Bringing Barley Back: Analysis of Heritage Varieties for Use as Germplasm Sources to Improve Resistance against the Most Devastating, Contemporary Disease in Canada, Fusarium Head Blight (Fusarium graminearum).

Fusarium head blight (FHB), caused by Fusarium graminearum, is currently the most devastating disease for barley (Hordeum vulgare) in Canada. Associated mycotoxins can compromise grain quality, where deoxynivalenol (DON) is considered particularly damaging due to its frequency of detection. Breeding barley with a lower DON content is difficult, due to the poor adaptation and malt quality of resistance sources. A set of European-derived heritage varieties were screened in an FHB nursery in Charlottetown, PE, with selections tested at Brandon, MB, between 2018-2022. Genetic evaluation demonstrated a distinct clustering of Canadian varieties from the heritage set. At Brandon, 72% of the heritage varieties ranked lower for DON content than did the moderately resistant Canadian check 'AAC Goldman', but resistance was associated with later heading and taller stature. In contrast with Canadian modern malting variety 'AAC Synergy', general deficiencies were observed in yield, enzyme activity, and extract, along with higher protein content. Nonetheless, several resistant varieties were identified with reasonable a heading date and yield, including 'Chevallier Chile', 'Domen', 'Djugay', 'Hannchen', 'Heils Franken', 'Moravian Barley', 'Loosdorfer' with 'Golden Melon', 'Nutans Moskva', and 'Vellavia', these being some of the most promising varieties when malting quality characteristics were also considered. These heritage resources could be used as parents in breeding to develop FHB-resistant malting barley varieties.

Genetic analyses of native Fusarium head blight resistance in two spring wheat populations identifies QTL near the B1, Ppd-D1, Rht-1, Vrn-1, Fhb1, Fhb2, and Fhb5 loci.

KEY MESSAGE: QTL analyses of two bi-parental mapping populations with AC Barrie as a parent revealed numerous FHB-resistance QTL unique to each population and uncovered novel variation near Fhb1. Fusarium head blight (FHB) is a destructive disease of wheat worldwide, leading to severe yield and quality losses. The genetic basis of native FHB resistance was examined in two populations: a recombinant inbred line population from the cross Cutler/AC Barrie and a doubled haploid (DH) population from the cross AC Barrie/Reeder. Numerous QTL were detected among the two mapping populations with many being cross-specific. Photoperiod insensitivity at Ppd-D1 and dwarfing at Rht-B1 and Rht-D1 was associated with increased FHB susceptibility. Anthesis date QTL at or near the Vrn-A1 and Vrn-B1 loci co-located with major FHB-resistance QTL in the AC Barrie/Reeder population. The loci were epistatic for both traits, such that DH lines with both late alleles were considerably later to anthesis and had reduced FHB symptoms (i.e., responsible for the epistatic interaction). Interestingly, AC Barrie contributed FHB resistance near the Fhb1 locus in the Cutler population and susceptibility in the Reeder population. Analyses of the Fhb1 candidate genes PFT and TaHRC confirmed that AC Barrie, Cutler, and Reeder do not carry the Sumai-3 Fhb1 gene. Resistance QTL were also detected at the expected locations of Fhb2 and Fhb5. The native FHB-resistance QTL detected near Fhb1, Fhb2, and Fhb5 do not appear to be as effective as Fhb1, Fhb2, and Fhb5 from Sumai-3. The presence of awns segregated at the B1 awn inhibitor locus in both populations, but was only associated with FHB resistance in the Cutler/AC Barrie population suggesting linkage caused the association rather than pleiotropy.

Current and future experimental strategies for structural analysis of trichothecene mycotoxins--a prospectus.

Fungal toxins, such as those produced by members of the order Hypocreales, have widespread effects on cereal crops, resulting in yield losses and the potential for severe disease and mortality in humans and livestock. Among the most toxic are the trichothecenes. Trichothecenes have various detrimental effects on eukaryotic cells including an interference with protein production and the disruption of nucleic acid synthesis. However, these toxins can have a wide range of toxicity depending on the system. Major differences in the phytotoxicity and cytotoxicity of these mycotoxins are observed for individual members of the class, and variations in toxicity are observed among different species for each individual compound. Furthermore, while diverse toxicological effects are observed throughout the whole cellular system upon trichothecene exposure, the mechanism of toxicity is not well understood. In order to comprehend how these toxins interact with the cell, we must first have an advanced understanding of their structure and dynamics. The structural analysis of trichothecenes was a subject of major interest in the 1980s, and primarily focused on crystallographic and solution-state Nuclear Magnetic Resonance (NMR) spectroscopic studies. Recent advances in structural determination through solution- and solid-state NMR, as well as computation based molecular modeling is leading to a resurgent interest in the structure of these and other mycotoxins, with the focus shifting in the direction of structural dynamics. The purpose of this work is to first provide a brief overview of the structural data available on trichothecenes and a characterization of the methods commonly employed to obtain such information. A summary of the current understanding of the relationship between structure and known function of these compounds is also presented. Finally, a prospectus on the application of new emerging structural methods on these and other related systems is discussed.

Secondary metabolites from anti-insect extracts of endophytic fungi isolated from Picea rubens.

The extracts of a selection of 150 foliar fungal endophytes isolated from Picea rubens (red spruce) needles were screened by LC-MS and assayed for toxicity. Three of these strains that were toxic to the forest pest Choristoneura fumiferana (eastern spruce budworm) in dietary bioassays were selected for further study. Their culture extracts were analyzed by LC-NMR spectroscopy, and the major metabolites were isolated by LC-MS-SPE or PTLC/column chromatography and characterized. The structures were elucidated by spectroscopic analyses including 2D NMR, HRMS and by comparison to literature data. Compounds 1 and 5-7 are hitherto unknown whereas compounds 2 and 3 are natural products described for the first time. Compound 4 is reported for the first time as a fungal metabolite and 8-9 were identified as known fungal metabolites in genera.

Characterization of polyketide metabolites from foliar endophytes of Picea glauca.

A collection of 250 foliar endophytes of Picea glauca (white spruce) yielded several isolates that produced metabolites toxic to Choristoneura fumiferana (spruce budworm). Three of these strains were selected for further study based on their ability to be cultured and produce secondary metabolites under laboratory conditions. The culture filtrate of each was extracted and analyzed by LC-MS and LC-NMR, and the major metabolites were isolated and characterized. Structures were elucidated by spectroscopic analyses including 2D NMR and HRMS and by comparison to literature data. In some cases the extract was methylated in order to facilitate separation, but the original natural structure was determined by comparing the NMR data of the isolated methylated product with that of the stop-flow NMR of the underivatized extract (i.e., 2a, 2b, and 4). Two of these metabolites, 1 and 2a, are new structures, 3 and 4 are reported here for the first time as fungal metabolites, and 5- 10 as known fungal metabolites from other species. Tyrosol (10) was the only common metabolite found in all three extracts but did not account for the observed toxicity to C. fumiferana.

Identification of synthetic regioisomeric lutein esters and their quantification in a commercial lutein supplement.

Synthetic mixtures of 24 mono- and diesters of the asymmetric hydroxylated carotenoid lutein with lauric, myristic, palmitic, and stearic acids were analyzed by liquid chromatography-ultraviolet/visible spectroscopy (LC-UV-vis) and characterized by LC-mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). These compounds were then used for identifying the composition of a commercial lutein supplement. This is the first report of chromatographic separation of mixed fatty acid lutein diesters. Preferential MS loss of fatty acids or water occurred initially at the 3'-hydroxy position in the epsilon-ionone ring and subsequently at the 3-hydroxy position in the beta-ionone ring. This selective fragmentation leads to facile assignment of the specific fatty acids to the appropriate regioisomeric ionone ring. A commercial lutein supplement contained low levels of two pairs of regioisomeric monoesters and nearly equal levels of three homogeneous diesters and five pairs of mixed diesters. Palmitic acid was the predominant fatty acid, with lower amounts of myristic, stearic, and lauric acids.

Isolation and structure elucidation by LC-MS-SPE/NMR: PR toxin- and cuspidatol-related eremophilane sesquiterpenes from Penicillium roqueforti.

Three eremophilane sesquiterpenes (1, 2, and 3) were isolated from Penicillium roqueforti DAOM 232127, and their structures were established. The new (3S)-3-acetoxyeremophil-1(2),7(11),9(10)-trien-8-one (3) is a likely biosynthetic precursor of PR toxin. 1-Hydroxyeremophil-7(11),9(10)-dien-8-one (1) is related to the immunosuppressant cuspidatol. The application of semihyphenated LC-MS-SPE/NMR to rapidly identify, purify, and elucidate the structures of 1, 2, and 3 is described.

Isolation and metabolite production by Penicillium roqueforti, P. paneum and P. crustosum isolated in Canada.

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20-30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (aw) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.