Abstract

Crown rot, caused by Phytophthora cactorum, is a devastating disease of strawberry. While most commercial octoploid strawberry cultivars (Fragaria × ananassa Duch) are generally susceptible, the diploid species Fragaria vesca is a potential source of resistance genes to P. cactorum. We previously reported several F. vesca genotypes with varying degrees of resistance to P. cactorum. To gain insights into the strawberry defence mechanisms, comparative transcriptome profiles of two resistant genotypes (NCGR1603 and Bukammen) and a susceptible genotype (NCGR1218) of F. vesca were analysed by RNA-Seq after wounding and subsequent inoculation with P. cactorum. Differential gene expression analysis identified several defence-related genes that are highly expressed in the resistant genotypes relative to the susceptible genotype in response to P. cactorum after wounding. These included putative disease resistance (R) genes encoding receptor-like proteins, receptor-like kinases, nucleotide-binding sites, leucine-rich repeat proteins, RPW8-type disease resistance proteins, and ‘pathogenesis-related protein 1’. Seven of these R-genes were expressed only in the resistant genotypes and not in the susceptible genotype, and these appeared to be present only in the genomes of the resistant genotypes, as confirmed by PCR analysis. We previously reported a single major gene locus RPc-1 (Resistance to Phytophthora cactorum 1) in F. vesca that contributed resistance to P. cactorum. Here, we report that 4–5% of the genes (35–38 of ca 800 genes) in the RPc-1 locus are differentially expressed in the resistant genotypes compared to the susceptible genotype after inoculation with P. cactorum. In particular, we identified three defence-related genes encoding wall-associated receptor-like kinase 3, receptor-like protein 12, and non-specific lipid-transfer protein 1-like that were highly expressed in the resistant genotypes compared to the susceptible one. The present study reports several novel candidate disease resistance genes that warrant further investigation for their role in plant defence against P. cactorum.

Abstract

Phytophthora cactorum has two distinct pathotypes that cause crown rot and leather rot in strawberry (Fragaria × ananassa). Strains of the crown rot pathotype can infect both the rhizome (crown) and fruit tissues, while strains of the leather rot pathotype can only infect the fruits of strawberry. The genome of a highly virulent crown rot strain, a low virulent crown rot strain, and three leather rot strains were sequenced using PacBio high fidelity (HiFi) long read sequencing. The reads were de novo assembled to 66.4–67.6 megabases genomes in 178–204 contigs, with N50 values ranging from 892 to 1,036 kilobases. The total number of predicted complete genes in the five P. cactorum genomes ranged from 17,286 to 17,398. Orthology analysis identified a core secretome of 8,238 genes. Comparative genomic analysis revealed differences in the composition of potential virulence effectors, such as putative RxLR and Crinklers, between the crown rot and the leather rot pathotypes. Insertions, deletions, and amino acid substitutions were detected in genes encoding putative elicitors such as beta elicitin and cellulose-binding domain proteins from the leather rot strains compared to the highly virulent crown rot strain, suggesting a potential mechanism for the crown rot strain to escape host recognition during compatible interaction with strawberry. The results presented here highlight several effectors that may facilitate the tissue-specific colonization of P. cactorum in strawberry.

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Abstract

Rubus idaeus L. (red raspberry), is a perennial woody plant species of the Rosaceae family that is widely cultivated in the temperate regions of world and is thus an economically important soft fruit species. It is prized for its flavour and aroma, as well as a high content of healthful compounds such as vitamins and antioxidants. Breeding programs exist globally for red raspberry, but variety development is a long and challenging process. Genomic and molecular tools for red raspberry are valuable resources for breeding. Here, a chromosome-length genome sequence assembly and related gene predictions for the red raspberry cultivar ‘Anitra’ are presented, comprising PacBio long read sequencing scaffolded using Hi-C sequence data. The assembled genome sequence totalled 291.7 Mbp, with 247.5 Mbp (84.8%) incorporated into seven sequencing scaffolds with an average length of 35.4 Mbp. A total of 39,448 protein-coding genes were predicted, 75% of which were functionally annotated. The seven chromosome scaffolds were anchored to a previously published genetic linkage map with a high degree of synteny and comparisons to genomes of closely related species within the Rosoideae revealed chromosome-scale rearrangements that have occurred over relatively short evolutionary periods. A chromosome-level genomic sequence of R. idaeus will be a valuable resource for the knowledge of its genome structure and function in red raspberry and will be a useful and important resource for researchers and plant breeders.

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Abstract

Denne rapporten oppsummerer foreløpige resultater fra 2020 i IPM-Golf-prosjektet "Ingergrated Management of Important Turfgrass Diseases and Insect Pests on European Golf-Courses" Feltforsøk på Microdochium flekk ble utført i Landvik, Norge og Bingley, Storbritannia. På Landvik viste resultatene at rulling ved lav N og sitronsyre, tilført fra aug.-okt. kan redusere Microdochium flekk til en viss grad blant de ikke-kjemiske behandlingene. Høy N resulterte i mer Mikrodochium flekk, men mindre antraknose. På Bingley viste resultatene at behandlingene som inneholder jernsulfat spesielt høyt jern, lyktes med å kontrollere sykdommen, men effekten varte ikke gjennom vinteren. Feltforsøkene ved Kjøpenhavns Golf Club viste at rulling to ganger i uken forbedret kvaliteten på greens gjennom vekstsesongen og at reduksjon av Microdochium flekk ble oppnådd ved å rulle fra august til desember. Feltforsøkene med UV-C-stråling ved Osnabrück Golf Club viste at denne metoden kunne kontrollere, men ikke bekjempe fullstendig dollar spot. Litteraturgjennomgangen om myrstankelbein og hageoldenborre viste at problemene varierer sterkt mellom år og de ulike landene.

Abstract

We used metabarcoding of ITS 1 and 2 to compare the mycobiome of Norwegian spring wheat seed lots of two commonly grown spring wheat varieties (Mirakel and Zebra) harvested in 2016 and 2017. The seed lots varied in germination and were grouped according to high and low germination (≥90% and <90% germinated seeds, respectively) determined by the ISTA germination method. In addition, the percentage of each seed lot infested by the most important wheat pathogens (Microdochium spp., Fusarium spp., and Parastagonospora nodorum) was determined using a plate-out test on PDA, and species-specific qPCR was used to quantify the amount of DNA of F. avenaceum, F. culmorum, F. graminearum, F. poae, M. majus, M. nivale, and P. nodorum. Our study indicated that the presence of Microdochium was most associated with poor germination (which is as expected), while P. nodorum; although present at relatively high levels, apparently had limited impact on germination. Among the species quantified by qPCR, M. majus was the most abundant, F. avenaceum was detected at low levels, whereas the other fusaria were barely detected. Metabarcoding data indicated a negative association between the presence of the fungal genus Neoascochyta and germination, while Pyrenophora and Alternaria species appeared positively associated with germination. Our results indicated some co-existence patterns between fungal species, including both pathogenic and non-pathogenic species, with some species combinations associated with the germination potential of wheat seed.

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Abstract

The plant pathogenic fungus Fusarium graminearum is known to produce a wide array of secondary metabolites during plant infection. This includes several nonribosomal peptides. Recently, the fusaoctaxin (NRPS5/9) and gramilin (NRPS8) gene clusters were shown to be induced by host interactions. To widen our understanding of this important pathogen, we investigated the involvement of the NRPS4 gene cluster during infection and oxidative and osmotic stress. Overexpression of NRPS4 led to the discovery of a new cyclic hexapeptide, fusahexin (1), with the amino acid sequence cyclo-(d-Ala-l-Leu-d-allo-Thr-l-Pro-d-Leu-l-Leu). The structural analyses revealed an unusual ether bond between a proline Cδ to Cβ of the preceding threonine resulting in an oxazine ring system. The comparative genomic analyses showed that the small gene cluster only encodes an ABC transporter in addition to the five-module nonribosomal peptide synthetase (NRPS). Based on the structure of fusahexin and the domain architecture of NRPS4, we propose a biosynthetic model in which the terminal module is used to incorporate two leucine units. So far, iterative use of NRPS modules has primarily been described for siderophore synthetases, which makes NRPS4 a rare example of a fungal nonsiderophore NRPS with distinct iterative module usage.

Abstract

Plants with roots and soil clumps transported over long distances in plant trading can harbor plant pathogenic oomycetes, facilitating disease outbreaks that threaten ecosystems, biodiversity, and food security. Tools to detect the presence of such oomycetes with a sufficiently high throughput and broad scope are currently not part of international phytosanitary testing regimes. In this work, DNA metabarcoding targeting the internal transcribed spacer (ITS) region was employed to broadly detect and identify oomycetes present in soil from internationally shipped plants. This method was compared to traditional isolation-based detection and identification after an enrichment step. DNA metabarcoding showed widespread presence of potentially plant pathogenic Phytophthora and Pythium species in internationally transported rhizospheric soil with Pythium being the overall most abundant genus observed. Baiting, a commonly employed enrichment method for Phytophthora species, led to an increase of golden-brown algae in the soil samples, but did not increase the relative or absolute abundance of potentially plant pathogenic oomycetes. Metabarcoding of rhizospheric soil yielded DNA sequences corresponding to oomycete isolates obtained after enrichment and identified them correctly but did not always detect the isolated oomycetes in the same samples. This work provides a proof of concept and outlines necessary improvements for the use of environmental DNA (eDNA) and metabarcoding as a standalone phytosanitary assessment tool for broad detection and identification of plant pathogenic oomycetes.

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Abstract

The genus Metarhizium is composed of species used in biological control programs of agricultural pests worldwide. This genus includes common fungal pathogen of many insects and mites and endophytes that can increase plant growth. Metarhizium humberi was recently described as a new species. This species is highly virulent against some insect pests and promotes growth in sugarcane, strawberry, and soybean crops. In this study, we sequenced the genome of M. humberi, isolate ESALQ1638, and performed a functional analysis to determine its genomic signatures and highlight the genes and biological processes associated with its lifestyle. The genome annotation predicted 10633 genes in M. humberi, of which 92.0% are assigned putative functions, and ∼17% of the genome was annotated as repetitive sequences. We found that 18.5% of the M. humberi genome is similar to experimentally validated proteins associated with pathogen–host interaction. Compared to the genomes of eight Metarhizium species, the M. humberi ESALQ1638 genome revealed some unique traits that stood out, e.g., more genes functionally annotated as polyketide synthases (PKSs), overrepresended GO-terms associated to transport of ions, organic and amino acid, a higher percentage of repetitive elements, and higher levels of RIP-induced point mutations. The M. humberi genome will serve as a resource for promoting studies on genome structure and evolution that can contribute to research on biological control and plant biostimulation. Thus, the genomic data supported the broad host range of this species within the generalist PARB clade and suggested that M. humberi ESALQ1638 might be particularly good at producing secondary metabolites and might be more efficient in transporting amino acids and organic compounds.

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Abstract

Background Sphingolipids are structural components and signaling molecules in eukaryotic membranes, and many organisms produce compounds that inhibit sphingolipid metabolism. Some of the inhibitors are structurally similar to the sphingolipid biosynthetic intermediate sphinganine and are referred to as sphinganine-analog metabolites (SAMs). The mycotoxins fumonisins, which are frequent contaminants in maize, are one family of SAMs. Due to food and feed safety concerns, fumonisin biosynthesis has been investigated extensively, including characterization of the fumonisin biosynthetic gene cluster in the agriculturally important fungi Aspergillus and Fusarium. Production of several other SAMs has also been reported in fungi, but there is almost no information on their biosynthesis. There is also little information on how widely SAM production occurs in fungi or on the extent of structural variation of fungal SAMs. Results Using fumonisin biosynthesis as a model, we predicted that SAM biosynthetic gene clusters in fungi should include a polyketide synthase (PKS), an aminotransferase and a dehydrogenase gene. Surveys of genome sequences identified five putative clusters with this three-gene combination in 92 of 186 Fusarium species examined. Collectively, the putative SAM clusters were distributed widely but discontinuously among the species. We propose that the SAM5 cluster confers production of a previously reported Fusarium SAM, 2-amino-14,16-dimethyloctadecan-3-ol (AOD), based on the occurrence of AOD production only in species with the cluster and on deletion analysis of the SAM5 cluster PKS gene. We also identified SAM clusters in 24 species of other fungal genera, and propose that one of the clusters confers production of sphingofungin, a previously reported Aspergillus SAM. Conclusion Our results provide a genomics approach to identify novel SAM biosynthetic gene clusters in fungi, which should in turn contribute to identification of novel SAMs with applications in medicine and other fields. Information about novel SAMs could also provide insights into the role of SAMs in the ecology of fungi. Such insights have potential to contribute to strategies to reduce fumonisin contamination in crops and to control crop diseases caused by SAM-producing fungi.

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Abstract

The bread-making quality of wheat depends on the viscoelastic properties of the dough in which gluten proteins play an important role. The quality of gluten proteins is influenced by the genetics of the different wheat varieties and environmental factors. Occasionally, a near complete loss of gluten strength, measured as the maximum resistance towards stretching (Rmax), is observed in grain lots of Norwegian wheat. It is hypothesized that the loss of gluten quality is caused by degradation of gluten proteins by fungal proteases. To identify fungi associated with loss of gluten strength, samples from a selection of wheat grain lots with weak gluten (n = 10, Rmax < 0.3 N) and strong gluten (n = 10, Rmax ≥ 0.6 N) was analyzed for the abundance of fungal operational taxonomic units (OTUs) using DNA metabarcoding of the nuclear ribosomal Internal Transcribed Spacer (ITS) region ITS1. The DNA quantities for a selection of fungal pathogens of wheat, and the total amount of fungal DNA, were analyzed by quantitative PCR (qPCR). The mean level of total fungal DNA was higher in grain samples with weak gluten compared to grain samples with strong gluten. Heightened quantities of DNA from fungi within the Fusarium Head Blight (FHB) complex, i.e. Fusarium avenaceum, Fusarium graminearum, Microdochium majus, and Microdochium nivale, were observed in grain samples with weak gluten compared to those with strong gluten. Microdochium majus was the dominant fungus in the samples with weak gluten. Stepwise regression modeling based on different wheat quality parameters, qPCR data, and the 35 most common OTUs revealed a significant negative association between gluten strength and three OTUs, of which the OTU identified as M. majus was the most abundant. The same analysis also revealed a significant negative relationship between gluten strength and F. avenaceum detected by qPCR, although the DNA levels of this fungus were low compared to those of M. majus. In vitro growth rate studies of a selection of FHB species showed that all the tested isolates were able to grow with gluten as a sole nitrogen source. In addition, proteins secreted by these fungi in liquid cultures were able to hydrolyze gluten substrate proteins in zymograms, confirming their capacity to secrete gluten-degrading proteases. The identification of fungi with potential to influence gluten quality can enable the development of strategies to minimize future problems with gluten strength in food-grade wheat.

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Abstract

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Abstract

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available.

Abstract

Endogenous antimicrobial peptides (AMPs) are evolutionarily ancient factors of innate immunity, which are produced by all multicellular organisms and play a key role in their protection against infection. Red king crab (Paralithodes camtschaticus), also called Kamchatka crab, is widely distributed and the best known species of all king crabs belonging to the family Lithodidae. Despite their economic importance, the genetic resources of king crabs are scarcely known and no fullgenome sequences are available to date. Therefore, analysis of the red king crab transcriptome and identifcation and characterization of its AMPs could potentially contribute to the development of novel antimicrobial drug candidates when antibiotic resistance has become a global health threat. In this study, we sequenced the P. camtschaticus transcriptomes from carapace, tail fap and leg tissues using an Illumina NGS platform. Libraries were systematically analyzed for gene expression profles along with AMP prediction. By an in silico approach using public databases we defned 49 cDNAs encoding for AMP candidates belonging to diverse families and functional classes, including buforins, crustins, paralithocins, and ALFs (anti-lipopolysaccharide factors). We analyzed expression patterns of 27 AMP genes. The highest expression was found for Paralithocin 1 and Crustin 3, with more than 8,000 reads. Other paralithocins, ALFs, crustins and ubiquicidins were among medium expressed genes. This transcriptome data set and AMPs provide a solid baseline for further functional analysis in P. camtschaticus. Results from the current study contribute also to the future application of red king crab as a bio-resource in addition to its being a known seafood delicacy.

Abstract

Invasive alien species and new plant pests are introduced into new regions at an accelerating rate, due to increasing international trade with soil, plants and plant products. Exotic, plant pathogenic oomycetes in soil from the root zone of imported plants pose a great threat to endemic ecosystems and horticultural production. Detecting them via baiting and isolation, with subsequent identification of the isolated cultures by Sanger sequencing, is labour intensive and may introduce bias due to the selective baiting process. We used metabarcoding to detect and identify oomycetes present in soil samples from imported plants from six different countries. We compared metabarcoding directly from soil both before and after baiting to a traditional approach using Sanger-based barcoding of cultures after baiting. For this, we developed a standardized analysis workflow for Illumina paired-end oomycete ITS metabarcodes that is applicable to future surveillance efforts. In total, 73 soil samples from the rhizosphere of woody plants from 33 genera, in addition to three samples from transport debris, were analysed by metabarcoding the ITS1 region with primers optimized for oomycetes. We detected various Phytophthora and Pythium species, with Pythium spp. being highly abundant in all samples. We also found that the baiting procedure, which included submerging the soil samples in water, resulted in the enrichment of organisms other than oomycetes, compared to non-baited soil samples.

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Abstract

Fungal non-ribosomal peptide synthetase (NRPS) clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one NRPS. However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in Fusarium graminearum. Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase kmt6, which induced the production of fusaoctaxin A.

Abstract

Complex communities of microorganisms influence plant and agroecosystem health and productivity. Bacteria and fungi constitute a major part of the wheat head microbiome. A microorganism’s ability to colonize or infect a wheat seed is influenced by interacting microbiome. In Norway, wheat seed lots are routinely analysed for the infestation by Fusarium head blight and seedling blight diseases, such as Fusarium and Microdochium spp., and glume blotch caused by Parastagonospora nodorum using traditional methods (plating grain on PDA, recording presence or absence of fungal colonies) The purpose is to decide if the seed quality is suitable for sowing and whether seed treatment is needed. This method is time consuming, require knowledge within fungal morphology, and do not facilitate identification to species level in all cases. Molecular methods such as sequencing could allow detection and quantification of “all” microbial DNA, only limited by the specificity of the primers. Microbial profiling (metabarcoding) can be very time and cost-effective, since a mixture of many samples can be analysed simultaneously for both fungi and bacteria, and other microbes if required. In our project “Phytobiome” we used metabarcoding to analyse microbial communities in wheat heads and verify this information with results from qPCR and plate studies for a more complete study. Around 150 spring wheat seed lots from the years 2016-2017 (including two cultivars) were selected for analysis. One of the main objectives was to find microorganisms associated with seed germination. We will present findings from this work, but also some challenges when using PCR-based sequencing methods, especially regarding Fusarium head blight fungi.

Abstract

Occasionally, high mycotoxin levels are observed in Norwegian oat grain lots. The development of moderate resistant oat cultivars is therefore highly valued in order to increase the share of high quality grain into the food and feed industry. The Norwegian SafeOats project (2016-2020) aims to develop resistance screening methods to facilitate the phase-out of Fusarium-susceptible oat germplasm. Furthermore, SafeOats will give new insight into the biology of F. langsethiae and HT2+T2 accumulation in oats. The relative ranking of oat varieties according to F. graminearum/DON versus F. langsethiae/HT2+T2 content has been explored in naturally infested as well as in inoculated field trials. Routine testing of the resistance to F. graminearum in oat cultivars and breeding lines has been conducted in Norway since 2007. We are currently working on ways to scale up the inoculum production and fine tune the methodology of F. langsethiae inoculation of field trials to be routinely applied in breeding programs. Through greenhouse studies, we have analysed the content of Fusarium DNA and mycotoxins in grains of selected oat varieties inoculated at different development stages. Furthermore, we are studying the transcriptome during F. langsethiae and F. graminearum infestation of oats. The project also focus on the occurrence of F. langsethiae in oat seeds and possible influence of the fungus on seedling development in a selection of oat varieties. On average, the fungus was observed on 5% of the kernels in 168 seed lots tested during 2016-2018. No indication of transmission of F. langsethiae from germinating seed to seedlings was found in a study with germination of naturally infected seeds. So far, the studies have shown that the ranking of oat varieties according to HT2+T2 content in non-inoculated field trials resembles the ranking observed in inoculated field trials. The ranking of oat varieties according to DON content is similar in non-inoculated and F. graminearum inoculated field trials. However, the ranking of oat varieties according to DON content does not resemble the ranking for HT2+T2. The results from SafeOats will benefit consumers nationally and internationally by providing tools to increase the share of high quality grain into the food and feed industry. The project is financed by The Foundation for Research Levy on Agricultural Products/Agricultural Agreement Research Fund/Research Council of Norway with support from the industry partners Graminor, Lantmännen, Felleskjøpet Agri, Felleskjøpet Rogaland & Agder, Fiskå Mølle Moss, Norgesmøllene, Strand Unikorn/Norgesfôr and Kimen Seed Laboratory.

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Abstract

The oomycete pathogen Phytophthora cactorum causes crown rot, a major disease of cultivated strawberry. We report the draft genome of P. cactorum isolate 10300, isolated from symptomatic Fragaria x ananassa tissue. Our analysis revealed that there are a large number of genes encoding putative secreted effectors in the genome, including nearly 200 RxLR domain containing effectors, 77 Crinklers (CRN) grouped into 38 families, and numerous apoplastic effectors, such as phytotoxins (PcF proteins) and necrosis inducing proteins. As in other Phytophthora species, the genomic environment of many RxLR and CRN genes differed from core eukaryotic genes, a hallmark of the two-speed genome. We found genes homologous to known Phytophthora infestans avirulence genes including Avr1, Avr3b, Avr4, Avrblb1 and AvrSmira2 indicating effector sequence conservation between Phytophthora species of clade 1a and clade 1c. The reported P. cactorum genome sequence and associated annotations represent a comprehensive resource for avirulence gene discovery in other Phytophthora species from clade 1 and, will facilitate effector informed breeding strategies in other crops.

Abstract

The proportion of Norwegian wheat used for food has varied significantly during the recent decade, mainly because of the instability of factors that are essential to baking quality (i.e. protein content and gluten functionality). During the same period, serious contamination of Fusarium spp. and mycotoxins was observed in some grain lots [1, 2]. A project was established to generate greater knowledge of the interface between gluten functionality and effects of Fusarium species and other microorganisms on Norwegian wheat quality. Instances of severe degradation of gluten proteins that resulted in an almost complete loss of gluten functionality were observed in some lots of Norwegian wheat. The degradation of the gluten appeared to be caused by exogenous proteases. Metabarcoding of fungi and bacteria in these grain lots identified fungi within the Fusarium Head Blight complex, as well as one bacterial species, as candidate species for influencing gluten functionality. Some of these candidates were inoculated on wheat during flowering [3]. Analysis of baking quality of the flour from this experiment revealed a reduced proportion of un-extractable polymeric proteins (%UPP) and severe reductions in the gluten’s resistance to stretching (RMAX) in wheat flour from plants inoculated with Fusarium graminearum. Flour from wheat inoculated with Fusarium avenaceum was generally less infested, and showed minimal or no reduction in gluten functionality and %UPP compared to flour from the F. graminearum infested samples. Flour from wheat inoculated with Michrodochium majus is yet to be analysed. References 1. Koga, S., et al., Investigating environmental factors that cause extreme gluten quality deficiency in winter wheat (Triticum aestivum L.). Acta Agriculturae Scandinavica, Section B—Soil & Plant Science, 2016. 66(3): p. 237-246. 2. Hofgaard, I., et al., Associations between Fusarium species and mycotoxins in oats and spring wheat from farmers’ fields in Norway over a six-year period. World Mycotoxin Journal, 2016. 9(3): p. 365-378. 3. Nielsen, K.A.G., Effect of microorganisms on gluten quality in wheat., in Faculty of Biosciences. 2017, Norwegian University of Life Sciences: Ås.

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Abstract

Helminthosporium solani causes silver scurf, which affects the quality of potato. The biocontrol agent Clonostachys rosea greatly limited the severity of silver scurf symptoms and amount of H. solani genomic DNA in laboratory experiments. Transcriptomic analysis during interaction showed that H. solani gene expression was highly reduced when coinoculated with the biocontrol agent C. rosea, whereas gene expression of C. rosea was clearly boosted as a response to the pathogen. The most notable upregulated C. rosea genes were those encoding proteins involved in cellular response to oxidative stress, proteases, G-protein signaling, and the methyltransferase LaeA. The most notable potato response to both fungi was downregulation of defense-related genes and mitogen-activated protein kinase kinase kinases. At a later stage, this shifted, and most potato defense genes were turned on, especially those involved in terpenoid biosynthesis when H. solani was present. Some biocontrol-activated defense-related genes in potato were upregulated during early interaction with C. rosea alone that were not triggered by H. solani alone. Our results indicate that the reductions of silver scurf using C. rosea are probably due to a combination of mechanisms, including mycoparasitism, biocontrol-activated stimulation of plant defense mechanisms, microbial competition for nutrients, space, and antibiosis.

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Abstract

Production of chrysogine has been reported from several fungal genera including Penicillium, Aspergillus, and Fusarium. Anthranilic acid and pyruvic acid, which are expected precursors of chrysogine, enhance production of this compound. A possible route for the biosynthesis using these substrates is via a nonribosomal peptide synthetase (NRPS). Through comparative analysis of the NRPSs from genome-sequenced producers of chrysogine we identified a candidate NRPS cluster comprising five additional genes named chry2–6. Deletion of the two-module NRPS (NRPS14 = chry1) abolished chrysogine production in Fusarium graminearum, indicating that the gene cluster is responsible for chrysogine biosynthesis. Overexpression of NRPS14 enhanced chrysogine production, suggesting that the NRPS is the bottleneck in the biosynthetic pathway.

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Abstract

The genus Microbacterium contains bacteria that are ubiquitously distributed in various environments and includes plant-associated bacteria that are able to colonize tissue of agricultural crop plants. Here, we report the 3,508,491 bp complete genome sequence of Microbacterium sp. strain BH-3-3-3, isolated from conventionally grown lettuce (Lactuca sativa) from a field in Vestfold, Norway. The nucleotide sequence of this genome was deposited into NCBI GenBank under the accession CP017674.

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Abstract

In fungi, distribution of secondary metabolite (SM) gene clusters is often associated with host- or environment-specific benefits provided by SMs. In the plant pathogen Alternaria brassicicola (Dothideomycetes), the DEP cluster confers an ability to synthesize the SM depudecin, a histone deacetylase inhibitor that contributes weakly to virulence. The DEP cluster includes genes encoding enzymes, a transporter, and a transcription regulator. We investigated the distribution and evolution of the DEP cluster in 585 fungal genomes and found a wide but sporadic distribution among Dothideomycetes, Sordariomycetes, and Eurotiomycetes. We confirmed DEP gene expression and depudecin production in one fungus, Fusarium langsethiae. Phylogenetic analyses suggested 6–10 horizontal gene transfers (HGTs) of the cluster, including a transfer that led to the presence of closely related cluster homologs in Alternaria and Fusarium. The analyses also indicated that HGTs were frequently followed by loss/pseudogenization of one or more DEP genes. Independent cluster inactivation was inferred in at least four fungal classes. Analyses of transitions among functional, pseudogenized, and absent states of DEP genes among Fusarium species suggest enzyme-encoding genes are lost at higher rates than the transporter (DEP3) and regulatory (DEP6) genes. The phenotype of an experimentally-induced DEP3 mutant of Fusarium did not support the hypothesis that selective retention of DEP3 and DEP6 protects fungi from exogenous depudecin. Together, the results suggest that HGT and gene loss have contributed significantly to DEP cluster distribution, and that some DEP genes provide a greater fitness benefit possibly due to a differential tendency to form network connections.

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Abstract

The aim of this study was to evaluate the natural occurrence of Beauveria spp. in soil, from infections in the stink bug Piezodorus guildinii, an important pest of common bean (Phaseolus vulgaris) and as endophytes in bean plant tissue. Twelve conventional and 12 organic common bean fields in the Villa Clara province, Cuba were sampled from September 2014 to April 2015. One hundred and fifty Beauveria isolates were obtained from soil samples, bean plant parts and stink bugs. The overall frequency of occurrence of Beauveria isolates in conventional fields (8.4%) was significantly lower than that in organic fields (23.6%). Beauveria were also obtained significantly more frequently from bean roots in organic fields (15.0%) compared to bean roots in conventional fields (3.3%). DNA sequencing of the intergenic Bloc region was performed for Beauveria species identification. All isolates where characterized as Beauveria bassiana (Balsamo-Crivelli) Vuillemin, and clustered with isolates of neotropical origin previously described as AFNEO_1. The Cuban B. bassiana isolates formed five clusters in the phylogeny. Isolates of two clusters originated from all four locations, organic and conventional fields, as well as soil, plants and stink bugs. Organic fields contained isolates of all five clusters while conventional fields only harbored isolates of the two most frequent ones. Mating type PCR assays revealed that mating type distribution was skewed, with MAT1/MAT2 proportion of 146/4, indicating limited potential for recombination. The present study is the first to report of B. bassiana as a naturally occurring endophyte in common bean. Further, it shows that B. bassiana occurs naturally in diverse environments of common bean fields, and constitutes a potential reservoir of natural enemies against pest insects particularly in organic fields.

Abstract

The genus Pectobacterium, which belongs to the bacterial family Enterobacteriaceae, contains numerous species that cause soft rot diseases in a wide range of plants. The species Pectobacterium carotovorum is highly heterogeneous, indicating a need for re-evaluation and a better classification of the species. PacBio was used for sequencing of two soft-rot-causing bacterial strains (NIBIO1006T and NIBIO1392), initially identified as P. carotovorum strains by fatty acid analysis and sequencing of three housekeeping genes (dnaX, icdA and mdh). Their taxonomic relationship to other Pectobacterium species was determined and the distance from any described species within the genus Pectobacterium was less than 94% average nucleotide identity (ANI). Based on ANI, phylogenetic data and genome-to-genome distance, strains NIBIO1006T, NIBIO1392 and NCPPB3395 are suggested to represent a novel species of the genus Pectobacterium, for which the name Pectobacterium polaris sp. nov. is proposed. The type strain is NIBIO1006T (=DSM 105255T=NCPPB 4611T).

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Abstract

Putative proton coupled di-peptide transporters, PTR2s, are found in filamentous fungi in different numbers and their function during fungal development and plant infection is unresolved. In Fusarium graminearum, the cause of head blight in cereals, we identified four putative PTR2 transporters (FgPTR2A-D). The genes did not cluster together in phylogenetic analyses and only FgPTR2A and FgPTR2C were able to complement a PTR2 deficient yeast mutant in uptake of di-peptides. All FgPTR2s are continuously expressed throughout the fungal lifecycle, although at different levels. In silico analyses of existing expression-data show that FgPTR2B is found at higher levels than the others in planta and during sexual development. Deletion mutants of FgPTR2A, FgPTR2C, and FgPTR2D had a higher production of deoxynivalenol (DON) and zearalenone and lower production of fusarielin H than the wild type. Perithecium development was reduced in these mutants but unaffected by deletion of FgPTR2B. Conidia production was reduced in the FgPTR2B mutant and unaffected by deletion of the other PTR2 transporters. Sexual development and secondary metabolite production are known to be linked at the regulatory level and the results suggest that PTR2s are active in nitrogen turnover and thereby influence signal processes.

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Abstract

Biosynthesis of the black perithecial pigment in the filamentous fungus Fusarium graminearum is dependent on the polyketide synthase PGL1 (oPKS3). A seven-membered PGL1 gene cluster was identified by over-expression of the cluster specific transcription factor pglR. Targeted gene replacement showed that PGL1, pglJ, pglM and pglV were essential for the production of the perithecial pigment. Over-expression of PGL1 resulted in the production of 6-O-demethyl-5-deoxybostrycoidin (1), 5-deoxybostrycoidin (2), and three novel compounds 5-deoxybostrycoidin anthrone (3), 6-O-demethyl-5-deoxybostrycoidin anthrone (4) and purpurfusarin (5). The novel dimeric bostrycoidin purpurfusarin (5) was found to inhibit the growth of Candida albicans with an IC50 of 8.0 +/− 1.9 μM. The results show that Fusarium species with black perithecia have a previously undescribed form of 5-deoxybostrycoidin based melanin in their fruiting bodies.

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Abstract

Fusarium langsethiae is a widespread pathogen of small grain cereals, causing problems with T-2 and HT-2 toxin contamination in grains every year. In an effort to better understand the biology of this fungus, we present a draft genome sequence of F. langsethiae Fl201059 isolated from oats in Norway. The assembly was fragmented, but reveals a genome of approximately 37.5 Mb, with a GC content around 48%, and 12,232 predicted protein-coding genes. Focusing on secondary metabolism we identified candidate genes for 12 polyketide synthases, 13 non-ribosomal peptide synthetases, and 22 genes for terpene/isoprenoid biosynthesis. Some of these were found to be unique compared to sequence databases. The identified putative Tri5 cluster was highly syntenic to the cluster reported in F. sporotrichioides. Fusarium langsethiae Fl201059 produces a high number of secondary metabolites on Yeast Extract Sucrose (YES) agar medium, dominated by type A trichothecenes. Interestingly we found production of glucosylated HT-2 toxin (Glu-HT-2), previously suggested to be formed by the host plant and not by the fungus itself. In greenhouse inoculations of F. langsethiae Fl201059 on barley and oats, we detected the type A trichothecenes: neosolaniol, HT-2 toxin, T-2 toxin, Glu-HT-2 and numerous derivatives of these.

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Abstract

The mycotoxin enniatin B, a cyclic hexadepsipeptide produced by the plant pathogen Fusarium, is prevalent in grains and grain-based products in different geographical areas. Although enniatins have not been associated with toxic outbreaks, they have caused toxicity in vitro in several cell lines. In this study, the cytotoxic effects of enniatin B were assessed in relation to cellular energy metabolism, cell proliferation, and the induction of apoptosis in Balb 3T3 and HepG2 cells. The mechanism of toxicity was examined by means of whole genome expression profiling of exposed rat primary hepatocytes. Enniatin B altered cellular energy metabolism and reduced cell proliferation in Balb 3T3 and HepG2 cell lines. Furthermore, the proportion of apoptotic cell populations of Balb 3T3 cells slightly increased. On the other hand, enniatin B caused necrotic cell death in primary hepatocytes. Gene expression studies revealed the alteration of energy metabolism due to effects on mitochondrial organization and function and the assembly of complex I of the electron transport chain.

Abstract

The proportion of Norwegian wheat used for food has recently been dramatically lower due to both reduced production and poor quality. Furthermore, the Norwegian milling and baking industries have experienced major challenges in utilizing Norwegian wheat due to the instability of factors, such as protein content and gluten functionality, that are of major importance for baking quality. The variation in the wheat quality can itself cause economic losses for the milling and baking industry due to uncertainty in the marketplace. In the same period as a large variation in baking quality was reported in Norwegian wheat, serious contamination of Fusarium spp. and mycotoxins were observed in some grain lots. We have revealed the severe degradation of gluten proteins in some Norwegian wheat samples leading to an almost complete loss in the gluten functionality. The degradation of the gluten appears to be caused by exogenous proteases, and was associated with the presence of Fusarium spp., and their metabolites, and other microorganisms in the wheat. Increased knowledge is needed to establish the cause of the poor gluten functionality and to develop control measures to reduce the amount of poor quality wheat entering the food value chain. In 2014, a new project was established to generate deeper knowledge in the interface between gluten functionality and effects of Fusarium spp. and other microorganism on wheat quality, and to better utilize Norwegian wheat grown in this challenging environment. A metagenomic analysis, designed to identify microorganisms associated with reduced baking quality, has been undertaken. To study the influence of the identified microorganisms and their metabolites on gluten functionality, wheat plants were inoculated with microorganisms, selected based upon the results of the metagenomics study. Fusarium species are among those microorganisms being tested.

Abstract

The aim of this study was to evaluate the effect of conventional versus organic common bean (Phaseolus vulgaris) production on natural occurrence of Beauveria spp. as entophytes in bean plant tissue, from soil and as infections in stink bugs (Hemiptera: Pentatomidae), an important pest of bean in Cuba. Twenty-four organic and conventionally managed bean fields were sampled from September 2014 to April 2015 and Beauveria spp. were isolated and DNA extracted. PCR amplification of the intergenic Bloc region was performed for the identification of Beauveria species. Eighty-seven isolates were obtained from the soil samples by using the Galleria mellonella baiting technique. Further, 45 isolates were obtained from endophytic tissues of bean plant parts and 18 isolates were acquired from stink bugs. Only Beauveria bassiana was identified by DNA sequencing in this material. B. bassiana was more prevalent in soil, plant and stink bugs sampled from organic fields (41% soil, 22% plant, 9% bugs) compared to conventional fields (17% soil, 8% plant, 2% bugs). All plant parts were colonized by B. bassiana, but a significantly higher occurrence of this fungus was found in roots (9%) compared to stems (6%), leaves (4%) and pods (2%) in organic fields. In conventional fields there was a significantly higher occurrence of B. bassiana acquired from root (4%) and stem (3%) compared to leaves (1%) and pods (1%). Mating type PCR assays revealed that each of the isolates carried single mating types, with frequencies of 146/150 (MAT1) and 4/150 (MAT2), indicating limited potential for recombination. Our findings show that B. bassiana occur naturally as endophytes in bean fields in Cuba and contribute to a better ecological understanding of B. bassiana in agriculture.

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Abstract

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

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Abstract

The estrogenic mycotoxin zearalenone (ZON) produced by some Fusarium spp. causes reproductive problems and hyperestrogenic syndromes in mammals. In an effort to elucidate the molecular pathways of ZON production, we present a comparative real-time quantitative polymerase chain reaction expression study of seven contiguous genes in the ZON biosynthetic cluster on sterile rice and during wheat and oat infection. Under ZON production on rice, the polyketide synthase (PKS) genes PKS4 and PKS13, alcohol oxidase FG12056 gene, and transcriptional regulator FG02398 gene showed similarly upregulated patterns, whereas the nonribosomal peptide synthetase (NPS) FG02394, the K+ channel beta subunit FG12015, and the protein kinase FG02399 displayed a variant pattern. During the same time period under wheat infection when no ZON was produced, the PKS genes and the NPS were downregulated relative to rice whereas the K+ channel beta subunit gene FG12015 was markedly upregulated, suggesting that it may play a role in the infection process. This is the first expression study of ZON biosynthetic genes in planta. The results give insight into the regulation and activities of the ZON gene cluster under different experimental systems and suggest a connection between ZON and a K+ channel that could reveal a novel function for ZON in Fusarium spp.

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Abstract

The Fusarium genus includes devastating plant pathogenic fungi that cause diseases in cereals around the world. They produce several mycotoxins, including the estrogenic compound zearalenone. To better understand the molecular mechanisms determining zearalenone production, we performed differential display RT-PCR under conditions where Fusarium graminearum and F. culmorum produced high amounts of zearalenone. We found 133 expressed sequence tags (ESTs) and 54 of these were considered to be up-regulated during high zearalenone production. Several of the ESTs were confirmed to be up-regulated by real-time qPCR, but none showed any significant down-regulation in the zearalenone negative mutant Delta PKS4-T9, or were similar to typical gene expression patterns of previously described zearalenone-related genes. Some of the up-regulated ESTs were similar to genes involved in secondary metabolite production, lipid metabolism, transcriptional activation, provision of precursors, signal transduction, transport or detoxification. Several of the ESTs were also located adjacent to one another in the genome and therefore might represent genes involved in the same biosynthetic pathway. Members of six such putative pathways could be found. All sequences were compared to the MIPS F. graminearum Genome Database to verify autocalled gene predictions experimentally and to introduce new exons and gene structures.