Jozef A. Van Assche

Zinc toxicity in ectomycorrhizal Pinus sylvestris

Six strains of ectomycorrhizal fungi were compared for their ability to increase zinc tolerance in Pinus sylvestris L. seedlings. Pioneer and ‘late-stage’ fungi as well as one strain collected from a Zn-polluted site were included in the study. The accumulation of zinc in the host plants was determined at two different sublethal substrate Zn concentrations. The mycobionts varied considerably in their protection of the autobionts against zinc toxicity. Several fungal species reduced zinc accumulation in the pine seedlings. A Thelephora terrestris (Ehrh.) Fr. Strain, however, increased the Zn concentration in its host plants. Specific zinc-retaining capacity of the mycelium and density of the extramatrical mycelium were important features determining the effectiveness of the zinc retention in the fungal symbiont.The experiments were carried out in a growth chamber where plants were cultivated in root observation chambers. The determination of the extent of substrate colonization showed that an elevated zinc concentration in a substrate might be an important stress factor for the growth of an ectomycorrhizal fungus. The growth of the extramatrical mycelium of some species was inhibited at the highest zinc concentration supplied. A determination of the in vitro zinc tolerance of the fungi could not always predict their tolerance as symbionts, where the latter characteristic did not determine the Zn retention capacity of their mycelium.In this paper we also discuss the possibility that on Zn-polluted soils ectomycorrhizal species and strains are selected that are Zn-tolerant and, in addition, are able to protect their own energy source, the autobiont, from toxicity. These mycorrhizal fungi act as a safety net, that can immobilize large amounts of zinc, thus preventing transport to the host plant.

Reversible and irreversible activation ofPhycomyces blakesleeanus spores

Phycomyces spores do not germinate in a suitable culture medium unless they are activated by one of a range of treatments. Heating the spores for 3 min at 44°C resulted in nearly complete germination in culture medium; however, when such spores were incubated in water, rapid deactivation occurred. Deactivation of spores treated for 3 min at 50°C was much slower and only partial. After reversible activation (44°C) RNA and protein synthesis increased rapidly in culture medium but not in water. Immediately after irreversible activation (50°C) uridine and leucine incorporation was severely reduced but increased rapidly upon further incubation in both water and culture medium. Activation of spores at 44 or 50°C in culture medium had a similar effect on nucleoside triphosphate content: the ATP level was high and did not change markedly after activation; the UTP and GTP content, however, showed a clear-cut increase shortly after activation. Spores incubated in water had a much lower nucleoside triphosphate content but upon irreversible activation (50°C) the pattern of the nucleoside triphosphates was similar to that in culture medium. Reversible activation (44°C) of spores incubated in water yielded only a temporary increase in ATP level. The pattern of respiration was the same for reversibly and irreversibly activated spores in culture medium. During incubation in water, however, irreversibly activated spores had a higher respiration than reversibly activated spores. This suggests that the irreversibility of activation at 50°C was caused by the occurrence of the initial phases of germination, even in water, whereas after reversible activation external carbon sources were required to start germination and to maintain the activated state. Respiration was insensitive to azide in dormant spores but became progressively more sensitive during germination.

The effects of cadmium and the cadmium-zinc interaction on the axenic growth of ectomycorrhizal fungi

Eleven strains of ectomycorrhizal fungi belonging to seven species have been cultured on a cadmium-contaminated growth medium in order to determine their in vitro cadmium tolerance. Four strains were collected from a zinc and cadmium-polluted soil. Radial growth rate was a sensitive parameter to detect Cd toxicity. A wide differential response to Cd was obtained between the individual species. A clear relation between Cd tolerance and site origin of the isolates did not exist, although such a relationship was found when strains are compared within one species. Cd-sensitive and Cd-tolerant strains of Suillus bovinus were studied in more detail. Two isolates were grown on media with combinations of two non-toxic zinc concentrations and three cadmium levels. Adding a higher Zn concentration to the medium resulted in a reduction of the toxic effect of Cd. This antagonistic effect also resulted in a lowered Cd concentration in the mycelium.

The comparative germination ecology of nine Rumex species

The germination requirements, dormancy cycle and longevity of nine Rumexspecies were studied in field conditions and laboratory experiments to show theadaptations of the related species to their specific habitat. Within one genus,rather striking differences were observed in germination ecology. However, theclosely related species, R. acetosa and R.scutatus, are very similar: they fruit in early summer; theirseeds can germinate immediately after dispersal, and they are nondormant andshort-lived. R. acetosella also has fruits insummer, but the seeds do not germinate the first season after dispersal. Theyare long-lived, but buried seeds do not show a dormancy cycle; they mightgerminate in different seasons after exposure to light. Seeds of four species (R. conglomeratus,R. maritimus, R. sanguineus andR. crispus) are long-lived and undergo aseasonal dormancy cycle, with a low level of dormancy in winter and early springand a deep dormancy in summer as was already known for R.obtusifolius. These seeds are shed in the autumn, and they germinatemainly in the spring in consecutive years. R. maritimusalso germinates in summer and autumn on drying muddy soils. The seeds of R. hydrolapathum only germinate onwaterlogged soils, which explains its growth at the edge of streams and ponds.Its seeds are rather short-lived. The seeds of the species on very wetplaces require a higher temperature for germination.

Germination ecology of eleven species of Geraniaceae and Malvaceae , with special reference to the effects of drying seeds

Germination and survival of water-impermeable seeds of 11 species of Geraniaceae and Malvaceae were monitored during dry storage and during burial in soil for up to 2.5 years. During dry storage, seeds of annual Geraniaceae became permeable and also lost their physiological dormancy. However, during burial in natural conditions, most seeds remained impermeable and viable, with no seasonal change in germination capacity. Germination in only one species ( Geranium robertianum ) was enhanced by daily alternating temperatures when seeds were exhumed in spring. Drying of exhumed seeds broke physical dormancy. Seeds of the perennial Geranium pratense gradually became permeable in a prolonged germination test of 31 weeks. Most seeds of Malva remained impermeable during dry storage. Buried seeds gradually germinated in situ , and exhumed seeds had a low germination capacity in all seasons. We concluded that dormancy of hard seeds in natural conditions may be broken by drying during summer, by specific temperature regimes or by gradual softening of the seed coat, ensuring the spread of germination over many seasons.

Trehalose metabolism in dormant and activated spores of Phycomyces blakesleeanus Burgeff

Evidence is obtained for the existence of two different localizations of trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) in Phycomyces spores: one inside the cell, and one in the periplasmic region. The latter enzyme is sensitive to 0.1 mol l-1 HCl treatment and its activity can be regulated by external pH changes. The periplasmic form of the enzyme is involved in the metabolism of added labelled trehalose. This sugar is hydrolyzed externally to glucose which is found mainly in the incubation medium and which is partly absorbed by the spores. During incubation trehalose leaks out from both dormant and activated spores and is subsequently hydrolyzed to glucose. The intracellular trehalase is probably involved in the breakdown of endogenous trehalose in spores. After heat activation the hydrolysis of endogenous trehalose is stimulated even without an important increase in activity of intracellular trehalase. Additional treatments which break dormancy of spores without a significant activation of trehalase are the following: heating of HCl-treated spores and treatment of spores with reducing substances (e.g. Na2S2O4 and NaHSO3).

Germination ecology of the holoparasite Cuscuta epithymum

Little is known about the seed ecology of annual holoparasites, such as Cuscuta (dodders), and how germination is timed to occur when seedlings can attach to a host. Seeds of Cuscuta epithymum , which is a rare and often threatened species in dry heaths of north-western Europe, were water-impermeable (physically dormant) at maturity in late summer. An 8-week period at 5°C, followed by incubation at 23, 15/6 or 30/20°C, resulted in 15–30% germination. Thus, in nature, only part of the seed population exposed to low winter temperatures and a subsequent warm spring potentially becomes water-permeable and can germinate in spring. When scarified seeds were cold-stratified at 5°C for 8 weeks and then incubated at 23°C, essentially all the viable seeds ( c. 65%) germinated. It is concluded that seeds have a combination of physical (PY) and physiological (PD) dormancy (the first to be reported in the Convolvulaceae ), which is interpreted as a double safety mechanism preventing germination at unfavourable times or places. After PY and PD were broken, seeds did not require light or fluctuating temperatures for germination. Some loss of PD (afterripening) can occur before PY is broken. These data indicate that a portion of the seed crop remains physically dormant in spring and potentially forms a persistent seed bank, a strategy that eliminates the risk of a total reproductive failure in a particular bad year.

Multiple environmental signals required for embryo growth and germination of seeds of Selinum carvifolia (L.) L. and Angelica sylvestris L. ( Apiaceae )

Germination and dormancy breaking requirements were studied in Selinum carvifolia (L.) L. and Angelica sylvestris L. ( Apiaceae ). Seeds of these two species have an underdeveloped embryo and are morpho-physiologically dormant. The embryo does not start to grow until physiological dormancy is broken by cold stratification. Incubating seeds at fluctuating temperatures in the light, after cold stratification, had a stimulating effect on embryo growth and seed germination. Seeds of S . carvifolia and A . sylvestris have non-deep simple morphophysiological dormancy (MPD), since gibberellic acid (GA 3 ) could substitute for cold stratification. This is the first report of non-deep simple MPD that is broken by cold stratification in the Apiaceae . Under natural conditions, physiological dormancy is broken by low temperature conditions during winter. Embryo growth and germination occur in a short time interval when temperatures start rising in early spring. Due to the fact that multiple environmental signals regulate dormancy, seedling emergence in these species is timed very accurately in spring.

The effect of gamma radiation on breaking of dormancy in Phycomyces spores

Dormant sporangiospores of Phycomyces blakesleeanus were activated by Cs-137 gamma rays. After a dose of about 300 krad, between 80 and 90% of the spores germinated normally. However, further development of the mycelium was inhibited. Even with dry spores a partial activation was obtained. Activation by gamma rays was not accompanied by an increase in trehalase activity.

Morphological and physiological dormancy in seeds of Aegopodium podagraria ( Apiaceae ) broken successively during cold stratification

A low-temperature requirement for dormancy break has been observed frequently in temperate-climate Apiaceae species, resulting in spring emergence of seedlings. A series of experiments was performed to identify dormancy-breaking requirements of Aegopodium podagraria , a nitrophilous perennial growing mainly in mildly shaded places. In natural conditions, the embryos in seeds of A. podagraria grow in early winter. Seedlings were first observed in early spring and seedling emergence peaked in March and April. Experiments using temperature-controlled incubators revealed that embryos in seeds of A. podagraria grow only at low temperatures (5°C), irrespective of a pretreatment at higher temperatures. Seeds did not germinate immediately after embryo growth was completed, instead an additional cold stratification period was required to break dormancy completely. Once dormancy was broken, seeds germinated at a range of temperatures. Addition of gibberellic acid (GA 3 ) had a positive effect on embryo growth in seeds incubated at 10°C and at 23°C, but it did not promote germination. Since seeds of A. podagraria have a low-temperature requirement for embryo growth and require an additional chilling period after completion of embryo growth, they exhibit characteristics of deep complex morphophysiological dormancy.