Map-based cloning identifies velvet A as a critical component of virulence in Fusarium pseudograminearum during infection of wheat heads

Although better known as a pathogen of wheat stem bases, Fusarium pseudograminearum also causes Fusarium head blight. A natural isolate of F. pseudograminearum was identified that showed severely reduced virulence towards wheat heads and a map-based cloning approach was undertaken to identify the genetic basis of this phenotype. Using a population of 95 individuals, a single locus on chromosome 1 was shown to be responsible for the low virulence. Fine mapping narrowed the region to just five possible SNPs of which one was in the F. pseudograminearum homologue of velvet A. Knockout mutants of velvet A, which were non-pathogenic towards wheat, confirmed that velvet A regulates virulence in this pathogen. The mutation in velvet A was only found in a single field isolate and the origin of the mutation is unknown.     

Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.     

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.     

Population Genetics of Three Important Head Blight Pathogens Fusarium graminearum, F. pseudograminearum and F. culmorum

Homothallic Fusarium graminearum (teleomorph Gibberella zeae) and anamorphic F. culmorum are destructive pathogens causing Fusarium head blight (FHB) of small‐grain cereals worldwide, while heterothallic F. pseudograminearum (G. coronicola) seems to be restricted to Australia as a FHB pathogen. In a comprehensive treatise of pathogen population genetics, this review summarizes global knowledge of genetic diversity among isolates sampled at various spatial and temporal scales, examines the mechanisms that generate this diversity and explores the implications of pathogen diversity and plasticity to resistance breeding. Despite their different modes of reproduction, there is large variation among isolates of all three species originating from different countries and continents. With a few exceptions, haplotype diversity ranges from 60 to 100% even within populations from individual fields. In F. graminearum, over 90% of the variation is found within populations, even when samples are collected from areas as small as 0.25 m². Variation among populations is low (4–8%) with negligible population subdivision. This indicates a high level of gene flow (N_m = 8–71) with linkage equilibrium for the majority of selectively neutral molecular marker loci analysed. These findings for F. graminearum point to large random mating populations driven by occasional outcrossing, high gene flow across large geographical distances and a relatively low host‐mediated directional selection. Similar conclusions can be drawn for the Canadian population of F. pseudograminearum, but not for populations from Australia, where different pathogen ecology may have reduced the frequency of sexual recombination. Phylogenetic analyses indicate delineation of lineages in F. graminearum, often along geographically separated lines, while the related F. pseudograminearum is a single recombining species with limited or no lineage development. The anamorphic F. culmorum shows no obvious clonal structure in its population as might have been expected. High levels of diversity within fields may have been caused by balancing selection from frequent alternation between saprophytic and parasitical life cycle and/or a hidden or recently extinct teleomorph. Other mechanisms including parasexual cycles or active transposable elements may also be involved but these have not been investigated as yet. Crosses between and among F. graminearum lineages have shown a rather simple, additive inheritance of pathogenicity and aggressiveness with frequent transgressive segregation in crosses among isolates with moderate aggressiveness. This raises the spectre of highly aggressive and/or toxigenic isolates evolving if a limited range of quantitative trait locus for FHB resistance is deployed on a large scale. Combining more than one genetically distinct sources of resistance, possibly with different modes of action against the pathogen, will be necessary to avoid severe FHB outbreaks in the future.     

Baseline sensitivity of Fusarium graminearum from wheat fields in Henan,China, to metconazole and analysis of cross resistance with carbendazim and phenamacril

The filamentous plant pathogenic fungus Fusarium graminearum is one of the most important pathogens causing Fusarium head blight (FHB) in wheat in the Henan Province of China. Metconazole is among the demethylation inhibitor (DMI) fungicides with a higher inhibitory activity on the mycelial growth of F. graminearum. In 2016 and 2017, 119 single spore isolates of F. graminearum, prior to being exposed to metconazole, were recovered from 52 wheat fields near 11 cities in Henan Province. The inhibitory activity of metconazole on the mycelia of the Henan F. graminearum population was determined, and EC₅₀ values were calculated. The range of EC₅₀ values of the Henan F. graminearum population to metconazole was 0.0103 to 0.0775 μg/ml with an average EC₅₀ value of 0.0293 ± 0.0114 μg/ml. The sensitivity frequency distribution curve presented a single peak in a narrow range. No cross-resistance was found between the DMI fungicide metconazole and the benzimidazole fungicide carbendazim or the cyanoacrylate fungicide phenamacril. Therefore, these sensitivity data could be used as the baseline of F. graminearum susceptibility to metconazole in the Henan Province and provide the basis for monitoring metconazole resistance in this area.     

FcRav2, a gene with a ROGDI domain involved in Fusarium head blight and crown rot on durum wheat caused by Fusarium culmorum

Fusarium culmorum is a soil‐borne fungal pathogen which causes foot and root rot and Fusarium head blight on small‐grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in the contamination of kernels with type B trichothecene mycotoxins. Our knowledge of the pathogenicity factors of this fungus is still limited. A transposon tagging approach based on the mimp1/impala double‐component system has allowed us to select a mutant altered in multiple metabolic and morphological processes, trichothecene production and virulence. The flanking regions of mimp1 were used to seek homologies in the F. culmorum genome, and revealed that mimp1 had reinserted within the last exon of a gene encoding a hypothetical protein of 318 amino acids which contains a ROGDI‐like leucine zipper domain, supposedly playing a protein–protein interaction or regulatory role. By functional complementation and bioinformatic analysis, we characterized the gene as the yeast Rav2 homologue, confirming the high level of divergence in multicellular fungi. Deletion of FcRav2 or its orthologous gene in F. graminearum highlighted its ability to influence a number of functions, including virulence, trichothecene type B biosynthesis, resistance to azoles and resistance to osmotic and oxidative stress. Our results indicate that the FcRav2 protein (and possibly the RAVE complex as a whole) may become a suitable target for new antifungal drug development or the plant‐mediated resistance response in filamentous fungi of agricultural interest.     

Response of Barley Genotypes to Fusarium Head Blight under Natural Infection and Artificial Inoculation Conditions

Forty-eight spring barley genotypes were evaluated for deoxynivalenol (DON) concentration under natural infection across 5 years at Harrington, Prince Edward Island. These genotypes were also evaluated for Fusarium head blight (FHB) severity and DON concentration under field nurseries with artificial inoculation of Fusarium graminearum by the grain spawn method across 2 years at Ottawa, Ontario, and one year at Hangzhou, China. Additionally, these genotypes were also evaluated for FHB severity under greenhouse conditions with artificial inoculation of F. graminearum by conidial suspension spray method across 3 years at Ottawa, Ontario. The objective of the study was to investigate if reactions of barley genotypes to artificial FHB inoculation correlate with reactions to natural FHB infection. DON concentration under natural infection was positively correlated with DON concentration (r = 0.47, P < 0.01) and FHB incidence (r = 0.56, P < 0.01) in the artificially inoculated nursery with grain spawn method. Therefore, the grain spawn method can be used to effectively screen for low DON. FHB severity, generated from greenhouse spray, however, was not correlated with DON concentration (r = 0.12, P > 0.05) under natural infection and it was not correlated with DON concentration (r = −0.23, P > 0.05) and FHB incidence (r = 0.19, P > 0.05) in the artificially inoculated nursery with grain spawn method. FHB severity, DON concentration, and yield were affected by year, genotype, and the genotype × year interaction. The effectiveness of greenhouse spray inoculation for indirect selection for low DON concentration requires further studies. Nine of the 48 genotypes were found to contain low DON under natural infection. Island barley had low DON and also had high yield.     

A high‐resolution genetic map of the cereal crown rot pathogen Fusarium pseudograminearum provides a near‐complete genome assembly

Fusarium pseudograminearum is an important pathogen of wheat and barley, particularly in semi‐arid environments. Previous genome assemblies for this organism were based entirely on short read data and are highly fragmented. In this work, a genetic map of F. pseudograminearum has been constructed for the first time based on a mapping population of 178 individuals. The genetic map, together with long read scaffolding of a short read‐based genome assembly, was used to give a near‐complete assembly of the four F. pseudograminearum chromosomes. Large regions of synteny between F. pseudograminearum and F. graminearum, the related pathogen that is the primary causal agent of cereal head blight disease, were previously proposed in the core conserved genome, but the construction of a genetic map to order and orient contigs is critical to the validation of synteny and the placing of species‐specific regions. Indeed, our comparative analyses of the genomes of these two related pathogens suggest that rearrangements in the F. pseudograminearum genome have occurred in the chromosome ends. One of these rearrangements includes the transposition of an entire gene cluster involved in the detoxification of the benzoxazolinone (BOA) class of plant phytoalexins. This work provides an important genomic and genetic resource for F. pseudograminearum, which is less well characterized than F. graminearum. In addition, this study provides new insights into a better understanding of the sexual reproduction process in F. pseudograminearum, which informs us of the potential of this pathogen to evolve.     

一株小麦赤霉病拮抗菌的筛选鉴定及防治效果分析

自小麦赤霉病发病田块土壤中,分离得到1株小麦赤霉病高效拮抗菌株。通过形态特征、生理生化特性和16S rDNA序列比对分析,将这株拮抗菌株鉴定为奇异变形杆菌(proteus mirabilis)。P. mirabilis DY05发酵液和无细胞上清均可显著抑制禾谷镰刀菌菌丝体生长(抑制率分别为79.50%和51.25%)和分生孢子萌发(抑制率均为100%),减少呕吐毒素产生(分别减少84.32%和82.82%)。田间赤霉病防治试验结果显示,接种DY05,可降低发病率52.13%,同时病情指数降低48.74%,显示出较好的防治效果。促生生理活性评价试验结果显示,菌株DY05可以产生铁载体和IAA,并具有溶磷作用和ACC脱氨酶活性,具有很好的促生长潜力。盆栽试验结果表明,菌株DY05对小麦植株生长具有显著的促进作用。与对照组相比,菌株DY05处理可以显著增加小麦幼苗茎高、根长、鲜重和干重,其中茎高、根长、鲜重和干重分别提高了22.21%、26.41%、44.77%和26.53%。分离得到的拮抗菌株DY05具有拮抗病原真菌和促进植物生长的双重功能,为开发禾谷镰刀菌生物防治制剂提供了菌种材料。     

Identification of 11 polymorphic simple sequence repeat loci in the phytopathogenic fungus Fusarium pseudograminearum as a tool for genetic studies

Simple sequence repeat (SSR) markers for Fusarium pseudograminearum with 2 to 3 bp repeat motifs were identified by screening the genome database of the related species Fusarium graminearum. Twelve SSRs amplified single loci in both F. graminearum and F. pseudograminearum. Forty F. pseudograminearum and six F. graminearum individual isolates were screened to determine levels of polymorphism, with all SSRs displaying three to 14 alleles across all isolates. Eleven SSRs were polymorphic across F. pseudograminearum isolates tested proving the usefulness of genome databases of closely related species in identifying genetic markers.     

The xylanase inhibitor TAXI‐III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants

The xylanase inhibitor TAXI‐III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI‐III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre‐incubation of xylanase FGSG_03624 with TAXI‐III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non‐transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI‐III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI‐III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI‐III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI‐III to avoid host cell death.     

Concentration and cultivar effects on efficacy of CLO-1 biofungicide in controlling Fusarium head blight of wheat

Fusarium head blight (FHB) is a destructive disease of wheat in Canada and Clonostachys rosea strain ACM941 has been identified as a promising biological control agent for managing FHB. In the present research the concentration and cultivar effects on the efficacy of CLO-1, a formulated product of C. rosea strain ACM941, in controlling FHB and deoxynivalenol (DON) contamination in wheat was studied. Of the eight concentrations ranging from 10⁴ to 10⁸ cfu mL⁻¹ evaluated, significant effects were generally observed for concentrations at or above 10⁶ cfu mL⁻¹ in the greenhouse and field trials in 2009 and 2010. In the greenhouse, CLO-1 reduced the area under the disease progress curve (AUDPC) by 65–83%, Fusarium damaged kernels (FDK) by 68–92%, and DON by 51–95%. Under field conditions, CLO-1 reduced FHB index by 30–46%, FDK by 31–39%, and DON by 22–33%. These effects were numerically lower but not significantly different from those of the registered fungicide Folicur® (tebuconazole) used in these trials. When applied onto wheat cultivars differing in resistance to FHB in field trials in 2009 and 2010, CLO-1 was most effective on the moderately resistant cultivar AC Nass (representing the highest level of resistance commercially available) and least effective on the highly susceptible cultivar AC Foremost. Results of this study suggest that CLO-1 is a promising biocontrol product that may be used in combination with cultivar resistance for managing FHB in wheat.     

Potential for control of seedling blight of wheat caused by Fusarium graminearum and related species using the bacterial endophyte Bacillus mojavensis

Fusarium-infected wheat seed decreases germination, seedling emergence, and causes post emergence seedling death, and can contribute to wheat scab and ear rot of maize, with consequent production of mycotoxins such as deoxynivalenol and zearalenone. Current seed treatments have proved ineffective in controlling seedling blight and scab. A patented endophytic bacterial strain, Bacillus mojavensis RRC 101, and several other strains of this species were studied to determine in vitro antagonism to some Fusarium species and to assess the potential of this bacterium to serve as an endophytic biocontrol for seedling blight of wheat produced by species within the F. graminearum complex, as well as other species of Fusarium. Seedling emergence and seed germination were two tests used as indicators of seedling blight. These tests were conducted in growth rooms with two wheat cultivars highly susceptible to scab, Norm and Pioneer 2552, and other cultivars with varying resistance to scab. The results indicated that all strains of this bacterium were antagonistic in vitro to the strains of F. graminearum and its seven related species, as well as four strains of F. pseudograminearum and the two strains of F. verticillioides. Germination of the highly scab susceptible cultivar 2552 was increased from 77 to 97% when planted in soil containing a mixed inoculum of F. graminearum and related species. Seedling emergence in the very susceptible wheat cultivar Norm increased from 20 to 82% when treated with the bacterium. The data indicated that inoculating wheat kernels with B. mojavensis reduced seedling blight of wheat produced by F. graminearum and related Fusarium species indicating the potential for this bacterium as a biocontrol under field condition.     

Aggressiveness spectrum and genetic variability of Fusarium spp. on rice and wheat varieties

Abstract

A total of 106 Fusarium spp. were isolated from infected roots and soil samples of wheat and rice. Of the 106 isolates, 32 from wheat, and 74 from rice, were isolated. Six Fusarium spp. (F. oxysporum, F. moniliforme, F. poae, F. graminearum, F. tricinctum and F. equiseti) were identified at specie level. In aggressiveness tests Fusarium spp. root rot causing fungi were screened out into different aggressiveness classes according to disease severity scales. The aggressiveness of Fusarium spp. was studied on wheat varieties (Inqalab-91 and chakwal-86) and on rice varieties (Basmati-385 and IRRI-6) under controlled conditions. The overall total number of aggressive isolates was higher in rice than in wheat. However, the percentage of severely aggressive isolates was high in wheat, whereas the percentage of moderately and slightly aggressiveness isolates was high in rice. In rice, five isolates were non-aggressive and on wheat 17 were non-aggressive. Random Amplified Polymorphism DNAs (RAPDs) were used to study the polymorphism and genetic variations within the population of Fusarium spp. that established to study correlation between taxonomical and genetical characters of fungi. Five random primers were used P1 (5′-AGGAGGACCC-3′), P2 (5′-ACGAGGGACT-3′), PE7 (5′-AGATGCAGCC-3′), P14 (5′-CCACAGCACG-3′) and PE20 (5′-AACGGTGACC-3′). Each of the 10-mer primers produced results based on the respective banding patterns they generated in present investigations. Primers distinguished the F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti. All the tested primers yielded amplification products, and that were reproducible. Although there was some intraspecific variation with primers, some strains were similar and some were different in banding pattern. In F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti were seen clustered close to one another but each primer separated them unambiguously. All primer (P1, P2, P14, PE7 and PE20) combination produced 62 bands. All primers have shown interspecific and intraspecific variations in banding patterns.     

Complex microcolinearity among wheat, rice, and barley revealed by fine mapping of the genomic region harboring a major QTL for resistance to Fusarium head blight in wheat

A major quantitative trait locus (QTL), Qfhs.ndsu-3BS, for resistance to Fusarium head blight (FHB) in wheat has been identified and verified by several research groups. The objectives of this study were to construct a fine genetic map of this QTL region and to examine microcolinearity in the QTL region among wheat, rice, and barley. Two simple sequence repeat (SSR) markers (Xgwm533 and Xgwm493) flanking this QTL were used to screen for recombinants in a population of 3,156 plants derived from a single F₇ plant heterozygous for the Qfhs.ndsu-3BS region. A total of 382 recombinants were identified, and they were genotyped with two more SSR markers and eight sequence-tagged site (STS) markers. A fine genetic map of the Qfhs.ndsu-3BS region was constructed and spanned 6.3 cM. Based on replicated evaluations of homozygous recombinant lines for Type II FHB resistance, Qfhs.ndsu-3BS, redesignated as Fhb1, was placed into a 1.2-cM marker interval flanked by STS3B-189 and STS3B-206. Primers of STS markers were designed from wheat expressed sequence tags homologous to each of six barley genes expected to be located near this QTL region. A comparison of the wheat fine genetic map and physical maps of rice and barley revealed inversions and insertions/deletions. This suggests a complex microcolinearity among wheat, rice, and barley in this QTL region.     

Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples

AIMS: The aim of the study was to develop a sensitive detection method of Fusarium culmorum contamination in cereal samples. METHODS AND RESULTS: A nested-PCR method using a single closed tube was developed for the detection of F. culmorum in infected cereal samples. The concentrations of the first primer pair was diluted 10,000 times compared to the concentration used for the second primer pair. Differing annealing temperatures allowed both first and second polymerase chain reaction (PCR) reactions to be performed subsequently in the same closed tube. The detection limit was 5-50 fg of purified target DNA and allowed the detection of 1% infected seeds of wheat in a mixture with uninfected grains. CONCLUSIONS: F. culmorum can be specifically detected in cereal samples by the highly sensitive method of nested-PCR in a single closed tube. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the detection of F. culmorum in cereal samples that is approximately 100 times more sensitive than previous PCR methods, involves low risk of cross contaminations between samples, low costs and reduced hands-on time as compared to standard nested-PCR protocols.     

3B染色体短臂小麦赤霉病抗性主效QTL的分析

采用区间作图和复合区间作图方法对重组自交系群体宁894037／Alondram、望水白／Alondra和苏麦3号／A1ondra进行了抗赤霉病QTL分析,结果表明,用在田间和温室的赤霉病抗性鉴定资料,在3个赤霉病抗源宁894037、望水白和苏麦3号的3B染色体短臂上均检测到主效QTL的存在。宁894037主效QTL位于标记BARCl33与Xgwm493之间的5．0cM的区间内,最高可解释42．8％的赤霉病抗性;望水白的主效QTL位于标记BARCl47与Xgwm493之间11．5cM的区间内,最高可解释15．1％的赤霉病抗性;苏麦3号的主效QTL位于Xgwm533a与Xgwm493之间13．0cM的区间内,最高可解释10．6％的赤霉病抗性。与赤霉病抗性主效QTL紧密连锁的标记均为SSR标记,可直接用于分子辅助育种。