The view that nutrient acquisition by most plants growing in natural ecosystems is mediated by mycorrhiza-forming symbiotic fungi is now largely accepted. With this perception comes the need to learn more about the identity of the organisms responsible for these key processes. Herein lies a challenge taken up by Vrålstad et al. on pp. 549–563 in this issue.

SUMMARY 367

I. INTRODUCTION 367

II. NUMBER 368

III. SIZE 379

IV. AIR SPACE IN THE SEEDS 381

V. FLOATATION AND DISPERSAL 383

1. Air 383

(a) Physical considerations 383

(b) Dispersal 387

(c) Birds 415

2. Water 416

(a) Physical considerations 416

(b) Dispersal 416

VI. CONCLUSIONS 417

Acknowledgements 417

References 418

Orchid seeds are very small, extremely light and produced in great numbers. Most range in length from c. 0.05 to 6.0 mm, with the difference between the longest and shortest known seeds in the family being 120-fold. The ‘widest’ seed at 0.9 mm is 90-fold wider than the ‘thinnest’ one, which measures 0.01 mm (because orchid seeds are tubular or balloon-like, ‘wide’ and ‘thin’ actually refer to diameter). Known seed weights extend from 0.31 lg to 24 μg (a 78-fold difference). Recorded numbers of seeds per fruit are as high as 4000000 and as low as 20–50 (80000–200000-fold difference). Testae are usually transparent, with outer cell walls that may be smooth or reticulated. Ultrasonic treatments enhance germination, which suggests that the testae can be restrictive. Embryos are even smaller: their volume is substantially smaller than that of the testa. As a result, orchid seeds have large internal air spaces that render them balloon-like. They can float in the air for long periods, a property that facilitates long-distance dispersal. The difficult-to-wet outer surfaces of the testa and large internal air spaces enable the seeds to float on water for prolonged periods. This facilitates distribution through tree effluates and/or small run-off rivulets that may follow rains. Due to their size and characteristics, orchid seeds may also be transported in and on land animals and birds (in fur, feathers or hair, mud on feet, and perhaps also following ingestion).

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50–25000 μg g−1) and cadmium (1–170 μg g−1). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.

The hydraulic conductance (L0) of detached, exuding root systems from melon (Cucumis melo cv. Amarillo oro) was measured. All plants received a half-strength Hoagland nutrient solution, and plants stressed either solely with NaCl (50 mM) or with NaCl (50 mM) following treatment (2 d) with CaCl2 (10 mM) were compared with controls and CaCl2-treated (10 mM) plants. The L0 of NaCl-treated plants was markedly decreased when compared to control and CaCl2-treated plants, but the decrease was smaller when NaCl was added to plants previously treated with CaCl2. A similar effect was observed when the flux of Ca2+ into the xylem and the Ca2+ concentration in the plasma membrane of the root cells were determined. In control, CaCl2- and NaCl + CaCl2-treated plants, HgCl2 treatment (50 μM) caused a sharp decline in L0 to values similar to those of NaCl-stressed roots, but L0 was restored by treatment with 5 mM DTT. However, in NaCl roots only a slight effect of Hg2+ and DTT were observed. The effect of all treatments on L0 was similar to that on osmotic water permeability (Pf) of individual protoplasts isolated from roots. The results suggest that NaCl decreased the passage of water through the membrane and roots by reducing the activity of Hg-sensitive water channels. The ameliorative effect of Ca2+ on NaCl stress could be related to water-channel function.

During the initial phases of elongation of pea internodes, oat and rice coleoptiles, oat mesocotyls, soybean hypocotyls and dandelion peduncles, net transverse orientation of cellulose wall microfibrils (Mfs) was found in the outer epidermal wall. This paper demonstrates that in all these axes, with the exception of rice coleoptile, net longitudinal orientation of microfibrils occurs in the outer epidermal wall in portions of the axes that were still elongating at the time of sampling. The timing of the transition to net longitudinal orientation and whether the transition proceeded acropetally or basipetally varied with the type of axis under study. The variability of the relationship between extension and the transition from net transverse to net longitudinal orientation suggests that factors other than extension are important in determining the transition. Layers of longitudinal wall microfibrils may be added to the extending epidermal wall to bolster its tensile strength commensurate with its function during and after extension. Attention is drawn to the parallels between the concept of tissue tension in growing axes and the concept that the epidermis functions as a stressed skin in the support of mature plant parts in primary growth.

Sorghum (Sorghum bicolor) synthesizes a complex mixture of 3-deoxyanthocyanidin phytoalexins in response to inoculation with the non-pathogenic fungus Bipolaris maydis. The anthocyanin cyanidin 3-dimalonyl glucoside, is also synthesized naturally in response to light. To determine the order and time of appearance of these compounds, etiolated sorghum mesocotyls were inoculated with B. maydis and tissue extracts were analysed by photodiode array-HPLC every 2 h for the first 24 h and at 48 h post inoculation (hpi). Uninoculated and inoculated etiolated mesocotyls were also allowed to incorporate L-[U-14C] phenylalanine. Apigeninidin appeared at 10 hpi, followed by luteolinidin and apigeninidin 5-O-arabinoside at 14 hpi. Luteolinidin 5-methylether was not detected until 18 hpi and apigeninidin 7-methylether not until 20 hpi. The concentrations of the primary phytoalexins, apigeninidin, luteolinidin and apigeninidin 5-O-arabinoside, rose steadily between 12 and 24 hpi, and the levels of apigeninidin and luteolinidin were approximately equivalent by 24 hpi. However, between 24 and 48 hpi luteolinidin and luteolinidin 5-methylether accumulated rapidly so that by 48 hpi the amounts of luteolinidin and luteolinidin 5-methylether had increased approximately twofold. Radiolabelling also showed that 14C was incorporated into the 3-deoxyanthocyanidins and cyanidin 3-dimalonyl glucoside. Several other unidentified phenolic compounds also accumulated radioactivity.

Nitrogen limitation compromises the realization of yield potential in cereals more than any other single factor. In rice, the world's most important crop species, the assumption has long been that only ammonium-N is efficiently utilized. Consequently, nitrate utilization has been largely ignored, although fragmentary data have suggested that growth could be substantial on nitrate. Using the short-lived radiotracer 13N, we here provide direct comparisons of root transmembrane fluxes and cytoplasmic pool sizes for nitrate- and ammonium-N in a major variety of Indica rice (Oryza sativa), and show that nitrate acquisition is not only of high capacity and efficiency but is superior to that of ammonium. We believe our results have implications for rice breeding and molecular genetics as well as the design of water-management and fertilization regimes. Potential strategies to harness this hitherto unexplored N-utilization potential are proposed.

Sloughing of root cap cells and exudation of mucilage plays an important role in the penetration of compacted soils by roots. For the first time we have quantified the rate of sloughing of root cap cells in an abrasive growth medium that was compacted to create mechanical impedance to root growth. The number of maize (Zea mays) root cap cells sloughed into sand increased as a result of compaction, from 1930 to 3220 d−1 per primary root. This represented a 12-fold increase in the number of cells sloughed per mm root extension (from 60 to >700). We estimated that the whole of the cap surface area was covered with detached cells in compacted sand, compared with c. 7% of the surface area in loose sand. This lubricating layer of sloughed cells and mucilage probably decreases frictional resistance to soil penetration. The total carbon deposited by the root was estimated at c. 110 μg g−1 sand d−1. Sloughed cells accounted for <10% of the total carbon, the vast majority of carbon being contained in mucilage exudates.

Recent research in whole-plant stomatal physiology, conducted largely with potted plants in controlled environments, suggests that stomatal conductance (gs) might be more closely linked to plant chemical variables than to hydraulic variables. To test this in a field situation, seasonal gs was examined in relation to a number of plant and environmental variables in 11 temperate, deciduous forest tree species. Stomatal conductance was generally better correlated with environmental variables (air temperature, vapor pressure deficit, PPFD) than with plant variables, and slightly better correlated with plant hydraulic variables (shoot water and osmotic potentials) than with plant chemical variables (xylem sap ABA concentration, xylem sap pH). We examined a model, developed previously for maize, which describes regulation of gs by xylem sap ABA concentration with leaf water status acting to modify stomatal sensitivity to the ABA signal. This model explained slightly more variation in seasonal gs in the forest trees than did single plant variables but not more variation than most single environmental variables. Response surface models, especially those incorporating environmental variables, were more consistently successful at explaining gs across species.

Patterns of molecular genetic variation were examined in seed collections of Plantago major and Plantago intermedia, used previously to investigate the variations in ozone (O3) resistance of these species across Europe. Total genomic DNA was amplified with random primers (random amplied polymorphic DNA (RAPD) and inter- single sequence repeats (SSR)) to produce 73 genetic markers. In addition, allozyme and chloroplast variations were surveyed. Genetic markers were examined for association with O3 resistance in 18 British populations of P. major as well as 27 continental European populations of P. major and P. intermedia. Two populations that exhibited increased resistance to O3 following several years' exposure to high O3 concentrations in the field showed decreased genetic variation over time. In addition, their genetic composition showed no drastic change, which suggests that the change in resistance to O3 was probably the result of selection on genotypes already present in local populations (selection in situ). It appears that selection for O3 resistance may occur in independent populations, and also may involve a number of genetically determined traits. Consequently the finding that plants with similar degrees of O3 resistance are not closely related was not unexpected. However, the finding of an association of several genetic markers with O3 resistance merits further investigation.

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under reference (current ambient, 360 μmol mol−1 CO2) and double ambient (720 μmol mol−1 CO2) carbon dioxide (CO2) mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density) and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO2 concentration on the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf variation. In general, a doubling of the atmospheric CO2 concentration enhanced plant growth and significantly increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated CO2 concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate. However, no significant differences were found for the maximum rate of carboxylation (Vcmax) and the light saturated rate of electron transport (Jmax) between the control and elevated CO2 treatment.

The processes of symbiotic germination and seedling development were analysed in the myco-heterotrophic orchid Corallorhiza trifida, seeds of which were buried in ‘packets’ either adjacent to or at varying distances from adult plants in defined communities of ectomycorrhizal tree species. Germination occurred within eight months of burial under Betula–Alnus and within seven months under Salix repens. It was always associated with penetration of the suspensor by a clamp-forming mycorrhizal fungus. Four distinct developmental stages were defined and the rates of transition through these stages were plotted. There was no evidence of a relationship between extent of germination or rate of development and the presence of naturally distributed plants of C. trifida at the spatial scale of 1 m. The best germination and the most rapid rate of development of C. trifida seedlings occurred in a Salix repens community located at a considerable distance from any extant C. trifida population. Determination of internal transcribed spacer (ITS) RFLPs and of gene sequences of the fungi involved in symbiotic germination and growth of C. trifida, revealed them to belong exclusively to the Thelephora–Tomentella complex of the Thelephoraceae. These fungi are known also to be ectomycorrhizal associates of trees. It is hypothesized that the rate of growth of the C. trifida seedlings is determined by the ability of the fungal symbionts to transfer carbon from their ectomycorrhizal co-associates.

Seedlings of the myco-heterotrophic orchid Corallorhiza trifida which had been germinated in the field in mesh bags developed hyphal links and mycorrhizas with Betula pendula and Salix repens, but not with Pinus sylvestris, when transplanted into soil microcosms. The fungus connecting the myco-heterotroph to Betula and Salix formed endomycorrhiza in the orchid with typical pelotons, but formed ectomycorrhizas with the autotrophs. The orchid plants, when linked to Betula and Salix by fungal hyphae, gained 6–14% in weight over 25–28 wk. In microcosms supporting P. sylvestris, and in control microcosms which lacked autotrophs, the Corallorhiza plants lost 13% of their weight over the same period. In the course of the 28-wk experimental period new Corallorhiza seedlings, in addition to those added as part of the experiment, appeared in the microcosms containing Salix and Betula but not in the Pinus microcosms. Shoots of Betula and Salix plants grown in association with Corallorhiza were fed with 14CO2, and the movement of the isotope was subsequently traced by a combination of digital autoradiography and tissue oxidation. Direct transfer of C from both autotrophs to the myco-heterotroph occurred in all cases where the associates had become connected by a shared fungal symbiont. Orchid seedlings lacking these hyphal connections, introduced to the microcosms as controls immediately before isotope feeding, failed to assimilate significant amounts of C. The results provide the first experimental confirmation that growth of Corallorhiza trifida can be sustained by supply of C received directly from an autotrophic partner through linked fungal mycelia.

The mycobionts of Piceirhiza bicolorata, a distinct ectomycorrhizal morphotype of conifers and hardwoods, have been identified by internal transcribed spacer 1 (ITS1) nuclear ribosomal DNA (rDNA) sequence comparison of the fungi involved. Samples of Piceirhiza bicolorata were obtained from seedlings of Picea abies, Pinus sylvestris, Betula pubescens, Populus tremula, Quercus robur and Salix phylicifolia. In an initial screening, the fungus amplified with universal ITS primers from ectomycorrhizal root samples of P. bicolorata shared approx. 95% ITS1 sequence identity with the ericoid mycorrhizal fungus Hymenoscyphus ericae. A total of 77 out of 88 (= 87.5%) DNA samples (i.e. 52/56 root samples and 25/32 axenic culture isolates) of P. bicolorata were successfully amplified with a taxon-selective primer designed for exclusive amplification of H. ericae-like strains. Forty-seven amplicons were sequenced, yielding 15 different ITS1 genotypes that differed by 1–14 nucleotide character state changes. An inferred ITS1 phylogeny (maximum parsimony) showed that a single major evolutionary lineage of P. bicolorata embraced the historically important H. ericae isolates in a 100% bootstrap-supported clade. The 15 P. bicolorata genotypes were positioned in four subclades, roughly corresponding to morphological groups of P. bicolorata isolates observed in axenic culture. Culture isolates of H. ericae and P. bicolorata share some common morphological features including slow, dense growth and formation of short aerial hyphal aggregates. Our results suggest that members of the H. ericae aggregate participate in the formation of the distinct ectomycorrhizal morphotype P. bicolorata. This opposes the widely accepted discrimination of ericoid and ectomycorrhizal mycobionts of the boreal forest ecosystem. The high prevalence of the P. bicolorata morphotype on pioneer seedlings of P. sylvestris, B. pubescens and S. phylicifolia at a copper mine spoil was remarkable. Hypotheses of possible nutrient mobilization and detoxification potentials of the fungal associates of P. bicolorata are discussed. We hypothesize that ericoid and ectomycorrhizal plants may share mycobionts of the H. ericae aggregate.

The effects of mycorrhiza formation in combination with elevated CO2 concentrations on carbon metabolism of Norway spruce (Picea abies) seedlings and aspen (Populus tremula × Populus tremuloides) plantlets were analysed. Plants were inoculated for 6 wk with the ectomycorrhizal fungi Amanita muscaria and Paxillus involutus (aspen only) in an axenic Petri-dish culture at 350 and 700 μl l−1 CO2 partial pressure. After mycorrhiza formation, a stimulation of net assimilation rate was accompanied by decreased activities of sucrose synthase, an increased activation state of sucrose-phosphate synthase, decreased fructose-2,6-bisphosphate and starch, and slightly elevated glucose-6-phosphate contents in source leaves of both host species, independent of CO2 concentration. Exposure to elevated CO2 generally resulted in higher net assimilation rates, increased starch as well as decreased fructose-2,6-bisphosphate (aspen only) content in source leaves of both mycorrhizal and nonmycorrhizal plants. Our data indicate only slightly improved carbon utilization by mycorrhizal plants at elevated CO2. They demonstrate however, that both factors which modulate the sink-source properties of plants increase the capacity for sucrose synthesis in source leaves mainly by allosteric enzyme regulation.

Most work on root proliferation to a localized nutrient supply has ignored the possible role of mycorrhizal fungi, despite their key role in nutrient acquisition. Interactions between roots of Plantago lanceolata, an added arbuscular mycorrhiza (AM) inoculum and nitrogen capture from an organic patch (Lolium perenne shoot material) dual-labelled with 15N and 13C were investigated, to determine whether root proliferation and nitrogen (N) capture was affected by the presence of AM fungi. Decomposition of the organic patch in the presence and absence of roots peaked in all treatments at day 3, as shown by the amounts of 13CO2 detected in the soil atmosphere. Plant N concentrations were higher in the treatments with added inoculum 10 d after patch addition, but thereafter did not differ among treatments. Plant phosphorus concentrations at the end of the experiment were depressed by the addition of the organic residue in the absence of mycorrhizal inoculum. Although uninoculated plants were also colonized by mycorrhizal fungi, colonization was enhanced at all times by the added inoculum. Addition of the AM inoculum increased root production, observed in situ by the use of minirhizotron tubes, most pronouncedly within the organic patch zone. Patch N capture by the end of the experiment was c. 7.5% and was not significantly different as a result of adding an AM inoculum. Furthermore, no 13C enrichments were detected in the plant material in any of the treatments showing that intact organic compounds were not taken up. Thus, although the added AM fungal inoculum benefited P. lanceolata seedlings in terms of P concentrations of tissues it did not increase total N capture or affect the form in which N was captured by P. lanceolata roots.