Head infection and accumulation of Fusarium toxins in kernels of 12 barley genotypes inoculated with Fusarium graminearum isolates of two chemotypes

Twelve barley genotypes were inoculated with two F. graminearum isolates of different chemotype I₁ #148 (producing nivalenol/deoxynivalenol) and I₂ #108 (deoxynivalenol/acetyldeoxynivalenol). For both I₁ and I₂ isolates, respectively, reductions (%) in number of kernels head^-1 10.6 and 14.3; yield 39.6 and 35.7; weight of 1000 kernels 36.9 and 23.2 were observed in inoculated plants from control values. Chemical analysis revealed the presence (average concentration mg kg^-1) of deoxynivalenol (1.3) and nivalenol (3.2) in kernels of all genotypes inoculated with the I₁ isolate, and zearalenone (0.2) in three samples. After inoculation with the I₂ isolate, deoxynivalenol (37.8) and zearalenone (0.4) were found in kernels of all genotypes, while 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol, respectively, were determined in five and four samples only. No significant correlation between examined characteristics was found for either the I₁ or I₂ isolate. The results obtained contribute information on the accumulation of toxins in cereal grain inoculated with F. graminearum isolates of different chemotypes.     

Common resistance to different <Emphasis Type="Italic">Fusarium</Emphasis> spp. causing <Emphasis Type="Italic">Fusarium</Emphasis> head blight in wheat

Different sets of wheat genotypes were tested under field conditions by spraying inocula of isolates of seven Fusarium spp. and Microdochium nivale (formerly F. nivale) in the period 1998–2002. The severity of Fusarium head blight (FHB), Fusarium-damaged kernels (FDK), the yield reduction and the deoxynivalenol (DON) contamination were also measured to describe the nature of the resistance. The degrees of FHB severity of genotypes to F. graminearum, F. culmorum, F. avenaceum, F. sporotrichioides, F. poae, F.␣verticillioides, F. sambucinum and M. nivale were very similar, indicating that the resistance to F.␣graminearum was similar to that for other Fusarium spp. listed. This is an important message to breeders as the resistance relates not only to any particular isolate of F. graminearum, but similarly to isolates of other Fusarium spp. This holds true for all the parameters measured. The DON contamination refers only to DON-producers F. graminearum and F. culmorum. Highly significant correlations were found between FHB, FDK, yield loss and DON contamination. Resistance components such as resistance to kernel infection, resistance to DON and tolerance were identified in the more susceptible genotypes. As compared with western European genotypes which produced up to 700 mg kg⁻¹ DON, the Hungarian genotypes produced only 100 mg kg⁻¹ at a similar FDK level. This research demonstrates the importance of measuring both FDK and DON in the breeding and selection of resistant germplasm and cultivars.     

Effect of Fungicides on Fusarium Head Blight and Deoxynivalenol Content in Durum Wheat Grain

In 1998–99 and 1999–2000 six trials were conducted to evaluate the effect of fungicides on Fusarium head blight in the field, on infected kernels and deoxynivalenol (DON) concentration in grain. A single application of prochloraz, tebuconazole, epoxiconazole or bromuconazole, applied to durum wheat varieties at the manufacturer's recommended dose at the beginning of anthesis stage, provided good control of the disease when infective pressure in the field was low to medium, and when the main pathogens were F. graminearum and F. culmorum. Kresoxim-methyl showed a low efficacy at controlling the disease. Tebuconazole, prochloraz and bromuconazole were effective at controlling F. graminearum and F. culmorum, while kresoxim-methyl was not effective in reducing Fusarium infected kernels. DON concentration in grain of cultivars inoculated with F. graminearum and F. culmorum was high, averaging 4.2mgkg^–1 (untreated control). Tebuconazole, prochloraz and bromuconazole reduced DON concentration by 43%, while epoxiconazole was ineffective. DON concentration in kernels of naturally infected cultivars was 1.95mgkg^–1, a concentration which exceeds the 1mgkg^–1 maximum level of contamination allowed in the United States. Furthermore prochloraz, bromuconazole and tebuconazole applications, in the naturally inoculated trials, reduced DON concentration from 73% to 96%, while epoxiconazole showed the lowest effectiveness. Moreover, a positive linear correlation between Fusarium infected grains and the DON concentration was observed.     

A comparison of aggressiveness and deoxynivalenol production between Canadian <Emphasis Type="Italic">Fusarium graminearum</Emphasis> isolates with 3-acetyl and 15-acetyldeoxynivalenol chemotypes in field-grown spring wheat

Twenty four isolates of Fusarium graminearum, half of which were 3-acetyldeoxynivalenol (3-ADON) and half 15-acetyldeoxynivalenol (15-ADON) chemotypes, were tested for their ability to produce deoxynivalenol and to cause Fusarium head blight (FHB) in spring wheat cultivars. The objectives of this study were to determine (1) whether 3-ADON isolates differ in aggressiveness, as measured by the FHB index, and DON production from 15-ADON isolates under field conditions, and (2) whether the performance of resistant host cultivars was stable across isolates. Field tests of all isolates were conducted with three replicates at each of two locations in Canada and Germany in 2008 with three host genotypes differing in FHB resistance level. The resistant host genotype showed resistance regardless of the chemotype or location. The differences between mean FHB indices of 3-ADON and 15-ADON isolates were not significant for any wheat genotype. In contrast, average DON production by the 3-ADON isolates (10.44 mg kg⁻¹) was significantly (P < 0.05) higher than for the 15-ADON isolates (6.95 mg kg⁻¹) at three of the four locations where moderately resistant lines were tested, and at both locations where susceptible lines were evaluated. These results indicate that 3-ADON isolates could pose a greater risk to food safety. However, as the mean aggressiveness and DON production of 3-ADON and 15-ADON chemotypes was similar on highly resistant lines, breeding and use of highly resistant lines is still the most effective measure of reducing the risks associated with DON in wheat.     

Scab Response and Moniliformin Accumulation in Kernels of Oat Genotypes Inoculated with Fusarium avenaceum in Poland

Inoculation experiments with 14 genotypes of oats (10 cultivars and 4 lines) were performed during 1996, 1997 and 1998 in Sitaniec, South-Eastern Poland. Panicles of oats were inoculated with a conidial suspension of Fusarium avenaceum, which caused a reduction in yield by 33% and in 1000 kernel weight (TKW) by 21%. During the period between inoculation and harvest, F. avenaceum was able to accumulate moniliformin (MON) in kernels at an average level of 0.13mgkg^–1 (gg^–1). The highest reduction of yield components caused by the F. avenaceum inoculation was found for cv. Santor, followed by lines CHD 1171, STH 2795 and cvs: Kwant and Farys, while cvs Slawko, Dukat, Borys and Komes exhibited the highest resistance to the disease in terms of TKW and yield reductions after inoculation.     

Genetic variation of aggressiveness in individual field populations ofFusarium graminearum andFusarium culmorum tested on young plants of winter rye

Fusarium graminearum andF. culmorum are capable of infecting winter cereals at all growth stages. From natural field epidemics of wheat head blight and rye foot rot, three fungal populations were collected with 21, 38 and 54 isolates, respectively; their aggressiveness was analyzed in comparison to collections ofF. graminearum (25 isolates) andF. culmorum (70 isolates) that represent a wide range of geographical locations and host species. All isolates were tested for aggressiveness on young plants of winter rye in the greenhouse and scored for disease severity on a 1–9 scale. Disease ratings of individual isolates ranged from 1.5 to 5.7 indicating quantitative variation of aggressiveness. Genotypic variance was highest in the twoFusarium collections. No substantial difference was found in the amount of genotypic variation betweenF. graminearum andF. culmorum. Individual field populations revealed 57–66% of the total genotypic variation of the collections. This implies a high degree of diversity of aggressiveness within single field populations ofF. graminearum andF. culmorum causing natural epidemics.     

Effect of benomyl on soil fungi associated with rye. 1. Effect on the incidence of sharp eyespot caused by Rhizoctonia cerealis

Effects of benomyl on incidence of pathogens affecting the culm base of rye were studied in field trials and growth chamber experiments. Spraying of the crop with the fungicide at a high dosage (2.4 kg.ha^–1) resulted in a tenfold increase of sharp eyespot caused byRhizoctonia cerealis and reduced foot rot symptoms caused by fusaria by 50%. In a field trial at a low dosage (0.24 kg.ha^–1) a slight increase of sharp eyespot was observed. In one year, probably because of wet conditions during the infection period, sharp eyespot did not occur in either benomyl-treated or untreated plots, but eyespot caused byPseudocercosporella herpotrichoides was abundant. Its occurrence was reduced from 74% affected culm bases in untreated plots to 8% and 1% in plots that received 0.24 and 2.4 kg.ha^–1 of the fungicide, respectively.In growth chambers seedlings were grown in two sandy soils inoculated withR. cerealis. The soil was kept dry at about 35% of the moisture holding capacity. In plots with benomyl (1 mg.kg^–1; moisture content 11% of fresh weight), fewer seedlings emerged than in plots without the fungicide. This result was highly significant (P<0.01) for one soil but not for the other. The number of seedlings that remained free of disease symptoms was higher (P<0.01) in untreated than in fungicide-treated plots of both soils.Isolates of pathogens obtained from diseased culms were tested for their sensitivity to benomyl. Growth of all of them includingR. cerealis was inhibited, although not always completely suppressed, at 10 g.ml^–1 on potato-dextrose agar. ED₅₀ values of most isolates ofR. cerealis were between 2.2 and 3.1 g.ml^–1. The fungus was slightly but consistently less sensitive thanF. culmorum. Mycelial growth ofF. nivale was appreciably more sensitive than that of the otherFusarium spp. from cereals.P. herpotrichoides andF. nivale were the most sensitive pathogens tested with ED₅₀ values of <1 g.ml^–1. Accordingly,F. nivale was absent on culms from treated plots. In a growth chamber experiment, seedlings were protected from infection by supplying the fungicide (1 mg.kg^–1) to previously inoculated soil.In a laboratory assay the effect of benomyl on microbial antagonism toR. cerealis was estimated for rhizosphere soil. Enhanced incidence of sharp eyespot in treated crops was associated with adverese effects of the fungicide on microbial antagonism. There is presumptive evidence thatR. cerealis is suppressed by bacteria after wet periods during the vegetation period of the crop and by fungi after dry periods. Only fungal antagonism, which may be less effective, is affected by benomyl. The response to benomyl of the microflora in different soils varied. Reasons for this inconsistency are suggested.Samenvatting In veldproeven en in een klimaatkamer werd de invloed van benomyl op het optreden van voetziekten in rogge onderzocht. In veldjes die bespoten waren met een hoge dosis van het fungicide (in totaal 2.4 kg.ha^–1) bleken tienmaal zoveel halmen met scherpe oogvlekken, veroorzaakt doorRhizoctonia cerealis, voor te komen dan in onbespoten veldjes. Daarentegen was voetrot veroorzaakt doorFusarium-soorten met 50% verminderd. In een volgende veldproef, waarbij een voor de praktijk geadviseerde dosis (0.24 kg.ha^–1) was toegepast, werd een lichte toename van scherpe oogvlekken waargenomen.In een ander jaar trad scherpe oogvlekkenziekte in het geheel niet op, ook niet in met benomyl behandelde veldjes. De vochtige omstandigheden tijdens de infectieperiode zijn daarvan waarschijnlijk de oorzaak. Daarentegen werd de oogvlekkenziekte, welke doorPseudocercosporella herpotrichoides werd veroorzaakt, veel aangetroffen. In de onbehandelde veldjes waren 74% van de halmen aangetast tegen 8 en 1% in de veldjes die met het fungicide waren behandeld in doseringen van 0.24 en 2.4 kg.ha^–1.De invloed van het fungicide op de aantasting van kiemplanten werd in klimaatkamerproeven onderzocht. Daartoe werden twee zandgronden metR. cerealis geënt. De grond werd droog gehouden (op 35% van het waterhoudend vermogen). In grond met fungicide (1 mg.kg^–1) was de opkomst minder dan in grond zonder fungicide. Dit was zeer significant (P<0.01) voor één van de beide zandgronden, maar niet voor de andere. Het aantal gezonde kiemplanten was in beide gevallen duidelijk hoger (P<0.01) voor de onbehandelde grond.De isolaten van ziekteverwekkers uit aangetaste halmen werden op hun gevoeligheid voor het fungicide getoetst. Op aardappel-glucoseagar werden alle isolaten in hun groei geremd bij een benomyl-concentratie van 10 g.ml^–1.R. cerealis was iets minder gevoelig danF. culmorum. Voor het overgrote deel van de isolaten vanR. cerealis lag de ED₅₀ waarde tussen 2,2 en 3,1 g.ml^–1. De myceliumgroei vanF. nivale werd meer geremd dan die van de andereFusarium-soorten.P. herpotrichoides enF. nivale waren met een ED₅₀ waarde van <1 g.m.^–1 de gevoeligste pathogenen die uit de halmvoeten werden geïsoleerd. Dat de populatie vanF. nivale in benomylhoudende grond wordt onderdrukt, blijkt uit (1) het feit dat de schimmel niet voorkwam op halmen uit behandelde veldjes en (2) de bescherming tegen infectie van kiemplanten als aan de besmette grond fungicide (1 mg.kg^–1) was toegevoegd.In laboratoriumproeven werd de invloed van benomyl op het microbiële antagonisme in rhizosfeergrond tegenR. cerealis bepaald. Een toename in het optreden van scherpe oogvlekkenziekte in behandelde gewassen bleek gepaard te gaan met een remming van het antagonisme tegen de ziekteverwekker. Er zijn sterke aanwijzingen datR. cerealis na vochtige perioden tijdens de vegetatieperiode door bacteriën wordt onderdrukt en na droge perioden door schimmels. Het antagonisme van de laatste groep lijkt minder effectief te zijn en alleen dit antagonisme wordt door benomyl verlaagd. Tenslotte wordt een mogelijke oorzaak aangegeven voor de ongelijke respons op het fungicide van het microbieel antagonisme in verschillende gronden.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Competition Effects Among Isolates of Fusarium culmorum Differing in Aggressiveness and Mycotoxin Production on Heads of Winter Rye

Fusarium culmorum is a phytopathogenic, toxigenic fungus causing seedling diseases, foot rot and head blight of cereals. For estimating competition effects in mixtures of two single-spore isolates, two winter rye single crosses were tested with either four isolates individually or four 1 : 1 mixtures of the same isolates in six field environments. Two isolates (FC46, FC64) were highly aggressive deoxynivalenol (DON) and 3-acetyl DON-producers, the other two (FC30, FC71) were medium aggressive nivalenol-producers. Rye heads were inoculated during flowering with conidia of pairs of isolates expressing similar (FC46 + FC64, FC30 + FC71) or contrary (FC46 + FC71, FC30 + FC64) levels of aggressiveness and similar or different concentrations and chemotypes of mycotoxins, respectively. Head blight rating and yield components relative to the non-inoculated plots were recorded as aggressiveness traits. Additionally, mycotoxin concentrations were measured in the rye grain. Random pathogen samples were re-isolated from heads at the onset of symptom development and analysed by molecular markers (RAPD–PCR) in one environment. Aggressiveness of the isolate mixtures was significantly lower than that of the isolates applied individually on both rye genotypes. Similarly, mycotoxin concentrations were significantly lower in the mixtures in seven out of eleven comparisons. Among the re-isolates, the component genotypes of a mixture significantly deviated from the inoculated 1 : 1 ratio when a particular isolate (FC46) was present in the mixture. This isolate displayed a superior competitive ability irrespective of the aggressiveness or mycotoxin profile of the mixing partner illustrating that pathogenic fitness is caused by additional factors that have not, as yet, been identified.     

Inhibition by aluminum of mycelial growth and of sporangial production and germination inPhytophthora infestans

Mycelial growth on clarified V8 agar of the potato late blight pathogenPhytophthora infestans was inhibited when either aluminum chloride (AlCl₃, 6 H₂O) or aluminum sulfate (Al₂(SO₄)₃, 18 H₂O) was added to the culture medium at concentrations of 2.5–100 mg.l^–1 Al³⁺. Toxicity of Al³⁺ varied among the fiveP. infestans isolates tested, but toxicity of sulfate and chloride salts was similar for a given isolate. Overall sporangial production was affected in all five isolates by both Al³⁺ forms. Al³⁺ also decreased sporangial germination at concentrations equal to or greater than 10 mg.l^–1 in two isolates. These data support the hypothesis of aluminum toxicity as a major factor in soil suppressiveness toP. infestans.     

Species composition,toxigenic potential and pathogenicity of Fusarium graminearum species complex isolates from southern Brazilian rice

This study aimed to assess the extent and distribution of Fusarium graminearum species complex (FGSC) diversity in rice seeds produced in southern Brazil. Four species and two trichothecene genotypes were detected among 89 FGSC isolates, based on a multilocus genotyping assay: F. asiaticum (69·6%) with the nivalenol (NIV) genotype, F. graminearum (14·6%) with the 15‐acetyldeoxynivalenol (ADON) genotype, and F. cortaderiae (14·6%) and F. meridionale (1·1%), both with the NIV genotype. Seven selected F. asiaticum isolates from rice produced NIV in rice‐based substrate in vitro, at levels ranging from 4·7 to 84·1 μg g^?1. Similarly, two F. graminearum isolates from rice produced mainly 15‐ADON (c. 15–41 μg g^?1) and a smaller amount of 3‐ADON (c. 6–12 μg g^?1). One F. meridionale and two F. cortaderiae isolates did not produce detectable levels of trichothecenes. Two F. asiaticum isolates from rice and two from wheat (from a previous study), and one F. graminearum isolate from wheat, were pathogenic to both crops at various levels of aggressiveness based on measures of disease severity in wheat spikes and rice kernel infection in a greenhouse assay. Fusarium asiaticum and the reference F. graminearum isolate from wheat produced NIV, and deoxynivalenol and acetylates, respectively, in the kernels of inoculated wheat heads. No trichothecene was produced in kernels from inoculated rice panicles by any of the isolates. These findings constitute the first report of FGSC composition in rice outside Asia, and confirm the dominance of F. asiaticum in rice agroecosystems.     

Characterisation of kernel resistance against <Emphasis Type="Italic">Fusarium</Emphasis> infection in spring wheat by baking quality and mycotoxin assessments

Fusarium head blight (FHB) of wheat heads by Fusarium culmorum causes serious yield losses and compromises the end-use quality by accumulation of mycotoxins and alteration of baking characteristics. The most promising control strategies against the disease combine adequate cropping techniques (i.e. crop rotation avoiding maize as a preceding crop) with the use of resistant varieties. Different types of resistance against this disease have been described such as the resistance to primary infection of the spikelets and the reduction of spread of the infection in other parts of the ear. In recent years, the ability of the kernels to prevent penetration of the fungus and mycotoxin accumulation has received increasing attention. Yet, the detection of kernel resistance for breeding purposes is rather difficult, as the corresponding resistance mechanisms are not fully understood. The aim of the present work is to compare different aspects of kernel resistance in order to define the most significant criteria for breeding purposes. The experimental set up included eight modern Swiss spring wheat varieties grown on small irrigated yield plots (3 × 1.5 m) inoculated at anthesis with a mixture of Fusarium culmorum isolates. Disease ratings from 7 to 28 days post-inoculation were completed with post-harvest analyses for the accumulation of the mycotoxin deoxynivalenol and different baking quality parameters. Results indicate that the accumulation of the mycotoxin deoxynivalenol in the kernels is correlated with visible symptoms on the ear before harvest. In terms of baking quality parameters, water absorption, dough softening and dough resistance are impaired in susceptible varieties after FHB infection, while resistant varieties are not affected. The results obtained here indicate that kernel resistance can be defined by low deoxynivalenol accumulation in the kernels and by stability of several baking quality parameters under conditions of high FHB infection pressure.     

Assessment of infection in wheat by <Emphasis Type="Italic">Fusarium</Emphasis> protein equivalent levels

Determination of the Fusarium protein equivalent (FPE) levels in kernels for better characterisation of genotypes showing Fusarium head blight (FHB) resistance, and better detection of susceptibility to kernel infection among genotypes with slight symptom expression was carried out. Twelve wheat cultivars and eight hexaploid winter wheat lines derived from a cross of Triticum aestivum with related species T. macha, T. polonicum, and T. dicoccoides were evaluated for levels of spike and kernel infection, the content of the mycotoxin deoxynivalenol (DON) and FPE in kernels after artificial inoculation with the fungus Fusarium culmorum in the field in 2006–2007. The ELISA immunochemical method was employed for the quantitative analyses of DON and FPE. Three wheat lines had a significantly low infection of spikes and kernels compared to cvs Sumai 3 and Nobeoka Bozu, indicating the presence of specific resistance mechanisms to FHB. The significantly low AUDPC (area under the disease progress curve) and the high level of FPE and DON content in kernels indicated a lack of resistance in one wheat line (crossed with T. polonicum). The results showed highly significant correlations (P < 0.01) between FPE and DON content and between FPE and AUDPC. In addition, correlations between FPE and reductions in yield components were also highly significant. Quantification of Fusarium spp. in wheat kernels can be helpful for evaluating wheat genotypes for their levels of resistance to FHB.     

Fusarium head blight and mycotoxin contamination of wheat,a review

Summary An infection of bread wheat by fusarium head blight contaminates the crop with mycotoxins, particularly deoxynivalenol (DON) and nivalenol (NIV). The toxicity and natural occurrence of these mycotoxins in wheat are reviewed. Based on 8 years data of fusarium head blight epidemics of wheat in the Netherlands, DON contamination of the grain was estimated. Fusarium head blight ratings averaged an infection of 1.7% of all spikelets; estimates for DON contamination averaged 0.9 mg kg^–1. Taking a guideline level for DON in uncleaned bread wheat of 2 mg kg^–1, in 1979 and 1982 a wheat crop was produced with estimated DON concentrations above the limit of tolerance. Human and animal exposure to mycotoxins in the Netherlands appears to be small but chronic. The information presented in this paper illustrates the need for an annual evaluation of the crop for fusarium head blight incidence and mycotoxin content, and the necessity of fusarium head blight resistant wheat cultivars.Samenvatting Aaraantasting van tarwe doorFusarium culmorum enFusarium graminearum leidt tot vorming van mycotoxinen in het graan, waarvan deoxynivalenol (DON) en nivalenol (NIV) de belangrijkste toxinen zijn. In dit artikel wordt een overzicht gegeven van de toxicologische aspecten, en het voorkomen van deze toxinen in tarwe. Informatie over DON en NIV in tarwe in West-Europa is schaars. Gebaseerd op gegevens vanFusarium epidemieën in de jaren 1979–1986 wordt een schatting gegeven van de concentratie DON in Nederlandse tarwe. Rekening houdend met de herkomst en verwerking van tarwe, blijken zowel in dierlijk als menselijk voedsel lage concentraties DON chronisch voor te komen. Op basis van een maximaal toelaatbare dagelijkse dosis DON van 3 g kg^–1 lichaamsgewicht is de schatting van de dagelijkse opname van DON in het jaar volgend op de oogst van 1982 net op de grens. Zowel een jaarlijkse inventarisatie vanFusarium aantasting en DON besmetting van het graan, als de ontwikkeling vanFusarium-resistente rassen zijn noodzakelijk.     

Characterization and genetic analysis of field isolates ofBotryotinia fuckeliana (Botrytis cinerea) resistant to dichlofluanid

Field isolates ofBotryotinia fuckeliana were collected from naturally infected plants. Their responses to the multisite fungicide dichlofluanid in mycelium growth test fell into three phenotypic classes, characterized by the following EC₅₀ (and MIC) values ing ml^–1: sensitivity, 1–3 (6–10); low resistance, 3–10 (> 100); high resistance, 10–30 (> 100). The corresponding values obtained for these classes in a spore germination test were respectively: 0.05 (0.2), 0.05–0.1 (0.5), 0.5–1 (0.9–1.5). Resistant isolates were crossed with two sensitive and two resistant strains of appropriate mating type to determine the genetic basis of resistance. Distribution of resistance phenotypes in ascospore progeny indicated that a gene, namedDic1, was probably responsible for the low or high resistance of 14 mutants selectively collected from experimental plots of greenhouse-grown gerbera sprayed several times with dichlofluanid or tolyfluanid. A second gene, namedDic2, was probably responsible for the low resistance displayed by two isolates (from grapevine and from carnation) maintained in the laboratory collection. As a result of the investigation, the use of dichlofluanid in integrated management programmes against grey mould is discussed.     

Characterisation of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low,medium and high degree of resistance to DMI-fungicides

Differential accumulation of [¹⁴C]imazalil and [¹⁴C]fenarimol by germlings of wild-type and DMI-resistant isolates ofPenicillium italicum was studied at various pH values. At pH 7 and 8 the low-resistant isolate E_300–3 accumulated 22% and 35%, respectively, less imazalil than the wild-type isolate W₅. Imazalil accumulation at pH 5 and 6 was similar. Isolate E_300–3 also accumulated less fenarimol as compared with the wild-type isolate. This difference was much more obvious than for imazalil and was observed at all pH values tested. Differences in accumulation of both imazalil and fenarimol between low (E_300–3), medium (H₁₇) and high resistant (I₃₃) isolates were not observed. These results suggest that decreased accumulation of DMIs is responsible for a low level of resistance only and that additional mechanisms of resistance might operate in isolates with a medium and high degree of resistance. With all isolates fenarimol accumulation was energy-dependent. This was not obvious for imazalil.The wild-type and DMI-resistant isolates had a similar plasma membrane potential as determined with the probe [¹⁴C]tetraphenylphosphonium bromide ([¹⁴C]TPP⁺). Various test compounds, among which ATPase inhibitors, ionophoric antibiotics and calmodulin antagonists, affected the accumulation of [¹⁴C]TPP⁺, [¹⁴C]imazalil and [¹⁴C]fenarimol. No obvious correlation between the effects of the test compounds on accumulation levels of the fungicides and [¹⁴C]TPP⁺ could be observed. These results indicate that the plasma membrane potential does not mediate the efflux of DMI fungicides byP. italicum.     

Comparison of Chinese and Japanese A1 isolates of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

The mating type, glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) genotypes, RG57 fingerprint, and mitochondrial DNA (mtDNA) haplotype of Chinese isolates of Phytophthora infestans collected in Hebei and Gansu in 1996 were compared with those of Japanese isolates collected during 1997–2000. The Chinese isolates were divided into four genotypes, one of which was identical to the dominant Japanese genotype, A1-A (mating type A1; Gpi 100/100; Pep 100/100; RG57 100010001100110100011001110: 1–25, 14a, and 24a; and mtDNA haplotype IIa). Comparison of the genotypes with reported data revealed that some completely and partially identical genotypes occur in Russia and parts of Europe. The other two A1 genotypes and one A2 genotype were also detected in Gansu (Gpi 100/100, Pep 100/100, and mtDNA haplotype Ia), which were regarded as unique to this region.     

Induction of oxidants in tomato leaves treated with <Emphasis Type="SmallCaps">DL</Emphasis>-β-Amino butyric acid (BABA) and infected with <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> ssp. <Emphasis Type="Italic">michiganensis</Emphasis>

Bacterial canker is an economically important disease of tomato. Resistance induced by DL-β-Amino butyric acid against bacterial canker caused by Clavibacter michiganensis ssp. michiganensis in tomato plants was investigated. Different doses of DL-β-Amino butyric acid (250–1000 μg ml⁻¹ doses) were tested on 3-week old plants inoculated with a 10⁸ CFU ml⁻¹ bacterial suspension, and disease development was evaluated after inoculation and treatment. Although in vitro growth of the bacteria was not affected by DL-β-Amino butyric acid treatment, foliage sprays of 500 μg ml⁻¹ DL-β-Amino butyric acid significantly suppressed disease development up to 54% by day 14 after inoculation at the four different doses tested. Bacterial populations were reduced by 84% in BABA-treated plants compared to water-treated plants by day 4 after inoculation. Inoculated BABA-treated plants showed significantly higher phenylalanine ammonia-lyase activity, peroxidase activity, and H₂O₂ concentration than inoculated water-treated plants during day 1 after treatment. These findings suggest that the DL-β-Amino butyric acid treatment resulted in an increase of these enzymes and in H₂O₂ concentration in planta, and was associated with induction of resistance to bacterial canker.     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

The Effect of Inoculation Treatment and Long-term Application of Moisture on Fusarium Head Blight Symptoms and Deoxynivalenol Contamination in Wheat Grains

Fusarium head blight (FHB) is an important disease of wheat, which can result in the contamination of grains with mycotoxins such as deoxynivalenol (DON). Artificial inoculation of flowering ears with conidial suspensions is widely used to study FHB diseases. Our goal was to compare four inoculation treatments in which a conidial suspension was sprayed on flowering ears and to study the effect of the application of moisture during kernel setting and filling with a mist-irrigation system. Ten wheat genotypes were inoculated with a DON-producing Fusarium culmorum strain. Inoculation treatments varied in time of application of the inoculum (morning or evening) and in the method of controlling humidity during inoculation (bagging or mist irrigation). A wet season was simulated with a mist-irrigation system, keeping the crop canopy wet for at least 26 days after flowering. The severity of FHB symptoms (area under disease progress curve (AUDPC)), yield loss and DON contamination in the grains were determined. AUDPC data obtained with the different inoculation treatments were highly correlated (r=0.85–0.95). Mist irrigation after inoculation resulted in a higher mean disease severity, but in a overall lower toxin contamination as compared to the non-irrigated treatments. Genotypic differences in DON accumulation were present: for one wheat line toxin contamination significantly increased when irrigated, while two genotypes accumulated significantly less toxin. The closest relationships (r=0.73–0.89) between the visual symptoms and the DON content were obtained under moderate mean infection pressure. This relation between visual symptoms and the DON content deteriorated at higher infection levels.