European agriculture is confronted with a growing demand of consumers for organic vegetables. For example, a leading supermarket chain in the UK has currently about over 400 organic products for sale, 60 of which are vegetables, fruits or salads. An estimated 2% of the total European vegetables (e.g. 6% to 10% in Denmark) and fruit market is now organically produced, and this number is expected to triple in the next few years based on an annual growth rate of up to 10% to 40% in Europe. These data are further underlined by the fact that the UK with its annual organic food market retail value is among the highest in the world at between 409 million euros and 455 million euros. The UK depends for 70% of its organic food supplies on imports. However, Austria is the European Country with the largest share of organic production in total agricultural output with 8% of its total agricultural area fully converted to organic production systems by the end of 2000. Consequently, a decrease in these above figure based on undesirable components such as mycotoxins, fungal poisons need to be prevented by setting suitable strategies in place to ensure a safe organic food supply by developing detection methods, anticipating mycotoxin risks, tracing the sources of contaminants in the food production chain, and eliminating the risk factors.
The overall objective of this project is to develop strategies to ensure a safe organic food supply by developing detection methods, anticipating mycotoxin risks, tracing the sources of contaminants in the food production chain, and eliminating the risk factors. The research will be done with the model carrot - Alternaria, the latter fungus being a known producer of harmful mycotoxins.
In the first year the work mainly focused on developing the methods needed throughout the project, in particular the methods for detection of the Alternaria fungus and its metabolites.
Suitable methods for detection of the mycotoxin-producing A. alternata and A. radicina on various types of carrot plant material, in particular seeds and roots, were developed and tested. Based on the results obtained, it was decided that for detection of A. alternata the blotter test will be the basis. A deep freezing step (overnight incubation of imbibed seeds at -20 C) will be included when testing seeds, in order to prevent germination. Seedling and root material can be placed directly on wet blotters. Evaluation will take place after 7 days of incubation at 20°C and a 12-h nUV cycle, based on morphological characteristics of conidia and conidiophores. For detection of A. radicina, seeds or samples of seedlings and roots will be plated on the selective ARSA (Alternaria Radicina Selective Agar) medium. For testing seedling and root material, the fungicide Botran can be omitted from the medium. Evaluation for fungal growth will take place after 7 and 14 days of incubation at 28 C. A PCR-based assay was developed for detection of both Alternaria species as well, because both the traditional deep-freeze-blotter method and plating on selective medium are time consuming methods and may not be sensitive enough. Specific primers for detection and identification of the Alternaria species on carrot seeds and roots were designed, based on sequences of ribosomal gene repeats of 45 different Alternaria isolates, and tested. The primers were sensitive and could differentiate between the three Alternaria species occurring on carrot, i.e. A. radicina, A. alternata and A. dauci. A. alternata and A. radicina could be detected in DNA isolated from carrot material (seeds and infected root material) applying the specific primers, even at low infection levels. Comparison with the blotter method and plating on ARSA medium (the latter for A. radicina) by testing naturally infected seed samples and root material, showed that results of the PCR-assay were similar for the detection of A. alternata and A. radicina. A positive correlati on was found between the percentage of seed infection established by the blotter method and the intensity of the amplified, specific product. A small-scale ring test with 5 participating laboratories was performed to evaluate the PCR-based assay developed. The results showed that several participants encountered problems, in particular with the DNA isolation and purification step. This shows that the PCR-based assay is not ready yet for routine testing of seed samples for the presence of Alternaria. According to the results of one project partner, isolating DNA from (infected) carrot root material was much easier to perform. This suggests that the PCR-based assay may be suited for testing of Alternaria contamination of harvested carrot roots.
For various purposes, suited analytical methods for the determination of the principal Alternaria mycotoxins alternariol (AOH), altertoxin I (ATX I), tenuazonic acid (TeA), and radicinin (RAD) were developed. An agar plug method will be applied in future work for preliminary determination of the toxigenicity of various Alternaria isolates, followed by a more extensive study of the toxigenicity by means of TLC and TLC-densitometric methods. Rice grains were shown to be a good natural substrate to study production of the Alternaria mycotoxins under various conditions. TLC and HPLC will used for analysis of this inoculated plant material. For the analysis of carrot plant samples, naturally infected or symptomless, HPLC will be applied. Different extraction and clean-up procedures as well as HPLC conditions had to be developed for the analysis of rice fungal cultures and carrot material respectively. For the analysis of rice fungal cultures, acceptable methods with regard to within-laboratory standard deviation, recovery, and limit of determination are now available. For analysis of carrot material, acceptable methods are available for determination of AOH, ATX I, RAD and AME. TeA could not be determined in the same analysis method, because it was not retained during the clean-up procedure. A separate HPLC method will have to be developed for TeA. Furthermore, the methods will need to be validated by testing spiked carrot material. In addition, a HPLC method for the simultaneous determination of AAL toxins (TA1 and TA2) and fumonisins (FB1 and FB2) in inoculated rice cultures was developed. This method will be used to test whether the Alternaria species occurring on carrot also are able to produce these mycotoxins.
Two tests, i.e. a root slice test and a petiole test, were developed to test carrot accessions for differences in resistance towards Alternaria radicina. The concept protocols are currently under discussion, and will be used in the next years to test various carrot accessions for Alternaria resistance. Accessions with high levels of resistance can be used, outside the scope of the project, in resistance breeding programmes. Furthermore, a method was developed to enable screening of microorganisms from the spermo- and rhizosphere of carrot for their ability to suppress Alternaria. This should lead to the selection of potential biological control agents that can be applied to seeds.