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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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. Copyright © 2009 Society of Chemical Industry     

Evidence for differential host preference in Microdochium nivale var. majus and Microdochiumnivale var. nivale

The pathogenicity of Microdochium nivale var. majus and var. nivale was tested on wheat, rye and oat seedlings using both visual disease scoring and quantitative PCR measurements. In an individual inoculation trial at 10°C var. majus and var. nivale were strongly pathogenic towards wheat and rye, with var. nivale causing slightly greater disease in rye. At this temperature only var. nivale caused significant disease of oats. In a further experiment M. nivale was inoculated as a series of mixtures of the two varieties and incubated at 15°C. The ratio of the varieties present in the inoculum and present at harvest was analysed by quantitative PCR and this enabled a coefficient of selection to be calculated for the varieties on each host. M. nivale var. majus showed a weak selective advantage over var. nivale on wheat (0.33 ± 0.08) and oat seedlings (0.35 ± 0.016) and M. nivale var. nivale showed a strong selective advantage over var. majus on rye seedlings (0.92 ± 0.26). The isolates were also compared for sensitivity to benzoxazolinone (BOA), a hydroxamic acid compound derived from rye leaves. M. nivale var majus was found to be significantly more sensitive to BOA than M. nivale var. nivale , indicating a possible mechanism for the selective advantage of var. nivale growing on rye. This is the first substantiated indication of a significant difference in host preference between Microdochium nivale var. majus and var. nivale .     

The effect of potential resistance inducers on development of <Emphasis Type="Italic">Microdochium majus</Emphasis> and <Emphasis Type="Italic">Fusarium culmorum</Emphasis> in winter wheat

The effect of potential resistance inducing chemicals on disease development of Fusarium head blight was studied in winter wheat (Triticum aestivum L.). As a pre-screening test, the effect of different treatments on development of Microdochium majus (syn. Microdochium nivale var. majus) was studied in detached leaves. Based on these tests, DL-3-aminobutyric acid, Bion (benzo-(1,2,3) thiadiazole-7-carbothioic acid S-methyl ester), and a foliar fertilizer containing potassium phosphite were selected for further studies. Greenhouse-grown winter wheat was sprayed with aqueous solutions of the potential resistance inducers 7 days prior to Fusarium culmorum point inoculation of the heads. Disease development was registered as number of bleached spikelets per inoculated spike. Spraying plants with the foliar fertilizer reduced the disease severity of F. culmorum by up to 40%. A reduced disease development of M. majus was also observed in detached leaves pre-treated with the foliar fertilizer. When the foliar fertilizer was added to the growth medium, a reduced in vitro growth of M. majus and F. culmorum was observed, indicating that the effect on disease development is at least partly due to a fungistatic effect. No significant reduction in disease development was observed in wheat pre-treated with DL-3-aminobutyric acid or Bion, although these compounds tended to reduce disease development, especially when applied in combination with other potential resistance inducers. We conclude that spraying winter wheat with a solution containing potassium phosphite can reduce development of M. majus and F. culmorum.     

Phosphite-mediated enhancement of defence responses in Agrostis stolonifera and Poa annua infected by Microdochium nivale

The ascomycete fungus Microdochium nivale is one of the most damaging pathogens of cool season turfgrass. Prevention of and recovery from infection is costly to many sports facilities each year. In recent years, use of many chemical plant protectants has been restricted and turfgrass managers have increasingly sought alternative measures for disease control. The use of phosphite has been shown to be effective in reducing M. nivale disease symptoms in Agrostis stolonifera and Poa annua. The aim of this research was to assess initial defence responses in M. nivale-infected turfgrass, specifically total phenolic content and hydrogen peroxide generation, to determine the effect phosphite treatment has on these responses and on suppression of symptoms. Phenolic compounds and H₂O₂ are shown to be components of host responses. Phosphite treatment led to enhanced accumulations of total phenolic content, and when applied sequentially or singly to greenhouse plants, it led to significant reductions in M. nivale disease symptoms compared to phosphate-treated plants or controls. H₂O₂ extractions indicated that while phosphite treatment increased H₂O₂ generation compared to controls, the effect was no different to the responses in phosphate-treated plants.     

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.     

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.     

A New Method for Producing Mycelium-Free Conidial Suspensions from Cultures of Microdochium nivale

A technique to improve the sporulation of Microdochium nivale in culture and to produce mycelium-free conidial suspensions was evaluated using cellophane-covered potato dextrose agar (PDA). Time to sporulation was significantly shorter on the cellophane-covered PDA (P < 0.001), yields of conidia were higher (P < 0.01) and conidial suspensions were produced virtually free of the mycelial fragments present in suspensions from PDA only. The conidial inoculum produced on cellophane had lower pathogenicity to wheat cv. Equinox in a detached leaf assay, showing significantly longer incubation periods (P < 0.05) and latent periods (P < 0.01), than conidia produced on PDA alone. However, the apparent decline in pathogenicity of conidial suspensions produced on cellophane compared to PDA alone was small.     

Differential Control of Head Blight Pathogens of Wheat by Fungicides and Consequences for Mycotoxin Contamination of Grain

Fusarium head blight of wheat is caused by a disease complex comprised of toxigenic pathogens, predominantly Fusarium spp., and a non-toxigenic pathogen Microdochium nivale, which causes symptoms visually indistinguishable from Fusarium and is often included as a causal agent of Fusarium head blight. Four field trials are reported here, including both naturally and artificially inoculated trials in which the effect of fungicide treatments were noted on colonisation by Fusarium and Microdochium, and on the production of deoxynivalenol (DON) mycotoxin. The pathogen populations were analysed with quantitative PCR and samples were tested for the presence of the mycotoxin DON. Application of fungicides to reduce Fusarium head blight gave a differential control of these fungi. Tebuconazole selectively controlled F. culmorum and F. avenaceum and reduced levels of DON, but showed little control of M. nivale. Application of azoxystrobin, however, selectively controlled M. nivale and allowed greater colonisation by toxigenic Fusarium species. This treatment also lead to increased levels of DON detected. nobreak Azoxystrobin application two days post-inoculation increased the production of DON mycotoxin per unit of pathogen in an artificially inoculated field trial. This result indicates the potential risk of increased DON contamination of grain following treatment with azoxystrobin to control head blight in susceptible wheat cultivars. This is the first study to show differential fungicidal control of mixed natural pathogen populations and artificial inoculations in field trials.     

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.     

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.     

Regional and varietal differences in the risk of wheat seed infection by fungal species associated with fusarium head blight in Italy

The incidence of seed infection by fungal species pertinent to the fusarium head blight complex was monitored from 1999 to 2002 in two soft and three durum wheat cultivars grown across the northern, central and southern production zones of Italy, in order to characterize the species composition at the seed level. The main species recovered were Fusarium graminearum, F. poae and Microdochium nivale. There was a marked influence of production zone on seed infection incidence for both durum and soft wheat cultivars, with incidence of infection generally decreasing from the northern to the southern zone. Incidence of seed infection by different species of Fusarium was twice to four times higher in durum compared with the soft wheat cultivars in the study. There were no significant differences in terms of seed infection incidence between the two soft wheat cultivars, but the durum cultivars differed in their levels of seed infection for some of the pathogens. The results demonstrated that the durum cultivars were more at risk of seed infection by pathogens associated with fusarium head blight, and that wheat grown in northern Italy is at higher risk of seed infection by these species.     

Suppression of the in vitro growth and development of Microdochium nivale by phosphite

The ascomycete fungus Microdochium nivale is a major pathogen of many species of the gramineae. Control measures rely heavily on chemical fungicides, making alternative means of disease reduction desirable. Phosphite (PO₃^3?), has proven efficacy in reducing susceptibility of different species of gramineae to oomycetes, and has adverse effects on the in vitro growth of numerous other pathogens. The effect of phosphorous acid (H₃PO₃), phosphoric acid (H₃PO₄), dihydrogen potassium phosphite (KH₂PO₃), dihydrogen potassium phosphate (KH₂PO₄) and potassium hydroxide (KOH) on the in vitro mycelial growth and development of M. nivale was determined. Radial growth on amended potato dextrose agar (PDA) was used to calculate mean daily growth and percentage inhibition. PO₃^3? had a significant inhibitory effect on mycelial growth, with EC₅₀ values ranging between 35.9 and 40.99 μg mL^?1, whilst PO₄^3? and KOH had no significant inhibitory effect. Microscopic examination of mycelia showed morphological deformities in hyphae growing on PO₃^3? amended PDA, whilst hyphal growth was normal on PO₄^3? and KOH amended PDA. Conidial germination of M. nivale was significantly reduced following immersion in solutions of 50, 100 and 250 μg mL^?1 of PO₃^3?, while PO₄^3? and KOH at the same concentrations induced no inhibitory affect. These results show that PO₃^3? is a significant inhibitor of the growth of M. nivale and may have the potential to be used as a chemical control agent in the field.     

Major Changes in Fusarium spp. in Wheat in the Netherlands

The re-emergence of fusarium head blight throughout the world and especially in Western Europe prompted a survey of the situation in the Netherlands. To allow for a high throughput screening of large numbers of samples, a diagnostic PCR method was developed to detect the most common species of Fusarium occurring on wheat. Seven primer pairs were tested for their ability to identify isolates of Fusarium avenaceum, F. culmorum, F. graminearum, F. poae, F. proliferatum and Microdochium nivale var. majus and M. nivale var. nivale. Each primer pair only generated a PCR product with the corresponding Fusarium species and all PCR fragments had different molecular sizes. This allowed the generation of these amplicons using a mixture of all seven primer pairs. The robustness of this multiplex PCR encouraged us to screen a large series of isolates collected in 2000 and 2001. In both years 40 fields were sampled leading to a collection of 209 isolates from 2000 and 145 isolates from 2001. The results of the multiplex PCR demonstrated that F. graminearum was the most abundant species in the Fusarium complex on wheat in both years. This is in sharp contrast to reports from the 1980s and early 1990s, which found F. culmorum as the predominant species. Primers derived from the tri7 and tri13 genes, which are implicated in the acetylation and oxygenation of the C-4 atom of the backbone of the trichothecene molecule, were used to discriminate between deoxynivalenol and nivalenol (NIV) producers. The populations of F. culmorum and F. graminearum both showed a slight increase in NIV-producers in 2001.     

Effect of cold‐induced changes in physical and chemical leaf properties on the resistance of winter triticale (×Triticosecale) to the fungal pathogen Microdochium nivale

This study showed that several mechanisms of the basal resistance of winter triticale to Microdochium nivale are cultivar‐dependent and can be induced specifically during plant hardening. Experiments and microscopic observations were conducted on triticale cvs Hewo (able to develop resistance after cold treatment) and Magnat (susceptible to infection despite hardening). In cv. Hewo, cold hardening altered the physical and chemical properties of the leaf surface and prevented both adhesion of M. nivale hyphae to the leaves and direct penetration of the epidermis. Cold‐induced submicron‐ and micron‐scale roughness on the leaf epidermis resulted in superhydrophobicity, restricting fungal adhesion and growth, while the lower permeability and altered chemical composition of the host cell wall protected against tissue digestion by the fungus. The fungal strategy to access the nutrient resources of resistant hosts is the penetration of leaf tissues through stomata, followed by biotrophic intercellular growth of individual hyphae and the formation of haustoria‐like structures within mesophyll cells. In contrast, a destructive necrotrophic fungal lifestyle occurs in susceptible seedlings, despite cold hardening of the plants, with the host epidermis, mesophyll and vascular tissues being digested and becoming disorganized as a result of the low chemical and mechanical stability of the cell wall matrix. This work indicates that specific genetically encoded physical and mechanical properties of the cell wall and leaf tissues that depend on cold hardening are factors that can determine plant resistance against fungal diseases.     

Evaluation of pathogenicity and aggressiveness of <Emphasis Type="Italic">F. langsethiae</Emphasis> on oat and wheat seedlings relative to known seedling blight pathogens

Fusarium and Microdochium species are causal agents of seedling blight of small-grain cereal crops where they may contribute to a significant reduction in crop establishment and final yield. Two experiments were carried out to investigate the potential pathogenicity and aggressiveness of F. langsethiae, a recently identified fungus linked with the contamination of cereals with high levels of the trichothecene mycotoxins, HT-2 and T-2. An artificial seed inoculation method involving conidial suspensions was used and the experiments conducted in a growth cabinet set at either 5 or 15°C with a 12 h photoperiod. Known seedling blight pathogens of the genus Fusarium and Microdochium were used for comparison. At 15oC, F. culmorum, M. nivale and M. majus caused seedling blight of oats and wheat with F. culmorum, on average being the most aggressive than the latter two. At 5oC, only F. culmorum and M. nivale caused seedling blight of oats and wheat. Under the experimental conditions employed, F. langsethiae and F. poae failed to produce seedling blight disease indicating that these two species are not pathogenic to oat and wheat cultivars, Gerald and Claire respectively, at the seedling stage of development. They are therefore unlikely to affect crop establishment and other yield components such as tiller number, grain yield per head as well as grain weight if there is no subsequent foot-rot and/or head blight where infected seeds are sown.     

Conidiogenesis of Fusarium nivale and Rhynchosporium oryzae and its taxonomic implications

Scanning electron microscopy has shown thatFusarium nivale Ces. ex Sacc., the anamorph ofMonographella nivalis (Schaffnit) E. Müll., has annellate conidiogenous cells and this character together with its amphisphaeriaceous teleomorph distinguishes it from other fusaria.F. nivale and its varietymajus are assigned to a new genus,Gerlachia, asG. nivalis var.nivalis andG. nivalis var.major. For the teleomorph of the latter the new combinationMonographella nivalis var.neglecta (Krampe) W. Gams & E. Müll. is proposed. The causal agent of rice leaf scald,Rhynchosporium oryzae Hashioka & Yokogi, has the same conidiogenesis and is transferred toGerlachia asG. oryzae (Hashioka & Yokogi) W. Gams and distinguished fromG. nivalis by conidial shape and higher cardinal temperatures.     

Development of Stem-base Pathogens on Different Cultivars of Winter Wheat Determined by Quantitative PCR

The progress of development of stem-base pathogens in crops of second winter wheat was plotted in nine experiments in three years. The amount of each pathogen present was determined by quantitative PCR. Where Tapesia yallundae was present in quantifiable amounts, it usually developed earlier than the other eyespot pathogen, T. acuformis. Both species were usually present in greater amounts on cultivars which are more susceptible to eyespot. The sharp eyespot pathogen, Rhizoctonia cerealis, developed more erratically than either of the Tapesia spp. and there were no consistent effects on different cultivars. Fusarium spp., the cause of brown foot rot, were rarely present in quantifiable amounts, but Microdochium nivale was usually present as one or both of the varieties nivale and majus. Late-season (after anthesis) decreases in M. nivale suggest that any brown foot rot symptoms attributable to this fungus would have fully developed earlier. Cultivar differences in amounts of M. nivale were most clear in stems during internode extension and when relatively large amounts of DNA were present. Such differences approximately reflected eyespot susceptibility, cv. Soissons containing most and cv. Lynx containing least DNA. The results emphasise the difficulty in relating diagnoses, by quantitative PCR or other means, at early growth stages when decisions to apply fungicides against stem-base disease are made, to later disease severity.     

Analysis of head and leaf reaction towards <Emphasis Type="Italic">Microdochium nivale</Emphasis>

This research examined the variation in the response of eight commercial wheat cultivars to Microdochium nivale isolates using both in vivo FHB tests (AUDPC and RHW measurements) and in vitro detached leaf assays (LGR). Irrespective of fungal variety, the two Italian cvs Fortore and Norba exhibited the greatest amount of visual disease symptoms (mean AUDPC=2.2 and 2.3, respectively), being significantly more susceptible than the other six cultivars (AUDPC 1.24) (P < 0.05). Irrespective of fungal variety, the Italian cv. Norba and the Irish cv. Falstaff were more susceptible than the other cultivars (except Fatima 2) in terms of RHW (P < 0.05), while the cvs Fortore, GK Othalom and Consort were more resistant than the other five cultivars (P < 0.05). In the detached leaf assay, the Hungarian cv. GK Othalom and the Italian cv. Norba were more susceptible (mean LGR=0.79 and 0.81 mm day^–1, respectively) to M. nivalethan the other six cultivars (mean LGR=0.51–0.72) (P < 0.05). Analysis of the relationship between head and leaf reaction to M. nivaleinfection revealed no significant correlation.     

Control of Microdochium majus in winter wheat with botanicals – from laboratory to the field

The fungal pathogen Microdochium majus, causing snow mould, seedling blight and foot rot, results in severe yield losses in small grain cereals. There are few options to control this pathogen in organic production. In this study, aqueous extracts or botanical powders prepared from chamomile, meadowsweet, thyme and Chinese galls were tested in vitro against M. majus conidia germination and mycelial growth, respectively. Subsequently, three botanicals were chosen, applied as powders with different seed coating adhesives, and tested for their effect on the incidence of M. majus from naturally infected wheat seed lots and on seedling emergence from soil under controlled environmental conditions. Furthermore, seed treatments with warm water, a bacterial product or one chosen botanical were tested in a growth chamber and in a field experiment over three consecutive years. Of the botanicals tested, Chinese galls showed the highest efficacy in controlling M. majus, reducing conidia germination and mycelial growth by up to 97 and 100%, respectively, and reducing the incidence from infested seeds by up to 59%. In two growth chamber experiments, total seedling emergence increased by up to 30 and 59% compared with the control treatments following an application with Chinese galls. Under field conditions, yield increase through Chinese galls, the bacterial product and the warm water treatment was 19, 10 and 37% compared with the untreated control, respectively. This study demonstrates the potential of Chinese galls to control M. majus in wheat. Options for improved formulations or combinations of heat‐based treatments with Chinese galls are discussed.