Microdochium Nivale Research Articles

Effect of azoxystrobin on activities of antioxidant enzymes and alternative oxidase in wheat head blight pathogens Fusarium graminearum and Microdochium nivale

Wheat head blight pathogens Fusarium graminearum and Microdochium nivale have distinct sensitivities to strobilurin fungicides, which inhibit activity of complex III in the mitochondrial electron transport chain. When mycelia were cultured in medium with the strobilurin fungicide azoxystrobin (AZ), F. graminearum increased its oxygen-consumption, but M. nivale, which is more sensitive than Fusarium species to strobilurins, did not. There was no increase in oxygen consumption in F. graminearum by the treatment with AZ and alternative oxidase (AOX) inhibitor n-propyl gallate. AZ enhanced the generation of intracellular H2O2 in both fungi. Activities of antioxidant enzymes in M. nivale were consistently higher than in F. graminearum. Quantification of AOX transcripts by real-time PCR indicated that transcription of AOX in F. graminearum and M. nivale was induced by AZ, suggesting that AOX activities in both fungi are regulated at the transcriptional level. AOX transcription in F. graminearum was rapidly induced by AZ and reached a maximum by 60 min after treatment. On the contrary, induction in M. nivale was low and slow. These results suggest that differential ability of F. graminearum and M. nivale to activate AOX transcription is involved in the difference in their sensitivity to AZ.

Crystallization of carbohydrate oxidase from Microdochium nivale.

Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 A resolution, with unit-cell parameters a = b = 55.7, c = 610.4 A and apparent space group P6(2)22. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 A, beta = 95.7 degrees . Data sets were collected to a resolution of 2.4 A. The structure was solved by the molecular-replacement method. Model refinement is currently in progress.

First occurrence of resistance to strobilurin fungicides in Microdochium nivale and Microdochium majus from French naturally infected wheat grains

Microdochium nivale (Fr.) Samuels & Hallet and Microdochium majus (Wollenweber) belong to the Fusarium ear blight (FEB) fungal complex affecting cereals. In 2007 and 2008, major Microdochium sp. infestations were observed in France, and the efficacy of strobilurins was found to be altered in some field trials. The aim of this study was to determine the sensitivity to strobilurins of French isolates of Microdochium and to characterise the possible mechanisms of resistance. Half of the strains collected in 2007 were resistant to strobilurins, and most also displayed strong resistance to benzimidazoles. Strobilurin resistance was found mostly in M. majus isolates. Positive cross-resistance was observed between all strobilurins tested, but not with the phenylpyrrole derivative fludioxonil and the various classes of sterol biosynthesis inhibitors (SBIs). In most strains, resistance was correlated with the G143A substitution in cytochrome b, the molecular target of strobilurins. Two other mechanisms were also detected at lower frequencies. This is the first report of strobilurin resistance in Microdochium. Several resistance mechanisms have evolved independently in populations and may have different impacts on field efficacy. This makes the accurate detection and quantification of QoI resistance difficult. The management of field resistance and efficacy must be adapted to take these findings into account.

Aggressiveness of Three Snow Mold Fungi on Creeping Bentgrass Cultivars under Controlled Environment Conditions

Snow molds are the most important winter diseases of turfgrass in the United States and Canada. Eight isolates of three snow mold fungal species (three isolates of Typhula ishikariensis, three of T. incarnata, and two of Microdochium nivale) were collected from infected turfgrasses on golf courses. The isolates were evaluated for their relative aggressiveness on three cultivars (L-93, Penncross, and Providence) of creeping bentgrass (Agrostis palustris) under the same controlled conditions. Four plant ages (15, 19, 23 and 27 week-old plants from germination to inoculation) were evaluated for their susceptibility to the three pathogens and for the recovery of the plants. Regardless of age or cultivar of the host plant, M. nivale was found to be more aggressive and faster to infect and colonize than Typhula species. After three weeks recovery, M. nivale-inoculated plants showed higher disease severity than plants inoculated with the two Typhula species. Plants infected by Typhula species displayed no significant difference in disease severity. As creeping bentgrass plants get older, the severity of disease caused by three snow molds gradually decreases. This effect was observed in all cultivars tested, suggesting expression of age-related resistance as the bentgrass plants matured.

Tolerance to simulated ice encasement and Microdochium nivale in USA selections of greens-type Poa annua

Lack of winter hardiness is a limitation to the use of annual meadowgrass (Poa annua f. reptans) on golf course putting greens in northern climates. Our objectives were 1) to assess the tolerance of 13 selections of annual meadowgrass to simulated ice encasement (SIE) and pink snow mould (PSM, Microdochium nivale); 2) to determine if these tolerances were interrelated or related to plant concentrations of water soluble carbohydrates (WSC); and 3) to determine if tolerance to SIE can be associated with accumulation of toxic substances. Samples of the annual meadowgrass selections were taken from an experimental green at University Park, Pennsylvania, on four dates from 23 Nov. 2005 until 27 Mar. 2006. Samples of creeping bentgrass (Agrostis stolonifera L.) ‘Penn A-4’ were included for comparison in studies on SIE and WSC. Samples were vacuum-sealed in plastic bags at 1°C for SIE and inoculated with three isolates of M. nivale for PSM tolerance. Small, but significant, differences were found among annual meadowgrass selections after 23 days of SIE, but all selections were dead after 33 days. Significant differences were detected in foliar blighting after inoculation with M. nivale, regrowth relative to uninoculated control being three times higher in the most tolerant than in the most susceptible selections. In late November, two annual meadowgrass selections contained significantly more WSC than did creeping bentgrass, but this was not correlated with tolerance to SIE or PSM, which were also not interrelated. A separate experiment with increasing duration of SIE to one of the annual meadowgrass selections and ‘Penn A-4’ showed that the critical exposure periods were 25–30 and 42–47 days, respectively. The concentrations of the phytotoxic compounds butan-l-ol and ethyl butyrate were two- to three-times higher in annual meadowgrass than in creeping bentgrass.

The occurrence and predominance ofFusariumspecies on barley kernels in Slovakia

This article focuses on the occurrence of Fusarium species on barley kernels in Slovakia. The study was conducted over two consecutive crop years (2004–2005) on three cultivars (potential malting varieties) at five locations in Slovakia. Fusarium spp. were detected in all locations and years ranging from 0.1 to 27% seed infestation. The highest infestation of kernels was recorded in 2005 for all localities and all cultivars. The following species were detected: Fusarium acuminatum, F. arthrosporioides, F. avenaceum, F. culmorum, F. chlamydosporum, F. equiseti, F. graminearum, F. langsethiae, F. nivale (syn. Microdochium nivale), F. oxysporum, F. poae, F. sambucinum, F. semitectum, F. solani, F. sporotrichioides, and F. tricinctum. The most common was F. poae in all localities and cultivars, with an isolation frequency ranging from 0.33 to 11.67%. This is the first report of F. langsethiae occurrence in Slovakia with an isolation frequency ranging from 0.33 to 6.67%. In 2005 a wider spectrum of Fusarium species, a higher level of kernel infestation, and a lower germinative activity was recorded than in 2004. The germinative activity of kernels was impaired the most in 2005 irrespective of cultivar. The decreasing of germinative activity in 2005 corresponded with higher kernel infestation with Fusarium fungi.

Genetic Diversity for Pink Snow Mold Resistance in Greens‐Type Annual Bluegrass

Unseeded annual bluegrass (Poa annua L.) is an important component of golf greens in many regions of Canada and the United States. Although this turfgrass species has desirable playing attributes, it suffers from susceptibility to environmental and biological stresses including subfreezing temperatures and snow molds. In this study, we compared 29 genotypes collected from golf greens located in Québec and Ontario for their resistance to pink snow mold (SM). Plants were inoculated with Microdochium nivale [(Fries) Samuels & Hallett], causal agent of SM, and incubated under controlled conditions. High levels of variation in SM resistance were detected within the collection and between genotypes. Analysis of the relationship between climatic parameters at the sites of origin and SM susceptibility revealed that level of resistance was positively correlated to the duration of snow cover. Genetic diversity within the Poa collection was estimated using the sequence related amplified polymorphism (SRAP) technique. The UPGMA (unweighted‐pair group method arithmetic average) dendrogram yielded two main clusters that differed markedly in their proportion of SM‐resistant genotypes. Our results show that SM disease is a major selection pressure for the generation of genetic diversity among annual bluegrass biotypes that evolved on golf greens in northern climates. SRAP polymorphisms between bulked genotypes with contrasting resistance to SM were identified and could be used as markers for SM resistance in annual bluegrass.

Mycotoxin‐producing Fusarium Species Occurring in Winter Wheat in Belgium (Flanders) During 2002–2005

AbstractDeoxynivalenol (DON) content and Fusarium spp. (Fusarium graminearum, Fusarium culmorum, Fusarium avenaceum, Microdochium nivale and Fusarium poae) of mycotoxin‐producing Fusarium species in winter wheat in Belgium (Flanders) were determined. Field trials were set up in the varietal testing network of Flanders Agricultural Centre for Small Grains (Roeselare – Beitem, Belgium) and followed up during growing seasons 2001–2002, 2002–2003, 2003–2004 and 2004–2005. Fusarium infection and DON contamination were mainly influenced by location and environmental parameters. Mean DON levels ranged from 0 to 15 mg/kg. Over the period of four growing seasons cvs Deben, Ordeal and Napier had the highest DON contamination. Seasonal and local weather conditions before and during flowering together with local crop husbandry measures (crop rotation, soil preparation) seemed to be of great importance in explaining the variation in results obtained. At Bottelare a positive correlation between disease index and DON content was found for the growing seasons 2001–2002 and 2002–2003, but not the season 2003–2004. Fusarium graminearum and F. culmorum were in general the most frequently occurring Fusarium spp. in Flanders over the 4 years but the composition of the Fusarium population varied strongly from location to location and from year to year. Fusarium graminearum predominated in areas especially where maize was cultivated, whereas in areas with more small cereals in the crop rotation more F. culmorum was present. Also temperature played a role in the composition of Fusarium spp.

Fungal Diversity in Composting Process of Pig Manure and Mushroom Cultural Waste Based on Partial Sequence of Large Subunit rRNA

Fungal diversity during composting was investigated by culture-independent rDNA sequence analysis. Composting was carried out with pig manure and mushroom cultural waste using a field-scale composter (Hazaka system), and samples were collected at various stages. Based on partial sequence analysis of large subunit (LSU) ribosomal RNA (rRNA) and sequence identity values, a total of 12 different fungal species were found at six sampling sites; Geotrichum sp., Debaryomyces hansenii, Monographella nivalis, Acremonium strictum, Acremonium alternatum, Cladosporium sphaerospermum, Myriangium durosai, Pleurotus eryngii, Malassezia globosa, Malassezia restricta, Rhodotorula glutinis, and Fusarium sporotrichioides. Geotrichum sp. of the class Saccharomycetes was the most predominant fungal species throughout the composting process (185 out of a total of 236 identified clones, or 78.4%), followed by Acremonium strictum (7.6%), Monographella nivalis (5.1%), and Pleurotus eryngii (3.8%). The prevalence of Geotrichum sp. was the lowest (61.1%) at the beginning of composting, and then gradually increased to 92.5% after 10 days of composting.

Correlation between black point symptoms and fungal infestation and seedling viability of wheat kernels

The level of occurrence of black point, the spectrum of fungal species and damage to wheat seedling vigour associated with it were assessed during 2003 and 2004 in the Slovak Republic. The incidence of black point kernels ranged between 0.2–2.4% in 2003 and 24.2–34.3% in 2004. The kernels’ fungal contamination varied from 60% to 100%. <i>Alternaria</i> spp., <i>F. poae</i> and <i>F. culmorum</i> were isolated from all localities and all subsamples. <i>Stemphylium vesicarium</i>, <i>Fusarium culmorum</i>, <i>F. graminearum</i>, <i>F. avenaceum</i>, <i>F. sporotrichioides</i>, <i>Microdochium nivale</i>, <i>Epicoccum nigrum</i>, <i>Penicillium</i> spp., <i>Aspergillus niger</i>, <i>Rhizopus nigricans</i> and <i>Cochliobolus sativus</i> were isolated less frequently. Fungi of the genus Alternaria were the most dominant, followed by <i>Fusarium</i> and <i>Microdochium</i> among which <i>F. poae</i> was dominant. Irrespective of incubation temperature, the germinative capacity and coleoptile growth rate of discolored kernels were affected more in the wet and cold year 2004. The inhibition of germination and seedling viability was more pronounced at the incubation temperature 22°C than at 15°C. Inhibition of coleoptile growth rate was 0.12–3.12% in black point kernels collected in 2003, and 0.24–9.28% in those collected in 2004.

Effects of sowing time on pink snow mould, leaf rust and winter damage in winter rye varieties in Finland

Disease infection in relation to sowing time of winter rye (Secale cereale) was studied in southern Finland in order to compare overwintering capacity of modern rye varieties and to give recommendations for rye cultivation. This was done by using three sowing times and four rye varieties in field trials conducted at three locations in 1999–2001. The early sown rye (beginning of August) was severely affected by diseases caused by Puccinia recondita and Microdochium nivale, whereas postponing sowing for two weeks after the recommended sowing time resulted in considerably less infection. The infection levels of diseases differed among rye varieties. Finnish rye varieties Anna and Bor 7068 were more resistant to snow mould and more winter hardy than the Polish variety Amilo, or the German hybrid varieties Picasso and Esprit. However, Amilo was the most resistant to leaf rust. In the first year snow mould appeared to be the primary cause of winter damage, but in the second year the winter damage was positively correlated with leaf rust. No significant correlation between frit fly infestation and winter damage or disease incidence of snow mould or leaf rust was established. The late sowing of rye (in the beginning of September) is recommended in Finland, particularly with hybrid varieties, to minimize the need for chemical plant protection in autumn.;

ABA level, proline and phenolic concentration, and PAL activity induced during cold acclimation in androgenic Festulolium forms with contrasting resistance to frost and pink snow mould ( Microdochium nivale)

The metabolic changes in foliar abscisic acid, proline and phenolic concentration, and PAL activity induced during cold acclimation in androgenic forms of Festulolium contrasting in terms of their resistance to frost and Microdochium nivale were studied. The frost-resistant and snow mould-resistant (cross-tolerant) genotype (561) was characterized by a higher ABA amount and higher PAL activity compared to those of the frost and snow mould-susceptible genotype (621) during cold acclimation. The obtained results suggest that both these parameters are involved in a cross-tolerance mechanism relating to frost and snow mould resistance.

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.

Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow mould

Biological control microorganisms have long been promoted as an alternative to conventional pesticides. Before registration of a microbial biocontrol product for commercial sale, it must be evaluated as regards potential spread and persistence after release. In this study, strainspecific sequence-characterized amplified region (SCAR) markers were developed to monitor the biocontrol candidate strain Pseudomonas brassicacearum MA250, which is effective against snow mould ( Microdochium nivale). One SCAR marker, OPA2-73, was used in quantitative real-time PCR (Q-PCR) on samples from a climate chamber experiment in which winter wheat seeds were treated with the bacterium or a chemical control agent, or left untreated. The results showed that MA250 persisted for up to 3 weeks after sowing on the kernel residues and also colonized the roots of treated seedlings. Total MA250 cell numbers on biocontrol treated seedlings after three weeks were approximately 10 6 cells, compared with the original inoculum of 10 6–10 7 cells per seed. Corresponding cell numbers of MA250 on chemically treated and untreated seedlings were below the detection limit. This study shows that SCAR marker OPA2-73 is a specific and sensitive tool for monitoring the biocontrol microorganism MA250 in environmental samples.

Specific interactions between arbuscular mycorrhizal fungi and plant growth-promoting bacteria: as revealed by different combinations

The interactions between two plant growth-promoting rhizobacteria (PGPR, Pseudomonas fluorescens SBW25 and Paenibacillus brasilensis PB177), two arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Glomus intraradices) and one pathogenic fungus (Microdochium nivale) were investigated on winter wheat (Triticum aestivum cultivar Tarso) in a greenhouse trial. PB177, but not SBW25, had strong inhibitory effects on M. nivale in dual culture plate assays. The results from the greenhouse experiment show very specific interactions; for example, the two AM fungi react differently when interacting with the same bacteria on plants. Glomus intraradices (single inoculation or together with SBW25) increased plant dry weight on M. nivale-infested plants, suggesting that the pathogenic fungus is counteracted by G. intraradices, but PB177 inhibited this positive effect. This is an example of two completely different reactions between the same AM fungus and two species of bacteria, previously known to enhance plant growth and inhibit pathogens. When searching for plant growth-promoting microorganisms, it is therefore important to test for the most suitable combination of plant, bacteria and fungi in order to achieve satisfactory plant growth benefits.

First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.

Genetic analysis and PCR-based identification of major Fusarium species causing head blight on wheat in Japan

Identifying the Fusarium species cause Fusarium head blight (FHB) and produces mycotoxins in wheat and other cereal is difficult and time consuming because of confusing phenotypic classification systems. In Japan, the F. graminearum complex, F. culmorum, F. avenaceum, and Microdochium nivale predominantly cause FHB. The internal transcribed spacer (ITS) and 5.8S of rDNA, a partial sequence of β-tubulin and mitochondrial cytochrome b (cytb) genes of the four species were PCR-amplified and analyzed. On the basis of the ITS, β-tubulin and cytb sequences, F. avenaceum and M. nivale are distinct from the F. graminearum complex and F. culmorum, whereas the F. graminearum complex is closely related to F. culmorum. Moreover, thiophanate–methyl-resistant isolates of the F. graminearum complex and F. culmorum did not have an amino acid substitution at amino acid codon 198 or 200 of β-tubulin. In contrast, very highly or highly thiophanate–methyl-resistant isolates of M. nivale had Glu (GAG) substituted with Ala (GCG) or Lys (AAG) at codon 198, respectively. The allele-specific PCR assay was used to identify the F. graminearum complex and F. culmorum, and these Fusarium species could be distinguished rapidly.

Colonization of winter wheat stem bases by communities of pathogenic fungi

The current study was focused on fungi colonizing the stem bases of wheat. Particularly, fungal species with significant harmful effect on wheat were studied. The dynamics of the stem base colonization by these fungi over the growing season were observed, and the association between individual pathogenic fungi was also evaluated. In addition, the fungal leaf sheaths penetration, which can cause physical disturbance, was assessed. The asymptomatic presence of fungal DNA in wheat tissue was confirmed. Microdochium nivale was found on the stem bases most frequently. Other fungi, such as Oculimacula spp., Rhizoctonia cerealis and Fusarium spp., were presented considerable less frequently. The statistical significant association was confirmed only between M. nivale var. nivale and M. nivale var. majus . Out of these, M. nivale tended to decrease in summer season, the frequency of occurrence of Oculimacula spp. and R. cerealis increased at later growth stages. Along with this, these species infected only plants...

The role of organic vs. conventional farming practice, soil management and preceding crop on the incidence of stem-base pathogens on wheat

A stem-base disease complex of winter wheat includes several causal agents, such as eyespot (Oculimacula yallundae and O. acuformis), sharp eyespot (Rhizoctonia cerealis) and brown foot rots (Fusarium graminearum, F. culmorum, F. avenaceum, F. po ae, Microdochium nivale vars. nivale and majus). Farming practices (organic and conventional), soil management (ploughing compared to min- and no-tillage) and the preceding crop (cereal, maize and oilseed rape) were studied as factors affecting the incidence of stem-base pathogens. The stem-base diseases were assessed visually and by molecular methods (PCR) on four winter wheat cultivars at four growth stages. The comparison of organic and conventional farming practices did not result in large differences in the relative incidence of stem-base pest species over the period of this study; nevertheless, a higher frequency of positive detection was recorded for three organisms (O. acuformis, O. yallundae and M. nivale var. nivale) under conventional farming. Other factors had none or only partial effects. Soil management didn’t affect the stem-base pathogens incidence significantly. The preceding crop maize reduced the incidence of M. nivale var. majus.

Evaluation of Foliar Application of Several Fungicides for Control of Wheat Scab Caused by Microdochium nivale

Evaluation of Foliar Application of Several Fungicides for Control of Wheat Scab Caused by <I>Microdochium nivale</I>