Genetic analysis and PCR-based identification of major <Emphasis Type="Italic">Fusarium</Emphasis> species causing head blight on wheat in Japan

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

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.     

Quantitative Detection of Fusarium Species in Wheat Using TaqMan

Fusarium head blight (FHB) of wheat and other small-grain cereals is a disease complex caused by several fungal species. To monitor and quantify the major species in the FHB complex during the growing season, real-time PCR was developed. TaqMan primers and probes were designed that showed high specificity for Fusarium avenaceum, F. culmorum, F. graminearum, F. poae and Microdochium nivale var. majus. Inclusion of an internal PCR control and serial dilutions of pure genomic DNAs allowed accurate determination of the concentration of fungal DNA for each of these species in leaves, ears as well as harvested grains of winter wheat. The DNA concentration of F. graminearum in grain samples correlated (r ²= 0.7917) with the incidence of this species on the grain as determined by isolation from individual kernels. Application of the TaqMan technology to field samples collected in 40 wheat crops in the Netherlands during the growing season of 2001 revealed that M. nivale var. majus predominated on leaves early in the season (GS 45-65). Ears and harvested grains from the same fields, however, showed F. graminearum as the major species. In 2002, grain samples from 40 Dutch fields showed a much wider range of species, whereas in ears from 29 wheat crops in France, F. graminearum was the predominant species. The concentration of DON correlated equally well with the incidence of the DON-producing species F. culmorum and F. graminearum in the grain samples (r ²= 0.8232) as well as with total DNA of both these species (r ²= 0.8259). The Fusarium TaqMan technology is an important tool to quantify and monitor the dynamics of individual species of the complex causing FHB in cereals during the growing season. This versatile tool has been applied in a comparison of different genotypes, but can also be applied to other disease management systems, e.g. fungicide treatments.     

Studies on in vitro Growth and Pathogenicity of European Fusarium Fungi

The effect of temperature on the in vitro growth rates and pathogenicity of a European Fusarium collection consisting of isolates of Fusarium graminearum, F. culmorum, F. avenaceum, F. poae and Microdochium nivale was examined. Irrespective of geographic origin, the optimum temperature for the growth of F. graminearum, F. culmorum and F. poae was 25 °C, while that for F. avenaceum and M. nivale was 20 °C. In general, the growth rates of F. graminearum, F. culmorum and F. poae increased between 10 and 25 °C and those of F. avenaceum and M. nivale increased between 10 and 20 °C. Pathogenicity tests were carried out by examining the effect of the five species on the in vitro coleoptile growth rate of wheat seedlings (cv. Falstaff). Irrespective of geographic origin, the temperature at which F. avenaceum, F. culmorum and F. graminearum caused the greatest retardation in coleoptile growth ranges 20–25 °C (>89.3% reduction), whilst for F. poae and M. nivale it was 10–15 °C (>45.6% retardation), relative to uninoculated control seedlings. In general, F. culmorum and F. graminearum were the most pathogenic of the five species, causing at least a 69% reduction in coleoptile growth at 10, 15, 20 and 25 °C. General linear model analysis (GLIM) showed that species accounted for 51.3–63.4% of the variation in isolate growth and from 19.5% to 44.3% of the variation in in vitro pathogenicity. Country of origin contributed from 22.6% to 51.9% to growth rate variation and from 0.73% to 7.61% to pathogenicity variation. The only significant correlation between in vitro growth and pathogenicity was that observed for M. nivale at 15 °C (r = -0.803, P < 0.05).     

Morphological and molecular identification of <Emphasis Type="Italic">Fusarium</Emphasis> species associated with head blight on wheat in East Croatia

Fusarium head blight (FHB) is an important fungal disease of wheat. The aim of this research was to determine the diversity of Fusarium species infecting winter wheat ears in East Croatia. In 2008 wheat kernels were obtained from three locations in the eastern part of Croatia (Tovarnik, Osijek, Pozega), and in 2009 from two additional locations (Slavonski Brod, Nova Gradiska). In total, 498 visually diseased kernels were selected for morphological identification of Fusarium spp. The identity of 226 selected isolates was further investigated by molecular techniques. The predominant species on wheat kernels in East Croatia in 2008 were F. graminearum, isolated and confirmed from more than 80% of sampled wheat kernels, followed by F. avenaceum (8%) and F. culmorum (7%). Incidence of F. poae was less than 2%. The most common species identified in 2009 were F. graminearum (50%), F. culmorum (13%), F. avenaceum (12%) and F. poae (7%). This is the first report on the identification of Fusarium species isolated from naturally infected wheat ears in Croatia.     

Comparison of pathogenicity of the Fusarium crown rot (FCR) complex (<Emphasis Type="Italic">F. culmorum</Emphasis>, <Emphasis Type="Italic">F. pseudograminearum</Emphasis> and <Emphasis Type="Italic">F. graminearum)</Emphasis> on hard red spring and durum wheat

Fusarium species involved in the Fusarium crown rot (FCR) complex affect wheat in every stage of development from seedling to grain fill. This study was designed to compare the aggressiveness of the FCR complex members including F. culmorum, F. pseudograminearum and F. graminearum in causing seedling blight, decreased plant vigour and crown rot. To assess their relative pathogenicity, two hard red spring wheat cultivars and two durum wheat cultivars were inoculated in the field with five isolates from each of the three species for two years. Significant differences in patterns of pathogenicity were identified. In particular, F. culmorum caused greater seedling blight while F. pseudograminearum and F. graminearum caused greater crown rot. Greatest yield reductions were caused by F. pseudograminearum. Cultivar differences were identified with respect to seedling disease and late season crown rot. No interactions were identified between cultivar performance and isolates or species with which they were challenged.     

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.     

Biodiversity of Fusarium species in ears and stalks of maize plants in Belgium

In order to investigate the pre-harvest contamination of maize plants by Fusarium species in Belgium, a three-year survey has been performed in five fields in which three hybrids differing in susceptibility to maize stalk rot were sampled at four different physiological stages. An extensive collection of 5,659 Fusarium isolates characterized at the species level was established during the 2005, 2006, and 2007 growing seasons, with a total of 23 different Fusarium species identified to occur on ears and stalks. A high number of plants was already contaminated by Fusarium spp. at the anthesis stage, although no symptoms were visible on ears or on stalks. As the season progressed, the incidence of Fusarium-infected maize plants reached 100% in several fields. At the end of the growing season, the most frequently isolated species in maize ears were F. graminearum, sometimes associated with F. avenaceum, F. crookwellense, F. culmorum, F. poae, and F. temperatum, a new species recently described on maize. The predominant Fusarium species detected in stalks at the end of the growing season were F. graminearum and F. crookwellense, often associated with F. culmorum and F. temperatum. Year-to-year variability observed for the incidence of F. graminearum can most likely be associated with differences in climatic conditions among the three years.     

Association of Fusarium Species in the Wheat Stem Rot Complex

Data from a national survey were analysed to investigate whether there was interdependence among the Fusarium species, which cause the stem rot complex of wheat. About 25 wheat stems were sampled from each of 260 sites over the main wheat growing areas in the UK. Occurrence of each Fusarium species on individual stems was determined. Fusarium culmorum, F. avenaceum and Microdochium nivale were the three dominant species, detected in 248, 185 and 239 out of the 260 sites. There were no interactions among species in the distribution of the three species over the 260 sites. Several statistical tests were used to determine whether there was interdependence among the three species on the same stem within each site. Of the three species, there was only limited evidence of competition between F. culmorum and F. avenaceum.     

Early PCR-based detection of Fusarium culmorum,F. graminearum,F. sporotrichioides and F. poae on stem bases of winter wheat throughout Poland

Foot rot and crown rot are fungal diseases of wheat caused by a complex of Fusarium species. They have a huge economic impact mainly due to yield reduction. A survey was conducted to identify four Fusarium species, occurring on wheat stem bases, using species-specific PCR assays in samples collected during spring of 2012. The dominant species was F. graminearum, which was identified in above 64 % of samples. F. culmorum was detected in 15.71 %, F. poae in 15.71 % and F. sporotrichioides in 5.71 % wheat fields. Most of the wheat fields in the eastern Poland were infected with at least one or two of Fusarium species, while in central Poland no Fusarium species were identified in most of the fields. The presence of F. graminearum tends to favor the presence of F. culmorum and this effect was visible also for F. poae and F. sporotrichioides. The frequency of F. graminearum and F. culmorum detections were highest where wheat crops were preceded by maize and in the samples from late sown fields. The opposite observation was made for F. poae and F. sporotrichioides, where the number of detections of these species was higher in samples from early sown fields. The number of detected Fusarium species was significantly lower in samples collected from fields protected with autumn herbicide in comparison to unprotected fields. The rate of autumn N fertilization did not affect the number of Fusarium detections.     

Molecular and Morphological Diversity of Fusarium Species in Finland and North-Western Russia

In 2001 the range of the total Fusarium contamination percentage of infected seeds was between 0% and 44%, while in 2002 the contamination level was 2–25% in naturally infected Finnish samples and 5–14.5% in six samples from northwestern Russia. The most common Fusarium species in barley were F. avenaceum, F. arthrosporioides, F. sporotrichioides and F. culmorum, while in spring wheat the most common Fusarium species were F. avenaceum, F. arthrosporioides, F. culmorum, F. sporotrichioides and F. graminearum. In most cases, molecular identification with species-specific primers corresponded to the morphological analyses and allowed the identification of degenerated and otherwise morphologically difficult cultures. It was even possible to separate most of the F. arthrosporioides isolates from Finland from the closely-related F. avenaceum isolates. In the phylogenetic analysis of combined β-tubulin, IGS and ITS sequences most European F. arthrosporioides formed a separate clade from most isolates of F. avenaceum and from all isolates of F. tricinctum. Most of the species-specific primers also amplified DNA extracted from grain samples. It was, for instance, possible to detect F. avenaceum in all barley samples with contamination levels higher than 1% and in all spring wheat samples with contamination levels higher than 3%. The detection level for F. graminearum was at a contamination level of 3–5% and that for F. culmorum at a contamination level of 1–5%. In addition, the first Finnish F. langsethiae isolate was found by means of species-specific primers.     

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.     

Das Artenspektrum der Gattung Fusarium an Weizen und Roggen in Bayern in den Jahren 2003 und 2004

During the years 2003 and 2004 grains of wheat and rye were examined for the occurrence of different Fusarium species in Bavaria. The data obtained indicate that rye is infected with Fusarium spp. on a lower level than wheat. Overall F. graminearum was the most important Desoxynivalenol (DON) producing species with infected kernels per sample up to 18,5% whereas F. culmorum recedes more in the background. In addition F. poae was detected in high amounts: up to 28,5% kernels per sample were infected. Also the incidence of F. avenaceum was high. In contrast F. equiseti, F. tricinctum, F. sporotrichioides, F. oxysporum and Microdochium nivale were detected to a much lower extent.     

Deoxynivalenol, Nivalenol and Moniliformin in Wheat Samples with Head Blight (scab) Symptoms in Poland (1998–2000)

Fusarium head blight (scab) epidemics of wheat occurred in uawy (Northern Poland) during 1998 and in Wielkopolska (West) and in Southern regions of Poland in 1999. Four species were identified in wheat heads with scab symptoms: Fusarium culmorum, Fusarium graminearum, Fusarium avenaceum and Microdochium nivale. A significant increase in the frequency of F. graminearum (between 23% and 38%), was observed, compared to about 10% during the previous decade. The mycotoxins deoxynivalenol (DON), nivalenol (NIV) and moniliformin (MON) in amounts up to 24.3, 14.2 and 1.72mgkg^–1respectively, were identified in kernels samples.     

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.     

Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis

Fusarium graminearum, Fusarium culmorum and Fusarium cerealis are major causal agents of Fusarium Head Blight (scab) which is a disease of global significance in all cereal growing areas. These fungi produce trichothecene mycotoxins, principally nivalenol (NIV) and deoxynivalenol (DON). Genes Tri13 and Tri7 from the trichothecene biosynthetic gene cluster convert DON to NIV (Tri13) and NIV to 4-acetyl-NIV (Tri7). We have developed positive–negative PCR assays based on these two genes, which accurately indicate a DON or NIV chemotype in F. graminearum, F. culmorum and F. cerealis. These assays are useful in assessing the risk of trichothecene contamination, and can be informative in epidemiological studies. All NIV chemotype isolates studied have functional copies of both Tri13 and Tri7, and all DON-producing isolates have both genes disrupted or deleted. We have identified several mutations in these genes, which are conserved across F. graminearum lineage, RAPD and SCAR groupings and between the three species. There appears to be evidence of inter-species hybridisation within the trichothecene biosynthetic gene cluster.     

Identification and pathogenicity of<Emphasis Type="Italic">Fusarium</Emphasis> spp. from stem bases of winter wheat in Erzurum,Turkey

Four-hundred-sixty-eightFusarium andFusarium-like isolates were obtained from crowns and subcrown internodes of winter wheat grown in Erzurum, Turkey. Of these isolates, 34.8% wereFusarium acuminatum, 32.3% wereF. equiseti, 16.9% wereF. oxysporum, 15.0% wereMicrodochium nivale (formerlyFusarium nivale), 0.6% wereF. tabacinum and 0.4% wereF. solani. In pathogenicity tests on wheat, the highest disease severity was caused by isolates ofM. nivale, whereas isolates ofF. acuminatum, F. equiseti, F. oxysporum andF. solani were slightly virulent; isolates ofF. tabacinum were nonpathogenic. This is the first report ofM. nivale andF. tabacinum from wheat in Turkey. http://www.phytoparasitica.org posting Jan. 29, 2003.     

Molecular and biological characterization of an isolate of <Emphasis Type="Italic">Tomato mottle mosaic virus</Emphasis> (ToMMV) infecting tomato and other experimental hosts in eastern Spain

Fusarium Head Blight is a major disease of wheat and an important contributor to the reduced cultivation of wheat in South Africa, where the crop often is grown under irrigation. We collected Fusarium isolates from 860 Fusarium Head Blight-infected wheat heads in seven irrigated wheat-growing areas of South Africa. Six Fusarium species, i.e., F. chlamydosporum, F. crookwellense, F. culmorum, F. equiseti, F. graminearum and F. semitectum were recovered, three of which, i.e., F. chlamydosporum, F. equiseti and F. semitectum, were not previously associated with Fusarium Head Blight in South Africa. Fusarium graminearum occurred at high frequencies at all seven locations. Based on polymerase chain reaction (PCR) assays of diagnostic sequences, more isolates were predicted to produce deoxynivalenol than nivalenol. Fusarium graminearum (sensu lato) appears to be the primary causal agent of Fusarium Head Blight in irrigated wheat in South Africa, which may not be the case for wheat cultivated under rain-fed conditions. Rotations of irrigated wheat with other graminaceous crops and maize could increase fungal inoculum and disease pressure. The establishment of Fusarium Head Blight in the irrigated wheat region of the country means that resistant lines and alternative agronomic practices are needed to limit disease severity, yield losses and mycotoxin contamination.     

Simultaneous identification of species and trichothecene chemotypes of <Emphasis Type="Italic">Fusarium asiaticum</Emphasis> and <Emphasis Type="Italic">F. graminearum</Emphasis> sensu stricto by multiplex PCR

A multiplex PCR assay was developed for simultaneous identification of the species and trichothecene chemotypes for Fusarium asiaticum and F. graminearum sensu stricto based on the genes related to trichothecene biosynthesis. PCR was carried out in a single reaction with three pairs of primers designed for the tri6 region and one pair of primers designed for tri3. We confirmed that the multiplex PCR was able to identify species and chemotypes for all tested strains of F. asiaticum and F. graminearum s. str. isolated in Japan. This technique would be a useful and rapid tool for diagnosis, epidemiology, and population structure studies of the F. graminearum complex in Japan.