Arne Holst-Jensen

Phylogeny and toxigenic potential is correlated in Fusarium species as revealed by partial translation elongation factor 1 alpha gene sequences

Partial translation elongation factor 1 alpha (TEF-1alpha) gene and intron sequences are reported from 148 isolates of 11 species of the anamorph genus Fusarium; F. avenaceum (syn. F. arthrosporioides), F. cerealis, F. culmorum, F. equiseti, F.flocciferum, F. graminearum, F. lunulosporum, F. sambucinum, F. torulosum, F. tricinctum and F. venenatum. The sequences were aligned with TEF-1alpha sequences retrieved from 35 isolates of F. kyushuense, F. langsethiae, F. poae and F. sporotrichioides in a previous study, and 39 isolates of F. cerealis, F. culmorum, F. graminearum and F. pseudograminearum retrieved from sequence databases. The 222 aligned sequences were subjected to phylogenetic analyses using maximum parsimony and Bayesian Markov Chain Monte Carlo maximum likelihood statistics. Support for internal branching topologies was examined by Bremer support, bootstrap and posterior probability analyses. The resulting trees were largely congruent. The taxon groups included in the sections Discolor, Gibbosum and Sporotrichiella sensu Wollenweber & Reinking (1935) all appeared to be polyphyletic. All species were monophyletic except F. flocciferum that was paraphyletic, and one isolate classified as F. cfr langsethiae on the basis of morphology that grouped with F. sporotrichioides. Mapping of toxin profiles, host preferences and geographic origin onto the DNA based phylogenetic tree structure indicated that in particular the toxin profiles corresponded with phylogeny, i.e. phylotoxigenic relationships were inferred. A major distinction was observed between the trichothecene and non-trichothecene producers, and the trichothecene producers were grouped into one clade of strictly type A trichothecene producers, one clade of strictly type B trichothecene producers and one clade with both type A and type B trichothecene producers. Furthermore, production of the type A trichothecenes T-2/HT-2 toxins are associated with a lineage comprising F. langsethiae and F. sporotrichioides. The ability to produce zearalenone was apparently gained parallel to the ability to produce trichothecenes, and later lost in a derived sublineage. The ability to produce enniatins is a shared feature of the entire study group, with the exception of the strict trichothecene type B producers and F. equiseti. The ability to produce moniliformin seems to be an ancestral feature of members of the genus Fusarium which seems to have been lost in the clades consisting of trichothecene/zearalenone producers. The aims of the present study were to determine the phylogenetic relationships between the different species of Fusarium commonly occurring on Norwegian cereals and some of their closest relatives, as well as to reveal underlying patterns such as the ability to produce certain mycotoxins, geographic distribution and host preferences. Implications for a better classification of Fusarium are discussed and highlighted.

Testing for genetically modified organisms (GMOs): Past, present and future perspectives

This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

A quantitative TaqMan® MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci.

Here we present the development and first validation of a TaqMan minor groove binder (MGB) real-time polymerase chain reaction (RT-PCR) method for quantitative and highly specific detection of Aphanomyces astaci, the causative agent of crayfish plague. The assay specificity was experimentally assessed by testing against DNA representative of closely related oomycetes, and theoretically assessed by additional sequence similarity analyses comparing the primers and probe sequences to available sequences in EMBL/GenBank. The target of the assay is a 59 bp unique sequence motif of A. astaci found in the internal transcribed spacer 1 of the nuclear ribosomal gene cluster. A standard curve for quantification was established by setting up a four-fold dilution series with genomic A. astaci DNA. The absolute limit of detection (LOD(abs)), defined as the lowest concentration yielding a false negative probability<5% was found to be approximately 5 PCR forming units (PFU<or=target template copies) equivalent to less than one A. astaci genome. The absolute limit of quantification (LOQ(abs)) was experimentally established as 10 times the LOD(abs). Assay performance was also assessed with samples of naturally infected and non-infected susceptible crayfish (Astacus astacus) and carrier crayfish (Pacifastacus leniusculus). The benefits and limitations of the method are discussed, and guidance to practical application and interpretation of analytical results is provided.

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20+ species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.

MOLECULAR PHYLOGENY AND EVOLUTION OF MONILINIA (SCLEROTINIACEAE) BASED ON CODING AND NONCODING RDNA SEQUENCES

The nuclear internal transcribed spacers, the 5.8S subunit, ~560 bp of the small subunit, and ~320 bp of the large subunit of the nuclear ribosomal DNA repeat from 17 species of Monilinia and eight species of closely related genera were sequenced. Phylogenies were constructed using maximum parsimony. The results support the hypothesis that Monilinia is not monophyletic. A fundamental distinction was found between the section Junctoriae and the section Disjunctoriae. Four evolutionary lineages were identified within the Disjunctoriae: one species on Crataegus, one group of species on dry stone fruits of rosaceous hosts, one group of species on capsular fruits of ericaceous hosts, and one group of species on sweet berry fruits of ericaceous hosts. Comparisons between branching topologies of hosts and Monilinia species suggest that although cospeciation among hosts and parasites has been the rule, several host jumps have taken place. Sclerotinia pirolae was determined to be a true member of the Disjunctoriae. The closest taxon groups to the Junctoriae were found to be Botrytis and Sclerotinia, with Ciborinia being the closest taxon group to the Disjunctoriae. There is evidence of an increased rate of ssrRNA evolution in the lineage of species that attack ericaceous berries.

Gene stacking in transgenic plants: towards compliance between definitions, terminology, and detection within the EU regulatory framework.

The combination or stacking of different traits or genes in plants is rapidly gaining popularity in biotech crop production. Here we review the existing terminology regarding gene stacking in plants, and its implications in relation to genetics, biosafety, detectability and European regulations. Different methods of production of stacked gene traits, as well as the status of their cultivation and approval, are reviewed. Related to the different techniques of transformation and production, including classical breeding, and to differences in global authorization and commercialization practices, there are many types, definitions, and perceptions of stacking. These include: (1) stacking of traits and (2) stacking of events, which are the most widely accepted perceptions of stacking, and (3) stacking of genes, which from the analytical and traceability point of view may be a more appropriate perception. These differences in perceptions and definitions are discussed, as are their implications for analytical detection and regulatory compliance according to (in particular) European Union (EU) regulations. A comprehensive terminology regarding gene stacking with regulatory relevance is proposed. The haploid genome equivalent is proposed as the prevailing unit of measurement at all stages throughout the chain, in order to ensure that terminology and definitions of gene stacks are adapted to analytical detection, traceability, and compliance with EU regulations.

The postfire discomycete Geopyxis carbonaria (Ascomycota) is a biotrophic root associate with Norway spruce (Picea abies) in nature

The hypothesis that the postfire discomycete Geopyxis carbonaria (Ascomycota, Pezizales, Pyronemataceae) has a biotrophic association with roots of Norway spruce (Picea abies) in nature was tested by isolation of fungal strains from fresh, brown, smooth mycorrhiza‐like root tips of Norway spruce collected from below the depth of detrimental heat penetration in a postfire site. The morphology of seven culture isolates originating from the smooth mycorrhiza‐like root tips of two different spruce trees was congruent with the morphology of axenic culture isolates obtained from ascospores of G. carbonaria. DNA sequences of the nuclear ribosomal internal transcribed spacers ITS1 and ITS2 from these root‐derived cultures and the ascosporic G. carbonaria culture isolates were found to be identical, further supporting the conclusion that the isolates were conspecific. The extensive ascocarp and ascospore formation of G. carbonaria which succeeds a forest fire may be explained in terms of a fungal escape from a moribund tree associate. Possible ecological adaptations of G. carbonaria to the pre‐ and postfire community are discussed.

Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments.

Aphanomyces astaci, a specialised parasite of North American freshwater crayfish, is the disease agent of crayfish plague that is lethal to European freshwater crayfish. The life cycle of A. astaci has been inferred from experimental laboratory studies, but less is known about its natural sustainability and ecology. To address such questions, tools for monitoring of A. astaci directly in aquatic environments are needed. Here, we present an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder real-time PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen.

Microarray-based method for detection of unknown genetic modifications

BackgroundDue to the increased use of genetic modifications in crop improvement, there is a need to develop effective methods for the detection of both known and unknown transgene constructs in plants. We have developed a strategy for detection and characterization of unknown genetic modifications and we present a proof of concept for this method using Arabidopsis thaliana and Oryza sativa (rice). The approach relies on direct hybridization of total genomic DNA to high density microarrays designed to have probes tiled throughout a set of reference sequences.ResultsWe show that by using arrays with 25 basepair probes covering both strands of a set of 235 vectors (2 million basepairs) we can detect transgene sequences in transformed lines of A. thaliana and rice without prior knowledge about the transformation vectors or the T-DNA constructs used to generate the studied plants.ConclusionThe approach should allow the user to detect the presence of transgene sequences and get sufficient information for further characterization of unknown genetic constructs in plants. The only requirements are access to a small amount of pure transgene plant material, that the genetic construct in question is above a certain size (here ≥ 140 basepairs) and that parts of the construct shows some degree of sequence similarity with published genetic elements.