Erik Lysøe

Forsker

Divisjon for bioteknologi og plantehelse

Bioteknologi og molekylær genetikk

(+47) 997 13 274
erik.lysoe@nibio.no

Sted
Ås - Bygg H7

Besøksadresse
Høgskoleveien 7, 1433 Ås

Sammendrag

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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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.

Sammendrag

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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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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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.

Sammendrag

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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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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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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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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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.

Sammendrag

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.

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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.

Sammendrag

Fusarium-toksiner i korn kan utgjøre en helserisiko for mennesker og dyr. For å kunne redusere dette problemet er det viktig å forstå hvordan soppinfeksjonen skjer og når, hvordan og hvor mye soppene produserer av de forskjellige toksinene. Det er nødvendig å utvikle gode metoder for å kunne detektere og kvantifisere toksinene, samt modeller for å kunne forutsi sannsynligheten for at kornet er kontaminert med mykotoksiner.