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

ABSTRACT: 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.

SUBMITTER: Comte A

PROVIDER: S-EPMC5336299 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Comte Alexia A Gräfenhan Tom T Links Matthew G MG Hemmingsen Sean M SM Dumonceaux Tim J TJ

PloS one 20170303 3

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 ide ...[more]

PMID: 28257512

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Project description:Abstract The ergot diseases of agricultural and nonagricultural grasses are caused by the infection of Claviceps spp. (Hypocreales, Ascomycota) on florets, producing dark spur?like sclerotia on spikes that are toxic to humans and animals, leading to detrimental impacts on agriculture and economy due to the downgrading of cereal grains, import–export barriers, reduced yield, and ecological concerns. At least seven phylogenetic lineages (phylogenetic species) were identified within the premolecular concept of C. purpurea s.l. (sensu lato) in agricultural areas and vicinities in Canada and the Western United States. Claviceps purpurea s.s (sensu stricto) remained as the most prevalent species with a wide host range, including cereal crops, native, invasive, and weedy grasses. The knowledge on genetic diversity and distribution of C. purpurea s.s. in North America is lacking. The objective of the present study was to shed light on genetic differentiation and evolution of the natural populations of C. purpurea s.s. Multilocus DNA sequences of samples from Canada and the Western USA were analyzed using a phylogenetic network approach, and population demographic parameters were investigated. Results showed that three distinct genetically subdivided populations exist, and the subdivision is not correlated with geographic or host differentiations. Potential intrinsic mechanisms that might play roles in leading to the cessation of gene flows among the subpopulations, that is, mating and/or vegetative incompatibility, genomic adaptation, were discussed. The neutrality of two house?keeping genes that are widely used for DNA barcoding, that is, translation elongation factor 1?? (TEF1??) and RNA polymerase II second largest subunit (RPB2), was challenged and discussed. Regardless of the frequent sexuality and multiple modes of dispersal, the population of the toxigenic plant pathogen, Claviceps purpurea, that infects wide range of agricultural and nonagricultural grasses, was subdivided in nature. The population structure shed light on the underline mechanisms that might have led to the cessation of gene flow among subdivided populations. House?keeping gene markers were not neutral in certain circumstances.

Project description:BackgroundErgot, caused by the fungal pathogen Claviceps purpurea, infects the female flowers of a range of cereal crops, including wheat. To understand the interaction between C. purpurea and hexaploid wheat we undertook an extensive examination of the reprogramming of the wheat transcriptome in response to C. purpurea infection through floral tissues (i.e. the stigma, transmitting and base ovule tissues of the ovary) and over time.ResultsC. purpurea hyphae were observed to have grown into and down the stigma at 24 h (H) after inoculation. By 48H hyphae had grown through the transmitting tissue into the base, while by 72H hyphae had surrounded the ovule. By 5 days (D) the ovule had been replaced by fungal tissue. Differential gene expression was first observed at 1H in the stigma tissue. Many of the wheat genes differentially transcribed in response to C. purpurea infection were associated with plant hormones and included the ethylene (ET), auxin, cytokinin, gibberellic acid (GA), salicylic acid and jasmonic acid (JA) biosynthetic and signaling pathways. Hormone-associated genes were first detected in the stigma and base tissues at 24H, but not in the transmitting tissue. Genes associated with GA and JA pathways were seen in the stigma at 24H, while JA and ET-associated genes were identified in the base at 24H. In addition, several defence-related genes were differential expressed in response to C. purpurea infection, including antifungal proteins, endocytosis/exocytosis-related proteins, NBS-LRR class proteins, genes involved in programmed cell death, receptor protein kinases and transcription factors. Of particular interest was the identification of differential expression of wheat genes in the base tissue well before the appearance of fungal hyphae, suggesting that a mobile signal, either pathogen or plant-derived, is delivered to the base prior to colonisation.ConclusionsMultiple host hormone biosynthesis and signalling pathways were significantly perturbed from an early stage in the wheat - C. purpurea interaction. Differential gene expression at the base of the ovary, ahead of arrival of the pathogen, indicated the potential presence of a long-distance signal modifying host gene expression.

Project description:Ergot alkaloids are vasoconstrictors frequently detected in low concentrations in livestock feed. The Canadian Food Inspection Agency permits up to 3,000 μg ergot alkaloids per kg cattle feed. The objective of this study was to examine the effects of feeding low concentrations of ergot alkaloids over 9-weeks on vascular dynamics in the caudal and internal iliac arteries of beef cows. A relationship between ergot alkaloid concentration in feed and hemodynamic changes in the caudal and internal iliac arteries was hypothesized. Periparturient beef cows were randomized into four groups and group fed mixed rations containing <15 μg ergot alkaloids per kg of dry matter intake (Control, n = 9), 48 μg/kg (Low, n = 9), 201 μg/kg (Medium, n = 8), and 822 μg/kg (High, n = 6). Three experimental periods comprised the study: pre-treatment (2 weeks), treatment (9 weeks), and post-treatment (3 weeks). B-mode and Doppler ultrasonography was performed weekly to measure hemodynamic endpoints. Plasma prolactin concentrations and rectal temperatures were measured weekly. Caudal artery diameter decreased (Treatment*Experimental Period i.e., Tx*EP, p < 0.001) by 14% in the High group during the treatment period. Reductions (Tx*EP, p < 0.001) in caudal artery blood flow (37%, 29%) and blood volume per pulse (29%, 11%) were recorded during the treatment period in the High and Medium groups. Internal iliac artery diameter and blood flow decreased (Tx*EP, p ≤ 0.004) by 13% and 40% during the treatment period in the Medium group. Moderate reductions (Tx*EP, p ≤ 0.042; 12-25%) in the mean blood velocity during the treatment and post-treatment periods and decreases (Tx*EP, p ≤ 0.01; 12-17%) in the peak systolic velocity of both arteries during the post-treatment period were also detected. Prolactin did not change in any group during the treatment period (p = 0.462). Rectal temperatures were within the normal physiological range for beef cows. In conclusion, we documented moderate vasoconstriction in the caudal artery and the internal iliac artery in cows fed 201-822 μg ergot alkaloids per kg of dry matter intake for 9-week period near parturition. The pattern of alterations was similar between the caudal and internal iliac arteries. Results of this study suggest that feeding up to 822 μg/kg produce reversible pharmacological changes in beef cow vasculature and warrant reconsideration of current regulations for cattle.

Project description:Rye is used as food, feed, and for bioenergy production and remain an essential grain crop for cool temperate zones in marginal soils. Ergot is known to cause severe problems in cross-pollinated rye by contamination of harvested grains. The molecular response of the underlying mechanisms of this disease is still poorly understood due to the complex infection pattern. RNA sequencing can provide astonishing details about the transcriptional landscape, hence we employed a transcriptomic approach to identify genes in the underlying mechanism of ergot infection in rye. In this study, we generated de novo assemblies from twelve biological samples of two rye hybrids with identified contrasting phenotypic responses to ergot infection. The final transcriptome of ergot susceptible (DH372) and moderately ergot resistant (Helltop) hybrids contain 208,690 and 192,116 contigs, respectively. By applying the BUSCO pipeline, we confirmed that these transcriptome assemblies contain more than 90% of gene representation of the available orthologue groups at Virdiplantae odb10. We employed a de novo assembled and the draft reference genome of rye to count the differentially expressed genes (DEGs) between the two hybrids with and without inoculation. The gene expression comparisons revealed that 228 genes were linked to ergot infection in both hybrids. The genome ontology enrichment analysis of DEGs associated them with metabolic processes, hydrolase activity, pectinesterase activity, cell wall modification, pollen development and pollen wall assembly. In addition, gene set enrichment analysis of DEGs linked them to cell wall modification and pectinesterase activity. These results suggest that a combination of different pathways, particularly cell wall modification and pectinesterase activity contribute to the underlying mechanism that might lead to resistance against ergot in rye. Our results may pave the way to select genetic material to improve resistance against ergot through better understanding of the mechanism of ergot infection at molecular level. Furthermore, the sequence data and de novo assemblies are valuable as scientific resources for future studies in rye.

Dataset Information

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

Publications

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

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