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- Materials and methods
- Conflict of Interest
- Supporting Information
The numerous plant pathogens of the genus Fusarium are responsible for significant losses in crop yield due to both loss of biomass and accumulation of mycotoxins in infiltrated parts. The major toxic compounds synthesized by divergent Fusarium isolates include the following: zearalenone, fumonisins, trichothecenes and their derivatives (D'Mello et al. 1999). While there is a growing body of work documenting biological significance of additional, emergent toxins (e.g. butenolide, fusarins, equisetin, beauvericin and enniatins), their estimated economic and biomedical importance is considerably lower (Desjardins and Proctor 2007).
Notably, the above-mentioned major toxins (fumonisins, trichothecenes, zearalenone and derivative compounds) are frequently not inactivated during food/feed processing and can be present in a masked form (plant-formed conjugates, i.e. glucosides, which can be activated by mammal gut microbiota—e.g. Berthiller et al. 2013; Dall'Erta et al. 2013), increasing health risks to farm animals and humans (Creppy et al. 2002). As more research results are collected, the estimates of health and economical risks (associated with long-term masked mycotoxin exposure) are revised upwards. The updated estimates lead to increasingly restrictive norms for toxin content for food and feed (e.g. European Commission Recommendation 2006/576/EC proposing norms for ochratoxin A, T-2 and HT-2 toxins as well as deoxynivalenol and zearalenone). This only serves to increase a need for efficient and quick methods of assessing possible sources of contamination, preferably by preventing losses in crop yield, via good farming practices including effective fungicide treatments.
The genetic determinants of fumonisin, trichothecene and zearalenone biosynthesis have been characterized in multiple plant pathogenic taxa. Characterization of both core biosynthetic genes (polyketide synthases, trichodiene synthase) and key accessory genes (such as transcription factors or key processing enzymes) enables construction of toxigenicity assays directly targeting the genetic basis of toxin production and accumulation. At the same time (Stepien et al. 2011), the biosynthetic gene alleles exhibit significant interspecific differences, which makes them useful for precise identification of infectious species/populations.
The zearalenone biosynthetic cluster spanning 25 kb of the genomic sequence has been characterized in Fusarium graminearum (Kim et al. 2005), with four principal genes required for toxin biosynthesis (zea1, zea2, zeb1, zeb2) and 3 other genes regulated in conjunction with zeb2 expression patterns (FG02394, FG02399 and FG012015—uncovered by qRT-PCR experiments described by Lysøe et al. (2009)).
Conversely, trichothecene biosynthesis constitutes a multistage process, controlled by at least 12 essential genes, forming a 25-kb-long cluster in F. graminearum (Brown et al. 2001; Kimura et al. 2003). The trichothecene cluster is linked to a key tri5 gene encoding trichodiene synthase, however, four genes segregate at separate loci (notably tri13 and tri14 controlled by a transcription factor encoded by tri10—Tag et al. (2001)). To date, the main cluster has been extensively characterized with numerous studies targeted especially at F. graminearum and F. sporotrichioides species (Kimura et al. 2007), as well as some members of the genus Trichoderma (Cardoza et al. 2011). There is considerable evidence for complex gene relocation scenarios underlying chemotype diversification leading to extant trichothecene type-A- and type-B-producing species (Proctor et al. 2009).
In the past decade, the fumonisin cluster structure (16 gene cluster spanning 42 kb length) has been determined for three toxigenic Fusarium species: F. verticillioides, F. oxysporum (FRC O-1890 strain) and F. proliferatum (Proctor et al. 2008). The interspecies differences between individual biosynthesis-related sequences encompass up to 20% of constituent residues. Notably larger differences are found in gene-flanking regions, an observation which suggests divergent evolutionary paths for cluster copies in different species. Here, the difference in species history and gene phylogeny has been attributed to complex birth/death evolution of the cluster (with independent sorting of copies) and/or horizontal gene transfer events (Proctor et al. 2013). During fumonisin biosynthesis, substitutions of polyketide synthase and/or termination factor can lead to significant changes in the specificity of polyketide condensation for fumonisin analogs (Zhu et al. 2008; Li et al. 2009).
As the broad, genetic basis of the biosynthetic pathway for three major Fusarium mycotoxins is known and multiple exemplar sequences are readily available, it is now possible to develop targeted diagnostic solutions. Through utilizing knowledge about disparate species for the design of degenerate cross-species-specific primers, it is possible to target well-conserved parts of coding sequence (corresponding to conserved parts of protein sequence). Especially for core, secondary metabolite biosynthetic genes, these regions of the coding sequence are unlikely to change in toxin-producing isolates (corroborated by recent evidence for purifying selection in secondary metabolism genes—e.g. Baker et al. 2012).
Current studies on the variability and diversity of the fungal populations make use of various genetic markers, such as the translation elongation factor (tef-1α) and internal transcribed spacer (ITS1/2), employed in assays of the genus Trichoderma (Chaverri et al. 2003; Blaszczyk et al. 2011) and conservative fragments of the genome such as a calmodulin gene (CaM) in Trichoderma and Fusarium populations (Chaverri et al. 2003; Mulè et al. 2004). Also, mitochondrial DNA (mtDNA) is used as a marker of genetic variation. Its relatively short length and the presence of conserved and variable regions allow the identification of closely related species (Ma and Michailides 2007). The sequence of the large subunit of the RNA polymerase II (Hibbett et al. 2007) can also be used to distinguish between divergent phytopathogenic species. Among so many molecular markers, the translation elongation factor (tef-1α) appears to be the most useful in taxonomic studies of fungi, especially in the genus Fusarium (Geiser et al. 2004; Kristensen et al. 2005). Recently, more attention is devoted to markers directly involved in the secondary metabolism (Proctor et al. 2009). Many researchers use genes from the FUM cluster as a good additional marker for phylogenetic and taxonomic studies of the fumonisin-producing Fusarium species (González-Jaén et al. 2004; Baird et al. 2008; Stepien et al. 2011).
The current line of research for the detection of toxigenic species involves simultaneous use of multiple genes belonging to different clusters responsible for toxin production, for example mPCR assays detecting aflatoxigenic, trichothecene- and fumonisin-producing and ochratoxigenic fungal isolates (Rashmi et al. 2013). The recent studies also aim to combine qualitative and quantitative methods for detecting the toxigenic potential. One of the approaches, based on multiplex real-time PCR, is able to detect and quantify mycotoxigenic species in cereal grains with the use of markers targeting the trichothecene synthase (tri5) gene in trichothecene-producing Fusarium sp. isolates, the rRNA gene in Penicillium verrucosum and the polyketide synthase gene (Pks) in Aspergillus ochraceus (Vegi and Wolf-Hall 2013).
The problem addressed in the proposed work was to design and standardize a diagnostic tool allowing the identification of toxigenic Fusarium isolates producing fumonisin B1, trichothecenes and zearalenone. The new protocol is applicable for both in vitro and field samples, with resolution sufficient for direct sequencing of amplified sequences.
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- Materials and methods
- Conflict of Interest
- Supporting Information
In this study, we present a novel approach to detect the toxigenic potential of various phytopathogenic fungi by partially multiplexed, degenerate primers based on the genes essential for biosynthesis of major Fusarium sp. mycotoxins (fumonisins, trichothecenes and zearalenone). Such tools are especially valuable when updated risk assessments concerning fungal toxin contamination lead to more restrictive norms regulating their acceptable levels in food and/or feed. These trends result in an increase in demand for efficient and rapid methods for the detection and assessment of potential sources of contamination which can be used also as a part of decision support systems (DSS). At the moment, DSS are primarily focused on the observation of the occurrence of pathogens on host plants (Evans et al. 2008), spores in the air (Kaczmarek et al. 2009) or the impact of weather conditions on the life cycles of pathogens (Dawidziuk et al. 2012).
The isolates of the F. oxysporum complex constitute a remarkable outlier in the results obtained for fumonisin-producing species. In this case, the trace amounts of fumonisin were found in cultures of several isolates, but mPCR markers were consistently absent. Previous works by Proctor et al. (2008, 2013) demonstrate possible divergent origins of the fumonisin clusters in distinct member species of F. oxysporum and F. fujikuroi complexes. Past research also shows that synthesis of the long reduced polyketide mycotoxins is controlled by accessory genes (i.e. fum8) under a scheme which permits complementation by different core/accessory genes (Zhu et al. 2008; —fum8 complementation for control of biosynthesis). As F. oxysporum is a species with high supernumerary chromosome content (c. 25%; Ma et al. 2010) likely stemming from past horizontal transfers, there is a possibility of different/highly divergent genetic basis complementing biosynthesis of low amounts of fumonisins and/or fumonisin-like compounds in the F. oxysporum complex. Notably, the molecular and morphological identification of isolates can be a grey area in some cases (e.g. newly characterized cryptic species like F. temperatum—Scauflaire et al. 2011; low resolution of broad barcode markers in complexes of related species—Blaszczyk et al. 2011). Current and future research is poised to demonstrate finer splits in the complexes of closely related species, previously characterized as monophyletic species (O'Donnell et al. 2013). The taxonomic identification is supplemented and supported by differences in chemotype and sequence of biosynthesis-related genes from closely related taxa—a process made easier by markers designed for direct sequencing of amplification products. Nevertheless, the problematic results do not apply to the most important economic, toxigenic Fusarium species occurring in cultivated high-yield crops (e.g. maize—F. verticillioides, wheat—F. graminearum, F. culmorum).
In related research, previously carried out by Rashmi et al. (2013), the researchers focused on diverse isolates (mainly toxigenic and non-toxigenic Fusarium, Aspergillus and Penicillium), demonstrating the applicability of multiplex PCR to detect ochratoxin-, fumonisin- and trichothecene-synthesizing isolates. However, Rashmi and co-workers did not attempt to provide a more detailed taxonomic identification of their cultures. In our approach, each pathogenic isolate was obtained by single-spore technique and its species assigned by both morphological and molecular methods. The test can efficiently detect the presence of the marker gene in five hundred picograms of template and about one infected kernel among hundred uninfected seeds and each obtained product can be validated by direct sequencing. Sensitivity on this level can significantly support the farmers for instance in the appropriate and rational use of fungicide treatments in the field. The developed diagnostic approach can directly be used in biological material obtained from the field (infected kernels) without the need for prior cultivation on artificial media. Unfortunately, such analysis is only possible in infected kernels. The DNA isolated from chaffs is not of sufficient quality to give reliable results, likely due to the presence of PCR inhibitors, such as polysaccharides (e.g. dextran sulphate, alginic acid—Demeke and Jenkins 2010). This could be alleviated by improvement in preparation procedures. There is a possibility of further extending the approach to direct quantitative studies of the mycotoxin-producing pathogens which (up to date) are typically focused on detection of specific fungal producers (F. graminearum, P. verrucosum, A. ochraceus) and not on assessing the toxigenic potential grounded in common genetic basis among related but distinct species (Vegi and Wolf-Hall 2013).
The multiplexed PCR assay used in the protocol allows for the detection of toxigenic potential in many species simultaneously and in a standardized way. The resulting quality of optimized PCRs allows for direct sequencing of amplification products. Additionally, the low cost (relative to HPLC analysis) of the assay allows easy coupling with simple, targeted techniques (e.g. ELISA) to quickly confirm presence of a specific toxin. Thus, the method can be easily adapted as early warning against mycotoxin contamination allowing much more effective application of fungicides and can serve as supplement conventional mycotoxin detection techniques. What is also very important is that, through the usage of the direct sequencing of the PCR products, the results from individually cultivated isolates should allow easy characterization of variability and phylogeny of infecting pathogen populations.