European agriculture faces a growing demand by consumers for organic products. Retailers and supermarkets continually introduce new fresh or processed organic products.
However, undesirable components such as mycotoxins, fungal poisons which can produce toxic syndromes in humans and animals, pose a continuous challenge to the safety and quality of agricultural products. In organic production systems there is an increased risk of contamination with mycotoxins, due to the lack of adequate control measures for fungal infection. In order to ensure a safe organic food supply, such mycotoxin risks have to be minimised.
A shared-cost RTD project entitled "Safe organic vegetables and vegetable products by reducing risk factors and sources of fungal contaminants throughout the production chain: the carrot - Alternaria model" was funded under the Fifth Framework Programme of the European Commission, Thematic programme Quality of Life and Management of Living Resources, key action Food, Nutrition and Health. Seven partners from five countries cooperate in this project (QLK1-1999-0986), which ran from January 2000 to June 2004.
The objective of the project was to develop strategies for a safe organic food supply by developing detection methods, identifying mycotoxin risks in the production chain, determining the critical control points, and developing preventive measures. The research was performed using the model system carrot – Alternaria. Carrot is one of the most popular organic vegetables bought by European consumers and is an important component of various organic food products, such as baby food and vegetable juices. The fungus Alternaria occurs on a wide variety of crops and is a known producer of harmful mycotoxins. It can be found throughout the whole carrot production chain, from seed to end-products.
The project consisted of eight major work packages, aimed at:
- Establishing rapid and precise methods for detection and quantification of Alternaria infection and mycotoxin contamination
- Determining the physiological and genetic basis of production and accumulation of Alternaria mycotoxins
- Monitoring mycotoxin production during carrot crop production, storage and processing, to determine critical control points in the production chain, and to enable risk assessment
- Developing tools to aid breeding for resistance to Alternaria diseases
- Developing control measures to improve the production of high quality, disease-free starting material under organic conditions
- Developing alternative seed treatments to reduce seed infection and to increase stress tolerance of seeds and seedlings
- Optimising storage conditions and developing post-harvest treatments to prevent Alternaria root rot and mycotoxin accumulation during storage
- Developing a total strategy to reduce the risk of mycotoxins in organic vegetables and derived products
The results of the project will aid in the production of safe organic carrots and processed products. This will contribute to improvement of the health of the European consumer and the promotion of organic farming. The obtained results can be applied to other organic crops and food products, such as cabbage, rice, cereals and oils of sunflower and rapeseed, where similar problems with Alternaria metabolites may occur. The results of this project will be used by EU regulatory and standardisation bodies, food authorities, seed producers, organic armers and industrial end-users.
Results and milestones achieved:
Establishing methods for detection of Alternaria on carrot
Suitable methods for detection of the mycotoxin-producing A. alternata and A. radicina on carrot material, in particular seeds and roots, were established. For detection of A. alternata, the so-called blotter test will be the standard, which allows determining the presence of the fungus based on morphological characteristics of the fungus. For detection of A. radicina, samples of plant material will be plated on a selective medium (ARSA, Alternaria Radicina Selective Agar), and examination for fungal growth takes place after 7-14 days of incubation. A PCR-based assay was also developed for detection of both Alternaria species, because both the blotter test 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 and shown to be sensitive and able to differentiate between the 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 infected carrot material, but comparison with the results of the blotter method and plating on ARSA medium showed that results of the PCR-assay were not always reliable. Further optimization of the method will have to take place outside the scope of this project.
Establishing methods for analysis of Alternaria mycotoxins
HPLC-based analytical methods for simultaneous determination of the principal Alternaria toxins altertoxin I (ATXI), alternariol (AOH), alternariol methyl ether (AME), tenuazonic acid (TeA) and radicinin (RAD) in rice cultures of A. radicina and A. alternata were developed. 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. To monitor mycotoxin accumulation in carrots, it was necessary to clean up the extracts on a polymeric based cartridge and a C18 cartridge for the determination of TeA and RAD + ATXI + AME, respectively, before HPLC analysis of the two purified extracts. The protocol for determination of radicinin, tenuazonic acid, altertoxin I, and alternariol methyl ether in carrots by high performance liquid chromatography and uv diode array detection is now available in CEN format. Recovery, repeatability, and the limit of quantification were acceptable for all toxins tested with the exception of the recovery of RAD, which was only 35%.
Establishing the basic understanding of Alternaria mycotoxin production
Various strains of A. radicina and A. alternata were isolated from seed samples of carrot, wild carrot, parsley and celery collected in various countries, and 28 A. radicina isolates and 23 A. alternata isolates were tested for their virulence and mycotoxin production. A. radicina isolates from carrot and wild carrot were more virulent than isolates from parsley and celery, but differences between A. alternata isolates were not significant. With regard to mycotoxin production, A. radicina isolates of carrot, celery and parsley could produce high amounts (> 1000 µg/g) of RAD when cultured on rice, and A. alternata isolates from carrot, wild carrot, and parsley produced high amounts of TeA and lesser amounts of AOH, AME, and ATX-I. In inoculated carrot slices, AOH and AME were always present, but fortunately TeA was only produced by a few A. alternata isolates and at low levels.
Compared to rice cultures, A. radicina strains showed a different metabolite profile when grown on carrot slices, meaning that the same fungus produced other compounds. In fact, one unknown compound could be identified as epi-radicinol (epi-ROH). This compound was shown to be non-toxic in Artemia salina and erythroblastoid human cell line testing and is, so far, not considered to be a threat to human health. Most of the A. radicina strains isolated from carrot produced epi-ROH after inoculation, besides ROH and RAD. The latter two were usually detected at lower levels.
The incubation temperature affected the mycotoxin production in carrot; in general the production was the highest at 20 °C, and at 1 °C no toxins were produced up to 1 month of incubation. This suggests that storage of Alternaria-contaminated carrots after harvest in cooled warehouses at low temperatures does not pose a risk with regard to toxin production during storage.
Monitoring mycotoxin accumulation in the production chain
On 3 locations in Europe (DK, NL, and F) organic carrots were produced from the same seed batches containing Alternaria contamination. The place of production had a large influence on the contamination of the carrot roots produced. In two subsequent seasons, production in NL resulted in high contamination levels of A. radicina, and production in F in high levels of A. alternata; production in DK gave intermediate results. A correlation between the initial seed contamination and edible root contamination has been established, suggesting that disease-free starting material is important. With regard to the analysis for mycotoxins, no A. alternata toxins were found in 254 organic carrot samples collected at harvest and during storage in 2 subsequent growing seasons. With respect to A. radicina toxins, only 3 out of 254 samples were contaminated with epi-ROH, of which one also contained RAD. Occasionally, carrot samples showing black rot symptoms caused by A. radicina were found contaminated with epi-radicinol and, less frequently, radicinin and radicinol. These 3 compounds have been tested and seem to be of a more phytotoxic nature than harmful to humans and animals.
Samples of carrot containing products, such as baby food and vegetable juices, of both organic and conventional origin were tested as well for presence of Alternaria toxins, but none could be detected in 87 carrot containing products. In case Alternaria toxins would have been present in carrots used for carrot juice production, it was shown that the production process, which included a blanching or steaming step, would reduce the amount of toxins considerably.
In post-harvest storage experiments at various temperatures, the mycotoxins which are considered to be the most risky for human health, i.e. tenuazonic acid, altertoxin-I, alternariol, and alternariol methyl ether, were not detected in artificially inoculated carrots. Accumulation of RAD (2.5 µg/g) and epi-ROH (1 µg/g) occurred after 17 weeks storage at 1°C in A. radicina inoculated carrots. These levels increased to 18 µg/g and 62 µg/g respectively, after continued storage for 1 month at 10°C and 3 weeks at 20°C at 95-100% relative humidity. These results suggest that storage of carrots at low temperatures, e.g. in cooled warehouses, diminishes the risk of Alternaria mycotoxin production.
Control measures to prevent the introduction of Alternaria in the carrot production chain and which are acceptable to organic farmers were studied. Bioassays to test carrot genotypes for differences in resistance towards A. radicina were developed, and will be used to select carrot accessions with improved levels of resistance for use in breeding programmes. Several accessions which vary in susceptibility were found, and can now be used as controls in future testing. Furthermore, antagonistic micro-organisms have been isolated from carrot habitats and were tested for their efficacy of controlling Alternaria. Coating or priming seed with selected Clonostachys isolates effectively controlled seed borne Alternaria spp. and improved field establishment. A combination of bioproming and a hot water treatment, generally enhanced the efficacy of Alternaria control on seeds. A plant-based compound (Biosept), which inhibited the growth of Alternaria in vitro, improved seedling emergence in green-house tests, and its efficacy was in general comparable to that of the fungicide control iprodione.
To prevent rot of carrots during storage due to Alternaria spp., post-harvest treatments with isolates of Geotrichum candidum isolated from cheese were tested, but were not able to suppress Alternaria present on the carrots. The same biocontrol organisms as selected for seed treatments could not be used due to their cytotoxic activity. Other treatments and conditions, such as storage at low temperatures and removal of damaged carrots, should be applied as preventive measures.
In seed and carrot production experiments the epidemiology of the disease was studied, and various measures based on culture conditions, monitoring steps, and treatments tested for effectiveness. Moreover, the critical control points in the organic production chain of carrots and carrot-derived products were determined. An overall strategy, based on these critical control points and the useful tools developed in all workpackages for controlling Alternaria, was devised, aimed at reduction of the sources of contaminants in the production chain of organic carrots. This resulted in a short manual consisting of 2 flow diagrams of the production process with the critical control points, accompanying explanations, and 2 decision tables. The decision tables show for each step in the production chain information on the recommended production system, what kind of preventive measures may be taken, the necessary monitoring actions, the interpretation of the monitoring results (including proposed threshold levels), and the recommended actions that should be taken based on the outcome of the monitoring. This instruction manual can be used by (organic) carrot growers and carrot breeders.
As a result of the basic understanding of Alternaria mycotoxin production and the strategies and materials developed to prevent the introduction of Alternaria spp. and accumulation of their (myco)toxins in the production chain, we now have the tools to produce safer organic carrots and processed products. This will contribute to improving the health of the European consumer, due to control of the accumulation of harmful mycotoxins, and protection of the environment, due to the use of several project results in conventional farming systems resulting in a decreased need for fungicide applications. Moreover, the results obtained will contribute to the promotion of organic farming systems, thereby supporting the development of more sustainable agricultural production systems.
The results of this project can be used by EU regulatory bodies (e.g. SCF), standardisation bodies, food authorities, industrial end-users and farmers.
Ir. R. DriessenRijk Zwaan B.V
De Lier, The Netherlands
Dr. O. M. Madsen
Dr. I.M.B. Knudsen
Royal Veterinary and Agricultural University
Frederiksberg C, Denmark
Dr. G. Simon
La Ménitré, France
Dr. M. Solfrizzo
CNR Institute of Sciences of Food Production (ISPA)
EC Scientific Officer
Dr. Achim Boenke
DG Research – Unit BI.1, SDME 8/18
Rue de la Loi 200, B-1049 Brussel, Belgium
Tel +32-2-296 0756
Fax +32-2-296 4322
For further information on this project please contact:
Dr. Ruud W. van den Bulk
Plant Research International
Droevendaalsesteeg 1, P.O.Box 16
6700 AA Wageningen, The Netherlands