R.M. Clear

Comparison of Canadian Fusarium graminearum isolates for aggressiveness, vegetative compatibility, and production of ergosterol and mycotoxins

Fusarium graminearum is the predominant pathogen causing fusarium head blight of cereals in North America. Fifteen Canadian isolates of Fusarium graminearum were highly diverse in terms of vegetative compatibility grouping (VCG) and varied for production of ergosterol and mycotoxin production in rice culture. Aggressiveness was assessed by scoring the disease severity incited in wheat spikes by each isolate. Two inoculation methods, single-floret injection and spray of entire spikes, were used to screen 4 wheat varieties for reaction to the F. graminearum isolates. All isolates were of broadly similar aggressiveness, with disease severity ranging from 17.2 to 39.1 for single floret injection, and 39.1 to 69.0 for spray inoculation. Disease severity, ergosterol production, and mycotoxin development were not correlated. Using nitrate non-utilizing mutants the 15 isolates were grouped into 14 VCGs. Deoxynivalenol (DON) was produced by all isolates in rice culture, at levels between 0.2 and 249 ppm. 15-acetyldeoxynivalenol was produced by 14 of the 15 isolates at levels between 0.4 and 44.6 ppm. These results reveal a high level of diversity for several characteristics among F. graminearum isolates from Canada.

Fusarium head blight pathogens isolated from fusarium-damaged kernels of wheat in western Canada, 1993 to 1998

Survey results from 1993 to 1998 for fungi isolated from fusarium-damaged kernels of wheat are presented. There is compelling evidence that Fusarium graminearum has recently been spreading westward from southeastern Manitoba, replacing less pathogenic Fusarium species as the principal fusarium head blight (FHB) pathogen. This movement has been accompanied by increasing economic losses from the effects of FHB. Environmental factors such as a lower average daily temperature in June and July may be influential in limiting the damage from F. graminearum in the western prairies, where F. graminearum currently is rare. However, precipitation levels at anthesis equal to those in the areas presently affected by economic levels of FHB occur in many western crop districts, suggesting that precipitation levels during the period of anthesis will likely promote further westward spread of this pathogen. The potential role of infected seed as a mechanism for long-distance dispersal of F. graminearum is considered

Prevalence of fungi and fusariotoxins on barley seed from western Canada, 1995 to 1997

The mycoflora and levels of deoxynivalenol (DON) on oat seed (Avena sativa) grown in western Canada was determined by analyzing a total of 511 grain samples collected from 39 crop districts in Alberta, Saskatchewan, and Manitoba during 1995-1997. Fungi representing a minimum of 85 species were recovered. Alternaria alternata was the most frequently isolated species from each province. Levels of A. alternata, Bipolaris sorokiniana, and Fusarium graminearum were highest in Manitoba and eastern Saskatchewan, whereas Cladosporium species and Drechslera avenacea were highest in samples from Alberta and western Saskatchewan. DON levels ≥ 0.10 ppm were found in seeds from two Manitoba crop districts in 1996 and three in 1997, with a maximum level of 0.34 ppm. In 1997, DON was also detected in composite samples of seed from three Saskatchewan and two Alberta crop district composites.

Relative aggressiveness and production of 3- or 15-acetyl deoxynivalenol and deoxynivalenol by Fusarium graminearum in spring wheat

Abstract Fusarium graminearum is the principal cause of fusarium head blight in North America, a disease that has caused severe losses in yield and quality of cereals. In North America, the vast majority of F. graminearum isolates produce 3- or 15-acetyl deoxynivalenol (ADON) in addition to DON. Until recently, 15-ADON isolates predominated, but a rapid shift from 15-ADON to 3-ADON producers in Canada and north central USA has been documented. In order to better understand the effect of this population shift on relative aggressiveness of isolates and mycotoxin accumulation, we tested a total of 58 isolates for 3- and 15-ADON production on two Canadian spring wheat cultivars, ‘Roblin’ (susceptible) and ‘5602 HR’ (moderately resistant). In Experiment 1, three isolates from the Canadian provinces of Manitoba, Saskatchewan and Alberta, each of which produced either 15-ADON or 3-ADON, were tested using spray inoculation. In Experiment 2, 20 isolates which produced 15-ADON and 20 which produced 3-ADON from Manitoba, were tested using point inoculation. There were no significant differences in aggressiveness among isolates based either on geographic origin or mycotoxin type. Analysis of seeds from inoculated heads by gas chromatography/mass spectrometry indicated that the 3-ADON producing isolates had significantly higher DON levels than the 15-ADON isolates in ‘Roblin’ after both spray and point inoculation and in ‘5602HR’ after point inoculation. DON levels following point inoculation by 15-ADON isolates were similar in the two cultivars. The 15-ADON isolates from Alberta produced less DON than 15-ADON isolates from Manitoba and Saskatchewan. Consistently, more ADON was produced by 15-ADON isolates than by 3-ADON isolates. The results of the study suggest that if the percentage of 3-ADON isolates in Canada increases, DON levels in cereals are likely to increase in epidemic years.

Relationships among deoxynivalenol, ergosterol and Fusarium exoantigens in Canadian hard and soft wheat.

Soluble extracellular components (exoantigens) from cultures of Fusarium graminearum and F. sporotrichioides were used to produce antisera from chickens for an indirect enzyme immunoassay. This immunoassay was used to estimate Fusarium exoantigen levels in 40 samples of fusarium head blight-infected hard red spring wheat from Manitoba, and in 50 samples of infected soft white winter wheat from Ontario. These wheat samples were also assayed for deoxynivalenol (DON), the predominant Fusarium mycotoxin, and for ergosterol, a metabolite reflecting fungal biomass. Using F. sporotrichioides antisera, the linear correlations between exoantigen level and DON content for the hard and soft wheats had coefficients of 0.80 and 0.76, respectively. With the same antisera, linear correlations between exoantigen level and total ergosterol concentration for the hard and soft wheats had coefficients of 0.66 and 0.81, respectively.

Fungal plant pathogens infecting barley and wheat seed from Alberta, 1995-1997

From 1995 to 1997, spikes were collected from a total of 160 barley and 188 wheat fields from the Peace River region to the Three Hills area of Alberta, Canada. After threshing, 100 seeds from each field were surface sterilized, incubated on potato dextrose agar, and examined for the presence of the following pathogens: Fusarium graminearum, other Fusarium spp., Cochliobolus sativus, Pyrenophora tritici-repentis, Pyrenophora graminea, Pyrenophora teres, and Stagonospora nodorum. Fusarium graminearum was not detected in any seed samples. The most common Fusarium species isolated was Fusarium avenaceum. Maximum seed infection levels with F. avenaceum in a single field were 51 and 37% for barley and wheat, respectively. Substantial levels of seed-borne P. teres were found in barley with maximum infection levels of 82, 81, and 89% in 1995, 1996, and 1997, respectively. Stagonospora nodorum was also commonly found in both barley and wheat seed with maximum infection levels of 61 and 54%, respectively. Pyrenophora tritici-repentis, P. graminea, and C. sativus were generally present at low levels.

Development of a specific TaqMan real-time PCR assay for quantification of Fusarium graminearum clade 7 and comparison of fungal biomass determined by PCR with deoxynivalenol content in wheat and barley.

A Fusarium graminearum clade 7 specific real-time quantitative PCR (qPCR) assay was developed in this study based on unique polymorphisms in sequences of the mating type protein (MAT) gene. PCR amplification was not observed in eight phylogenetic lineages of the F. graminearum complex and four other closely related Fusarium species. Accuracy of the quantification of the real-time PCR assay was verified with wheat DNA spiked with F. graminearum clade 7 DNA. Wheat samples representing two Canadian wheat classes, CWRS (Canadian Western Red Spring) and CWRW (Canadian Western Red Winter) were used to determine the relationships among F. graminearum DNA, deoxynivalenol (DON) and Fusarium damaged kernel (FDK). The amount of DON and F. graminearum DNA remaining after removal of FDK varied among samples, but was sometimes substantial. Positive correlations were observed between F. graminearum clade 7 DNA (in picograms) and DON as well as FDK. There was also a strong correlation between FDK and DON in CWRS and CWRW wheat composite samples, but the inherent variability in individual producer samples precluded a definitive correlation. For barley, a positive correlation was observed between Fusarium DNA and DON values. Real-time PCR assays can be a valuable tool for barley as there are no reliable symptoms to visually assess the level of Fusarium head blight in this crop.

The characterization of chicken antibodies raised against Fusarium spp. by enzyme-linked immunosorbent assay and immunoblotting

Immunoassays for the detection of three Fusarium species have been developed. Chickens were immunized with the soluble antigens from within the mycelia (mycelia-soluble antigens) and the exoantigens of Fusarium sporotrichioides, Fusarium poae and Fusarium graminearum and the antigens were characterized using enzyme-linked immunosorbent assays, immunoblotting and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The cross-reactivities of antisera with eleven species of Fusarium, eleven species of fungi from other genera and the buffer-extracts of grain were determined. Antisera to the exoantigens and mycelia-soluble antigens did not cross-react with buffer-extracts of grain. The antisera against the mycelia-soluble antigens tended to cross-react with the antigens from other genera whereas the antisera against the exoantigens were genus specific. The antiserum raised against exoantigens from F. poae was species specific. The molecular weights of the immunodominant antigens from the Fusaria were above 28 kilodaltons. Antigens from Fusaria showed specific bands in SDS-PAGE gel that can be used to produce more specific antibodies. The results suggest that exoantigen immunoassay can be developed to identify Fusarium genus, whereas mycelia-soluble antigens are not suitable for immunoidentification among genus.

Evaluation of the pathogenicity of Fusarium graminearum and Fusarium pseudograminearum on soybean seedlings under controlled conditions

Fusarium graminearum, the cause of fusarium head blight of small-grain cereals and of gibberella ear rot of corn, has recently been reported to attack soybean, causing root rot and pod blight. A morphologically similar species, Fusarium pseudograminearum, is also an important pathogen of the roots and crown of cereals, but its pathogenicity against soybean has not been tested yet. Pathogenicity tests were conducted under controlled conditions to compare aggressiveness within and between isolates of F. graminearum and F. pseudograminearum in causing root rot of soybean. In each experiment, agar plugs (2 mm in diameter) from 7-day-old fungal cultures were placed on the primary root at the VE (vegetative emergence) growth stage. Twenty seedlings of each of three soybean cultivars were inoculated separately with six isolates each of the two Fusarium spp. All isolates caused visible infection 10 d after inoculation, but F. graminearum isolates were significantly more aggressive than those of F. pseudograminearum. The two species caused average disease severities of 2.3 and 1.1, reduced shoot lengths by 28.9% and 6.9%, and reduced plant dry masses by 12.8% and 7.9%, respectively. Significant differences (P < 0.05) were found among cultivars for all parameters and among isolates of F. graminearum in disease severity and shoot length, but there were no significant cultivar × isolate interactions. Further research is needed to verify, in soybean, genotypic difference and possible resistance to the two species.

Effect of dry heat treatment on seed-borne Fusarium graminearum and other cereal pathogens

Pathogen levels and seed viability of two samples each of barley (Hordeum vulgare) (B1, B2), Canada western red spring wheat (Triticum aestivum) (RS1, RS2), and Canada western amber durum wheat (AD1, AD2) were assessed after heating seed at 50 or 70°C for up to 14 days. RS2 and B2, with an initial incidence of 23 and 84% of Fusarium graminearum, respectively, were also heated at 60°C for 24 days and 80°C for 10 days. Pathogen levels and seed viability were assessed by plating seed onto potato dextrose agar and wet filter paper, respectively. Fusarium graminearum was eliminated from RS2 after 15 days at 60°C, 5 days at 70°C, or 2 days at 80°C. In B2, F. graminearum was eliminated after 21 days at 60°C, 9 days at 70°C, or 5 days at 80°C. After heating at 50 or 70°C, the observed frequency of Cochliobolus sativus in B1 declined slightly but significantly over time, whereas it substantially increased in B2. A significant decline in the incidence of C. sativus was observed in B2 after heating at 80°C. Pyrenophora teres was observed significantly more often in B1 after heating at 50 or 70°C, whereas Pyrenophora tritici-repentis in AD1 and AD2 was unaffected by heating at 50°C. However, the detection of P. tritici-repentis did significantly increase over time in AD2 when heated at 70°C. In AD1, heating at 70°C initially increased, then decreased the observed incidence of this pathogen. Cochliobolus sativus, P. teres and P. tritici-repentis were still viable in the samples after 14 days of heating at 70°C, but C. sativus was not detected after 10 days at 80°C. Germination of wheat tempered to 12, 14, and 16% moisture content was unaffected by heating at 70°C for 7 days, whereas barley tempered to the same moisture content had a slight decline in germination. Germination rates in most samples were unaffected by the treatment times and temperatures sufficient to eradicate F. graminearum, but a significant decline in viability was recorded for AD2 and B1 heated at 70°C. The germination of B2 increased when heated at 70°C, but declined when heated at 80°C. It is recommended that thermotherapy be applied to control national and international movements of F. graminearum and other heat-sensitive pathogens in germplasm used for research and breeding purposes.Key words: thermotherapy, Cochliobolus sativus, Pyrenophora teres, Pyrenophora tritici-repentis.