Summary 373

I. Introduction: Arum-types and Paris-types 374

II. Possible functional implications 375

III. Extent of the two classes in the plant kingdom 377

1. Bryophytes and Pteridophytes 377

2. Gymnosperms 379

3. Angiosperms 379

IV. Is the distinction between classes useful? 383

V. The structural basis 383

VI. The role of the fungal genome 384

VII. Physiology revisited 384

VIII. Conclusions 385

Acknowledgements 386

References 386

This review describes diversity in the structure of (vesicular)–arbuscular (VA) mycorrhizas, i.e. endomycorrhizas formed by Glomalean fungi. In particular, we consider the extent in the plant kingdom of the two classes first described by Gallaud (1905). These are: (1) the Arum-type, defined on the basis of an extensive intercellular phase of hyphal growth in the root cortex and development of terminal arbuscules on intracellular hyphal branches; (2) the Paris-type, defined by the absence of the intercellular phase and presence of extensive intracellular hyphal coils. Arbuscules are intercalary structures on the coils. However, there have been many reports that in Paris-types arbuscules are relatively few in numbers, small, or absent altogether.

A survey of the literature has revealed that Paris-types occur more frequently in the plant kingdom than Arum-types and predominate in ferns, gymnosperms and many wild angiosperms. The cultivated herbs that are the subject of much experimental work are mostly Arum-types. Although evidence is still limited, there are differences at the family level. In 41 angiosperm families there are records of only Paris-type VA mycorrhizas and in 30 families records of only Arum-types. Another 21 families have examples of both classes, or intermediates between them. Accordingly, we consider whether the original division into two classes is still useful. We conclude that it is when considering the physiology of the symbiosis and especially the issue of whether different fungus/host interfaces have specialized roles in transfer of inorganic nutrients and organic carbon between the partners. If there is no such specialization between hyphal coils and arbuscules, then the latter might not be necessary for the function of Paris-types. This would account for reports of the infrequency or absence of arbuscules in this class. The control exerted on structures by the genomes of host and fungus, and possible reasons (anatomical and physiological) for the existence of the VA mycorrhizal structures, are discussed. The presence or absence of extensive intercellular spaces and differences in the wall structure of cortical cells might be particularly important in determining which type of VA mycorrhiza is formed.

Cloned cuttings of Betula pendula Roth were grown in field fumigation chambers at Birmensdorf throughout one growing season in filtered air with either <3 (control) or 90/40 nl l−1 O3 (day/night; ozone generated from pure oxygen). Each ozone regime was split into high and low soil nutrient regimes by watering plants with either a 0·05% or a 0·005% solution of a fertilizer which contained macronutrients and micronutrients.

Fertilization had a strong effect on plant growth, enzyme activities and the expression of ozone-induced effects at the biochemical level. The activities of PEPC and Rubisco were enhanced about threefold in the plants with high fertilization (HF). Significant effects of ozone were in most cases found only in the older leaves of the plants with low fertilization (LF). There, sucrose, glucose and fructose levels were enhanced. In both fertilization treatments, the number of starch granules along the minor veins was increased. These ozone effects point to a decreased or inhibited phloem loading. The increased PEPC activity and the enhanced malate levels in the ozone-exposed plants might be the result of a redirection of carbon flow from sucrose synthesis and translocation towards anapleurotic processes, which can feed detoxification and repair of ozone injury as indicated by enhanced respiration. These findings agree well with the observed effects of ozone in lowering the root[ratio ]shoot biomass ratio. Although there was a marked reduction in the O3/LF plants, O3/HF plants showed no significant response. Inositol was decreased under ozone exposure in both fertilizer treatments, contrasting with the pattern for carbohydrates.

These results demonstrate the role of fertilization as an important modifier of ozone-induced effects at the plant biochemical level. Well fertilized plants appear to cope better with the impact of ozone on metabolism.

Young spruce (Picea abies [L.] Karst; 2-yr) and beech trees (Fagus sylvatica L.; current-year and 1-yr-old seedlings) were grown under field conditions at a rural site near the city of Basel (Schönenbuch; 400 m above sea level) and 2-yr-old spruce trees at an elevated montainous site (Wengernalp; 1890 m above sea level). The plants were exposed in open-top-chambers (OTC) either to charcoal-filtered air or ambient air with ozone being the predominant air pollutant at both sites. The exposures lasted up to 1 yr. At the end of the growth period the plants were harvested and the pool sizes of the major non-structural carbohydrates were determined. Exposure to ambient air compared with filtered air controls caused an increase of glucose, fructose, pinitol, starch and, in some cases, sucrose in needles whereas carbohydrate contents in roots and twigs of spruce were reduced, in particular at Wengernalp. In beech saplings at Schönenbuch, a significant rise of sucrose and, in some cases, of raffinose, together with a significant reduction of glucose and fructose was observed in buds, leaves, twigs, and roots in ambient air compared with filtered air. In phloem exudates of current year beech seedlings, a 54–160% higher content of carbohydrates was found in ambient air than in filtered air. In roots of the beech saplings, the amount of soluble carbohydrates increased and starch contents mostly decreased. In general, starch storage in roots and stems was more abundant in beech trees. Spruce, especially at the mountainous site, accumulated high amounts of soluble carbohydrates in needles, in particular glucose.

‘Tiny Tim’ tomato plants were exposed to five CO2 treatments (375 (ambient), 450, 525, 600 or 675 μmol mol−1) in combination with O3 (0 or 80 nmol mol−1). Biomass was evaluated following 3, 5, 7 and 13 wk exposure. Biomass following 13 wk exposure also included weekly harvests of mature tomato fruit beginning week 8. Carbon dioxide enrichment significantly enhanced total vegetative plant d. wt at each harvest, as well as cumulative yield of mature fruit, whereas O3 significantly suppressed total vegetative plant d. wt at each harvest and reduced total cumulative fruit yield. The magnitude of these changes varied with the development of tomato from early growth to mature fruit yield. Carbon dioxide enrichment reduced the detrimental effects of O3 on total vegetative plant d. wt of tomato following 3, 5, 7 and 13 wk exposure. Final mature fruit yield was 24% higher under enriched CO2 treatments than in ambient CO2. Ozone suppressed final yield by 31% following exposure to 80 nmol mol−1 O3 when compared with exposure to charcoal-filtered (CF) air. The impacts of both CO2 and O3 on yield were, however, dependent upon the presence or absence of the other gas. In the absence of O3, yields were very similar for the ambient and elevated CO2 treatments, but in the presence of O3, yields under ambient CO2 were greatly suppressed whereas yields under elevated CO2 were similar to those in the absence of O3. Thus, enriched CO2 ameliorated most of the suppressive effect of O3 on yield of mature fruit.

Three perennial grass species, Lolium perenne L., Agrostis capillaris L. and Festuca ovina L., were homogeneously labelled in phytotrons with 14CO2 at two CO2 concentrations (350 and 700 μl l−1). Plants were grown under two nitrogen regimes: one with a minor addition of 8 kg N ha−1, the other with an addition of 278 kg N ha−1. Carbon allocation over the different compartments of the plant/soil systems was measured: shoots, roots, rhizosphere soil (soil solution, microbial biomass and soil residue), and bulk soil. Elevated CO2 increased total net 14C recovery in all species by 14%, and significantly enhanced the below-ground 14C allocation by 26%, this enhancement was 24%, 39% and 21% for root, rhizosphere soil and bulk soil, respectively. Within the rhizosphere soil, the 14C amounts in the soil solution (+69%) and soil residue (+49%) increased significantly. Total microbial biomass-C in the rhizosphere soil was also increased (15%) by the elevated CO2 treatment, but only in proportion to the increased root mass. No interactions were observed between the elevated CO2 and N treatments. The N treatment increased total net 14C recovery by more than 300% and 14C was preferentially allocated to the shoots, leading to a significant increase in shoot-to-root ratio. However, N fertilization also increased (+111%) the absolute amount of 14C in soil. The three species behaved differently, but no interactions were observed between CO2 treatment and plant species. These results show that elevated CO2 induces an increased C input into soil for all three grass species at both N levels. However, the highest absolute amounts were found in the soils of the fastest growing species and at the highest N level.

We investigated the effect of elevated CO2 on the growth and mycorrhizal colonization of three tree species native to north-eastern American forests (Betula papyrifera Marsh., Pinus strobus L. and Tsuga canadensis L. Carr). Saplings of the tree species were collected from Harvard Forest, Massachusetts, and grown in forest soil under ambient (c. 375 ppm) and elevated (700 ppm) atmospheric CO2 concentrations for 27–35 wk.

In all three species there was a trend to increasing whole-plant, total-root and fine-root biomass in elevated CO2, and a significant increase in the degree of ectomycorrhizal colonization in B. papyrifera and P. strobus, but not in T. canadensis. However, in T. canadensis the degree of colonization with arbuscular mycorrhizas increased significantly. In both the ambient and elevated environments, on the roots of B. papyrifera and P. strobus 12 distinct ectomycorrhizal morphotypes were identified. Distinct changes in the ectomycorrhizal morphotype assemblage of B. papyrifera were observed under CO2 enrichment. This change resulted in an increase in the frequency of ectomycorrhizas with a higher incidence of emanating hyphae and rhizomorphs, and resulted in a higher density of fungal hyphae in a root exclusion chamber.

In Nicotiana sylvestris Spegazzini and Comes (Solanaceae), we examined the relationships among wounding, endogenous leaf jasmonic acid (JA) pools, and whole-plant (WP) nicotine accumulation over a range of wounding intensities and spatial distributions, in order to explore optimal defence (OD) theory predictions. We quantitatively wounded one or four leaves and then quantified: (1) JA in damaged and undamaged leaves 90 min after wounding; (2) WP nicotine concentration after 5 d (the times when JA and nicotine attain the largest wound-induced concentrations). We find: (1) statistically significant, positive relationships on a leaf-by-leaf basis among the number of leaf punctures, endogenous leaf JA, and WP nicotine accumulation; (2) that young, undamaged leaves have a higher concentration of JA than do older, undamaged leaves, and produce a greater amount of JA per puncture than older leaves, but that all leaves have the same JA content (ng JA per leaf); and (3) that a damaged leaf produces less JA when other leaves in the canopy are wounded than when it is the only wounded leaf in the canopy, but that when it is the only wounded leaf, the phylotactically adjacent, undamaged leaves do not increase their JA concentrations. The observation that younger leaves produce more JA per puncture than do older leaves is consistent with OD theory predictions. The observation that a small amount of damage localized to a single leaf is as effective as a larger amount of damage dispersed across the canopy in increasing leaf JA and WP nicotine accumulation shows the plant's ability to differentiate between dispersed and localized damage. Because the quantity of JA in a wounded leaf 90 min after wounding is a reliable indicator of the WP nicotine response to wounding, this trait provides insight into how plants integrate information about environmental insults and tailor their defence responses.

Carbodioxide uptake, oxygen evolution and chlorophyll fluorescence of leaves of Lobelia rhynchopetalum Hemsl., a giant rosette plant of the tropical alpine regions of Ethiopia, were studied under field conditions at 4000 m above sea level. Our objective was to investigate the photosynthetic adaptation to the combination of wide fluctuation in diurnal temperature, high photon flux densities (PFD) and low CO2 partial pressure encountered in these regions. At an ambient CO2 partial pressure of c. 17 Pa, maximal rates of CO2 uptake were low, ranging between 4 and 6 μmol m−2 s−1. Such rates, however, required high PFDs and were observed only at levels of 1500 μmol photons m−2 s−1. Carbon dioxide uptake was significantly inhibited when PFD was >2000 μmol photons m−2 s−1. On the other hand, at saturating CO2 levels, maximal photosynthetic oxygen evolution was higher (30 μmol O2 m−2 s−1), saturating at the same PFD as CO2 uptake. Quantum efficiency of CO2 uptake (0·006 mol CO2 mol photons−1, at high altitude and a low CO2 partial pressure of 17 Pa) and even of oxygen evolution under CO2-saturating conditions in the leaf O2 electrode (0·05 mol O2 mol photons−1) indicated reduced photosynthetic efficiency. Electron transport rate (ETR) was strongly correlated with the leaf temperature. Non-photochemical quenching (NPQ) responded inversely to leaf temperature and stomatal conductance.

The results indicated that in the morning, when the sun irradiates the partly frozen leaves with closed stomata, NPQ is the principal mechanism by which Lobelia leaves protect their photosynthetic apparatus. However, during the day, the predominant upright inclination of the leaves significantly contributes to protecting the leaves from excess light absorption. A comparison of the chlorophyll fluorescence of young and old leaves revealed that the former had high ETR and quantum efficiency of photosynthetic electron transport but a lower capacity for NPQ. Extremely high NPQ values but low ETR and low quantum efficiency were recorded for the old leaves. Thus, in the course of maturation the leaves apparently lose photosynthetic efficiency but increase their capability for protective non-photochemical quenching.

The photosynthetic and respiratory activities of the leaves of a tropical tree, cacao, Theobroma cacao L., grown under shading, were determined in relation to leaf age and light conditions within the canopy, in order to gain a deeper insight into characteristics of the leaf population composing a canopy.

The specific leaf weight and leaf water content varied with the height of leaves from the ground and/or the irradiance. The net photosynthetic rate also varied markedly depending on leaf age and irradiance. The leaves in full or nearly full irradiance (<70% full daylight) attained the maximum rate c. 60 d after the leaves emerged, and the rate decreased to nearly zero at a leaf age of c. 270 d.

The nocturnal leaf respiratory activity was proportional to the photosynthetic activity during the day, but the proportional ratio was not constant throughout the survival period of leaves. Within the closed canopy, the upper leaves, which much higher ratios of respiratory to photosynthetic activity, under full or nearly full irradiance, tended to have much shorter mean longevities (c. 160 d) than the lower leaves under a lower irradiance (c. 310 d).

Cuticular water permeance was manipulated in Corylus avellana L., Hypericum androsaemum L. and Populus tremula L. by (1) long-term application of low doses of various systemic herbicides inhibiting biosynthesis of cuticular waxes, (2) very short-term application of organic solvents to the leaf surface, and (3) exposure to natural strong winds. Treatment effects were very variable, but increased the natural range of permeances by a factor of 10 or so in undamaged leaves. All species had hypostomatous leaves. Relative change of leaf conductance (g) in response to stepwise increases of leaf-to-air water vapour pressure difference (VPD) was measured for individual leaves (Corylus) or groups of leaves at the shoot or branch tip. Adaxial cuticular water permeance (P) was determined for the same leaves after measurement of the VPD-response. A proportional measure of relative change of g with VPD, d(logeg)/dVPD, was then plotted against P. No increase in the strength of the closing response to increasing VPD was found with increasing P, as would have been expected if water loss through the cuticle was involved in stomatal response to changes in VPD via a direct effect on guard cell turgor. By contrast, high P coincided, most clearly in Corylus, with a reduced strength of the stomatal closing response to increasing VPD, i.e. less negative d(logeg)/dVPD. As the responses were non-linear, the value of d(logeg)/dVPD changed with VPD. With rising VPD, all three species and a fourth one previously studied showed a decline in the value of d[d(logeg)/dVPD]/d(log P), reaching negative values in one species. This is interpreted in terms of two independent and antagonistic effects of increased cuticular water permeance on guard cell response to VPD, one acting by reducing the backpressure exerted on guard cells by the epidermis, and the other one possibly causing greater depression of guard cell turgor through delivery of more chemical messengers (such as abscisic acid) to the guard cells with the cuticular transpiration stream.

A strain of non-pathogenic Fusarium oxysporum Schlecht. emend. Snyd. & Hans. has been selected for its capacity to reduce the incidence of Fusarium wilt of tomato. Among the possible modes of action of this strain, competition with the pathogen for the colonization of the root surface and tissues has been proposed. In order to study the pattern of root colonization, young Lycopersicon esculentum Miller (tomato) plants grown in a nutrient solution were inoculated by a suspension of F. oxysporum microconidia and processed at time-intervals for microscopic observations. The fungal strain was transformed with the Gus reporter gene to facilitate the observations. Within 24 h of inoculation the root surface was colonized by a dense network of hyphae, with the exception of the apex, which was colonized only after 48 h. A few hyphae were observed penetrating into the epidermis, leading to the internal colonization of the root cortex. This colonization was always discontinuous, since defence reactions of the plant limited the extension of the fungus. The barrier formed by thickenings and coilings of the cell walls and hypertrophied cells was most frequently observed in the external cortex and, sometimes, deeper in the internal cortex, close to the vessels which were never colonized. Typical defence reactions such as wall appositions, intercellular plugging and intracellular osmiophilic deposits, were frequently observed. This is the first report, based on microscopic observations, of the capacity of a non-pathogenic strain of F. oxysporum to colonize roots of tomato.

Actinomycetes isolated from carrot rhizosphere were screened for their in vitro and in vivo antagonism to Pythium coloratum Vaartaja, a causal agent of cavity-spot disease of carrots (Daucus carota L.). Forty-five streptomycete and non-streptomycete actinomycete isolates were screened for in vitro antagonism in a carrot bioassay. Of these, seven which reduced or prevented lesion formation were identified using cultural, morphological, physiological, biochemical and cell wall characteristics as Streptomyces janthinus, S. cinerochromogenes, Streptoverticillium netropsis, Actinomadura rubra, Actinoplanes philippinensis, Micromonospora carbonaceae, and Streptosporangium albidum. All seven isolates tested produced non-volatile antifungal metabolites, but failed to produce inhibitory volatile compounds. Actinoplanes philippinensis and M. carbonacea grew epiphytically on the hyphae and oospores of P. coloratum. The external surface of the oospores of the pathogen was heavily colonized by both hyperparasites, their hyphae were found to coil tightly around the oospore wall, and frequently caused cytoplasmic collapse of oospores. Sporangia of A. philippinensis were often seen to emerge from the colonized hyphae and oospores of P. coloratum. None of the other actinomycete isolates showed hyperparasitism.

All seven isolates significantly reduced the incidence of cavity spot in soil artificially infested with the pathogen in the glasshouse. Streptomyces janthinus and Strepto. albidum were the most effective in reducing the disease in inoculated plants. In addition, all the actinomycetes species except Ac. rubra and M. carbonacea, in the presence or absence of the pathogen, significantly (P<0·05) increased mean fresh root weight compared to the treatment which included P. coloratum only. This study shows that these actinomycetes have considerable potential for future use as biocontrol agents of cavity spot under natural field conditions. This is the first report of cavity-spot disease of carrots being controlled by microbial antagonists, and is the first report of non-streptomycete actinomycetes to control a Pythium disease.

IAA production by the ectomycorrhizal fungus Paxillus involutus grown in pure culture was investigated by testing IAA accumulation or IAA-synthesizing activity. All the Paxillus involutus strains studied released indole compounds either when incubated with 0·5 mm tryptophan or in its absence. HPLC analyses of indole compounds all yielded two main peaks identified by reference to controls as being IAA and indole-3-carboxylic acid and a small peak of indole-3-aldehyde. Mass spectrometry unequivocally confirmed IAA production in the culture filtrates and in the incubation solution used to study in vitro IAA-synthesizing activity. The IAA-synthesizing activity was found to vary widely, from 7·6 to 137·4 nmol IAA h−1 g−1 f. wt, between strains. The mycorrhizal activity of these strains with seedlings of Pinus sylvestris cultured under aseptic conditions was tested. Fungal strains that were characterized in pure culture conditions by high IAA-synthesizing activity induced more fine roots and significantly higher numbers of mycorrhizas than strains which showed a low activity of IAA synthesizing enzymes. The increased ectomycorrhiza formation was generally accompanied by an increased growth of seedlings.

Seedlings of Pseudotsuga menziesii (Mirb.) Franco, Pinus ponderosa Dougl. ex Laws, Arbutus menziesii Pursh., and cuttings of Arctostaphylos uva-ursi (L.) Spreng were grown in monoculture and in conifer-hardwood dual-culture combinations in the glasshouse and inoculated with spore slurries of six Rhizopogon species. The primary objectives were to assess and compare the pattern of host specificity between symbionts and to study the influence of co-cultured plants on ectomycorrhiza development. The Rhizopogon spp. ranged from genus-specific to multiple-host compatible. In monoculture, four Rhizopogon sp. (R. ellenae Smith, R. occidentalis Zeller & Dodge, R. smithii Hosford and R. subcaerulescens Smith) formed ectomycorrhizas with Pinus ponderosa, and two Rhizopogon sp. (R. parksii Smith and R. vinicolor Smith) formed ectomycorrhizas with Pseudotsuga menziesii. None of the fungi tested developed ectomycorrhizas on Arbutus menziesii or Arctostaphylos uva-ursi in monoculture. In dual culture, three of the four Rhizopogon species (R. ellenae, R. occidentalis and R. subcaerulescens) that formed ectomycorrhizas on Pinus ponderosa, formed some ectomycorrhizas on Arbutus menziesii and Arctostaphylos uva-ursi. Rhizopogon parksii and R. vinicolor only formed ectomycorrhizas on Pseudotsuga menziesii.

Interspecific C transfer was studied in laboratory microcosms containing pairs of 6-month-old Betula papyrifera Marsh. and Pseudotsuga menziesii (Mirb.) Franco seedlings growing in individual, root-restrictive (28 μm pore size) pouches filled with field soil. Interspecific transfer was examined by reciprocal labelling of seedlings with 13CO2(gas) and 14CO2(gas). At the time of labelling, the root zones of ectomycorrhizal (EM) B. papyrifera and P. menziesii were interconnected by an extensive network of EM mycelium. Carbon transferred through EM connections was distinguished from that through soil pathways by comparing microcosms where interconnecting hyphae were left intact vs. those where they were severed immediately before labelling.

Transfer was bidirectional, and represented 5% of total isotope uptake by both B. papyrifera and P. menziesii together. P. menziesii received on average 50% more 14C and 66% more 13C from paper birch than vice versa, however, differences between species were not statistically significant. Neither net nor bidirectional transfer differed between severing treatments, leaving in question the relative importance of EM hyphae versus soil transfer pathways. The tendency for P. menziesii to receive more isotope than B. papyrifera corresponded with a 10-fold greater net photosynthetic rate per seedling and two-fold greater foliar N concentration of B. papyrifera than P. menziesii.

Copper and zinc tolerances of 10 micropropagated birch (Betula pendula and B. pubescens) clones were studied in hydroponic culture. Tolerance indices were determined, based on the mean growth rate of the longest root in 1 wk. A seed-derived clone (142A), from a lead/Zn-contaminated site showed more tolerance to Cu and Zn than bud-derived clones (HA02 and HA18) from a Cu/nickel-contaminated site or an ozone-tolerant clone (KL-2-M) from an uncontaminated area. For Cu, the EC50 values were 30, 14, 8 and 11 μm in clones 142A, HA02, HA18 and KL-2-M, respectively. For Zn, the EC50s were 4000 and 350 μm in clones 142A and KL-2-M, respectively. The relative Cu and Zn tolerances of the other clones were estimated by growing the plants in 30 μm CuSO4, and in 2000 or 350 μm ZnSO4, respectively. It is of interest that the Zn-tolerant clone 142A was tolerant to Cu, although this metal was present at a very low concentration in the soil where the parent tree grows. Another clone (142B), from another seed of the same parent tree, was tolerant neither to Zn nor Cu. Compared with their own EC50s for root growth for Cu, 142A took up more Cu than KL-2-M, suggesting that the higher tolerance of the former clone is not explained by reduced uptake of Cu. The Zn uptake in clones 142A and KL-2-M was studied at 4000 μm and 800 μm Zn, respectively. Interestingly, the roots of both clones contained the same amount of Zn, even though clone 142A was exposed to a fivefold concentration of Zn.

A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd2+, Pb2+, Zn2+ and Sb3−, measured as an inhibition of fungal biomass production. Isolates were grown in liquid media amended with the metals, individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd2+ and Zn2+ was recorded, while Pb2+ and Sb3− individually had little effect. The presence of Pb2+ and Sb3− in the media did however, ameliorate Cd2+ and Zn2+ toxicity in some circumstances. Interactions between Cd2+ and Zn2+ were investigated further over a range of concentrations. Zn2+ was found to significantly ameliorate the toxicity of Cd2+ to three of the four isolates tested. The influence of Zn2+ varied between ECM species and with the concentrations of metals tested.