Early detection and control of toxigenic fusarium species and ochratoxigenic fungi in plant products

This result is a PCR method to detect the main lineage within Fusarium verticillioides, the main species responsible for fumonisin contamination of crops, particularly maize, and an important maize pathogen. This method allows detection in fungal cultures and DNA extracted from cereal or other commodities samples. This method can be performed in the same assay than the previous result. It is highly sensitive since it is based on multycopy sequence. The method will be published within peer-reviewed scientific journal. The method has direct commercial use as a PCR method or further developed in hybridisation based kits. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The result is a method for identification of diagnostic sequence motifs from a matrix of related DNA sequences. It builds on a four step approach: sequence alignments, character based phylogenetic analysis, mapping of features of particular interest and relevance (host specificity, toxin profiles, geographical origin, pathogenicity), and finally tracing the diagnostic sequence motifs from the phylogenetic tree(s). The method can be used to develop reliable diagnostic assays for practically any group of biological organisms, provided sufficient DNA sequence data is available. Mining, exploring and exploiting the present large sequence databases, is a logical step in the post genomic area. The approach is described and exemplified in scientific papers to be published in inter nation peer-review journals. The method has no direct commercial use potential but may be used as a tool for development of methods that can be commercialised. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

This result is a PCR method to detect the main species responsible for fumonisin contamination and for several diseases in crops, particularly maize. This method allows detection in fungal cultures and DNA extracted from cereals or other commodities. It is highly sensitive since it is based on a multicopy sequence. The method will be published within peer-reviewed scientific journal. The method has direct commercial use potential as a PCR method and to be developed in hybridisation based kits. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The method is based on a pair of PCR primers which was designed upon sequence information of a fragment specifically encountered in AFLP patterns prepared from genomic DNA of Aspergillus ochraceus. The fragment was sequenced and a pair of primers was designed. PCR using the said primer pair (OCA-V/OCA-R) yielded a 258bp product with genomic DNA isolated from various strains of A. ochraceus but not with DNA isolated from other closely related fungal species. The system was optimised to result in highest sensitivity and specificity. It may be commercially exploited in applications where this ochratoxin a producing fungus can be detected in pure cultrures and in contaminated samples, e.g. green coffee. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The result is a set of PCR methods to detect the major Fusarium species responsible for causing head blight of small grain cereals and contaminating grain with mycotoxins. The species include F. sporotrichioides, F. langsethiae, F. armeniacum, F. acuminatum and F equiseti. The method has been developed to permit detection of these species, within cereal plant tissues and other matrices, using a single PCR protocol. The protocol is identical to a previously developed method to detect additional Fusarium species, F. culmorum, F. graminearum, F. avenaceum and F. poae. All the significant Fusarium pathogens of cereals occurring in Europe can be detected with this highly sensitive, specific method using a single universal protocol. The method has revealed the presence of F. langsethiae across Europe whereas it had previously only been identified from Scandinavian samples. The method will be published within peer-reviewed scientific journals. Licence agreements are foreseen with laboratories both within and outside Europe. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

A Real Time RT PCR system has been set up against a putative polyketide synthase gene involved in ochratoxin biosynthesis of P. nordicum. It was possible to monitor and quantify the expression of this gene in vitro and in a food matrix like wheat. Expression of this gene could be detected about 36 h before the first ochratoxin could be determined analytically. The system is very specific and can be used to monitor the induction of an ochratoxin biosynthetic gene under relevant environmental conditions. The system may be helpful to identify critical control points (CCP´s) in HACCP concept aginst P. nordicum. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

Fusarium proliferatum, F. subglutinans and F. verticillioides are the most important Fusarium species occurring on maize worldwide, capable of producing a wide range of mycotoxins which are a potential health hazard for animals and humans. The ribosomal internal transcribed spacer and a portion of the calmodulin gene were sequenced and analysed in order to design species-specific primers useful for diagnosis. The primer pairs were based on a partial calmodulin gene sequence. Three pairs of primers (PRO1/2, SUB1/2 and VER 1/2) produced PCR products of 585, 631 and 578bp for F. proliferatum, F. subglutinans and F. verticillioides, respectively. Primer specificity was confirmed by analyzing DNA of 150 strains of these species, mostly isolated from maize in Europe and USA. The sensitivity of primers was 12.5pg when the pure total genomic DNA of each species was analysed. The developed PCR assay should provide a powerful tool for the detection of toxigenic fungi in maize kernels. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The result is a set of PCR methods to detect Fusarium species that produce trichothecenes and/or enniatins. Assays to the gene encoding trichodiene synthase provide generic detection of trichothecene producing species while assays to the gene encoding enniatin synthetase provide generic detection of enniatin producing species. Assays to the gene tri4, encoding the second step in trichothecene biosynthesis broadly differentiate between producers of type A and type B trichothecenes, the former being significantly more toxic to humans than the latter. The result also includes assays to differentiate between isolates that produce nivalenol and those that produce deoxynivalenol (both type B trichothecenes). The former is significantly more toxic to humans than the latter. This set of assays provides a powerful tool to incorporate into crop disease management and risk assessment models for mycotoxin occurrence across Europe and elsewhere. The set of assays has been developed to permit highly sensitive detection of species able to produce the relevant toxins, within cereal plant tissues and other matrices. The method will be published within peer-reviewed scientific journals. Licence agreements are foreseen with laboratories both within and outside Europe. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The result is a PCR method to detect the main fumonisin producing Fusarium species responsible for fumonisin contamination of crops and food products (Fusarium verticillioides, Fusarium proliferatum and Fusarium nygamai). This method allows detection in fungal cultures and DNA extracted from cereal or other commodities. This method can be further developed in hybridisation based kits and in quantitative PCR assays. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

Immunodiagnostic methods for OTA in coffee: The methods of analysis to be employed for the detection of ochratoxin A are not specified by the EU legislation. Antibodies to OTA were produced and assays with a greater sensitivity than those currently available were developed. These antibodies were successfully incorporated into a rapid one step LFD assay, one application of this assay is the rapid detection of ochratoxin A in coffee. Immunodiagnostic methods for OTA in wine: Antibodies to OTA were produced and assays with a greater sensitivity than those currently available were developed. These antibodies were successfully incorporated into a rapid one step LFD assay, one application of this assay is the rapid detection of ochratoxin A in wine. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The result is a modular DNA array system for multiplex detection of Fusarium species. The system can be expanded to include additional taxa, including non-Fusarium species. The system is built on technology owned by Eppendorf Array Technology (EAT), and is developed in close collaboration with EAT and the University of Namur, Belgium. Specific hybridisation probes are developed on the basis of unique sequence motifs identified from character based phylogenetic analyses of one or more genes. The gene(s) can be PCR amplified with "universal" primers, and the resulting population of amplification products is analysed qualitatively by array hybridisation. Diagnostic probes will be the intellectual property of the developers, and the EAT technology will be used to provide cheap and affordable arrays and readers for diagnostics. The result will provide a fast, reliable and affordable alternative to conventional microbiological testing (culturing, microscopy) and qualitative PCR (specific primers, gel electrophoresis) and may be particularly useful to screen agricultural crops for presence of potential pathogens and toxigenic fungi. The system is likely to be further developed into a (semi)quantitative system. The system is under expansion and optimisation and will be subjected to large scale validation prior to commercialisation. Licence agreements are foreseen with laboratories in- and outside Europe. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

A molecular PCR method for detection, quantification and differentiation of ochratoxigenic Penicillia has been developed. It differentiates between P. verrucosum and P. nordicum. Both species are able to produce ochratoxin and are morphologically very similar. For differentiation two primer pairs are used giving rise to two specific fragments for P. nordicum, but only one fragment for P. verrucosum. Based on the specific fragment for P. nordicum a quantitative Real Time PCR system has been developed. The system is very robust and it has been shown that it is functional in food matrices like wheat. It could be used to monitor the growth kinetics of P. nordicum in wheat. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The method uses a specific lysis buffer and commercially available DNA extraction columns to prepare genomic DNA from agar plugs cut from a living fungal culture. DNA is pure enough to be directly used as template in PCR. Time needed for DNA extraction is less than 10 min per sample. The method described was demonstrated to be useful in connection with method 9092 for identification of pure cultures of Aspergillus ochraceus. The method may be useful in several similar applications when incorporated into a PCR assay system for fungal organisms. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

Influence of culture parameters (trace metals, carbon source, nitrogen source) on the production of ochratoxin A in cultures of A. ochraceus has been intensively studied. No correlation between biomass production and OTA biosynthesis was observed under neither condition. OTA production depended on initial pH of the culture medium at inoculation. Production of OTA was observed reproducibly at an initial pH of 6.5 whereas no OTA was produced in cultures inoculated at pH 5.0. Biomass production was not depending on the initial pH at inoculation. Observations could be used to trigger production of OTA in a non-producing culture by shifting pH to 6.5 during cultivation. Onset of toxin production was observed with a time delay of about 20 h after pH shift. The result has no direct commercial application but has serious impact on basic research in the field of biosynthesis of OTA as well as on studies of its genetics and regulation. A practical output from this result may be that processing of food and feed in which OTA production by A. ochraceus might be critical, should be run under OTA suppressive conditions, e.g. at pH lower than 5.0 at start of the process. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

A cDNA-AFLP approach was developed to identify differentially regulated genes in mycotoxin producing and non-producing growth stages of Fusarium proliferatum (Gibberella fujikuori MP-D). By using 160 PCR-primer combinations a number of fragments, which showed strikingly different intensities depending on growth stage were cloned, subjected to Northern analysis and sequenced. Two fragments, amplified from genes up-regulated during spore germination gave significant sequence homology to an amino acid trasporter gene from Neurospora crassa and a GAL4-like transcriptional activator, respectively. Among the cDNAs derived from late growth stage transcripts, fragments of genes involved in polyol metabolism and cell cycle regulation, respectively were identified. This research also resulted in the isolation of additional cDNAs (without significant homologies to known sequences), which clearly differentiated between mycotoxin producing and non-producing growth stages of the fungus. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

The result is a real-time PCR method for quantification of DNA derived from the T-2/HT-2 producing Fusarium species F. langsethiae and F. sporotrichioides. The method determines a relative ratio between cereal derived DNA and DNA from the two fungal species. The method can be used to determine the infection level in a wide range of cereal matrices, and may prove particularly useful for the farmer at pre-harvest, and for food and feed manufacturers and retailers, and competent authorities at post-harvest. The method is much faster and several times more sensitive than conventional microbiological methods, and a high degree of correlation has been observed between infection levels (determined with the method) and toxin levels (determined by chemical analyses). The method will be published in a peer-reviewed scientific journal. A patent covering the specific oligonucleotides (primers and probes) is foreseen. Manufacturing and licence agreements are foreseen with laboratories in- and outside Europe. More information on the DETOX-FUNGI project can be found at http://detox.ba.cnr.it.

A robust, multiplex PCR assay was developed for the rapid identification of mating type in the Gibberella fujikuori species complex. The diagnostic value of this technique was clearly proven on eight pairs of mating type tester strains (A-H) of G. fujikuroi, as well as on a number of field isolates. In order to assess the potential for mating in Fusarium species with no known sexual stage, diagnostic PCR-primers were developed and tested on a number of strains of F. avenaceum, F. camptoceras, F. cerealis, F. culmorum, F. poae, F. semitectum and F. sporotrichioides. All strains contained one of the two conserved MAT regions. RT-PCR experiments showed, that the mating type genes are transcribed in these asexual fungi. These techniques help to select potential partners for laboratory crossing experiments. Assessing the potential for mating by toxigenic strains of Fusarium would increase our understanding of the genetic mechanisms that maintain intraspecific diversity, and biological and evolutionary species integrity. The frequency of sexual reproduction also is an important parameter when designing strategies to control plant pathogens, since these strategies are often different for clonally and for sexually reproducing organisms. Three papers and six conference presentations have been published in this work. The method can be used by breeding companies and plant protection services. More information on the DETOX-FUNGI project can be found at: http://detox.ba.cnr.it.

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