Cause Of Fusarium Head Blight Research Articles

First report ofFusarium meridionalecausing Fusarium head blight of wheat in Henan Province, China.

Fusarium graminearum and F. asiaticum have been found as a major cause of Fusarium head blight (FHB) of wheat (Triticum aestivum L.), especially in Henan Province of China (Zhang et al. 2014; Xu et al. 2021). In May 2021, a survey to determine the composition of Fusarium species infecting wheat heads was conducted in commercial fields in Henan. A total of 395 diseased spikes with premature whitening symptom were collected from 31 commercial fields in Henan. Symptomatic spikelets were excised, surface-sterilized for 10 s in 70% ethanol followed by 1 min in 3% sodium hypochlorite, rinsed three times with autoclaved distilled water, and then plated onto potato dextrose agar (PDA) medium. Isolated colonies that resembled Fusarium species were transferred to fresh PDA plates and purified using a single spore method. Species were identified based on sequence analysis of the translation elongation factor-1α (TEF) and trichothecene 3-Oacetyltransferase (Tri 101) gene (Proctor et al. 2009). The results indicated that F. graminearum (43.3%), F. asiaticum (47.8%), F. pseudograminearum (6.6%) were the main causal agents of FHB in Henan. However, nine isolates (2.3%) were found to be identical to F. meridionale by sequence comparison in GenBank, and eight isolates of which came from three fields with 1% to 2% diseased spikes near Reservoir Luhun (34.1255° N, 112.1111° E, altitude: 388 m above sea level), Songxian County of Henan. The isolates of F. meridionale were transferred onto carnation leaf agar (CLA) and incubated at 20℃ under black light blue illumination. Macroconidia were abundant, relatively slender, curved to almost straight, commonly six- to seven-septate, and 27.0 to 61.0 (average 44.0) μm × 3.2 to 6.8 (average 5.3) μm. Microconidia were not observed. The TEF sequences (Accession nos. OM460748 to OM460756) and the Tri 101 sequences (OM460759 to OM460767) of the nine isolates showed 99 to 100% similarity with the TEF and Tri 101 sequences of F. meridionale NRRL 28436 and NRRL 28723 (AF212435 and AF212436 (TEF); AF212582 and AF212683 (Tri 101)). To complete Koch's postulates, the pathogenicity of the fungus was tested by using the single floret inoculation method by injecting 20-μl conidial suspension (5 × 105 conidia per milliliter) into healthy inflorescences of wheat cultivar Bainong 207 at anthesis in the field. Another 30 healthy inflorescences were injected with sterile distilled water. The heads were covered with polyethylene bags that were removed after 2 days. Twenty days after inoculation, while control inflorescences were asymptomatic, the F.meridionale-inoculated inflorescences showed 12% bleached spikelets per spike. By using the methodology described above, the fungus was re-isolated from infected spikelets of inoculated wheat heads but not from the controls. Although F.meridionalehas frequently been reported in association with Fusariumear rot (FER) of maize in Chongqing City and Gansu Province (Zhang et al. 2014; Zhou et al. 2018), and riceFER in Sichuan Province (Dong et al. 2020), to our knowledge, this is the first report of F. meridionale from diseased wheat heads in Henan, China. Further investigation is needed to gain a better understanding of this species by collecting isolates from different cropping system in Henan, which maize-wheat and rice-wheat rotation fields have coexisted in the region.

First Report of Fusarium graminearum Causing Flower Blight On Hemp (Cannabis sativa) in Kentucky.

In October of 2020, a grower in Boyle County, KY, reported mold and blight symptoms on flowers of field-grown hemp. Plants were approaching harvest, and the mold was affecting 100% of the cultivar 'White CBG' being grown for cannabinoid (CBD) extraction. Mycelium colonized the flower heads and any seeds within bracts. Affected flower bracts were necrotic, and mycelium and necrosis in the most severe cases also encompassed adjacent (sugar) leaves. Necrotic symptomatic tissue was collected, disinfested in 10% bleach for one minute, and cultured on acidified potato dextrose agar (APDA). Each isolate was single-spored, transferred to PDA, stored in 15% glycerol at -80°C and maintained at room temperature under blacklight blue and fluorescent bulbs on a 12-hour light-dark cycle. Colonies produced white-pink mycelia with a dark red pigment on the undersides. Conidia collected after 7-9 days were falcate and septate (5 to 6). No microconidia were produced. Macroconidia measured 35.4-49.7 µm x 3.4-5.8 µm (n=50). The strains produced blue-black fertile perithecia on carrot agar when induced according to the method of (Bowden and Leslie, 1999). To confirm pathogenicity, flowers of hemp cultivars 'Lifter', 'Trump Towers', 'Wife' and 'White CBG' were inoculated in the greenhouse with a representative fungal strain (20Hemp010). Plants were inoculated at two different stages: when the styles were still green or after they had become senescent. Macroconidia were collected from 7- to 9-day-old cultures grown under a 12-hour light-dark cycle. Plants were spray-inoculated with a 5 x 105 per ml conidial suspension in 0.05% Tween 20 until runoff. Flower heads were individually covered with clear plastic bags and incubated for 72 h at 95-100% humidity under greenhouse benches to avoid direct light. Bags were removed after 72 h and returned to the bench. Greenhouse conditions were 23-25°C with a 14-hour photoperiod and 50% RH. Symptoms developed 7 dai in 1% of the flowers inoculated when styles were green, and 36% of the flowers that had senescent styles. Symptoms were similar to those initially noticed in Boyle County, including necrotic flower bracts and sugar leaves, and visible fungal growth. Symptoms were more severe on plants inoculated when styles were necrotic. Recovered fungi were morphologically similar to 20Hemp010. Genomic DNA was extracted from the mycelium with the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit. A fragment of the translation elongation factor 1-alpha 1 gene was amplified with primers EF1 and EF2 as described by (O'Donnell et al. 1998). Amplicons were sequenced and the consensus (MZ407909) was compared with the NCBI GenBank Refseq database by BLASTn. The top hit was Fusarium graminearum with 100% identity (JF270185.1). Pairwise alignments via MycoBank Fusarium MLST and Fusarium-ID also revealed a top hit of F. graminearum with 100% identity (AY452957.1). Conidial and colony morphology were also consistent with F. graminearum (Leslie and Summerell, 2006), thus we conclude that this species was the causal agent of the flower blight and mold. The same disease was subsequently confirmed on hemp in Breathitt and Franklin Counties in KY in 2020. This is the first report of this disease in KY, although F. graminearum has been reported previously causing a similar flower blight on hemp in NY and NC (Bergstrom et al., 2019, Thiessen et al. 2020). Fusarium graminearum is common in KY as a cause of Fusarium head blight on wheat and Gibberella ear rot on corn. In cereals, fungal infection is facilitated by the production of the mycotoxin deoxynivalenol (DON), which is harmful to humans and livestock (Desjardins and Hohn, 1997). As hemp production in Kentucky continues to rise for production of CBD products and edible grains, accumulation and concentration of DON in these products could become a concern.

Early transcriptomic response of the mycoparasite Sphaerodes mycoparasitica to the mycotoxigenic Fusarium graminearum 3-ADON, the cause of Fusarium head blight

Mycoparasites are an assemblage of biotrophic and necrotrophic fungi that occur on plant pathogenic fungal hosts. Biotrophic mycoparasites are often overlooked in transcriptomic-based biocontrol studies. Sphaerodes mycoparasitica (S.m.) is a specific biotrophic mycoparasite of plant pathogenic Fusarium graminearum (F.g.), a devastating Fusarium head blight (FHB) disease in small-grain cereals. To understand the biotrophic mycoparasitism comprehensively, we performed Illumina RNA-Seq transcriptomic study on the fungus–fungus interaction in vitro. The aim is to identify the transcript-level mechanism related to the biotrophic S.m. mycoparasitism, particularly its ability to effectively control the F.g. 3-ADON chemotype. A shift in the transcriptomic profile of the mycoparasite was triggered in response to its interaction with F.g. during recognition (1.5 days) and colonization (3.5 days) steps. RNA-Seq analysis revealed ~ 30% of annotated transcripts with "function unknown". Further, 14 differentially expressed genes functionally linked to the biotrophic mycoparasitism were validated by quantitative real-time PCR (qPCR). The gene expression patterns of the filamentous haemagglutinin/adhesin/attachment factor as well as cell wall-degrading glucanases and chitinases were upregulated by host interaction. Besides, mycoparasitism-associated antioxidant resistance genes encoding ATP-binding cassette (ABC) transporter(s) and glutathione synthetase(s) were upregulated. However, the thioredoxin reductase was downregulated which infers that this antioxidant gene can be used as a resistance marker to assess S.m. antifungal and antimycotoxigenic activities. The interactive transcriptome of S. mycoparasitica provides new insights into specific mycoparasitism and will contribute to future research in controlling FHB.Graphic

Comparative Genomics of Eight Fusarium graminearum Strains with Contrasting Aggressiveness Reveals an Expanded Open Pangenome and Extended Effector Content Signatures

Fusarium graminearum, the primary cause of Fusarium head blight (FHB) in small-grain cereals, demonstrates remarkably variable levels of aggressiveness in its host, producing different infection dynamics and contrasted symptom severity. While the secreted proteins, including effectors, are thought to be one of the essential components of aggressiveness, our knowledge of the intra-species genomic diversity of F. graminearum is still limited. In this work, we sequenced eight European F. graminearum strains of contrasting aggressiveness to characterize their respective genome structure, their gene content and to delineate their specificities. By combining the available sequences of 12 other F. graminearum strains, we outlined a reference pangenome that expands the repertoire of the known genes in the reference PH-1 genome by 32%, including nearly 21,000 non-redundant sequences and gathering a common base of 9250 conserved core-genes. More than 1000 genes with high non-synonymous mutation rates may be under diverse selection, especially regarding the trichothecene biosynthesis gene cluster. About 900 secreted protein clusters (SPCs) have been described. Mostly localized in the fast sub-genome of F. graminearum supposed to evolve rapidly to promote adaptation and rapid responses to the host’s infection, these SPCs gather a range of putative proteinaceous effectors systematically found in the core secretome, with the chloroplast and the plant nucleus as the main predicted targets in the host cell. This work describes new knowledge on the intra-species diversity in F. graminearum and emphasizes putative determinants of aggressiveness, providing a wealth of new candidate genes potentially involved in the Fusarium head blight disease.

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.

FgEaf6 regulates virulence, asexual/sexual development and conidial septation in Fusarium graminearum.

Fusarium graminearum is a destructive fungal pathogen and a major cause of Fusarium head blight (FHB) which results in severe grain yield losses and quality reduction. Additionally, the pathogen produces mycotoxins during plant infection, which are harmful to the health of humans and livestock. As it is well known that lysine acetyltransferase complexes play important roles in pathogenesis, the roles of the Eaf6 homolog-containing complex have not been reported in fungal pathogen. In this study, a Eaf6 homolog FgEaf6 was identified in F. graminearum. To investigate the functions of FgEaf6, the gene was deleted using the split-marker method. ΔFgEaf6 mutant exhibited manifold defects in hyphal growth, conidial septation, asexual and sexual reproduction. Moreover, the virulence of the ΔFgEaf6 mutant was drastically reduced in both wheat heads and wheat coleoptiles. However, the FgEaf6 gene deletion did not impact DON production. An FgEaf6-gfp fusion localized to the nucleus and a conserved coiled-coil (C-C) domain was predicted in the sequence. Mutants with deletions in the C-C domain displayed similar defects during development and virulence as observed in the ΔFgEaf6 mutant. Moreover, the truncated gene was cytoplasm localized. In conclusion, the FgEaf6 encodes a nuclear protein, which plays key regulatory roles in hyphal growth, conidial septation, asexual/sexual reproduction, and the virulence of F. graminearum. The C-C is an indispensable domain in the gene. This is the first report on Eaf6 homolog functioning in virulence of fungal pathogen.

Selection of an Endophytic Streptomyces sp. Strain DEF09 From Wheat Roots as a Biocontrol Agent Against Fusarium graminearum.

Selection of biological control agents (BCA) profits from an integrated study of the tripartite interactions occurring among the BCA, the plant and the pathogen. The environment plays a crucial role in the efficacy of BCA, therefore, the selection process shall utmost mimic naturally occurring conditions. To identify effective biocontrol strains against Fusarium graminearum, the major cause of Fusarium head blight (FHB) in wheat and deoxynivalenol (DON) accumulation in grains, a workflow consisting of in vitro and in vivo assays was set up. Twenty-one Streptomyces strains, 16 of which were endophytes of different plants, were analyzed. In vitro and in vivo tests characterized their plant growth promoting (PGP) traits. Biocontrol activity against F. graminearum was firstly assessed with a dual culture assay. An in vivo germination blotter assay measured Fusarium foot rot and root rot symptoms (FFR-FRR) reduction as well as growth parameters of the plant treated with the Streptomyces strains. A selected subset of Streptomyces spp. strains was then assessed in a growth chamber measuring FFR symptoms and growth parameters of the wheat plant. The approach led to the identification of an effective Streptomyces sp. strain, DEF09, able to inhibit FHB on wheat in controlled conditions by blocking the spread of the pathogen at the infection site. The results were further confirmed in field conditions on both bread and durum wheat, where DEF09 decreased disease severity up to 60%. This work confirms that FRR and FFR pathosystems can be used to identify BCA effective against FHB.

First Report of the Fusarium tricinctum Species Complex Causing Fusarium Head Blight of Wheat in Brazil

Fusarium graminearum species complex (FGSC) is the main cause of Fusarium head blight (FHB) in Brazil. However, during surveys conducted from 2008 to 2011, 26 wheat kernel-borne or airborne (air above wheat canopy) strains failed using a FGSC-specific multilocus genotyping assay (Del Ponte et al. 2015). Eight spike-borne strains originating from south of Parana (PR) state (two locations), 11 airborne strains from one field at Passo Fundo (RS), and seven kernel-borne strains each from a different location in northern RS state were subjected to amplification and sequencing of the translation elongation factor 1-alpha gene (primers EF1/EF2, O’Donnell et al. 1998). Maximum parsimony- and maximum likelihood-based methods were fitted using MEGA X (Kumar et al. 2018). Eleven strains were also sequenced for the second largest subunit of RNA polymerase II (primers 5f2/7cr, O’Donnell et al. 2008). The genetic analyses used either the single- or the two-gene datasets. Sequences were deposited in GenBank (MK572748 to MK572784). Three species of the Fusarium tricinctum species complex (FTSC) were identified: 15 F. avenaceum, nine FTSC 11, and two F. reticulatum. Although the two most frequent species were either airborne or kernel-borne, F. reticulatum was only airborne. Among the strains from PR, FTSC 11 was represented by one strain and the rest by F. avenaceum. In RS, FTSC 11 and F. avenaceum, airborne or kernel-borne, were in similar frequency. Eighteen strains representative of all species were subjected to pathogenicity assays on an FHB-susceptible spring wheat (BR18). Two standard FGSC strains, F. graminearum (CML 3066) and F. meridionale (CML 3344), were included for comparison. Three potted wheat plants (replicates) were used. Fungi were grown on Spezieller Nahrstoffarmer agar for 7 days at 25°C with a 12-h photoperiod. A macroconidia suspension (1 × 10⁴ macroconidia/ml) was applied uniformly onto wheat heads at flowering using a household handheld sprayer (2 ml/head). Plants were covered with a plastic bag for 48 h and remained in the greenhouse until harvest (35 days). Disease severity was assessed visually at 4 and 8 days postinoculation (dpi) on each inoculated head. The assay was repeated once. All strains were pathogenic to wheat, and the reisolation of at least two strains from infected spikes confirmed their identity. FHB severity at 4 dpi ranged from 11.7 to 82.8% for F. avenaceum, from 14.1 to 46.2% for F. reticulatum, and from 14.1 to 90.2% for FTSC 11. Similarly, a large intraspecies variation of mean FHB severity was observed at 8 dpi for F. avenaceum (33 to 94.5%), F. reticulatum (32.6 to 79.6%), and FTSC 11 (42.2 to 97.8%). Although evidence for a species effect was not strong (P = 0.08), some strains of all species induced FHB at levels similar to FGSC. FTSC members are known to prevail in cooler climates such as Canada and northern Europe (Bottalico and Perrone 2002; Grafenhan et al. 2013). They were reported in Kentucky (U.S.A.) wheat, including F. reticulatum strains that were weakly pathogenic toward wheat (Bec et al. 2015). In Brazil, barley from the 2016 growing season was found to harbor FTSC strains (Piacentini et al. 2019). Further work is needed to understand whether FTSC is an emergent FHB pathogen, especially because of their known ability to produce enniatins and beauvericin mycotoxins, which are not regulated in Brazil.

Susceptibility of non-cereal crops to Fusarium graminearum complex and their role within cereal crop rotation as a source of inoculum for Fusarium head blight

Fusarium graminearum, the cause of Fusarium head blight (FHB), is an important cereal pathogen. Moreover, some non-graminaceous crops are also known to be susceptible to F. graminearum infection. This study assessed the presence of F. graminearum species complex on non-cereal plants, grown in a cereal crop rotation and evaluated its pathogenicity to non-cereal plants in vitro and to spring wheat under field conditions. The relative density of Fusarium species isolated from oilseed rape, pea, potato and sugar beet plants was assessed in 2015 and 2016. A total of 403 isolates of Fusarium spp. were obtained from non-cereal plants and only 5% of the isolates were identified as F. graminearum. The pathogenicity test revealed that isolates of F. graminearum from spring wheat and non-cereal plants caused discolourations on leaves of faba bean, fodder beet, oilseed rape, pea, potato and sugar beet. The pea was the crop most susceptible to F. graminearum isolated from spring wheat. The pathogenicity of F. graminearum from sugar beet, oilseed rape, pea and potato to the same hosts differed depending on isolate and inoculated plant. Under field conditions, F. graminearum isolates from pea, potato, oilseed rape and wild viola were able to cause typical FHB symptoms in spring wheat. Based on the information generated in this study, we conclude that under congenial conditions, growing faba bean, pea, sugar beet, fodder beet, oilseed rape and potato plants in a cereal crop rotation may serve as alternative or reservoir hosts for F. graminearum pathogens.

Population genomics of Fusarium graminearum reveals signatures of divergent evolution within a major cereal pathogen.

The cereal pathogen Fusarium graminearum is the primary cause of Fusarium head blight (FHB) and a significant threat to food safety and crop production. To elucidate population structure and identify genomic targets of selection within major FHB pathogen populations in North America we sequenced the genomes of 60 diverse F. graminearum isolates. We also assembled the first pan-genome for F. graminearum to clarify population-level differences in gene content potentially contributing to pathogen diversity. Bayesian and phylogenomic analyses revealed genetic structure associated with isolates that produce the novel NX-2 mycotoxin, suggesting a North American population that has remained genetically distinct from other endemic and introduced cereal-infecting populations. Genome scans uncovered distinct signatures of selection within populations, focused in high diversity, frequently recombining regions. These patterns suggested selection for genomic divergence at the trichothecene toxin gene cluster and thirteen additional regions containing genes potentially involved in pathogen specialization. Gene content differences further distinguished populations, in that 121 genes showed population-specific patterns of conservation. Genes that differentiated populations had predicted functions related to pathogenesis, secondary metabolism and antagonistic interactions, though a subset had unique roles in temperature and light sensitivity. Our results indicated that F. graminearum populations are distinguished by dozens of genes with signatures of selection and an array of dispensable accessory genes, suggesting that FHB pathogen populations may be equipped with different traits to exploit the agroecosystem. These findings provide insights into the evolutionary processes and genomic features contributing to population divergence in plant pathogens, and highlight candidate genes for future functional studies of pathogen specialization across evolutionarily and ecologically diverse fungi.

Molecular Characterization and Functional Analysis of PR-1-Like Proteins Identified from the Wheat Head Blight Fungus Fusarium graminearum.

The group 1 pathogenesis-related (PR-1) proteins originally identified from plants and their homologs are also found in other eukaryotic kingdoms. Studies on nonplant PR-1-like (PR-1L) proteins have been pursued widely in humans and animals but rarely in filamentous ascomycetes. Here, we report the characterization of four PR-1L proteins identified from the ascomycete fungus Fusarium graminearum, the primary cause of Fusarium head blight of wheat and barley (designated FgPR-1L). Molecular cloning revealed that the four FgPR-1L proteins are all encoded by small open reading frames (612 to 909 bp) that are often interrupted by introns, in contrast to plant PR-1 genes that lack introns. Sequence analysis indicated that all FgPR-1L proteins contain the PR-1-specific three-dimensional structure, and one of them features a C-terminal transmembrane (TM) domain that has not been reported for any stand-alone PR-1 proteins. Transcriptional analysis revealed that the four FgPR-1L genes are expressed in axenic cultures and in planta with different spatial or temporal expression patterns. Phylogenetic analysis indicated that fungal PR-1L proteins fall into three major groups, one of which harbors FgPR-1L-2-related TM-containing proteins from both phytopathogenic and human-pathogenic ascomycetes. Low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteolytic assays indicated that the recombinant FgPR-1L-4 protein exists as a monomer and is resistant to subtilisin of the serine protease family. Functional analysis confirmed that deletion of the FgPR-1L-4 gene from the fungal genome results in significantly reduced virulence on susceptible wheat. This study provides the first example that the F. graminearum-wheat interaction involves a pathogen-derived PR-1L protein that affects fungal virulence on the host.

Fitness Traits of Deoxynivalenol and Nivalenol-Producing Fusarium graminearum Species Complex Strains from Wheat.

Fusarium graminearum of the 15-acetyl-deoxynivalenol (15-ADON) chemotype is the main cause of Fusarium head blight (FHB) of wheat in southern Brazil. However, 3-ADON and nivalenol (NIV) chemotypes have been found in other members of the species complex causing FHB in wheat. To improve our understanding of the pathogen biology and ecology, we assessed a range of fitness-related traits in a sample of 30 strains representatives of 15-ADON (F. graminearum), 3-ADON (F. cortaderiae and F. austroamericanum), and NIV (F. meridionale and F. cortaderiae). These included perithecia formation on three cereal-based substrates, mycelial growth at two suboptimal temperatures, sporulation and germination, pathogenicity toward a susceptible and a moderately resistant cultivar, and sensitivity to tebuconazole. The most important trait favoring F. graminearum was a two times higher sexual fertility (>40% perithecial production index [PPI]) than the other species (<30% PPI); PPI varied among substrates (maize > rice > wheat). In addition, sensitivity to tebuconazole appeared lower in F. graminearum, which had the only strain with effective fungicide concentration to reduce 50% of mycelial growth >1 ppm. In the pathogenicity assays, the deoxynivalenol producers were generally more aggressive (1.5 to 2× higher final severity) toward the two cultivars, with 3-ADON or 15-ADON leading to higher area under the severity curve than the NIV strains in the susceptible and moderately resistant cultivars, respectively. There was significant variation among strains of the same species with regards asexual fertility (mycelial growth, macroconidia production, and germination), which suggested a strain- rather than a species-specific difference. These results contribute new knowledge to improve our understanding of the pathogen-related traits that may explain the dominance of certain members of the species complex in specific wheat agroecosystems.

Genome-Wide Analysis of Small Secreted Cysteine-Rich Proteins Identifies Candidate Effector Proteins Potentially Involved in Fusarium graminearum-Wheat Interactions.

Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (FHB), a devastating disease of wheat. We report here a comprehensive analysis of SSCPs encoded in the genome of this fungus and selection of candidate effector proteins through proteomics and sequence/transcriptional analyses. A total of 190 SSCPs were identified in the genome of F. graminearum (isolate PH-1) based on the presence of N-terminal signal peptide sequences, size (≤200 amino acids), and cysteine content (≥2%) of the mature proteins. Twenty-five (approximately 13%) SSCPs were confirmed to be true extracellular proteins by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis of a minimal medium-based in vitro secretome. Sequence analysis suggested that 17 SSCPs harbor conserved functional domains, including two homologous to Ecp2, a known effector produced by the tomato pathogen Cladosporium fulvum. Transcriptional analysis revealed that at least 34 SSCPs (including 23 detected in the in vitro secretome) are expressed in infected wheat heads; about half are up-regulated with expression patterns correlating with the development of FHB. This work provides a solid candidate list for SSCP-derived effectors that may play roles in mediating F. graminearum-wheat interactions. The in vitro secretome-based method presented here also may be applicable for identifying candidate effectors in other ascomycete pathogens of crop plants.

Species composition and genetic structure of Fusarium graminearum species complex populations affecting the main barley growing regions of South America

Members of the Fusarium graminearum species complex (FGSC), such as F. graminearum and F. asiaticum, are the main cause of fusarium head blight (FHB) of wheat and barley worldwide. In this study, 117 FGSC isolates obtained from commercial barley grain produced in Argentina (n = 43 isolates), Brazil (n = 35), and Uruguay (n = 39) were identified to species and trichothecene genotypes, and analysed using amplified fragment length polymorphism (AFLP) and sequence‐related amplified polymorphism (SRAP) markers. In addition, reductase (RED) and trichothecene 3‐O‐acetyltransferase (Tri101) were sequenced for a subset of 24 isolates. The majority of the isolates (n = 103) were identified as F. graminearum, which was the only species found in Argentina. In Uruguay, only one F. cortaderiae isolate was found among F. graminearum isolates. In Brazil, F. graminearum also dominated the collection (22/35), followed by F. meridionale (8/35), F. asiaticum (2/35), F. cortaderiae (2/35) and F. austroamericanum (1/35). Species were structured by trichothecene genotype: all F. graminearum were of the 15‐acetyldeoxynivalenol (ADON), F. meridionale, F. asiaticum and F. cortaderiae were of the nivalenol (NIV), and F. austroamericanum was of the 3‐ADON genotype. Both AFLP and SRAP data showed high levels of genetic variability, which was higher within than among countries. Isolates were not structured by country of origin. SRAP analysis grouped F. graminearum in a separate cluster from the other species within the complex. However, AFLP analysis failed to resolve the species into distinct clades with partial clustering of F. meridionale, F. austroamericanum, F. asiaticum and F. graminearum isolates.

Development of an FgMito assay: A highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto

An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r2-values of >0.99. We further showed that three low concentrations of fungal template 2pg, 0.6pg and 0.2pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.

Ratio of 3-ADON and 15-ADON isolates of Fusarium graminearum recovered from wheat kernels in Manitoba from 2008 to 2012

Fusarium head blight (FHB) of wheat, caused principally by Fusarium graminearum in Canada, results in accumulation of mycotoxins in the grain. The fungus produces deoxynivalenol (DON) and its acetylated forms 3-ADON or 15-ADON. Increasing numbers of F. graminearum isolates collected in Manitoba between 1998 and 2004 were of the 3-ADON chemotype, whereas prior to 1998, the 15-ADON chemotype was considered the only significant cause of FHB in North America. Between 2008 and 2012, a series of studies were conducted at Carman (2008, 2009) and Glenlea, Manitoba (2008–2012) to monitor the ratio of 3-ADON to 15-ADON chemotypes recovered after wheat plots were artificially inoculated, usually in a 1:1 ratio of 3-ADON:15-ADON isolates of F. graminearum. Additional studies were conducted under controlled conditions. In 2008, the majority of isolates recovered from both Carman and Glenlea were of the 3-ADON chemotype. In subsequent years, however, the ratio was closer to 1:1 or sometimes with a slight predominance of 15-ADON isolates. Under controlled conditions, recovery of 15-ADON isolates predominated at lower incubation temperatures (18–22°C) whereas at 28°C, 3-ADON isolates were more prevalent. At moderate temperature (24°C), results were mixed – seven samples had a 1:1 ratio, three had higher recovery of 3-ADON isolates, and two had higher recovery of 15-ADON. However, an examination of weather variables over the 5-year period of the field studies showed no correlation between recovery of chemotype and temperature or precipitation.

Regional differences in species composition and toxigenic potential among Fusarium head blight isolates from Uruguay indicate a risk of nivalenol contamination in new wheat production areas

Members of the Fusarium graminearum species complex (FGSC) are the primary cause of Fusarium head blight (FHB) of wheat, and frequently contaminate grain with trichothecene mycotoxins that pose a serious threat to food safety and animal health. The species identity and trichothecene toxin potential of 151 FGSC isolates collected from wheat in Uruguay were determined via multilocus genotyping. Although F. graminearum with the 15ADON trichothecene type accounted for 86% of the isolates examined, five different FGSC species and all three trichothecene types were identified in this collection. This is the first report of Fusarium asiaticum, Fusarium brasilicum, Fusarium cortaderiae, and Fusarium austroamericanum from Uruguay. In addition, we observed significant (P<0.001) regional differences in the composition of FGSC species and trichothecene types within Uruguay. Isolates of F. graminearum with the 15ADON type were the most prevalent in western provinces (95%), while F. asiaticum (43%) and the NIV type (61%) predominated in the new wheat production zone in Cerro Largo along Uruguay's eastern border with Brazil. F. graminearum isolates (15ADON type) were significantly (P<0.005) more aggressive on wheat than were isolates from the other species examined (NIV or 3ADON types). However, F. graminearum isolates (15ADON type) were significantly (P<0.05) more sensitive to tebuconazole than isolates from other species (NIV type). These results document substantial heterogeneity among the pathogens responsible for FHB in Uruguay. In addition, the regional predominance of the NIV trichothecene type is of significant concern to food safety and indicates that additional monitoring of nivalenol levels in grain may be required.

Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity

Type II myosin is required for cytokinesis/septation in yeast and filamentous fungi, including Fusarium graminearum, a prevalent cause of Fusarium head blight in China. A type II myosin gene from the Chinese F. graminearum strain 5035, isolated from infected wheat spikes, was identified by screening a mutant library generated by restriction enzyme-mediated integration. Disruption of the Myo2 gene reduced mycelial growth by 50% and conidiation by 76-fold, and abolished sexual reproduction on wheat kernels. The Δmyo2 mutants also had a 97% decrease in their pathogenicity on wheat, and mycotoxin production fell to just 3.4% of the normal level. The distribution of nuclei and septa was abnormal in the mutants, and the septal ultrastructure appeared disorganized. Time-lapse imaging of septation provided direct evidence that Myo2 is required for septum initiation and formation, and revealed the dynamic behavior of GFP-tagged Myo2 during hyphal and macroconidia development, particularly in the delimiting septum of phialides and macroconidial spores. Microarray analysis identified many genes with altered expression profiles in the Δmyo2 mutant, indicating that Myo2 is required for several F. graminearum developmental processes and biological activities.

Heat- and cold-shock responses in Fusarium graminearum 3 acetyl- and 15 acetyl-deoxynivalenol chemotypes

Fusarium graminearum Schwabe is the primary cause of Fusarium head blight (FHB) in North America. Chemically distinct F. graminearum sub-populations can be identified based on the type or composition of deoxynivalenol (DON) mycotoxin derivatives, including 3-acetyl (3-ADON) and 15-acetyl (15-ADON). The evaluation of randomly selected 3-ADON and 15-ADON isolates, collected from spring wheat throughout Canada, was performed using thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ice-nucleation activity (INA), and heat and cold tolerance tests conducted within a temperature range of -70°C to 65°C. The results indicated that the 3-ADON sub-population, which is responsible for the highest disease severity and has rapidly displaced the 15-ADON sub-population, produces more DON and zearalenone (ZEA) than the 15-ADON sub-population when exposed to heat and cold. Following exposures (1 and 2 h) to extremely high or low temperatures, 3-ADON isolates exhibited faster mycelial growth than 15-ADON isolates. In addition, the warmest temperature at which INA activity occurred was in 3-ADON (-3.6°C) vs. 15-ADON (-5.1°C). Taken together, these features suggest that the newly emerging 3-ADON sub-population is more resilient than the resident 15-ADON sub-population. Overall, the differences between the two sub-populations could provide new insights into FHB epidemiology and if validated under field conditions, may provide important information for predicting future FHB epidemics.

Genetic Diversity in Fusarium graminearum from a Major Wheat-Producing Region of Argentina

The Fusarium graminearum species complex (FGSC) is a group of mycotoxigenic fungi that are the primary cause of Fusarium head blight (FHB) of wheat worldwide. The distribution, frequency of occurrence, and genetic diversity of FGSC species in cereal crops in South America is not well understood compared to some regions of Asia, Europe and North America. Therefore, we examined the frequency and genetic diversity of a collection of 183 FGSC isolates recovered from wheat grown during multiple growing seasons and across a large area of eastern Argentina, a major wheat producing region in South America. Sequence analysis of the translation elongation factor 1−α and β-tubulin genes as well as Amplified Fragment Length Polymorphism (AFLP) analyses indicated that all isolates were the FGSC species F. graminearum sensu stricto. AFLP analysis resolved at least 11 subgroups, and all the isolates represented different AFLP haplotypes. AFLP profile and geographic origin were not correlated. Previously obtained trichothecene production profiles of the isolates revealed that the 15-acetyldeoxynivalenol chemotype was slightly more frequent than the 3-acetyldeoxynivalenol chemotype among the isolates. These data extend the current understanding of FGSC diversity and provide further evidence that F. graminearum sensu stricto is the predominant cause of FHB in the temperate main wheat-growing area of Argentina. Moreover, two isolates of F. crookwellense and four of F. pseudograminearum were also recovered from wheat samples and sequenced. The results also suggest that, although F. graminearum sensu stricto was the only FGSC species recovered in this study, the high level of genetic diversity within this species should be considered in plant breeding efforts and development of other disease management strategies aimed at reducing FHB.