Covariation of Ergot Severity and Alkaloid Content Measured by HPLC and One ELISA Method in Inoculated Winter Rye across Three Isolates and Three European Countries.

Ergot caused by Claviceps purpurea is a problem for food and feed security in rye due to the occurrence of toxic ergot alkaloids (EAs). For grain elevators and breeders, a quick, easy-to-handle, and cheap screening assay would have a high economic impact. The study was performed to reveal (1) the covariation of ergot severity (= percentage of sclerotia in harvested grain) and the content of 12 EAs determined by high performance liquid chromatography (HPLC) and (2) the covariation between these traits and results of one commercial enzyme linked immunosorbent assays (ELISA). In total, 372 winter rye samples consisting of a diverse set of genotypes, locations from Germany, Austria, and Poland over two years, and three isolates were analyzed. Ergocornine and α-ergocryptine were detected as major EAs. Ergocristinine occurred as a minor component. Claviceps isolates from different countries showed a similar EA spectrum, but different quantities of individual EAs. A moderate, positive covariation between ergot severity and EA content determined by HPLC was observed across two years (r = 0.53, p < 0.01), but large deviation from the regression was detected. ELISA values did neither correlate with the HPLC results nor with ergot severity. In conclusion, a reliable prediction of the EA content based on ergot severity is, at present, not possible.

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

The 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), ß-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.

Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR.

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = -0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = -0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = -0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination.

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.

Ergot species of the Claviceps purpurea group from South Africa.

Results of a survey and study of the Claviceps purpurea group of species in South Africa are being presented and five new species are described. Morphological descriptions are based on the anamorphs and four nuclear genetic loci. Claviceps fimbristylidis sp. nov. on Fimbristylis complanata was discovered wide-spread across five provinces of the country associated with water and represents the fourth Claviceps species recorded from the Cyperaceae. Claviceps monticola sp. nov. is described from Brachypodium flexum growing in mountain forests in Mpumalanga Province, as well as the northern Drakensberg southwards into the Eastern Cape Province. Claviceps pazoutovae sp. nov. is recorded from Stipa dregeana var. dregeana and Ehrharta erecta var. erecta, also associated with these mountain ranges. Claviceps macroura sp. nov. is recorded from Cenchrus macrourus from the Eastern Cape and Claviceps capensis sp. nov. from Ehrharta villosa var. villosa is recorded from the Western Cape Province. Claviceps cyperi, only recorded from South Africa is included in the study. Ergot alkaloid profiles of all species are provided and showed similarity to C. purpurea. Only C. cyperi and in lesser degree C. capensis, C. macroura, and C. pazoutovae produced ergot alkaloids in clinically significant amounts. Several reported species infect invasive grass species, native to South Africa, and thus represent potentially invasive species.

Contamination with ergot bodies (Claviceps purpurea sensu lato) of two horse pastures in Northern Germany.

Because the occurrence of Claviceps in European pastures may have been overlooked to cause serious health problem for grazing animals, we documented the degree of Claviceps contamination in two horse pastures and estimated whether the horses could have ingested a critical quantity of alkaloids. We counted the Claviceps sclerotia and determined alkaloid levels using high performance liquid chromatography with fluorescence detection. Depending on the location, the number of sclerotia varied from 0.09 to 0.19 per square meter (central area) and from 0.23 to 55.8 per square meter (border strips). Alkaloid levels in individual sclerotia also varied in different genera of grasses, ranging from 0.98 ± 0.17 µg/kg in Agrostis sp. to 25.82 ± 9.73 µg/kg in Dactylis sp., equivalent to 0.98 µg/kg and 7.26 mg/kg. Sclerotia from Dactylis contained high levels of ergosine (0.209 % ± 0.100 %) and ergocristine (0.374 % ± 0.070 %). Depending on the localization in pastures, alkaloid levels in forage (dry matter, DM) ranged from 16.1 to 45.4 µg/kg in central areas and from 23.9 to 722 µg/kg in border strips. The amount of alkaloids that a horse could have ingested depended on its daily DM uptake, which was higher in the central areas (5.85 kg/day) than in the border strips (2.73 or 0.78 kg/day). In the central areas, this amount of alkaloids ranged from 94.2 to 265.9 µg/day; and in the border strips, from 65.3 (in 2.73 kg DM/day) to as much as 563.8 µg/day (in 0.78 kg DM/day). All these amounts are higher than the European averages for alkaloids ingested by horses via feedstuffs.

Evaluation of a triplex real-time PCR system to detect the plant-pathogenic molds Alternaria spp., Fusarium spp. and C. purpurea.

This article describes the development of a triplex real-time PCR system for the simultaneous detection of three major plant-pathogenic mold genera (Alternaria spp., Fusarium spp. and the species Claviceps purpurea). The designed genus-specific primer-probe systems were validated for sensitivity, specificity and amplification in the presence of background DNA.

Occurrence of Ergot and Ergot Alkaloids in Western Canadian Wheat and Other Cereals.

A new method was developed to analyze 10 ergot alkaloids in cereal grains. Analytes included both "ine" and "inine" type ergot alkaloids. Validation of the method showed it performed with good accuracy and precision and that minor enhancement due to matrix effects was present during LC-MS/MS analysis, but was mitigated by use of an internal standard. The method was used to survey durum and wheat harvested in 2011, a year in which ergot infection was particularly widespread in western Canada. A strong linear relationship between the concentration of ergot alkaloids and the presence of ergot sclerotia was observed. In addition, shipments of cereals from 2010-2012 were also monitored for ergot alkaloids. Concentrations of total ergot alkaloids in shipments were lower than observed in harvest samples, and averaged from 0.065 mg/kg in barley to 1.14 mg/kg in rye. In shipments, the concentration of ergot alkaloids was significantly lower in wheat of higher grades.

Delimitation of cryptic species inside Claviceps purpurea.

Claviceps purpurea is an ovarian parasite infecting grasses (Poaceae) including cereals and forage plants. This fungus produces toxic alkaloids and consumption of contaminated grains can cause ergotism in humans and other mammals. Recent molecular genetics studies have indicated that it included three cryptic species (G1, G2, G3). In this study, reproductive isolation amongst these groups and among material from Phragmites and Molinia was tested using gene flow statistics for five polymorphic loci, and to support these data, phylogenetic affiliations based on gene trees and a multigene phylogeny were used. The four recognized species are characterized based on morphology and host spectrum and formal taxonomic names are proposed. Claviceps purpurea sensu stricto (G1 group) represents a typical rye ergot, but infects various other grasses. Typical hosts of Claviceps humidiphila (new name for G2 species), like Phalaris arundinacea, belong to grasses preferring humid locations. Claviceps spartinae (G3) is specific to chloridoid grasses from salt barches. The material from Phragmites and Molinia can be authenticated with the species Claviceps microcephala for which the new name Claviceps arundinis is proposed here. The divergence time between species was estimated and the tools for species identification are discussed.

Relationship between lutein and mycotoxin content in durum wheat.

Levels of lutein and a number of mycotoxins were determined in seven varieties of durum wheat (Triticum durum) and two varieties of common wheat (Triticum aestivum) in order to explore possible relationships amongst these components. Durum wheat cultivars always showed both higher lutein and mycotoxin contents than common wheat cultivars. The mycotoxins detected in both common and durum wheat cultivars were produced by the genera Fusarium, Claviceps, Alternaria and Aspergillus. Fusarium was the major producer of mycotoxins (26 mycotoxins) followed by Claviceps (14 mycotoxins), which was present only in some cultivars such as Chevalier (common wheat), Lupidur and Selyemdur (both durum wheat), Alternaria (six mycotoxins) and Aspergillus (three mycotoxins). Positive correlations between the levels of lutein and mycotoxins in durum wheat cultivars were found for the following mycotoxins: deoxynivalenol (DON), its derivative DON-3-glucoside, moniliformin, culmorin and its derivatives (5-hydroxyculmorin and 15-hydroxyculmorin).

Human and cattle ergotism since 1900: symptoms, outbreaks, and regulations.

Ergotism in humans and cattle are caused by several species of Claviceps that infect rye and other cereal grains. Symptoms in humans vary greatly and are generally classified as convulsive, gangrenous, or gastrointestinal (enteric). Cattle are particularly susceptible to both gangrenous and hyperthermic ergotism (also called summer syndrome). The prevalence of ergotism has decreased as knowledge of the fungus has increased, mainly through implementation of regulations and advances in milling procedures. However, outbreaks in humans have recently occurred in lower socioeconomic populations of Ethiopia (1977 and 2001) and India (1975) with devastating results. Prominent outbreaks in cattle have occurred in Australia (1987), the United States (1996), South Africa (1996-1997), and Brazil (1999) and, as opposed to human cases, they do not appear to be bound by economic development. This review provides a detailed summary of all major ergot epidemics since 1900 in both humans and cattle. Special attention is devoted to the ergotism symptoms and to the regulations surrounding the control of ergot in the food supply.

Online detection and quantification of ergot bodies in cereals using near infrared hyperspectral imaging.

The occurrence of ergot bodies (sclerotia of Claviceps purpurea) in cereals presents a high toxicity risk for animals and humans due to the alkaloid content. To reduce this risk, the European Commission fixed an ergot concentration limit of 0.1% in all feedstuffs containing unground cereals, and a limit of 0.05% in 'intervention' cereals destined for humans. This study sought to develop a procedure based on near infrared hyperspectral imaging and multivariate image analysis to detect and quantify ergot contamination in cereals. Hyperspectral images were collected using an NIR hyperspectral line scan combined with a conveyor belt. All images consisted of lines of 320 pixels that were acquired at 209 wavelength channels (1100-2400 nm). To test the procedure, several wheat samples with different levels of ergot contamination were prepared. The results showed a correlation higher than 0.99 between the predicted values obtained using chemometric tools such as partial least squares discriminant analysis or support vector machine and the reference values. For a wheat sample with a level of ergot contamination as low as 0.01 %, it was possible to identify groups of pixels detected as ergot to conclude that the sample was contaminated. In addition, no false positives were obtained with non-contaminated samples. The limit of detection was found to be 145 mg/kg and the limit of quantification 341 mg/kg. The reproducibility tests of the measurements performed over several weeks showed that the results were always within the limits allowed. Additional studies were done to optimise the parameters in terms of number of samples analysed per unit of time or conveyor belt speed. It was shown that ergot can be detected using a speed of 1-100 mm/s and that a sample of 250 g can be analysed in 1 min.

[Isolation, identification and insecticidal activity of endophyte from Achnatherum inebrians].

OBJECTIVE: To study endophyte species of Achnatherum inebrians and to screen strains with insecticidal activity against cotton insect. METHODS: We isolated endophytic from roots,stems,leaves and seeds of health A. inebrians by grinding separation method and identified by a dual approach of morphological and physiological observation and 16S rDNA gene (for bacteria) and ITS sequence (for fungi) based molecular identification. Then,those endophytes were inoculated into liquid media for fermentation and the crude extracts were used to test insecticidal activities by slide disc immersion and nebulization methods. RESULTS: We isolated bacteria species classified into 8 genera of Bacillus, Streptomyces, Corynebacterium, Phyllobacterium, sphingomonnas, Paenibacillus, Pseudomonas, Acinetobacter and 2 fungi of Claviceps purpure and Claviceps Chaetomium. Of them, the strain Streptomyces rochei (GA) and Claviceps purpurea (PF-2) had more than 85% of mortality to cotton aphis. CONCLUSION: Two strains of PF-2 and GA associated within the A. inebrians had significant insecticidal activity to cotton aphis (Aphis gossypii), which may provide a new biological resource to explore new microbial insecticide.

Multigene analysis suggests ecological speciation in the fungal pathogen Claviceps purpurea.

Claviceps purpurea is an important pathogen of grasses and source of novel chemical compounds. Three groups within this species (G1, G2 and G3) have been recognized based on habitat association, sclerotia and conidia morphology, as well as alkaloid production. These groups have further been supported by Random Amplification of Polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, suggesting this species may be more accurately described as a species complex. However, all divergent ecotypes can coexist in sympatric populations with no obvious physical barriers to prevent gene flow. In this study, we used both phylogenetic and population genetic analyses to test for speciation within C. purpurea using DNA sequences from ITS, a RAS-like locus, and a portion of beta-tubulin. The G1 types are significantly divergent from the G2/G3 types based on each of the three loci and the combined dataset, whereas the G2/G3 types are more integrated with one another. Although the G2 and G3 lineages have not diverged as much as the G1 lineage based on DNA sequence data, the use of three DNA loci does reliably separate the G2 and G3 lineages. However, the population genetic analyses strongly suggest little to no gene flow occurring between the different ecotypes, and we argue that this process is driven by adaptations to ecological habitats; G1 isolates are associated with terrestrial grasses, G2 isolates are found in wet and shady environments, and G3 isolates are found in salt marsh habitats.

A new species of Epichloë symbiotic with Chinese grasses.

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea.

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.

Oligosaccharides produced by submerged cultures of Claviceps africana and Claviceps sorghi.

Oligosaccharides produced by submerged cultures of C. africana and C. sorghi were isolated by semipreparative HPLC. Structure of 6-O-beta-D-fructofuranosyl-D-glucopyranose (blastose), 1,6-bis-O-(beta-D-fructofuranosyl)-alpha-D-glucopyranoside (neokestose) and two sugar alcohols, 1-O-beta-D-fructofuranosyl-D-mannitol (fructosylmannitol) and 1,6-bis-O-(beta-D-fructofuranosyl)-D-mannitol (bisfructosylmannitol) was determined by NMR spectrometry. MALDI TOF MS analysis revealed molecular ions [M+Na]+ that indicate the presence of other tetra- and pentasaccharides (m/z = 689.4 and 851.5, respectively) and corresponding sugar alcohol (m/z = 691.4). Rapid conversion of sucrose into series of oligosaccharides and corresponding sugar alcohols was observed in all tested strains.

Geographic distribution and diversity in Claviceps purpurea from salt marsh habitats and characterization of Pacific coast populations.

Claviceps purpurea specific to grasses in salt marsh habitats (Group G3) has previously been identified on Spartina spp. in two locations: New Jersey, USA and southern England. We have identified this subgroup of C. purpurea (G3) in 11 distinct populations including western Europe, South America, and along the Atlantic and Pacific Coasts of the USA. In addition, G3 C. purpurea was discovered on a new host grass genus, Distichlis. Unweighted pair group mean analyses of AFLP and RAPD data reveal distinct structure in G3 C. purpurea populations. Pacific coast populations show little diversity, suggesting they may have been introduced recently in that region. 43 isolates, representing 11 populations were identified as G3 based on the presence of an EcoRI restriction site in the 5.8S ribosomal DNA, and a clear genetic separation from isolates representing the other two C. purpurea subgroups (G1 and G2). In addition, all isolates originating from Spartina densiflora, S. foliosa, S. alterniflora, and S. anglica were identified as belonging to G3. RAPD and AFLP analyses supported the recognition of three discrete groups within C. purpurea and revealed high genetic variability between groups, with only 1.8% of polymorphic markers shared across all isolates. Similarly, analysis of molecular variation (AMOVA) revealed that genetic variability was mainly due to variations between groups (63.5%) rather than within groups (28.5%) or within populations (7.96%). G3 isolates were 35% similar, Pacific coast isolates 83% similar. Ninety percent similarity among isolates from the San Francisco Bay Area suggests this is a recently introduced population.

Effect of soil temperature and moisture on survival and infectivity of Metarhizium anisopliae to four tephritid fruit fly puparia.

The infectivity of 4 isolates of Metarhizium anisopliae to puparia of Ceratitis capitata treated as late third-instar larvae in unsterilized soil was investigated in the laboratory under controlled temperature and moisture. At 20-30 degrees C, mortality in puparia was highest at water potential of -0.1 and -0.01 mega Pascal (MPa) and lowest at water potential of -0.0055 and -0.0035 MPa in all the isolates. In wetter soil however, isolates ICIPE 20 and 60 caused significantly higher mortality than ICIPE 18 and 69. The survival of conidia in drier soil (-0.1 MPa) was not adversely affected at all temperatures. However, in wet soil (-0.0035 MPa) there was drastic reduction in colony counts in ICIPE 18 and 69 at 25 and 30 degrees C but conidial density in ICIPE 20 and 60 remained at the initial level at 14 days after inoculation at all temperatures. When ICIPE 20 was evaluated against three other fruit fly species (Ceratitis cosyra, Ceratitis rosa, and Ceratitis fasciventris), significant reduction in adult emergence and higher pupal mortality occurred in C. cosyra and C. fasciventris than in C. rosa at a combination of 15 and 20 degrees C and -0.1 and -0.0035 MPa. However, at higher temperature and the same moisture level, the isolates were equally pathogenic across the 3 species. It is probable that in addition to pathogen cycling and multiplication from dead infected insects in the soil, a balance between microbial degradation and replenishment of inoculum of virulent isolates occur through fluctuations in, and intricate interactions between temperature and moisture levels. This study is indicative of the potential of using isolate ICIPE 20 for soil inoculation against pupariating third-instar larva of fruit flies, thus providing a novel alternative to chemical soil application.