Development and function of Pisolithus and Scleroderma ectomycorrhizas formed in vivo with Allocasuarina,Casuarina and Eucalyptus

The effect of inoculating seedlings of Eucalyptus grandis, Allocasuarina littoralis and Casuarina equisetifolia with two isolates of Pisolithus and two isolates of Scleroderma from under eucalypts was examined in a glasshouse trial. Ectomycorrhizas formed extensively on Eucalyptus (23–46% fine roots ectomycorrhizal) and Allocasuarina (18–51% fine roots ectomycorrhizal). On Casuarina, the fungi were either unable to colonize the rhizosphere (one isolate of Pisolithus), or sheathed roots, resembling ectomycorrhizas, formed on 1–2% of the fine roots. Colonization of roots by one isolate of Scleroderma resulted in the death of Casuarina seedlings. Inoculation with fungi increased shoot dry weight by up to a factor of 32 (Eucalyptus), 4 (Allocasuarina) and 3 (Casuarina). Ectomycorrhizas formed in associations with Eucalyptus and Allocasuarina had fully differentiated mantles and Hartig nets in which the host and fungal cells were linked by an extensive fibrillar matrix. Sheathed roots in Casuarina lacked a Hartig net, and the epidermis showed a hypersensitive reaction resulting in wall thickening and cell death. The sheaths are described as mantles since the density and arrangement of the hyphae in the sheaths was similar to that in mantles of the eucalypt ectomycorrhizas. The intercellular carbohydrate matrix was not produced in the Casuarina mantle in association with Pisolithus, hence the mantle was not cemented to the root. These structures differ from poorly compatible associations described previously for Pisolithus and Eucalyptus. The anatomical data indicate that ectomycorrhizal assessment based on surface morphological features may be misleading in ecological studies because compatible and incompatible associations may not be distinguishable.     

The ability of 16 ectomycorrhizal fungi to increase growth and phosphorus uptake of Eucalyptus globulus Labill. and E. diversicolor F. Muell.

The effectiveness of 16 fungal isolates in forming ectomycorrhizas and increasing the growth and phosphorus uptake of Eucalyptus globulus Labill. and E. diversicolor F. Muell. seedlings was examined in the glasshouse. Seedlings were grown in yellow sand at 2 phosphorus levels (4 and 12 mg P kg^-1 sand). At the time of harvest (100 days), the non-inoculated seedlings and seedlings inoculated with Paxillus muelleri (Berk.) Sacc. and Cortinarius globuliformis Bougher had a low level of contamination from an unknown mycorrhizal fungi. Seedlings inoculated with Thaxterogaster sp. nov. and Hysterangium inflatum Rodway had developed mycorrhizas of the superficial type whereas Hydnangium carneum Wallr. in Dietr., Hymenogaster viscidus Massee & Rodway, Hymenogaster zeylanicus Petch, Setchelliogaster sp. nov., Laccaria laccata (Scop. ex. Fr.) Berk., Scleroderma verrucosum (Vaillant) Pers., Amanita xanthocephala (Berk.) Reid & Hilton, Descolea maculata Bougher and Malajczuk and Pisolithus tinctorius (Pers.) Coker & Couch formed typical pyramidal ectomycorrhizas. The dry weight of non-inoculated and inoculated E. globulus seedlings at 12 mg P kg^-1 sand did not differ, whereas several isolates caused growth depression of E. diversicolor. By contrast, at 4 mg P kg^-1 sand growth increases ranged from 0–13 times above that of non-inoculated seedlings. P. tinctorius produced the largest growth increase on both eucalypt species. In general, isolates which developed more extensive mycorrhizas on roots produced the largest growth responses to inoculation. Isolates which increased plant growth also increased phosphorus uptake by the plant. Seedlings inoculated with L. laccata and S. verrucosum retained more phosphorus in their roots than plants inoculated with the other fungal isolates.     

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     

The effect of soil pH on the ability of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill.

We examined the effect of two levels of soil pH (5 and 6) on the ability (effectiveness) of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill. at a deficient supply of P. Plants were inoculated with one of six fungal isolates [Laccaria laccata (Scop. ex Fr.) Berk. and Br. (isolates A and B), Pisolithus tinctorius (Pers.) Coker and Couch (isolates A and B), Descolea maculata Bough. and Mal. and Setchelliogaster sp. nov.] and were grown in a P-deficient sand, in pots, in a temperature-controlled glasshouse. Seedlings were harvested 89 days after planting and were assessed for dry matter production, tissue P concentrations, ectomycorrhizal colonization of roots and hyphal development in soil.Uninoculated plants had less than 5% of their fine root length colonized by ectomycorrhizal fungi. In contrast, inoculated plants had 30% or greater of their fine root length ectomycorrhizal. Inoculation increased the uptake of P and growth of plants for all isolates and at both levels of soil pH, although growth responses to inoculation were greater at pH 6, particularly for the two L. laccata isolates. Isolates which colonized roots most extensively increased plant growth to the greatest extent. D. maculata was the most effective fungal isolate at pH 5, and both D. maculata and L. laccata A were most effective at pH 6. The effects of soil pH on plant growth were also related to some extent to the effects of soil pH on colonized root length. Growth responses to inoculation were related less well to hyphal development in soil. The L. laccata isolates formed more hyphae in soil (on a per pot, per m of fine root, and per m of colonized fine root basis) than other fungal isolates, but were not always more effective in increasing plant grown.     

The survival and development of inoculant ectomycorrhizal fungi on roots of outplanted Eucalyptus globulus Labill

The survival and development of two inoculant ectomycorrhizal fungi (Hebeloma westraliense Bough. Tom. and Mal. and Setchelliogaster sp. nov.) on roots of outplanted Eucalyptus globulus Labill. was examined at two expasture field sites in the south-west of Western Australia. Site 1 was a gravelly yellow duplex soil, and Site 2 was a yellow sandy earth. Plants were grown in steamed or unsteamed soil, in root bags designed as field containers for young growing trees. Three, 6 and 12 months after outplanting, plants were removed from these bags and assessed for dry weights of shoots and ectomycorrhizal colonization of roots.The inoculant ectomycorrhizal fungi (identified on the basis of the colour and morphology of their mycorrhizas) survived on roots of E. globulus for at least 12 months after outplanting at both field sites. At Site 1, however, colonization of new fine roots by the inoculant fungi was low (less than 20% of fine root length). Inoculation had no effect on the growth of E. globulus at this site. In contrast, at Site 2 the inoculant ectomycorrhizal fungi colonized up to 30–50% of new fine root length during the first 6 months after outplanting. There was a corresponding growth response to ectomycorrhizal inoculation at this site, with a close relationship (r²=0.82^**) between plant growth at 12 months and root colonization at 3 months. Plant growth at 12 months was related less closely with root colonization at 6 or 12 months. Root colonization by resident ectomycorrhizal fungi increased with time at both field sites. At Site 2, this increase appeared to be at the expense of colonization by the inoculant fungi, which was reduced to less than 10% of fine root length at 12 months. Steaming the soil had little effect on colonization by the inoculant ectomycorrhizal fungi at either field site, but decreased colonization by the resident ectomycorrhizal fungi.     

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).     

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     

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     

Soil fumigation and phosphorus supply affect the formation of Pisolithus-Eucalyptus urophylla ectomycorrhizas in two acid Philippine soils

To examine the effects of microbial populations and external phosphorus supply of two Philippine soils on mycorrhizal formation, Eucalyptus urophylla seedlings were inoculated with two Pisolithus isolates and grown in fumigated, reinfested and unfumigated soil fertilized with four rates of phosphorus. The Pisolithus isolates used were collected from under eucalypts in Australia and in the Philippines. Soils were infertile acid silty loams collected from field sites in Pangasinan, Luzon and Surigao, Mindanao.Significant interaction was observed between inoculation, soil fumigation and phosphorus supply on mycorrhizal formation by the Australian isolate in Surigao soil but not in Pangasinan soil. Soil fumigation enhanced mycorrhizal formation by the Australian isolate but did not affect root colonization by the Philippine isolate. Root colonization by the Australian isolate was highest in the reinfested soil while for the Philippine isolate it was highest in the unfumigated soil. The Australian isolate was more effective than the Philippine isolate in promoting growth and P uptake of E. urophylla seedlings in both soils. Total dry weight and P uptake of E. urophylla seedlings inoculated with the Australian isolate were maximum in fumigated and in the reinfested Pangasinan and Surigao soils supplied with 8 mg P kg^-1 soil. In the unfumigated soil, growth of seedlings inoculated with the Australian isolate was significantly reduced. Seedlings inoculated with the Philippine isolate had the largest dry weights and P contents in unfumigated Pangasinan and Surigao soils supplied with 8 mg P kg^-1 soil.These results indicate that the performance of the Australian Pisolithus isolate was markedly affected by biological factors in unfumigated soil. Thus, its potential use in the Philippines needs to be thoroughly tested in a variety of unfumigated soils before its widespread use in any inoculation programme.     

Inhibition of intestinal uptake of amino acids by unconjugated bile salt.

The unconjugated bile salt, sodium deoxycholate, at a concentration of 0.5 mM was shown to inhibit the intestinal uptake of the amino acids L-glycine, L-leucine, L-proline, L-lysine and L-tyrosine in rats in vitro. This effect was acutely reversible except for the basis amino acid L-lysine and is therefore not simply due to tissue damage. These results, and the recent finding that sodium deoxycholate inhibits intestinal absorption of amino acids in vivo, suggest that impaired intestinal amino acid transport may contribute to hypoproteinaemia in patients with bacterial overgrowth in the upper small intestine in whom deoxycholate is present in the small intestinal lumen in excessive concentrations.