Nivale Infection Research Articles

Cold-modulated leaf compounds in winter triticale DH lines tolerant to freezing and Microdochium nivale infection: LC-MS and Raman study.

Tolerance to freezing and seedling diseases caused by Microdochium spp. is an essential trait for the wintering of triticale (×Triticosecale Wittmack) and other cereals. Preceding multi-year studies indicate that after long-term exposure to the low temperature, cereal seedlings acquire a genotype-dependent cross-tolerance to other subsequent stresses. This paper presents the first non-gel protein profiling performed via high performance liquid chromatography coupled with Mass Spectrometry as well as Fourier Transform-Raman spectroscopy measurements performed directly on leaves of triticale seedlings growing under different conditions. The research used doubled haploid lines selected from the mapping population, with extreme tolerance/susceptibility to freezing and M. nivale infection. These non-targeted methods led to the detection of twenty two proteins cold-accumulated in the most tolerant seedlings in relation to susceptible ones, classified as involved in protein biosynthesis, response to different stimuli, energy balancing, oxidative stress response, protein modification, membrane structure and anthocyanin synthesis. Additionally, in seedlings of the most freezing- and M. nivale -tolerant line, cold-hardening caused decrease of the carotenoid and chlorophyll content. Moreover, a decrease in the band intensity typical for carbohydrates as well as an increase in the band intensity characteristic for protein compounds were detected. Both studied lines revealed a different answer to stress in the characteristics of phenolic components.

Alterations in the Transcriptome of Rye Plants following the Microdochium nivale Infection: Identification of Resistance/Susceptibility-Related Reactions Based on RNA-Seq Analysis.

Microdochium nivale is a progressive and devastating phytopathogen that causes different types of cereal crop and grass diseases that are poorly characterized at the molecular level. Although rye (Secale cereale L.) is one of the most resistant crops to most of the phytopathogens, it is severely damaged by M. nivale. The recent high-quality chromosome-scale assembly of rye genome has improved whole-genome studies of this crop. In the present work, the first transcriptome study of the M. nivale-infected crop plant (rye) with the detailed functional gene classification was carried out, along with the physiological verification of the RNA-Seq data. The results revealed plant reactions that contributed to their resistance or susceptibility to M. nivale. Phytohormone abscisic acid was shown to promote plant tolerance to M. nivale. Flavonoids were proposed to contribute to plant resistance to this pathogen. The upregulation of plant lipase encoding genes and the induction of lipase activity in M. nivale-infected plants revealed in our study were presumed to play an important role in plant susceptibility to the studied phytopathogen. Our work disclosed important aspects of plant-M. nivale interactions, outlined the directions for future studies on poorly characterized plant diseases caused by this phytopathogen, and provided new opportunities to improve cereals breeding and food security strategies.

Quantitative Trait Loci and Candidate Genes Associated with Cold-Acclimation and Microdochium nivale Tolerance/Susceptibility in Winter Triticale (x Triticosecale).

Tolerance to pink snow mold caused by Microdochium nivale appears after a cold-hardening period and it is an essential, genotype-dependent, complex quantitative trait for the wintering of triticale (x Triticosecale) and other cereals. Despite long-term studies, a marker for the selection of the tolerant genotypes is still insufficiently recognized. Chlorophyll fluorescence has been reported as a sensitive indicator of stress effects on photosynthesis and can be used to predict plant tolerance. In this study, the genomic regions (QTLs) associated with the level of winter triticale seedlings damage caused by M. nivale infection as well as photosynthesis quantum efficiency and chlorophyll a fluorescence parameters were identified in seedlings of mapping population of 89 doubled haploids lines (DHs) derived from F1 hybrid of cv. ‘Hewo’ and cv. ‘Magnat’ accompanied with the genetic map consisting of 20 linkage groups with a total map length 4997.4 cm. Independent experiments performed in controlled conditions revealed 13 regions identified by a composite interval mapping, located on 7A, 1B, 2B, 6B, 7B, 3R, 5R, and 6R linkage groups and related to the PI, PIABS, TRo/CS, ABS/CS, ABS/CSm, ABS/RC, and Qy values as well as M. nivale tolerance T and susceptibility level P expressed by the seedling damage index. Additionally, candidate genes were in silico identified with the sequence position on wheat (2B and 7B) and rye (5R) chromosomes, where relevant QTL regions were found. The most important candidate genes indicated for M. nivale tolerance of cold-hardened triticale seedlings include those coding: sterol 3-beta-glucosyltransferase UGT80A2-like, transcription factor NAI1-like, and flavonol3-sulfotransferase-like proteins on chromosomes 2B and 5R.

Rapid increases in β-1,3-glucanase and chitinase activities are markers of resistance to Microdochium nivale in grasses of the Lolium‑Festuca complex

Microdochium nivale causes pink snow mould - a destructive disease of seedlings, stem bases, and ears of winter grasses and cereals. Glucanase and chitinase belong to pathogenesis-related proteins and exhibit a specific activity in response to pathogens. The aim of this study was to investigate changes in the activity of these enzymes in the leaves of chosen forage grasses during Microdochium nivale infection. Different cultivars of Festuca arudinacea, F. pratensis, Festulolium brauni, Lolium multiflorum, and L. perenne were prehardened at 12 °C and hardened at 2 °C and then inoculated with M. nivale mycelium. Leaf samples were collected before infection, as well as two, four, and eight days after inoculation. Each cultivar showed a specific pattern of changes in the enzymatic activities in response to M. nivale infection. We conclude that a rapid increase in glucanase and chitinase activities after M. nivale attack enhanced resistance to this pathogen and may be recognized as a physiological marker of grass resistance to this snow mould.

Changes in phenolic acid abundance involved in low temperature and Microdochium nivale (Samuels and Hallett) cross-tolerance in winter triticale (x Triticosecale Wittmack)

Tolerance to the pink snow mould resulting from Microdochium nivale infection is an essential trait of triticale (x Triticosecale) for winter survival. In the present study, we aimed to verify whether the presence and concentration of free and cell wall-bound phenolic acids are important factors in triticale responses to M. nivale infection. Based on 3 years’ testing of triticale tolerance, 2 out of 92 doubled haploid triticale lines derived from ‘Hewo’ × ‘Magnat’ F1 hybrid were selected, which are the most tolerant and the most sensitive to M. nivale infection. Plants were grown along with their parents under controlled conditions, pre-hardened and cold-hardened, while non-hardened plants served as the control. Hardened plants were covered with the artificial snow-imitating covers and inoculated with M. nivale mycelium, while the control plants were treated the same way except the infection. The aim of the study was to identify differences in the initial content and composition of phenolics under the influence of applied stresses. Conducted HPLC analysis showed that the most abundant were ferulic, rosmarinic, chlorogenic, sinapic, and trans-cinnamic acids. The contents of most of phenolics depended on genotype and growth conditions. Two cell wall-bound sinapic and trans-cinnamic acids, could be indicated as potentially related to the increased snow mould tolerance of winter triticale seedlings. A correlation between the total phenolic levels with the tolerance was not found; however, the proportion between the total levels of cell wall-bound and free phenolic compounds under low temperature could play a role prior to M. nivale infection.

Changes in antioxidants activity in grasses from complex Lolium-Festuca as a response to Microdochium nivale infection

Pink snow mould is a disease caused by Microdochium nivale affecting winter cereals and grasses. In response to pathogen attack, plants initiate oxygen burst within which hydrogen peroxide increases rapidly in a huge amount. We have investigated the activity of catalase (CAT), non-specific peroxidases (PX) and isoforms of superoxide dismutase (SOD) in leaves of grasses from complex Lolium-Festuca 2, 4 and 8 days after inoculation with M. nivale. Each plant species showed a specific pattern of enzymatic kinetics during infection. We concluded that increased activity of PX and decreased activity of CAT may be recognized as the markers of the M.nivale resistance.

The effects of cold-hardening and Microdochium nivale infection on oxidative stress and antioxidative protection of the two contrasting genotypes of winter triticale

Pink snow mould resulting from Microdochium nivale (Samuels and I.C. Hallett) regularly infects winter triticale (× Triticosecale, Wittm.). Therefore, in the present paper we aimed to identify biochemical markers potentially involved in the increased resistance against M. nivale of this valuable crop. In our experiment, we used two doubled haploid (DH) lines with the highest and the lowest cold-induced snow mould resistance, previously identified in cold chamber tests. The impact of cold-hardening (4 weeks at 4 °C) as well as covering plants with an artificial system to mimic snow covers and inoculation with M. nivale mycelium in leaves of seedlings, different genotypes on the thiobarbituric acid-reactive substance (TBARS) level and superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) activities was assessed. Our studies indicate that, indeed, various genotypes differ in their response to the treatments in regard to the measured parameters. The leaf TBARS content was always lower in seedlings of snow mould-resistant DH1 line in comparison with susceptible DH92 ones, suggesting that tissue damage was more pronounced in plants of the second genotype. The activity of SOD and CAT had no correlation with the level of snow mould resistance. The cold-enhanced POX I activity was higher in DH1 seedlings under the snow-imitating covers with or without M. nivale inoculation in comparison with DH92 plants. Moreover, POX II activity was higher after cold-hardening in the resistant genotype. POX III activity was enhanced by abiotic stress in leaves of DH1 seedlings, while it was not detected in susceptible genotype in any experimental term.

Fusarium seedling blight of wheat and oats: effects of infection level and fungicide seed treatments on agronomic characters

The effects of seed-borne Fusarium spp. and Microdochium nivale infection in spring wheat, winter wheat, and oats in Sweden was investigated in field trials for the agronomic characters yield, thousand kernel weight, grain volume weight, gluten, protein, starch, straw strength, and plant density. Seed with high and low levels of infection was mixed to obtain six infection levels in the proportions of 100/0, 80/20, 60/40, 40/60, 20/80, and 0/100 percent. The seed was untreated or treated with Celest Extra Formula M (CEFM, difenoconazole + fludioxonil) or Celest Formula M (CFM, fludioxonil). In the field trials using untreated seed, there were significant differences between infection levels only for some agronomic characters and levels. Fungicide seed treatment with CEFM in spring wheat had no significant effect on most agronomic characters including yield. In winter wheat and oats, seed treatment with CFM increased yield by 7–11% and plant density by up to 33% while having no effect on other characters. The percentage discoloration of crown roots and stem bases due to Fusarium/Microdochium spp. was also investigated visually in winter wheat and oats and found to increase with higher infection level. Fungicide seed treatment thus mainly increased plant emergence in seed lots with low-to-moderate Fusarium/Microdochium spp. infection and had little or no effects on other agronomic characters.

Identifying QTLs for cold-induced resistance to Microdochium nivale in winter triticale

Snow mould caused by Microdochium nivale (Fr.) Samuels & Hallett is the most widespread seedling disease in winter cereals. Due to the complexity of the resistance mechanisms, a poorly understood genetic background and strong interaction with winter weather conditions, it is difficult to assess the resistance of triticale cultivars via conventional inoculation methods. Genetic resistance is the most economical and environmental friendly way to control M. nivale infection; therefore, the objective of this study was to detect the quantitative trait loci (QTLs) associated with resistance components of winter triticale in a mapping population derived from a cross of the ‘Modus’ (partly resistant) and ‘SaKa 3006’ (sensitive) varieties. High-resolution mapping was conducted by using 1518 molecular markers (diversity arrays technology, simple sequence repeat and amplified fragment length polymorphism). Partial resistance components assessed in this study, i.e. candidate QTLs, were detected on chromosomes 1B, 2A, 3A, 3B, 5A, 5B, 6A, 6B and 7B, whereas QTLs describing overall seedling vitality in non-infected control plants were located on chromosomes 1B, 2B, 3A, 5A, 7B and 7R.

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 H 2 O 2 level are correlated with a higher resistance to low temperature, low light intensity and pink snow mould in triticale seedlings. The higher H 2 O 2 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.

Microdochium nivale (Fr., Samuels & Hallett): cytological analysis of the infection process in triticale (×Triticosecale 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.

Cold-hardening of winter triticale (x Triticosecale Wittm.) results in increased resistance to pink snow mould Microdochium nivale (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.

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 Loliummultiflorum and Festucapratensis genomes, was characterised by changes in biochemical parameters similar to the resistant meadow fescue and tall fescue.

Changes in abscisic acid, salicylic acid and phenylpropanoid concentrations during cold acclimation of androgenic forms of Festulolium (Festuca pratensis × Lolium multiflorum) in relation to resistance to pink snow mould (Microdochium nivale)

AbstractWe investigated changes in concentrations of abscisic (ABA) and salicylic acid (SA), phenolic compounds and phenylalanine ammonia‐lyase (PAL) activity in relation to cold‐induced tolerance of four androgenic genotypes of Festulolium (Festuca × Lolium hybrids) to frost and to the snow mould fungus Microdochium nivale. Cold acclimation increased frost tolerance and resistance to snow mould. Resistant genotypes were characterized by higher ABA concentrations during the first 54 h of cold acclimation and lower concentrations of SA than susceptible genotypes. After cold acclimation, the content of phenolics was significantly lower in genotypes tolerant to frost and M. nivale infection than in susceptible genotypes, while PAL activity was significantly higher. Signalling networks controlling cold acclimation to frost (abiotic) and mould infection (biotic) appears to involve increases in foliar concentrations of ABA and decreases in the SA level during successful cold acclimation. Higher PAL activity and lower concentrations of phenolic compounds also appear to be associated with enhanced tolerance to frost and fungal attack.

The effect of a mixture of seed-borne Microdochium nivale var. majus and Microdochium nivale var. nivale infection on Fusarium seedling blight severity and subsequent stem colonisation and growth of winter wheat in pot experiments

Greenhouse experiments were conducted in order to determine the impact of seed-borne Microdochium nivale var. nivale and var. majus inoculum, and seed treatment with a carboxin+thiram mixture, on the development of seedling blight, and on subsequent stem colonisation and growth of winter wheat (cv. Cadenza). Experiments were conducted at temperatures favourable (3°C) and unfavourable (22°C) to M. nivale. Seed-borne inoculum resulted in seedling blight symptom development when plants were grown at 3°C, but not when plants were grown at 22°C. For seedlings grown at 3°C, plants arising from heavily blighted seedlings developed more severe symptoms of stem colonisation, when compared with those arising from seedlings from carboxin+thiram treated seeds. In addition, the vigour of such plants (assessed by determining the number of tillers and ears per plant, stem length, green leaf area, dry weight and yield) was also significantly lower than for plants arising from carboxin+thiram treated seeds. Microdochium nivale var. majus and var. nivale appeared to have little effect on plant vigour from seedlings grown at 22°C. This is the first recorded incidence of seedling blight affecting subsequent plant growth. Microdochium nivale var. majus and var. nivale stem colonisation increased from growth stage (GS) 40–49 to harvest in plants raised from seedlings grown at both temperatures. Microdochium nivale var. majus and var. nivale were isolated from the second node at GS 40–49 and the third node at harvest of plants from seedlings grown at 3°C. For plants from seedlings raised at 22°C, M. nivale var. majus and var. nivale were isolated from the first node at GS 40–49 and the second node at harvest. Carboxin+thiram seed treatment decreased the extent and severity of stem colonisation on plants from seedlings grown at 22°C.

The effects of fungicides on Fusarium spp. and Microdochium nivale and their associated trichothecene mycotoxins in French naturally-infected cereal grains

Chemicals applied to wheat at the flowering stage are used to reduce yield losses associated with Head Blight (HB) and trichothecene contamination of infected grains. Efficacy of fungicide treatments at anthesis were assessed against Fusarium spp. ( Fusarium graminearum, F. avenaceum, F. poae, F. culmorum and F. tricinctum were the most frequent Fusarium species) and Microdochium nivale and on Fusarium mycotoxin concentration over three years on naturally infected fields of soft wheat, durum wheat and barley. Deoxynivalenol (DON) and nivalenol (NIV) were the most frequently detected mycotoxins. Treatments including azoxystrobin significantly decreased M. nivale infection levels, while infection levels of F. graminearum and F. culmorum were unaffected. Treatments that included metconazole or tebuconazole significantly decreased F. graminearum or F. culmorum infection levels, but the effect was variable across the years and type of fungicide applied. In 2002, DON concentration in wheat treated with tebuconazole and metconazole was reduced by 46% and 48%, respectively, while NIV concentration was unaffected. Yet, in 2000 and 2001, significant reduction in F. graminearum or F. culmorum infection levels were not always associated with significant reductions in DON and NIV mycotoxins. The fungicides we applied in naturally infected fields did not always reduce mycotoxin producers or alternatively the trichothecenes produced. Different explanations for this phenomenon are discussed.

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

AbstractThe 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.

The relationship between wheat seed weight, infection by Fusarium culmorum or Microdochium nivale, germination and seedling disease

The distribution of seeds by weight for three lots of winter wheat cv. Avalon infected by Fusarium culmorum and three lots of winter wheat cv. Riband infected by Microdochium nivale was determined. The distribution of infected seeds within each seed lot was then determined by isolating F. culmorum from seeds on moist filter paper and M. nivale from seeds on potato dextrose agar. The distribution of M. nivale infected seeds between seeds of different weight was similar to that of the seed lot as a whole, whereas the distribution of F. culmorum was greater in light seeds than heavy seeds. The percentage germination of infected seeds decreased with seed weight. A similar situation was found with respect to seedling emergence in compost for F. culmorum infected seeds. However, with M. nivale infection, similar numbers of seedlings emerged from both light and heavy infected seeds. Seed treatment with guazatine increased seedling emergence for both light and heavy seed infected by M. nivale. However, seedling emergence from F. culmorum infected seed was poor even following treatment with guazatine. Poor emergence was most evident from light seed.

Yield‐loss Relationships in Cereals

SUMMARYThe results of six studies using precision methods to obtain quantitative yield‐loss relationships are reported. Losses ranged from 100 per cent for Pyrenophora graminea on barley, to 51 per cent in a simulated grazing trial on wheat, 20 and 8 per cent respectively for Fusarium nivale and Rhizoctonia solani causing culm lesions on wheat, and 12 per cent for yellow rust on barley. Evidence of interactions between seed treatment with mercury, F. nivale infection and mildew on barley is also presented, in which seed treatment was beneficial regardless of seed infection, and F. nivale intensified the loss due to mildew.