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Risk assessment and integrated ochratoxin a (ota) management in grape and wine


The number and type of isolates and their ability to produce OTA was determined at harvest, after 7 days at 20ºC and after 30 d at 0ºC in the presence of SO2 generator pads and 2 days at 20ºC, with and without ethanol dips. The results show that ethanol dips did not reduce the number of Aspergillus isolates that were recovered from the berries at any of the storage regimes. An increase in the number of isolates was observed in 2 of the samples at storage of 20ºC indicating neither the ethanol treatment and storage conditions were capable of coping with a situation of decay development. In experiments hold under commercial simulation of storage, grapes of cv. Superior were stored at 0ºC for 45 days. Control, untreated grapes suffered decay and the healthy berries were sampled in addition to ethanol-treated berries and berries which were exposed to SO2 generator pads. No isolates could be recovered from the SO2-treated berries. Both the ethanol and control treatments contained A. niger and A. carbonarius on the berries and most of the later isolates were OTA positive. Berries of cv. In addition, Red Globe were exposed to different levels of SO2. The results of the natural in vivo contamination of the berries showed that the control untreated grapes were contaminated by both biseriate isolates and A. carbonarius. An low level of 0.5 ppm SO2 in the package reduced the amount of isolates that were recovered from the berries whereas a level of 5 ppm no isolates could be recovered from the berries. In this experiment, Petri dishes containing A. niger and A. carbonarius were introduced to packages immediately after spreading the spores on Rose-Bengal medium. After 12 days at 0ºC storage plates were incubated at 25ºC and the number of fungal colonies was counted. The results show that a level of 0.5 ppm SO2 reduced the amount of A. carbonarius to quarter and A. niger to half of the control. No isolates could develop on plates that were exposed to 5 ppm of SO2.
A collection of 156 strains representing OTA producing strains of A. carbonarius and the A. niger aggregate isolated from vineyards of six countries of the Mediterranean Basin (France, Portugal, Spain, Italy, Greece and Israel) was created. All strains have been given an IMI number. The isolates have been cryo-preserved using two methods namely, storage in straws under liquid nitrogen, and freeze-dried in ampoules.From the scientific point of view, the IMI (CABI Bioscience) Culture Collection created with the strains collected during the project in the Mediterranean Basin will be Ĝopenĝ and available for research studies by other scientists.
In disease surveys of field crops, a common practice is to collect samples at a constant interval along a path of predetermined shape. The sampling design along the X-shaped path of the field, used in this research to choose plants for estimating the mean OTA content of the vineyard, is considered by plant pathologists to be one of the best approaches. High variability was observed in OTA content both among bunches and among plants in the two vineyards with different contamination levels. Very high amounts of OTA were detected in 1 or 2 strongly contaminated bunches in both vineyards. This variability is totally random; in fact, no relation was found between OTA content and the position of a bunch on the branch or the location of the plant in the vineyard. Sampling design markedly influenced the assessment of must contamination. The best results were obtained when sampling involved 1 bunch per plant, in a pre-defined position, from at least 10 plants. The difference from reference OTA contamination decreased when the number of sampled plants increased. A reduction of variability was also obtained, as expected, increasing the number of sampled bunches. Despite the large variability among OTA levels detected in bunches, estimated means obtained following different sampling design did not differ significantly from reference OTA content in both vineyards. If the true contamination in a vineyard is 2 µg Kg-1, the limit fixed in Europe for OTA content in must and wine, the range of OTA content assessed using the described sampling designs, should lie between 5 and 0.8 µg Kg-1 in the worst situation, when bunches were collected from 1-2 plants (SSC, n=10), and 2.9 and 1.4 in the best situation, e.g. when the basal bunch of the central shoot was collected (SSB_a, n=10), the position commonly suggested for the evaluation of quality characters at ripening. The accuracy could be considered acceptable, even if there is a trend towards overestimation; in this case corrective actions must be taken and good indications can be obtained for the management of the must.
Based on data available, critical control points were defined both during grape production and processing. Early veraison is a crucial stage to control berry status, checking for damage, both mechanical and that caused by parasites, and visible black mould. Close to ripening it is necessary to repeat these checks, also supported by OTA analysis, particularly when the vineyard is situated in a high risk area, is managed in high risk years or has visible black mould. Detection of OTA content close to ripening can enable a better planning of post-harvest management. Because of the uneven distribution of OTA in vineyards, a sampling protocol is crucial to guarantee the representativeness of the sample. During wine making, control points could be all unit operation, but when must has a low OTA content the wine can be considered safe. A further control can be planned after alcoholic or malo-lactic fermentation if OTA level in must is above the legal limit. Two leaflets summarizing the main actions possible in pre- and post- harvest to minimize the presence of OTA in grapes and wine were prepared in collaboration with all the partners. In the leaflets some tips on the background of the problem, some information on OTA in vineyards and in wine, a short code of good manufacture practices, critical control points and the possible corrective actions are reported. These leaflets will be useful for all the operators of the grape-wine industry. The aim is to produce final suggestions for all actors in grape product, to satisfy the limit fixed by EC.
Several trials were performed with different adjuvants and with different amounts of each adjuvant. Five red wines with different content of OTA were used in trials with different fining agents: Atos, Micosorb, charcoal+kalium caseinate, Toxical. The results showed that products containing charcoal are efficient in decreasing the level of ochratoxin A in the wine and that when the concentration of OTA is high it isn t possible to achieve an acceptable level of OTA in the wine with any of the tested adsorbents. The reduction obtained with Atos appears related to both the contamination level of wine and the amount of adsorbent used; for example the addition of 0.2 g/L produces a decrease of 55% in the content of OTA and the addition of 0.5 g/L adsorbent leads at least to a 90% reduction. If OTA content is low, the amount of adjuvant to be used can be low: so the colour of red wine is not compromised. Otherwise, if the OTA content is high and a significant amount of charcoal must be used, the color intensity decreases significantly. Other experiments tested also the possibility to reduce OTA content with fining corrective actions. Charcoal was the fining agent more able to reduce the OTA level. The effect of charcoal on OTA level seems to be good at high concentration considering short time (24 hours). In fact a reduction of 81% was proved. Considering longer time (2.5 days), the effect of high and low concentration of the fining agent resulted the same. After 48 hours of contact PVPP and cellulose removed a similar quantity of OTA, but the amounts required for cellulose are lesser. Gluten and pea proteins have a similar behavior, but gluten is more effective at low amounts. In order to evaluate reduction kinetics by yeast walls, 100 g/hL of them have been added to 5 aliquots of wine containing an OTA level of 0.54 ug/L. The maximum OTA reduction by means of yeasts and their by-products is generally of 8 days at 25°C, only Mycosorb permits a fast reduction (60 hours). The amounts to have 40-50 % OTA decrease at 8 days of contact are about: - 100 g/hL for yeast walls; - 40 g/hL for yeast hulls; - 200 g/hL for active dry yeast; - 10 g/L for yeast crème. High adsorbent action is produced at 25 g/hL by Mycosorb derived from S. cerevisiae. Several bacterial strains were tested for their ability in reducing OTA content in musts and wine. Among the L. plantarum strains tested the most relevant activity was registered for strains belonging to the group V. The different Oenococcus oeni commercial strains tested showed different abilities in reducing OTA. In particular, R1 was the least efficient (16.1% reduction), while R4 allowed the best result (45.9% reduction) which is also similar to that obtained with L. plantarum V 22.
Sampling managed before ripening confirmed the absence of OTA till to veraison, also when it was relevant at harvesting. This is a relevant point because the period veraison- ripening can be definitely considered crucial for OTA accumulation in grapes. OTA is a problem that originates in the vineyard. Damaged berries, by abiotic and/or biotic causes, are a favourable substrate; in fact, the efficiency of black aspergilli in producing OTA increases when wounds favour grape invasion. OTA is detected in symptomless bunches, but mouldy berries are associated with higher toxin levels. Because the period between early veraison and harvesting is considered as crucial, factors able to influence fungal growth and OTA synthesis, in particular meteorological conditions, need to be monitored carefully at this time. This period is slightly different in different countries; a delay was noticed in Europe respect to Israel. Data obtained during the field sampling were a very good base for further studies and data analysis mainly finalised to the development of a Decision Support System (DSS) to minimise OTA content in grapes.
In order to characterize by molecular methods responsible species involved in the ochratoxin A (OTA) contamination of grapes from Europe and Israel, a total of 189 strains of black aspergilli, including A. carbonarius and uniseriate species (A. aculeatus, A. japonicus), were studied. Sixty six strains were morphologically identified as belonging to the uniseriate species and 123 as A. carbonarius. None of the uniseriate species were able to produce OTA. From the A. carbonarius strains, 96.7% were OTA producers (0.1 to 654.3 mg/g). We characterized 53 strains of A. carbonarius from different countries by RAPD and ITS-5.8S rDNA sequencing analysis. Forty nine strains have a similar RAPD pattern and identical ITS-5.8S rDNA sequences. They produce OTA at different levels. No correlation was determined between the obtained clusters and OTA production level or origin. Only four strains, morphologically identified as A. carbonarius, were not able to produce OTA. These strains showed a different RAPD pattern and the section of DNA sequenced differed from the sequence of the other 49 strains. These OTA-non producing strains represent a new species in the Aspergillus section Nigri. This study also confirms the high percentage of OTA-producing strains in A. carbonarius and the inability of the uniseriate black Aspergillus species to produce OTA.
The geographic area studied lies between 8.63° East and 35.66° West longitude and 31.57° and 48.09° North latitude. The incidence of berries infected by black aspergilli was significantly correlated with geographic coordinates, both at early veraison and ripening. A positive correlation was obtained with longitude, indicating that incidence increased going from West to East, while the correlation with latitude was negative, showing a positive gradient going towards the South of Europe. Incidence of A. carbonarius was also positively correlated to longitude, both at early veraison and harvesting. The prediction map for A. Section Nigri in 2001 showed a fungal incidence higher than 50% in southern Spain and Portugal, and in Israel. The percentage of berries colonised by black aspergilli was between 10 and 25% in Northern-central Italy, while in all the other areas it was between 25 and 50%. Fungal incidence in the whole area monitored was lower in 2002 with respect to the previous year and it exceeded 50% only in Israel. Section Nigri was more frequently isolated in 2003. An incidence higher than 85% was obtained in most of Israel and in southern France; these areas were surrounded by an area with incidence higher than 50% which included the border area of Italy (Piemonte), South-East of Italy (southern Puglia) and Greece. The prediction map for A. carbonarius at harvesting in 2003 showed a gradient very similar to that for black aspergilli, even if with a lower incidence. The highest incidence, between 20 and 40%, occurred in the regions of South-East France, most of Greece and Israel, while at early veraison it was predicted only in northern Greece.
In order to characterise by molecular methods the Aspergillus niger aggregate species involved in the ochratoxin A (OTA) contamination of European wine grapes and table grapes from Israel, a total of 173 strains were studied. The ITS-5.8S rDNA fragments of 173 A. niger agreggate strains from grapes included in this study were amplified and their PCR amplicons were RsaI digested in order to classify the strains in the RFLP types, N and T. This technique was previously defined in our laboratory. All of the strains belonging to the A. niger aggregate were classified into the two RFLP types previously defined: type N (43%) and type T (57%). Twenty out of the 173 strains of A. niger aggregate produced OTA (0.1 to 10.5 mg g-1). All the OTA producing species belonged to the N-RFLP type. This RFLP characterization has been proposed as a technique for screening possible OTA producing A. niger aggregate strains due to the fact that all the OTA-producing isolates whose RFLP pattern is known have been classified as type N, whereas none of the type T strains are able, at the moment, to produce this mycotoxin.
The study managed in South Europe and Israel defined clearly fungi responsible for OTA presence in grapes. They were all belonging to A. section Nigri, with A. carbonarius playing a main role.The inoculum of black aspergilli is always present in vineyards and fungi can be isolated from bunches starting from the early stages of the berries development even if their incidence is relevant from early veraison.OTA is a problem that originates in the vineyard. Damaged berries, by abiotic and/or biotic causes, are a favourable substrate; in fact, the efficiency of black aspergilli in producing OTA increases when wounds favour grape invasion. OTA is normally detected in symptomless bunches, but mouldy berries are associated with higher toxin levels. The period between early veraison and harvesting can be considered as crucial and factors able to influence fungal growth and OTA synthesis, in particular meteorological conditions, need to be monitored carefully during this time. Data obtained during the field sampling were a very good base for further studies and data analysis mainly finalised to the development of a Decision Support System (DSS) to minimise OTA content in grapes.
The study examined, for the first time, the detailed effect of temperature x aw conditions on germination and germ tube extension of spores of A.carbonarius. This is critical in defining the capacity of contaminant spores to become established on grapes and subsequently grow and produce OTA. Germination occurs very rapidly in vitro suggesting that a wide range of aw x temperature levels over which infection could occur. Studies on grape tissue and grape skin confirmed that at >85% ERH conducive conditions occur for spore germination and establishment on grapes if insect damage or cracking of the skin surface occurs. This study has been the first to confirm that optimum environmental conditions for OTA production by A.carbonarius isolates are very different from the optimum for germination and growth. Thus optimum OTA production was at 15-20°C depending on isolate, with optimum aw conditions of 0.95-0.98. The amount of OTA produced varied markedly between isolates from different countries. The fact that partitioning of OTA results in a greater accumulation in spores than fungal biomass or medium suggests that sometimes the presence of large numbers of spores on the grape surface but largely passively present could result in OTA contamination despite no skin damage.
All available information were used to draw the relational diagram of A. carbonarius-grape pathosystem. The other fungi were not considered because of minor interest. The inoculum was always observed on berries; it was not considered a limiting factor and its quantification was considered not relevant. Spore germination has been studied on grape juice agar, grape flesh and grape skin and interesting equations were found able to predict correctly germination. The basic function was a logistic and it was fitted to data considering 2 factors, time and temperatures, or 3 factors, when relative humidity was added. Growth rate of A. carbonarius in function of temperature is available and it will be improved, if necessary with further data collected in different humidity conditions. The mycelium of A. carbonarius can stay on skin and colonise berries or grow inside and infect berries. Colonisation and infection rates are influenced by the same variable. Temperature is always a relevant factor because, as described before, influence the growth rate, but berries status and grape varieties are definitely relevant. Taking about berries status (BS) the main aspect regards presence/absence of damages on skin. In case of damages, it is very easy for the fungus to develop inside the berry and it was confirmed in many trials that infection is very rapid. Many factors can determine skin damages, but rain (R), especially during ripening, and pest and disease (P&D) presence play a main role. A correlation was found between OTA content in berries and Lobesia botrana damages, probably related to both wounds and spore dissemination. Larvae probably contribute to spore dispersal; they can act as vectors, trapping conidia in the cuticle ornamentation, and facilitating rapid penetration tunnelling berries. Among pathogens, powdery mildew seemed the most conducive for black aspergilli. Infected berries often are misshapen or have rusty spots on the surface and severely affected fruit often split open, mainly during ripening when the inoculum of black aspergilli is more relevant. Grape variety is a further relevant factor that can favour berries infection and its relevance was shown both in vitro and in field trials. This factor needs to be studied deeper, because the reasons that make a variety more or less susceptible were not understood. It is not easy to quantify colonisation or infection of berries, but a yes/no response seem sufficient to run the model, at least for the first validation. Ochratoxin A seems more concentrated in the outer part of the berry, in the skin and very close layers, but till now its production without berry infection was not demonstrated. For that reason, only infected berries were considered to quantify OTA production.
The winemaking process causes an OTA decrease. The stage that causes the most important reduction is the malo-lactic fermentation. In wines that aren t intended to the malo-lactic fermentation, the OTA level can be reduced by means of corrective actions with charcoal-base adjuvants. If wines have a slight OTA contamination, charcoal-base adjuvants can be used on the end-product in concentrations of 10 g/hL maximum, to reduce the OTA concentration without reducing the wine colour. If wines have an important OTA contamination, charcoal-based adjuvants can be used on the must before maceration, so it is possible to use concentrations of the product higher than 10 g/hL, able to reduce the OTA concentration without reducing wine quality, wine colour in particular. When ochratoxin A contamination occurs in grapes, some practices may be implemented to reduce its carry-over to wine. In all cases, it is recommended to determine the ochratoxin A content in must. In the case of significant OTA contamination the following procedures should be evaluated: - Avoid or reduce skin maceration; - Adapt pressing rate to the health status of grapes (reduce time of operation) - In case of red grapes, treat must before fermentation (be aware of possible losses in aromatic and polyphenolic compounds) - Avoid the use of pectolytic enzymes to aid clarification, filtration or centrifugation are preferable - Use fining agents known to be effective againts OTA (be aware of regulation 1622/2000, laying down rules for the use of fining agents in white and red vinification) - Use yeast and bacteria known to be effective againts OTA (either by its adsorption or transformation) Operations unit such as solid-liquid separation and the fermentative processes are effective in reducing OTA. In order to manage the hazards of OTA in winemaking and to verify if OTA content in wine is lower than the legal limit of 2 µg/L defined by European Commission (EC regulation N° 123/2005 of 26 January 2005), OTA analysis in must and in wine at the end of alcoholic fermentation would be enough, since the following phases reduce OTA content.
The effectiveness of Switch (where fludioxonil and cyprodinil are the active ingredients) in controlling the incidence of sour rot caused by Aspergillus spp. in raisin and wine producing vineyards in two consecutive growing seasons and geographical areas was evidenced. Switch significantly reduced the level of Aspergillus populations on the collected berries and late Switch applications are more efficient compared to the early ones. Fludioxonil is also the most effective active ingredient against Aspergillus spp., in the field if compared with Chorus (where cyprodinil is the active ingredient) and Carbentazim and offers a promising base for planning spray programs to reduce or control sour rot of berries in raisin or wine producing vineyards and underlined the relevance of a good management of the vineyard also against other parasites to have the best efficacy of the product.Aureobasidium pullulans strain K4 and Cryptococcus laurentii strain GY18 were equally effective to Switch on reducing the percentage of sour rot and the percentage of the Aspergillus population on the collected berries.
The most relevant variables to discriminate low and high incidence of black aspergilli in vineyards were soil type and grape variety. The most favourable soil for high incidence was clay and local varieties grown in France, Greece, Italy, Portugal and Spain resulted in higher susceptibility. Further studies are necessary to define the role of grape variety because this result regards mainly local varieties and the reason for higher susceptibility was not found. Data on the role of pests and diseases are limited, and they were not enough to be included in the statistical analysis, but a good management of them in vineyards certainly guarantees a relevant decrease in OTA content at harvesting, as demonstrated in several trials. The main disease able to favour A. carbonarius is powdery mildew but a major role is played by Lobesia botrana comparing neighbouring vineyards, managed with different approaches to crop protection. Specific sprays to control black aspergilli are possible and the mixture cyprodinil+fludioxonil gave the most promising results.

Exploitable results

The incredible explosion of knowledge production in Biology during the two last decades has created a critical need for bioinformatics instruments able to manage data and facilitate their retrieval and analysis. Hundreds of biological databases have been produced and integration of biological data from these different resources is extremely necessary when we want to focus our efforts towards the study of a particular layer of biological knowledge. Molecular sequences and biological data on gene and their functional products are publicly available from a wide variety of databases. A part from primary and genomic databases (Embl, Swissprot, Ensembl, etc.), in which these kind of information are dispersed among all the others, many specialized databases have been developed collecting data from external heterogeneous data resources, including literature references, and in some cases also adding information coming from in-silico and/or experimental studies of gene and/or protein functional characterization. As far as those databases dedicated to sequences, no information are available on databases dedicated to AFLP automated data, in collaboration with a bioinformatics society, we have developed a specific database named “AFLP Manager” able to recognize and download directly the ABI files generated from the GeneScan Collection software (PE Applied Biosystems) after the separation on ABI Prism 310 automated DNA sequencer. The AFLP Manager after loading the ABI prism data is able to recognize each information on the sample (peaks, time of run, areas of peaks, etc.). This database was developed in a DBMS free in MSQL and it is able to collect, modify, compare data, make query and export a final data set in a binary matrix useful for statistical analyses. Actually the database is available on the web at the following URL:
The highly conserved regions of calmodulin gene were considered as target sequences for the assay. In an alignment of sequences representing Aspergillus spp. occurring on grapes, primers and probes sequences were chosen. The primers and probe sequences were completely conserved among all sequences examined within the A. carbonarius strains. Primer/probe combination successfully amplified DNA extracted from pure cultures of A. carbonarius fungal species, whereas no fluorescence could be measured with any of the other species within the Aspergillus genera. Occasionally, the fluorescence value of negative control (containing water instead template) started to increase after 36 - 38 cycles. This is not unusual when use SYBR Green and is caused by primer-dimers formation (especially at low level of target template). Even though this effect was almost eradicated by reducing the primer concentration, any increase in fluorescence after this point was deemed unreliable and not used to detect or quantify samples. It was avoided using a sequence specific assay, as TaqMan method. The TaqMan real time PCR is based on the cleavage of an internal probe by the 5’-/3’ endonuclease activity of the Taq polymerase. During each cycle of the extension phase one molecule of the reporter dye is released for each molecule amplified resulting in generation of fluorescent emission of the reporter dye. The threshold cycle (Ct values) indicates the increase of reporter fluorescence and it is the number of cycles before the fluorescence emitted passes a fixed limit. The log10 of the number of targets initially present is proportional to the Ct value and can be measured using a standard curve. To develop a standard curve for the quantitative PCR reactions were used serial dilutions of A. carbonarius strains. A pure culture of A. carbonarius was used to extract the DNA and make two fold dilutions for obtaining the standard curve useful for Real Time PCR. Using the TaqMan method, large numbers of samples can be processed allowing a much more intensive monitoring of the population dynamics. To evaluate intra-assay variability, each dilution was assayed in triplicate. Relative standard deviations of the Ct values within the same sample was lower than 1.5%. Application of this method to a known conidial suspension (CFU/ml) provided a positive correlation between CFU and haploid genome weight. Genomic DNA weight of A. carbonarius (not known) was assumed to be similar to that of A. niger (4.13 x 10-5ng, for a haploid genome) reported in the website tml. To verify this assumption an absolute quantification of total DNA was made for DNA extracted from a known conidial suspension (3x102 CFU/ul) of A. carbonarius, by real-time quantitative PCR assay. For a suspension of 3x105 conidia 10.95ng of total DNA was obtained, corresponding to 3.65x10-5 ng for a single CFU or conidium, which is very close to the genome weight of A. niger (35.9 Mb). In order to study the usefulness of the TaqMan PCR assay as a tool in food quality/safety control the quantification of A. carbonarius DNA was carried out in triplicate on 15 samples of grapes having a known content of OTA. The regression curve of A. carbonarius DNA content versus OTA content showed a very good correlation (R2=0.92), despite the finding of low levels of DNA in 4 ochratoxin-free samples.
The visual inspection of the aligned calmodulin partial gene sequences readily identified unique regions within the amplified fragment, (e.g. in the range of 350-420 base pairs) that has been exploited in the development of generic PCR-based assay for the detection of A. carbonarius and A. japonicus/aculeatus. Using the genetic sequence variation founded in this region, all primers were designed to operate at high annealing temperatures (58°C), thereby preventing the co-amplification of non specific target of DNA. In calmodulin sequences, we observed 99,98 % identity for strains of A. carbonarius, and 99,40 % identity for strains of A. japonicus and A. aculeatus analysed. Specific primer pairs amplified in A. carbonarius a fragment of 371 bp length and in A. japonicus a fragment of 583 bp length. To determine the diagnostic value of the primer set in a large scale, experiments were started to test its usability on a large number of isolates: 30 strains each of the target species, 42 strains of other closely related species within the black aspergilli and others main fungal/yeast species occurring on grapes as Botrytis cinerea, Saccharomyces cerevisae and other toxigenic fungi such as some Fusarium species. Aspergillus japonicus strains were not amplified by A. carbonarius-specific PCR primers (CARBO1/2), and A. carbonarius strains were not amplified by A. japonicus -specific PCR primers (JAPO1/2). None of the others Aspergillus section Nigri were amplified by the two sets of primers with the exception of the four strains of A. aculeatus which were amplified by the specific primers JAPO1/2. No product was amplified from any fungal/yeast tested. The primers were also tested against others main fungal/yeast species occurring on grapes as Botrytis cinerea, Saccharomyces cerevisae and other toxigenic fungi such as some Fusarium species, without any amplifications
The data of AFLP analysis performed on 300 strains with the primer A showed a clear pattern’s peaks for the three species main occurring on grapes. So we are able to construct a dendrogram of similarity that evidenced the grouping in three distinct clusters of the strains belonging to A. carbonarius, A. niger group and A. japonicus/A. aculeatus . These result were successively confirmed by the AFLP analyses made with other three primers (B, C and D), all the results obtained gave good fingerprints useful to construct dendrograms that clearly separate the three main groups of the Black Aspergilli (Fig. 5). The AFLP banding pattern was variable from 20-25 bands for A. carbonarius and A. japonicus aggregate to 30-40 bands for A. niger aggregate, confirming the higher genetic variability among this species aggregate as was showed by sequence analyses. On the basis of these great variability found in the A. niger aggregate strains the AFLP analysis revealed the presence of two main clusters within this species aggregate that seem clearly separated, suggesting the possible split in two different species of this two groups of A. niger strains.