Growth of ectomycorrhizal fungi stimulated by lipids from a pine root exudate

Summary The content of fatty acids was analysed in an exudate from roots of pine seedlings grown axenically in vermiculite with a synthetic nutrient medium. The dominating fatty acdis were fewer in the exudate than in the roots. Unsaturated fatty acids were predominant. The total lipid fraction of the exudate promoted mycelial growth in two of the three ectomycorrhizal fungi tested.     

Effect of ectomycorrhizal fungi on chestnut ink disease

 Seedlings of Castanea sativa were inoculated at transplanting time with four ectomycorrhizal (ECM) fungi, Laccaria laccata, Hebeloma crustuliniforme, H. sinapizans and Paxillus involutus. At the end of the first vegetative season, 7 months after sowing, half of the mycorrhizal and nonmycorrhizal seedlings were challenged with a zoospore suspension of Phytophthora cambivora and the other half with P. cinnamomi. Five months later, mycorrhizal plants infected with P. cambivora or P. cinnamomi showed no sign of pathogen infection. The ECM fungi increased plant biomass also in the presence of the pathogen. Mycorrhizal seedlings inoculated with the pathogens showed greater shoot and root development than nonmycorrhizal chestnut plants. All the fungi tested reduced the negative effect of the ink disease pathogens on the plant host in vivo. The mechanisms by which the ECM fungi protect chestnut seedlings are discussed. Accepted: 20 May 1999     

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     

Tissue density and growth response of ectomycorrhizal fungi to nitrogen source and concentration

 Amanita rubescens Pers., Lactarius affinis Pk., Leccinum aurantiacum (Fr.) S.F. Gray, Tylopilus felleus (Bull. ex Fe.) Karsten, and two isolates of Suillus intermedius (Smith & Thiers) Smith & Thiers collected from an approximately 55-year-old Pinus resinosa Ait. plantation, and Pisolithus tinctorius (Pers.) Coker & Couch obtained from another source, were tested for their abilities to grow with protein as the primary source of nitrogen. Protein plates contained 63 mg l^–1 N as bovine serum albumen and 7 mg l^–1 N as arginine. Control plates contained only 7 mg l^–1 N as arginine. All isolates except Leccinum aurantiacum and one isolate of S. intermedius attained greater dry weight with protein as the primary source of N. Lactarius affinis, Leccinum aurantiacum, P. tinctorius, and both isolates of S. intermedius had higher tissue densities on protein medium. Amanita rubescens had lower tissue density. To determine if increase in tissue density was an effect of total N concentration or an effect of N source (protein versus arginine), we performed a second experiment in which arginine concentration was increased (7 mg l^–1 N versus 70 mg l^–1 N). The second experiment also included Cenococcum geophilum Fr. but excluded T. felleus. Higher tissue densities with increased nutrients were found in C. geophilum, Lactarius affinis, Leccinum aurantiacum, and both isolates of S. intermedius. Only A. rubescens and P. tinctorius did not have increased densities. The results suggest that these ectomycorrhizal fungi alter their growth forms according to N concentration. At low N concentrations, a growth form likely to promote exploitation of a large volume of medium for a given biomass is produced. At high concentrations, a growth form likely to promote exploitation of a rich source of N is produced. Whether ectomycorrhizal fungi growing in association with roots would act in a similar fashion is not known. Accepted: 30 July 1998     

Biotite and microcline as potassium sources in ectomycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

 The aim of this study was to investigate the role of plants colonised by two ectomycorrhizal fungi, Paxillus involutus and Suillus variegatus, in mobilising potassium (K) from biotite and microcline, two minerals common in acid to medium-acid bedrock. This was carried out in a 33-week pot study with seedlings of Pinus sylvestris growing in symbiosis with the fungi, where no K was added or where K was added in the form of biotite or microcline. The mineral additions were similar to those found in natural soils. All seedlings, including non-mycorrhizal, were able to access the K in biotite, leading to stimulated growth and K uptake relative to controls. Microcline addition induced growth depression in all seedlings except those colonised by P. involutus, which were stimulated. The soil solution from S. variegatus-colonised seedlings grown with biotite had higher concentrations of citric and oxalic acid. Citric acid concentration was positively correlated to the fungal biomass (ergosterol) in the soil, as well as to the foliar K in S. variegatus-colonised seedlings. Seedlings growing without K addition had low K concentrations in the shoot. Magnesium (Mg) concentrations were enhanced in seedlings with severe K shortage, indicating that Mg can substitute for K, while calcium concentrations did not vary significantly. Accepted: 18 January 1999     

Mycorrhiza formation and growth of Eucalyptus globulus seedlings inoculated with spores of various ectomycorrhizal fungi

 As many eucalypts in commercial plantations are poorly ectomycorrhizal there is a need to develop inoculation programs for forest nurseries. The use of fungal spores as inoculum is a viable proposition for low technology nurseries currently producing eucalypts for outplanting in developing countries. Forty-three collections of ectomycorrhizal fungi from southwestern Australia and two from China, representing 18 genera, were tested for their effectiveness as spore inoculum on Eucalyptus globulus Labill. seedlings. Seven-day-old seedlings were inoculated with 25 mg air-dry spores in a water suspension. Ectomycorrhizal development was assessed in soil cores 65 and 110 days after inoculation. By day 65, about 50% of the treatments had formed ectomycorrhizas. By day 110, inoculated seedlings were generally ectomycorrhizal, but in many cases the percentage of roots colonized was low (<10%). Species of Laccaria, Hydnangium, Descolea, Descomyces, Scleroderma and Pisolithus formed more ectomycorrhizas than the other fungi. Species of Russula, Boletus, Lactarius and Hysterangium did not form ectomycorrhizas. The dry weights of inoculated seedlings ranged from 90% to 225% of the uninoculated seedlings by day 110. Although plants with extensively colonized roots generally had increased seedling growth, the overall mycorrhizal colonization levels were poorly correlated to seedling growth. Species of Laccaria, Descolea, Scleroderma and Pisolithus are proposed as potential candidate fungi for nursery inoculation programs for eucalypts. Accepted: 7 May 1998     

Effect of organic and inorganic nitrogen sources on endogenous polyamines and growth of ectomycorrhizal fungi in pure culture

 The abilities of three ectomycorrhizal fungi, Paxillus involutus, Suillus variegatus and Lactarius rufus, to utilize organic and inorganic nitrogen sources were determined by measuring the growth and endogenous free polyamines (putrescine, spermidine and spermine) of pure culture mycelium. Differences were found in the utilization of the nitrogen sources and in the polyamine concentrations between the fungal species and between isolates of L. rufus. All the fungi grew well on ammonium and on several amino acids. Endogenous polyamine levels varied with the nitrogen source. Spermidine was commonly the most abundant polyamine; however, more putrescine than spermidine was found in P. involutus growing on inorganic nitrogen or arginine. Low amounts of spermine were found in S. variegatus and some samples of L. rufus. None or only a trace of spermine was found in P. involutus mycelium. In all fungi, putrescine concentrations were higher with ammonium than with the nitrate treatment. The total nitrogen content of peat did not determine the ability of L. rufus strains isolated from peatland forest sites to utilize organic nitrogen. Accepted: 27 November 1998     

Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery

 The coinoculation efficacy of the ectomycorrhizal fungi Laccaria laccata and Thelephora terrestris on the growth and mycorrhizal development of Pinus patula seedlings was studied and compared to individual inoculation of these fungi in a nursery. The total number of mycorrhizas was higher in seedlings inoculated with the combined inoculum than with the individual inocula. The colonization by T. terrestris was higher than L. laccata when the seedlings were inoculated with the two fungi simultaneously. Coinoculation significantly increased the height and dry weight of the seedlings compared with individual inoculation, both in steam-sterilized and unsterilized soil. Accepted: 12 May 1997     

Field performance in northern Spain of Douglas-fir seedlings Inoculated with ectomycorrhizal fungi

 Experimental plantations were established in northern Spain to determine the effects of different ectomycorrhizal fungi on growth and survival of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) under field conditions. Douglas-fir seedlings were inoculated with Laccaria bicolor S238 mycelia in two bareroot nurseries in central France or with spore suspensions of three hypogeous ectomycorrhizal species: Melanogaster ambiguus, Rhizopogon colossus and R. subareolatus, in a Spanish containerised nursery. The effects of ectomycorrhizal inoculation on plant survival after outplanting were limited, being only significant at the Guipuzkoan (Spain) site, when plants inoculated with L. bicolor S238 were compared to non-inoculated plants grown in non-fumigated soil. L. bicolor S238 had a significant effect on plant growth during the phase of bareroot nursery growth and this difference was maintained after field outplanting. Nursery inoculations with M. ambiguus, R. colossus and R. subareolatus improved plant growth during the first 2 and 3 years after field outplanting. The positive effects of the inoculation treatment on seedling height, root collar diameter and stem volume persisted after 5 years of field growth. Inoculation with these ectomycorrhizal fungi may improve the field performance of Douglas-fir seedlings in northern Spain. Accepted: 12 February 1999     

Eucalyptus root and shoot chitinases, induced following root colonization by pathogenic versus ectomycorrhizal fungi, compared on one- and two-dimensional activity gels

The present work deals with the effect of root fungal colonization on chitinases activities in Eucalyptus seedlings. Plant chitinases indiced during pathogenic infection are thought to be directed against the fungus, but chitinases induced by ectomycorrhizal fungi may contribute to ectomycorrhizal ontogenesis. Plant responses were compared to determine whether plants induce different chitinases activities in contact with symbionts and pathogens, and whether chitinases are induced systemically in both cases. Despite 2-D analysis of Eucalyptus root chitinolytic activities, induced following root colonization by pathogenic or ectomycorrhizal fungi, it was not possible to differentiate between both infections. Moreover, ectomycorrhizal colonization, as pathogenic infections, led to systemic induction of chitinase activities far from the site of inoculation. Contrasting with roots, the chitinase activities induced in shoots were not positively correlated with ectomycorrhizal strain aggressiveness. The differential stimulation of root chitinase activity by aggressive or non-aggressive ectomycorrhizal strains was related to induction of five additional isoforms in response to contact with the most aggressive strains.     

Cultivation of mushrooms of edible ectomycorrhizal fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis

Edible mushroom fungi in the genera Lyophyllum, Tricholoma, Leucopaxillus, Suillus, Rhizopogon, Lactarius, and Morchella were tested for mycorrhization with Pinus densiflora in vitro. Most of the tested fungi in the genera Lyophyllum, Tricholoma, Suillus, Rhizopogon, and Lactarius formed ectomycorrhizas 2–4 months after fungal inoculation. Mycorrhizal seedlings were then acclimatized in open-pot soil under growth-chamber conditions. Almost all mycorrhizal seedlings sustained their symbiont and developed new mycorrhizas for 8–9 months after transplantation. Under these conditions, more than half of the tested species formed primordia and Tricholoma flavovirens, Rhizopogon rubescens, and Lactarius akahatsu developed basidiocarps with young host plants. Accepted: 28 November 2000     

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     

Presence of arbuscular mycorrhizas in typically ectomycorrhizal host species from Cameroon and New Zealand

 Ectomycorrhizas (EcM) and arbuscular mycorrhizas (AM) were screened for in saplings of 14 EcM tree species from the N'Dupé and Korup National Park rainforests, SW Cameroon, belonging to Caesalpiniaceae and Uapacaceae. The pattern of EcM and AM colonisation of a dual mycorrhizal species from this rainforest (Uapaca staudtii, Uapacaceae) was compared with dual EcM/AM colonisation of Leptospermum scoparium (Myrtaceae) from New Zealand. Both species were collected in a range of habitats. EcM and AM colonisation differed among species in the Korup National Park rainforest: 12 species belonging to the Caesalpiniaceae (Amherstieae) were consistently EcM, and AM structures occurred occasionally in six of them; two other species belonging to Caesalpiniaceae (Afzelia bipindensis) and Uapacaceae (U. staudtii) were dual mycorrhizal with variable levels of colonisation by both EcM and AM fungi. EcM and AM dual colonisation varied with both habitat and identity of the partners. The presence of EcM fungi in most of the root samples of U. staudtii and a negative relationship between AM and EcM colonisation within the same root system suggested a greater EcM affinity of this species. In contrast, most root samples of L. scoparium were colonised by AM, but only a few by EcM. Genuine dual EcM/AM associations in root samples of U. staudtii where the two mycorrhizal types co-occurred could be attributed to an AM-EcM succession. However, differences between predicted and observed frequencies of genuine dual EcM/AM associations in several samples of both U. staudtii and L. scoparium indicated that other factors influenced dual EcM/AM associations. The results of this study showed the importance of the identity of the host species in determining the pattern of dual EcM and AM colonisation. Accepted: 18 September 1998     

Interaction between embryogenic cultures of Scots pine and ectomycorrhizal fungi

 Embryogenic cell masses of three Scots pine (Pinus sylvestris) cell lines K779, K884 and K1009 were cultivated with the ectomycorrhizal (ECM) fungi Laccaria bicolor, L. proxima, Pisolithus tinctorius, Paxillus involutus and two strains of Suillus variegatus. The average growth ratio of the slowly proliferating cell line K1009 was improved by L. proxima and S. variegatus strain H, while of the rapidly proliferating lines K779 and K884 the non-mycorrhizal controls grew best. The fungi caused two distinct reactions in embryogenic cultures. In the positive reaction, the shape and light yellow colour of the cultures resembled the controls, while in the negative reaction the embryogenic cells became brown and necrotic and the fungi grew aggressively over them. These reactions to the fungi did not correlate completely with effects on the growth ratio. All the cell lines enhanced the radial growth of S. variegatus H and of P. tinctorius, while the Laccaria species and S. variegatus strain 1 thrived better alone. This study shows that early-stage embryogenic cells of Scots pine and ECM fungi are able to interact. As some fungi produced a positive reaction or even increased proliferation, they could be used to enhance somatic embryogenesis of Scots pine. Specific fungi might be used to induce the growth of slowly proliferating cell lines, and knowledge of positive cell line-fungus interactions could be useful in work with later stages of somatic embryogenesis, such as rooting. Accepted: 16 July 1998     

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     

Relationships between fungal uptake of ammonium, fungal growth and nitrogen availability in ectomycorrhizal Pinus sylvestris seedlings

 Nitrogen deposition and intentional forest fertilisation with nitrogen are known to affect the species composition of ectomycorrhizal fungal communities. To learn more about the mechanisms responsible for these effects, the relations between fungal growth, nitrogen uptake and nitrogen availability were studied in ectomycorrhizal fungi in axenic cultures and in symbiosis with pine seedlings. Effects of different levels of inorganic nitrogen (NH₄) on the mycelial growth of four isolates of Paxillus involutus and two isolates of Suillus bovinus were assessed. With pine seedlings, fungal uptake of ¹⁵N-labelled NH₄ was studied in short-term incubation experiments (72 h) in microcosms and in long-term incubation experiments (3 months) in pot cultures. For P. involutus growing in symbiosis with pine seedlings, isolates with higher NH₄ uptake were affected more negatively at high levels of nitrogen availability than isolates with lower uptake. More NH₄ was allocated to shoots of seedlings colonised by a high-uptake isolate, indicating transfer of a larger fraction of assimilated NH₄ to the host than with isolates showing lower NH₄ uptake rates. Thus low rates of N uptake and N transfer to the host may enable EM fungi avoid stress induced by elevated levels of nitrogen. Seedlings colonised by S. bovinus transferred a larger fraction of the ¹⁵N label to the shoots than seedlings colonised by P. involutus. Seedling shoot growth probably constituted a greater carbon sink in pot cultures than in microcosms, since the mycelial growth of P. involutus was more sensitive to high NH₄ in pots. There was no homology in mycelial growth rate between pure culture and growth in symbiosis, but N uptake in pure culture corresponded to that during growth in symbiosis. No relationship was found between deposition of antropogenic nitrogen at the sites of origin of the P. involutus isolates and their mycelial growth or uptake of inorganic nitrogen. Accepted: 18 September 1998     

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 effects of Laccaria proxima and fibrous pulp waste on the growth of nine container-grown conifer seedling species

 Japanese larch (Larix kaempferi), white spruce (Picea glauca), black spruce (Picea mariana), red spruce (Picea rubens), jack pine (Pinus banksiana), mugo pine (Pinus mugo), red pine (Pinus resinosa), Japanese black pine (Pinus thunbergii) and Douglas-fir (Pseudotsuga menziesii var. menziesii), were inoculated to test the effective host range of the ectomycorrhizal fungus Laccaria proxima and the possibility of utilizing pulp waste as a potting medium for containerized seedling production. Laccaria proxima tended to improve the container growth of Japanese black pine and white spruce, and significantly improved that of jack pine, mugo pine, black spruce, red spruce and Douglasfir. The growth of red pine and Japanese larch were only slightly improved with L. proxima. Pulp waste (33% by volume) had negative effects on tree seedling growth, except for Douglasfir (no significant effect). The interactions of Laccaria proxima and pulp waste varied; the hosts were significantly positive (P<0.01) in the case of jack pine and black spruce, but there was no significant effect for the rest. Negative effects were found with Japanese black pine. Use of pulp waste in seedling production of jack pine, black spruce, mugo pine, red spruce and Douglasfir inoculated with L. proxima and of Japanese black pine both with and without L. proxima is feasible, but further research is necessary to determine the optimal percentage of pulp waste that can be utilized in seedling production of tree species and the field performance of these seedlings. Accepted: 30 August 1995