Evaluation of mycelial inocula of edible<Emphasis Type="Italic"> Lactarius</Emphasis> species for the production of<Emphasis Type="Italic"> Pinus pinaster</Emphasis> and<Emphasis Type="Italic"> P. sylvestris</Emphasis> mycorrhizal seedlings under greenhouse conditions

Different methods to inoculate seedlings of Pinus pinaster and P. sylvestris with edible Lactarius species under standard greenhouse conditions were evaluated. Fungal inoculations were performed both under pure culture synthesis in vitro, followed by transplantation of acclimatized seedlings, and directly in the greenhouse using different techniques for inocula production (mycelial slurries, vegetative inoculum grown in peat-vermiculite and alginate-entrapped mycelium). In vitro inoculations with L. deliciosus produced thoroughly colonized seedlings. However, a sharp decrease in mycorrhizal colonization was detected on transplanted seedlings after 4 month's growth in the greenhouse. On the other hand, all the inocula applied directly in the greenhouse, except the alginate-entrapped mycelium, produced a variable number of mycorrhizal seedlings and colonization rates after the first growing season, depending on the plant-fungal combination and the inoculation method. Inoculations with vegetative inocula of the strain 178 of L. deliciosus were the most effective in producing mycorrhizal seedlings. All the seedlings inoculated with this strain were colonized although the colonization rates were relatively low. The commercial feasibility of the different inoculation methods for the production of seedlings colonized with edible Lactarius species is discussed.     

Effects of experimental conditions on mycorrhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-body formation of the Saffron milk cap under controlled soilless conditions

The mycorrhizal relationships between pines and two edible species of Lactarius sect. Dapetes were investigated by optimizing the experimental conditions of mycelial growth and of mycorrhizal colonization of pine seedlings. In vitro mycelial growth of Lactarius deliciosus and L. sanguifluus was improved on a buffered medium containing glucose, amino acids, and vitamins. Two methods of mycorrhization of pines with Lactarius deliciosus were tested. The mycorrhizal colonization was rapid and intense under non-aseptic conditions with a low nutrient supply and without exogenous glucose. A positive influence of mycorrhizal colonization on Pinus sylvestris growth was subsequently observed. Under axenic conditions and with a high nutrient supply, mycorrhization was stimulated at 10 g/L of exogenous glucose, irrespective of the phosphorus concentration. At high phosphorus level (1 mM) and 0.1, 1.0, or 10.0 g/L glucose, growth of Pinus sylvestris was reduced by inoculation. Stability and development of Pinus spp./Lactarius deliciosus symbioses were assayed in a climatic chamber using containers filled with a synthetic substrate. Over a 2-year culture period, the root systems of the pine seedlings were heavily colonized by Lactarius deliciosus. One year following inoculation, Lactarius deliciosus fruit-body primordia appeared associated with Pinus sylvestris seedlings. Six months later, two mature basidiomata were obtained. This is the first report of soilless fruit-body formation of this edible mushroom.     

Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition

Real-Time PCR has been applied to quantify extraradical soil mycelium of the edible ectomycorrhizal fungus Lactarius deliciosus in an interspecific competition experiment under greenhouse conditions. Couples of Pinus pinea seedlings inoculated with either L. deliciosus, Rhizopogon roseolus, or non-inoculated (control) were transplanted into pots filled with two types of soil in all the possible combinations. Total DNA was extracted from soil samples at 3 and 6 months after transplantation to perform real-time PCR analysis. DNA extractions from soil mixed with known amounts of mycelium of L. deliciosus were used as standards. Six months after transplantation, the percentage of mycorrhizas of L. deliciosus and seedling growth were significantly affected by the soil type. Extraradical soil mycelium of L. deliciosus was positively correlated with the final percentage of mycorrhizas and significantly affected by the sampling time and soil depth. The competition effect of R. roseolus was not significant for any of the measured parameters, probably due to the sharp decrease of the mycorrhizal colonization by this fungus. We conclude that real-time PCR is a powerful technique for extraradical mycelium quantification in studies aimed at evaluating the persistence of introduced strains of L. deliciosus in field plantations.     

6种外生菌根真菌对西南桦幼苗的接种效应

【背景】西南桦是兼具内生、外生菌根的典型菌根营养型树种,菌根化育苗是其壮苗培育的有效措施。【目的】揭示外生菌根真菌对西南桦无性系幼苗生长和养分含量的影响,为其菌根化育苗筛选优良外生菌根真菌提供科学依据。【方法】以BY1、FB4、FB4+和A5等4个西南桦优良无性系为研究对象,选用土生空团菌(Cenococcumgeophilum)、松乳菇(Lactariusdeliciosus)、黄硬皮马勃(Scleroderma flavidum)、多根硬皮马勃(S. polyrhizum)、褐环乳牛肝菌(Suillus luteus)和红绒盖牛肝菌(Xerocomuschrysenteron)6个外生菌根真菌进行盆栽接种试验,分析接种处理间及无性系间苗高、地径、生物量以及养分含量差异。【结果】6个菌种均能与西南桦无性系幼苗形成外生菌根共生体,接种多根硬皮马勃与黄硬皮马勃显著促进了幼苗生长和养分吸收(P0.05),说明其与幼苗的亲和力明显优于其它菌种。尽管菌根侵染率在4个无性系之间无显著差异(P≥0.05),但各菌种对FB4、BY1幼苗生长的促进作用显著强于其它2个无性系。【结论】多根硬皮马勃和黄硬皮马勃可作为西南桦菌根化育苗的优选菌种。     

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

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

The effect of aluminium on uptake and distribution of magnesium and calcium in roots of mycorrhizal Norway spruce seedlings

Spruce seedlings ( Picea abies (L.) Karst.) colonized with Lactarius rufus (Scop.) Fr. or Lactarius theiogalus Fr. were grown in an axenic silica sand culture system with frequently renewed nutrient solution. After successful mycorrhizal colonization the seedlings were exposed to 800 μ M Al(NO₃)₃ (pH 3.9) for 13 or 17 weeks. Concentrations of Al, Mg, and Ca in the tissues of the mycorrhizal root tips were determined by X-ray microanalysis. After 13 or 17 weeks of exposure to Al, high Al concentrations were found in cell walls of all mycorrhizal tissues except the stele tissues. Compared to the controls Mg levels in most of the mycorrhizal structures were reduced by Al treatment. Calcium levels in cortex cell walls of root tips colonized with L. rufus were reduced by exposure to Al. However, in cell walls of the stele Ca levels were significantly increased. No differences in Al or Mg levels were detected in structures of mycorrhizal and non-mycorrhizal root tips from the same individual seedlings. These results suggest that (1) the endodermis is the primary barrier to radial Al transport and (2) the presence of a hyphal sheath did not prevent Al from reaching the root cortex and from displacing Mg and Ca.     

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

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

The combined effects of Pseudomonas fluorescens and Tuber melanosporum on the quality of Pinus halepensis seedlings

The ecological, economic and social values of the ectomycorrhizal fungi of the black truffle found in the rural Mediterranean are well known. The inoculation of Pinus halepensis seedlings with mycorrhizal fungi and rhizobacteria can improve the morphology and physiology of the seedlings and benefit the regeneration of arid regions and the reintroduction of inocula of mycorrhizal fungi into these areas. Some rhizobacteria can improve the establishment and functioning of ectomycorrhizal symbiosis. In this study, seedlings of P. halepensis were inoculated with the mycorrhizal fungus Tuber melanosporum and the rhizobacteria Pseudomonas fluorescens CECT 844 under non-limiting greenhouse conditions. Five months after inoculation, we analysed the growth, water parameters (osmotic potential at saturation, osmotic potential at turgor loss and modulus of elasticity), concentrations of mycorrhizal colonies, nutrient concentration and nutrient contents (N, P, K, Ca, Mg and Fe) in roots and aerial parts of the seedlings. Subsequently, tests were performed to estimate the root growth potentials. None of the treatments changed the water parameters or growth potentials of the roots. The inoculations improved the growth and nutrient uptake of the seedlings, although the combination of P. fluorescens CECT 844 and T. melanosporum did not generally lead to a significant improvement over the positive effects of a simple inoculation of T. melanosporum; however, the addition of P. fluorescens CECT 844 did double the rate of the mycorrhization of T. melanosporum. These results may be promising for enhancing the cultivation of truffles.     

The effect of soil phosphorus on the external mycelium growth of arbuscular mycorrhizal fungi during the early stages of mycorrhiza formation

The effects of soil P amendments and time of application on the formation of external mycelium by different arbuscular mycorrhizal (AM) fungi were studied. In the first experiment the external mycelium produced in the soil by the AM fungus Glomus etunicatum Beck. and Gerd., during the early stages of root colonization (7 and 14 days after inoculation), was quantified by the soil-agar film technique. A Brazilian Oxisol was used with three different phosphate levels, varying from deficient to supra-optimal for the plant. Significant differences were observed in the phosphate and inoculation treatments for plant dry weight, P content in the tissue, root length and root colonization, at fourteen days after planting. At 7 days, mycelium growth, root colonization and their relationship were reduced at supra-optimal P concentrations. Applications of P one week after planting reduced mycelium growth and root colonization more than when applied to the soil before planting. In a second experiment the arbuscular mycorrhizal (AM) fungi, Scutellospora heterogama (Nicol. and Gerd.) Walker and Sanders and E3 were tested and compared with Glomus etunicatum. For the species studied, the length of external hyphae per unit of colonized root length was affected by small P additions but no further significant differences were observed at high P levels. The three AM endophytes showed marked differences in their response to P in the soil: Scutellospora heterogama, although producing external mycelium more profusely than the Glomus spp., showed a higher sensitivity to soil P supply.     

Intraspecific variability of Lactarius deliciosus isolates: colonization ability and survival after cold storage

Intraspecific variability in root colonization, extraradical growth pattern, and survival after cold storage of Lactarius deliciosus isolates was determined in pure culture conditions using Pinus pinaster as a host plant. The ectomycorrhizal ability of L. deliciosus at 30, 45, and 60 days from inoculation was highly variable among isolates and was negatively correlated to the age of the culture (time elapsed from isolation). The formation of rhizomorphs was related to colonization ability, but no relationship was found between colonization and formation of extraradical mycelium. The final colonization achieved at 60 days from inoculation was not related to the tree species under which the sporocarps were collected. However, isolates from sporocarps collected under P. pinaster colonized more rapidly the seedlings than those collected under other pine species. The climatic range of the sporocarps from which the isolates were obtained (maritime vs. continental) was not related to the formation of mycorrhizas at 60 days from inoculation. However, isolates from sporocarps collected from a maritime climate area colonized more rapidly the P. pinaster seedlings than those collected from a continental zone. Tolerance to cold water storage of L. deliciosus was also isolate dependent. Growth revival in agar was obtained from most of the isolates after 28 months of cold storage at 4°C, but only 10 out of 29 isolates showed unaffected growth. The ITS rDNA alignment of all the L. deliciosus isolates showed a low variability with identities over 99%. Most of the variation was detected in the ITS1 region and consisted in single nucleotide changes and/or punctual indel mutations. The number of base differences per sequence from averaging over all sequence pairs was 1.329, which is in the low range when compared with other ectomycorrhizal species. No ITS pattern due to geographical origin of the isolates could be discerned.     

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     

Sources of inocula influence mycorrhizal colonization of plants in restoration projects: a meta‐analysis

Inoculation may influence mycorrhizal colonization and provide benefits to plants in restoration projects. However, it is unclear whether inoculation has consistent effects across ecosystem types, if it has long‐term effects on colonization, and whether sources of inocula differ in their effectiveness. To address these issues, we performed a meta‐analysis of published restoration studies across a variety of ecosystems to examine the effects of mycorrhizal inoculation on mycorrhizal establishment and plant growth under field conditions. Although we included trials from a variety of geographic locations, disturbance types, and ecosystem types, the majority were based in temperate ecosystems in the Northern Hemisphere, and fewer trials were from tropical ecosystems. Across ecosystem types, we found that inoculation consistently increased the abundance of mycorrhizal fungi in degraded ecosystems, and thus improved the establishment of plants. These benefits did not significantly attenuate over time. Moreover, inocula from different sources varied in their effects on mycorrhizal colonization. Inocula sourced from reference ecosystems and inocula with specific fungal species yielded higher increases in mycorrhizal colonization than did inocula from commercial sources. These results suggest that inocula source matters, and that an initial investment into mycorrhizal inoculation could provide lasting benefits for facilitating the establishment of the below‐ and aboveground components of restored ecosystems.     

Hrp Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an omega-Km interposon within the hrp gene cluster of each strain. The resulting Hrp mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.     

Culture of mycorrhizal tree seedlings under controlled conditions: Effects of nitrogen and aluminium

A sand culture system was developed for growth of mycorrhizal seedlings under monoxenic conditions, with frequently renewed nutrient solution The composition of the nutrient solution resembled that of a forest soil solution, based on long-term measurements from forest sites at Soiling, northern Germany. Seedlings of Picea abies (L.) Karst. inoculated with Lactarius rufus (Scop.) Fr. were grown in this culture system. Plants developed rapidly, having almost totally mycorrhizal root systems. Nitrate at 2.7 mM in the nutrient solution and applied over a 13 week period had no negative effect on mycorrhizal development. Ammonium at 2.7 mM reduced the degree of mycorrhizal infection slightly, in such a way that the degree of mycorrhizal infection was reduced to a much less extent than the total number of root lips. Hence, the impact of NH⁺₄ may be primarily on root development and not on mycorrhizal fungal colonization. When the concentrations of NO-₃ and NH⁺₄ used in the present study are compared to those found in forest soil solutions, NO-₃ and NH₄⁺ would not appear to influence mycorrhizal development negatively under natural conditions. Aluminium at 0.8 mM and applied over a 13 week period reduced Mg uptake into roots and needles by 52 and 64%, respectively, resulting in needle chlorosis and strongly reduced photosynthetic activity. From a comparison of this study with others, no major difference in physiological response to aluminium exposure between non-mycorrhizal seedlings and seedlings colonized with Lactarius rufus was found.     

Differential effects of tropical arbuscular mycorrhizal fungal inocula on root colonization and tree seedling growth: implications for tropical forest diversity

The potential for mycorrhizae to influence the diversity and structuring of plant communities depends on whether their affinities and effects differ across a suite of potential host species. In order to assess this potential for a tropical forest community in Panama, we conducted three reciprocal inoculation experiments using seedlings from six native tree species. Seeds were germinated in sterile soil and then exposed to arbuscular mycorrhizal fungi in current association with naturally infected roots from adults of either the same or different species growing in intact forest. The tree species represent a range of life histories, including early successional pioneers, a persistent understory species, and emergent species, typical of mature forest. Collectively, these experiments show: (i) the seedlings of small-seeded pioneer species were more dependent on mycorrhizal inocula for initial survival and growth; (ii) although mycorrhizal fungi from all inocula were able to colonize the roots of all host species, the inoculum potential (the infectivity of an inoculum of a given concentration) and root colonization varied depending on the identity of the host seedling and the source of the inoculum; and (iii) different mycorrhizal fungal inocula also produced differences in growth depending on the host species. These differences indicate that host–mycorrhizal fungal interactions in tropical forests are characterized by greater complexity than has previously been demonstrated, and suggest that tropical mycorrhizal fungal communities have the potential to differentially influence seedling recruitment among host species and thereby affect community composition.     

Mobilization and transfer of nutrients from litter to tree seedlings via the vegetative mycelium of ectomycorrhizal plants

The ability of the mycorrhizal fungus Paxillus involutus to mobilize nitrogen and phosphorus from discrete patches of beech ( Fagus sylvatica ), birch ( Betula pendula ) and pine ( Pinus sylvestris ) litter collected from the fermentation horizon of three forest soils, and to transfer the nutrients to colonized B. pendula Roth seedlings, was investigated in transparent observation chambers. The mycelium of P. involutus foraged intensively in all three types of litter, leading to a significant decline in their phosphorus contents after 90 d. Over the same period only one of the litter types, beech, showed more than a 10% loss of its N contents. Exploitation of the litter led to invigoration of the vegetative mycelium of the fungus throughout the chambers as well as to significant increases of biomass production and leaf area in seedlings grown in the plus litter (+L) relative to those in minus litter (−L) systems. The yield increases were associated with gains in whole plant tissue content and concentration of P, but in content only in the case of N. Calculations suggest that a major proportion of the phosphorus lost from litter originated in its organic fraction. The possible basis of the discrepancy between values of N loss from litter and gain by the plant is discussed and the extent to which the distinctive pattern of nutrient mobilization is a feature peculiar to this fungus-plant combination is considered. It is concluded that nutrient mobilization from natural organic substrates in the fermentation horizon of forest soils may be a key function of the vegetative mycelium of mycorrhizal systems. The need for experimental analyses of a greater range of fungus-plant partnerships is stressed.     

Measurement of a rugulosin-producing endophyte in white spruce seedlings

Previous studies conducted in growth chambers had demonstrated the successful experimental inoculation of white spruce seedlings with anti-insectan toxin producing needle endophytes. Needles colonized with the rugulosin-producing endophyte 5WS22E1 (DAOM 229536) contained rugulosin in concentrations that impaired spruce budworm growth. Here we report experimental inoculations conducted under nursery conditions. To improve the reliability of detecting successful colonization, a polyclonal assay was developed for the target endophyte 5WS22E1. It was able to reliably detect the fungus in 500 ng subsamples of colonized needles. Seventeen months post-inoculation, 330 seedlings from 1235 inoculated were colonized. A random selection of 113 colonized seedlings was analyzed for rugulosin. Needles of most (90%) contained detectable concentrations of rugulosin. The range and distribution of the rugulosin concentrations was similar to that found in earlier tests done in growth chambers.     

Inoculating wheat (Triticum aestivum L.) with the endophytic bacterium Serratia sp. PW7 to reduce pyrene contamination

This research was conducted to find an optimal inoculation way for a pyrene-degrading endophytic Serratia sp. PW7 to colonize wheat for reducing pyrene contamination. Three inoculation ways, which are soaking seeds in inocula (TS), dipping roots of seedlings in inocula (TR), and spraying inocula on leaves of seedlings (TL), were used in this study. Inoculated seedlings and noninoculated seedlings (CK) were, respectively, cultivated in Hoagland solutions supplemented with pyrene in a growth chamber. The results showed that strain PW7 successfully colonized the inoculated seedlings in high numbers, and significantly promoted the growth of seedlings (TS and TR). More importantly, strain PW7 reduced pyrene levels in the seedlings and the Hoagland solutions. Compared to the noninoculated seedlings, the pyrene contents of the inoculated seedlings were decreased by 35.7-86.3% in the shoots and by 26.8–60.1% in the roots after 8-day cultivation. By comparing the efficiencies of decreasing pyrene residues, it can be concluded that TR was an optimal inoculation way for endophytic strains to colonize the inoculated plants and to reduce the pyrene contamination. Our findings provide an optimized inoculation way to reduce organic contamination in crops by inoculating plants with functional endophytic bacteria.     

The ectomycorrhizal symbiosis between Lactarius deliciosus and Pinus sylvestris in forest soil samples: symbiotic efficiency and development on roots of a rDNA internal transcribed spacer-selected isolate of L. deliciosus

The effect on plant growth of pre-inoculation of Pinus sylvestris with the ectomycorrhizal (ECM) edible basidiomycete Lactarius deliciosus (isolate D45) under controlled conditions, and the development on roots of this basidiomycete, were investigated in gamma-irradiated and unsterilized containers containing different forest soil cores or a perlite-vermiculite mixture. Five months after planting, L. deliciosus mycorrhizal plants exhibited greater growth than the non-mycorrhizal ones in all soil types, i.e. up to a 325% increase in shoot height in the sterilized soils. The experiment demonstrated the dependency of P. sylvestris seedlings upon ECM symbiosis for their survival in gamma-irradiated, microbiologically disturbed soil samples. Furthermore, in two soils, the growth of L. deliciosus-inoculated seedlings was greater in the sterilized soil samples than in the non-sterilized ones, i.e. 46% and 132% increase in shoot height under sterilized soil conditions. In containers randomly sampled from each soil type, the degree of root colonization by the inoculated isolate, calculated as the number of mycorrhizal root tips divided by the total number of root tips x100, ranged from 80% to 35%. Within the short term, the inoculated isolate developed rapidly on roots, dominated, and hampered ectomycorrhiza formation by various unidentified (but not Lactarius) resident ECM fungi in unsterilized soil types. Results indicate that the ECM species L. deliciosus is worth investigating to ascertain if other isolates benefit pine growth like the isolate D45, and are therefore also attractive candidates for forestry applications in the Mediterranean area.     

Coinoculation of containerized Douglas-fir (Pseudotsuga menziesii) and maritime pine (Pinus pinaster) seedlings with the ectomycorrhizal fungi Laccaria bicolor and Rhizopogon spp.

 Coinoculations with mycelium of Laccaria bicolor and spores of Rhizopogon spp. included in alginate gel have been carried out to determine: (1) the ability of the mixed inoculum to produce dual-colonized containerized Douglas-fir and maritime pine planting stocks and (2) the colonization pattern of the two fungi in individual root systems. For both tree species, the maximal proportion of dual-colonized seedlings obtained almost never exceeded 50%. The rest of the seedlings remained colonized by a single fungus or were non-colonized. In Douglas-fir inoculations, the relationship between the dual-colonized seedlings obtained and the initial dose of the two fungi was highly significant. The highest proportion of dual-colonized seedlings was obtained when the highest dose of R. subareolatus was used (10⁶ spores/seedling), regardless of the dose of L. bicolor. Among the treatments producing 25% or more dual-colonized seedlings, differences in the proportion of Laccaria/Rhizopogon mycorrhizas and total root colonization percentages were not clearly related to the initial combination of doses. The proportion of dual-colonized maritime pine seedlings was not significantly related to the initial inoculation doses of the two fungi. The proportion of Laccaria/Rhizopogon mycorrhizas was not significantly different among treatments with 25% or more dual-colonized seedlings, whereas total colonization percentages ranged from 37% with the combination 0.08/10⁴ (g L. bicolor / spores R. roseolus per seedling) to 74% with the combination 0.08/10⁶, this difference being statistically significant. Accepted: 15 September 1998