Tom Gräfenhan

Simultaneous profiling of seed‐associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds

In order to address the hypothesis that seeds from ecologically and geographically diverse plants harbor characteristic epiphytic microbiota, we characterized the bacterial and fungal microbiota associated with Triticum and Brassica seed surfaces. The total microbial complement was determined by amplification and sequencing of a fragment of chaperonin 60 (cpn60). Specific microorganisms were quantified by qPCR. Bacteria and fungi corresponding to operational taxonomic units (OTU) that were identified in the sequencing study were isolated and their interactions examined. A total of 5477 OTU were observed from seed washes. Neither total epiphytic bacterial load nor community richness/evenness was significantly different between the seed types; 578 OTU were shared among all samples at a variety of abundances. Hierarchical clustering revealed that 203 were significantly different in abundance on Triticum seeds compared with Brassica. Microorganisms isolated from seeds showed 99–100% identity between the cpn60 sequences of the isolates and the OTU sequences from this shared microbiome. Bacterial strains identified as Pantoea agglomerans had antagonistic properties toward one of the fungal isolates (Alternaria sp.), providing a possible explanation for their reciprocal abundances on both Triticum and Brassica seeds. cpn60 enabled the simultaneous profiling of bacterial and fungal microbiota and revealed a core seed-associated microbiota shared between diverse plant genera.

ATP citrate lyase 1 (acl1) gene-based loop-mediated amplification assay for the detection of the Fusarium tricinctum species complex in pure cultures and in cereal samples.

The combined data set of the acl1 and tef-1α gene sequences of 61 fungal strains assigned to Fusarium tricinctum, Fusarium avenaceum, Fusarium acuminatum, Fusarium arthrosporioides, Fusarium flocciferum and Fusarium torulosum were used to study the phylogenetic relations between taxa. F. tricinctum, F. acuminatum and F. avenaceum formed distinct clades. Members of the F. tricinctum/F. acuminatum clade fall into three well supported lineages, of which the largest includes the epitype of F. tricinctum. Loop-mediated isothermal amplification (LAMP) was used to amplify a 167 bp portion of the acl1 gene in F. tricinctum (Corda) Saccardo. DNA amplification was detected in-tube by indirect calcein fluorescence under black light after 60 min of incubation at 65.5 °C. The assay had a detection limit of 0.95 pg of purified genomic DNA of F. tricinctum CBS 410.86 per reaction, corresponding to ca. 18 genomic copies of the acl1 gene. Specificity of the assay was tested using purified DNA from 67 species and subspecies of Fusarium as well as 50 species comprising 22 genera of other filamentous fungi and yeasts. The assay detected 21 of the 23 F. tricinctum strains tested. Cross reactivity was observed with eight out of 13 strains in F. acuminatum but with none of 17 F. avenaceum strains tested. Specificity was further confirmed by conventional PCR with primers designed from the same gene. Detection of F. tricinctum from culture scrapings directly added to the reaction master mix, in DNA extracts from wheat, in single barley grains or in washings of bulk grain samples are proposed as possible applications showing the suitability of the method for food analysis. Finally it was demonstrated that the LAMP reaction can be run using simple lab equipment such as a heating block, water bath, hybridization oven or household equipment, e.g. a microwave oven.

Deoxynivalenol levels and chemotype frequency in barley cultivars inoculated with two chemotypes of Fusarium graminearum

Abstract Corn kernels colonized by Fusarium graminearum were spread between lines of barley (Hordeum vulgare) in an irrigated Fusarium head blight (FHB) nursery at a 2 : 1 ratio of isolates producing 15-acetyldeoxynivalenol (15 ADON) to isolates producing 3-acetyldeoxynivalenol (3 ADON) (in 2008) or 1 : 1 ratio (in 2009, 2010). In 2008, it was the first use in this field of a 3 ADON isolate after 9 years of artificial inoculation with exclusively 15 ADON isolates. Sets of three 2-row and three 6-row cultivars were used as checks throughout the nursery, representing a range of susceptibility to FHB. Composites of these cultivars were prepared and analysed for per cent F. graminearum infection, chemotype frequency and levels of deoxynivalenol (DON), 3 ADON and 15 ADON. Seed infection by F. graminearum ranged from 83.5% (2009) to 98.5% (2010), with little difference across the nursery. In 2008, the chemotype distribution of F. graminearum and DON levels on infected kernels showed a several-fold difference from the eastern side (67% 3 ADON isolates and 46 ppm DON) to the western end of the field (16% 3 ADON isolates and 13 ppm DON), but not in the subsequent 2 years. In both 2009 and 2010, the 3 ADON chemotype was recovered from only 18% and 13% of the seeds, and overall average DON levels were 18 ppm and 24 ppm, respectively. In 2008, the highest DON and 3 ADON levels were associated with the highest frequency of the 3 ADON chemotype. In all years, the recovery of isolates did not reflect the ratio of the initial inoculum, suggesting that other factors influenced the ultimate infection of the seed. However, the relative relationship among the barley cultivars was consistent throughout all 3 years: DON levels were much lower in the most resistant cultivars compared with the most susceptible cultivars in all years and composites. Genotypic response to DON accumulation did not show strong interaction with 3 ADON frequency, suggesting that the underlying genetic resistance of the barley cultivars is cross-applicable between chemotypes.

Fusarium praegraminearum sp. nov., a novel nivalenol mycotoxin-producing pathogen from New Zealand can induce head blight on wheat

Abstract We report on the molecular and morphological characterization of a novel type B trichothecene toxin-producing species (i.e. B clade) recovered from litter in a maize field near Wellington, New Zealand, which is described as Fusarium praegraminearum sp. nov. This species was initially identified as F. acuminatum based on morphological characters. However, it differs from this species by producing longer, slightly asymmetrically curved macroconidia in which the apical cell is not as pointed and by its much faster colony growth rate on agar. Molecular phylogenetic analyses of portions of 13 genes resolved F. praegraminearum as the most basal species within the B clade. Mycotoxin analyses demonstrated that it was able to produce 4-acetylnivalenol and 4,15-diacetylnivalenol trichothecenes, the nontrichothecene sesquiterpenes culmorin and hydroxy-culmorins, and the estrogen zearalenone in vitro. Results of a pathogenicity experiment revealed that F. praegraminearum induced moderate head blight on wheat.

Rapid screening of Alternaria mycotoxins using MALDI-TOF mass spectrometry.

BACKGROUND Members of the Alternaria genus produce various toxins whose occurrence in agricultural commodities is a major concern for humans and the environment. The present study developed a simple and efficient matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the rapid detection of Alternaria toxins. RESULTS A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) by MALDI-TOF MS was developed. Different solid phase extraction (SPE) clean-up methods were tried to optimize the purification of wheat matrix, and an optimal extraction method was designed to recover the three Alternaria toxins. In addition, various MALDI matrices were examined and α-cyano-4-hydroxycinnamic acid (CHCA) matrix gave good repeatability for all three Alternaria toxins. CONCLUSION This is the first study to report the detection of three important Alternaria toxins concurrently using MALDI-TOF MS and opens up the possibility of rapid screening of Alternaria toxins in several other cereals and food products.

Draft Genome Sequence of Alternaria alternata ATCC 34957

ABSTRACT We report the draft genome sequence of Alternaria alternata ATCC 34957. This strain was previously reported to produce alternariol and alternariol monomethyl ether on weathered grain sorghum. The genome was sequenced with PacBio technology and assembled into 27 scaffolds with a total genome size of 33.5 Mb.

Fusarium Diseases of Canadian Grain Crops: Impact and Disease Management Strategies

The genus Fusarium, first described in the early nineteenth century, is composed of a wide range of soil-borne saprophytic and pathogenic fungi. More than a few hundred different phylogenetic species of Fusarium have been identified to date. Plant species are the main target of Fusarium pathogenicity, although some species, including F. chlamydosporum, F. oxysporum and F. verticillioides, have been shown to infect immune-compromised humans. It is said that most plant species are susceptible to at least one disease caused by Fusarium fungi. Fusarium species can cause vascular wilt diseases, for which a broad range of host plants are susceptible, involving fungal colonization of the xylem via the roots and the growing mycelium eventually causes vessel obstruction, blocking transport of water to the aerial parts of the plant. In dicots, over 100 formae speciales of F. oxysporum have been identified as causative agents in vascular wilt, including F. oxysporum ff. spp. lycopersici, phaseoli and pisi, which infect tomato, beans and pea crops, respectively. Fusarium species also cause root rots and stem rots of various field crops worldwide, including peas and related pulse crops. In cereals and corn (maize) Fusarium crown rot (FCR) and Fusarium stalk (stem) rot, respectively, are caused by a different group of Fusarium pathogens from those responsible for diseases in dicots, and include F. graminearum, F. culmorum, F. avenaceum, F. verticillioides and F. pseudograminearum. In addition to root and stem rot diseases, Fusarium species also infect the inflorescence structures, causing Fusarium head blight (FHB; also known as scab) in cereals and Fusarium ear blight (sometimes referred to as FEB) in maize, and leads to damage and yield loss in developing kernels. There is an overlap of species responsible for Fusarium crown and stalk rots with those responsible for Fusarium head and ear blights. Many of these species produce harmful mycotoxins, including trichothecenes and fumonisins, which accumulate in the kernels of infected heads. In this chapter, we will start with an introduction to Fusarium species, their classification and genetics, provide a review of the Fusarium diseases of three groups of Canadian field crops (cereals, maize and pulses), followed by sections on disease management strategies, and Fusarium toxin quantification methods.

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.