Rhizopogon Roseolus Research Articles

Effects of pH, water stress and temperature on in vitro cultures of ectomycorrhizal fungi from Mediterranean forests

Abstract A comparative study of the growth of eight species of ectomycorrhizal fungi [ Hebeloma edurum Metrod ex Bon, Lactarius deliciosus (L.: Fr.) S.F. Gray., Rhizopogon roseolus (Corda) Th.M.Fr., Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.: Fr.) Roussel, Suillus luteus (L.: Fr.) Roussel, Tricholoma focale (Fr.) Ricken, Tricholoma striatum (Schaeff.) Sacc.] in pure culture at different pH values, water potentials and temperatures was carried out. The biomass production and mycelial growth of each strain were assessed and characterised morphologically. H. edurum and L. deliciosus showed a clear affinity for neutral or slightly basic media. Most species had the lowest colony diameters and biomass production at pH 2.5. Only H. edurum, R. roseolus and S. collinitus grew at the highest water stress levels (−13.85 and −15.45 bar). All species grew at the lowest values of temperature whereas only R. roseolus and S. collinitus did at 30 °C.

Inoculation of containerized Pinus pinea L. seedlings with seven ectomycorrhizal fungi

Containerized Pinus pinea L. seedlings are commonly used for reforestation in the Mediterranean area. While there is an increasing knowledge of the potential ectomycorrhizal fungi associated with Pinus pinea, few studies exist of inoculation techniques with selected ectomycorrhizal fungi. We tested seven ectomycorrhizal fungi for their effectiveness with containerized Pinus pinea seedlings. Hebeloma crustuliniforme, Laccaria laccata and Pisolithus tinctorius were applied as vegetative inocula while Melanogaster ambiguus, Pisolithus tinctorius, Rhizopogon luteolus, Rhizopogon roseolus and Scleroderma verrucosum were tested as spore inocula. The inoculum of each fungus was tested at several application rates. Among the fungi tested as vegetative inocula, the highest percentages of ectomycorrhizas were obtained with H. crustuliniforme at all rates tested. The ectomycorrhizas formed by L. laccata varied from 11% to 40% depending on the inoculum rate applied. Vegetative inoculum of Pisolithus tinctorius was only effective at the highest inoculum rates and gave mycorrhization percentages around 60%. Pisolithus tinctorius applied as a spore inoculum formed ectomycorrhizas at a frequency of about 50% at the effective inoculum rates. The rest of the fungi applied as spore inocula produced more than 50% of ectomycorrhizas at the effective spore concentrations. These included the highest percentages of ectomycorrhizas (>80%) obtained with both Rhizopogon species. Differences in growth due to inoculation with the different fungi were not detected and in some cases inoculation even reduced the total biomass accumulated by seedlings. All seedlings reached a size suitable for transplantation.

Continuous measurement of stem-diameter growth response of Pinus pinea seedlings mycorrhizal with Rhizopogon roseolus and submitted to two water regimes

Linear variable differential transformer (LVDT) sensors were used to detect continuous diameter growth responses of Pinus pinea (stone pine) seedlings inoculated with the ectomycorrhizal fungus Rhizopogon roseolus. Colonised and non-colonised seedlings provided with sensors were submitted to different water regimes in two consecutive experiments established in a controlled-temperature greenhouse module (cycle1), and in an adjacent module without temperature control (cycle2). Under regular irrigation, colonised seedlings showed significantly higher growth than non-colonised seedlings. Water-stressed seedlings showed no benefit from inoculation in terms of growth. Also, seedlings with a high colonisation level recovered more slowly from water stress than control seedlings. A significant positive relationship between maximum daily shrinkage (amplitude of the daily stem contraction) and global radiation was observed only in the first water-stress period in cycle1 and in regularly irrigated seedlings in both cycles. However, no differential responses due to inoculation were observed. The mycorrhizal colonisation of the seedlings at the end of the experiment was related with the initial colonisation level. Mycorrhizal colonisation by R. roseolus in old roots was maintained at significantly higher levels in seedlings which had an initial colonisation level >50% than in seedlings with <50% initial colonisation. Also, more newly formed roots became colonised in seedlings which had an initial colonisation level >50% than in seedlings with an initial colonisation <50%, which had almost no new root colonisation. From the results obtained, it can be concluded that LVDT sensors can be used to detect a differential response of plants according to water supply, mycorrhizal status and, in some cases, to their colonisation level. The results are discussed in relation to the predictive possibilities of the method for the selection of efficient mycorrhizal fungi for the promotion of plant growth.

Effects of nitrogen and phosphorus fertilization on mycorrhizal formation of two poplar clones(Populus trichocarpa andP. tremula x tremuloides)

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Organic acids produced by mycorrhizal Pinus sylvestris exposed to elevated aluminium and heavy metal concentrations

A cultivation method was developed to enable exposure of ectomycorrhizal plants with intact extramatrical mycelium to solutions containing different concentrations of aluminium or heavy metals. Pinus sylvestris seedlings colonized by Suillus variegatus (two isolates), Rhizopogon roseolus or Paxillus involutus (two isolates) were used. Seedlings were transferred to Petri dishes containing glass beads and exposed to elevated concentrations of Al, Cd, Cu, or Ni in two ways: immediately following transfer; and after allowing mycorrhizal seedlings to develop an extraradical mycelium that colonized the interface between the upper surface of the beads and the metal‐ containing solution. Production of organic acids in mycorrhizal and non‐mycorrhizal systems was measured by withdrawing samples from the solution and analyzing by HPLC. In most experiments, levels of oxalic acid were significantly higher in mycorrhizal treatments than in non‐mycorrhizal controls. The measured levels of organic acids were variable, but the results obtained suggest that production of oxalic acid is stimulated by exposure to elevated Al in mycorrhizal seedlings colonized by S. variegatus and R. roseolus. Elevated Al concentrations also increased oxalic acid production by non‐mycorrhizal seedlings significantly in two of four Al experiments performed, but the measured concentrations were significantly lower than in corresponding mycorrhizal treatments in both cases. Malonic acid was found in the culture solution of non‐mycorrhizal and P. involutus‐ colonized seedlings, but only trace amounts were found in S. variegatus or R. roseolus‐infected seedlings. Citric, shikimic, lactic, acetic, propionic, fumaric, formic, iso‐butyric and butyric acid were found in variable concentrations. Production of oxalic acid by seedlings colonized by S. variegatus BL or P. involutus was not stimulated by exposure to 0.44 μM Cd or 17 μM Ni. Exposure to 0.157 mM Cu in two separate experiments using P. involutus 87.017 and two strains of S. variegatus (BL and I59) appeared to stimulate production of oxalic acid irrespective of mycorrhizal status or species.

Chromatographic properties of the cadmium induced SOD isoform in Rhizopogon roseolus

Exposure of Rhizopogon roseolus mycelia to 15 mmol·dm−3 cadmium for 24 h induces a different pattern of Mn-SOD on polyacrylamide gels, probably being a changed form of an originally existing one. The presence of cadmium affects the chromatographic properties of this enzyme and its mobility through the acrylamide gel. This new isoform was purified using DEAE Trisacryl chromatography. Cadmium induced isoform adsorbed stronger to the ligands and was eluated with a Tris-HCl buffer containing 0.1 mol·dm−3 NaCl. SOD from control samples (not treated with cadmium) was eluated with the same buffer without NaCl.

Inoculation of containerized Pseudotsuga menziesii and Pinus pinaster seedlings with spores of five species of ectomycorrhizal fungi

Container-grown Pseudotsuga menziesii and Pinus pinaster seedlings were inoculated with water suspensions of spores of five ectomycorrhizal fungi commonly found in northeastern Spain. Pseudotsuga menziesii seedlings were inoculated with basidiospores of Melanogaster ambiguus, or Rhizopogon subareolatus, or with ascospores of Tuber maculatum. Pinus pinaster seedlings were inoculated with basidiospores of Melanogaster ambiguus, Rhizopogon roseolus or Scleroderma citrinum. The spore concentrations were 102–107 spores per seedling for Melanogaster ambiguus (in Pseu dotsuga menziesii) and Rhizopogon subareolatus, 103–107 for Melanogaster ambiguus (in Pinus pinaster), Rhizopogon roseolus, and Scleroderma citrinum, and 102–104 for Tuber maculatum. Melanogaster ambiguus colonized more short roots in a larger proportion of plants at 107 spores per seedling than at any other rate. The highest colonization by Rhizopogon subareolatus was obtained at 104 spores per seedling and higher, and all inoculated plants became infected at 106 spores per seedling and higher. Tuber maculatum colonized a high percentage of short roots at all rates tested; the proportion of infected plants was over 80% at 103–104 spores per plant, decreasing to 50% at 102 spores per plant. Rhizopogon roseolus colonized the highest number of short roots on nearly all the inoculated plants when applied at 105 spores per seedling and higher. Scleroderma citrinum colonized a high percentage of short roots on all inoculated plants when applied at 105 spores per seedling and higher. The abundance of sporocarps of Melanogaster ambiguus, Rhizopogon subareolatus, R hizopogon roseolus and Scleroderma citrinum and their colonization ability at relatively low rates allows these spores to be used as ectomycorrhizal inocula on a large scale.

Effect of sulphite on superoxide dismutase activity in the mycorrhizal fungus Rhizopogon roseolus

Sulphite at a concentration of 1 mM did not strongly affect the growth of mycelium. Higher concentrations of 5–20 mM almost completely inhibited the growth of mycelium and superoxide dismutase (SOD) [EC.1.15.1.1.] activity. The activity of this enzyme was not detectable on polyacrylamide gels. The lack of induction of SOD and the resulting oxidative stress may in part be responsible for the growth inhibition caused by high concentrations of sulphite.

Examination of spores and young mycelia of Rhizopogon roseolus by scanning electron microscopy

Rhizopogon Fr. is one oi' the most important without development ofyoung mycelium. After 60 genera of ectomycorrhizal fungi. Although basi­ days sorne spores produced spherical germ vesicles, diospore inocula have been used in nurseries for without protrusion of a germ tube or development sorne years, few reports of attempts to germinate of young mycelium. One or several germ-hyphae basidiospores oi' Rhizopogon are found in the grew out from the vesicle. In many cases, the literature. Bulmer (1964) appears to be the first hyphal apices ceased elongation and started investigator to report germination experiments swelling up forming one or more vesicles. Deacon with species of this genus. (1988) reported that the process of interruption­ Fries (1941l developed a method to induce spore swelling-ramification is frequently observed germination in several species of gasteromycetes during apical growth in a great number offungi. and ectomycorrhizal hymenomycetes. The first To obtain more information, we studied the spore fruitful experiment was the result of a happy morphology under the scanning electron micro­ coincidence: on a malt extract agar plate with scope (SEM), using both dried material and agar spores of Lycoperdon umbrinum Pers.: Pers. sorne cultures from the same fruitbody. The principal contamination appeared, and close to one uniden­ problem, to select the best method to prepare tified yeast colony a few germinating Lycoperdon samples for SEM, is discussed in this paper. spores were detected. This was the first step to developing a method based on the introduction ofa Methods germination-inducing colony ofyeast, for instance Dried fragments ofthe fruitbody used in germina­ Rhodotorula glutinis (Fr.i Harrison, aIl10ng the tion experiments were prepared following three spores plated on to agar media (Fries, 1943). The different protocols (A-C): (A) Mounted directly on same author improved Laccaria laccata (Scop.: Fr.) standard SEM stubs using double-sided adhesive Berk. germination 10 to 20% by adding activated tape and coated with gold (Martin et al., 1993). (B) charcoal to the agar media (Fries, 1977). Fixed with 70% ethanol. Rehydrated 15 min in Following the methodology of Fries one of us distilled water, 15 min in ammonia and 15 min in (MPM) had the opportunity of observing spore distilled water. Dehydrated in a graded ethanol germination and different patterns ofdevelopment series (50%,60% 70%, 80%, 90%, 95%, 95%, 100%, of young mycelia, from spore cultures of Rhizopo­100%) in 15 min exchanges. Passed through four gon roseolus (Corda) Th. MFr., collected in Teruel, exchanges (15 min) of a graded isoamyl acetate Spain (herbarium number BCC-MPM 1859). series and dehydrated with critical point drying Under the light microscope, spores of R. roseo­(CPD), using carbon dioxide as the drying fluid, lus are cylindrico-ellipsoid to fusoid (6.5-8 x 2.5­ mounted on standard SEM stubs using alu­ 3.5 !-lm), smooth and with one or two strongly minium tape and coated with gold (Martin & light-refractive droplets. One sign ofthe germina­ Rocabruna, 1988). (C) Hydrated in saturated tion process is the increment of the spore length, chloral hydrate for 24 h, directly fixed and accompanied by the coalescence of its droplets. dehydrated in 95% ethanol for 15 min, exchanged Twenty three days afer plating, direct germina­ to 100% ethanol for 15 mino Passed through four tion occurred by an apical germ tube, with or exchanges (15 min) of a graded isoamyl acetate

Effect of mycorrhizal inoculation and soil restoration on the growth of Pinus halepensis seedlings in a semiarid soil

An experiment was carried out to evaluate the growth of mycorrhizal Pinus halepensis seedlings planted in a semiarid soil amended with urban refuse in southeast Spain. Three fungal species were used: Pisolithus tinctorius, Rhizopogon roseolus, and Suillus collinitus. After 8 months, inoculated seedlings grown under controlled conditions did not differ significantly from controls with regard to plant height and nutrient assimilation. Other features such as root development and stem dry weight showed that the plants grew better in the absence of mycorrhizal inoculation. The mycorrhizal seedlings and the controls were planted in three experimental plots treated with urban refuse (0, 6, and 12 Kg m-2). After 1 year of growth under field conditions the results showed that the type of fungus inoculated significantly influenced P. halepensis development. This effect varied with the dose of urban refuse. Plant growth was encouraged by the application of refuse but only at the lowest dose. Under these conditions P. tinctorius was the most effective fungus and R. roseolus yielded poorer plant development. The highest application of urban refuse led to notably worse results and a significant decrease in seedling growth compared to controls. In the control plot (without refuse) S. collinitus was the most effective fungus in plant growth improvement. The smallest application of urban refuse had a positive effect on the assimilation of N, P, and K in seedlings inoculated with P. tinctorius and S. collinitus.

Basidiospore viability in stored slurries

The viability and activity of basidiospores corresponding to 14 species of ectomycorrhizal fungi stored in slurries was studied. The methods used were the aceto-iron-haematoxylin nuclear stain to determine basidiospore viability and the FDA stain to determine basidiospore activity and dormancy. The basidiospores of Cortinarius sp., Hebeloma sp., Inocybe sp., Laccaria bicolor, Laccaria laccata, Lactarius deliciosus, Lactarius sanguifluus, Rhizopogon roseolus, Russula delica, Russula sanguinea, Suillus collinitus, Suillus granulatus, Suillus variegatus and Tricholoma terreum were suspended in slurries in duplicate experiments and half were stored at 3–4 °C and the other half at −15°. Spore viability and activity was tested after 180 days of storage. Loss of viability was least in species of Rhizopogon and Suillus, but data showed an almost total loss of viability in both refrigerated and frozen slurries of all fungi.

Implication of ectomycorrhizal fungi in the cytokinin relations of loblolly pine (Pinus taeda L.)*

SUMMARYSeven ectomycorrhizal fungi were screened by a soybean cell division bioassay to determine their potential for cytokinin synthesis. Fungal isolates of Rhizopogon roseolus (Corda) Hollos and Suillus punctipes (Peck) Sing, released significant quantities of cytokinin‐like substances into the culture medium. In contrast, Cenococcum geophilum (Fr.), Hebeloma crustuliniforme (Bull. ex. St. Amans) Qual., Pisolithus tinctorius (Pers.) Coker and Couch, Suillus granulatus (L. ex. Fr.) O. Kuntze, and Thelephora terrestris (Ehrh.) Fr. failed to elicit a positive cytokinin response in the soybean bioassay. Purification of S. punctipes culture filtrates by cation exchange, organic solvent partitioning, and separation on Sephadex LH‐20 revealed the presence of compounds with the same retention volume as zeatin and zeatin riboside. Greenhouse studies with loblolly pine (Pinus taeda L.) indicated that, together with improved plant growth and enhanced mineral nutrition, P. tinctorius mediated a significant effect on host cytokinin relations. Cytokinin activity of extracted needles increased 44 and 30% for 12‐ and 14‐month‐old seedings, respectively, following ectomycorrhizal establishment. Axenically grown seedlings colonized by 5. punctipes showed no alterations in growth, nutrition, or cytokinin concentration, compared to non‐inoculated controls, even though mycorrhizal formation exceeded 30%. The ability of ectomycorrhizal fungi to affect host cytokinins was not dependent upon an ability to synthesize cytokinins in pure culture. Results are discussed in relation to improved mineral nutrition, enhanced root growth, and increased short‐root proliferation in the ectomycorrhizal association.

Degradation of 14C-labelled lignin and dehydropolymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi

The ability of ericoid and ectomycorrhizal fungi to utilize 14C-labelled lignin and O14CH3-labelled dehydropolymer of coniferyl alcohol as sole C sources has been assessed in pure culture studies. The results indicate that ericoid mycorrhizal fungi are more effective in degrading lignin than ectomycorrhizal fungi. Amongst the ectomycorrhizal fungi the facultative mycorrhizal fungus Paxillus involutus degraded lignin more readily than those which are normally considered to be obligately mycorrhizal fungi such as Suillus bovinus and Rhizopogon roseolus. The importance of these lignin degrading capabilities is discussed in relation to the predominance of specific mycorrhiza forms along a gradient of increasing organic matter and hence lignin content of soil.

Mycelial uptake, translocation and assimilation of nitrogen from 15N‐labelled ammonium by Pinus sylvestris plants infected with four different ectomycorrhizal fungi

summaryThe uptake and assimilation of 16N‐labelled ammonium was followed in Pinus sylvestris L. plants infected with four different ectomycorrhizal fungi, Rhizopogon roseolus Fr. Suillus bovinus (Fr.) O. Kuntze, Pisolithus tinctorius (Fr.) Fr. and Paxillus involutus (Mich, ex Pers.) Cohen &amp; Couch. Plants were grown in flat perspex observation chambers or in Petri dishes containing non‐sterile peat; in each case the fungal mycelium growing from a host plant was allowed to cross a barrier and to colonize an area of peat from which roots had been excluded. Labelled ammonium was fed to the mycelium, and the shoot, root and mycelial tissues analysed for total and 15N‐labelled free amino acid contents after a feeding period of 72 h. High proportions of 15N‐labelled glutamate/glutamine, aspartate/asparagine, and alanine were found in the fungal mycelia of all species except Pax. involutus where labelled aspartate/asparagine was not found. Lower proportions of labelled serine, threonine, tyrosine, lysine, ornithine and arginine were also found in the mycelium. The degree of 15N enrichment declined throughout the transport pathway but between 5 and 50% of the amino acids were 15N‐labelled in the plant shoots. In total, at least 2–3 % of the nitrogen supplied was assimilated as labelled amino acid during the 3 day feeding period, the largest amounts of labelling occurred in glutamic acid/gultamine and aspartic acid/asparagine.

Sulphur oxidation by soil fungi including some species of mycorrhizae and wood-rotting basidiomycetes

The mycorrhizal fungi Amanita muscaria, Paxillus involutus, Hymenoscyphus ericae, Pisolithus tinctorius, Rhizopogon roseolus, and Suillus bovinus oxidized elemental sulphur to thiosulphate and sulphate in vitro. In some, but not all cases, tetrathionate was also formed. Limited oxidation of elemental sulphur by R. roseolus also occurred when growing in association with Pinus contorta in unsterilized peat. Although yeasts capable of oxidizing sulphur could not be isolated from a wide range of soils, a yeast-like fungus (Monilia sp.) isolated from deciduous woodland soil oxidized elemental sulphur to sulphate, forming thiosulphate, but not tetrathionate. This fungus also oxidized tetrathionate to sulphate but showed only limited ability to oxidize thiosulphate to tetrathionate. Both Aspergillus niger and Trichoderma harzianum oxidized elemental sulphur in mixed culture with Mucor flavus. Larger amounts of sulphate were initially formed in mixed, compared to single culture; but by week 5 of the incubation period sulphate formation was greatest in single culture. The wood-rotting fungi, Hypholoma fasciculare and Phanerochaete velutina showed a limited ability to oxidize elemental sulphur in vitro but were incapable of oxidizing the element when growing as mycelial cords in non-sterilized soils. The relevance of these results to the possibility that fungi play a role in sulphur oxidation in soils is commented upon.

Sulphur oxidation by soil fungi including some species of mycorrhizae and wood-rotting basidiomycetes

The mycorrhizal fungi Amanita muscaria, Paxillus involutus, Hymenoscyphus ericae, Pisolithus tinctorius, Rhizopogon roseolus, and Suillus bovinus oxidized elemental sulphur to thiosulphate and sulphate in vitro. In some, but not all cases, tetrathionate was also formed. Limited oxidation of elemental sulphur by R. roseolus also occurred when growing in association with Pinus contorta in unsterilized peat. Although yeasts capable of oxidizing sulphur could not be isolated from a wide range of soils, a yeast-like fungus (Monilia sp.) isolated from deciduous woodland soil oxidized elemental sulphur to sulphate, forming thiosulphate, but not tetrathionate. This fungus also oxidized tetrathionate to sulphate but showed only limited ability to oxidize thiosulphate to tetrathionate. Both Aspergillus niger and Trichoderma harzianum oxidized elemental sulphur in mixed culture with Mucor flavus. Larger amounts of sulphate were initially formed in mixed, compared to single culture; but by week 5 of the incubation period sulphate formation was greatest in single culture. The wood-rotting fungi, Hypholoma fasciculare and Phanerochaete velutina showed a limited ability to oxidize elemental sulphur in vitro but were incapable of oxidizing the element when growing as mycelial cords in non-sterilized soils. The relevance of these results to the possibility that fungi play a role in sulphur oxidation in soils is commented upon.

Decomposition of 14C-labelled lignin, holocellulose and lignocellulose by mycorrhizal fungi

Five different species of known ecto-mycorrhizal fungi: Cenococcum geophilum, Amanita muscaria, Tricholoma aurantium, Rhizopogon luteolus and Rhizopogon roseolus were studied for their ability to metabolize the major components of plant cell walls. All strains were able to decompose 14C-labelled plant lignin, 14C-lignocellulose and 14C-DHP-lignin at a rate which was lower than the one observed for the known white rot fungi Heterobasidion annosum and Sporotrichum pulverulentum. Also 14C-(U)-holocellulose was relatively less degradable for the mycorrhizal fungi than for the white rotters. On the other hand, aromatic monomers like 14C-vanillic acid were decomposed to a much higher extent by two species of mycorrhizal fungi compared to the activity observed for Heterobasidion annosum. The results of the experiments reveal that these stains of mycorrhizal fungi are well able to utilize the major components of plant material and thus can contribute to litter decomposition in the forest floor.

Inhibition of growth of nine ectomycorrhizal fungi by cadmium, lead, and nickel in vitro

Growth of Amanita muscaria, Cenococcum graniforme, Laccaria laccata, Pisolithus tinctorius, Rhizopogon roseolus, Suillus brevipes, S. grevellei, S. luteus, and Thelephora terrestris on Hagem Nutrient Agar as modified by Modess at 20°C for 28 days was inhibited by cadmium, lead, and nickel. All fungi were arrested by 350 μg cadmium per ml (ppm) or less. Lead arrested five species at 200 ppm or less; Cenococcum graniforme, L. laccata, and S. luteus were arrested at 2,000 ppm lead. Nickel arrested growth of six fungi at 20 ppm or less. Amanita muscaria, S. luteus, and L. laccata were arrested at 40, 175 and 225 ppm nickel, respectively. Metal concentrations that did not arrest delayed growth for 7–21 days after which the growth rate was comparable to the control.

INHIBITION OF GROWTH OF ECTOMYCORRHIZAL FUNGI IN CULTURE BY PHOSPHATE

SUMMARYThe influence of pH on the growth of, and carbohydrate utilization by, three ectomycorrhizal fungi in culture was assessed by growing them on glucose, sucrose and raffinose in the presence and absence of 0‐05 M phosphate buffer and 0‐05 M 2‐(N‐morpholino) ethane‐sulphonic acid buffer (MES). This concentration of phosphate significantly inhibited growth of Cenococcum geophilwn and Rhizopogon roseolus on all carbon sources. Suillus bovinus was more tolerant, exhibiting uninhibited growth on glucose, though adaptive growth on both sucrose and raffinose was significantly less in phosphate buffer than in the other two systems. MES had a superior buffering capacity and was less inhibitory to growth of the three fungi than phosphate buffer. The growth of C. geophilum on all carbon sources was uninhibited in MES, although this buffer significantly inhibited adaptive growth by the other two species on sucrose and raffinose, possibly by inhibiting induction or activity of invertase. The use of phosphate buffers by several workers in tests of carbohydrate utilization by ectomycorrhizal fungi will undoubtedly have affected their growth. This may, to some extent, explain differences in patterns of utilization of carbon between isolates of the same species which have been reported by different investigators. These have previously been attributed to variability between strains in utilization of carbohydrate rather than in sensitivity to phosphate. Possible ways by which phosphate inhibits growth are discussed.

Survival potential of sexual and asexual spores of ectomycorrhizal fungi

In developing methods for the inoculation of pine seed with ectomycorrhizal fungi, the effects of some environmental conditions on the storage and germination of various types of propagule of seven mycorrhizal fungi were investigated. Chlamydospores of three highly effective unidentified mycorrhizal symbionts, the oidia of Xerocomus subtomentosus , (L. ex Fr.) Quel., and the basidiospores of Rhizopogon roseolus (Corda) T. M. Fr., Suillus granulatus (L. ex Fr.) O. Kuntze and Pisolithus tinctorius (Pers.) Coker & Couch were tested. Chlamydospores had low heat tolerance and lost viability in storage even under optimal conditions of humidity. For germination they showed narrow ranges of tolerance to temperature and pH. They thus appear to have little survival value. Oidia and basidiospores showed high heat tolerance and germinated well after storage for 60 days at relative humidities near 50 %. They also germinated over wider ranges of temperature and substrate pH than chlamydospores. Basidiospores of P. tinctorius showed particularly broad tolerances to environmental conditions during both storage and germination. Basidiospores thus show great promise as seed inoculants for direct sowing programmes.