Ectomycorrhizal Development Research Articles

Pinus patulaestablishment problem associated with poor ectomycorrhizal development in previously cultivated soils

SYNOPSIS A field survey of Pinus patula Schlecht. et Cham. plantations at Mondi's Mooi River Estate, Kwazulu-Natal, South Africa was conducted to assess the levels of ectomycorrhizal (EM) fungal colonisation. The objective was to determine whether the establishment problem (old land syndrome) is related to the quality and quantity of EM fungal colonisation of P. patula roots. EM colonisation was assessed in previously cultivated soils and compared to that in virgin soils at four sites. Mycorrhizal colonisation was higher in roots from virgin soil than in previously cultivated soils, although this difference was statistically significant at only two sites. Basidiomata of three EM fungi (Thelephora terrestris, Laccaria laccata and Boletus edulis) were collected from the plantations in virgin soil and one (T. terrestris) from the previously cultivated soil. Three types of EM fungal roots were identified from the survey area. These findings suggest that poor mycorrhizal development in previously cultivated so...

Influence of laser light on mycelial growth of Hebeloma mesophaeum and ectomycorrhizal development on Scots pine

The effects of exposure to helium-neon (He-Ne) or Argon (Ar) laser light (λ=632.8 nm and 514 nm, respectively) on the growth of Hebeloma mesophaeum mycelium in pure culture were studied. Growth rates were highest after exposure to the He-Ne laser (1×60 s)+Ar laser (2×60 s) and to the He-Ne laser for 3×30. Container-grown Pinus sylvestris (pine) seedlings were inoculated with a water suspension of H. mesophaeum mycelium previously exposed to different kinds of laser light. After 3 months, the percentage of mycorrhizal associations on pine roots was 34.3% higher after He-Ne laser treatment and 47.1% higher after Ar laser treatment than in the controls with untreated fungus. However, seedlings infected with treated fungus were smaller than the control. Overall, laser light stimulated growth of H. mesophaeum mycelia in pure culture and enhanced mycorrhizal development on Scots pine seedlings.

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.

Growth and ectomycorrhizal colonization of ponderosa pine seedlings supplied different levels of atmospheric CO 2 and soil N and P

Individual and interactive effects of atmospheric CO 2 enrichment and soil N and P fertility on above- and below-ground growth and mycorrhizal colonization of juvenile ponderosa pine ( Pinus ponderosa Dougl. ex Laws.) were examined. Seedlings were grown from seed in atmospheres with 700 μl l −1, 525 μl l −1, or ambient CO 2 concentrations. High and low soil N treatments were created by adding sufficient (NH 4) 2SO 4 to an infertile soil mixture to establish total N concentrations of 500 μg g −1 and 400 μg g −1, respectively, while high and low P treatments consisted of 68 μg g −1 and 43 μg g −1 concentrations, respectively, of extractable P created by additions of CaHPO 4. All seedlings were inoculated with the mycobiont Pisolithus tinctorius (Pers.) Coker and Couch shortly after emergence. Three whole-seedling harvests at four month intervals permitted assessment of treatment effects on shoot and root growth and ectomycorrhizal development. Initially, 525 μl l −1 CO 2 and high N and P were all influential factors in above-ground growth, with each of these treatments increasing shoot weight while the latter increased height, diameter and volume. Stimulation of root growth was evident in dry weight and length measurements at the first harvest, with N and P main treatment effects again evident, but the response to elevated CO 2 was most pronounced in the 700 μl l −1 atmosphere. After eight months, soil P was of little consequence above- or below-ground, but high N increased shoot dimensions, volume, and weight and root weight and length. Furthermore, the 525 μl l −1 CO 2 treatment emerged as the dominant stimulatory atmosphere both above- and below-ground, as seedlings grown in intermediate CO 2 exhibited the largest shoot diameters, greatest shoot and root weights, and the longest root systems at the second harvest. At the final harvest, interactive effects of 525 μl l −1 CO 2 and high N were prominent, as this treatment combination produced the largest shoot dimensions, volume and weight, and the greatest root volume and coarse and fine root weights. Intermediate CO 2 also produced the longest root systems after 12 months. Shoot/root ratios were lowered by growth in 700 μl l −1 CO 2 after four months and by both enriched atmospheres after eight months, but this effect was no longer evident at the final harvest. Greater numbers of mycorrhizae were formed by seedlings grown in 700 μl l −1 CO 2 after four months and by those grown in 525 μl l −1 CO 2 after eight months. Both enriched atmospheres increased mycorrhizal counts after 12 months, and seedlings grown in high CO 2 and low N exhibited the highest percentage of total root system length colonized at the final harvest as well. Overall, these results indicate that CO 2 enrichment stimulates shoot and root growth of juvenile ponderosa pine, a response dependent on soil N rather than P availability, and that the magnitude of the growth increase is greater in 1.5× ambient than in 2× ambient CO 2.

Exponential fertilization increases nutrient uptake and ectomycorrhizal development of black spruce seedlings

Intensive fertilization may inhibit adequate mycorrhizal development for forest nursery stock production. Containerized black spruce (Picea mariana (Mill.) BSP) seedlings exposed to four fertilization regimes (one conventional and three exponential at 12.5, 12.5, 25, and 50 mg N/seedling, respectively) and two ectomycorrhizal inoculations (Hebeloma crustuliniforme (Bull. ex St-Amans) Quel. and Laccaria bicolor (R. Mre.) Orton) were grown from seed to assess mycorrhizal formation and nutrition of young trees under intensive greenhouse culture. Exponentially increasing fertilizer additions stimulated ectomycorrhizal development (49-85%) compared with conventional constant-rate fertilization (22-26%). Exponential fertilization also increased seedling N (13-34%) and P (5-18%) uptake, although dry matter production was reduced (17-25%) at the lowest exponential addition rate. Ectomycorrhizal inoculation did not affect seedling biomass but increased uptake of N (6-17%), P (5-20%), and K (4-18%), demonstrating a potential for nutrient loading by fungi. Higher ectomycorrhizal formation found under exponential fertilization regimes was attributed to lower initial nutrient levels maintained in the growing media. Results indicate that high exponential fertilization combined with fungal inoculation may be effective for producing both nutrient-loaded and ectomycorrhizally infected planting stock.

Atmospheric CO 2 enrichment and soil N fertility effects on juvenile ponderosa pine: Growth, ectomycorrhizal development, and xylem water potential

Interactive effects of elevated atmospheric CO 2 and soil N fertility on above- and below-ground growth, mycorrhizal colonization, and water relations of juvenile ponderosa pine ( Pinus ponderosa Dougl. ex Laws.) were investigated. One-year-old seedlings were planted in undisturbed field soil within open-top chambers which permitted creation of atmospheres with 700 μl l −1, 525 μl l −1, or ambient CO 2 concentrations. High and medium soil N treatments were imposed by incorporating sufficient (NH 4) 2SO 4 to increase total N by 200 μg g −1 and 100 μg g −1, respectively, while unamended soil, which had a total N concentration of approximately 900 μg g −1, constituted the low N treatment. Following each of two consecutive field growing seasons, whole seedlings of every combination of CO 2 and N treatment were harvested to permit assessment of shoot and root growth and quantification of ectomycorrhizal development. Late in the second growing season, a simulated drought episode was imposed by withholding irrigation during which predawn and midday xylem water potential and soil water potential were measured. The initial harvest revealed that coarse and fine root weights were increased by CO 2 enrichment during the first growing season. This result was most apparent in the 525 μl l −1 CO 2 treatment and high soil N, which produced the greatest root volume as well. Shoot/root ratio decreased with increasing CO 2 at the first harvest. After two growing seasons, elevated CO 2 increased seedling diameter, shoot and root volume, and shoot and coarse root weight, again most prominently in high N. Unlike the initial results, however, stimulation of seedling growth by the 700 μl l −1 CO 2 atmosphere exceeded that in 525 μl l −1 CO 2 after two growing seasons, and shoot/root ratio was unaffected by either CO 2 or N. At both harvests, seedlings grown in the enriched atmospheres generally had higher mycorrhizal counts and greater percentages of colonized root length, but differences among treatments in ectomycorrhizal development were nonsignficant regardless of quantification method. During the imposed drought episode, xylem water potential of seedlings grown in elevated CO 2 descended below that of seedlings grown in the ambient atmosphere as soil water potential decreased, most notably in the predawn measurements. These results suggest that CO 2 enrichment stimulates shoot and root growth of juvenile ponderosa pine under field conditions, a response somewhat dependent on soil N availability. However, below-ground growth is not increased proportionally more than that above ground, which may predispose this species to greater stress when soil water is limited.

Exponential fertilization increases nutrient uptake and ectomycorrhizal development of black spruce seedlings

Intensive fertilization may inhibit adequate mycorrhizal development for forest nursery stock production. Containerized black spruce (Picea mariana (Mill.) BSP) seedlings exposed to four fertilization regimes (one conventional and three exponential at 12.5, 12.5, 25, and 50 mg N/seedling, respectively) and two ectomycorrhizal inoculations (Hebeloma crustuliniforme (Bull. ex St-Amans) Quel. and Laccaria bicolor (R. Mre.) Orton) were grown from seed to assess mycorrhizal formation and nutrition of young trees under intensive greenhouse culture. Exponentially increasing fertilizer additions stimulated ectomycorrhizal development (49-85%) compared with conventional constant-rate fertilization (22-26%). Exponential fertilization also increased seedling N (13-34%) and P (5-18%) uptake, although dry matter production was reduced (17-25%) at the lowest exponential addition rate. Ectomycorrhizal inoculation did not affect seedling biomass but increased uptake of N (6-17%), P (5-20%), and K (4-18%), demonstrating a potential for nutrient loading by fungi. Higher ectomycorrhizal formation found under exponential fertilization regimes was attributed to lower initial nutrient levels maintained in the growing media. Results indicate that high exponential fertilization combined with fungal inoculation may be effective for producing both nutrient-loaded and ectomycorrhizally infected planting stock.

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria spp. with Eucalyptus diversicolor seedlings. Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L. bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2–3 months).

Root Response of Mature Live Oaks in Coastal South Carolina to Root Zone Inoculations with Ectomycorrhizal Fungal Inoculants

New fine root growth and ectomycorrhizal development were determined for mature live oak {Quercus virginiana) growing in a stressed landscape after soil injection of ectomycorrhizal fungal {Pisolithus tinctorius Pt) spores with or without fertilizer (12-48-8) or after vertical mulch application of a mix of Pt spores, soil-enriching bacteria, various organics, and water-managing gels. After 6 months, the injected spores stimulated more ectomycorrhizal development than the fertilizer or water-only control treatments. When spores and fertilizer were applied together, they increased by four times the fine root biomass and by three times the ectomycorrhizal development than observed in the water-only control. The estimated relative root absorbing potential of the roots was 14 times that of the control. After 4 or 6 months, the vertical mulch treatment stimulated six times more fine roots and twice the ectomycorrhizal development found in the control treatment. These differences reflected an 8 to 12 fold increase in the relative root absorbing potential of the fine roots and ectomycorrhizae. In both studies, Pt ectomycorrhizae accounted for over half of the ectomycorrhizal development. Natural ectomycorrhizal development ranged from 18 to 38 % of fine roots in control treatments. The results demonstrated that Pt spore and bacterial inoculants can be successfully introduced into the root system of mature live oaks in a stressed environment, increase fine root growth, and form abundant ectomycorrhizae.

Ectomycorrhizal root growth in scots pine stands in response to manipulation of litter and humus layers

The effect on ectomycorrhizal root growth in a nitrogen-enriched planted stand of Scots pine (Pinus sylvestris L.) on podzolic sandy soil to manipulation of litter and humus layers (removal, doubling and control treatments) was examined, and compared to ectomycorrhizal root growth in an untreated naturally established Scots pine stand on nutrient-poor non-podzolic sandy soil. Half a year after manipulation of litter and humus layers in the planted stand, ingrowth-cores to a depth of 60 cm were installed in both stands. Scots pine roots were sampled four times during two growing seasons. Ectomycorrhizal roots were found at all sampled soil depths to 60 cm in all plots. Root growth and ectomycorrhizal development were greater in the naturally established stand than in all plots in the planted stand. Numbers of ectomycorrhizal root tips in the litter and humus removal plots were generally higher than in the control plots in the planted stand until May 1992. Doubling litter and humus did not significantly affect root length or the numbers of ectomycorrhizal root tips. The N dissolved , NH4 + and NO3 – concentrations and the organic matter content in the upper 5 cm of the mineral soil in the planted stand on podzolic sandy soil were generally higher and the pH significantly lower than in the naturally established stand on non-podzolic sandy soil. Root growth and ectomycorrhizal development in the secondary stand may have been negatively affected by the chemical composition of the podzolic sandy soil.

Fungal adhesion pad formation and penetration of root cuticle in early stage mycorrhizas ofPicea abies andLaccaria amethystea

Primary events during the establishment of the fungus-root symbiosis in ectomycorrhizas are still little understood. No attention has been paid so far to the adhesion of hyphae to the root cuticle and penetration of this barrier, although the importance of the cuticle has been shown for pathogen-plant interactions. Early developmental stages of in vitro mycorrhization ofLaccaria amethystea onPicea abies after short periods of incubation in growth chambers under elevated CO2 concentrations were studied by light and transmission electron microscopy. No structural changes in mycorrhization related to elevated CO2 were found, but fine roots and mycorrhizas developed faster. Adhesion pad formation was observed at hyphal tips in contact with the root cuticle. The adhesion pad was connected to the outer cell wall layer of the hypha and reacted positively to the Swift reaction for cysteine rich proteins. Although the reaction cannot be considered as totally specific, findings are discussed in respect to hydrophobins, which have recently been found to be expressed during early steps in ectomycorrhizal development. The root cuticle was dissolved and penetrated by fungal tips of the fingerlike branching mycelium attached to the root surface. The findings are compared with well documented pathogenic fungus-plant interactions at the cuticle. The possibility of restriction of hyphal attack to that part of the cuticle covering cell junctions is discussed.

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.

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

SUMMARYThe early development of indigenous and introduced Scots pine (Pinus sylvestris L.) ectomycorrhiza in natural forest humus was examined using molecular fingerprinting techniques. Non‐mycorrhizal or mycorrhizal (Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) Fr.) seedlings were placed in transparent two‐dimensional microcosms on a thin layer of sieved humus to allow observation of ectomycorrhizal development and fungal growth. Twelve ectomycorrhizal morphotypes, mainly based on colour, gross morphology and outer mantle structure, were identified over a 3‐month period. No successional trends in mycorrhiza formation were observed. For further characterization, individual ectomycorrhiza representing the different morphotypes were subjected to analyses of esterase (EST) isozyme profiles and RFLPs of the internal transcribed spacer (ITS) from fungal rRNA genes following amplification using PCR. All 10 morphotypes analysed displayed different esterase isozyme profiles, and the characteristic s. bovinus species diagnostic band (S. b. EST8) was detected in two white morphotypes. Ten Pink and Black ectomycorrhiza were all separated into one and three groups, respectively. A fast‐running plant‐specific polymorphic locus (Z) was also confirmed in most ectomycorrhizal morphotypes. Diagnostic species‐specific (S. bovinus and Pink) EST bands were detected in intact external mycelium colonizing soil. Successful amplification of the ITS from individual ectomycorrhiza, of eight different morphotypes, was found to be mainly influenced by the DNA template concentration. Different RFLPs (Hinf I, Mbo I and Hha I) of the ITS placed the white morphotypes into two groups, one corresponding to S. bovinus, in agreement with the esterase fingerprinting. By contrast, Pink and Beige morphotypes displayed different EST profiles but nearly identical RFLP fingerprints. The ITS amplification success rate of standardized DNA template concentrations from 10 individual Pink and Black ectomycorrhiza was 90 and 50%, respectively. In all successful amplifications, the black morphotype yielded the smallest ITS fragment, similar to earlier reports for Cenococcum geophilum, that gave identical RFLPs. The ecological significance of the observed mycorrhizal diversity and combined application of these two identification methods is discussed.

Site preparation alters soil distribution of roots and ectomycorrhizae on outplanted western white pine and Douglas-fir

This report documents root and ectomycorrhizal development on container-produced (1-0), outplanted, western white pine and Douglas-fir seedlings growing in site-prepared forest soils typical of the Inland Northwestern US. The following site preparations were used: 1) mounding organic and surface mineral horizons; 2) mounding with subsequent physical removal or chemical control of competing vegetation; 3) scalping to reduce competing vegetation; and, 4) a control or no post-harvest disturbance. Treatments were applied on relatively harsh and moderate sites in northern Idaho. Most ectomycorrhizae on the seedling population were found in the mineral substrates that dominated planting sites. However, compared to mineral substrates, highest seedling ectomycorrhizal tip counts were recorded in organic matter, particularly decayed wood or mixtures containing decayed wood. Strong ectomycorrhizal development was characteristic of western white pine. It supported highest ectomycorrhizal activity in organic substrates on the harshest treatments (scalps). Douglas-fir showed even stronger relative increases of ectomycorrhizae in organic substrates on harsh treatments. Three of the four common ectomycorrhizal morphological types were concentrated in mineral substrates with all treatments. A treatment-induced change of behavior was shown by the principal pine type. It occurred at highest numbers in organic substrates of the mound with competing vegetation treatment and in mineral substrates with the control. If relative availability to seedling roots was considered, organics (especially decomposed wood) were generally equal or superior to mineral substrates for supporting ectomycorrhizal activity on planted seedlings.

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.

Site preparation alters biomass, root and ectomycorrhizal development of outplanted western white pine and Douglas-fir

This report documents impacts from manipulations of native forest soils and competing vegetation on development and ectomycorrhizal formation of outplanted western white pine and Douglas-fir in the Inland Northwest. Treatments were: 1) mounding surface horizons with competition left in place, 2) mounding surface horizons with subsequent physical or chemical control of competing vegetation, 3) scalping for control of competing vegetation, and 4) a control, or no postharvest disturbance. Treatments were applied on a low-altitude, relatively harsh site and on a higher altitude, more moderate site. Wich established seedlings, mounding with no competition control generally produced small seedlings (5–15g; both species) with low numbers of ectomycorrhizal short roots (21). Mounding with competition control produced large seedlings (20–48g) with moderate numbers of short roots (25). Scalping produced small seedlings (8–16g) with high numbers of short roots (41). The control also produced small seedlings (8–13g) but with a moderate number of short roots (27). Douglas-fir produced more short roots on the harsh than the moderate site. Western white pine produced high root to shoot ratios (above 0.60) in the mound with competition on the harsh site. Douglas-fir produced very high root to shoot ratios (above 1.0) in both the mound with no competition of the harsh site and in the mound with competition of the moderate site. Throughout, western white pine produced more ectomycorrhizae more rapidly than Douglas-fir. Ectomycorrhizal development and root and shoot weight of both conifers were adversely affected by competing vegetation.

Changes in protein biosynthesis during the differentiation ofPisolithus–Eucalyptus grandisectomycorrhiza

Protein biosynthesis in Pisolithus – Eucalyptus grandis ectomycorrhiza was related to the stage of ectomycorrhizal development using two-dimensional polyacrylamide gel electrophoresis of proteins labelled by in vivo incorporation of35S radiolabelled amino acids. Nineteen-day-old seedlings were radiolabelled and the primary root was divided into 1-cm segments. With increasing distance from the tip of the primary root, the lateral roots developed as follows: segment 1, no lateral tips; segment 2, three lateral tips, 1–4 days old; segment 3, five lateral tips, 3–8 days old; segment 4, five lateral tips, 7–12 days old. Six-day-old ectomycorrhizas were fully formed with a mantle and Hartig net. During ectomycorrhizal development, there was a decrease in all plant proteins and differential accumulation of fungal proteins. The apical segment of the primary root had a biosynthesis profile very similar to that of noninoculated roots. By contrast, the other segments of the primary root, with attached lateral roots, had biosynthesis profiles that were similar to those of the free-living hyphae. Thus, plant biosynthesis was shown to be predominantly associated with the primary root meristem. The domination of the fungal partner in the protein biosynthesis of developing ectomycorrhiza is probably a consequence of stimulated fungal growth and the corresponding decrease in plant meristematic activity. Ectomycorrhizal development was associated with a differential accumulation of fungal polypeptides and the appearance of a group of symbiosis-related acid fungal polypeptides between 27 and 37 kDa. As the polypeptides were present in a similar magnitude throughout ectomycorrhizal development (lateral tips 1–12 days old), it is suggested that they function as structural proteins associated with mantle formation. Keywords: ectomycorrhizal development, Eucalyptus, Pisolithus, protein biosynthesis, symbiosis-related polypeptides.

Ectomycorrhizal development on Scots pine (Pinus sylvestris L.) seedlings in different soils

In the present study ectomycorrhizal development of Laccaria bicolor, Rhizopogon luteolus and Suillus bovinus associated with Scots pine (Pinus sylvestris) seedings was studied as affected by primary stand humus, secondary stand humus, podsolic sandy soil or peat in perspex growth chambers. After 9 weeks, ectomycorrhizal development with S. bovinus was significantly greater in peat and primary stand humus than in secondary stand humus or podsolic sandy soil. Ectomycorrhizal development with R. luteolus in secondary stand humus was higher than in primary stand humus. Degree of ectomycorrhizal development of L. bicolor, R. lutuelus and S. bovinus on Scots pine was related to potassium concentration, organic matter content and pH of the soils suggesting that chemical composition of the soils affects ectomycorrhizal development.

The effect of soil desiccation on the nutrient status of Eucalyptus regnans F. Muell seedlings

The poor growth of young Eucalyptus regnans seedlings in undried soil from the mature forest of E. regnans can be overcome by previously air-drying the soil or by adding sufficient amounts of complete soluble fertilizer or equivalent concentrations of P (as NaH2PO4) and N (as NaNO3). A factorial pot experiment in which phosphate and nitrate were added to undried soil indicated that P was the primary deficiency for young seedlings and that response to N did not occur until this lack was satisfied. In dried soil, seedlings also responded to additions of complete fertilizer but most of this effect was due to N rather than P. Field trials in the mature forest also indicated greater growth in dried soil than undried soil and confirmed a response of young seedlings to superphosphate. In pot experiments, the concentration of P and N per g plant dry weight after four months was relatively constant irrespective of the final size of the plant. Seedlings in dried soil extracted up to 15 times more P than did those grown in undried soil. In general, chemical analysis of soil indicated more extractable P and N from dried soil although this was not always consistently so. Soil desiccation resulted in an increase in soil surface area due to the fragmentation of larger peds and to an increase in the number of microfractures which remained in the soil crumbs after rewetting. Mycorrhiza are likely to be important since the differentiation of the growth response of seedlings in dried and undried soil, which occurred at 5–6 weeks, corresponded with the establishment of full ectomycorrhizal development (80% root tips). The factors concerned with the increase in fertility after air-drying are discussed.

Growth and ectomycorrhizal formation of container-grown Douglas-fir seedlings inoculated with Laccariabicolor under four levels of nitrogen fertilization

Container-grown Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco) seedlings were inoculated at the time of sowing with a Laccariabicolor (Maire) Orton mycelial suspension produced in a fermentor. They were grown in a peat moss–vermiculite substrate under four levels of N fertilization (7.2, 14.4, 21.6, and 28.7 mg/seedling per season (N1, N2, N3, and N4, respectively)) to determine the N level suitable for both ectomycorrhizal development and seedling growth. After 18 weeks in the greenhouse, seedlings inoculated with L. bicolor had 44%, 32%, 44%, and 5% of their short roots mycorrhizal when fertilized with N1, N2, N3, and N4, respectively. Only when they were fertilized with N4 did the L. bicolor seedlings have significantly greater shoot height than the controls. For the other growth parameters, they were not significantly different from control seedlings for any of the N levels. After 18 weeks, regardless of the level of N, seedlings inoculated with L. bicolor had significantly lower N concentrations (%) and contents (mg/seedling) than the uninoculated ones. Consequently, for the same production of biomass, the mycorrhizal seedlings had taken up less N than the nonmycorrhizal ones. The efficiency of applied N, expressed in terms of produced biomass, decreased when the N fertilization increased; mycorrhizal and nonmycorrhizal seedlings did not tend to be different. The efficiency of the absorbed N also decreased with the level of applied N, but less rapidly, and tended to be greater for the mycorrhizal seedlings than for the nonmycorrhizal ones. Therefore, the mycorrhizal infection improved the utilization of the absorbed N. N3 was the best of the four N levels used, since it was the only one that maximized both the ectomycorrhizal formation and the growth of the seedlings. In other words, a total seedling N concentration of 1.6% and a substrate fertility of 52 ppm N are appropriate to optimize both the ectomycorrhizal development and the growth of Douglas-fir seedlings.