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Safe organic vegetables and vegetable products by reducing risk factors and sources of fungal contaminants throughout the production chain: the carrot - alternaria model

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Hyperspectral image analysis offers the potential to measure a part of the spectrum of an object in a very detailed way. In combination with pattern recognition methods, this offers the possibility to discriminate between different objects using both spectral and spatial information. In this study, it was shown that spectral differences were present between healthy and Alternaria-infected carrots. Although the results should be used with care, the feasibility of this new technology for discriminating between good and spoiled food products, such as organic carrots, was proven. Beside the potential use of this technology in carrot breeding programmes for determining resistance levels towards Alternaria, it could also be used in-line to detect and sort out carrots (or other food products)with unwanted traits or symptoms. It may be possible to directly test for the presence of mycotoxin-containing products, when the presence of mycotoxins can be determined based on hyperspectral image analysis.
With this method light infections can be distinguished from heavy infections of seed. Other materials, such as carrot root or seedling material, can be tested on the ARSA-medium as well. The diagnosis involves observation of morphology of colonies produced by the fungus on carrot seeds, seedlings and root discs incubated on the Alternaria Radicina Selective Agar (ARSA).Based on the results from a ring test with 5 laboratories and 4 levels of seed contamination/infection, it was concluded that the method provides reliable information on the seed contamination/infection with Alternaria radicina and allows discrimination between light and heavy infection which in turn, gives the prognosis for infection at the seed germination and root storage stages. The advantage of the method lies in its higher specificity as well as easier and less time-consuming diagnosis step as compared with the deep-freezing-blotter-method.
Antagonistic fungi Clonostachys rosea and C. solani restrained production of toxins by Alternaria radicina and A. alternata in carrot root discs. Of these, C. rosea was more effective than C. solani. The results indicate the need for determination not only efficacy of a prospect biocontrol organism against growth of a target fungus, but also their ability to inhibit toxin production. However, growth character and toxigenic properties of antagonists must be taken into consideration as well, especially in case if a prospect biocontrol agent is to be applied in carrot for human/animal consumption.
The fate of alternariol and alternariol methyl ether was investigated during carrot processing at laboratory scale starting from fresh carrot roots that were artificially contaminated with alternariol and alternariol methyl ether. The principal steps were: sample preparation, blanching (with boiling water or steam), maceration (with pectinase and hemicellulase enzymes), juice extraction (pressing) and pasteurisation. The entire processing cycle caused a reduction >98% for both toxins in the final juice, independently of the blanching procedure used during the process. Most of the toxins was recovered in the water used during blanching (boiling water and cooling water). The fate of the phytoalexin 6-methoxymellein (6-MM) during carrot juice processing was also investigated by using different enzyme formulations for maceration and blanching procedures. A reduction of 6-MM by 85 or 94% was obtained after the entire cycle of carrot juice processing, depending of the blanching procedure used.
The testing of samples from organically grown carrots and of carrot containing products, such as baby food and vegetable juices, showed absence of A. alternata mycotoxins and sporadically presence of A. radicina toxins. A total of 356 carrot samples have been analysed by HPLC for their Alternaria toxins content. These samples included 266 carrot samples from different cultivars (Tempo, Royal Chantenay Rola, Kira, Nanco, Aston) produced in France, The Netherlands and Denmark in two consecutive seasons under organic conditions and collected at harvesting and during storage, and 87 carrot-based commercial products (soups, vegetable sauces, carrot puree, whole canned baby carrots, vacuum-packed sliced carrots, pudding, baby foods) collected in France, The Netherlands, Denmark, Poland and Italy. Out of 356 samples, six samples belonging to stored carrot samples and showing rot symptoms, were found contaminated with either epi-radicinol, radicinin and radicinol whereas no A. alternata mycotoxins were found in any tested samples. Although Alternaria alternata has been frequently found in organic carrots none of the mycotoxins produced by this fungus were detected in carrot roots or in carrot commercial products. These samples were also analysed for their 6-methoxymellein (6-MM)content. Levels of 6-MM in carrot samples and carrot based products were generally below or rarely close to the "just noticeable difference" level for this compound (48-71µg/g). In particular 6-MM was found in 69% of 356 tested samples at levels ranging from 0.14 to 76µg/g in fresh carrots and from 0.04 to 15.64µg/g in processed carrot products. These levels of 6-MM does not represent a problem for the consumers in terms of sensory quality.
The toxigenic profile of Alternaria radicina isolated from carrot and other umbelliferous plants has been determined by growing the fungus on rice and carrot discs. Dried and finely ground rice or carrot cultures were extracted with a mixture of acidic acetonitrile, methanol and water. After filtration the culture extracts were appropriately diluted with water, filtered and analysed by reversed phase liquid chromatography with ultraviolet diode array detector. When cultured on rice sixteen out of eighteen isolates produced radicinin and radicinol at levels ranging from 1 to 3921µg/g and from 1 to 44µg/g, respectively, whereas no isolate produced epi-radicinol. Ten out of the eighteen isolates were also cultured on carrot discs, confirming the production of radicinin (19-2840µg/g). Five of these isolates produced epi-radicinol (at levels of 26-223µg/g) and four also produced radicinol (at levels of 3-15µg/g). The results of this investigation confirm that radicinols are important elements in the metabolic profile of A. radicina isolates from carrots and other umbelliferous plants such as celery and parsley. Moreover, they confirm that epi-radicinol is produced by A. radicina on carrot discs, but not on rice. This supports the peculiarity of A. radicina as a carrot pathogen that can easily develop and colonise carrot tissues and produce toxins in vivo.
Post-harvest storage experiments performed at various temperatures showed that 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 up to 25 weeks storage in both control carrots and carrots artificially inoculated with toxigenic Alternaria alternata. Only low levels of alternariol (0.1- 1µg/g) were detected in carrots stored for 32-48 weeks at room temperature. Accumulation of the phytotoxins radicinin, radicinol and epi-radicinol occurred in carrots artificially inoculated with toxigenic Alternaria radicina strains and stored at various temperatures or in control carrots stored at temperatures >10°C. Accumulation of radicinols and radicinin in stored carrots was stimulated by successive temperature rises from 1°C to 10°C and from 10°C to 20°C, reaching maximum levels of 60µg/g epi-radicinol and 26µg/g radicinin. Wounded carrots resulted more susceptible to rot symptoms development and phytotoxins (radicinin and radicinols) accumulation during storage at various temperatures. These results suggest that storage of carrots at low temperatures, e.g. in cooled warehouses, and prevent injuries diminishes the risk of Alternaria toxins accumulation. Storage of carrots at 1°C was suitable to maintain low levels of 6-methoxymellein (6-MM) for a period of at least 17 weeks whereas storage at 20°C increased 6-MM content up to 6.5 fold. No effect of Alternaria spp. infection was observed on 6-MM accumulation. The effect of temperature was amplified by the length of storage and the physiological state of carrots.
Testing Alternaria spp. for their toxigenicity at 1, 10 and 20 degrees Celsius on PDA, rice and carrot root discs revealed that although the fungi were capable of producing toxins on PDA and rice, none were detected on carrot until 28 days of incubation. This indicates that a relatively short term storage of carrot at low temperature seems to be not hazardous for consumers, even in case of contamination with any of the two Alternaria spp. tested.
This result can be applied to the seeds after harvest, as a measure to reduce seed-borne infections with Alternaria radicina. It is based on 2 subsequent treatments (or processing steps) after threshing: - sorting out the seeds with a low specific gravity with a gravity sorter, and - followed by sorting out the most immature seeds with chlorophyll fluorescence sorting equipment. With these post-harvest treatments it is possible to remove bad quality and Alternaria infected seeds, thereby improving the overall quality of the organically produced carrot seed lots and reducing the chance of introducing Alternaria in the production chain of organic carrots. This also lowers the risk of (myco)toxins in the production chain. Implementation of the result should lead to a better supply of high-quality organic carrot seeds for organic farmers, thereby closing the organic production cycle and guaranteeing a better harvest of the organic produce. The results can also be applied in conventional seed production systems, thereby reducing the amount of fungicides used to chemically control the disease.
The method is based on high performance liquid chromatography (HPLC) and UV diode array detection. A liquid chromatographic method for the determination of Alternaria radicina and A. alternata toxins in carrots was developed. Toxins were extracted from carrot with an acidified mixture of water, methanol and acetonitrile. The filtered extract was divided in two parts that were purified by solid phase extraction on a C18 column for the analysis of radicinin (RAD), altertoxin-I (ATX-I), alternariol (AOH), and alternariol methyl ether (AME) and on a polymeric Oasis HLB column for tenuazonic acid (TeA), respectively. Toxins were quantified by reversed phase liquid chromatography with ultraviolet diode array detector by using two consecutive isocratic mixtures of acetonitrile-sodium dihydrogen phosphate solution. Mean recoveries of TeA, ATX-I, AME, RAD, and AOH from carrots spiked at levels between 0.5 and 3.0µg/g were 69, 71, 90, 36, and 78%, with mean within-laboratory repeatability of 14, 5, 4, 6, and 18%, respectively. The mean between-laboratory reproducibilities for the determination of TeA, ATX-I, AME, and RAD in spiked samples were 25, 22, 6, and 12%, respectively. Limits of detection (signal-to-noise ratio of 3) for RAD, TeA, ATX-I, AME, and AOH were 0.006, 0.02, 0.02, 0.01, and 0.005µg/g, respectively. This method proved to be applicable to the analysis of 6-methoxymellein in carrot samples. This compound is a phytoalexin which is produced by the carrot itself in response to microbial infection or other form of stress and is associated with the bitterness of stressed carrots. For the analysis of carrot-based products (baby foods, carrot purees, puddings, sauces and vegetables soups) the method was appropriately modified. In particular, carrot-based products were diluted with water, centrifuged and the filtered extract purified by C18 column and analysed by reversed phase HPLC by using a gradient of acetonitrile in water as mobile phase. Detection limits were 0.02 and 0.01µg/g for carrots and carrot-based products, respectively.
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 under the project for controlling Alternaria, was devised, aimed at the reduction of the sources of contaminants in the production chain of organic carrots. The result is 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/seed producers. It contributes to the overall goal of the project to minimise the risk of introducing Alternaria and its toxic metabolites in the organic production chain of carrots and carrot-derived products. Implementation of the results should lead to a better supply of high-quality organic carrot seeds for organic farmers, thereby closing the organic production cycle and guaranteeing a better harvest of the organic produce. The results can also be applied in conventional seed production systems, thereby reducing the amount of fungicides used to chemically control the disease.
A collection of microorganisms with antagonistic properties against Alternaria spp. were obtained following the selection criteria listed below: - Naturally occurring microorganisms being indigenous to the carrot environment. - Biocontrol efficacy tested in a hierarchic assessment scheme - first on naturally infected seed, secondly on artificially inoculated seed. - Preference to specific temperatures. - Easily propagated at room temperature and unable to grow at 37°C. - Different taxa of microorganisms were included to maximise the probability of success. - Productivity: culturable in inexpensive media. - Storage capacity of formulated preparation: at least 6 month. - Risk assessment. 105 microorganisms were investigated. Four antagonists, Plectosporium tabacinum (IK1755), Clonostachys rosea f. catenulata (IK1878), C. rosea (IK1871) and C. solani (IK1889) efficiently controlled seedborne Alternaria spp. The efficacy of the selected antagonists was comparable to that of the fungicide Iprodione. The selected effective candidates can be further developed and exploited for biocontrol of Alternaria diseases in carrots. Moreover the well described collection consisting of 105 potential antagonists can be evaluated and exploited in other diseases/crops systems.
Specific primers for detection and identifica-tion of the Alternaria species on carrot seeds and roots were designed and shown to be highly sensitive and able to differentiate between the three Alternaria species occurring on carrot, i.e. A. radicina, A. alternata and A. dauci. The A. alternata primers cross-reacted with A. longipes and A. lini, however these species do not occur on carrot. 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. A positi-ve correlation was found between the percentage of seed infection established by the blotter method and the intensity of the amplified, specific product. Based on a small-scale ring test with 5 participating laboratories, the PCR method was found not suited for application on carrot material, in particular seeds. The difficulties experienced are related to problems with the DNA isolation and extraction procedure, likely as the result of interfering oily substances in the seeds. For analysis of carrot root material, the PCR assay developed seems to be useful. This PCR-based assay needs to be further optimised. Nonetheless, when the problems with interfering components can be solved, this assay will speed up the analysis time and the sensitivity for Alternaria detection. A faster detection method then the methods currently in use is requested by seed companies and inspection services, because an analysis time of 7-14 days may hamper trade. The method developed so far has been published and members of the International Seed Health Initiative (seed companies and inspection services) and the International Seed Testing Association have shown interest in the method. It is likely that seed companies and inspection services will test it, and jointly optimise the method for future use.
The development of Clonostachys rosea during seed germination and plant emergence and the interaction between C. rosea and A. radicina on carrot seed were studied in a time course experiment. A gfp transformant of a wildtype isolate of C. rosea was included in Fluorescence microscopy studies of the interaction. The results showed that Plectosporium tabacinum (IK1755), Clonostachys rosea f. catenulata (IK1878), C. rosea (IK1871) and C. solani (IK1889) multiply on seeds during biopriming to levels which are suitable for biocontrol of diseases. C. rosea grows and sporulates intensively on Alternaria infected seed, it coils around Alternaria spores and mycelium and restrict hyphal extension and sporulation of A. radicina. In conclusion: C. rosea is a suitable biocontrol agent for controlling A. radicina. Based on microscopic evaluation of the growth and distribution of the antagonist during priming, C. rosea colonized the whole surface of the pericarp, including the apex of the seed where the primary root emerges. This is important for protecting the seedling against inoculum of pathogenic Alternaria spp. as well as other seedborne pathogens located on or in the pericarp. Furthermore, it provides an opportunity to protect the seedling from soilborne pathogens attacking the emerging seedling. The results can explain the efficacy of the biocontrol agents and support knowledge about mechanisms of biocontrol by C. rosea. This insight is an important background for further biocontrol studies in other disease/crop systems, especially for biological seed treatment in combination with other physiologically treatments.
The method is based on a blotter test and involves examination of the fungal morphology after 7 days of incubation. For testing seeds a deep-freeze step to prevent germination is necessary. The diagnosis involves microscopic observation of the fungus sporulation on incubated carrot seeds, seedlings and roots. The test is completed within 7 days which is advantageous in terms of work planning in a laboratory. Based on the results from a ring test with 5 laboratories and 4 levels of seed contamination, it was concluded that the deep-freezing-blotter-test provides reliable information on the seed contamination with Alternaria alternata.
The assay was set up to check the toxigenicity of A. alternata isolated from carrots and other umbelliferous plants and cultured on rice material. The fungus Alernaria alternata produce several mycotoxins harmful for humans and animals. The occurrence of Alternaria alternata on carrots produced in Europe is quite common. The determination of the mycotoxins profile of A. alternata isolated from carrots can help in predicting the risk of a possible mycotoxins contamination of the crop. It was previously reported that some strains of A. alternata were able to produce both fumonisin B1 and AAL toxins. A method was developed for the simultaneous determination of fumonisins (FB1, FB2) and AAL-toxins (TA, a mixture of two structural isomers TA1 and TA2) in inoculated rice cultures of A. alternata. Toxins were extracted from test samples with a mixture of water and acetonitrile. The filtered extract was diluted with a solution of potassium chloride and purified by solid phase extraction on a polymeric Oasis HLB column. The purified sample extract was submitted to pre-column derivatisation with OPA reagent and analysed by C18 reversed phase HPLC with fluorometric detection. Mean recoveries and repeatability for FB1, FB2 and TA were 89% (6%), 89% (9%) and 70% (23%), respectively. Quantification limits (signal to noise 6/1) were 0.1µg/g for TA and FB1 and 0.5µg/g for FB2.
A protocol for testing efficacy of fungal and yeast antagonists in plant bioassays was developed. The test was based on: - Carrot seeds naturally infected with Alternaria radicina and A. dauci. - Coating infected seeds with a defined number of spores from potential antagonists. - Assessment of plant emergence and seedling health in sand. In a second screening the best isolates were further tested on seeds artificially inoculated with A. radicina using two inoculum levels of the pathogen. The method can be used to study and evaluate efficacy of biocontrol candidates and botanicals. Furthermore the bioassay offers a fast and low cost method to test carrot resistance to seedborne Alternaria spp.
Treatment of carrot roots with methanolic solutions of radicinol (RAD) epi-radicinol (epi-ROH),the toxins produced by A. radicina and alternariol (AOH) and alternariol methyl ether (AME), which are produced by A. alternata, resulted in changes in their cell structures visible under transmission electron microscope. Plication of cell membranes, and partly cell walls, accumulation of multivesicular bodies and numerous vesicles in cytoplasm were observed. Moreover, microvacualisation and distension of mitochondrion cristae occurred. Responses of carrot root cells to treatments with particular toxins were specific. No changes were noticed in the structure of nucleus, Golgi bodies and endoplasmatic reticulum. The data obtained led to a better understanding of phytotoxic properties of the RAD, epi-ROH, AOH and AME, as well as disease processes caused by Alternaria spp. on ultrastructure cell level.
Organic carrots were produced from the same seed batches containing Alternaria contamination in 3 locations in Europe (DK, NL, and F) in two subsequent seasons. Sampling was performed 2 months after sowing, at the end of the growing season, after 1 month storage, and after 3 months storage. The place of production had a large influence on the contamination of the carrot roots produced. Production in NL resulted in high contamination levels of A. radicina, whereas production in F resulted in high levels of A. alternata contamination. 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 Alternaria alternata mycotoxins were found in 254 organic carrot samples collected at harvest and during storage in the 2 subsequent growing seasons. With respect to Alternaria radicina phytotoxins, only 3 out of 254 samples were contaminated with epi-radicinol, and one also contained radicinin. Occasionally, carrot samples showing black rot symptoms caused by Alternaria radicina were found contaminated with epi-radicinol and, less frequently, radicinin and radicinol. These compounds were tested on human cell lines, Artemia salina brine shrimps, and carrot seedlings and showed a phytotoxic activity other than harmful to humans and animals.
Biocontrol agents were produced on solid media in order to obtain fungal spores with high vitality and a stable shelf life. The protocol was based on: - Two growth media: sterilised rice or a peat/bran mixture moistened with water and inoculated with the biocontrol agent. - Two propagation periods. - Air-drying and milling of preparation. - Assessment of germination speed of dried conidia. - Assessment of viability of dried conidia in stored preparations. The four antagonists, Plectosporium tabacinum (IK1755), Clonostachys rosea f. catenulata (IK1878), C. rosea (IK1871) and C. solani (IK1889) are all readily produced on a relatively low cost solid peat bran medium during a 14-21 days incubation period and have a shelf life stable up to 8 months when storage at 4°C or 20°C. These product characteristics are important for commercial up-scaling of biocontrol agents.
Alternaria radicina is a seedborne fungal pathogen responsible for black rot disease of carrots and is a known producer of the phytotoxin radicin. Although the pathogenicity of A. radicina against carrot has been previously described, no data are available on the possible involvement of radicinin as pathogenicity or virulence factors of A. radicina versus this vegetable. Alternaria radicina cultured on carrot slices produced radicinin together with another compound having an UV spectrum similar to that of radicinol. The phytotoxin epi-radicinol, a diastereomer of radicinol, was isolated from large cultures of Alternaria radicina cultured on carrot slices and identified by GC/MS, LC/MS, 1H NMR and 13C NMR. Radicinin and epi-radicinol showed a phytotoxic effect on carrot seedlings producing root growth inhibition. The natural occurrence of epi-radicinol and, to a lesser extent, of radicinol and radicinin has been demonstrated in carrots affected by A. radicina black rot. These results together with the finding of higher levels of these phytotoxins in carrot cultivars more susceptible to A. radicina suggest that radicinin and radicinols could play a role in the pathogenicity of A. radicina against carrot.
A diversity in toxin production by the isolates of Alternaria alternata and A. radicina from carrot, parsley, celery and wild carrot was demonstrated. A. alternata produced tenuazonic acid (TeA), alternariol (AOH), alternariol methyl ether (AME) and altertoxin I (ATX I) in PDA and rice cultures, whereas mainly AOH and AME on carrot root discs. In any media tested, A. alternata produced neither AAL-toxins (TA1 and TA2)nor fumonisins (FB1 and FB2). Isolates from carrot produced AOH and AME on carrot at lower levels as compared with those from parsley and celery. A. radicina produced mainly radicinin (RAD)in PDA and rice cultures whereas mainly RAD and epi-radicinol (epi-ROH)on carrot discs. Isolates from carrot and wild carrot were more toxigenic than those from parsley and celery. The isolates collected may serve as a tool for breeding works.
53 isolates of Alternaria alternata and A. radicina from carrot, parsley, celery and wild carrot were tested for their toxigenicity on rice, potato-dextrose-agar (PDA), and carrot root discs. A diverse toxigenicity of the fungi was demonstrated. A. alternata produced tenuazonic acid (TeA), alternariol (AOH), alternariol methyl ether (AME) and altertoxin I (ATX I) in rice and PDA cultures, whereas mainly AOH and AME on carrot root discs. In any media tested, A.alternata produced neither AAL-toxins (TA1 and TA2) nor fumonisins (FB1 and FB2) which is of importance for consumers. Isolates from carrot produced AOH+AME on carrot at lower levels as compared with those from parsley and celery. A. radicina produced mainly radicinin (RAD)in rice and PDA cultures whereas mainly RAD and epi-radicinol (epi-ROH)on carrot discs. Isolates from carrot and wild carrot were more toxigenic than those from parsley and celery which may be an additional evidence for a closer relation of the fungus to Daucus carota as compared with Petroselinum hortense and Apium graveolens. Significant positive correlations between the level of RAD and TeA produced by Alternaria spp. isolates on PDA and the disease index in seedlings in the plant assay and the percentages of diseased seedlings and seeds in the germination test, respectively were found. These may indicate involvement of both toxins in the disease processes either in seedlings or at seed germination stage.

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