Isozyme expression of nutrient-mobilizing and defence-related enzymes were examined in different functional components of intact Scots pine (Pinus sylvestris L.) ectomycorrhizal root systems developed in natural unsterilized forest humus. Scots pine seedlings colonized by Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) strains were grown on humus in two-dimensional Perspex[target] microcosms to allow development of complete ectomycorrhizal root systems. Soluble proteins from uncolonized short roots, whole mycorrhizal root tips or dissected mantle and core fractions, fungal strands and the outermost soil colonizing fine hyphae were subjected to native polyacrylamide gel electrophoresis and stained to detect esterase, acid phosphatase, peroxidase and different polyphenoloxidase isozyme activities. Tissue-specific esterase isozymes were detected in all components and most, including unique Hartig net isozymes, were of fungal origin. High fungal acid phosphatase activities were detected in mycorrhiza and fine hyphae of S. bovinus, supporting earlier findings of active phosphatase activity at the fungal interface in the Hartig net region and in the fine hyphal margins of extramatrical mycelium actively colonizing the humus. All compartments in the P. involutus mycorrhizosphere had weaker acid-phosphatase activities. Peroxidases formed a large part of the soluble-protein content in non-mycorrhizal short roots. The amount of peroxidases, on a tissue f. wt basis, was similar in mycorrhizal and non-mycorrhizal roots, but differential isozyme expression was detected, indicating a change in root peroxidase activities following mycorrhiza formation. The expression of DL-3,4-dihydroxyphenylalanine metabolizing polyphenol oxidase enzymes was reduced in mycorrhizas compared with non-mycorrhizal short roots. In the extramatrical mycelial components, fine hyphae expressed the highest polyphenoloxidase activity. P. involutus displayed some polyphenol-oxidizing isoenzymes not detected in S. bovinus systems. Laccase activity was not detected in the plant and fungal components examined. It is concluded that enzyme activities and isozyme expression are differentially regulated in the different functional components of Scots pine mycorrhizospheres.

Light, fluorescence and electron microscopy were employed to follow the development of the endodermis in cluster roots and lateral roots of Grevillea robusta A. Cunn. ex R. Br. Endodermal cells had three different origins: rootlet endodermis arose from the rootlet meristem; endodermis covering the primordium shortly after initiation came from division of parental endodermis; cells at the junction between parent and rootlet endodermis developed from re-differentiated rootlet cortical cells. In the cluster root, the Casparian band formed in three ways, and was not initially present opposite the two sets of single xylem elements in the rootlet stele. A new clearing technique was developed that allowed visualization of xylem, suberized endodermis, Casparian band formation and phenolic compounds. In lateral roots, endodermal differentiation was asynchronous, but was related to position relative to protoxylem poles. However, the observed delay began before these poles had differentiated. At the tip of mature rootlets, which are determinate, the endodermis terminates in a ‘dome’ of cells, with the initial cell differentiating as an endodermal cell. Results are discussed in terms of determinate development in roots and the spatial and temporal contexts within which this development takes place.

Seedlings of Betula pendula Roth. were grown in the presence of Paxillus involutus (Batsch) Fr., and metabolic changes during mycorrhiza formation were examined by measuring organic acid and amino acid pools and related enzyme activities, following sequential harvests. Glutamine, aspartate and asparagine pools were always lower in infected roots than in non-infected roots, especially during Hartig net initiation and formation. Glutamate concentration was similar in both tissues. Citrate and malate were the two major organic acids detected and their concentrations were equal in infected and non-infected roots. Aspartate aminotransferase, glutamine synthetase, NAD-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activities were higher in infected roots than non-infected roots. For all enzymes revealed on polyacrylamide gels, both root and fungal isoforms were present in infected roots. Quantitative changes in enzyme capacities and metabolite pools indicated that mycorrhiza formation caused a re-arrangement of the main metabolic pathways during the very early stages following contact, which might be related to the structural changes.

We have studied the chitin and ergosterol contents of ectomycorrhizal roots in three sets of experiments to evaluate them as indicators of fungal biomass. The first set of experiments showed that ageing had a marked effect on ergosterol concentrations. The ergosterol content of 7-month-old, brown, shrunken Pinus sylvestris L.–Paxillus involutus (Fr.) Fr. mycorrhizas was found to be only 10% of that found in white, turgid, 1- or 4-month-old specimens. This supports the hypothesis that the compound is a good indicator of living fungal biomass. Ageing had a lesser effect on chitin concentrations since the chitin levels found in 7-month-old mycorrhizas were still 60% of the levels found in 1- and 4-month-old specimens.

Consequently, the chitin[ratio ]ergosterol ratio increased from about 14 to 19 in 1- and 4-month-old mycorrhizas respectively to about 110 in 7-month-old mycorrhizas. In the second set of experiments, we found that variation in plant growth had no effect on the chitin[ratio ]ergosterol ratio in whole root systems of either Alnus incana (L.) Moench or Pinus sylvestris mycorrhizal with Paxillus involutus. In the third set of experiments, we found a constant relationship between the two marker concentrations in 10-month-old root systems of Pinus sylvestris, regardless of fungal species involved, using Paxillus involutus, Piloderma croceum Erikss. & Hjorts and Suillus variegatus (Fr.) O. Kuntze as test organisms. Taken together, the results of this study suggest that both chitin and ergosterol give reliable, but different, relative measures of fungal biomass in mycorrhizal roots. Furthermore, we demonstrate that, in combination, the two chemical markers can be used to estimate both total and living fungal biomass (derived from the chitin[ratio ]ergosterol ratio).

Azolla filiculoides Lam. causes serious weed problems in Britain, but its long-term survival might be limited by winter death. The aim of this study was to establish the low temperature responses and limitations of A. filiculoides sporophytes.

In the laboratory, normal vegetative growth was shown to continue at 5°C. Reddening of plants was a response to low temperature and high light conditions which could be prevented by shading. Adult plants died after short (18 h) exposure to −4°C but survived sub-zero temperatures >−4°C. Evidence was found of seasonal changes in chill tolerance, but not in freeze tolerance.

In outdoor culture, plants survived encasement in ice and air temperatures to −5°C. Additional evidence suggested that natural populations can readily survive air temperatures much lower than this. Microclimatic effects are likely to be responsible for this discrepancy between laboratory and outdoor culture results.

Three phenotyes were identified; survival, colonizing and mat forms.

Sixty-two isolates of Pisolithus were obtained from a carpophore population collected from a 90-km2 area of New South Wales, Australia. Random amplified polymorphic DNA (RAPD) analysis was used initially to screen isolates for variation, and restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region and ITS sequence analysis were subsequently performed on selected isolates. ITS–RFLP and ITS sequence analysis indicated that two isolates (LJ07 and WM01) from different sites were considerably different from all other isolates, a strict consensus tree generated by bootstrap analysis of sequence data strongly suggesting that isolates LJ07 and WM01 represent a separate Pisolithus species. Although the general morphology of carpophores and basidiospores of the majority of collected isolates was consistent with previous descriptions of P. tinctorius (Pers.) Coker and Couch, basidiospore characteristics suggest that isolates LJO7 and WMO1 might represent a Pisolithus species distinct from P. tinctorius and the previously described Australian species, P. microcarpus (Cooke & Massee) G. Cunn.

Variation within the internal transcribed spacer (ITS) of the ribosomal RNA gene of 15 isolates representing seven European Armillaria species, was examined by sequencing of the PCR-amplified products. The analysis of an 744-bp region showed that the 5.8S gene appeared to be highly conserved in the 15 isolates and in other Basidiomycetes and Ascomycetes, whereas ITS1 and especially ITS2 spacers exhibited polymorphisms due to base substitutions, insertions or deletions of up to eight nucleotides. An initial dendrogram for the full sequence was drawn using cluster analysis (UPGMA), and a tree was constructed using the maximum parsimony method. Both methods indicated that the isolates could be divided into four major groups. One group, consisting of A. ectypa, was distinct from all the other species. Examination of the other groups indicated that A. tabescens and A. mellea were in a separated cluster, with a significant variation between the two isolates of the latter species. A. gallica and A. cepistipes constituted another closely related group distinguishable from A. ostoyae and A. borealis, these latter two species exhibiting the highest similarity. The results are consistent with, and discussed in regard to, the relationships estimated previously by pairing tests, morphological and physiological comparisons, as well as by restriction fragment length polymorphism of the rDNA.

A recent re-analysis of the data of Salisbury (1974) claims his data do not support the hypothesis that seeds of species from shaded habitats are heavier than those from unshaded habitats, partly because the original analysis was inappropriate and partly because of bias in the dataset. We show first that the re-analysis itself contains errors, and second that the charge of bias is based largely on a misunderstanding. We also show that analysis of a larger dataset, drawn from Salisbury (1942) and from Salisbury (1974), provides convincing support for the hypothesis and suggests that the relationship is independent of life history.

Sporulation in the floating fern Azolla filiculoides Lam. is both frequent and widespread in Britain and might therefore play a greater part in the population dynamics of the species than has been suggested by earlier reports. In laboratory experiments, increasing plant density and/or phosphorus supply resulted in increased sporulation. It was estimated that a thick mat of 8 kg m2 fresh biomass can produce 380000 microsporocarps and 85000 megasporocarps per m2.

Light and temperatures >10°C were necessary for sporocarp germination. Sporocarps could survive exposure to both low temperatures (5°C for at least 3 months) and sub-zero temperatures (−10°C for at least 18 d). Sporocarps were found to survive storage in water for 3 yr and to germinate from mud samples collected in the field. In laboratory culture, sporeling growth and survival were optimal at 15°C.

There is some evidence that A. filiculoides might have adapted to the British climate since its introduction.

Studies on whether or not species which need more light for establishment have smaller seeds should be made between plants of similar mature height living in the same soil and macroclimate. Salisbury's classic comparisons confused differences in light-requirement during establishment with differences in mature height and in the supply of soil resources.