Cereal Diseases Research Articles

Using mushroom-forming fungi in preventing and reducing mycotoxins in cereal products

Background: Biological control for cereal diseases caused by mycotoxigenic fungi is part of the alternative methods to be developed to secure food and feed production, recognizing the negative effects these fungi have on crop yield and their potential production of mycotoxins.
 Objective: Update recent knowledge about the potential of mushroom-forming fungi as biological control agents of mycotoxigenic fungi species, acting as antagonists on crop debris, as source of active extracts with antifungal and/or antimycotoxin properties and as detoxifying agents in solid-state fermentation processes of contaminated cereals.
 Methods: A literature review of the published works was carried out, the main topics were analyzed, as well as the species with the greatest potential of biological control agents of mycotoxingenic fungi.
 Results and conclusions: Mushroom forming fungi could be used as antagonists on crop debris or grains, as sources of active extracts with antifungal and/or antimycotoxin properties, and as detoxifying agents. An integration of these three potentials gives rise to an environment friendly process for production of both safe grains and edible mushrooms.

Fusarium culmorum ile Enfekte Edilen Arpa Çeşitlerinde WRKY Transkripsiyon Faktörlerinin Analizi

One of the most critical problems of cereal breeding is Fusarium crown rot disease caused by various Fusarium species. Fusarium culmorum is one of the predominant pathogen in Turkey and causes serious product losses. In this study, the early response of barley cultivars upon F. culmorum infection were analyzed by disease severity and gene expression patterns of WRKY transcription factors. In that context, firstly, disease severities of 9 barley cultivars (Hordeum vulgare L. cvs. Epona, Escadre, Gazda, Oliver, Avcı 2002, Burakbey, Tarm 92, Manava, and Ramata) infected with F. culmorum were determined with disease index percentages. After 7 days of infection, Epona was more sensitive than the other cultivars while the lowest disease index was seen in Gazda. Total RNA extractions were performed at 72 hai from the root tissues of Epona and Gazda. Expression analysis of HvWRKY6, HvWRKY9, HvWRKY24, HvWRKY25, HvWRKY33, HvWRKY34, HvWRKY42, and HvWRKY46 genes were conducted by qPCR. As a result of pathogen stress, it was observed that the transcript levels of HvWRKY33 was significantly upregulated in both cultivars. HvWRKY6, HvWRKY34 and HvWRKY46 genes were increased in Epona while upregulation of HvWRKY25 and HvWRKY34 genes were detected in Gazda. No significant decreases were detected in any cultivars. This study is important in terms of providing an association between WRKY genes and pathogen stress response.

Breeding for Fusarium head blight resistance in wheat—Progress and challenges

AbstractFusarium head blight is among the most extensively studied fungal diseases of wheat and other small grain cereals due to its impact on yield and quality, but particularly due to its potential to produce mycotoxins, which are harmful to humans and animals. Since our last comprehensive review on QTL mapping and marker‐assisted selection for FHB resistance in wheat in 2009, numerous studies have been conducted to identify, validate or fine‐map resistance QTL. The main aim of this review is to update and summarize findings on FHB resistance breeding of wheat published during the last decade. Furthermore, we compiled a user‐friendly table listing FHB resistance QTL data providing a valuable resource for further FHB resistance research. The role of morphological and phenological traits on FHB resistance and possible consequences for resistance breeding are discussed. This review concentrates current knowledge on breeding for FHB resistance and suggests strategies to enhance resistance by deploying molecular breeding methods, including marker‐assisted and genomic selection.

Use of Competitive Filamentous Fungi as an Alternative Approach for Mycotoxin Risk Reduction in Staple Cereals: State of Art and Future Perspectives.

Among plant fungal diseases, those affecting cereals represent a huge problem in terms of food security and safety. Cereals, such as maize and wheat, are very often targets of mycotoxigenic fungi. The limited availability of chemical plant protection products and physical methods to control mycotoxigenic fungi and to reduce food and feed mycotoxin contamination fosters alternative approaches, such as the use of beneficial fungi as an active ingredient of biological control products. Competitive interactions, including both exploitation and interference competition, between pathogenic and beneficial fungi, are generally recognized as mechanisms to control plant pathogens populations and to manage plant diseases. In the present review, two examples concerning the use of competitive beneficial filamentous fungi for the management of cereal diseases are discussed. The authors retrace the history of the well-established use of non-aflatoxigenic isolates of Aspergillus flavus to prevent aflatoxin contamination in maize and give an overview of the potential use of competitive beneficial filamentous fungi to manage Fusarium Head Blight on wheat and mitigate fusaria toxin contamination. Although important steps have been made towards the development of microorganisms as active ingredients of plant protection products, a reasoned revision of the registration rules is needed to significantly reduce the chemical based plant protection products in agriculture.

Trichothecenes in Cereal Grains - An Update.

Trichothecenes are sesquiterpenoid mycotoxins produced by fungi from the order Hypocreales, including members of the Fusarium genus that infect cereal grain crops. Different trichothecene-producing Fusarium species and strains have different trichothecene chemotypes belonging to the Type A and B class. These fungi cause a disease of small grain cereals, called Fusarium head blight, and their toxins contaminate host tissues. As potent inhibitors of eukaryotic protein synthesis, trichothecenes pose a health risk to human and animal consumers of infected cereal grains. In 2009, Foroud and Eudes published a review of trichothecenes in cereal grains for human consumption. As an update to this review, the work herein provides a comprehensive and multi-disciplinary review of the Fusarium trichothecenes covering topics in chemistry and biochemistry, pathogen biology, trichothecene toxicity, molecular mechanisms of resistance or detoxification, genetics of resistance and breeding strategies to reduce their contamination of wheat and barley.

Use of Botanicals to Suppress Different Stages of the Life Cycle of Fusarium graminearum.

Fusarium head blight (FHB) is one of the most important cereal diseases worldwide, causing yield losses and contamination of harvested products with mycotoxins. Fusarium graminearum is one of the most common FHB-causing species in wheat and barley cropping systems. We assessed the ability of different botanical extracts to suppress essential stages of the fungal life cycle using three strains of F. graminearum (FG0410, FG2113, and FG1145). The botanicals included aqueous extracts from white mustard (Sinapis alba) seed flour (Pure Yellow Mustard [PYM] and Tillecur [Ti]) as well as milled Chinese galls (CG). At 2% concentration (wt/vol), PYM and Ti completely inhibited growth of mycelium of all F. graminearum strains whereas, at 1%, CG reduced the growth by 65 to 83%, depending on the strain. While PYM and Ti reduced the germination of both conidia and ascospores at 2% (wt/vol), CG was only effective in reducing conidia germination. Perithecia formation of FG0410 but not FG2113 was suppressed by all botanicals. Moreover, application of botanicals on mature perithecia led to a two- to fourfold reduction in discharge of ascospores. Using liquid chromatography (LC) with diode array detection, we quantified the principal glucosinolate component sinalbin of PYM and Ti. LC time-of-flight mass spectrometry was used to demonstrate that the bioactive matrix of CG contains different gallotannins as well as gallic and tannic acids. Possible antifungal mechanisms of the botanical matrices are discussed. The results of this study are promising and suggest that PYM, Ti, and CG should be explored further for efficacy at managing FHB.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

T64SHARED GENETIC ETIOLOGY BETWEEN OBSESSIVE-COMPULSIVE DISORDER, OBSESSIVE-COMPULSIVE SYMPTOMS IN THE POPULATION, AND INSULIN SIGNALING

Fusarium head blight (FHB) is one of the most prevalent diseases of wheat and other small grain cereals that is predominantly caused by the fungal pathogen Fusarium graminearum. Extraction of total proteins were from tissues of A061-3 and A061-4 plants. Three biological replicates were carried out for each line at four time points. Samples were performed using iTRAQ (Isobaric tags for relative and absolute quantification). This data is being made available to increase the understanding of FHB resistance proteomics. The data from this study are related to the research article “Isobaric tags for relative and absolute quantification-based proteomic analysis of defense responses triggered by the fungal pathogen Fusarium graminearum in wheat” [1].

In vitro studies of 3-O-Methylquercetin against phytopathogenic fungi of major cereals

Maize, wheat, and rice are major cereal crops in the world, which provide more caloric energy than any other type of cereal class. In recent years, the yield of these cereal grains has stagnated due to a myriad of constraints, including infestation by pests and pathogens. Plant pathogens, especially agricultural phytopathogenic fungi, may be resistant to the available chemical fungicides which may have detrimental effects and are expensive. This study investigated whether natural products isolated from Linzia glabra Steetz of family Asteraceae could be used as alternative antifungals for control of cereal diseases caused by agricultural fungi. A single pure fraction was separated and identified from L. glabra methanolic flower crude extract using high performance liquid chromatography and high resolution mass spectrometry methods. The pure fraction isolated was tested for inhibition of spore germination in vitro against Fusarium graminearum, Fusarium oxysporum, Fusarium verticillioides, Zymoseptoria tritici, Ustilago maydis, and Pyricularia oryzae. The fraction was identified as a flavonol, 3-O-Methylquercetin using high resolution mass spectrometry. Among the fungi tested, spores of Z. tritici were the most susceptible to 3-O-Methylquercetin (44% spore inhibition), followed by F. oxysporum (33% inhibition) at 27 μg/mL. Azoxystrobin, a standard commercialized fungicide, gave a complete inhibition of 100% against Z. tritici and 58% inhibition against F. oxysporum at 5 μg/mL. L. glabra contains flavonols with antifungal activity, but the modest and weak antifungal activity of 3-O-Methylquercetin compared to a standard such as azoxystrobin demonstrates the challenge to produce highly potent natural product fungicides. There is need for continued search for antifungals with new modes of action as solutions to challenges in agricultural production.

Control of powdery mildew (Blumeria graminis spp.) in cereals by Serenade®ASO (Bacillus amyloliquefaciens (former subtilis) strain QST 713)

Powdery mildew (Blumeria graminis f.sp. tritici, f.sp. hordei, f.sp. avenae) is an important disease in cereal crops causing significant yield reductions, if not effectively controlled. The biofungicide Bacillus amyloliquefaciens (former subtilis) strain QST 713 suspension concentrate (Serenade®ASO) was investigated for its potential as a control agent against powdery mildew in winter wheat, spring barley, oats and triticale. Serenade®ASO reduced the severity of powdery mildew, providing moderate control in the range of 20–65%. The efficacy was highest at the earlier assessments and less pronounced at later assessments. With a few exceptions, no clear dose-response was seen between using 1, 2, 4, 6 or 8 L/ha Serenade®ASO applied four times at 8–10-day intervals. The efficacy was more variable compared with chemical references. Yield responses were positive following treatments. However, the yield increase by Serenade®ASO was insignificant and significantly inferior to that of prothioconazole. In a few cases, the use of Serenade®ASO in combination with the adjuvant Silwet®Gold improved the efficacy, but the results were inconsistent. The preventive and curative application of Serenade®ASO was tested at three dose rates in an artificially inoculated wheat and barley greenhouse pot trial. These trials confirmed the lack of a clear dose-response but confirmed that timing has a major impact on control, with the best control obtained when application took place on the day of inoculation in wheat and 0–2 days after inoculation in barley. This study revealed that powdery mildew challenges Serenade®ASO as a stand-alone control measure. The results show that further research is needed for the promotion of biofungicides as part of an integrated disease management approach in cereal diseases.

Comparing forward to inverse frameworks to obtain hip joint kinematics during a squat task

Fusarium head blight (FHB) is one of the most prevalent diseases of wheat and other small grain cereals that is predominantly caused by the fungal pathogen Fusarium graminearum. Extraction of total proteins were from tissues of A061-3 and A061-4 plants. Three biological replicates were carried out for each line at four time points. Samples were performed using iTRAQ (Isobaric tags for relative and absolute quantification). This data is being made available to increase the understanding of FHB resistance proteomics. The data from this study are related to the research article “Isobaric tags for relative and absolute quantification-based proteomic analysis of defense responses triggered by the fungal pathogen Fusarium graminearum in wheat” [1].

Deletion of the benzoxazinoid detoxification gene NAT1 in Fusarium graminearum reduces deoxynivalenol in spring wheat.

Benzoxazinoid (Bx) metabolites produced by wheat and other members of the Poaceae have activity against Fusarium sp. that cause cereal diseases including Fusarium head blight (FHB) on wheat and barley. Certain Bx metabolites can be detoxified by Fusarium sp. with the arylamine N-acetyltransferase NAT1. Investigation of this pathway may reveal strategies for increasing FHB resistance, such as selection for higher levels of Bx metabolites within existing germplasm and/or engineering fungal susceptibility via host induced silencing of NAT1. We assessed the reactions of fifteen wheat cultivars or breeding lines adapted to the Northwestern United States to infection with F. graminearum Δnat1 mutants that should be sensitive to Bx metabolites. Significant differences were noted in disease severity and deoxynivalenol (DON) among the cultivars 21 d after inoculation with either mutant or wildtype (PH1) strains. Mutant vs. wildtype strains did not result in significant variation for infection severity (as measured by % infected florets), but inoculation with Δnat1 mutants vs. wildtype resulted in significantly lower DON concentrations in mature kernels (p < 0.0001). Of the cultivars tested, HRS3419 was the most resistant cultivar to PH1 (severity = 62%, DON = 45 ppm) and Δnat1 mutants (severity = 61%, DON = 30 ppm). The cultivar most susceptible to infection was Kelse with PH1 (severity = 100%, DON = 292 ppm) and Δnat1 mutants (severity = 100%, DON = 158 ppm). We hypothesized that sub-lethal Bx metabolite levels may suppress DON production in F. graminearum Δnat1 mutants. In vitro assays of Bx metabolites BOA, MBOA, and DIMBOA at 30 μM did not affect growth, but did reduce DON production by Δnat1 and PH1. Although the levels of Bx metabolites are likely too low in the wheat cultivars we tested to suppress FHB, higher levels of Bx metabolites may contribute towards reductions in DON and FHB.

Biocontrol of Cereal Crop Diseases Using Streptomycetes.

A growing world population and an increasing demand for greater food production requires that crop losses caused by pests and diseases are dramatically reduced. Concurrently, sustainability targets mean that alternatives to chemical pesticides are becoming increasingly desirable. Bacteria in the plant root microbiome can protect their plant host against pests and pathogenic infection. In particular, Streptomyces species are well-known to produce a range of secondary metabolites that can inhibit the growth of phytopathogens. Streptomyces are abundant in soils and are also enriched in the root microbiomes of many different plant species, including those grown as economically and nutritionally valuable cereal crops. In this review we discuss the potential of Streptomyces to protect against some of the most damaging cereal crop diseases, particularly those caused by fungal pathogens. We also explore factors that may improve the efficacy of these strains as biocontrol agents in situ, as well as the possibility of exploiting plant mechanisms, such as root exudation, that enable the recruitment of microbial species from the soil to the root microbiome. We argue that a greater understanding of these mechanisms may enable the development of protective plant root microbiomes with a greater abundance of beneficial bacteria, such as Streptomyces species.

Biofabrication of Zinc Oxide Nanoparticles With Syzygium aromaticum Flower Buds Extract and Finding Its Novel Application in Controlling the Growth and Mycotoxins of Fusarium graminearum

Fusarium graminearum is a leading plant pathogen that causes Fusarium head blight, stalk rot, and Gibberella ear rot diseases in cereals and posing the immense threat to the microbiological safety of the food. Herein, we report the green synthesis of zinc oxide nanoparticles from Syzygium aromaticum (SaZnO NPs) flower bud extract by combustion method and investigated their application for controlling of growth and mycotoxins of F. graminearum. Formation of SaZnO NPs was confirmed by spectroscopic methods. The electron microscopic (SEM and TEM) analysis revealed the formation of triangular and hexagonal shaped SaZnO NPs with size range 30–40 nm. The synthesized SaZnO NPs reduced the growth and production of deoxynivalenol and zearalenone of F. graminearum in broth culture. Further analysis revealed that treatment of mycelia with SaZnO NPs resulted in the accumulation of ROS in the dose-dependent manner. Also, SaZnO NPs treatment enhanced lipid peroxidation, depleted ergosterol content, and caused detrimental damage to the membrane integrity of fungi. Moreover, SEM observations revealed that the presence of diverged micro-morphology (wrinkled, rough and shrank surface) in the macroconidia treated with SaZnO NPs. Taken together, SaZnO NPs may find a potential application in agriculture and food industries due to their potent antifungal activity.

Phytotoxins produced by pathogenic fungi of agrarian plants

This review, covering the literature from 1965 to present (2018), treats of phytotoxins produced by fungi responsible of heavy diseases of important agrarian crops, including legumes, cereals, fruit trees, vegetables and crops for oil seed production. The symptoms induced on the infected plants are reported and the heavy economical losses caused by the diseases were also described. The chemical characterization and biological activity of the fungal phytotoxins, belonging to the different classes of natural compounds, is reported. In some cases, the probable role played by phytotoxins in the induction of plant disease symptoms is described as well as the results of structure–activity relationship and mode of action studies. The potential application in agriculture and in medicine for some of them is also discussed highlighting the increase in safety and the practical advantages.

Using Yield Response Curves to Measure Variation in the Tolerance and Resistance of Wheat Cultivars to Fusarium Crown Rot.

The disease crown rot, caused predominantly by the fungal pathogen Fusarium pseudograminearum, is a major disease of winter cereals in many regions of the world, including Australia. A methodology is proposed, using response curves, to robustly estimate the relationship between grain yield and increasing crown rot pathogen burdens. Using data from a field experiment conducted in northern New South Wales, Australia in 2016, response curves were derived for five commercial wheat cultivars exposed to six increasing rates of crown rot inoculum, where the rates served to establish a range of crown rot pathogen burdens. In this way, the response curve methodology is fundamentally different from alternate approaches that rely on genetic or environmental variation to establish a range in pathogen burdens over which yield loss relationships are estimated. By manipulating only the rates of crown rot inoculum and, thus, pathogen burden directly, the number of additional confounding factors and interactions are minimized, enabling the robust estimation of the rate of change in yield due to increasing crown rot pathogen burdens for each cultivar. The methodology revealed variation in the rate of change in yield between cultivars, along with the extent of crown rot symptoms expressed by the cultivars. Variation in the rate of change in yield between cultivars provides definitive evidence of differences in the tolerance of commercial Australian wheat cultivars to crown rot caused by F. pseudograminearum, while variation in the extent of crown rot symptoms signifies differences in the resistance of the cultivars to this disease. The response curve methodology also revealed variation in how the different mechanisms of tolerance and resistance act to limit yield losses due to crown rot for different cultivars.

Asymptomatic weeds are frequently colonised by pathogenic species of Fusarium in cereal‐based crop rotations

SummarySeveral Fusarium species cause harmful cereal diseases, such as fusarium head blight and crown rot, which, during pathogenesis, may result in significant grain yield and quality losses. Several species of agricultural weed are believed to be alternative and reservoir hosts for Fusarium spp.; however, studies have not comprehensively evaluated those weed species in cropping systems that may harbour these fungi. The objective of this study was to determine weed species in cereal‐based crop rotations that are asymptomatically colonised by Fusarium spp. We sampled all species of weed present in fields that were managed under six different crop sequences in 2015 and 2016. The study yielded 2326 single‐spore isolates of Fusarium spp. derived from various organs of asymptomatic weeds. Isolates were identified morphologically and then confirmed using PCR with species‐specific primers and/or sequencing of tef1α gene fragments. Isolates of nine Fusarium spp. were obtained from 689 of the 744 individuals collected that represented 56 weed species. Each weed species harboured at least one species of Fusarium, and &gt;80% were colonised by 3–9 Fusarium spp. In total, we identified 27 dicotyledonous weed species that were previously undocumented as Fusarium hosts and 251 new weed × Fusarium species combinations were revealed. Consequently, there is a greater risk of negative Fusarium impacts on cereal crops than was previously thought. We suggest effective weed management and inversion soil tillage may help mitigate these impacts.

First Scientific Report of Pyricularia oryzae Causing Gray Leaf Spot Disease on Perennial Ryegrass (Lolium perenne) in France

The fungus Pyricularia oryzae causes blast diseases of rice, wheat, and other cereals (e.g., millets). In addition, P. oryzae is also responsible for the gray leaf spot disease of ryegrass, a major problem for golf courses in the United States (Farman 2002). A host jump from perennial ryegrass (Lolium perenne) onto wheat was recently reported (Farman et al. 2017) and is suspected to be at the origin of strains causing severe epidemics on wheat in South America (Gladieux et al. 2017; Inoue et al. 2017). In 2016, disease on lawns of several football stadiums in France and Switzerland was reported in the nonscientific press. In 2017, P. oryzae was said to be the causal agent of this disease in the stadium of Geneva, Switzerland (Visentini 2017). In September 2017, perennial ryegrass plants presenting symptoms of disease were collected in a stadium (stade de la Mosson) in Montpellier, France. After 24- to 48-h incubation on moist filter paper (27°C; 12-h light/dark), diseased symptomatic samples produced spores morphologically similar to Pyricularia sp., and four monosporic strains were isolated from leaves and roots and were grown on rice flour agar (RFA) medium. Four ryegrass varieties were grown for 2 weeks in 7 × 7-cm pots filled with peat soil (Neuhaus S, Klasmann-Deilmann, Bourgoin Jallieu, France) in the greenhouse. After inoculation, plants were kept 1 week in a growth chamber (12-h light at 26°C/12-h dark at 21°C; humidity maintained below 70%). Spores were collected from 1-week-old colonies grown on RFA medium (27°C, 12-h light/dark) and used to prepare a spore suspension containing 5 × 10⁴ spores/ml in 0.5% gelatin. Groups of four pots, each containing a different variety, were inoculated by spraying a 10-ml suspension representing a single strain. The negative control consisted of plants sprayed with a solution of 0.5% gelatin and managed as the other treatments before and after inoculation. Three independent experiments were conducted. All four strains caused symptoms similar to those observed in the original samples and associated with gray leaf spot. Lesions collected from inoculated plants exhibiting symptoms and incubated 24 h in a Petri dish on moist filter paper (27°C; 12-h light/dark) produced typical spores of P. oryzae. The DNA of two strains (FR1067 and FR1069) was extracted from 5-day actively growing mycelium in liquid culture (2 yeast extract glucose [2YEG] medium: 10 g/liter of glucose and 2 g/liter of yeast extract), and the genome sequence was produced following published protocols (Gladieux et al. 2017). A phylogenetic analysis was conducted using additional genome sequences of P. oryzae strains from different hosts previously used by Gladieux et al. (2017): 10 from Setaria spp., 23 from Oryza sativa, 3 from Hordeum vulgare, 8 from Eleusine spp., 2 from Bromus spp., 1 from Avena sp., 1 from Eragrostis sp., 2 from Brachiaria spp., 1 from Festuca sp., 22 from Triticum spp., and 12 from Lolium spp. The two French strains from perennial ryegrass were highly similar and clustered in a clade gathering nine out of the 10 strains from Lolium. Altogether these results show that the epidemic of gray leaf spot on ryegrass in Montpellier’s football stadium was caused by P. oryzae. To our knowledge, this is the first scientific report of P. oryzae on ryegrass in France. We recommend monitoring the presence of this species on ryegrass because it could be the source of epidemics on wheat, a major crop in France and throughout Europe.

Non-canonical fungal G-protein coupled receptors promote Fusarium head blight on wheat.

Fusarium Head Blight (FHB) is the number one floral disease of cereals and poses a serious health hazard by contaminating grain with the harmful mycotoxin deoxynivalenol (DON). Fungi adapt to fluctuations in their environment, coordinating development and metabolism accordingly. G-protein coupled receptors (GPCRs) communicate changes in the environment to intracellular G-proteins that direct the appropriate biological response, suggesting that fungal GPCR signalling may be key to virulence. Here we describe the expansion of non-classical GPCRs in the FHB causing pathogen, Fusarium graminearum, and show that class X receptors are highly expressed during wheat infection. We identify class X receptors that are required for FHB disease on wheat, and show that the absence of a GPCR can cause an enhanced host response that restricts the progression of infection. Specific receptor sub-domains are required for virulence. These non-classical receptors physically interact with intracellular G-proteins and are therefore bona fide GPCRs. Disrupting a class X receptor is shown to dysregulate the transcriptional coordination of virulence traits during infection. This amounts to enhanced wheat defensive responses, including chitinase and plant cell wall biosynthesis, resulting in apoplastic and vascular occlusions that impede infection. Our results show that GPCR signalling is important to FHB disease establishment.

Phylogeny of Canadian ergot fungi and a detection assay by real-time polymerase chain reaction

ABSTRACTThe ergot disease of cereals has become increasingly important in agricultural areas of Canada since 1999. Generally, this disease is considered to be caused by Claviceps purpurea, but the taxonomy of Claviceps from these areas has not been well studied. The objectives of this study were (i) to determine the phylogenetic lineages (phylogenetic species) present in agricultural areas of Canada and (ii) to develop a molecular assay that can separate the lineages on crops from other lineages. Genetic diversity of Claviceps collected from agriculture areas in Canada were investigated using multilocus sequence typing. The loci sequenced include nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), partial fragments of translation elongation factor 1-α (TEF1), RNA polymerase II second largest subunit (RPB2), β-tubulin (tubB), and two ergot alkaloid synthesis genes (easA, easE). Based on individual locus and concatenated alignments, phylogenetic analyses revealed seven lineages within the premolecular concept of C. purpurea, of which five corresponded with undescribed species (G2b and G4–7). Although lineages G2–7 had narrow host ranges, lineage G1 (= C. purpurea s.s.) had a broad host range that overlapped with other lineages. A molecular diagnostic quantitative polymerase chain reaction (qPCR) assay was developed and validated with 185 samples from a wide range of host plants and geographic origins, including 10 phylogenetic species in C. sect. Claviceps, 8 in C. sect. Pusillae, 1 in C. sect. Citrinae, and 1–2 species from Alternaria, Fusarium, and Penicillium. The assay can detect lineage G1 at a concentration of 7.5 pg/μL and distinguish it from other Claviceps species and lineages. This facilitates disease management by detecting the inocula from nonagriculture host plants.

The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA.

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.