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 protect the cells from frost damage, and certain fungal-specific 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 degrees C. In a separate experiment, mycelia were frozen to -5 degrees C after pre-conditioning at either 2 degrees C or 22 degrees 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-conditioning enhanced recovery of mycelia following freezing. In four out of eight strains, this was paralleled by increases in mannitol and trehalose concentration at low temperature that presumably contribute towards cryoprotection. The results are discussed in an ecological context with regard to mycelial overwintering in soil.

Laccases and other polyphenol oxidases in ecto- and ericoid mycorrhizal fungi.

Polyphenol oxidases are known to be produced by a range of ectomycorrhizal (ECM) and ericoid mycorrhizal fungi. These enzymes include laccase (EC 1.10.3.2), catechol oxidase (EC 1.10.3.1) and tyrosinase (EC 1.14.18.1), between which there exists considerable overlap in substrate affinities. In this review we consider the nature and function of these enzymes, along with the difficulties associated with assigning precise enzymatic descriptions. The evidence for production of laccase and other polyphenol oxidases by ECM and ericoid mycorrhizal fungi is critically assessed and their potential significance to the mycorrhizal symbioses discussed.

Arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae.

• Differential resistance to arsenate (AsO4 3- ) is demonstrated here among populations of the ericoid mycorrhizal fungus Hymenoscyphus ericae isolated from Calluna vulgaris in natural heathland soils and soils contaminated with AsO4 3- . • Isolates (c. 25) of the fungus from each of two As and Cu mine sites, and a natural heathland site, were screened for AsO4 3- and Cu2+ resistance by growing isolates in media containing a range of AsO4 3- and Cu2+ concentrations. • H. ericae populations from the mine sites demonstrated resistance to AsO4 3- compared with the heathland population; the mine-site populations producing significant growth at the highest AsO4 3- concentration (4.67 mol m-3 ), whereas growth of the heathland population was almost completely inhibited. EC50 values for mine-site isolates were estimated to be 5-41-times higher than the heathland population. All isolates produced identical responses to increasing Cu2+ concentrations, with no differences observed between mine-site and heathland isolates. • Populations of H. ericae on the contaminated mine sites have developed adaptive resistance to AsO4 3- . By contrast, Cu2+ resistance appears to be constitutive.

Genetic diversity of root-associated fungal endophytes from Calluna vulgaris at contrasting field sites.

A total of 107 putative ericoid mycorrhizal endophytes were isolated from hair roots of Calluna vulgaris from two abandoned arsenic/copper mine sites and a natural heathland site in southwest England. The endophytes were initially grouped as 14 RFLP types, based on the results of ITS-RFLP analysis using the restriction endonucleases Hinf I, Rsa I and Hae III. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. The majority of endophyte isolates (62-92%) from each site were identified as Hymenoscyphus ericae, but a number of other less common mycorrhizal RFLP types were also identified, all of which appear to have strong affinities with the order Leotiales. None of the less common RFLP types was isolated from C. vulgaris at more than one field site. Neighbour-joining analysis indicated similarities between the endophytes from C. vulgaris and mycorrhizal endophytes isolated from other Ericaceae and Epacridaceae hosts in North America and Australia.

Physiological heterogeneity within fungal mycelia: an important concept for a functional understanding of the ectomycorrhizal symbiosis.

Individual mycelia of filamentous fungi display considerable heterogeneity at the physiological level. Important physiological processes such as nutrient absorption, extracellular enzyme secretion and solute translocation occur differentially within an individual mycelium, and vary according to spatio-temporal changes in patterns of gene expression as the mycelium develops and senesces. In ectomycorrhizal (ECM) fungi, gene expression appears to be strongly influenced by interaction with the soil environment and the host root. The ECM mycelium is thus a complex and dynamic entity wherein discrete regions display particular physiological attributes. Physiological heterogeneity is important in the overall functioning of the symbiosis. In the particular case of movement of phosphorus from soil to host root in the ECM symbiosis, heterogeneity might provide the driving force for the integrated processes of absorption, translocation and transfer. It is suggested that it is only by considering the sum of the seemingly disparate physiological processes within the heterogeneous mycelium that mycorrhizal functioning can be fully understood.

A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. II. Carbohydrate allocation in ageing Picea sitchensis/Tylospora fibrillosa (Burt.) Donk ectomycorrhizas.

Translocation of 14 C-labelled photosynthate was studied in seedlings of Picea sitchensis (Bong.) Carr. ectomycorrhizal with Tylospora fibrillosa (Burt.) Donk grown in Perspex observation chambers. Although translocation to young and older mycorrhizas was observed, accumulation of current 14 C-labelled photosynthate was reduced in older compared with young mycorrhizas in the same root system. Non-structural carbohydrate components of P. sitchensis/T. fibrillosa and undefined beech ectomycorrhizas collected from the field were quantified by gas-liquid chromatography. Relative differences in accumulated carbohydrates were identified between P. sitchensis/T. fibrillosa and beech mycorrhizas. Levels of total soluble and insoluble carbohydrate were lower in older compared with young P. sitchensis/T. fibrillosa mycorrhizas. The soluble sugars arabitol, glucose and mannitol were detected in significantly lower quantities in older mycorrhizas whilst fructose and trehalose content was similar to that of young mycorrhizas. These results are discussed in relation to the physiology of the ageing mycorrhiza.

A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. III. Phosphate absorption and transfer in ageing Picea sitchensis/Tylospora fibrillosa (Burt.) Donk ectomycorrhizas.

Phosphate absorption and transfer to the host was studied in seedlings of Picea sitchensis (Bong.) Carr. ectomycorrhizal with Tylospora fibrillosa (Burt.) Donk grown in Perspex root observation chambers. Phosphate absorption into excised mycorrhizas was reduced in older compared with young mycorrhizas, kinetic analysis indicating physiological differences in phosphate absorption between the two age classes over a range of external concentrations up to 1000µM. Individual young mycorrhizas labelled directly with 32 P transferred more 32 P to the host than older mycorrhizas in the same intact root system. There was variation in the ratio (young:older) suggesting a progressive reduction in nutrient transfer to the host as the mycorrhiza ages. The results are discussed in relation to nutrient accumulation in the field.

A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. I. Morphological and cellular changes in mycorrhizas formed by Tylospora fibrillosa (Burt.) Donk and Paxillus involutus (Batsch. ex Fr.) Fr.

An age sequence of mycorrhizas formed by Paxillus involutus (Batsch. ex Fr.) Fr. and Tylospora fibrillosa (Burt.) Donk was sampled from 6-9-month-old seedlings of Picea sitchensis (Bong.) Carr. grown on peat in root observation chambers. Information about the ageing process was obtained from co-ordinated studies of mycorrhizal morphology, anatomy, ultrastructure and cell vitality (indicated by FDA-staining). Newly produced mycorrhizas were pale and turgid over most of their length with obvious extramatrical mycelium. Most mycorrhizas over 50 d old had a light turgid apical portion and a darkened wrinkled proximal portion: extramatrical mycelium was less obvious. Many remained in this state until the sampling stopped at day 142, but some were completely darkened before day 50. The morphological appearance of a mycorrhiza was not a good indicator of its chronological age. Most mycorrhizas displayed periodic busts of growth which added portions of young turgid cortex to an ageing axis. The percentage of cortical cells showing nuclei in 1 µm sections of the apical 1 mm declined from 40-50 % in mycorrhizas < 25 d old to 5-15 % in mycorrhizas 110-140 d old. Cortical cells became more vacuolate as they aged and the number of organelles appeared to decline. Senescence proceeded from the outer to the inner cortex and from proximal to distal regions. Degenerate cortical cells were present in the Hartig net zone of mycorrhizas over 25 days old. Cortical cell degeneration preceded, but was closely followed by, degeneration of adjacent Hartig net. The FDA study supported the general pattern and timing of cell death interpreted from the morphological and ultrastructural study. Cortical/Hartig net fluorescence declined markedly in mycorrhizas over 70-85 d old. The stele was the last tissue in the mycorrhiza to degenerate. It is suggested that a mycorrhiza ceases to function in nutrient and water uptake when no living cortical/Hartig net interface remains. In this study a few mycorrhizas became non-functional by day 31. For most mycorrhizas the major decline in function took place after 85 days. This estimate is in broad agreement with the estimates of mycorrhizal life span obtained from biomass studies in the field.

Distribution of photosynthetically fixed carbon within root systems of Eucalyptus pilularis plants ectomycorrhizal with Pisolithus tinctorius.

In intact seedlings of Eucalyptus pilularis Stn. ectomycorrhizal with Pisolithus tinctorius (Pers.) Coker & Couch in growth pouches, and labelled photosynthetically with 14 C4 more radioactivity was translocated into mycorrhizal than to non-mycorrhizal roots. Results from autoradiography of intact root systems were supported by data from scintillation counting of individual root tips. Greater 14 C accumulation occurred in young than in older mycorrhizas. It is proposed that individual mycorrhizal roots act most strongly as carbon sinks early in their life and that there is a progressive reduction in the amount of photosynthate translocated to them as they age.

THE PHYSIOLOGY OF BASIDIOMYCETE LINEAR ORGANS: I. PHOSPHATE UPTAKE BY CORDS AND MYCELIUM IN THE LABORATORY AND THE FIELD.

Phosphate uptake as a function of external medium concentration has been determined for mycelium grown in the laboratory, segments of cords collected from the field and cords in the field for a range of wood-decay basidiomycetes. Hofstee plots in all cases can be interpreted as indicating the presence of two uptake systems. Uptake of phosphate by mycelium is reduced by increasing the concentration of phosphate in the growth medium from 10 µM to 10 mM. The major portion absorbed by cords in the field remains within the segment exposed to radioactive solution, suggesting conversion of the phosphate to an immobile form unavailable for translocation.