The use of species-specific PCR-based assays to analyse Fusarium ear blight of wheat

Polymerase chain reaction (PCR) assays for the detection of various Fusarium species and Microdochium nivale subspecies were compared with conventional visual disease assessment using a field plot of wheat in which the central subplot was inoculated with F. culmorum . Visual disease assessment was performed on a range of samples taken from each of 15 subplots at growth stage 80. At harvest, each sample was divided into its component parts, i.e. grain, glume and rachis, and species-specific PCR analysis was used to detect the presence of F. culmorum , F. poae , F. avenaceum , F. graminearum , M. nivale var. majus and M. nivale var. nivale . Within the inoculated subplot there was good correlation between visual disease assessment and PCR analysis, both techniques indicating a high incidence of F. culmorum in this region. According to the visual disease assessment results, there was also a relatively high incidence of F. culmorum in most other regions of the field plot. However, according to PCR analysis the incidence of F. culmorum in many of the other subplots was relatively low and F. poae , M. nivale var. majus and var. nivale , and F. avenaceum were detected within the grain, glume and rachis tissues of many of the ear samples from these subplots. F. poae predominated in the glume component of ears and M. nivale var. majus and var. nivale in the rachis component. M. nivale PCR results revealed that 64% of infected samples involved var. majus , and 36% var. nivale . PCR analysis has highlighted some difficulties that may arise when using visual assessment for studying disease complexes.     

Visual disease and PCR assessment of stem base diseases in winter wheat

Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four-week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum , F. culmorum , both varieties of Microdochium nivale , both eyespot-causing species of Tapesia and Rhizoctonia cerealis . PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R-type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var . majus , accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.     

Fusarium head blight of cereals in Denmark: species complex and related mycotoxins

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.     

Towards the Development of a Novel In Vitro Strategy for Early Screening of Fusarium Ear Blight Resistance in Adult Winter Wheat Plants

A novel in vitro bioassay is described for screening Fusarium ear blight (FEB) resistance in adult winter wheat plants. Seven winter wheat cultivars were assessed for components of partial disease resistance as 28 day-old detached leaf segments in the laboratory using isolates of Microdochium nivale var. nivale and M. nivale var. majus. Results were compared with disease data obtained at anthesis using the same cultivars as whole plants and the same isolates under glasshouse conditions. Significant cultivar differences were observed using detached leaves, with cv. Avalon (a Fusarium culmorum ear susceptible cultivar) having the shortest leaf incubation period, greatest leaf lesion development and shortest leaf latent period compared to cv. Spark (a Fusarium culmorum ear resistant cultivar), which had the longest leaf incubation period, least leaf lesion development and longest leaf latent period. Using whole plants, cv. Avalon had the shortest ear incubation period and greatest ear disease severity, whilst cv. Spark had the longest incubation period and least ear disease severity. Overall, cultivars of intermediate F. culmorum ear resistance expressed intermediate responses to M. nivale isolates, using both detached leaves and whole plants. Significant correlations were found with ear disease severity and ear incubation period in whole plants and components of partial disease resistance in detached leaves, with significant correlations obtained between leaf incubation period and ear disease parameters using the M. nivale var. nivale isolate. In addition, leaf latent period and leaf lesion size showed significant correlations with whole plant reactions using M. nivale var. nivale and var. majus isolates. The in vitro screening of cultivars as detached leaves using M. nivale isolates may offer a real possibility of a rapid bioassay for the early screening of FEB resistance in wheat and other cereals.     

Relationship between brown foot rot and DNA of Microdochium nivale, determined by quantitative PCR, in stem bases of winter wheat

Relationships between the incidence and severity of brown foot rot and of pathogenic fungi, determined by diagnostic and quantitative PCR, were investigated during the growth of nine winter wheat crops in three cropping seasons. Microdochium nivale vars nivale and majus were the only brown foot rot pathogens present in significant amounts. Relationships between disease symptoms and amounts of pathogen DNA were often weak in early spring (when shoot-base symptoms are usually most difficult to ascribe to particular pathogens by visual examination) because of indistinct symptoms and small amounts of pathogen. Relationships were strongest during stem elongation. The amount of M. nivale in the tissues tended to decline in the summer as the plants matured, apparently disappearing partially from necrotic lesions to which it contributed, resulting in a weakened relationship between symptoms and pathogen DNA. Regression analyses of brown foot rot on amounts of M. nivale DNA for different wheat cultivars generally produced lines with similar slopes but were often most significant for the cultivar with most eyespot resistance (i.e. with least confounding eyespot) or most apparently genuine brown foot rot. DNA of Fusarium spp. was rarely present in amounts sufficient to quantify.     

Fungicide seed treatment efficacy against Microdochium nivale and M. majus in vitro and in vivo

BACKGROUND: Seed-borne Microdochium majus (Wollenweber) and M. nivale Fries are the primary pathogens responsible for Fusarium seedling blight in the UK. The two species show differences in pathogenicity, host preference and sensitivities to temperature, but their relative sensitivities to fungicide seed treatments are unknown. The aim was firstly to determine the efficacy of fungicide seed treatments towards single-spore isolates of M. majus and M. nivale using in vitro experiments, and subsequently to determine efficacy in vivo over a range of temperatures. RESULTS: Differences in EC(50) values between all seed treatments were evident from the in vitro experiments and ranged from 0.028 mg L(-1) for fludioxonil to 22.8 mg L(-1) for carboxin + thiram. The two seed treatments that showed best performance in vitro were used to examine efficacy towards seed-borne infection in vivo at 4, 8, 12 and 16 degrees C. Generally, seedling emergence improved and the severity of stem-base disease symptoms on emerged seedlings was reduced for both species through the use of the fungicides. The combination of fludioxonil + difenconazole showed improved performance compared with fludioxonil alone. Significantly less severe symptoms were observed through the use of fludioxonil and fludioxonil + difenconazole compared with bitertanol + fuberidazole at 12 degrees C and for all except one M. nivale infected seed lot at 8 degrees C. CONCLUSIONS: Differences in fungicide sensitivity between the two species in vitro were not evident in vivo. This is the first report of the effect of fungicide seed treatments on the control of seedling blight caused by M. majus and M. nivale.     

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.     

Suppression of wheat-seedling diseases caused by Fusarium culmorum and Microdochium nivale using bacterial seed treatment

Snow mould, caused by Microdochium nivale , and seedling blight caused by members of the Fusarium complex, are cereal diseases of great economic importance in many temperate zones. In a glasshouse bioassay designed to enhance disease, about 600 plant-associated bacterial isolates obtained by different methods were screened for suppressive effects in wheat against infection caused by Fusarium culmorum . Although most of the isolates tested had a neutral effect on test plants and disease development, a few were synergistic to the pathogen and about one-fifth showed > 80% disease suppression. During five consecutive growing seasons, 164 bacterial isolates were tested in field experiments against both F. culmorum and M. nivale as causal agents of seedling blight. Tests for effects on yield in experiments with spring and winter wheat, performed in different climatic regions of Sweden, showed that disease-suppressive effects were repeatable. The most efficient isolates, three fluorescent pseudomonads and a species of Pantoea , suppressed disease equal to that of the fungicide guazatine, both with respect to crop stand and yield. Seed treatment with Pantoea sp. (isolate MF 626) increased yield by an average of more than 500 kg ha⁻¹ in six field experiments.     

Screening winter rye cultivars for snow mould (Microdochium nivale) resistance

The snow mould ( Microdochium nivale ) resistance of 13 winter rye cultivars was studied in field trials and in three different laboratory tests: snow mould chamber tests, enzymatic assay tests and leaf segment tests. On the basis of the results, it is suggested that both the field trials and the snow mould chamber tests describe more the general winterhardiness of plants involved in the survival of the crown tissue of plants during prolonged incubation under the snow cover, than the snow mould resistance. The results from the enzymatic assay and the leaf segment tests indicate that there are other, more specialized snow mould resistance mechanisms in the plant that act also at the single leaf level. At least some of these resistance reactions seem to be induced by the lytic enzymes secreted by M. nivale.     

Enhanced field control of wheat take-all using cold tolerant isolates of Gaeumannomyces graminis var. graminis and Phialophora sp. (lobed hyphopodia)

Cold tolerant isolates of Gaeumannomyces graminis var. graminis ( Ggg ) and Phialophora sp. (lobed hyphopodia), which produced at least comparable growth rates at 5°C to those of pathogenic G. graminis var. tritici ( Ggt ), were shown to control take-all disease in wheat effectively in 2 years of field experiments in New South Wales, Australia. The addition of oat inoculum of these fungi at the rate of 60 kg/ha to the seeding furrow significantly ( P  ≤ 0.05) reduced disease and increased grain yields by 33–45% compared to the Ggt alone treatment. The use of 30 kg/ha of oat inoculum also significantly ( P  ≤ 0.05) reduced disease and increased grain yields by 21–44%. These high levels of take-all control were obtained consistently from four field experiments on three different soil types with different pHs. A treatment inoculated with Ggg alone showed no disease symptoms and produced grain yields similar to that of untreated wheat. This fungus is, therefore, non-pathogenic to wheat. At high rates of inoculation of Ggg and Phialophora sp. (lobed hyphopodia), 65–80% of tillering wheat plants (GS 32) had root systems colonized by these fungi. In contrast, two Pseudomonas spp. and an isolate each of Ggg and Phialophora sp. (lobed hyphopodia), which did not grow at 5°C, were ineffective in controlling take-all. Take-all assessments during heading (GS 61-83) were highly correlated ( R ²=0.6047, P ≤0.0005) with the relative yield increase or decrease of inoculated treatments compared to the Ggt alone treatment. The use of a Ggg isolate (90/3B) and a Phialophora sp. (lobed hyphopodia) isolate (KY) for take-all control has been patented. These fungi are being developed for commercial use.     

Population Dynamics of Fusarium spp. and Microdochium nivale in Crops and Crop Residues of Winter Wheat

ABSTRACT Naturally occurring populations of Fusarium avenaceum, F. culmorum, F. graminearum, F. poae, and Microdochium nivale were studied in two field experiments from anthesis in June 2003 until harvest in crops of winter wheat, and subsequently during 10 months after harvest until June 2004 on their residues exposed on the soil surface under field conditions. The dynamics of the different pathogens were estimated by quantifying the amount of DNA present in wheat tissues using TaqMan-polymerase chain reaction. While colonization of grain by Fusarium spp. and M. nivale was low, high amounts of DNA of F. avenaceum, F. graminearum, and F. culmorum were found in ear residues, internodes, and nodes of the mature crop. Amounts of DNA of pathogens decreased significantly during the following 10 months in residues of internodes and nodes, but not in residues of stem bases. Knowledge on population dynamics of pathogens will help to develop preventive measures aimed at reduction of inoculum sources of head blight pathogens.     

Evaluation of type I fusarium head blight resistance of wheat using non-deoxynivalenol-producing fungi

A series of experiments was conducted to determine whether type I resistance (resistance to initial infection) to fusarium head blight (FHB) in wheat could be assessed using fungal species/isolates that do not produce deoxynivalenol (DON), a mycotoxin critical to the spread of Fusarium graminearum in the wheat spike. It was shown that, while the non-toxin-producing species Microdochium nivale and M. majus could infect following spray inoculation of wheat spikes, they were unable to spread within the spike following point inoculation. However, although these species might reveal type I resistance, they are not highly pathogenic towards wheat. A nivalenol (NIV)-producing isolate of F. graminearum caused high levels of disease following spray inoculation, but spread only very slowly within the spike and rarely induced bleaching above the point of inoculation. It is proposed that spray inoculation with an appropriate, aggressive, non-DON-producing FHB pathogen may be used to characterize type I resistance to complement point inoculation with a DON-producing isolate to assess type II resistance (resistance to spread within the spike).     

The contribution of ground-level inoculum of Fusarium culmorum to ear blight of winter wheat

In a series of field experiments in eastern England over 5 years, severe ear blight developed only in plots of winter wheat that were inoculated by spraying with conidial suspensions of Fusarium culmorum during anthesis, and in which infection was encouraged by rainfall or mist irrigation. In the absence of artificial inoculation of the ears, F. culmorum caused less extensive ear blight, and only where soil-surface inoculum was available after its application on infested plant material (colonized oat grains) up to 3–4 weeks before anthesis; it then developed most where significant rainfall occurred close to the time of anthesis. A warm, dry period following application of inoculum to the ground in late March contributed to increased infection of grain by F. culmorum , although ear blight was not increased. Ear infection therefore depended on adequate viable inoculum on infested plant debris within the crop, and conditions tending to favour brown foot rot development as well as, subsequently, rainfall and moist conditions during anthesis. These conditions did not occur together naturally during this period. Seedling infection by F. culmorum or Microdochium nivale made no significant contribution to ear blight. Inoculation of ears at anthesis with M. nivale or a locally obtained isolate of F. langsethiae did not produce ear blight symptoms. Possibilities for minimizing the availability of inoculum of F. culmorum and the implications for various options for ear-blight control are discussed.     

Pathogenicity of Gaeumannomyces graminis isolates to wheat and rye seedlings

A total of 187 isolates of Gaeumannomyces graminis , obtained from diseased cereal roots between 1979 and 1983, mostly from the UK, were compared for their ability to produce disease on wheat and rye seedlings in the glasshouse. Isolates varied continuously in pathogenic adaptation to rye. from those infecting wheat substantially and rye slightly (designated normal or N-isolates) to those infecting both wheat and rye substantially (designated R-isolates). The degree of adaptation to rye was a consistent characteristic of individual isolates. All were morphologically similar to G. graminis var. tritici R-isolates were obtained from all the areas sampled, they did not appear to predominate on a particular host species and occurred together with N-isolates and intermediates in a single field.     

Comparison of commercial European wheat cultivars to Fusarium infection of head and seedling tissue

Susceptibility of eight commercial European wheat cultivars to fusarium head blight (FHB) disease caused by Fusarium graminearum , F. culmorum , F. poae and Microdochium nivale (formerly known as Fusarium nivale ) was compared under controlled environment conditions (16°C). FHB did not differentially affect cultivars in terms of disease symptoms, fungal DNA content of grain or deoxynivalenol (DON) contamination. However, the Hungarian-grown cultivars GK-Othalom and Fatima 2 (of Romanian origin) showed greater type V resistance (yield tolerance) to FHB than did the others. Also, nivalenol was produced by F. poae in these two cultivars and in Italian cultivar Norba, but not in other cultivars. Overall, significant relationships were found between the FHB and seedling blight resistance in vitro of these eight cultivars, but such relationships were generally highly dependent on cultivar, and therefore it is likely that the in vitro test is at best measuring components of FHB resistance and/or genotype-specific resistance components.     

The incidence of Fusarium spp. in stem bases of selected crops of winter wheat in the Midlands, UK

The incidence of pathogenic Fusarium and Microdochium species in stem bases of winter wheat was investigated in a total of nine crops in 3 years, i.e. 1987, 1988 and 1989. Four Fusarium species were isolated: F. nivale ( = Microdochium nivale ), F. avenaceum , F. culmorum and F. graminearum . The predominant species was F. nivale followed by F. avenaceum and F. culmorum . Isolations of F. graminearum were made only from shoots collected during August 1989. The highest incidence of F. nivale occurred during April 1989 in the cultivar Brock when the fungus was isolated from 65% of the shoots sampled. The highest incidence of F. avenaceum was 60% (August 1988, cv. Slejpner) and F. culmorum 37% (August 1989, cv. Mercia). A delay in the isolation of Fusarium spp. during 1987 was attributed to the low January temperatures, and an upsurge of F. culmorum and F. graminearum during 1989 to the warm dry summer. The incidence of F. nivale fluctuated during the 1988 and 1989 seasons, particularly during spring. The effects of fungicide spray programmes and the growth and development of the wheat crop are discussed as possible contributory factors to this.     

A selective medium to isolate airborne spores of Microdochium nivale,causing winter wheat scab

Winter wheat scab in Hokkaido, Japan is caused predominantly by Gibberella zeae and Microdochium nivale and can result in significant yield losses. A selective medium for isolation of G. zeae was previously developed, but not for M. nivale. The purpose of this study therefore was to develop a selective medium for isolation of airborne spores of M. nivale. Based on the basic composition of Komada’s Fusarium-selective medium, carbon and nitrogen sources and the most suitable vitamin B component for the basal composition were examined. Hyphal growth of M. nivale was promoted when galactose was replaced with lactose and combined with L-asparagine, while aerial hyphal formation increased with thiamine hydrochloride as the vitamin B source. In antimicrobial composition, colony formation of other filamentous fungi was greatly inhibited by spiroxamine. Thiophanate methyl, to which M. nivale shows resistance, selectively inhibited the growth of Fusarium spp. only. Spore trapping using the selective medium was subsequently performed in a wheat field. M. nivale formed characteristic pinkish colonies on the selective medium in the case of contamination with other filamentous fungi, making differentiation easy. Overall, the findings show that LATTS medium developed in this study is effective for isolation of airborne spores of M. nivale.     

Stem-base disease and fungal colonisation of winter wheat grown in compost inoculated with Fusarium culmorum, F. graminearum and Microdochium nivale

Fungal colonisation of winter wheat cv. Cadenza by Fusarium culmorum, F. graminearum and Microdochium nivale was studied under conditions designed to avoid the splash dispersal of conidia from infested compost, to evaluate the possibility that systemic growth may transfer infection from the stem-base to the head. At decimal growth stages 33, 59, 77–87 and 95 the extent of fungal growth was assessed using a sample of 72 plants, by the recovery of fungal species from the stem-base, from each node and from the ear. Each of the fungi was recovered from stem tissues above soil level in some, apparently symptomless, plants. Symptoms of Fusarium foot rot were seen in an increasing proportion of plants during grain-fill and desiccation. There was an inverse relationship between recovery and the height above stem-base from which the stem tissue was excised. F. culmorum was the most frequently isolated fungus and it was also recovered from the highest position in plants. Only 3% of plants were colonised above the second node and none of the fungal species were recovered from either the fifth node or the ear. This suggests that colonisation and systemic growth from Fusarium infested compost is unlikely to contribute to the development of ear blight symptoms in winter wheat.     

Studies on the host range of the barley strain of Wheat dwarf virus using an agroinfectious viral clone

Comparative analysis of the host ranges of the barley and wheat strains of Wheat dwarf virus (WDV; family Geminiviridae ; genus Mastrevirus ) in Europe has been severely hampered by the lack of an infectious clone of the barley strain. To remedy this situation an agroinfectious clone of a Hungarian isolate of the barley strain (WDV-Bar[HU]) was constructed and its virulence tested in barley ( Hordeum vulgare ), wheat ( Triticum aestivum ), rye ( Secale cereale ) and oat ( Avena sativa ) by agroinoculation. Although all four species could be systemically infected by the isolate, infections were asymptomatic in the rye and oat cultivars tested. WDV-Bar[HU] induced chlorosis and stunting symptoms typical of WDV in barley, while in wheat low infection rates but high mortality of infected seedlings were observed. In contrast, a much higher percentage of wheat plants agroinoculated with a wheat-strain isolate (WDV-[Enk1]) became systemically infected. WDV-[Enk1] in wheat caused symptoms similar to those caused by WDV-Bar[HU] in barley. WDV-Bar[HU] was leafhopper-transmissible to barley seedlings, in which it caused typical WDV symptoms; geminate virus particles were isolated from the infected leaves. Comparison of the genomic sequences of 11 barley strain isolates from Europe and Turkey revealed that whereas WDV-Bar[HU] represents a typical barley-strain isolate that is not detectably recombinant, the Turkish barley isolate (WDV-Bar[TR]) is probably a recombinant between a barley-strain isolate and an as-yet-undescribed WDV-like mastrevirus species.     

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.