Phosphate supply and arsenate toxicity in ectomycorrhizal fungi

Three species of ectomycorrhizal fungi (Hebeloma crustuliniforme, Suillus variegatus and Cenococcum geophilum) were grown in axenic culture amended with range of AsO43– concentration under three different PO43– regimes. The fungi exhibited different growth responses to AsO43– that varied with PO43– concentration. Suillus variegatus showed the greatest sensitivity to AsO43–, with growth almost completely inhibited in the presence of AsO43– under the lower two PO43– treatments. Under the highest PO43– treatment however, growth was enhanced and S. variegatus was able to persist at AsO43– concentrations of up to 4 mM. Hebeloma crustuliniforme also showed high sensitivity to AsO43– especially at low PO43– concentration. The two higher PO43– treatments had an ameliorating effect on AsO43– toxicity in H. crustuliniforme. This demonstrates the ability of PO43– to alleviate AsO43– toxicity. The response from S. variegatus and H. crustuliniforme, both basidiomycetes, was in contrast to the ascomycete C. geophilum. This fungus demonstrated tolerance to AsO43– when grown in culture solution and PO43– did not have an ameliorating effect on AsO43– toxicity in C. geophilum.

Ectomycorrhizal symbiosis can enhance plant nutrition through improved access to discrete organic nutrient patches of high resource quality

It is known that roots can respond to patches of fertility; however, root proliferation is often too slow to exploit resources fully, and organic nutrient patches may be broken down and leached, immobilized or chemically fixed before they are invaded by the root system. The ability of fungal hyphae to exploit resource patches is far greater than that of roots due to their innate physiological and morphological plasticity, which allows comprehensive exploration and rapid colonization of resource patches in soils. The fungal symbionts of ectomycorrhizal plants excrete significant quantities of enzymes such as chitinases, phosphatases and proteases. These might allow the organic residue to be tapped directly for nutrients such as N and P. Pot experiments conducted with nutrient-stressed ectomycorrhizal and control willow plants showed that when high quality organic nutrient patches were added, they were colonized rapidly by the ectomycorrhizal mycelium. These established willows (0.5 m tall) were colonized by Hebeloma syrjense P. Karst. for 1 year prior to nutrient patch addition. Within days after patch addition, colour changes in the leaves of the mycorrhizal plants (reflecting improved nutrition) were apparent, and after I month the concentration of N and P in the foliage of mycorrhizal plants was significantly greater than that in non-mycorrhizal plants subject to the same nutrient addition. It seems likely that the mycorrhizal plants were able to compete effectively with the wider soil microbiota and tap directly into the high quality organic resource patch via their extra-radical mycelium. We hypothesize that ectomycorrhizal plants may reclaim some of the N and P invested in seed production by direct recycling from failed seeds in the soil. The rapid exploitation of similar discrete, transient, high-quality nutrient patches may have led to underestimations when determining the nutritional benefits of ectomycorrhizal colonization.

Low-temperature-induced changes in trehalose, mannitol and arabitol associated with enhanced tolerance to freezing in ectomycorrhizal basidiomycetes ( Hebeloma spp.)

Ectomycorrhizal fungi have been shown to survive sub-zero temperatures in axenic culture and in the field. However, the physiological basis for resistance to freezing is poorly understood. In order to survive freezing, mycelia must synthesise compounds that pro-tect the cells from frost damage, and certain fungal-spe-cific soluble carbohydrates have been implicated in this role. Tissue concentrations of arabitol, mannitol and trehalose were measured in axenic cultures of eight Hebeloma strains of arctic and temperate origin grown at 22, 12, 6 and 2°C. In a separate experiment, mycelia were frozen to –5°C after pre-conditioning at either 2°C or 22°C. For some, especially temperate strains, there was a clear increase in specific soluble carbohydrates at lower growth temperatures. Trehalose and mannitol were present in all strains and the highest concentrations (close to 2.5% and 0.5% dry wt.) were recorded only after a cold period. Arabitol was found in four strains only when grown at low temperature. Cold pre-condi-tioning enhanced recovery of mycelia following freez-ing. In four out of eight strains, this was paralleled by increases in mannitol and trehalose concentration at low temperature that presumably contribute towards cryopro-tection. The results are discussed in an ecological con-text with regard to mycelial overwintering in soil.

Temperature regulation of extracellular proteases in ectomycorrhizal fungi (Hebeloma spp.) grown in axenic culture

Strains of Hebeloma representative of different climatic zones were grown in axenic culture at either 2 °C and 22° or 6° and 22°. Culture filtrates were assayed for proteolytic activity using FITC labelled BSA as a substrate. Assays were run between 0–37°. Growth at low temperature induced greater proteolytic activity (g−1 D.W. mycelium). Many of the strains produced protease(s) which retained significant activity at temperatures as low as 0°, and a thermal optimum between 0–6° with a second optimum at higher temperature. The results are discussed in relation the nutrient acquisition potential of ectomycorrhizal fungi at low temperature and the contribution such cold active proteases might make to the soil enzyme pool.

The effect of temperature and inorganic phosphorus supply on growth and acid phosphatase production in arctic and temperate strains of ectomycorrhizal Hebeloma spp. in axenic culture

Acid phosphatase production by 12 Hebeloma strains was usually derepressed when inorganic phosphorus in the growth medium was limited, but appeared to be constitutive in some strains. At low temperatures (≤ 12°) arctic strains produced more extracellular and wall-bound acid phosphatase, yet grew more slowly than the temperate strains. We suggest that low growth rates in arctic strains may be a physiological response to cold whereby resources are diverted into carbohydrate accumulation for cryoprotection. At near freezing temperatures, increased extracellular phosphatase production may compensate for a loss of enzyme activity at low temperature and serve to hydrolyse organic phosphorus in frozen soil over winter.

Utilization of organic nitrogen by ectomycorrhizal fungi (Hebeloma spp.) of arctic and temperate origin

Arctic and temperate strains of Hebeloma spp. were grown in axenic culture on glutamic acid, alanine, lysine and NH4+ as sole sources of nitrogen (N), with excess carbon (C) or deficient C (supplied as glucose). Their ability to utilize seed protein as a natural N source was also assessed. All strains tested had the capacity to assimilate amino acids and generally utilized alanine and glutamic acid more readily than NH4+. Some strains were able to utilize amino C when starved of glucose C, and could mineralize amino-N to NH3-N. Arctic strains, in particular, appeared to be pre-adapted to the utilization of seed protein N and glutamic acid N, which is often liberated in high concentrations after soil freezing. The results are discussed in relation to their possible ecological importance.

Induction of cold active acid phosphomonoesterase activity at low temperature in psychrotrophic ectomycorrhizal Hebeloma spp.

Hebeloma strains of arctic and temperature origin, grown at 22° or 6°, were assayed for wall-bound and extracellular acid phosphomonoesterase (pNPPase) across a temperature range 2-37°. Only when grown at 6° was a cold active extracellular pNPPase induced in all the arctic strains and most of the temperature strains tested. Such enzymes are suggested to be a adaptation to low soil temperatures, and are discussed in the context of ectomycorrhizal access to soil PO4− monoesters at low temperature.

Long-term storage of ectomycorrhizal basidiomycetes (Hebeloma spp.) at low temperature

A method for maintaining viable cultures of ectomycorrhizal Hebeloma strains in cold liquid culture medium is described. Isolates of Hebeloma spp., collected over a wide geographic range, were stored at 2 °C for a period of three years. All cultures survived this storage period, a greater time period and success rate than has previously been reported for the long term storage of ectomycorrhizal basidiomycetes. The method may prove useful for long-term storage of other basidiomycete genera.

Comparative growth of ectomycorrhizal fungi (Hebeloma spp.) on organic and inorganic nitrogen

In the largely organic soils in which ectomycorrhizas are commonly found, a preference for absorbing organic nitrogen over mineral forms is likely to be an advantage, especially where mineralisation rates are low. To determine rates of both independent and preferential growth of ectomycorrhizal basidiomycetes on organic and inorganic nitrogen, strains of Hebeloma were grown on nutrient agar media containing either NH4+ or glutamic acid as the sole source of nitrogen, on both single medium and split plate Petri dishes. Growth rates on the split plate Petri dishes, where the fungi had access to both nitrogen sources, were generally greater than on the single medium dishes. Growth on glutamic acid was at least equal to, and usually greater than, that on NH4+. In some cases growth on NH4+ alone appeared severely inhibited, a condition that was partially alleviated by access to glutamic acid on the split plates Petri dishes. This highlights a potential pitfall of single nitrogen source growth studies. The greater growth of most strains on glutamic acid suggests an adaptation to organic nitrogen utilisation in these strains. If this is so in soils with low mineralisation rates, direct uptake of amino acids by ectomycorrhizal plants could by-pass the bottle neck that requires mineral nitrogen to be made available for plant uptake.

Global patterns of plant root colonization intensity by mycorrhizal fungi explained by climate and soil chemistry

Aim Most vascular plants on Earth form mycorrhizae, a symbiotic relationship between plants and fungi. Despite the broad recognition of the importance of mycorrhizae for global carbon and nutrient cycling, we do not know how soil and climate variables relate to the intensity of colonization of plant roots by mycorrhizal fungi. Here we quantify the global patterns of these relationships. Location Global. Methods Data on plant root colonization intensities by the two dominant types of mycorrhizal fungi world-wide, arbuscular (4887 plant species in 233 sites) and ectomycorrhizal fungi (125 plant species in 92 sites), were compiled from published studies. Data for climatic and soil factors were extracted from global datasets. For a given mycorrhizal type, we calculated at each site the mean root colonization intensity by mycorrhizal fungi across all potentially mycorrhizal plant species found at the site, and subjected these data to generalized additive model regression analysis with environmental factors as predictor variables. Results We show for the first time that at the global scale the intensity of plant root colonization by arbuscular mycorrhizal fungi strongly relates to warm-season temperature, frost periods and soil carbon-to-nitrogen ratio, and is highest at sites featuring continental climates with mild summers and a high availability of soil nitrogen. In contrast, the intensity of ectomycorrhizal infection in plant roots is related to soil acidity, soil carbon-to-nitrogen ratio and seasonality of precipitation, and is highest at sites with acidic soils and relatively constant precipitation levels. Main conclusions We provide the first quantitative global maps of intensity of mycorrhizal colonization based on environmental drivers, and suggest that environmental changes will affect distinct types of mycorrhizae differently. Future analyses of the potential effects of environmental change on global carbon and nutrient cycling via mycorrhizal pathways will need to take into account the relationships discovered in this study.

Ecto- and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings

In common with many plants native to low P soils, jarrah (Eucalyptus marginata) develops toxicity symptoms upon exposure to elevated phosphorus (P). Jarrah plants can establish arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations, along with a non-colonizing symbiosis described recently. AM colonization is known to influence the pattern of expression of genes required for P uptake of host plants and our aim was to investigate this phenomenon in relation to P sensitivity. Therefore, we examined the effect on hosts of the presence of AM and ECM fungi in combination with toxic pulses of P and assessed possible correlations between the induced tolerance and the shoot P concentration. The P transport dynamics of AM (Rhizophagus irregularis and Scutellospora calospora), ECM (Scleroderma sp.), non-colonizing symbiosis (Austroboletus occidentalis), dual mycorrhizal (R. irregularis and Scleroderma sp.), and non-mycorrhizal (NM) seedlings were monitored following two pulses of P. The ECM and A. occidentalis associations significantly enhanced the shoot P content of jarrah plants growing under P-deficient conditions. In addition, S. calospora, A. occidentalis, and Scleroderma sp. all stimulated plant growth significantly. All inoculated plants had significantly lower phytotoxicity symptoms compared to NM controls 7 days after addition of an elevated P dose (30 mg P kg−1 soil). Following exposure to toxicity-inducing levels of P, the shoot P concentration was significantly lower in R. irregularis-inoculated and dually inoculated plants compared to NM controls. Although all inoculated plants had reduced toxicity symptoms and there was a positive linear relationship between rank and shoot P concentration, the protective effect was not necessarily explained by the type of fungal association or the extent of mycorrhizal colonization.