Polyphosphate granules are an artefact of specimen preparation in the ectomycorrhizal fungusPisolithus tinctorius

Summary Polyphosphate granules are precipitated in the vacuoles of the ectomycorrhizal fungusPisolithus tinctorius (Pers.) Coker & Couch by various treatments, including conventional specimen preparation. Granules are not produced by glutaraldehyde fixation but appear at early stages of ethanol dehydration and are visible with Nomarski DIC microscopy. They show -metachromasy with toluidine blue O at low pH, are extracted by cold trichloroacetic acid and contain phosphorus and calcium as demonstrated by X-ray microanalysis. The granules are surrounded by electron-lucent areas that do not contain these elements at detectable levels. In contrast, vacuoles of freeze-substituted hyphae contain evenly dispersed flocculent material. Phosphorus and potassium are distributed more or less uniformly throughout, but calcium is not detected. This indicates that polyphosphate is present in the vacuole of living hyphae in soluble form and is precipitated to form granules by various treatments. It is thought that granules form when membranes, including the tonoplast, become leaky and there is an influx of precipitating ions such as calcium.Abbreviations DIC differential interference contrast - GMA glycol methacrylate - MMN modified Melin Norkrans - NMR nuclear magnetic resonance - P_i inorganic phosphate - STEM scanning transmission electron microscope     

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

In vitro weathering of phlogopite by ectomycorrhizal fungi

The ways in which ectomycorrhizal fungi benefit tree growth and nutrition have not been fully elucidated. Whilst it is most probably due to improved soil colonization, it is also likely that ectomycorrhizal fungi could be directly involved in nutrient cycling of soil reserves. This study assessed access by two species of ectomycorrhizal fungi to soil nonexchangeable K⁺ reserves. The incubation of ectomycorrhizal fungi in bi-compartment Petri dishes with phlogopite led to cation exchange reactions and to crystal lattice weathering. Paxillus involutus COU led to irreversible phlogopite transformations, while Pisolithus tinctorius 441 led to reversible ones. Simultaneous depletion in K⁺ and Mg²⁺ led to an enhanced weathering of phlogopite by P. tinctorius 441. The observation of phlogopite evolution shows that some specific Al³⁺ immobilization occurred under P. tinctorius 441. The data suggest that these bio-weathering mechanisms could be related to the release of fungal organic acids or other complex forming molecules.     

Genetic diversity of the ectomycorrhizal fungus Pisolithus tinctorius based on RAPD-PCR analysis

 Twenty Pisolithus tinctorius isolates from different geographic locations and different hosts were characterized by the random amplified polymorphic DNA technique. Thirteen arbitrary primers generated 87 DNA fragments, all of them polymorphic. These data were used to calculate genetic distances among the isolates. The pairwise genetic distances ranged from 1 to 100%, with an average of 58.7%. Cluster analysis based on the amplified fragments grouped the isolates according to their host and geographical origins. Group I contained isolates collected in Brazil and group II those collected in the Northern Hemisphere. In addition to the diversity seen at the molecular level, the isolates also showed host specificity. Greenhouse experiments demonstrated that isolates from the Northern Hemisphere colonized mainly Pinus whereas isolates from Brazil colonized only Eucalyptus. The molecular data suggest that the Pisolithus tinctorius isolates analyzed belong to two distinct groups. The data also suggest new guidelines for future investigations on the taxonomy and systematic of this important fungus species. Furthermore, these results support future experiments aimed at the selection and development of improved isolates of P. tinctorius. Accepted: 3 October 1997     

Genetic variability and taxonomic position of ectomycorrhizal fungus Pisolithus from India

Eight ectomycorrhizal fungal isolates of Pisolithus associated with Eucalyptus species in different parts of India were collected and the genetic variability of these isolates was studied by ITS-RFLP and ITS sequencing. All the isolates showed same RFLP patterns with each restriction enzyme, indicating all these isolates of Pisolithus are of the same genotype. The sequence comparison of KN6 of Indian isolate showed high sequence similarities with the isolates of Pisolithus associated with Eucalyptus from Australia. Phylogeny analysis showed that all the isolates compared in this study clustered into four main groups The Indian isolate (KN6) clustered with Pisolithus albus isolates of group I, which are associated with Eucalyptus. These results suggested that Pisolithus isolates found in India are P. albus.     

Histochemical demonstration of heavy metal tolerance in ectomycorrhizal fungi

Summary By the use of a specific histochemical staining method evidence was obtained that tolerance to heavy metals in ectomycorrizal fungi is based on the presence of metallothionein-like proteins. The implication that tolerance in these fungi should be induced by sublethal concentrations of heavy metals has been confirmed by us. Induction of metallothionein in ectomycorrizal fungi could possibly be helpful in protection of their host plants in areas polluted by heavy metals. In comparison with biochemical methods the histochemical method is able to locate the metal tolerance and has the added advantage that it may also be applied to mycorrhizas (root and fungus).     

Mycorrhizal associations in Pinus massoniana Lamb. and Pinus elliottii Engel. inoculated with Pisolithus tinctorius

Dichotomous mycorrhizas were induced in Pinus massoniana and Pinus elliottii seedlings inoculated with Pisolithus tinctorius growing under non-axenic conditions. Six months after inoculation, Pinus massoniana seedlings exhibited a higher degree of infection, bore more mycorrhizas and had developed more abundant extramatrical mycelium than seedlings of Pinus elliottii. Nevertheless, seedlings of Pinus massoniana were stunted and exhibited chorosis of the needles, indicating a possible nutrient deficiency. Histological examination of these pine mycorrhizas showed an ectomycorrhizal association typical of gymnosperms with an intercellular Harting net penetrating between several layers of cortical cells close to the endodermis. However, strong polyphenolic reactions, intracellular hyphae and wall modifications were occasionally observed, indicating that both host-tissue incompatibility and ectendomycorrhizal association can occur in pine species under stressed conditions.     

In vitro weathering of phlogopite by ectomycorrhizal fungi

Oxalate accumulation in external medium under hyphal mats of two ectomycorrhizal species is strongly stimulated (1.7 to 35 fold) by a simultaneous depletion of available K⁺ and Mg²⁺. Pisolithus tinctorius strain 441 accumulates oxalate both on NH₄–N and on NO₃–N whereas Paxillus involutus strain COU only accumulates oxalate on NO₃–N. On NO₃–N, under a simultaneous K⁺ and Mg²⁺ deficiency, P. involutus COU is a very active oxalate producer compared to P. tinctorius 441. The present results could explain the various mineralogical evolutions of a phlogopite mica previously recorded under P. involutus COU or P. tinctorius 441 and suggest a key role for fungal oxalic acid during mineral weathering in response to nutrient deficiency.     

The use of Box-Behnken design of experiments to study in vitro salt tolerance by Pisolithus tinctorius

The ectomycorrhizal fungus Pisolithus tinctorius was grown in vitro with different concentrations and combinations of three different sodium salts viz., sodium chloride, sodium sulphate and trisodium citrate (NaCl, Na₂SO4 and Na₃C₆H₅O₇) for 30 days. At the end of the experiment the mycelial biomass and protein content of the fungus was evaluated. Based on the salts tested the combination of NaCl and Na₂SO₄ in optimum concentrations actually promoted the growth of P. tinctorius. Box-Behnken design with three variables like NaCl, Na₂SO₄ and Na₃C₆H₅O₇ was used to identify a significant correlation between the effect of these variables on mycelial biomass production. The experimental values are in good agreement with predicted values, the correlation coefficient being 0.9880.     

Changes in the fungus-specific,soluble-carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius

A simple and convenient culture system has been developed for the analysis of ectomycorrhiza formation under controlled conditions. Rapid and synchronous mycorrhiza synthesis was observed when thin and even layers of Pisolithus tinctorius (Pers.) hyphae were brought at once into contact with the entire root system of 3-month-old Picea abies (L. Karst) plants. Suitable fungal layers were grown on cardboard with limiting glucose supply in the medium to maximize radial growth. The glucose was almost consumed by the time the fungus had spread over the whole cardboard and was ready for inoculation of the roots. At this stage, the fungus contained trehalose and arabitol as the main soluble carbohydrates. A few hours after the assembly of the culture system, contacts between roots and aerial hyphae were observed and a sheath was formed 3 days later, suggesting very rapid ectomycorrhiza formation under these conditions. The pool of soluble carbohydrates of the inoculum, i.e. the extramatrical mycelium, declined after inoculation of the roots and was almost zero after 2 weeks. The supply of carbon by the plant was then sufficient for the fungus to expand the soluble pool efficiently in both the mycorrhizas and the extramatrical mycelium. The kinetics of the carbohydrate pool and the observed differentiation of the short roots to mycorrhizas imply that in our culture system fully functional symbiosis was established no later than 14 days after the plants were inoculated with the fungus.     

Release of a reducing substance by the ectomycorrhizal fungi Pisolithus tinctorius and Paxillus involutus

Culture solutions of the ectomycorrhizal fungi Pisolithus tinctorius and Paxillus involutus are shown to reduce a high valency oxide of manganese at a range of pH values from 3.0 to 13.0. Manganese reduction in unmoculated culture media was confined to pH values below 5.0 and above 10.0. The results are consistent with the release of a reducing substance from the fungal mycelium.     

Changes in root growth patterns of (Picea abies) spruce roots by inoculation with an ectomycorrhizal fungusPisolithus tinctorius and jasmonic acid treatment

Symbiosis between fungi and plant roots forming a mycorrhiza involves extensive interactions at the molecular level between both partners. The role of plant hormones in the regulation of mycorrhizal infection is not known to involve jasmonates. Their endogenous levels increase during pathogen attack; however, little has been done on their involvement in mycorrhizae. In our recent work, root growth patterns of 2-month-old spruce seedlings after inoculation withPisolithus tinctorius and/or jasmonic acid (JA) treatment were studied using a paper-sandwich technique. Changes in root length, the degree of branching, presence and length of root hairs, and infection parameters were followed using a stereomicroscope. The first mycorrhizal contact of hyphae with roots was significantly accelerated upon treatment with 0.5 M JA. Interactions between root hairs and fungal hyphae were seen by scanning electron microscopy. The multiplication of root hairs of non-mycorrhized seedlings treated with 5.0 M JA and changes of the root surface were observed by the same technique.     

An overview of Cistus ectomycorrhizal fungi

The genus Cistus comprises a group of about 20 shrub species found in wide areas throughout the whole Mediterranean region to the Caucasus. Being one of the main constituents of the Mediterranean-type maquis, this plant genus is peculiar in that it has developed a range of specific adaptations to resist summer drought and frequent disturbance events, such as fire and grazing. In addition, it can form both ectomycorrhizas and arbuscular mycorrhizas. In this paper, we review the information available on the ectomycorrhizal fungi of Cistus across its entire geographic range, as gathered and critically sifted from both published literature sources and personal observations. Although the resulting data matrix was based primarily on accounts of sporocarp inventories in the field, existing knowledge on the features of Cistus natural and synthesized ectomycorrhizas was also included and discussed. In total, more than 200 fungal species belonging to 40 genera have been reported so far to be associated with Cistus. An analysis of the pattern of ectomycorrhizal diversity and host specificity revealed that members of the Cortinariaceae and Russulaceae make the most of both Cistus-aspecific and Cistus-specific mycobionts. Further studies are needed to expand our preliminary knowledge of the mycorrhizal ecology and biology of Cistus and its fungal associates, focusing on topics such as mycobiont diversity, host specificity, fungal succession, mycorrhizal influence on stress tolerance, and impact of disturbances, while comparing the findings with those from other ecosystems.     

New microsatellite markers for the ectomycorrhizal fungus Pisolithus tinctorius sensu stricto reveal the genetic structure of US and Puerto Rican populations

The ectomycorrhizal fungus Pisolithus tinctorius has been introduced to many areas around the world as a source of inoculum for pine plantations. However, little is known about the genetic structure of fungal populations in their introduced habitats. To study the genetics of exotic P. tinctorius populations, we developed and employed seven new microsatellite markers and compared samples from Puerto Rico (exotic range) and the eastern United States (native range). Bayesian cluster analysis, neighbor joining analysis and F_ST values all strongly separated Puerto Rican populations from North American populations. Consistent with a founder effect, populations from Puerto Rico had reduced allelic richness when compared to samples from the United States. The genetic structure of P. tinctorius populations in Puerto Rico is weak to modest and is not correlated with geographic distance reflecting anthropogenic movement of inoculum with forestry practices.     

Production of antifungal metabolites by the ectomycorrhizal fungusPisolithus tinctorius strain SMF

Summary An ectomycorrhizal fungus,Pisolithus tinctorius strain SMF, isolated from a basidiocarp removed from the roots of a recently fallen old growth fir in the Smoky Mountains of Tennessee, was characterized for its in vitro production of antifungal metabolites. On solid mediumP. tinctorius SMF strongly inhibited growth of strains ofFusarium solani, Geotrichum candidum, Phanerochaete chrysosporium, andVerticillium dahliae, all species known to be plant pathogens. Evidence from paired colony growth inhibition studies on agar plates indicated that production of antifungal agents byP. tinctorius SMF may be enhanced by close physical contact with other fungi. The antifungal activity ofP. tinctorius SMF was much greater than that of several culture collection strains ofP. tinctorius. The culture collection strains either showed no or very limited activity. The antifungal activity was associated with an apparently inducible metabolism ofP. tinctorius SMF and with the production of darkly colored water soluble phenolic metabolites. Small scale fermentation studies showed that the phenolics are readily producible by submerged culture fermentation. This is the first report of submerged culture production of antifungal metabolites by an ectomycorrhizal fungus.     

Extracellular and cellular mechanisms sustaining metal tolerance in ectomycorrhizal fungi

This review focuses on recent evidence that identifies potential extracellular and cellular mechanisms that may be involved in the tolerance of ectomycorrhizal fungi to excess metals in their environment. It appears likely that mechanisms described in the nonmycorrhizal fungal species are used in the ectomycorrhizal fungi as well. These include mechanisms that reduce uptake of metals into the cytosol by extracellular chelation through extruded ligands and binding onto cell-wall components. Intracellular chelation of metals in the cytosol by a range of ligands (glutathione, metallothioneins), or increased efflux from the cytosol out of the cell or into sequestering compartments are also key mechanisms conferring tolerance. Free-radical scavenging capacities through the activity of superoxide dismutase or production of glutathione add another line of defence against the toxic effect of metals.     

Salt stress affects in vitro growth and in situ symbioses of ectomycorrhizal fungi

Ten isolates of six species of ectomycorrhizal fungi were grown in vitro at nine concentrations of three sodium salts (NaCl, Na₂SO₄, Na₃C₆H₅O₇) for 4 weeks. Colony diamater, biomass and protein content of fungi were evaluated. Isolates of Pisolithus tinctorius and Suillus luteus were more tolerant of NaCl and Na₂SO₄ than of Na₃C₆H₅O₇. Fungi in the genera Cenococcum, Laccaria, and Thelephora were highly intolerant of Na₃C₆H₅O₇ and Na₂SO₄ in vitro. Biomass and protein content of fungi generally declined with increasing substrate salinity in solution culture. In situ ectomycorrhizal colonization by Laccara laccata and P. tinctorius and the dry weight of Pinus taeda seedlings were significantly reduced by 80 mM NaCl after 14 weeks. Only select ectomycorrhizal fungi appear capable of growth and symbiosis in saline soils.     

In situ and in vitro colonization of Cathaya argyrophylla (Pinaceae) by ectomycorrhizal fungi

Cathaya argyrophylla, a critically endangered conifer, is found to grow at four isolated areas located in subtropical mountains of China. To examine the involvement and usefulness of mycorrhizas for sustaining the population of this tree, we compared the root system, morphology, and structure of mycorrhizal roots of C. argyrophylla, which were collected from a natural stand and an artificial stand, each grown at a different location. More mycorrhizal roots were found for trees from an artificial stand. The presence of extramatrical mycelium, mantle, and Hartig net revealed that C. argyrophylla formed an ectomycorrhizal association in both sampling sites. Starch granules were found in mycorrhizal roots collected only from a natural stand. The aseptic synthesis of C. argyrophylla and Cenococcum geophilum was established for the first time in vitro. Typical ectomycorrhizas formed on seedlings on RM medium containing 0.1 g/l glucose, 5 weeks after inoculation. By light microscopy, the synthesized mycorrhizas showed a thin mantle from which emanated extramatrical hyphae and highly branched Hartig net. A simple, rapid, and convenient mycorrhiza synthesis system was developed, which facilitates further studies on ectomycorrhizal development of C. argyrophylla.     

Vacuole motility and tubule-forming activity inPisolithus tinctorius hyphae are modified by environmental conditions

Summary Motile tubular vacuole systems have been visualised using DIC optics in living hyphae ofPisolithus tinctorius without loading of any fluorescent tracer. Adding new medium, with or without the tracer CFDA, alters the motility of this system and increases the number of tubules. This response has been shown in individual hyphal tip cells and quantified in populations of tip cells. Vacuoles with motile tubules are also demonstrated in more basal cells of the hyphae, within 600 m of the growing hyphal front. The vacuoles in these cells show more limited motility, but similarly respond to addition of new medium by increased motility and tubular activity. This demonstration that the vacuole system in more mature regions is both motile and interconnected as in the tips, and similarly responds to changes in external conditions, supports the hypothesis that the vacuole system may play a role in long-distance transport. Vacuoles in the most mature cells, more than 600 m behind the hyphal growth zone are not motile. They do not respond to these stimuli and remain spherical and isolated. There are many explanations for this and the present lack of response does not exclude the transport hypothesis. The findings further support the concept that tubular vacuole systems are equivalent to animal endosomal/lysosomal systems and have implications for their motility, especially their plasticity in response to external stimuli, such as fluorescent tracers.Abbreviations CFDA 6-carboxyfluorescein diacetate - DIC differential interference contrast - MMN modified Melin-Norkrans medium - SEM standard error of the mean