Trichothecene mycotoxin genotypes of Fusarium graminearum sensu stricto and Fusarium meridionale in wheat from southern Brazil

A total of 82 fungal isolates was obtained from wheat kernel samples affected by fusarium head blight collected from 20 locations in southern Brazil. Polymerase chain reaction (PCR) assays were used to characterize trichothecene mycotoxin genotypes [deoxynivalenol (DON), nivalenol (NIV) and two acetylated derivatives of DON]. To identify isolates that producing DON and NIV, portions of the Tri13 gene were amplified. To identify 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) genotypes, portions of Tri3 and Tri12 were amplified. Nearly all of the isolates studied (76/82) were of the DON/15-ADON genotype. Six of the isolates were of the NIV genotype. The DON/3-ADON genotype was not observed. Portions of three genes were sequenced from representative isolates of the NIV and DON/15-ADON genotypes and compared with sequences from curated reference isolates of Fusarium in GenBank. blast queries for individual gene sequences and pairwise comparisons of percentage identity and percentage divergence based on 1676 bp of concatenated DNA sequence suggested that the isolates representing the DON/15-ADON genotype were Fusarium graminearum sensu stricto and the isolates representing the NIV genotype were Fusarium meridionale . This is the first detailed report of trichothecene mycotoxin genotypes of F. graminearum and F. meridionale in Brazil.     

Genetic population structure and trichothecene genotypes of Fusarium graminearum isolated from wheat in southern Brazil

A sample of 140 Fusarium graminearum isolates from Rio Grande do Sul, Brazil, representing three populations at least 150 km from one another, were examined for trichothecene genotype based on PCR amplification of portions of the Tri3 and Tri12 genes and a species‐specific (Fg16F/R) primer pair. Genetic diversity was assessed in a sample of 103 F. graminearum lineage 7 (F. graminearum sensu stricto) isolates using amplified fragment length polymorphism (AFLP) markers. The 15‐ADON genotype was dominant, followed by the NIV genotype (2–18% prevalence), across all three populations. All NIV‐type isolates were in lineage 2 (F. meridionale) and all 15‐ADON‐type isolates were in lineage 7. Isolates with the same haplotype were rare and genotypic diversity was uniformly high (≥98% of the count), suggesting that recombination has played a significant role. The number of migrants (N_m) was estimated between 5 and 6 across all loci and all populations, but the high frequency of private alleles (up to 30%) suggests a historical, rather than contemporary, gene flow. Regarding linkage disequilibrium, 0·8, 1·5 and 2·2% of the locus pairs from the three populations were in disequilibrium, which is lower than values reported in other locations. Thus, Brazilian populations differ from those found in Europe, North America and most of Asia in the presence of a significant frequency (7·8%) of isolates of the NIV genotype in lineage 2.     

Genetic variation and covariation for aggressiveness, deoxynivalenol production and fungal colonization among progeny of Gibberella zeae in wheat

Gibberella zeae (anamorph Fusarium graminearum ) causes head blight of cereals and contaminates grains with mycotoxins such as deoxynivalenol (DON). To determine the correlations among aggressiveness traits, fungal colonization and DON production, 50 progeny from a segregating population of G. zeae were inoculated onto a susceptible winter wheat cultivar in three field environments (year–location combinations). Aggressiveness traits were measured as head-blight rating and plot yield relative to noninoculated plots. Fungal colonization, measured as Fusarium exoantigen (ExAg) content, and DON production were analysed with two ELISA formats. Disease severity was moderate to high based on head-blight rating and relative plot yield. Fusarium ExAg content and DON production ranged from 0·26–1·41 units and from 4·18–43·70 mg kg⁻¹, respectively. Significant ( P  = 0·01) genotypic variation was found for all traits. Heritability for Fusarium ExAg content was rather low because of high progeny–environment interaction and error. DON/ Fusarium ExAg ratio did not vary significantly ( P  > 0·1) among progeny. Correlation between DON production and Fusarium ExAg content across environments was high ( r  = 0·8, P  = 0·01), but no covariation existed between aggressiveness traits and DON/ Fusarium ExAg content ratio.     

小麦赤霉病特大流行年不同品种(品系)间抗性试验分析

田间对比试验结果表明,赤霉病特大流行年不同小麦品种对赤霉病的抗性存在极显著差异,可分为高感、中感和中抗3个类型。因此,生产上应以中抗品种扬麦158作为当家品种,适当示范种植扬麦11、扬麦13等抗病、耐病性较强、产量较高的品种,并加强测报,适时开展药剂防治,综合控制病害的流行为害。     

Ordinal regression models for predicting deoxynivalenol in winter wheat

Deoxynivalenol (DON) is one of the most prevalent toxins in Fusarium‐infected wheat samples. Accurate forecasting systems that predict the presence of DON are useful to underpin decision making on the application of fungicides, to identify fields under risk, and to help minimize the risk of food and feed contamination with DON. To this end, existing forecasting systems often adopt statistical regression models, in which attempts are made to predict DON values as a continuous variable. In contrast, this paper advocates the use of ordinal regression models for the prediction of DON values, by defining thresholds for converting continuous DON values into a fixed number of well‐chosen risk classes. Objective criteria for selecting these thresholds in a meaningful way are proposed. The resulting approach was evaluated on a sizeable field experiment in Belgium, for which measurements of DON values and various types of predictor variables were collected at 18 locations during 2002–2011. The results demonstrate that modelling and evaluating DON values on an ordinal scale leads to a more accurate and more easily interpreted predictive performance. Compared to traditional regression models, an improvement could be observed for support vector ordinal regression models and proportional odds models.     

Phenotypic and molecular characterization of the resistance to azoxystrobin and pyraclostrobin in Fusarium graminearum populations from Brazil

Wheat farmers rely on fungicides to protect fields against several foliar and flowering diseases, including Fusarium head blight (FHB). A range of active ingredients is used in isolation or in dual premixes that include a dimethylation inhibitor (DMI) or a quinone outside inhibitor (QoI) fungicide. Comprehensive information about fungicide resistance in F. graminearum is available for DMIs, while for QoIs the data are scarce. We characterized 225 strains obtained from two states in southern Brazil, Rio Grande do Sul (RS) and Paraná (PR), in relation to their response to two QoIs. The median EC₅₀ (effective concentration leading to 50% inhibition of conidial germination) value for azoxystrobin (n = 25 isolates) was 2.20 μg/ml in the PR population and 4.04 μg/ml in the RS population. For pyraclostrobin (n = 50), the median EC₅₀ was 0.28 μg/ml in the PR population and 0.24 μg/ml in the RS population. Evidence of cross-resistance could not be detected. Screening using a discriminatory dose (DD) for azoxystrobin in a larger number of isolates from PR (n = 75) and RS (n = 100) states allowed the detection of 50% and 28% sensitive strains, respectively. Using the DD for pyraclostrobin, 33% and 18.8% were classified as less sensitive in the PR and RS isolates, respectively. In RS, the frequency of less-sensitive isolates increased over time (2007–2011). No point mutation at any of the target spots (F129L, G137R, G143A) was detected. Our results represent an important step towards the establishment of a sensitivity profile for two of the most commonly used QoIs in commercial premixes targeting FHB control.     

Quantifying cropping practices in relation to inoculum levels of Fusarium graminearum on crop stubble

This study was conducted in 58 producer‐field locations in Manitoba from 2003 to 2006 to understand how cropping practices influence Fusarium graminearum inoculum levels on stubble of various crops, including wheat, collected from the soil surface. Colonies per m² (CN) were determined and converted to base‐10 logarithm values (log₁₀CN). Mean log₁₀CN of the sampled field for various crops and groups of crops grown in the 3 years prior to sampling were tested to find significant differences. Average log₁₀CN values were also used to determine significant differences between tillage systems and the effect of number of years. Average log₁₀CN values for zero and minimum tillage systems were not different but were significantly higher than values for conventional tillage. A series of three crop rotation scenarios were tested using weighted log₁₀CN values for crop, tillage, their interaction and their squared terms in step‐wise regression models to identify which model was the best predictor of log₁₀CN. This was selected as the cropping practice index (CPI) model and was expressed as: CPI = 1·98423 + 0·55975 (C₂ × C₁ × T)² + 0·4390 (C₂ × T)², where C₁, C₂ and T represent the weighted log₁₀CN values for crops grown 1 and 2 years previously and tillage system, respectively. R² value for this model was 0·933 (P < 0·0001). The reliability of the CPI model was tested using jack‐knife full cross‐validation regression. The resulting R² was 0·899. The CPI model was tested using data collected from seven wheat fields in 2006 (R² = 0·567). The relationship between CPI and FHB index (R² = 0·715) was significant.     

Geographic distribution and genetic diversity of Fusarium graminearum and F. asiaticum on wheat spikes throughout China

A large number of Fusarium graminearum and F. asiaticum isolates were collected from wheat spikes from all regions in China with a history of fusarium head blight (FHB) epidemics. Isolates were analysed to investigate their genetic diversity and geographic distribution. Sequence characterized amplified region (SCAR) analyses of 437 isolates resolved both species, with 21% being F. graminearum (SCAR type 1) and 79% being F. asiaticum (SCAR type 5). AFLP profiles clearly resolved two groups, A and B, that were completely congruent with both species. However, more diversity was detected by AFLP, revealing several subgroups within each group. In many cases, even for isolates from the same district, AFLP haplotypes differed markedly. Phylogenetic analyses of multilocus DNA sequence data indicated that all isolates of SCAR type 1, AFLP group A were F. graminearum , whilst isolates of SCAR type 5, AFLP group B were F. asiaticum , demonstrating that it is an efficient method for differentiating these two species. Both species seem to have different geographic distributions within China. Fusarium graminearum was mainly obtained from wheat growing in the cooler regions where the annual average temperature was 15°C or lower. In contrast, the vast majority of F. asiaticum isolates were collected from wheat growing in the warmer regions where the annual average temperature is above 15°C and where FHB epidemics occur most frequently. This is the first report of the distribution of, and genetic diversity within, F. graminearum and F. asiaticum on wheat spikes throughout China.     

The contribution of ground-level inoculum of Fusarium culmorum to ear blight of winter wheat

In a series of field experiments in eastern England over 5 years, severe ear blight developed only in plots of winter wheat that were inoculated by spraying with conidial suspensions of Fusarium culmorum during anthesis, and in which infection was encouraged by rainfall or mist irrigation. In the absence of artificial inoculation of the ears, F. culmorum caused less extensive ear blight, and only where soil-surface inoculum was available after its application on infested plant material (colonized oat grains) up to 3–4 weeks before anthesis; it then developed most where significant rainfall occurred close to the time of anthesis. A warm, dry period following application of inoculum to the ground in late March contributed to increased infection of grain by F. culmorum , although ear blight was not increased. Ear infection therefore depended on adequate viable inoculum on infested plant debris within the crop, and conditions tending to favour brown foot rot development as well as, subsequently, rainfall and moist conditions during anthesis. These conditions did not occur together naturally during this period. Seedling infection by F. culmorum or Microdochium nivale made no significant contribution to ear blight. Inoculation of ears at anthesis with M. nivale or a locally obtained isolate of F. langsethiae did not produce ear blight symptoms. Possibilities for minimizing the availability of inoculum of F. culmorum and the implications for various options for ear-blight control are discussed.     

Genotypic diversity in Fusarium pseudograminearum populations in Australian wheat fields

Despite being closely related to Fusarium graminearum, which has been extensively characterized in many countries around the world, the population biology of Fusarium pseudograminearum, the main causal agent of crown rot of wheat in Australia and many other wheat‐growing regions, has been comparatively poorly studied to date. A simple sequence repeat analysis of 163 F. pseudograminearum isolates from three field sites in NSW, Australia identified 128 distinct multilocus genotypes. Observed genetic diversity within fields was high, whilst genetic variation between fields was low. Across all fields genetic linkage disequilibrium was detected, but of the three individual fields, only one also displayed linkage disequilibrium. These results indicate that the isolates obtained were part of the same, highly diverse, population. However, this population may not be freely interbreeding. Whilst isolation incidence of F. pseudograminearum was found to be spatially aggregated within fields, spatial aggregation of genotypes within fields was weak. The study suggests that processes influencing population dynamics may operate at a scale larger than the narrow geographical scale covered in the fields sampled.     

Effects of plant height on type I and type II resistance to fusarium head blight in wheat

By carefully separating type I and type II resistances, the possible effects of plant height on fusarium head blight (FHB) resistance in wheat were assessed using near‐isogenic lines (NILs) for several different reduced‐height (Rht) genes. Tall isolines all gave better type I resistance than their respective dwarf counterparts when assessed at their natural heights. These differences largely disappeared when the dwarf isolines were physically raised so that their spikes were positioned at the same height as those of their respective tall counterparts. The effects of plant height on type II resistance was less clear. For those NIL pairs which showed significant differences, it was the dwarf isolines which gave better resistance. As the Rht genes involved in these NILs locate at different genomic regions, the differences in FHB between the dwarf and tall isolines are unlikely to be the result of linkages between each of the different Rht loci with a beneficial or a deleterious gene affecting type I or type II resistance. Rather, the different FHB resistances are probably caused by direct or indirect effects of height difference per se, and microclimate may have contributed to the better type I resistance of the tall plants. Thus, caution should be exercised when attempting to exploit any of the FHB resistant loci co‐located with Rht genes.     

Inverse effects of Arabidopsis NPR1 gene on fusarium seedling blight and fusarium head blight in transgenic wheat

In this study, the Arabidopsis thaliana NPR1 (non‐expressor of PR genes) gene was integrated into an elite wheat cultivar, and the response of the transgenic wheat expressing NPR1 to inoculation with Fusarium asiaticum was analysed. With seedling inoculation, the transgenic lines showed significantly increased fusarium seedling blight (FSB) susceptibility, whereas floret inoculation resulted in enhanced fusarium head blight (FHB) resistance. Quantitative real‐time PCR revealed that expression of two defence genes, PR3 and PR5, was associated with susceptible reactions to FSB and FHB, whereas the PR1 gene was activated in resistance responses. This inverse modulation by the constitutively expressed NPR1 gene suggests that NPR1 has a bifunctional role in regulating defence responses in plants. Therefore, it is unsuitable for improving overall resistance to FSB and FHB in wheat.     

Vegetative Compatibility and Mycotoxin Chemotypes among Fusarium graminearum (Gibberella zeae) Isolates from Wheat in Argentina

Gibberella zeae (anamorph Fusarium graminearum) is the main pathogen causing Fusarium head blight of wheat in Argentina. The objective of this study was to determine the vegetative compatibility groups (VCGs) and mycotoxin production (deoxynivalenol, nivalenol and 3-acetyl deoxynivalenol) by F. graminearum populations isolated from wheat in Argentina. VCGs were determined among 70 strains of F. graminearum isolated from three localities in Argentina, using nitrate non-utilizing (nit) mutants. Out of 367 nit mutants generated, 41% utilized both nitrite and hypoxanthine (nit1), 45% utilized hypoxanthine but not nitrite (nit3), 9% utilized nitrite but not hypoxanthine (NitM) and 5% utilized all the nitrogen sources (crn). The complementations were done by pairing the mutants on nitrate medium. Fifty-five different VCGs were identified and the overall VCG diversity (number of VCGs/number of isolates) averaged over the three locations was 0.78. Forty-eight strains were incompatible with all others, thus each of these strains constituted a unique VCG. Twenty-two strains were compatible with other isolates and were grouped in seven multimembers VCGs. Considering each population separately, the VCG diversity was 0.84, 0.81 and 1.0 for San Antonio de Areco, Alberti and Marcos Juarez, respectively. Toxin analysis revealed that of the 70 strains of F. graminearum tested, only 90% produced deoxynivalenol, 10% were able to produce deoxynivalenol and very low amounts of 3-acetyldeoxynivalenol. No isolate produced nivalenol. The results indicate a high degree of VCG diversity in the F. graminearum populations from wheat in Argentina. This diversity should be considered when screening wheat germplasm for Fusarium head blight resistance.     

Comparative population structure and trichothecene mycotoxin profiling of Fusarium graminearum from corn and wheat in Ontario,central Canada

Fusarium graminearum causes fusarium head blight (FHB) of wheat and gibberella ear rot (GER) of corn in Canada and also contaminates grains with trichothecene mycotoxins. Very little is known about trichothecene diversity and population structure of the fungus from corn in Ontario, central Canada. Trichothecene genotypes of F. graminearum isolated from corn (n = 452) and wheat (n = 110) from 2010 to 2012 were identified. All the isolates were deoxynivalenol (DON) type. About 96% of corn isolates and 98% of wheat isolates were 15‐acetyl deoxynivalenol (15ADON) type. The fungal population structures from corn (n = 313) and wheat (n = 73) were compared using 10 variable number tandem repeat (VNTR) markers. The fungal populations and subpopulations categorized based on host, cultivar groups, years and geography showed high gene (H = 0.818–0.928) and genotypic (GD = 0.999–1.00) diversity. Gene flow was also high between corn and wheat population pairs (Nm = 8.212), and subpopulation pairs within corn (Nm = 7.13–23.614) or wheat (Nm = 19.483) populations. Phylogenetic analysis revealed that isolates from both hosts were F. graminearum clade 7. These findings provide baseline data on 3‐acetyl deoxynivalenol (3ADON) and 15ADON profiles of F. graminearum isolates from corn in Canada and are useful in evaluating mycotoxin contamination risks in corn and wheat grains. Understanding the fungal genetic structure will assist evaluation and development of resistant cultivars/germplasm for FHB on wheat and GER on corn.     

Transfer of the β‐tubulin gene of Botrytis cinerea with resistance to carbendazim into Fusarium graminearum

BACKGROUND: Resistance to carbendazim and other benzimidazole fungicides in Botrytis cinerea (Pers. ex Fr.) and most other fungi is usually conferred by mutation(s) in a single chromosomal β‐tubulin gene, often with several allelic mutations. In Fusarium graminearum Schwade, however, carbendazim resistance is not associated with a mutation in the corresponding β‐tubulin gene. RESULTS: The β‐tubulin gene conferring carbendazim resistance in B. cinerea was cloned and connected with two homologous arms of the β‐tubulin gene of F. graminearum by using a double‐joint polymerase chain reaction (PCR). This fragment was transferred into F. graminearum via homologous double crossover at the site where the β‐tubulin gene of F. graminearum is normally located (the β‐tubulin gene of F. graminearum had been deleted). The transformants were confirmed and tested for their sensitivity to carbendazim. CONCLUSION: The β‐tubulin gene conferring carbendazim resistance in B. cinerea could not express this resistance in F. graminearum, as transformants were still very sensitive to carbendazim. Copyright © 2010 Society of Chemical Industry     

Evaluation and characterization of resistance to fusarium head blight caused by Fusarium culmorum in UK winter wheat cultivars

Fusarium head blight (FHB) resistance of 50 cultivars from the National List of winter wheat cultivars approved for sale (or were undergoing trails for approval in 2003) in the UK was compared with 21 reference cultivars from continental Europe which had previously been characterized for resistance. Only three UK National List cultivars (Soissons, Spark and Vector) had stable resistance over trial sites that was significantly greater than that of the FHB susceptible cultivar Wizard. In addition, under moderate disease pressure, 21 of the National List cultivars had levels of the trichothecene mycotoxin deoxynivalenol (DON) above the proposed European Union limit of 1·25 ppm in grain. Surveys show that levels of FHB and DON in the UK crop are currently very low, however, should disease pressure increase for any reason, then an improvement in the overall levels of FHB resistance of UK winter wheat germplasm will be required. In order to infer the origin of resistance and to identify potentially novel resistance, allele sizes of microsatellite (simple sequence repeat, SSR) markers linked to quantitative trait loci (QTL) for FHB resistance were compared between the test cultivars and known, characterized resistance sources. The major FHB resistance QTL from the Chinese cv. Sumai-3 (3BS, 5A and 6B), the Romanian cv. Fundulea F201R (1B and 5A) and the French cv. Renan (5AL) were screened with 17 SSRs. No National List cultivar had haplotypes similar to any of these QTL. However, the highly resistant German reference cultivar Petrus had an identical haplotype to cv. Fundulea F201R on 1B indicating that this cultivar has an allelic FHB resistance QTL at that location.     

Characterization and expression of Fusarium graminearum endo-polygalacturonases in vitro and during wheat infection

This study investigated the expression and characterization of two polygalacturonases (PG1 and PG2) of Fusarium graminearum during infection of wheat spikelets; after purification, these were demonstrated to be products of two unique endo - pg genes annotated in the genome database of F. graminearum . Both genes ( Fgpg1 and Fgpg2 ) were expressed in vitro and during spike infection. PG1 had a greater specific activity, with a maximum at pH 5–7, was largely secreted in liquid culture and clearly detectable in the infected ovary tissue. PG2 was more active at pH 7–7·8, was poorly secreted in liquid culture and faintly detectable in infected ovaries. Both PG-encoding genes were maximally expressed 24 h after wheat spikelet infection, paralleling the expression of a pectin lyase ( Fgpnl1 ) gene; they anticipated the expression of a xylanase gene ( FgxylA ) that was induced only 48 h after infection with a maximum at 96 h. These data strongly indicate F. graminearum -secreted PG activity at an early stage of wheat infection.