<Emphasis Type="Italic">Fusarium</Emphasis> species complex on maize in Switzerland: occurrence,prevalence, impact and mycotoxins in commercial hybrids under natural infection

The Fusarium species complex of maize kernels and stem pieces as well as mycotoxin contamination of commercial grain maize hybrids for animal feed were evaluated in Switzerland. Throughout 2 years, natural Fusarium infection varied significantly between the years and the locations and it ranged from 0.4% to 49.7% for kernels and from 24.2% to 83.8% for stem pieces. Using the agar plate method, 16 different Fusarium species were isolated from kernels and 15 from stem pieces. The Fusarium species composition, prevalence and impact differed between the north and the south and between kernel and stem piece samples. The dominant species on kernels in the north were F. verticillioides (32.9%), F. graminearum (31.3%), F. proliferatum (7.3%) and F. crookwellense (7.1%), in the south F. verticillioides (57.1%), F. subglutinans (24.6%), F. proliferatum (14.8%) and F. graminearum (1.5%) and on stem pieces F. equiseti (36.0%), F. verticillioides (20.1%), F. graminearum (9.5%), F. crookwellense (6.2%) and F. subglutinans (6.2%). In the south, fumonisin concentration of most hybrids exceeded guidance values for animal feed. Other Fusarium species isolated were F. avenaceum, F. culmorum, F. oxysporum, F. poae, F. sambucinum, F. semitectum, F. sporotrichioides, F. solani, F. tricinctum and F. venenatum. Maize hybrids varied in their susceptibility to Fusarium infection. Because of the high diversity of Fusarium species encountered in Switzerland representing a high toxigenic potential, we propose to screen maize hybrids for resistance against various Fusarium species and examine maize produce for several mycotoxins in order to ensure feed safety.     

Population structure of plant-pathogenic Fusarium species in overwintered stalk residues from Bt-transformed and non-transformed maize crops

Bt-transformed maize contains genes from Bacillus thuringiensis encoding for insecticidal crystal proteins. Less insect damage on Bt maize stalks can cause a reduced infection by Fusarium species through plant injuries. This could affect the presence of plant-pathogenic Fusarium species on maize residues which serve as an inoculum source for subsequent crops. We collected overwintered maize stalks of four different Bt maize hybrids and their corresponding non-Bt lines in two consecutive years in a field trial in Germany. Fusarium spp. were isolated from 67% of 648 collected maize stalks. Identification with new multiplex PCR assays showed that F. graminearum, F. avenaceum, and F. proliferatum were the most abundant Fusarium species, isolated from 42%, 26%, and 15% of the stalks, respectively. Species abundances varied between varieties and collection years. No consistent difference was found between Bt and non-Bt stalks. Fusarium graminearum isolates were subject to a population genetic structure analysis with eight newly developed microsatellites. Significant association of loci and overrepresentation of repeated multilocus haplotypes indicated a substantial asexual component of reproduction, supporting selection of haplotypes. The data suggested selection of particular F. graminearum haplotypes by collection years but not by maize Bt transformation. Haplotypic changes between years caused no divergence in the distribution of alleles, suggesting that gene flow beyond the field scale prevented substructuring. We present evidence for gene flow between our saprophytic F. graminearum population on maize residues and a wheat-pathogenic population from a field 100 km distant.     

Fusarium miscanthi and other Fusarium species as causal agents of Miscanthus × giganteus rhizome rot

In 2010, Miscanthus × giganteus rhizomes were collected from Belgian fields showing emergence reduction and were analyzed for detection and identification of fungal species. All rhizomes were infected by at least one Fusarium species, of which F. culmorum, F. miscanthi, F. crookwellense and F. graminearum were the most abundant. Results of the pathogenicity tests by soil inoculation demonstrate the ability of F. miscanthi, F. culmorum and F. avenaceum to cause rhizome rot disease under greenhouse conditions. This is the first report of F. miscanthi and other Fusarium species isolated from M. × giganteus rhizomes in Belgium.     

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.     

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.     

<Emphasis Type="Italic">Fusarium</Emphasis> species and mycotoxin profiles on commercial maize hybrids in Germany

High year-to-year variability in the incidence of Fusarium spp. and mycotoxin contamination was observed in a two-year survey investigating the impact of maize ear rot in 84 field samples from Germany. Fusarium verticillioides, F. graminearum, and F. proliferatum were the predominant species infecting maize kernels in 2006, whereas in 2007 the most frequently isolated species were F. graminearum, F. cerealis and F. subglutinans. Fourteen Fusarium-related mycotoxins were detected as contaminants of maize kernels analyzed by a multi-mycotoxin determination method. In 2006, a growth season characterized by high temperature and low rainfall during anthesis and early grain filling, 75% of the maize samples were contaminated with deoxynivalenol, 34% with fumonisins and 27% with zearalenone. In 2007, characterized by moderate temperatures and frequent rainfall during the entire growth season, none of the 40 maize samples had quantifiable levels of fumonisins while deoxynivalenol and zearalenone were detected in 90% and 93% of the fields, respectively. In addition, 3-acetyldeoxynivalenol, 15-acetyldeoxnivalenol, moniliformin, beauvericin, nivalenol and enniatin B were detected as common contaminants produced in both growing seasons. The results demonstrate a significant mycotoxin contamination associated with maize ear rots in Germany and indicate, with regard to anticipated climate change, that fumonisins-producing species already present in German maize production may become more important.     

Fusarium temperatum, a mycotoxin-producing pathogen of maize

In a recent study, a population of Fusarium strains isolated from maize in Belgium was described as a new species, F. temperatum, that is morphologically similar and phylogenetically closely related to F. subglutinans, a species in the American clade of the Gibberella fujikuroi species complex. In fields, the F. temperatum:F. subglutinans ratio was very high, suggesting that F. temperatum outcompetes its sister species F. subglutinans. This raised the question whether this novel species contributes to the final rot symptoms observed on maize plants at harvest, as well as to the potential mycotoxin contamination. Results of the pathogenicity tests by soil and toothpick inoculation demonstrate the ability of F. temperatum to cause seedling malformation and stalk rot under greenhouse conditions. Screening of 15 Fusarium mycotoxins showed the ability of F. temperatum to produce moniliformin, beauvericin, enniatins and fumonisin B₁. The results indicate that F. temperatum can produce mycotoxins and cause maize diseases and, therefore, poses a potential risk to maize production and to the safety of human food and animal feed.     

Occurrence and Pathogenicity of Fusarium Species in Cereals in Finland1

Several Fusarium species occurred abundantly in spring cereal seed samples studied in 1966, 1968 and 1972 in Finland. A total of 17 species of Fusarium were isolated and identified. The most frequently isolated species were F. avenaceum (Fr.) Sacc., F. culmorum (W. G. Sm.) Sacc., F. poae (Peck) Wr. and F. tricinctum (Cda) Sacc. These species were found in more than 40% of the seed samples from 1972. The same 4 Fusarium species in addition to F. graminearum Schwabe were used in the pathogenicity test. Artificially inoculated spring wheat and barley seeds were grown in the field during the growing season of 1973. Of these 5 species F. culmorum was clearly the most pathogenic. This species significantly lowered the shooting percentage and the yield of both cereals. Foot rot assessment also revealed F. culmorum as the most damaging species. Natural soil-borne infection obviously increased the foot and root rot, and thus the soil-borne F. culmorum strongly affected all treatments. In the harvested seed F. poae dominated in all treatments, but F. culmorum still occurred most abundantly in the seed lot harvested from plots originally inoculated with this species.     

Genetic Analysis of the Role of Trichothecene and Fumonisin Mycotoxins in the Virulence of Fusarium

The phytotoxicity of the Fusarium trichothecene and fumonisin mycotoxins has led to speculation that both toxins are involved in plant pathogenesis. This subject has been addressed by examining virulence of trichothecene and fumonisin-nonproducing mutants of Fusarium in field tests. Mutants were generated by transformation-mediated disruption of genes encoding enzymes that catalyze early steps in the biosynthesis of each toxin. Two economically important species of Fusarium were selected for these studies: the trichothecene-producing species Fusarium graminearum, which causes wheat head blight and maize ear rot, and the fumonisin-producing species F. verticillioides, which causes maize ear rot. Trichothecene-non-producing mutants of F. graminearum caused less disease than the wild-type strain from which they were derived on both wheat and maize, although differences in virulence on maize were not observed under hot and dry environmental conditions. Genetic analyses of the mutants demonstrated that the reduced virulence on wheat was caused by the loss of trichothecene production rather than by a non-target mutation induced by the gene disruption procedure. Although the analyses of virulence of fumonisin-non-producing mutants of F. verticillioides are not complete, to date, the mutants have been as virulent on maize ears as their wild-type progenitor strains. The finding that trichothecene production contributes to the virulence of F. graminearum suggests that it may be possible to generate plants that are resistant to this fungus by increasing their resistance to trichothecenes. As a result, several researchers are trying to identify trichothecene resistance genes and transfer them to crop species.     

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.     

<Emphasis Type="Italic">Fusarium temperatum</Emphasis> isolated from maize in France

The presence of Fusarium temperatum in France was investigated by analyzing 40 Fusarium strains, previously isolated from infected maize ears in 2011 and formerly identified as Fusarium subglutinans based on morphological characteristics. In this study, 26 strains out of the 40 were identified as F. temperatum and 14 as F. subglutinans based on sequencing of the translation elongation factor 1α gene. The phylogenetic analysis showed that the two species represented two clades strongly supported by bootstrap values. The pathogenicity of F. temperatum strains was confirmed on maize ears. This study provides new information about F. temperatum isolated from maize in France.     

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.     

Quantitative detection of Fusarium pathogens and their mycotoxins in South African maize

The distribution and co‐occurrence of four Fusarium species and their mycotoxins were investigated in maize samples from two susceptible cultivars collected at 14 localities in South Africa during 2008 and 2009. Real‐time PCR was used to quantify the respective Fusarium species in maize grain, and mycotoxins were quantified by multi‐toxin analysis using HPLC‐MS. In 2008, F. graminearum was the predominant species associated with maize ear rot in the eastern Free State, Mpumalanga and KwaZulu‐Natal provinces, while F. verticillioides was predominant in the Northwest, the western Free State and the Northern Cape provinces. In 2009, maize ear rot infection was higher and F. graminearum became the predominant species found in the Northwest province. Fusarium subglutinans was associated with maize ear rot in both years at most of the localities, while F. proliferatum was not detected from any of the localities. Type B trichothecenes, especially deoxynivalenol, and zearalenone were well correlated with the amount of F. graminearum, fumonisins with F. verticillioides, and moniliformin and beauvericin with F. subglutinans. This information is of great importance to aid understanding of the distribution and epidemiology of Fusarium species in South Africa, and for predicting mycotoxin contamination risks and implementing preventative disease management strategies.     

Epidemiology of Fusarium Diseases and their Mycotoxins in Maize Ears

Fusarium species cause two distinct diseases on ears of maize, Fusarium ear rot (or pink ear rot) and Gibberella ear rot (or red ear rot), both of which can result in mycotoxin contamination of maize grain. The primary causal agent for Fusarium ear rot is Fusarium verticillioides, but F. subglutinans and F. proliferatum are also important. Gibberella ear rot is caused primarily by F. graminearum, but F. culmorum can also be important, especially in Europe. Aspects of the epidemiology of both diseases have been studied for decades, but only recently have efforts been made to synthesize this information into comprehensive models of disease development. Much of the work on F. graminearum has focused on Fusarium head blight of small-grain crops, but some of the results obtained are also relevant to maize. The primary mycotoxins produced by these fungi, fumonisins and deoxynivalenol, have differing roles in the disease-cycle, and these roles are not completely understood, especially in the case of fumonisins. Progress is being made toward accurate models for risk assessment of both diseases, but key challenges remain in terms of integrating models of pre- and post-infection events, quantifying the roles of insects in these diseases, and characterizing interactions among competing fungi and the environment.     

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.     

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.     

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.     

Toxigenic Fusarium species and Mycotoxins Associated with Head Blight in Small-Grain Cereals in Europe

The Fusarium species predominantly found associated with Fusarium head blight (FHB) in wheat and other small-grain cereals all over Europe are F. graminearum, F. avenaceum and F. culmorum. Among the less frequently encountered species are several others which are less pathogenic or opportunistic, but also toxigenic. These include F. poae, F. cerealis F. equiseti F. sporotrichioides F. tricinctum and, to a lesser extent, F. acuminatum F. subglutinans F. solani F. oxysporum F. verticillioides F. semitectum and F. proliferatum. The species profile of FHB is due to several factors, primarily climatic conditions, particularly rain and the temperature at flowering stage, but also agronomic factors, such as soil cultivation, nitrogen fertilization, fungicides, crop rotation, and host genotype. The most frequently encountered Fusarium mycotoxins in FHB in Europe has proved to be deoxynivalenol and zearalenone produced by F. graminearum and F. culmorum with the former more common in southern (warmer) and the latter in northern (colder) European areas. Nivalenol was usually found associated with deoxynivalenol and its derivatives (mono-acetyldeoxynivalenols), together with fusarenone-X, formed by F. graminearum F. cerealis F. culmorum and, in northern areas, by F. poae. Moreover, from central to northern European countries, moniliformin has been consistently reported, as a consequence of the widespread distribution of F. avenaceum whereas the occurrence of T-2 toxin derivatives, such as T-2 toxin and HT-2 toxin, and diacetoxyscirpenol have been recorded in conjunction with sporadic epidemics of F. sporotrichioides and F. poae. Finally, beauvericin and various enniatins have recently been found in Finnish wheat colonized by F.avenaceum and F. poae.     

Characterisation of novel <Emphasis Type="Italic">Fusarium graminearum</Emphasis> microsatellite markers in different <Emphasis Type="Italic">Fusarium</Emphasis> species from various countries

Fifteen novel microsatellite markers were isolated from Fusarium graminearum. The level of polymorphism at these novel and 13 previously published microsatellite markers was analysed in 33 F. graminearum strains from Europe, North America, and Nepal. The number of alleles for each of the novel markers ranged from 4 to 20 and gene diversity from 0.417 to 0.962. In comparison with the previously published markers, the resolution for distinguishing among different strains was slightly increased. Twenty-seven markers were also detectable in three F. culmorum strains and one F. crookwellense strain. None of the markers was detected in three F. avenaceum and four F. poae strains, underlining the potential use of these microsatellite markers for species differentiation.