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.