13C incorporation into signature fatty acids as an assay for carbon allocation in arbuscular mycorrhiza

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1omega5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating (13)C enrichment of 16:1omega5 and compared it with (13)C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [(13)C]glucose. The (13)C enrichment of neutral lipid fatty acid 16:1omega5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for (13)C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1omega5 than for the root specific neutral lipid fatty acid 18:2omega6,9. We labeled plant assimilates by using (13)CO(2) in whole-plant experiments. The extraradical mycelium often was more enriched for (13)C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between (13)C enrichment in neutral lipid fatty acid 16:1omega5 and total (13)C in extraradical mycelia in different systems (r(2) = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the (13)C enrichment of 16:1omega5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.     

13C Incorporation into Signature Fatty Acids as an Assay for Carbon Allocation in Arbuscular Mycorrhiza

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1ω5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating ¹³C enrichment of 16:1ω5 and compared it with ¹³C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [¹³C]glucose. The ¹³C enrichment of neutral lipid fatty acid 16:1ω5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for ¹³C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1ω5 than for the root specific neutral lipid fatty acid 18:2ω6,9. We labeled plant assimilates by using ¹³CO₂ in whole-plant experiments. The extraradical mycelium often was more enriched for ¹³C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between ¹³C enrichment in neutral lipid fatty acid 16:1ω5 and total ¹³C in extraradical mycelia in different systems (r² = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the ¹³C enrichment of 16:1ω5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.     

Mycorrhizal infection and ageing affect element localization in short roots of Norway spruce [Picea abies (L) Karst.]

Norway spruce [Picea abies (L.) Karst.] seedlings, nonmycorrhizal of mycorrhizal with Laccaria laccata or Paxillus involutus were grown in a quartz sand-nutrient solution system for 6 months and then treated with 5 M Pb for 4 days. Element contents of cortex cell wall of young, medium and old short roots were determined by X-ray microanalysis of longitudinal thin sections. The Pb content was influenced neither by age nor by the distance from the root tip (up to 1.7 mm) but was significantly lower in the P. involutus mycorrhizae than in the L. laccata mycorrhizae or in nonmycorrhizal short roots. In the P. involutus mycorrhizae, the P content of the cortex cell walls was twice as high in young mycorrhizae than in old mycorrhizae. In the nonmycorrhizal short roots and the L. laccata mycorrhizae, P content was influenced neither by age nor by distance from the root tip. The Ca and Fe contents of the cortex cell walls increased with age in the nonmycorrhizal short roots and the mycorrhizae. It is concluded that the element content of the cortex cell walls of short roots is strongly influenced by age, while the distance from the root tip seems to be of minor importance.     

Extraradical mycelium network of arbuscular mycorrhizal fungi allows fast colonization of seedlings under in vitro conditions

Actively growing extraradical hyphae extending from mycorrhizal plants are an important source of inoculum in soils which has seldom been considered in vitro to inoculate young plantlets. Seedlings of Medicago truncatula were grown in vitro in the extraradical mycelium network extending from mycorrhizal plants. After 3, 6, 9, 12, and 15 days of contact with the mycelium, half of the seedlings were harvested and analyzed for root colonization. The other half was carefully transplanted in vitro on a suitable growth medium and mycelium growth and spore production were evaluated for 4 weeks. Seedlings were readily colonized after 3 days of contact with the mycelium. Starting from 6 days of contact, the newly colonized seedlings were able to reproduce the fungal life cycle, with the production of thousands of spores within 4 weeks. The fast mycorrhization process developed here opens the door to a broad range of in vitro studies for which either homogenous highly colonized seedlings or mass-produced in vitro inoculum is necessary. Liesbeth Voets and Ivan Enrique de la Providencia contributed equally to this work. MUCL is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

Structural and functional interactions between extraradical mycelia of ectomycorrhizal Pisolithus isolates

Extraradical mycelia from different ectomycorrhizal (ECM) roots coexist and interact under the forest floor. We investigated structural connections of conspecific mycelia and translocation of carbon and phosphorus between the same or different genets. Paired ECM Pinus thunbergii seedlings colonized by the same or different Pisolithus isolates were grown side by side in a rhizobox as their mycelia contacted each other. (14)CO(2) or (33)P-phosphoric acid was fed to leaves or a spot on the mycelium in one of the paired seedlings. Time-course distributions of (14)C and (33)P were visualized using a digital autoradiographic technique with imaging plates. Hyphal connections were observed between mycelia of the same Pisolithus isolate near the contact site, but hyphae did not connect between different isolates. (14)C and (33)P were translocated between mycelia of the same isolate. In (33)P-fed mycelia, accumulation of (33)P from the feeding spot toward the host ECM roots was observed. No (14)C and (33)P translocation occurred between mycelia of different isolates. These results provide direct evidence that contact and hyphal connection between mycelia of the same ECM isolate can cause nutrient translocation. The ecological significance of contact between extraradical mycelia is discussed.     

Mucoid mutants of the biocontrol strain pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots

Extracellular polysaccharides play an important role in the formation of bacterial biofilms. We tested the biofilm-forming ability of two mutant strains with increased production of acidic extracellular polysaccharides compared with the wild-type biocontrol strain Pseudomonas fluorescens CHA0. The anchoring of bacteria to axenic nonmycorrhizal and mycorrhizal roots as well as on extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices was investigated. The nonmucoid wild-type strain P. fluorescens CHA0 adhered very little on all surfaces, whereas both mucoid strains formed a dense and patchy bacterial layer on the roots and fungal structures. Increased adhesive properties of plant-growth-promoting bacteria may lead to more stable interactions in mixed inocula and the rhizosphere.     

Molecular diversity of fungi from ericoid mycorrhizal roots

In order to investigate the diversity of fungal endophytes in ericoid mycorrhizal roots, about 150 mycelia were isolated from surface-sterilized roots of 10 plants of Calluna vulgaris. Each mycelium was reinoculated to C. vulgaris seedlings under axenic conditions, and the phenotype of the plant-fungus association assessed by light and electron microscopy. Many isolates that were able in vitro to produce typical ericoid mycorrhizae did not form reproductive structures under our culture conditions, whereas others could be identified as belonging to the species Oidiodendron maius. Morphological and molecular analysis of the fungal isolates showed that the root system of a single plant of C. vulgaris is a complex mosaic of several populations of mycorrhizal and non mycorrhizal fungi. PCR-RFLP techniques, used to investigate the mycorrhizal endophytes, revealed up to four groups of fungi with different PCR-RFLP patterns of the ITS ribosomal region from a single plant. Some of the mycorrhizal fungi sharing the same PCR-RFLP pattern showed high degree of genetic polymorphism when analysed with the more sensitive RAPD technique; this technique may prove a useful tool to trace the spread of individual mycorrhizal mycelia, as it has allowed us to identify isolates with identical RAPD fingerprints on different plants.     

Carbon Cost of the Fungal Symbiont Relative to Net Leaf P Accumulation in a Split-Root VA Mycorrhizal Symbiosis

Translocation of ¹⁴C-photosynthates to mycorrhizal (+ +), half mycorrhizal (0+), and nonmycorrhizal (00) split-root systems was compared to P accumulation in leaves of the host plant. Carrizo citrange seedlings (Poncirus trifoliata [L.] Raf. × Citrus sinensis [L.] Osbeck) were inoculated with the vesicular-arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith. Plants were exposed to ¹⁴ CO₂ for 10 minutes and ambient air for 2 hours. Three to 4% of recently labeled photosynthate was allocated to metabolism of the mycorrhiza in each inoculated root half independent of shoot P concentration, growth response, and whether one or both root halves were colonized. Nonmycorrhizal roots respired more of the label translocated to them than did mycorrhizal roots. Label recovered in the potting medium due to exudation or transport into extraradical hyphae was 5 to 6 times greater for (+ +) versus (00) plants. In low nutrient media, roots of (0+) and (+ +) plants transported more P to leaves per root weight than roots of (00) plants. However, when C translocated to roots utilized for respiration, exudation, etc., as well as growth is considered, (00) plant roots were at least as efficient at P uptake (benefit) per C utilized (cost) as (0+) and (+ +) plants. Root systems of (+ +) plants did not supply more P to leaves than (0+) plants in higher nutrient media, yet they still allocated twice the ¹⁴C-photosynthate to the mycorrhiza as did (0+) root systems. This indicates there is an optimal level of mycorrhizal colonization above which the plant receives no enhanced P uptake yet continues to partition photosynthates to metabolism of the mycorrhiza.     

Boron uptake by ectomycorrhizas of silver birch

Boron (B) is an essential micronutrient for plants but it is thought not to be essential for fungi. We studied whether the extraradical mycelia of Paxillus involutus in symbiosis with silver birch (Betula pendula) take up B and transport it to the host plant. We grew mycorrhizal plants in flat microcosms with a partitioning wall, below which there was only extraradical mycelium. A boric acid solution enriched in ¹⁰B was applied to these mycelia. Increased ¹⁰B/¹¹B isotope ratios were subsequently measured in birch leaves, stems, and roots plus mycorrhizas in the upper compartment. Boron was therefore taken up by the mycorrhizal mycelia and transported to the host plant in this species combination.     

Effect of mycorrhiza and nitrate nutrition on nitrate reductase activity in Scots pine seedlings

In vivo nitrate reductase (EC 1.6.6.1) activity was measured in seedlings of Scots pine ( Pinus sylvestris L.) inoculated with Cenococcum geophilum (Sow.) Ferd. & Winge, Paxillus involutus (Batsch:Fr) Fr, Piloderma croceum Erikss, & Hjortst, and Suillus variegatus (Fr.) O. Kuntze. The activity was higher in the mycorrhizal pine roots than was previously found in the fungus symbiont alone, but lower than in the roots of nonmycorrhizal pine seedlings. The differences observed in a previous study between the fungal species under pure culture conditions were not found in the present work for mycorrhiza synthezised with the same fungal species. An increase in the nitrate concentration of the nutrient solution increased the proportion of the nitrate reductase activity in the needles. The mycorrhizal root tips had higher nitrate reductase activity than nonmycorrhizal root tips in the same root system.     

Fungal lipid accumulation and development of mycelial structures by two arbuscular mycorrhizal fungi

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1omega5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1omega5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1omega5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.     

Distribution of lead in mycorrhizal and non-mycorrhizal Norway spruce seedlings

Non-mycorrhizal spruce seedlings (Picea abies Karst.) and spruce seedlings colonized with Lactarius rufus (Scop.) Fr. or two strains of Paxillits involutus (Batsch) Fr. were grown in an axenic silica sand culture system with frequently renewed nutrient solution. After successful mycorrhizal colonization, the seedlings were exposed to 1 μM PbCI₂ for 19 weeks. The degree of infection in all of the mycorrhizal treatments approached 100% during the experiment and was not affected by exposure to Pb. However, the number of root tips per root dry weight and the shoot: root ratio, both in the non-mycorrhizal and the mycorrhizal seedlings, had decreased after the 19 week treatment with PbCl₂ Using X-ray microanalysis, the distribution and concentration of Pb in the tissues of mycorrhizal and non-mycorrhizal root tips were compared. In the mycorrhizae of seedlings exposed to Pb no significant accumulation of Pb in the hyphal mantle or in fungal cell walls of the Hartig net were detected. Lead accumulated primarily in the cortex cell walls both of non-mycorrhizal and mycorrhizal root tips. No significant difference of Pb concentrations in root cortex cell walls of non-mycorrhizal and mycorrhizal seedlings was found; except for seedlings colonized with Paxillus involutus strain 537. However, at the endodermis no effect of mycorrhizal fungal colonization on the Pb tissue concentration was detected. The presence of the fungal sheath did not prevent Pb from reaching the root cortex. The endodermis acted as a barrier to Pb radial transport in both mycorrhizal and non-mycorrhizal seedling roots.     

Phosphorus and carbon availability regulate structural composition and complexity of AM fungal mycelium

The regulation of the structural composition and complexity of the mycelium of arbuscular mycorrhizal (AM) fungi is not well understood due to their obligate biotrophic nature. The aim of this study was to investigate the structure of extraradical mycelium at high and low availability of carbon (C) to the roots and phosphorus (P) to the fungus. We used monoxenic cultures of the AM fungus Rhizophagus irregularis (formerly Glomus intraradices) with transformed carrot roots as the host in a cultivation system including a root-free compartment into which the extraradical mycelium could grow. We found that high C availability increased hyphal length and spore production and anastomosis formation within individual mycelia. High P availability increased the formation of branched absorbing structures and reduced spore production and the overall length of runner hyphae. The complexity of the mycelium, as indicated by its fractal dimensions, increased with both high C and P availability. The results indicate that low P availability induces a growth pattern that reflects foraging for both P and C. Low C availability to AM roots could still support the explorative development of the mycelium when P availability was low. These findings help us to better understand the development of AM fungi in ecosystems with high P input and/or when plants are subjected to shading, grazing or any management practice that reduces the photosynthetic ability of the plant.     

Neighbouring weeds influence the formation of arbuscular mycorrhiza in grapevine

Grapevine (Vitis vinifera L.) and two selected weeds from Mediterranean Croatian vineyards (Plantago lanceolata L. and Tanacetum cinerariifolium (Trevir.) Sch.Bip.) were examined in pot culture experiments, individually or when combined, to see whether multiple hosts influenced the formation of the symbiosis with arbuscular mycorrhizal fungi (AMF). The results after six-month period showed that plant identity and density significantly influenced development of mycorrhizal intra- and extraradical mycelium and/or sporulation. Grapevine and T. cinerariifolium individually and in combination resulted in a greater development of arbuscular mycorrhizae in terms of spore production, extraradical mycelium length and root colonization compared with pots containing P. lanceolata. Herbaceous weed species seemed to promote a different set of dominant AMF, potentially providing a wider spectrum of AMF for colonising grapevine roots. This indicates the value of encouraging host plant diversity in vineyards. AMF sequences obtained in this study are the first data reported for soils in Croatia.     

Early events in the life of apple roots: variation in root growth rate is linked to mycorrhizal and nonmycorrhizal fungal colonization

Early events of mycorrhizal and nonmycorrhizal fungal colonization in newly-emerging roots of mature apple (Malus domestica Borkh) trees were characterized to determine the relationship of these events to fine root growth rate and development. New roots were traced on root windows to measure growth and then collected and stained to quantify microscopically the presence of mycorrhizal and nonmycorrhizal fungal structures. Most new roots were colonized by either mycorrhizal or nonmycorrhizal fungi but none less 25 days old were ever internally colonized by both. Compared to nonmycorrhizal colonization, mycorrhizal colonization was associated with faster growing roots and roots that grew for a longer duration, leading to longer roots. While either type of fungi was observed in roots as soon as 3 days after root emergence, intraradical colonization by mycorrhizal fungi was generally faster (peaking at 7 to 15 days) than that by nonmycorrhizal fungi and often occurred more frequently in younger roots. Only 15 to 35% of the roots had no fungal colonization by 30 days after emergence. This study provides the first detailed examination of the early daily events of mycorrhizal and nonmycorrhizal fungal colonization in newly emerging roots under field conditions. We observed marked discrimination of roots between mycorrhizal and nonmycorrhizal fungi and provide evidence that mycorrhizal fungi may select for faster growing roots and possibly influence the duration of root growth by non-nutritional means.     

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.     

Pathway and sink activity for photosynthate translocation in <Emphasis Type="Italic">Pisolithus</Emphasis> extraradical mycelium of ectomycorrhizal <Emphasis Type="Italic">Pinus thunbergii</Emphasis> seedlings

The purpose of this study was to identify the pathway and sink activity of photosynthate translocation in the extraradical mycelium (ERM) of a Pisolithus isolate. We labelled ectomycorrhizal (ECM) Pinus thunbergii seedlings with ¹⁴CO₂ and followed ¹⁴C distribution within the ERM by autoradiography. ¹⁴C photosynthate translocation in the ERM resulted in ¹⁴C distribution in rhizomorphs throughout the ERM, with ¹⁴C accumulation at the front. When most radial mycelial connections between ECM root tips and the ERM front were cut, the whole allocation of ¹⁴C photosynthates to the ERM was reduced. However, the overall pattern of ¹⁴C distribution in the ERM was maintained even in regions immediately above and below the cut, with no local ¹⁴C depletion or accumulation. We inferred from this result that every portion in the ERM has a significant sink activity and a definite sink capacity for photosynthates and that photosynthates detour the cut and reach throughout the ERM by translocation in every direction. Next, we prepared paired ECM seedlings, ERMs of which had been connected with each other by hyphal fusion, alongside, labelled the left seedling with ¹⁴CO₂, and shaded none, one or both of them. ¹⁴C photosynthates were acropetally and basipetally translocated from the left ERM to ECM root tips of the right seedling through rhizomorphs in the left and right ERMs, respectively. With the left seedling illuminated, ¹⁴C translocation from the left to the right ERM increased by shading the right seedling. This result suggests that reduced photosynthate transfer from the host to its ERM increased sink activity of the ERM.     

Defoliation increases carbon limitation in ectomycorrhizal symbiosis of <Emphasis Type="Italic">Betula pubescens</Emphasis>

Boreal forest trees are highly dependent on root-colonizing mycorrhizal fungi. Since the maintenance of mycorrhizal symbiosis implies a significant carbon cost for the host plant, the loss of photosynthetic leaf area due to herbivory is expected to reduce the host investment in mycorrhizae. We tested this hypothesis in a common garden experiment by exposing ectomycorrhizal white birch (Betula pubescens Ehrh.) seedlings to simulated insect defoliation of 50 or 100% intensity during either the previous or the current summer or repeatedly during both seasons before harvest. The shoot and root growth of the seedlings were distinctly reduced by both 100% defoliation and repeated 50% defoliation, and they were more strongly affected by previous-year than current-year defoliation. The root to shoot ratio significantly decreased after 100% defoliation, indicating reduced proportional allocation to the roots. Ergosterol concentration (i.e. fungal biomass) in the fine roots decreased by 100% defoliation conducted either in the year of harvest or in both years. No such decrease occurred following the 100% defoliation conducted in the previous year, indicating the importance of current photosynthates for fungal symbionts. The trend was similar in the colonization percentage of thick-mantled mycorrhizae in the roots, the most marked decline occurring in the repeatedly defoliated seedlings. The present results thus support the prediction that the plant investment in ectomycorrhizae may decline as a response to foliage loss. Moreover, the colonization percentage of thick-mantled mycorrhizae correlated positively with the ratio of leaf to heterotrophic plant biomass in the defoliated birch seedlings, but not in the control ones. This tends to indicate a stronger carbon limitation of ectomycorrhizal colonization in defoliated seedlings.     

Characterization of culturable bacterial populations associating with Pinus sylvestris--Suillus bovinus mycorrhizospheres

Bacterial isolations were carried out on Pinus sylvestris--Suillus bovinus mycorrhizospheres obtained directly from boreal pine forest. When samples were taken during dry weather, the numbers of bacterial colony-forming units were significantly higher in uncolonized short roots and external mycelia than in mycorrhizal roots and soil outside the mycorrhizosphere. In contrast, the colony-forming unit counts were similar in all hypogeous samples after rainy weather. Culturable bacteria were absent from most Suillus bovinus sporocarps. The bacteria isolated from all types of mycorr hizo sphere samples, i.e. short roots, mycorrhizal roots, and external mycelia, consisted primarily of Burkholderia spp., whereas most isolates from soil outside the mycorrhizosphere were identified as Paenibacillus spp. This study shows that mycorrhizal external mycelia can expand the habitat favourable for common rhizosphere bacteria into the soil far from the immediate rhizosphere. Some of these bacteria may help the trees with nitrogen acquisition, since potentially diazotrophic bacteria harbouring nitrogenase reductase (nifH) genes were isolated from mycorrhizal root tips.     

Dynamics of lead accumulation in mycorrhizal and non-mycorrhizal Norway spruce (Picea abies (L.) Karst.)

Twelve-week-old seedlings of Norway spruce (Picea abies (L.) Karst), non-mycorrhizal or mycorrhizal with Laccaria laccata, Paxillus involutus or Pisolithus tinctorius were exposed to 5 M Pb for either 32 or 42 days in a quartz sand-nutrient solution system. Ultrathin sections of mycorrhizal and non-mycorrhizal short roots were examined by X-ray microanalysis. After 42 days Pb treatment, the Pb content of the cortex cell walls was lower in the non-mycorrhizal short roots and in the P. involutus mycorrhizae than in the mycorrhizae of L. laccata or P. tinctorius. The Pb content of the cell walls of the hyphal mantle was higher in P. involutus than in L. laccata or P. tinctorius. The short term experiment over 32 days showed that the Pb content of the cortex cell walls strongly increased during the first 16 days in the non-mycorrhizal roots and the L. laccata mycorrhizae, whereas it increased more slowly in the P. involutus mycorrhizae. After 32 days Pb treatment, the Pb content in the cortex cell walls in the P. involutus mycorrhizae was similar to that in the non-mycorrhizal roots. P. involutus also decreased Pb translocation from the roots to the stems. Mycorrhizal infection was not affected by Pb but with P. involutus, the amount of extramatrical mycelium was reduced by 50% on day 32 compared to day 16. The extramatrical mycelium of L. laccata was not reduced by Pb. It is concluded that ectomycorrhizal fungi differ in their effect on Pb accumulation in the roots of Norway spruce. The binding capacity of the extramatrical mycelium seems to be an important factor.