β-1,3-glucanase and chitinase activities in winter triticales during cold hardening and subsequent infection by Microdochium nivale

The accumulation of pathogenesis-related proteins such as β-1,3-glucanases and chitinases was studied in cold induced snow mould resistance in two Polish cultivars of winter triticale, cv. Hewo and cv. Magnat that substantially differ in resistance to Microdochium nivale. The plants were pre-hardened at 12°C for 10 days and hardened at 4°C for 28 days. Subsequently, cold hardened plants were inoculated with fungal mycelium (M. nivale) and incubated at 4°C for 7 days in dark. Cold acclimatisation resulted in suppression of the total glucanase and chitinases activities in the resistant Hewo as well as sensitive Magnat cultivars that possibly coincides with altered metabolism. However, upon infection with M. nivale the chitinases were markedly induced in the cv. Hewo. At the same time, total β-1,3 glucanases activities did not seem to be affected by fungus in any of the tested triticale cultivars. The pattern and/or the activity of chitinases in plants might be indicative for the resistance/susceptibility against M. nivale.     

Cold-hardening of winter triticale (<Emphasis Type="Italic">x Triticosecale</Emphasis> Wittm.) results in increased resistance to pink snow mould <Emphasis Type="Italic">Microdochium nivale</Emphasis> (Fr., Samuels &; Hallett) and genotype-dependent chlorophyll fluorescence modulations

The resistance of triticale (x Triticosecale Wittm.) to infection of snow mould Microdochium nivale (Fr., Samuels & Hallett) was examined under different temperature pre-treatment regimes. The results of laboratory “cold chamber” resistance tests correlated with the breeders’ report from field experiments. Studied genotypes differed substantially in their resistance to infection. Two cultivars: ‘Magnat’ (susceptible) and ‘Hewo’ (relatively resistant) were further studied as a plant model to test the role of pre-hardening and cold-hardening induction of resistance expression. Both model cultivars were susceptible to M. nivale infection without cold pre-treatment and gained genotype-depended level of resistance after 4 weeks treatment at 4°C, moreover the resistance grew gradually. Simultaneously to the resistance tests, the measurements of chlorophyll fluorescence parameters were taken. The results showed that higher vitality index Rfd of cold-hardened triticale seedlings correlated with increased pink snow mould resistance while differences in other parameters of fluorescence were not distinctly significant. Establishment of Rfd in 4 weeks hardened triticale seedlings could be used for a large scale screening of breeding material in order to select potentially resistant genotypes. Such analyses have not been reported for triticale before.     

Physiological Basis for Differences in Resistance to Microdochium nivale (Fr.) Samuels and Hallett in Two Androgenic Genotypes of Festulolium Derived from Tetraploid F1 Hybrids of Festuca pratensis × Lolium multiflorum (Festulolium)

The aim of this study was to investigate the physiological basis for differences in resistance to pink snow mould (Microdochium nivale) in two androgenic genotypes of Festulolium (Festuca pratensis × Lolium multiflorum) which differed in terms of their resistance to M. nivale. Genotype 716 was more resistant than genotype 729. The study consisted of two experiments. The aim of the first experiment was to estimate the ability of the plants to survive winter conditions. The aim of the second experiment was to find physiological markers of resistance to snow mould. Festulolium plants were infected with M. nivale mycelium after pre‐hardening and hardening. After 2 weeks in the dark at 2°C, there was a sharp increase in the phenolic content in both genotypes. The increase was greater in the more resistant genotype 716 than in genotype 729. Phenolics therefore may play a very important role in overwintering in grasses, similar to carbohydrates. Based on the differences between the two genotypes, potential indicators of resistance to M. nivale in Festulolium include increased soluble carbohydrate content, increased phenolic content, increased hydrogen peroxide accumulation, decreased catalase activity, increased abscisic acid content and reduced heat emission.     

<Emphasis Type="Italic">Microdochium nivale</Emphasis> (Fr., Samuels &; Hallett): cytological analysis of the infection process in triticale (×<Emphasis Type="Italic">Triticosecale</Emphasis> Wittm.)

According to regular reports, one of the most serious diseases of winter cereal and grass varieties in moderate and cold climatic areas is pink snow mould caused by Microdochium nivale. Currently, the resistance of the economically important cereal species as triticale is not satisfactory. Moreover, there is no efficient strategy of protection against this pathogen and the understanding of plant resistance mechanisms is rather poor. Presented paper for the first time shows the cytological analysis of M. nivale infection in model triticale varieties by the use of fluorescent and light microscopy in combination with fluorescent dyes and hydrogen peroxide staining. Both, the infection level and the dynamic of the process varied for tested genotypes confirming the field and laboratory data of their different resistance to this pathogen. Moreover, our analysis showed that in both cultivars cold-hardening of seedlings delayed the mycelium growth. In both cultivars, hyphal walls and fungal penetration sites were visualized in crowns, leaf sheaths and leaves of hardened and non-hardened inoculated seedlings. For the first time the presence of the haustoria produced by M. nivale was confirmed in those tissues. Single infection hyphae usually penetrated into the host tissues via stomatal apparatuses were accompanied by the efflux of hydrogen peroxide. The data show a great potential of fluorescence techniques in studying the host plant–pathogen interactions providing a better insight into plant defence reactions that may allow elaboration of the efficient breeding strategies aimed at increasing resistance to this pathogenic fungus.     

Metabolites from <Emphasis Type="Italic">Pseudomonas brassicacearum</Emphasis> with activity against the pink snow mould causing pathogen <Emphasis Type="Italic">Microdochium nivale</Emphasis>

Bioassay-guided fractionation of cell-free culture supernatants of the bacterium Pseudomonas brassicacearum MA250 yielded three bioactive compounds (1–3). Compound 1 was identified as the unsaturated fatty acid γ-lactone piliferolide A, compound 2 as the not previously described open acid form of 1, and 3 as the compound SB-253514, which is an imide of a 3-O-rhamnosyl fatty acid and a bicyclic carbamate. All three compounds displayed moderate activity towards the pink snow mould causing pathogen Microdochium nivale, and may thus contribute to the previously observed biological control of this strain on M. nivale on wheat. Compound 1 further exhibited activity towards the human pathogen Aspergillus fumigatus, while compound 3 showed antifungal as well as antibacterial activity.     

Isolates of Microdochium nivale and M. majus Differentiated by Pathogenicity on Perennial Ryegrass (Lolium perenne L.) and in vitro Growth at Low Temperature

Pink snow mould is a serious disease on grasses and winter cereals in cold and temperate zones during winter. To better understand the basis for the variation in pathogenicity between different isolates of Microdochium nivale and M. majus and to simplify selection of highly pathogenic isolates to use when screening for resistance to pink snow mould in perennial ryegrass, we sought traits correlated with pathogenicity. Isolates of M. nivale were more pathogenic on perennial ryegrass than isolates of M. majus, as measured by survival and regrowth of perennial ryegrass after infection and incubation under simulated snow cover. Pathogenicity as measured by relative regrowth was highly correlated with fungal growth rate on potato dextrose agar (PDA) at 2°C. Measuring fungal growth on PDA therefore seems to be a relatively simple method of screening for potentially highly pathogenic isolates. In a study of a limited number of isolates, highly pathogenic isolates showed an earlier increase and a higher total specific activity of β‐glucosidase, a cell wall‐degrading enzyme, compared with less pathogenic isolates. None of the M. majus isolates was highly pathogenic on perennial ryegrass. Our results indicate biological differences between M. nivale and M. majus and thus strengthen the recently published sequence‐based evidence for the elevation of these former varieties to species status.     

Production of low temperature active lipase from the pink snow mold, Microdochium nivale (syn. Fusarium nivale)

Summary The pink snow mold, Microdochium nivale (syn. Fusarium nivale) SUF 1377 strain produced an extracellular low temperature active lipase during growth at 4°C. The lipase had the highest activity at 20°C, and retained 19% of its maximum activity at 0°C.     

Effect of Microdochium nivale and Low Temperature on Winter Survival of Perennial Ryegrass

Winter survival of perennial ryegrass (Lolium perenne L.) depends mainly on the plant resistance to low freezing temperature and to snow mould fungi (Microdochium nivale). Field evaluation of these plant characters gave irreproducible results. A comparison of field trials with laboratory tests pointed to the close correlation between the yield level of examined varieties and strains and their resistance to Microdochium nivale and frost. A lack of correlation between snow mould resistance and frost tolerance was shown.     

The Effect of Chitosan and Bion on Resistance to Pink Snow Mould in Perennial Ryegrass and Winter Wheat

The effects of chitosan on resistance to pink snow mould (Microdochium nivale) were studied in young winter wheat (Triticum aestivum L.) and perennial ryegrass (Lolium perenne L.) under controlled environmental conditions. In perennial ryegrass, the putative defence activator Bion was also tested. Resistance was measured as regrowth of plants after inoculation with M. nivale and incubation in darkness at 2°C. In winter wheat, pre‐treatment with chitosan at 1000 μg per plant increased resistance to subsequent infection by M. nivale, but this effect was less significant in a replicate experiment. Chitosan‐treated winter wheat plants expressed the gene for the pathogenesis‐related protein chitinase at higher levels than non‐treated plants. Chitinase gene expression was also stimulated by M. nivale infection in winter wheat. Perennial ryegrass pre‐treated with Bion or chitosan and inoculated with M. nivale did not display better regrowth after incubation than non‐treated, inoculated plants. Rather, regrowth was reduced in some of the Bion‐treated plants after incubation. We speculate that the cost or the mechanism of induced resistance makes Bion non‐effective in plants that are not actively growing. Bion at concentrations of 10, 100 and 1000 μg active ingredient per ml, and the highest concentration of chitosan used (2000 μg per ml) reduced in vitro growth of the pathogen, suggesting that both defence activators possess antifungal activity.     

Growth and morphology variation as a response to changing environmental factors in two Arctic species of Raphidonema (Trebouxiophyceae) from snow and soil

Two Arctic species of Raphidonema, a weakly filamentous green alga, were compared on the basis of their growth and morphology response to varied environmental factors. The strain of R. nivale was isolated from snow and R. sempervirens from soil collected in Svalbard. Morphological variability was evaluated in both strains, with emphasis laid on pleiomorphism of R. nivale. Growth characteristics in varied temperature and light were compared. Field observations were performed in summer 2003 at Werenskiold glacier, southwest Spitsbergen. In both strains, temperature and light were found to affect the shape and size of the cells, and in R. nivale also the number of cells in filament, probably by modifying growth and division rates. The strain of R. nivale grew best at 12°C and 200 mol m^–2 s^–1, R. sempervirens at 18°C and 50 mol m^–2 s^–1. In snow, R. nivale only appeared after katabatic wind had brought soil on the snow surface, and its concentrations decreased very rapidly. Cells of R. nivale in snow showed great signs of damage. On the basis of obtained results, it is suggested here that R. nivale is a soil species well adapted to soil environment, and only occasionally brought on snow.     

Components of Pink Snow Mould Resistance in Winter Wheat are Expressed Prior to Cold Hardening and in Detached Leaves

Resistance to pink snow mould, caused by Microdochium nivale, was investigated in four resistant winter wheat lines from the USDA World Cereal Collection (CI9342, CI14106, PI173440 and PI181268) and three Nordic wheat lines (Bjørke, Rida and V1004). Pink snow mould resistance was tested in non‐hardened and cold‐hardened plants incubated under artificial snow cover and in detached leaf segments mounted on water agar and incubated at either 3°C in darkness or at room temperature with light during the day. The wheat lines CI9342, CI14106 and PI181268 were more resistant than the Nordic lines, both before and after cold hardening. Thus, although cold hardening strongly increases the level of snow mould resistance in all the wheat lines, some resistance mechanisms are also present prior to cold hardening in some of the resistant lines. CI9342, CI14106 and PI181268 also had a higher level of resistance than the other lines in the detached leaf assay, indicating that these lines have some resistance mechanisms acting in the leaves. The resistance of PI173440 was expressed only in intact hardened plants and not in non‐hardened plants or in detached leaves. This indicates that this line relies on cold hardening‐related changes in the crown for its resistance. In the detached leaf assay the rate of lesion development varied greatly between leaves of different order. The highest correlation with the whole plant test was obtained when using secondary leaves and incubation at 3°C in the dark.     

Cold acclimation of forage grasses in relation to pink snow mould (Microdochium nivale) resistance

The aim of our work is to investigate the changes in phenolic level, PAL activity and heat production rate induced during pre-hardening at 12°C and cold acclimation at 2°C of the forage grasses Festulolium, meadow fescue, tall fescue and Italian ryegrass in relation to their resistance to snow mould caused by Microdochium nivale. Meadow fescue and tall fescue were most resistant to M. nivale infection, while Italian ryegrass demonstrated the least resistance to this fungus inoculation. Festulolium, meadow fescue and tall fescue responded similarly to low temperature, while Italian ryegrass demonstrated considerable disturbance of energy balance and lower phenolic concentration, which could explain a higher susceptibility of the latter species to infection by M. nivale. The enhanced level of phenolic compounds, probably utilised for cell wall lignification as well as equilibrium of the metabolic activity observed in meadow fescue and tall fescue, is very important for both cold and pathogen-resistance mechanisms. The studied Festulolium cultivar ‘Felopa’, a hybrid of the Lolium multiflorum and Festuca pratensis genomes, was characterised by changes in biochemical parameters similar to the resistant meadow fescue and tall fescue.     

Biological control of snow mould (Microdochium nivale) in winter cereals by Pseudomonas brassicacearum, MA250

Seed lots of winter wheat and rye, naturally infested with Microdochium nivale and Fusarium spp., were treated with an isolate of Pseudomonas, which was recovered from roots of Brassica napus. Seeds were treated with bacterial fermentate and dried before sowing or they were directly sprayed in the furrow-opener at the moment of sowing. Besides field experiments, parallel climate chamber bioassays were performed to assess the effect of bacterial treatment on snow mould caused by seed-borne M. nivale and Fusarium spp. The biocontrol effect was assessed by plant density counts and by measuring yield. Significant biocontrol activity, measured by plant density counts, was detected both in field and climate chamber experiments sown with wheat. Biocontrol effect after spray application at sowing was less pronounced, although a slight increase in plant density was observed. The cell concentration required to obtain adequate biocontrol effect was 10⁹ CFU per ml for the dose used. The bacterial isolate was identified by 16S rDNA sequencing and biochemical tests as a Pseudomonas brassicacearum strain.     

Carbon and nitrogen partitioning during the post‐anthesis period is conditioned by N fertilisation and sink strength in three cereals

Further knowledge of the processes conditioning nitrogen use efficiency (NUE) is of great relevance to crop productivity. The aim of this paper was characterise C and N partitioning during grain filling and their implications for NUE. Cereals such as bread wheat (Triticum aestivum L. cv Califa sur), triticale (× Triticosecale Wittmack cv. Imperioso) and tritordeum (× Tritordeum Asch. & Graebn line HT 621) were grown under low (LN, 5 mm NH₄NO₃) and high (HN, 15 mm NH₄NO₃) N conditions. We conducted simultaneous double labelling (¹²CO₂ and ¹⁵NH₄¹⁵NO₃) in order to characterise C and N partitioning during grain filling. Although triticale plants showed the largest total and ear dry matter values in HN conditions, the large investment in shoot and root biomass negatively affected ear NUE. Tritordeum was the only genotype that increased NUE in both N treatments (NUE_total), whereas in wheat, no significant effect was detected. N labelling revealed that N fertilisation during post‐anthesis was more relevant for wheat and tritordeum grain filling than for triticale. The study also revealed that the investments of C and N in flag leaves and shoots, together with the ‘waste’ of photoassimilates in respiration, conditioned the NUE of plants, and especially under LN. These results suggest that C and N use by these plants needs to be improved in order to increase ear C and N sinks, especially under LN. It is also remarkable that even though tritordeum shows the largest increase in NUE, the low yield of this cereal limits its agronomic value.     

Auswinterungsschäden in Raigras-Reinbeständen, die Bedeutung pilzlicher Krankheitserreger und ihre mögliche Eindämmung

Winterkilling in puresown ryegrass, the importance of fungal pathogens and possibilities for their control I. Results from field trials with natural infection Rotten plants, incomplete stands, a reduced number of shoots and a decline in dry matter yield were observed after hibernations of perennial and Italian ryegrass. Isolations from damaged plants showed that the main pathogen was the causal agent of snow mould Monographella nivalis (Schaffn.) E. Müll., asexual stage Gerlachia nivalis (Ces. ex Sacc.) W. Gams et E. Müll., synonym Fusarium nivale Ces. ex Sacc. The following fungi were also isolated: Laetisaria fuciformis(Mc Alp.) Burds., synonym Corticium fuciforme (Berk.) Wakef., Gibberella avenacea Cook, asexual stage Fusarium avenaceum (Fr.) Sacc. and Fusarium culmorum (W. G. Sm.) Sacc. The field trials were located at 440 and 630 m a.s.l. These elevations correspond to plain and mid-mountain climates, respectively. The tetraploid varieties ‘Lipo’, ‘Tetila’, ‘Citadel’ and ‘Bastion’ showed a higher level of resistance to snow mould compared to the diploid varieties ‘Ursus’, ‘Lemtal’, ‘Melino’ and ‘Pablo’. Tetraploid ryegrasses contaminated the soil with Gerlachia nivalis to a lesser extend than diploid ryegrasses. The influence of the date of cutting and of nitrogen fertilization in autumn on snow mould was also tested. Lowest values at both locations were observed when the last cutting occurred at the end of October. Severity was higher when the last cutting, took place earlier (mid September, end of September, mid October). Since parts of stands of the last cutting were strongly damaged by the cold, last cutting should take place in the middle of October, although infection with snow mould will not be as much reduced as if the last cutting was done at the end of October. The plant rotting caused by Gerlachia nivalis was increased with 80 kg N/ha in autumn compared with 40 kg N/ha.     

Susceptibility to take‐all of cereal and grass species,and their effects on pathogen inoculum

Two field trials were conducted to investigate different herbage grasses and cereals for their susceptibility to the disease take‐all, for their impact on concentrations of the pathogen, Gaeumannomyces graminis var. tritici (Ggt), in soil and for their effect on development of take‐all in a subsequent wheat crop. In the herbage grass trial, Bromus willdenowii was highly susceptible to Ggt, produced the greatest post‐senescence Ggt concentrations in soil and highest incidence of take‐all in following wheat crop. Lolium perenne, Lolium multiflorum and Festuca arundinacea supported low Ggt soil concentrations and fallow the least. The relationship between susceptibility to Ggt and post‐senescence concentrations in soil differed between pasture grasses and cereals. In a trial in which Ggt was added to half the plots and where wheat, barley, triticale, rye or fallow were compared, the susceptibility of the cereals to take‐all was not clearly linked to post‐harvest soil Ggt concentrations. In particular, triticale and rye had low and negligible take‐all infection respectively, but greater post‐harvest soil Ggt concentrations than barley or wheat. This indicates that low Ggt concentrations on roots may build up during crop senescence on some cereals. Soil Ggt concentrations were greater following harvest in inoculated plots sown to cereals, but in the second year there was more take‐all in the previously non‐inoculated than inoculated plots. Thus, the grass and cereal species differed in susceptibility to take‐all, in their impact on Ggt multiplication and in associated take‐all severity in following wheat crop.     

Phytotoxity and pathogenicity of Fusarium nivale towards cereal seelings

Strong phytotoxic activity towards whew seedling has been derected in culatural extracts of 12 Fusarium nivale strains. The exmined isolates have been found strong pathogens of rye. triticale and wheat seedlings. However, their phytotoxity was not corrclued with their pathogenicity.     

Obligatory short‐day plant,Perilla frutescens var. crispa can flower in response to low‐intensity light stress under long‐day conditions

An obligatory short‐day plant, Perilla frutescens var. crispa was induced to flower under long‐day conditions when grown under low‐intensity light (30 µmol m^?2 s^?1). Plant size was smaller under lower light intensity, indicating that the low‐intensity light acted as a stress factor. The phenomenon is categorized as stress‐induced flowering. Low‐intensity light treatment for 4 weeks induced 100% flowering. The plants responded to low‐intensity light immediately after the cotyledons expanded, and the flowering response decreased with increasing plant age. The induced plants produced fertile seeds, and the progeny developed normally. The plants that flowered under low‐intensity light had greener leaves. This greening was because of the decrease in anthocyanin content, and there was a negative correlation between the anthocyanin content and percent flowering. Treatment with L‐2‐aminooxy‐3‐phenylpropionic acid, an inhibitor of phenylalanine ammonia‐lyase (PAL), did not induce flowering under non‐inductive light conditions and inhibited flowering under inductive low‐intensity light conditions. The metabolic pathway regulated by PAL may be involved in the flowering induced by low‐intensity light.     

Impact of fusaproliferin on primary roots of maize cultivars differing in their susceptibility to <Emphasis Type="Italic">Fusarium</Emphasis>

Effects of fusaproliferin (FUS) on membrane potential (E _M), electrolyte leakage, enzymes activity and respiration of roots, were studied in two maize cultivars (Zea mays L.), differing in their susceptibility to this toxin. In short-term experiments (≤ 6 h), E _M has been rapidly and significantly depolarized by FUS. The rapidity of E _M depolarization in tolerant cv. Lucia was more expressive in comparison with susceptible cv. Pavla, but the extent of E _M depolarization was higher in cv. Pavla. In both maize cultivars, higher depolarization of E _M was registered in cells of root zone I. In long-term experiments after the first E _M depolarization, which occurred during the first 6 h of FUS treatment, gradual depolarization continued up to 24 h and was represented not only by the active component (E _P) but also by the passive component (E _D) of E _M. The decrease in E _M and E _D was followed by a loss of K⁺ ions from FUS treated roots of both cultivars. The leak of K⁺ ions from the root cells of both root zones as well as both maize cultivars increased with the time of FUS treatment and was significantly higher in susceptible cv. Pavla than in tolerant cv. Lucia. FUS treatment of maize roots resulted in a significant decrease of root respiration which was higher in susceptible cv. Pavla than in tolerant cv. Lucia.     

Photosynthetic and phenological responses of dwarf shrubs to the depth and properties of snow

Snow is known to have a major impact on vegetation in arctic ecosystems, but little is known about how snow affects plants in boreal forests, where the snowpack is uneven due to canopy impact. The responses of two dwarf shrubs, the evergreen Vaccinium vitis‐idaea and the deciduous V. myrtillus, to snow conditions were studied in a snow manipulation experiment in southern Finland. The thermal insulation of the snowpack was expected to decrease with partial removal or compression of the snow, while addition of snow was expected to have the opposite effect. The penetration of light was manipulated by partial removal of snow or by formation of an artificial ice layer in the snowpack. CO₂ exchange measurements that were carried out at the time of maximum snow depth in late March indicated significant photosynthetic activity in the leaves of V. vitis‐idaea under snow. Net gain of CO₂ was observed in the daytime on all the manipulation plots, excluding the snow addition plots, where light intensity was very low. The subnivean photosynthesis compensated for a substantial proportion (up to 80%) of the respiratory CO₂ losses. Chlorophyll fluorescence measurements indicated reduced potential capacity of photosystem II in the leaves of V. vitis‐idaea on those plots where snow cover was thin. Neither V. vitis‐idaea nor V. myrtillus suffered from frost damage (assessed as electrolyte leakage) when thermal insulation was reduced by means of snow manipulations. No phenological responses were observed in V. vitis‐idaea, but in V. myrtillus bud burst, leaf unfolding and flowering were advanced by 1–3 days on the addition plots. The results of the present study show that dwarf shrubs respond to not only the thickness of snow but also the physical properties of snow, both of which are expected to change due to climatic warming.