Spatiotemporal distribution of fruitbodies of ectomycorrhizal fungi in an Abies firma forest

 Spatial associations between ectomycorrhizal (ECM) fungi and their presumed host trees, and spatiotemporal associations among ECM fungi were surveyed for 3 years in an Abies firma-dominated forest in central Japan. A total of 39 species in 13 genera of ECM fungi were recorded, with more species in the Russulaceae than any other family. Russula ochroleuca, Russula sp.1 and Strobilomyces confusus tended to produce their fruitbodies on the forest floor directly under the crown of A. firma, whereas those of Inocybe cincinnata, Gomphus floccosus and G. fujisanensis were aggregated in limited areas outside the A. firma crown. Interspecific spatial associations were analysed for Russula sp.1, which was the most dominant species, and three other frequent species, I. cincinnata, S. confusus and R. ochroleuca. Pairwise, Russula sp.1 with I. cincinnata, with S. confusus or with R. ochroleuca showed an association which was exclusive, overlapping or independent, respectively. Fruiting phenologies differed in that S. confusus showed a peak density in the summer, whereas the other three species peaked in the autumn. These results suggest that the formation of ECM fruitbodies can be partitioned among the species both spatially and temporally. Accepted: 7 July 1998     

The diversity of ectomycorrhizal fungi associated with introduced Pinus spp. in the Southern Hemisphere, with particular reference to Western Australia

Accepted: 15 June 1998     

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi

The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller’s method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.     

Viability of ectomycorrhizal fungi following cryopreservation

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at −130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Fertilization alters nitrogen isotopes and concentrations in ectomycorrhizal fungi and soil in pine forests

To assess how nitrogen (N) availability affected ectomycorrhizal functioning and to test a theoretical model of ectomycorrhizal ¹⁵N partitioning, we measured C/N and δ¹⁵N in soils and nine fungal taxa in two Swedish N addition experiments. Sporocarp C/N and soil C/N decreased with fertilization, implying that N uptake per unit fungal growth increased. The S horizon was more responsive than the F and H horizons to changes in N addition, with N turnover for these horizons of 24, 57, and 57 y, respectively. Fungal and soil δ¹⁵N patterns identified fungal N sources, with N acquisition primarily from the S, F, or H horizon for two, five, and two taxa, respectively. With increasing N availability, sporocarp ¹⁵N enrichment increased in five taxa, in agreement with our model of fungal-plant N partitioning. However, it decreased in Lactarius rufus and Russula aeruginea, perhaps indicating shifts towards greater inorganic N uptake in these two taxa. This may relate to the generally lower sensitivity of these taxa to N deposition compared to the Cortinarius and Suillus taxa that fit our model of ¹⁵N partitioning.     

Competition between ectomycorrhizal fungi colonizing Pinus densiflora

 Interactive competition of Pisolithus tinctorius (Pers.) Coker et Couch with an unidentified species Tanashi 01 and Suillus luteus (L.: Fr.) S. F. Gray was investigated using a rhizobox. Pinus densiflora Sieb. et Zucc. was used as the host plant and mycelia were distinguished by hyphal color. The speed of mycelial spread differed between the fungi;P. tinctorius and Tanashi 01 grew faster than S. luteus. A P. tinctorius mycorrhizal seedling and a Tanashi 01 mycorrhizal seedling were transplanted on opposite sides of the rhizobox. The mycelia and mycorrhizae of P. tinctorius were overgrown by Tanashi 01 hyphae and development of P. tinctorius was gradually inhibited. The areas occupied by mycelia and mycorrhiza of P. tinctorius decreased by 52% and 37%, respectively, 154 days after transplantation relative to that at 91 days. In the overlap area of P. tinctorius and Tanashi 01, the latter fungus infected new root tips emerging from P. tinctorius mycorrhiza, which lacked a mantle of P. tinctorius hyphae, and formed a composite mycorrhizal structure. P. tinctorius mycorrhizae were progressively replaced by Tanashi 01 mycorrhizae. Mycelial spread of P. tinctorius and S. luteus were naturally inhibited but there was no interaction in mycorrhizal formation. Accepted: 18 June 1999     

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).     

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     

Induced hydrolytic enzymes of ectomycorrhizal fungi against pathogen Rhizoctonia solani

Interactions between ectomycorrhizal fungi (Suillus laricinus, S. tomentosus, Amanita vaginata and Gomphidius viscidus) and the pathogen Rhizoctonia solani in co-culture were studied using both light and scanning electron microscopy. S. laricinus, S. tomentosus and A. vaginata inhibited the growth of the pathogen. Moreover, A. vaginata exhibited coiling around and penetration of the hyphae into R. solani was observed in the interaction zone. Furthermore, the production of chitinases, beta-1,3-glucanases and beta-glucosidases by these ectomycorrhizal fungi on colloidal chitin or cell walls of R. solani was evaluated: chitinases were not induced by colloidal chitin but all three enzymes were induced by R. solani cell walls. No correlation between inhibition rate and production of lytic enzymes was found.     

Morphological analysis of early contacts between pine roots and two ectomycorrhizal Suillus strains

 Selection of ectomycorrhizal strains for application in forestry is mostly based on the evaluation of symbiotic performance in small-scale experiments. Two Suillus collinitus strains isolated from a Mediterranean and an alpine area were inoculated onto two pine tree species (Pinus pinea and P. nigra ssp. laricio var. corsicana) typical of these two environments. The early events during contact between the cell surfaces of plant and fungal partners were analysed morphologically using ultrastructural and immunocytochemical techniques. All four plant-fungus combinations led to a similar degree of mycorrhizal infection and to a similar colonization pattern. The first contact of fungal hyphae with root cap cells usually involved breakdown of the outermost electron-opaque layer of the plant cell walls. Hyphae further developed between this layer and the underlying wall strata. Ultrastructural observations revealed that S. collinitus strain J3-15-24, isolated from a Mediterranean area, induced a defence reaction in the roots of P. nigra, which grows typically in alpine areas. These observations suggest functional differences between the two fungal strains in their mycorrhizal capabilities. Accepted: 3 April 1998     

Fruiting of putative ectomycorrhizal fungi under blue gum (Eucalyptus globulus) plantations of different ages in Western Australia

 The species richness of putative ectomycorrhizal (EM) fungi fruiting in blue gum (Eucalyptus globulus Labill.) plantations in Western Australia was investigated in relation to plantation age. Eleven plantations, 1–8 years old, were selected for study and two native Eucalyptus forest sites in the same region were chosen for comparison. Sporocarps of 44 species of putative EM fungi were collected from the 13 sites. Of these, 30 species were found in blue gum plantations. The number of fungal species was highly positively correlated with plantation age and inversely correlated with soil pH. Young plantations (1–5 years) had 2–9 fungal species and were overwhelmingly dominated by species of Laccaria and Scleroderma. In older plantations (6–8 years), the relative abundance of sporocarps of each species within the fungal community decreased, accompanied by an increase in the number of fungal species (12–17 per site). A brief survey of the two native eucalypt forests in this region revealed a much higher number of fungal species than that observed in plantations. In plantations, species of Descolea, Laccaria, Pisolithus and Scleroderma typically fruited in young plantations. Species of epigeous fungi of the genera Boletus, Cortinarius, Hydnum, Inocybe, Lactarius, Paxillus, Russula and hypogeous fungi, including species of Descomyces, Hysterangium and Mesophellia, were found only in older plantations, or in native forests. Some of the fungi that fruit in young plantations are now being evaluated for use in commercial spore inoculation programs to increase the species diversity of EM fungi in exotic eucalypt plantations. Accepted: 8 October 1998     

Effect of ectomycorrhizal fungi on nitrogen mineralisation and the growth of Scots pine seedlings in natural peat

The aim of this study was to investigate the potential of different isolates of ectomycorrhizal (EM) fungi to enhance the growth of Pinus sylvestris seedlings in five natural peat substrates with different nitrogen concentrations, and the effect of the Scots pine seedlings and fungal inoculum on the formation of dissolved inorganic and organic nitrogen in peat. Utilization of different organic nitrogen compounds by microbial community in the peat was also investigated using Biolog MT MicroPlates. Inoculation of the seedlings with EM fungi enhanced seedling growth. Piloderma croceum increased root growth especially, whereas Lactarius rufus increased needle growth and Suillus variegatus I, II and III improved both root and needle growth. All the EM fungi also significantly affected stem growth. Nitrogen concentration of the peat did not affect seedling growth as much as the EM fungi. At the lowest peat N concentration (1.17%) NH ₄ ⁺ mineralisation was lower and DON (dissolved organic nitrogen) accumulation higher than at higher peat N concentrations. The EM fungal isolates had different effects on NH ₄ ⁺ and DON accumulation/degradation in peat. The EM fungal isolates significantly increased NH ₄ ⁺ formation in the peat, whereas L. rufus and P. croceum had an opposite effect on DON accumulation. S. variegatus I significantly decreased the DON concentrations during peat incubation. The N concentration of the peat slightly affected the utilization of amino acids and polyamines by the microbial community, whereas inoculation with S. variegatus I, II or III had no effect.     

Effects of manipulation of litter and humus layers on ectomycorrhizal colonization potential in Scots pine stands of different age

Effects of manipulation of litter and humus layers (removal, doubling and control treatments) on the colonization potential of ectomycorrhizal fungi were studied in two secondary stands of Pinus sylvestris (5 and 18 years old) in The Netherlands. Five-mont-hold, sterile-grown Scots pine seedlings, inoculated with Laccaria bicolor, Paxillus involutus or Rhizopogon luteolus and noninoculated seedlings were used as baits. The seedlings were harvested after one growing season. For comparison, sporocarps of ectomycorrhizal fungi were also investigated. Genus composition on the seedlings was independent of initial inoculum, but determined by both treatment and age of the stands. In both stands, removal of litter and humus layers increased, and addition of organic material decreased the number of ectomycorrhizal types on the seedlings. Not all indigenous genera were observed by either outplanting seedlings or sporocarp surveys.     

Protozoan communities around conifer roots colonized by ectomycorrhizal fungi

Protozoan communities around roots with different types of ectomycorrhizae were distinct. These protozoan communities differed both qualitatively and quantitatively with the host (Pinus ponderosa, Pseudotsuga menziesii, Picea sitchensis, Tsuga heterophylla and Abies grandis) and the ectomycorrhizal fungal species. Based on the species identified and the numbers of individuals of each species, six communities of protozoa were found associated with specific ectomycorrhizae. Previous researchers have shown that mycorrhizal colonization of roots alters the amounts and types of exudates produced by roots, which in turn alters the bacterial community present. Most likely, mycorrhizal colonization of roots influences the protozoan community around roots by controlling the bacterial community. However, the protozoan community may in turn influence the successional dynamics of ectomycorrhizal fungi on different host root systems by a variety of mechanisms. These mechanisms could include: (1) preying upon individuals and perhaps removing particular species of bacteria from the mycorrhizosphere; and (2) controlling nitrogen mineralization in the rhizosphere. Further work needs to be performed to determine the interaction between these quadrate (plant-bacteria-fungi-protozoa) associations.     

Transgene expression in regenerating cotyledons and embryogenic cultures of Scots pine

Regenerating cotyledons and embryogenic cultures of Scots pine (Pinus sylvestris L.) were used as targets for gene delivery via particle bombardment. Both target tissues differed in their response to selective agents, regulative sequences of transferred genes, and conditions optimal for particle bombardment. Of the gene constructs tested, the pB1410 including CaMV 35S promoter, AMV-translation enhancer and gusA:npt reporter and selectable marker gene fusion was the most suitable for cotyledon transformation, resulting on average in 3.7 ( 0.1 SE) -glucuronidase (GUS) expression units per cotyledon. In embryogenic cell masses the pB1221.1 gene construct including the gusA reporter gene driven by the 35S promoter gave the highest transient expression, 63 (plusmn; 15 SE) GUS expression units of g^-1 fresh weight of embryogenic cell mass. Kanamycin and geneticin concentrations suitable for selection of cotyledons were 10-15 mg dm^-3 and 0.5-1.0 mg dm^-3, respectively. Kanamycin (10 mg dm^-3) and phosphinotricin (1 mg dm^-3) as selectors caused a significant decline in growth, but geneticin did not significantly affect the growth of the embryogenic cultures during the 8 week cultivation period. The production of transgenic plantlets seems to be more dependent on the regeneration and multiplication efficiency of organogenesis and somatic embryogenesis of Scots pine than on gene delivery into regenerating tissues.     

Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid-gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the host's cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.     

Receptiveness of forest soils to ectomycorrhizal association:

 Soil receptiveness to a mycorrhizal association can be estimated by standard bioassay from a dose-response relationship. The method was developed using the association Pinus pinaster or Pseudotsuga menziesii with Laccaria bicolor as a model and was successfully used to characterize the receptiveness of two forest soils. From a physical and chemical point of view, both soils were receptive to the Laccaria bicolor association. Our results show that microbial factors are very important in the receptiveness of soil to ectomycorrhizal association. Ectomycorrhizal development on seedlings at outplanting sites is discussed in relation to soil receptiveness and the ecological competence of selected strains. Accepted: 11 October 1996     

In vitro synthesis of Pisolithus-Eucalyptus ectomycorrhizae: synchronization of lateral tip emergence and ectomycorrhizal development

 A simple and reproducible in vitro system is described for the synthesis of Pisolithus-Eucalyptus grandis ectomycorrhizae. Hyphal discs from actively growing colonies were placed in large petri dishes containing minimum nutrient agar overlaid with cellophane and allowed to grow for 7 days. Seeds were then surface sterilized and placed above the expanding fungal colonies and the plates slanted. Seedlings that germinated and grew in the presence of fungal hyphae had twice as many lateral root tips as seedlings that germinated before they were transferred onto hyphal mats. In addition, the lateral root tips of inoculated seedlings had a faster maturation rate and emerged closer to the primary root apex than non-inoculated seedlings. All lateral tips emerged in contact with fungal hyphae and the differentiation of ectomycorrhizae was followed by examining lateral tips basipetally along a single primary root. Typical ectomycorrhizae had formed on 4-day-old lateral tips, i.e. a mantle, radially elongated epidermal cells and a Hartig net commencing about 0.3 mm behind the lateral root apex. Thereafter, the mantle continued to thicken and the apical meristem diminished. The Hartig net often surrounded the apex of 11- to 12-day-old lateral root tips. This model system will facilitate detailed studies on synchronized ectomycorrhizal development and associated molecular and biochemical changes. Accepted: 12 January 1996