia avoided the ash patches. Possible differences between these fungi in patterns of carbon allocation were investigated by labelling seedlings with 14CO(2). Piloderma sp. 1 mycelia allocated significantly more 14C to ash patches than P. croceum. P. croceum allocated relatively more 14C to control patches than to the ash patches. The possible role of ectomycorrhizal fungi in mobilisation of nutrients from wood ash is discussed.     

Colonisation of spruce roots by two interacting ectomycorrhizal fungi in wood ash amended substrates

Interactions between two ectomycorrhizal fungal species, Piloderma croceum Erikss. and Hjortst. and Piloderma sp. 1 (found to colonise spruce roots and wood ash granules in the field), were investigated in wood ash amended substrates. The comparative ability of these fungi to colonise roots of non-mycorrhizal spruce (Picea abies (L.) Karst.) seedlings was studied in relation to factorial combinations of wood ash and N fertilisation. Non-mycorrhizal spruce seedlings (bait seedlings) were planted together with spruce seedlings colonised by P. croceum or Piloderma sp. 1. The growth substrate was a sand-peat mixture with wood ash or no ash and supplied with two levels of N, so that four substrate combinations were obtained. Piloderma sp. 1 mycelia colonised around 60% of the fine roots of bait seedlings in ash treatments regardless of N level and around 20-26% in treatments without ash. P. croceum only colonised 8% of the root tips in the presence of ash but 56% of the root tips in the low-N treatment without ash. However, in the high-N treatment without ash the colonisation level was reduced to around 30%. Total numbers of root tips per seedling did not vary significantly between the treatments. Possible reasons for the competitive advantage of Piloderma sp. 1 in wood ash fertilised substrate are discussed.     

Ectomycorrhizal weathering of the soil minerals muscovite and hornblende

Ectomycorrhizal fungi are hypothesized to enhance mineral weathering in forest soils. Several studies have shown an increased uptake of mineral-derived nutrients by trees when in symbiosis with ectomycorrhizal fungi. However, it is difficult to determine from these studies if the improved nutrient uptake is the result of increased weathering or better exploitation of the substrate by the ectomycorrhizal fungi. In a pot experiment, Pinus sylvestris (Scots pine) seedlings were grown with or without ectomycorrhizal fungi, and with or without the mineral muscovite as the only potassium (K) source or the mineral hornblende as the only magnesium (Mg) source. After 27 wk, all pools of non-mineral-bound K or Mg were determined. The ectomycorrhizal fungus Paxillus involutus increased weathering of muscovite but not hornblende. The other ectomycorrhizal fungi tested, Piloderma croceum and Suillus bovinus, did not increase weathering of either muscovite or hornblende compared with the nonmycorrhizal trees. The P. involutus-mediated mobilization of K from muscovite resulted in increased K content of root plus adhering hyphae, but not of shoots. In conclusion, ectomycorrhizal fungi may increase weathering of minerals in response to nutrient deficiencies, but this response is species specific.     

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes

ABSTRACT: BACKGROUND: Studies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner. RESULTS: None of the fifteen Streptomyces isolates inhibited all seven tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum). The growth of only one of the tested fungi, the mycorrhiza-forming fungus Laccaria bicolor, was stimulated by the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol) and siderophores (e.g. ferulic acid, desferroxiamines). Plant disease resistance was activated by a single streptomycete strain only. CONCLUSIONS: Our results show that the primary characteristic of mycorrhiza associated streptomycetes is to inhibit the growth of fungi and bacteria. In parallel, our study indicates that Streptomyces strains which are not general antagonists may produce previously un-described metabolites.     

Discrimination of <Emphasis Type="Italic">Tricholoma</Emphasis> species by species-specific ITS primers

The ITS region of ectomycorrhizal fungi was analyzed, and species-specific PCR primers were designed for 8 ectomycorrhizal Tricholoma species. Although a high degree of intraspecific homology was observed, interspecific variation was sufficient to design species-specific primers based on sequence of the ITS region. PCR amplification with the specific primers generated fragments of the expected sizes from DNA extracted from the strains of each species but gave no amplified products from the strains of the other 16 species in eight genera. These results suggest that sequence of the ITS region is appropriate to be used for species-level identification of ectomycorrhizal fungi.     

Real-Time PCR Assays for Monitoring Anaerobic Fungal Biomass and Population Size in the Rumen

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.     

Apatite as a P source in mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

The objectives of the study are firstly to test the ability of ectomycorrhizal pine seedlings to use apatite as a P source in comparison with non-mycorrhizal pine seedlings and secondly, to determine if there is a relation between exudation of organic acids and the ability to use apatite as a P source. Non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 4 different isolates of ectomycorrhizal fungi were grown for 220 days in sand/peat filled pots with apatite (Ca₅(F,OH)(PO₄)₃) as the sole P source. In an additional experiment, non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 2 different isolates of ectomycorrhizal fungi were grown without any P source for 250 days. All other nutrients were supplied in a balanced nutrient solution.Ectomycorrhizal seedlings grew less than non-mycorrhizal seedlings but ectomycorrhizal seedlings produced a large external mycelium not included in the biomass estimates. All seedlings in the present study had low shoot:root ratios compared to seedlings growing under optimal conditions. All seedlings grown with apatite as P source had higher foliar P concentrations (0.71–2.11 mg/g) than seedlings growing without any P source (0.57–0.75 mg/g) indicating a significant ability to use apatite as a P source. Seedlings colonized by Suillus variegatus and Paxillus involutus had higher concentrations and total contents of P in shoots compared with non-mycorrhizal seedlings, indicating significant improvement of P uptake by these fungi in comparison with non-mycorrhizal seedlings or seedlings colonized Piloderma croceum.No clear relationship between exudation of organic acids and uptake of P was found. Seedlings colonized by S. variegatus reduced the pH of the soil more than seedlings colonized by P. involutus or non-mycorrhizal seedlings. It is suggested that S. variegatus colonization improves the P uptake by reducing the pH of the soil while P. involutus improves P uptake by having a greater ability to absorb dissolved phosphate than non-mycorrhizal roots or roots colonized by the other fungi used in the study.     

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.     

Simultaneous monitoring of two fungal genotypes on plant roots by single nucleotide polymorphism quantification with an innovative KASPar quantitative PCR

An innovative quantitative PCR-based method derived from the Kompetitive Allele Specific PCR Assay Reagent (KASPar) system was developed to quantify the genomic DNA from two coexisting genotypes on the same tissues of a host-plant. For this purpose, the classical end-point KASPar method was evolved to a real-time method thanks to the addition of an adapted measurement step after each PCR cycle. It was applied to the quantification of the two genotypes G1 and G2 of the Gaeumannomyces graminis var. tritici (Ggt) soilborne fungus, pathogenic on wheat roots. Specific primers targeting a single nucleotide polymorphism from the ITS sequence were used allowing simultaneous quantification of both genotypes in the same reaction. The assays were applied to quantify fungal DNA of each genotype, aside or mixed together, after DNA extraction from fungal pure cultures and from single or co-inoculated roots in artificial medium or in soil. The detection and quantification lower limits for the two genotypes were 1.25 pg and 5 pg for DNA from fungal pure cultures, and 1.8 pg and 7 pg for DNA from fungal inoculated roots. The advantages of this cost-effective method are the high levels of specificity, sensitivity and reproducibility. Moreover, the accuracy of the method is independent of the copy numbers of the target sequences. The method is the first one to adapt the non-quantitative genotyping KASPar system to a quantitative application of two known genotypes of a species simultaneously and is suitable for simultaneous genotype-specific quantification of any other organisms (fungi, bacteria, plants).     

Organic anion exudation by ectomycorrhizal fungi and Pinus sylvestris in response to nutrient deficiencies

Low molecular weight organic anions (LMWOA) can enhance weathering of mineral grains. We tested the hypothesis that ectomycorrhizal (EcM) fungi and tree seedlings increase their exudation of LMWOA when supply of magnesium, potassium and phosphorus is low to enhance the mobilization of Mg, K and P from mineral grains. Ectomycorrhizal fungi and Pinus sylvestris seedlings were cultured in symbiosis and in isolation on glass beads with nutrient solution or with sand as a rooting medium, with a complete nutrient supply or with Mg, K, P or N in low supply. Concentrations of all dicarboxylic LMWOA in the rooting medium were measured. Nonmycorrhizal seedlings released predominantly malonate. Colonization with Hebeloma longicaudum decreased the amount of organic anions exuded, whereas Paxillus involutus and Piloderma croceum increased the concentration of oxalate but not the total amount of LMWOA. Phosphorus deficiency increased the concentration of LMWOA by nonmycorrhizal and EcM seedlings. Magnesium deficiency increased the concentration of oxalate by nonmycorrhizal and EcM seedlings, but not the concentration of total LMWOA. Paxillus involutus grown in pure culture responded differently to low nutrient supply compared with symbiotic growth. Ectomycorrhizal fungi did not increase the total concentration of LMWOA compared with nonmycorrhizal seedlings but, depending on the fungal species, they affected the type of LMWOA found.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.     

Influence of long-term repeated prescribed burning on mycelial communities of ectomycorrhizal fungi

To demonstrate the efficacy of direct DNA extraction from hyphal ingrowth bags for community profiling of ectomycorrhizal (ECM) mycelia in soil, we applied the method to investigate the influence of long-term repeated prescribed burning on an ECM fungal community. DNA was extracted from hyphal ingrowth bags buried in forest plots that received different prescribed burning treatments for 30 yr, and denaturing gradient gel electrophoresis (DGGE) profiles of partial fungal rDNA internal transcribed spacer (ITS) regions were compared. Restriction fragment length polymorphism (RFLP) and sequence analyses were also used to compare clone assemblages between the treatments. The majority of sequences derived from the ingrowth bags were apparently those of ECM fungi. DGGE profiles for biennially burned plots were significantly different from those of quadrennially burned and unburned control plots. Analysis of clone assemblages indicated that this reflected altered ECM fungal community composition. The results indicate that hyphal ingrowth bags represent a useful method for investigation of ECM mycelial communities, and that frequent long-term prescribed burning can influence below-ground ECM fungal communities.     

Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a <Emphasis Type="Italic">Quercus</Emphasis> woodland

The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fungal molecular diagnostics: a mini review

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.     

Manipulation of the onset of ectomycorrhiza formation by indole-3-acetic acid, activated charcoal or relative humidity in the association between oak microcuttings and Piloderma croceum: influence on plant development and photosynthesis

A Petri dish system in which development of oak (Quercus robur L.) microcuttings is stimulated by the late stage ectomycorrhizal (EM) fungus Piloderma croceum J. Erikss. & Hjortst. in a long pre-symbiotic stage was optimised to allow synchronous, rhythmic plant growth. Addition of indole-3-acetic acid or activated charcoal to the medium caused an early and more intensive EM formation coupled with suppression of most developmental effects of P. croceum. Leaf area, chlorophyll fluorescence, and content were compared in inoculated and uninoculated plants grown at two relative humidity levels (45 and 95%) and under consideration of three possible answers to inoculation, that is, no or EM formation after the 1st or the 2nd shoot flush. The culture conditions for uninoculated plants were suboptimal, leading toward photochemical stress reflected by a non photochemical quenching (qE) increase and a reduced Chl content at the end of the assay. Prior to EM formation, inoculation itself enhanced the optimal (Fv/Fm) and effective (phiPSII) quantum yield in leaves of the 1st shoot flush under reduced relative humidity. It also fully protected the plants against stress during the complete assays. The results indicate that inoculated plants only form EM once they have acquired a sufficient development level and C-providing capacity. However, the fungus actively improves the development and photosynthesis of plants up to the pre-mycorrhizal stage, helping them to reach this capacity.     

Genetic manipulation in vesicular-arbuscular mycorrhizal fungi

The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages. The work discussed here focuses on the application of Polymerase Chain Reaction (PCR) technology and the potential of promoting hyphal growth in the absence of the plant. These techniques are being used to study VAM fungi in two main areas. The first concerns the determination of the DNA sequences coding for the SSU ribosomal RNA of two VAM fungi. This approach has allowed the design of specific primers for the rapid identification and quantification of VAM fungi. The second area of research concerns the potential use of PCR technology to study selective expression of specific genes during fungal spore development in defined in vitro conditions. The achievement of this future prospect depends on the ability to prepare PCR-based cDNA libraries from small amounts of fungal material after stimulation of hyphal growth with CO₂ and plant flavonols.