Tracking mycorrhizas and extraradical mycelium of the edible fungus <Emphasis Type="Italic">Lactarius deliciosus</Emphasis> under field competition with <Emphasis Type="Italic">Rhizopogon</Emphasis> spp

The objective of this study is to evaluate the field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus in competition with two ubiquitous soil fungi. Couples of plants inoculated with either L. deliciosus, Rhizopogon roseolus, or R. luteolus were transplanted, 10 cm apart, in two different sites at the following combinations: L. deliciosus-R. roseolus, L. deliciosus-R. luteolus, L. deliciosus-control (non-inoculated), control-R. roseolus, control-R. luteolus, and control-control. Eight months after transplantation, root colonization and extraradical soil mycelium for each fungal species were quantified. For mycelium quantification, soil cores equidistant to the two plants in each couple were taken, and total deoxyribonucleic acid (DNA) was extracted. Real-time polymerase chain reaction analysis was performed using specific primers and TaqMan Minor groove binding (MGB) probes designed in the ribosomal DNA internal transcribed spacer region of each fungal species. Field site significantly influenced persistence of both mycorrhizas and extraradical mycelium of L. deliciosus. Extraradical mycelium quantity was positively correlated with the final percentage of ectomycorrhizas for the three fungal species. Different competitive pressure between the two Rhizopogon species on L. deliciosus persistence was observed, with R. luteolus having no effect on L. deliciosus survival. Negative correlation between the final percentage of mycorrhizas of L. deliciosus and R. roseolus was observed. However, no relationship was determined between extraradical mycelia of both fungal species. The results obtained suggest that competition between L. deliciosus and R. roseolus takes place in the root system, for ectomycorrhiza formation in available roots, rather than in the extraradical phase.     

Molecular identification of the edible ectomycorrhizal fungus Lactarius deliciosus in the symbiotic and extraradical mycelium stages

Specific rDNA ITS amplifications, microsatellite-primed PCR and ITS-SSCP analysis were applied to identify and characterize pre-selected isolates of the edible ectomycorrhizal fungus Lactarius deliciosus in different stages of the life cycle. Sampling was performed from pure cultures, mycorrhizas and soil from experimental plots established with nursery-inoculated pine seedlings. A newly-designed reverse primer (LDITS2R) combined with the universal forward ITS1 allowed to perform specific amplifications of L. deliciosus from all the samples. Microsatellite-primed PCR using the (GTG)5 oligonucleotide as a primer showed clear polymorphisms among the different L. deliciosus isolates. The patterns of mycorrhiza samples showed additional bands corresponding to the plant DNA. Single strand conformation polymorphism (SSCP) analysis of the specific rDNA ITS fragment amplified from 18 L. deliciosus isolates showed nine clearly different patterns. Mycorrhiza and soil samples showed coincident patterns with their respective fungal isolates. Specific rDNA ITS amplifications had not been previously used for SSCP analysis of ectomycorrhizas and extraradical mycelium. This relatively simple and inexpensive technique allows tracking L. deliciosus isolates in different stages of the fungus development. Specific ITS-SSCP analysis is promising in studies of the persistence of inoculated L. deliciosus isolates and their competitiveness with native ectomycorrhizal fungi, especially at the extraradical mycelium stage.     

Quantification of extraradical mycelium of Tuber melanosporum in soils from truffle orchards in northern Spain

Quantification of extraradical mycelium of black truffle (Tuber melanosporum) has been carried out in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites, within the natural distribution area of the black truffles in Navarre (northern Spain). Specific primers and a Taqman® probe were designed to perform real-time PCR with DNA extracted from soil samples. Amplification of T. melanosporum DNA was obtained from 131 out of the 160 soil samples. The detection limit of the technique was 1.48 μg mycelium/g of soil. The extraradical mycelium biomass detected in the soil from the natural truffle ground was significantly greater (up to ten times higher) than the mycelium biomass detected in any of the orchards. Soil from productive, nonirrigated orchards in the Tierra Estella site contained significantly more extraradical mycelium than the rest of orchards irrigated, productive of T. brumale, or nonproductive. The comparison of soil mycelium biomass in nonirrigated evergreen oak orchards in both sites showed significantly more mycelium biomass in the Tierra Estella site. This study is the first attempt to quantify extraradical mycelium of T. melanosporum in the soil using Taqman® probes. The obtained quantitative results are of special interest to evaluate the fungal response to cultural treatments and to monitor the dynamics of the extraradical mycelium of T. melanosporum in the soil.     

Shoots, roots and ectomycorrhiza formation of pine seedlings at elevated atmospheric carbon dioxide

The effect of elevated atmospheric CO₂ concentration on the growth of shoots, roots, mycorrhizas and extraradical mycorrhizal mycelia of pine (Pinus silvestris L.) was examined. Two and a half-month-old seedlings were inoculated axenically with the mycorrhizal fungus Pisolithus tincto-rius (Pers.) by a method allowing rapid mycorrhiza formation in Petri dishes. The plants were then cultivated for 3 months in growth chambers with daily concentrations of 350 and 600 μmol mol^?1 CO₂ during the day. Whereas plants harvested after 1 and 2 months did not differ appreciably between ambient and increased CO₂ concentrations, after 3 months they developed a considerably higher root biomass (%57%) at elevated CO₂, but did not increase significantly in root length. The mycorrhizal fungus Pisolithus tinctorius, which depended entirely on the plant assimilates in the model system, grew much faster at increased CO₂: 3 times more mycorrhizal root clusters were formed and the extraradical mycelium produced had twice the biomass at elevated as at ambient CO₂. No difference in shoot biomass was found between the two treatments after 91 d. However, since the total water consumption of seedlings was similar in the two treatments, the water use efficiency was appreciably higher for the seedlings at increased CO₂ because of the higher below-ground biomass.     

Field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics

Pinus pinea plants were inoculated with different strains of the edible ectomycorrhizal fungus Lactarius deliciosus. The inoculated plants were established in six experimental plantations in two sites located in the Mediterranean area to determine the effect of the initial colonization level and the inoculated strain on fungal persistence in the field. Ectomycorrhizal root colonization was determined at transplantation time and monitored at different times from uprooted plants. Extraradical soil mycelium biomass was determined from soil samples by TaqMan® real-time polymerase chain reaction (PCR). The results obtained indicate that the field site played a decisive role in the persistence of L. deliciosus after outplanting. The initial colonization level and the selection of the suitable strain were also significant factors but their effect on the persistence and spread of L. deliciosus was conditioned by the physical–chemical and biotic characteristics of the plantation soil and, possibly, by their influence in root growth. Molecular techniques based on real-time PCR allowed a precise quantification of extraradical mycelium of L. deliciosus in the field. The technique is promising for non-destructive assessment of fungal persistence since soil mycelium may be a good indicator of root colonization. However, the accuracy of the technique will ultimately depend on the development of appropriate soil sampling methods because of the high variability observed.     

Contrasting phosphate acquisition of mycorrhizal fungi with that of root hairs using the root hairless barley mutant

Comparisons between plant species or cultivars differing in root hair length have indicated a major impact of root hairs on the mycorrhizal dependency of plants with respect to phosphate (P) uptake. The current study aimed to investigate this relationship by comparing directly the mycorrhizal dependency of a spontaneous root hairless mutant, brb , in Hordeum vulgare cv Pallas and its wild type. Both brb and wild type were grown at different soil P levels in association with different mycorrhizal fungi. P uptake of brb and wild type was similar at high P levels, but P uptake by non-mycorrhizal brb plants at low P levels was substantially lower than that of the non-mycorrhizal wild-type plants. However, P uptake of the mutant was much increased by mycorrhizas and with one fungus, the additional P uptake was effectively translated into increased plant growth. Roots of the mutant contained typical colonization structures and a radioactive tracer confirmed P transport by the extraradical mycelium. This is the first direct evaluation of the relative effectiveness of root hairs and mycorrhizas. Mycorrhizas effectively substituted root hairs in P uptake, whereas the additional P was most often used less effectively in promoting plant growth than P provided by root hairs.     

The detection of Glomus spp. (arbuscular mycorrhizal fungi) forming mycorrhizas in three plants, at different stages of seedling development, using mycorrhiza-specific isozymes

A series of glasshouse experiments was used to determine mycorrhiza-specific isozymes (MSIs) produced by five species of Glomus colonizing roots of a desert shrub legume ( Anthyllis cytisoides L.), Thymus vulgaris L. and Allium porrum L. over time. Extracts of colonized roots were electrophoresed on non-denaturing polyacrylamide gels (PAGE) and stained for 10 different enzymes. Staining protocols for esterase, glutamate oxaloacetate transaminase, alkaline phosphatase and malate dehydrogenase provided MSIs for the mycorrhizas formed by different arbuscular mycorrhizal (AM) fungi that had colonized roots of the three host plants. There was no apparent correlation between levels of colonization or arbuscular intensities, at or between each sampling, and the presence of MSIs. The development of colonization by the AM fungi differed little between the three plants when assessed with two methods of estimating fungal biomass. The variety of MSIs detected might reflect the diversity of metabolic activities of these Glomus species and, possibly, differing ecological functions. The high-level induction of two alkaline phosphatase MSIs in the mycorrhizas of Anthyllis cytisoides colonized by Glomus microaggregatum BEG56 was used to track the fate of this fungus when the same plant was inoculated and transplanted into a semi-arid site in south-east Spain. The probable fungal origin of the isozyme was indicated by detection of the same isozyme in the extraradical mycelium formed by Glomus microaggregatum BEG56 on Allium porrum . The use of MSIs to detect the mycorrhizas of species of Glomus in colonized roots is discussed.     

Rapid typing of truffle mycorrhizal roots by PCR amplification of the ribosomal DNA spacers

DNA analyses were developed to type mycorrhizas of two Tuber species of commercial value (T. melanosporum, T. borchii) and a competitive fungus (Sphaerosporella brunnea) which forms ectomycorrhizas with plants usually considered hosts for truffles. Polymerase chain reaction (PCR) amplification of DNA isolated from fruitbodies, mycelia, mycorrhizas and leaves of host plants, was performed with a primer pair for an internal transcribed spacer ITS1-4. ITS amplification followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products clearly distinguished the two Tuber species at the fruitbody, mycorrhiza and mycelium levels. Accepted: 6 September 1996     

Nitrogen transfer and assimilation between the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith and Ri T-DNA roots of Daucus carota L. in an in vitro compartmented system

Nitrogen metabolism was examined in monoxenic cultures of carrot roots (Daucus carota L.) colonized with the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. Glutamine synthetase and glutamate dehydrogenase activities were significantly increased in mycorrhizal roots for which only the extraradical mycelium had exclusive access to NH4NO3 in a distinct hyphal compartment inaccessible to the roots. This was in comparison with the water controls but was similar to the enzyme activities of non-arbuscular-mycorrhizal (non-AM) roots that had direct access to NH4NO3. In addition, glutamate dehydrogenase activity was significantly enhanced in AM roots compared with non-AM roots. Carrot roots took up 15NH4+ more efficiently than 15NO3-, and the extraradical hyphae transfered 15NH4+ to host roots from the hyphal compartment but did not transfer 15NO3-. The extraradical mycelium was shown, for the first time, to have a different glutamine synthetase monomer than roots. Our overall results highlight the active role of AM fungi in nitrogen uptake, transfer, and assimilation in their symbiotic root association.     

Extraradical development and contribution to plant performance of an arbuscular mycorrhizal symbiosis exposed to complete or partial rootzone drying

Sweet potato plants were grown with or without Glomus intraradices in split-root pots with adjacent root compartments containing a soil with a low availability of phosphate. One fungal tube, from which root growth was excluded, was inserted into each root compartment. During 4 weeks before harvest, the soil moisture level in either both or only one of the two root-compartments of each pot was decreased. Controls remained well watered. Low soil moisture generally had a negative effect on the amount of extraradical mycelium of G. intraradices extracted from the fungal tubes. Sporulation in the fungal tubes was much higher compared with the soil in the root compartment, but remained unaffected by the soil moisture regime. Concentrations of P in extraradical mycelium were much lower than usually found in plants and fungi, while P concentrations in associated mycorrhizal host plant tissues were in an optimum range. This suggests efficient transfer of P from the extraradical mycelium to the host plant. Despite the negative effect of a low soil moisture regime on extraradical G. intraradices development, the symbiosis indeed contributed significantly to P uptake of plants exposed to partial rootzone drying. The possibility that extraradical arbuscular mycorrhizal fungal development was limited by P availability under dry soil conditions is discussed.     

Lipids in roots of Pinus sylvestris seedlings and in mycelia of Pisolithus tinctorius during ectomycorrhiza formation: changes in fatty acid and sterol composition

The composition of fatty acids and sterols in soil lipid fractions is often used as a global indicator for the status and changes of soil microbial communities. In order to validate such analyses in the context of ectomycorrhizal communities, an experiment was performed in which seedlings of Pinus sylvestris and the fungus Pisolithus tinctorius were grown separately, or combined to form ectomycorrhiza under axenic conditions. Fatty acids of the neutral lipid fraction (NLFAs) and the phospholipid fraction (PLFAs) as well as sterols were identified and quantified by gas chromatography–mass spectrometry. When grown separately, the two organisms differed strongly with respect to the sterol composition. Sterols had a much higher relative abundance in the fungus in comparison with the plant, and the two main fungal sterols, ergosterol and 24‐ethyllanosta‐8,24(24′)‐diene‐3beta,22zeta‐diol (Et lano 8,24), as well as six minor fungal sterols were not found in the plant. On the other hand, the three sterols found in plant roots were absent from the fungus. With regard to fatty acids, the lipids of both organisms contained the same three major PLFAs, namely n16:0, 18:2–9,12c, and 18:1–9c. However, plant lipids contained, in addition, eight PLFAs and five NLFAs that were not present in the fungus. On the other hand, the fungus contained two PLFAs and two NLFAs that were not present in the plant. When the fungus and the plant were brought together, there was a drastic change in the lipid composition of the root: within a day, all the saturated fatty acids in the NLFA fraction increased very strongly and then slowly decreased but remained at an elevated level throughout the experiment. All these saturated fatty acids also started to appear in the extraradical fungal mycelium; they increased steadily and reached their highest levels at the end of the experiment. These results indicate that in symbiosis, the fungus transports plant lipids from the symbiotic interface to the extraradical mycelium. Concerning sterols, the extraradical mycelium acquired only a small amount of plant‐specific sterols. However, its ergosterol content steadily decreased whereas the content of Et lano 8,24 remained high, causing the ratio of these two sterols to decrease from 1 : 7 to 1 : 20, whereas in the ectomycorrhizal root, the opposite phenomenon occurred, so that the ratio increased to a value of almost 1 : 1. The marked changes in the composition of the extraradical mycelium were well reflected in a principal component analysis of all lipid components. The present results show that a given ectomycorrhizal fungus may display markedly different lipid compositions in its intraradical and extraradical parts. In addition, they highlight a potential role of plant lipid transfer from the root to the fungus in the functioning of the ectomycorrhizal symbiosis.     

Metabolic and Genotypic Fingerprinting of Fluorescent Pseudomonads Associated with the Douglas Fir-Laccaria bicolor Mycorrhizosphere

A collection of 300 isolates of fluorescent pseudomonads was established from Douglas fir-Laccaria bicolor mycorrhizas and mycorrhizosphere and from adjacent bulk soil. These isolates were first phenotypically characterized with the Biolog method. Taxonomic identification assigned 90% of the isolates to the different biovars of Pseudomonas fluorescens, with inverted frequencies of biovars V and I from the bulk soil to the mycorrhizas, suggesting that the mycorrhizas exert a selective stimulation of the P. fluorescens bv. I and a counterselection of the P. fluorescens bv. V present in the soil. Multivariate analyses of the carbon source utilization data led to the definition of homogenous metabolic groups and to the identification of the most discriminating substrates for each group. The isolates from the mycorrhizosphere and from the mycorrhizas seem to preferentially utilize carbohydrates, in particular trehalose, which is the most abundant carbohydrate accumulated in the mycelium of L. bicolor. The results suggest that L. bicolor exerts a trehalose-mediated selection on the fluorescent pseudomonads present in the vicinity of the mycorrhizas. Isolates of P. fluorescens from the mycorrhizosphere and mycorrhizas were then genotypically characterized by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes and enterobacterial repetitive intergenic consensus-PCR DNA fingerprinting. Both methods revealed a high genetic polymorphism within the population studied, which was well correlated with the phenotypic characterization.     

Influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structure

Plant root systems colonized by arbuscular mycorrhizal (AM) fungi have previously been shown to influence soil bacterial populations; however, the direct influence of the AM extraradical mycelium itself on bacterial growth and community composition is not well understood. In this study, we investigated the effects of exudates produced by AM extraradical mycelia on the growth and development of an extracted soil bacterial community in vitro. The chemical composition of the mycelial exudates was analysed using proton nuclear magnetic resonance spectrometry. Following the addition of exudates to a bacterial community extracted from soil, bacterial growth and vitality were determined using a bacterial vitality stain and fluorescence microscopy. Changes in community composition were also analysed at various times over the course of 3 days by terminal restriction fragment length polymorphism analysis, in combination with cloning and sequencing of 16S rRNA genes. Mycelial exudates increased bacterial growth and vitality and changed bacterial community composition. Several Gammaproteobacteria, including a taxon within the Enterobacteriaceae, increased in frequency of occurrence in response to AM mycelial exudates. This study is the first attempt to identify carbohydrates from the extraradical mycelium of an AM fungus, and demonstrates the direct effects of mycelial exudates on a soil bacterial community.     

Fungal biomass production in response to elevated atmospheric CO2 in a Glomus mosseae–Prunus cerasifera model system

Biomass and length of intraradical and extraradical mycorrhizal mycelium under ambient (aCO₂) and elevated (eCO₂ ) atmospheric CO₂ was investigated using a non-destructive in vivo experimental model system. Time-course experiments allowed measurements of intact extraradical mycelium spreading from mycorrhizal roots of Prunus cerasifera micropropagated plants inoculated with the arbuscular mycorrhizal fungus Glomus mosseae, in controlled environmental chambers. The length of extraradical mycelium was significantly increased at the highest CO₂ concentration, ranging from 10.7 to 20.3 m at aCO₂ and eCO₂, respectively. The biochemical determination of mycelial glucosamine content allowed the evaluation of intraradical and extraradical fungal biomass, which were 2 and 3 times larger at eCO₂ than at aCO₂. Present data show that Glomus mosseae responds to increases of CO₂ concentrations producing larger mycorrhizal networks which may potentially represent carbon sink agents in soil ecosystems.     

Phosphorus Effects on the Mycelium and Storage Structures of an Arbuscular Mycorrhizal Fungus as Studied in the Soil and Roots by Analysis of Fatty Acid Signatures

The distribution of an arbuscular mycorrhizal (AM) fungus between soil and roots, and between mycelial and storage structures, was studied by use of the fatty acid signature 16:1(omega)5. Increasing the soil phosphorus level resulted in a decrease in the level of the fatty acid 16:1(omega)5 in the soil and roots. A similar decrease was detected by microscopic measurements of root colonization and of the length of AM fungal hyphae in the soil. The fatty acid 16:1(omega)5 was estimated from two types of lipids, phospholipids and neutral lipids, which mainly represent membrane lipids and storage lipids, respectively. The numbers of spores of the AM fungus formed in the soil correlated most closely with neutral lipid fatty acid 16:1(omega)5, whereas the hyphal length in the soil correlated most closely with phospholipid fatty acid 16:1(omega)5. The fungal neutral lipid/phospholipid ratio in the extraradical mycelium was positively correlated with the level of root infection and thus decreased with increasing applications of P. The neutral lipid/phospholipid ratio indicated that at high P levels, less carbon was allocated to storage structures. At all levels of P applied, the major part of the AM fungus was found to be present outside the roots, as estimated from phospholipid fatty acid 16:1(omega)5. The ratio of extraradical biomass/intraradical biomass was not affected by the application of P, except for a decrease at the highest level of P applied.     

Interactions between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and nontransformed tomato roots of either wild-type or AM-defective phenotypes in monoxenic cultures

Monoxenic symbioses between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and two nontransformed tomato root organ cultures (ROCs) were established. Wild-type tomato ROC from cultivar “RioGrande 76R” was employed as a control for mycorrhizal colonization and compared with its mutant line (rmc), which exhibits a highly reduced mycorrhizal colonization (rmc) phenotype. Structural features of the two root lines were similar when grown either in soil or under in vitro conditions, indicating that neither monoxenic culturing nor the rmc mutation affected root development or behavior. Colonization by G. intraradices in monoxenic culture of the wild-type line was low (<10%) but supported extensive development of extraradical mycelium, branched absorbing structures, and spores. The reduced colonization of rmc under monoxenic conditions (0.6%) was similar to that observed previously in soil. Extraradical development of runner hyphae was low and proportional to internal colonization. Few spores were produced. These results might suggest that carbon transfer may be modified in the rmc mutant. Our results support the usefulness of monoxenically obtained mycorrhizas for investigation of AM colonization and intraradical symbiotic functioning.     

Measurement of the extraradical mycelium of a vesicular-arbuscular mycorrhizal fungus in soil by chitin determination

Summary Development of a vesicular-arbuscular mycorrhizal (VAM) fungus in association with soybean was determined in a greenhouse soil mix by chitin assay. Samples were sieved to eliminate hexosamine-containing contaminants. This preparation reduced the interference caused by extraneous soil substances and permitted quantitative measurement of extraradical VAM fungal mycelium in the soil mix by colorimetric assay. Recovery of added chitin, used as an internal standard, was greater in the soil mix than in an inert medium indicating that some hexosamine was stabalized from chemical degradation by other soil components.