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.     

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.     

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.     

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.     

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.     

<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.     

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.     

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.     

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.     

The Responses of Pro‐ and Antioxidative Systems to Cold‐hardening and Pathogenesis Differ in Triticale (x Triticosecale Wittm.) Seedlings Susceptible or Resistant to Pink Snow Mould (Microdochium nivale Fr., Samuels & Hallett)

Two winter triticale (x Triticosecale Wittm.) cultivars, Magnat (susceptible to pink snow mould) and Hewo (relatively resistant), were used in a model system to test the effect of prehardening and different cold‐hardening regimes on pro‐ and antioxidative activity in seedling leaves. The concentration of hydrogen peroxide and the activity of total superoxide dismutase, catalase, peroxidase and ascorbic peroxidase were analysed spectrophotometrically. As there has been no previous analysis of the pro/antioxidative reaction of cereals to Microdochium nivale infection has been undertaken to‐date, this is the first in the series describing our results. We confirmed that both exposure to abiotic stress of low temperature and subsequent low light intensity, as well as biotic stress of M. nivale infection, change the pro‐ and antioxidative activity in model plants. Genotypes differed substantially in their hydrogen peroxide content: susceptible cv. Magnat generally showed higher levels during all the experiments. This result can lead to the conclusion that cv. Magnat is also more susceptible to low temperature and low light intensity than cv. Hewo. Simultaneous measurements of antioxidative activity indicated that the increased activity of catalases and peroxidases and the consequent lower H₂O₂ level are correlated with a higher resistance to low temperature, low light intensity and pink snow mould in triticale seedlings. The higher H₂O₂ level observed in the susceptible line is likely to be derived from the imbalance of reactive oxygen species production and consumption in this genotype under stress conditions.     

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.     

Local and systemic regulation of PSII efficiency in triticale infected by the hemibiotrophic pathogen Microdochium nivale

Microdochium nivale is a fungal pathogen that causes yield losses of cereals during winter. Cold hardening under light conditions induces genotype‐dependent resistance of a plant to infection. We aim to show how photosystem II (PSII) regulation contributes to plant resistance. Using mapping population of triticale doubled haploid lines, three M. nivale strains and different infection assays, we demonstrate that plants that maintain a higher maximum quantum efficiency of PSII show less leaf damage upon infection. The fungus can establish necrotrophic or biotrophic interactions with susceptible or resistant genotypes, respectively. It is suggested that local inhibition of photosynthesis during the infection of sensitive genotypes is not balanced by a supply of energy from the tissue surrounding the infected cells as efficiently as in resistant genotypes. Thus, defence is limited, which in turn results in extensive necrotic damage. Quantitative trait loci regions, involved in the control of both PSII functioning and resistance, were located on chromosomes 4 and 6, similar to a wide range of PSII‐ and resistance‐related genes. A meta‐analysis of microarray experiments showed that the expression of genes involved in the repair and de novo assembly of PSII was maintained at a stable level. However, to establish a favourable energy balance for defence, genes encoding PSII proteins resistant to oxidative degradation were downregulated to compensate for the upregulation of defence‐related pathways. Finally, we demonstrate that the structural and functional integrity of the plant is a factor required to meet the energy demand of infected cells, photosynthesis‐dependent systemic signalling and defence responses.     

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.     

Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary

Summer frost resistance and ice nucleation temperatures for 33 alpine plant species were measured in situ to avoid the shortcomings of laboratory tests. Species were selected to investigate the relationship between plant stature and upper distribution boundary, and frost resistance and freezing patterns. The species tested in situ were on average 1.1 K (± 0.2, SE) frost hardier than in laboratory tests. Frost resistance (LT₅₀) ranged from ?4.5 to ?14.6 °C and appeared insufficient to protect against air temperature minima, corroborating reports of natural frost damage. All species tolerated extracellular ice formation (recorded at ?1.9 ± 0.2 °C; E1). Initial frost damage occurred at average temperatures 4.9 K below E1. In 64% of the species a second exotherm (E2) and frost damage were recorded between ?3.7 and ?9.4 °C. In the highest ranging species E2 was not detectable. Frost resistance increased with increasing upper distribution boundary (0.4 K per 100 m), corresponding well with the altitudinal decrease in air temperature minima. No relationship between plant stature and frost resistance was found. Graminoids were significantly frost hardier than other growth forms. Frost survival at high altitudes will depend not only on altitudinal increase in frost resistance but also on freezing avoidance strategies, snow cover protection and a high recuperation capacity.     

Benzoxazinoid concentrations show correlation with Fusarium Head Blight resistance in Danish wheat varieties

Fusarium Head Blight (FHB) is a destructive disease that affects the grain yield and quality of cereals. The relationship between the natural defense chemicals benzoxazinoids and the FHB resistance of field grown winter wheat varieties was investigated. FHB resistance was assessed by the inoculation of wheat ears with mixtures of Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, and Microdochium nivale.     

Analysis of Microdochium nivale isolates from wheat in the UK during 1993

A total of 144 isolates of Microdochium nivale from stem bases of winter wheat were taken from 30 sites and 91 isolates from grain were taken from seven sites throughout the UK. Identification by polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region followed by restriction enzyme digestion of the PCR product revealed that 70% of stem base isolates were M. nivale var. majus and 30% var. nivale while 93% grain isolates were var. majus and 7% var. nivale. Almost all isolates were resistant to the benzimidazole fungicide benomyl. Perithecial production in vitro was more common in var. majus isolates and occurred in almost all grain isolates, but was less common in stem base isolates. The implications of the findings in terms of epidemiology and chemical control of this important cereal pathogen are discussed.     

Identification of the extracellular polysaccharide produced by the snow mold fungus Microdochium nivale

A water-insoluble, extracellular polysaccharide was isolated from the culture medium of the snow mold fungus, Microdochium nivale, that had been cultivated in potato/dextrose broth. The polysaccharide consisted of glucose only. Its Fourier transform infrared spectrum showed a beta configuration of the C1 position of glucose. Linkage analysis of the polysaccharide showed that it had a linear structure of -(14)-linked glucose. The polysaccharide was therefore identified as cellulose. This is the first report of extracellular cellulose occurring in fungi.     

A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, Microdochium nivale

A novel cold-induced cystatin cDNA clone (TaMDC1) was isolated from cold acclimated winter wheat crown tissue by using a macroarray-based differential screening method. The deduced amino acid sequence consisted of a putative N-terminal secretory signal peptide of 37 amino acids and a mature protein (mTaMDC1) with a molecular mass of 23 kDa. The mTaMDC1 had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited partial similarity to the cystatin domain. The recombinant mTaMDC1 was purified from Escherichia coli and its cysteine proteinase inhibitory activity against papain was analyzed. The calculated Ki value of 5.8×10⁻⁷ M is comparable to those reported for other phytocystatins. Northern and western blot analyses showed elevated expression of TaMDC1 mRNA and protein during cold acclimation of wheat. In addition to cold, accumulation of the TaMDC1 message was induced by other abiotic stresses including drought, salt and ABA treatment. Investigation of in vitro antifungal activity of mTaMDC1 showed strong inhibition on the mycelium growth of the snow mold fungus Microdochium nivale. Hyphae growth was totally inhibited in the presence of 50 μg/ml mTaMDC1 and morphological changes such as swelling, fragmentation and sporulation of the fungus were observed. The mechanisms of the in vitro antifungal effects and the possible involvement of TaMDC1 in cold induced snow mold resistance of winter wheat are discussed.     

Snow-Mold-Induced Apoplastic Proteins in Winter Rye Leaves Lack Antifreeze Activity

During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.