Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia

A survey of Fusarium head blight (FHB)-contaminated wheat in Ethiopia recovered 31 isolates resembling members of the Fusarium graminearum species complex. Results of a multilocus genotyping (MLGT) assay for FHB species and trichothecene chemotype determination suggested that 22 of these isolates might represent a new species within the Fg complex. Phylogenetic analyses of multilocus DNA sequence data resolved the 22 Ethiopian isolates as a novel, phylogenetically distinct species. The new species also appears to be novel in that MLGT probe data and sequence analysis of both ends of the TRI-cluster identified 15ADON and NIV recombination blocks, documenting inter-chemotype recombination involving the chemotype-determining genes near the ends of the TRI-cluster. Results of pathogenicity experiments and analyses of trichothecene mycotoxins demonstrated that this novel Fg complex species could induce FHB on wheat and elaborate 15ADON in planta. Herein the FHB pathogen from Ethiopia is formally described as a novel species.     

Use of the volatile trichodiene to reduce Fusarium head blight and trichothecene contamination in wheat

Fusarium graminearum is the primary cause of Fusarium head blight (FHB), one of the most economically important diseases of wheat worldwide. FHB reduces yield and contaminates grain with the trichothecene mycotoxin deoxynivalenol (DON), which poses a risk to plant, human and animal health. The first committed step in trichothecene biosynthesis is formation of trichodiene (TD). The volatile nature of TD suggests that it could be a useful intra or interspecies signalling molecule, but little is known about the potential signalling role of TD during F. graminearum-wheat interactions. Previous work using a transgenic Trichoderma harzianum strain engineered to emit TD (Th + TRI5) indicated that TD can function as a signal that can modulate pathogen virulence and host plant resistance. Herein, we demonstrate that Th + TRI5 has enhanced biocontrol activity against F. graminearum and reduced DON contamination by 66% and 70% in a moderately resistant and a susceptible cultivar, respectively. While Th + TRI5 volatiles significantly influenced the expression of the pathogenesis-related 1 (PR1) gene, the effect was dependent on cultivar. Th + TRI5 volatiles strongly reduced DON production in F. graminearum plate cultures and downregulated the expression of TRI genes. Finally, we confirm that TD fumigation reduced DON accumulation in a detached wheat head assay.     

Structural and functional characterization of the TRI101 trichothecene 3-O-acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum: kinetic insights to combating Fusarium head blight

Fusarium head blight (FHB) is a plant disease with serious economic and health impacts. It is caused by fungal species belonging to the genus Fusarium and the mycotoxins they produce. Although it has proved difficult to combat this disease, one strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro this has been shown to reduce the toxicity of the toxins by approximately 100-fold but has demonstrated limited resistance to FHB in transgenic cereal. To understand the molecular basis for the differences between in vitro and in vivo resistance the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes toward B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three-dimensional structures for the ternary complexes for both of these enzymes with coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein.     

Evaluation of genetic diversity of Fusarium head blight resistance in European winter wheat

Genetic diversity in relation to Fusarium head blight (FHB) resistance was investigated among 295 European winter wheat cultivars and advanced breeding lines using 47 wheat SSR markers. Twelve additional wheat lines with known FHB resistance were included as reference material. At least one SSR marker per chromosome arm, including SSR markers reported in the literature with putative associations with QTLs for FHB resistance, were assayed to give an even distribution of SSR markers across the wheat genome. A total of 404 SSR alleles were detected. The number of alleles per locus ranged from 2 to 21, with an average of 8.6 alleles. The polymorphism information content of the SSR markers ranged from 0.13 (Xwmc483) to 0.87 (Xwmc607), with an average of 0.54. Cluster analysis was performed by both genetic distance-based and model-based methods. In general, the dendrogram based on unweighted pair-group method with arithmetic averages showed similar groupings to the model-based analysis. Seven clusters were identified by the model-based method, which did not strictly correspond to geographical origin. The FHB resistance level of the wheat lines was evaluated in field trials conducted over multiple years or locations by assessing the following traits: % FHB severity, % FHB incidence, % diseased kernels, in spray inoculation trials, and % FHB spread and % wilted tips, in point inoculation trials. Association analysis between SSR markers and the FHB disease traits detected markers significantly associated with FHB resistance, including some that have not been previously reported. The percentage of variance explained by each individual marker was, however, rather low. Haplotype analysis revealed that the FHB-resistant European wheat lines do not contain the 3BS locus derived from Sumai 3. The information generated in this study will assist in the selection of parental lines in order to increase the efficiency of breeding efforts for FHB resistance.     

Inactivation of a cytochrome P-450 is a determinant of trichothecene diversity in Fusarium species

Species of the genus Fusarium produce a great diversity of agriculturally important trichothecene toxins that differ from each other in their pattern of oxygenation and esterification. T-2 toxin, produced by Fusarium sporotrichioides, and nivalenol (NIV), produced by some strains of F. graminearum, contain an oxygen at the C-4 position. Deoxynivalenol (DON), produced by other strains of F. graminearum, lacks a C-4 oxygen. NIV and DON are identical except for this difference, whereas T-2 differs from these trichothecenes at three other carbon positions. Sequence and Northern analyses of the F. sporotrichioides genomic region upstream of the previously described core trichothecene gene cluster have extended the cluster by two genes: TRI13 and TRI14. TRI13 shares significant similarity with the cytochrome P-450 class of enzymes, but TRI14 does not share similarity with any previously characterized proteins. Gene disruption and fermentation studies in F. sporotrichioides indicate that TRI13 is required for the addition of the C-4 oxygen of T-2 toxin, but that TRI14 is not required for trichothecene biosynthesis. PCR and sequence analyses indicate that the TRI13 homolog is functional in NIV-producing strains of F. graminearum but nonfunctional in DON-producing strains of the fungus. These genetic observations are consistent with chemical observations that biosynthesis of T-2 toxin and NIV requires a C-4 hydroxylase while biosynthesis of DON does not.     

Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production

Background  

The fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB) disease on wheat which can lead to trichothecene mycotoxin (e.g. deoxynivalenol, DON) contamination of grain, harmful to mammalian health. DON is produced at low levels under standard culture conditions when compared to plant infection but specific polyamines (e.g. putrescine and agmatine) and amino acids (e.g. arginine and ornithine) are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during FHB are unknown, but plant derived polyamines could contribute to DON induction in infected heads. However, the temporal and spatial accumulation of polyamines and amino acids in relation to that of DON has not been studied.     

Suppression of wheat Fusarium head blight by novel amphiphilic aminoglycoside fungicide K20

K20 is a novel amphiphilic aminoglycoside capable of inhibiting many fungal species. K20's capabilities to inhibit Fusarium graminearum the causal agent wheat Fusarium head blight (FHB) and to this disease were examined. K20 inhibited the growth of F. graminearum (minimum inhibitory concentrations, 7.8–15.6 mg L^?1) and exhibited synergistic activity when combined with triazole and strobilurin fungicides. Application of K20 up to 720 mg L^?1 to wheat heads in the greenhouse showed no phytotoxic effects. Spraying wheat heads in the greenhouse with K20 alone at 360 mg L^?1 lowered FHB severity below controls while combining K20 with half–label rates of Headline (pyraclostrobin) improved its disease control efficacy. In field trials, spraying K20 at 180 mg L^?1 and 360 mg L^?1 combined with half-label rates of Headline, Proline 480 SC (prothioconazole), Prosaro 421 SC (prothioconazole + tebuconazole), and Caramba (metconazole) reduced FHB indices synergistically. In addition, the K20 plus Proline 480 SC combination reduced levels of the mycotoxin deoxinivalenol by 75 % compared to the control. These data suggest that K20 may be useful as a fungicide against plant diseases such as FHB particularly when combined with commercial fungicides applied at below recommended rates.     

A genetic and biochemical approach to study trichothecene diversity in Fusarium sporotrichioides and Fusarium graminearum

The trichothecenes T-2 toxin and deoxynivalenol (DON) are natural fungal products that are toxic to both animals and plants. Their importance in the pathogenicity of Fusarium spp. on crop plants has inspired efforts to understand the genetic and biochemical mechanisms leading to trichothecene synthesis. In order to better understand T-2 toxin biosynthesis by Fusarium sporotrichioides and DON biosynthesis by F. graminearum, we compared the nucleotide sequence of the 23-kb core trichothecene gene cluster from each organism. This comparative genetic analysis allowed us to predict proteins encoded by two trichothecene genes, TRI9 and TRI10, that had not previously been described from either Fusarium species. Differences in gene structure also were correlated with differences in the types of trichothecenes that the two species produce. Gene disruption experiments showed that F. sporotrichioides TRI7 (FsTRI7) is required for acetylation of the oxygen on C-4 of T-2 toxin. Sequence analysis indicated that F. graminearum TRI7 (FgTRI7) is nonfunctional. This is consistent with the fact that the FgTRI7 product is not required for DON synthesis in F. graminearum because C-4 is not oxygenated.     

Haplotype diversity and population structure in cultivated and wild barley evaluated for Fusarium head blight responses

Fusarium head blight (FHB) is a threat to barley (Hordeum vulgare L.) production in many parts of the world. A number of barley accessions with partial resistance have been reported and used in mapping experiments to identify quantitative trait loci (QTL) associated with FHB resistance. Here, we present a set of barley germplasm that exhibits FHB resistance identified through screening a global collection of 23,255 wild (Hordeum vulgare ssp. spontaneum) and cultivated (Hordeum vulgare ssp. vulgare) accessions. Seventy-eight accessions were classified as resistant or moderately resistant. The collection of FHB resistant accessions consists of 5, 27, 46 of winter, wild and spring barley, respectively. The population structure and genetic relationships of the germplasm were investigated with 1,727 Diversity Array Technology (DArT) markers. Multiple clustering analyses suggest the presence of four subpopulations. Within cultivated barley, substructure is largely centered on spike morphology and growth habit. Analysis of molecular variance indicated highly significant genetic variance among clusters and within clusters, suggesting that the FHB resistant sources have broad genetic diversity. The haplotype diversity was characterized with DArT markers associated with the four FHB QTLs on chromosome 2H bin8, 10 and 13 and 6H bin7. In general, the wild barley accessions had distinct haplotypes from those of cultivated barley. The haplotype of the resistant source Chevron was the most prevalent in all four QTL regions, followed by those of the resistant sources Fredrickson and CIho4196. These resistant QTL haplotypes were rare in the susceptible cultivars and accessions grown in the upper Midwest USA. Some two- and six-rowed accessions were identified with high FHB resistance, but contained distinct haplotypes at FHB QTLs from known resistance sources. These germplasm warrant further genetic studies and possible incorporation into barley breeding programs.     

Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum

Fusarium head blight is one of the most important diseases of wheat worldwide due to crop losses and the contamination of grains with trichothecene mycotoxins. The biosynthesis of trichothecenes by Fusarium spp. is highest during infection, but relatively low levels are produced from saprophytic growth in axenic culture. A strain of Fusarium graminearum was constructed where the promoter from the TRI5 trichothecene biosynthesis gene was fused to GFP. Using this strain in large-scale nutrient profiling, a variety of amines were identified that significantly induce TRI5 expression. Analysis of trichothecene levels in the culture filtrates revealed accumulation of the toxin to over 1000 ppm in response to these inducers, levels either greater than or equivalent to those observed during infection. From this work, we propose that products of the arginine-polyamine biosynthetic pathway in plants may play a role in the induction of trichothecene biosynthesis during infection.     

Exploiting selective genotyping to study genetic diversity of resistance to Fusarium head blight in barley

Numerous barley cultivars from around the world have been identified as potential sources of Fusarium head blight (FHB) resistance genes. All of these cultivars exhibit partial resistance, and several mapping studies have shown that resistance to FHB is controlled by multiple genes. Successful development of barley cultivars with high levels of FHB resistance will require combining genes from multiple sources. We characterized five potential new sources of FHB resistance (AC Oxbow, Atahualpa, HOR211, PFC88209, and Zhedar#1) to determine if they contain new FHB resistance genes. Cluster analysis, using a set of 80 SSR markers distributed throughout the genome, showed that most of the new sources of resistance were not similar to three cultivars that have been used in previous FHB mapping studies (Chevron, Frederickson, and Gobernadora), with Atahualpa and HOR211 being the most dissimilar. By selective genotyping, we determined whether markers linked to six known FHB resistance quantitative trait loci (QTLs), discovered in other genotypes, explained variation for resistance in advanced breeding populations created from the new sources of resistance. Markers linked to four of the six known QTLs were associated with FHB severity in at least one of the populations. However, none of the six QTL regions were associated with variation for FHB severity in populations derived from crosses that utilized sources of resistance HOR211 or PFC88209. Selective genotyping is an efficient method for breeders to utilize current QTL information about disease resistance to search for new resistance genes.     

Evaluation of Fusarium head blight in barley infected by Fusarium graminearum

Fusarium head blight, which is primarily caused by Fusarium graminearum, is a devastating disease in the barley field. A real-time PCR protocol was developed to evaluate the growth of this pathogen in the host plant tissues. All four strains harbored the gene encoding ATP-BINDING CASSETTE TRANSPORTER (FgABC; FGSG_00541) as a single copy within their genomes. Our Southern blot result was identical with the genomic data for F. graminearum strain PH-1. Based on the crossing point (CP) values obtained in our TaqMan real-time PCR analysis, two standard curves describing the relationship among the CP value, FgABC copy number, and amount of fungal DNA were constructed. Chronological enumeration of fungal growth was coincided with the symptom development.     

Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians

Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk.     

Fusarium head blight: distribution in wheat in Latvia

Fusarium head blight (FHB) of wheat has, in recent years, been a very important worldwide disease in intensive growing of cereal. The objectives of this study were to evaluate the occurrence of FHB in wheat in Latvia and to identify the Fusarium species involved. This paper describes the distribution of Fusarium species that were isolated from samples representing winter and spring wheat varieties in Latvia, identified both by the classical morphological analyses of J. Leslie and B. Summerell (2006) and by PCR. The FHB incidence range in winter wheat was 1-20%, in spring wheat was 1-42%. The most significant factor affecting the incidence of fusarial head blight in wheat in Latvia was heightened temperature at the time of an thesis of wheat. In winter wheat 9 Fusarium species caused FHB: F. culmorum, F. avenaceum, F. graminearum, F. equiseti, F. poae, F. oxysporum, F. cerealis, F. sporotrichoides and F. verticillioides were identified by morphological characterization, and 5 were confirmed by PCR-analysis. After experience of 5 years, it can be concluded that the most frequent in winter wheat were F. poae and F. culmorum. In spring wheat from F. culmorum was dominant among 8 Fusarium species. Among 13 varieties of spring wheat, three were sensitive ('Chamsin', 'W 166', 'Azurite') and one was resistant ('Granny') to FHB in conditions of high natural infection in 2009. The monitoring surveys demonstrate a significant presence of FHB in spring wheat in conditions of heightened temperature at the time of flowering in Latvia.     

Weed species within cereal crop rotations can serve as alternative hosts for Fusarium graminearum causing Fusarium head blight of wheat

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) of small grain cereals, but the importance of weeds in the FHB disease cycle and the establishment of F. graminearum in agroecosystems are still not fully understood. The objective of this study was to determine the potential role of weeds present within cereal crop rotations as alternative hosts. F. graminearum was isolated from different organs of asymptomatic weeds sampled from six fields with cereal-crop rotations in Lithuania for two consecutive years (2015 and 2016). The fungi were identified using morphological and molecular methods. Out of 57 weed species that were investigated, 41 (71.9%) harboured F. graminearum isolates. Twenty five weed species were identified as new, previously undocumented, hosts. The majority (73.3%) of the isolates of F. graminearum from this study belonged to the 15ADON genotype while a smaller proportion (23.4%) belonged to the 3ADON genotype. All F. graminearum isolates that were assessed induced FHB symptoms on artificially inoculated spring wheat tested in the field.     

Molecular mapping of novel genes controlling Fusarium head blight resistance and deoxynivalenol accumulation in spring wheat.

Fusarium head blight of wheat is an extremely damaging disease, causing severe losses in seed yield and quality. The objective of the current study was to examine and characterize alternate sources of resistance to Fusarium head blight (FHB). Ninety-one F1-derived doubled haploid lines from the cross Triticum aestivum 'Wuhan-1' x Triticum aestivum 'Maringa' were examined for disease reaction to Fusarium graminearum by single-floret injection in replicated greenhouse trials and by spray inoculation in replicated field trials. Field and greenhouse experiments were also used to collect agronomic and spike morphology characteristics. Seed samples from field plots were used for deoxynivalenol (DON) determination. A total of 328 polymorphic microsatellite loci were used to construct a genetic linkage map in this population and together these data were used to identify QTL controlling FHB resistance, accumulation of DON, and agronomic and spike morphology traits. The analysis identified QTL for different types of FHB resistance in four intervals on chromosomes 2DL, 3BS, and 4B. The QTLs on 4B and 3BS proximal to the centromere are novel and not reported elsewhere. QTL controlling accumulation of DON independent of FHB resistance were located on chromosomes 2DS and 5AS. Lines carrying FHB resistance alleles on 2DL and 3BS showed a 32% decrease in disease spread after single-floret injection. Lines carrying FHB resistance alleles on 3BS and 4B showed a 27% decrease from the mean in field infection. Finally, lines carrying favourable alleles on 3BS and 5AS, showed a 17% reduction in DON accumulation. The results support a polygenic and quantitative mode of inheritance and report novel FHB resistance loci. The data also suggest that resistance to FHB infection and DON accumulation may be controlled, in part, by independent loci and (or) genes.     

A novel quantitative trait locus for Fusarium head blight resistance in chromosome 7A of wheat

A Chinese Spring-Sumai 3 chromosome 7A disomic substitution line (CS-Sumai 3-7ADSL) was reported to have a high level of Fusarium head blight (FHB) resistance for symptom spread within a spike (Type II) and low deoxynivalenol accumulation in infected kernels (Type III), but a quantitative trait locus (QTL) on chromosome 7A has never been identified from this source. To characterize QTL on chromosome 7A, we developed 191 7A chromosome recombinant inbred lines (7ACRIL) from a cross between Chinese Spring and CS-Sumai 3-7ADSL and evaluated both types of resistance in three greenhouse experiments. Two major QTL with Sumai 3 origin, conditioning both Type II and III resistance, were mapped in the short arm of chromosomes 3B (3BS) and near the centromere of chromosome 7A (7AC). The 3BS QTL corresponds to previously reported Fhb1 from Sumai 3, whereas 7AC QTL, designated as Fhb7AC, is a novel QTL identified from CS-Sumai 3-7ADSL in this study. Fhb7AC explains 22% phenotypic variation for Type II and 24% for Type III resistance. Marker Xwmc17 is the closest marker to Fhb7AC for both types of resistance. Fhb1 and Fhb7AC were additive, and together explained 56% variation for Type II and 41% for Type III resistance and resulted in 66% reduction in FHB severity and 84% reduction in deoxynivalenol (DON) content. Haplotype analysis of Sumai 3 parents revealed that Fhb7AC originated from Funo, an Italian cultivar. Fhb7AC has the potential to be used in improving wheat cultivars for both types of resistance.     

Sampling schemes for estimating spikelet incidence of Fusarium head blight

Fusarium head blight (FHB) is an important disease of wheat crops. The incidence of diseased spikelets in the population is most naturally estimated by sampling ears of wheat and counting the number of infected spikelets, X, and the total number of spikelets, Y, on each ear. However, there is usually much more variation from ear to ear in the number of infected spikelets than in the total number of spikelets and in practice it is common to obtain a larger sample of X values than Y values. In this paper, we investigate the relative efficiencies of these alternative sampling procedures. Based on historical data, we show that for the levels of disease incidence typically encountered, the latter sampling scheme is indeed more efficient. The results are relevant to estimating disease incidence in other crops.