Influence of environmental parameters on mycotoxin production by Alternaria arborescens.

Alternaria arborescens has been reported as a common fungal species invading tomatoes and is capable of producing several mycotoxins in infected plants, fruits and in agricultural commodities. Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA) are some of the main Alternaria mycotoxins that can be found as contaminants of food. This species can produce these toxic metabolites together with AAL toxins (Alternaria alternata f. sp. lycopersicum toxins), which can act as inhibitors of sphingolipid biosynthesis. The objective of this study was to determine the effect of water activity (aw, 0.995, 0.975, 0.950) and temperature (6, 15, 20, 25 and 30 °C) on mycotoxin production by A. arborescens on a synthetic tomato medium. The optimum production of AOH and AME occurred at 0.975 aw after 40 days of incubation at 30 °C. The maximum TeA accumulation was observed at 0.975 aw and 25 °C and at 0.950 aw and 30 °C. AAL TA was produced in higher quantities at 0.995 aw and 30 °C. At 6 °C no quantifiable levels of AOH or AME were detected, but significant amounts of TeA were produced at 0.975 aw. In general, high aw levels and high temperatures were favorable for mycotoxin production. The greatest accumulation of all four toxins occurred at 0.975 aw and 30 °C. The results obtained here could be extrapolated to evaluate the risk of tomato fruits and tomato products contamination caused by these toxins.

Toxigenic Alternaria species from Argentinean blueberries.

Blueberries are traditionally consumed in North America, some European countries and Japan. In Argentina, the blueberry crop is profitable because production starts in November, when the northern hemisphere lacks fresh fruit. Fungal contaminants can grow and produce mycotoxins in fresh fruit. The aims of this work were to identify the main genera of the mycobiota of blueberries grown in Argentina and to determine the toxicogenic potential, pathogenicity and host specificity of the species isolated. The genus Alternaria was the main component of the blueberry mycobiota (95%); minor proportions of Phoma spp. (4%) and Penicillium spp. (1%) were also isolated. According to their sporulation patterns, 127 Alternaria isolates belonged to the Alternaria tenuissima species-group, 5 to the Alternaria alternata species-group and 2 to the Alternaria arborescens species-group. The last mentioned species-group was not isolated at 5°C. Of the 134 isolates, 61% were toxicogenic in autoclaved rice; 97% of these produced alternariol (AOH) in a range from 0.14 to 119.18 mg/kg, 95% produced alternariol methylether (AME) in a range from 1.23 to 901.74 mg/kg and 65% produced tenuazonic acid (TA) in a range from 0.13 to 2778 mg/kg. Fifty two isolates co-produced the three mycotoxins. According to the size of the lesion that they caused on blueberries, the isolates were classified as slightly pathogenic, moderately pathogenic and very pathogenic. No significant differences in pathogenicity were found on different blueberry varieties. In this work, high incidence and toxicogenic potential of the Alternaria isolates from blueberries were demonstrated. Thus, more studies should be done to evaluate the health risk posed by the presence of the Alternaria toxins in blueberries and in the manufactured by-products.

Water activity and temperature effects on mycotoxin production by Alternaria alternata on a synthetic tomato medium.

Alternaria spp. have been reported to be the most frequent fungal species invading tomatoes. Certain species, in particular the most common one, A. alternata, are capable of producing several mycotoxins in infected plants and in agricultural commodities. Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA) are some of the main Alternaria mycotoxins that can be found as contaminants of food. The objective of this study was to determine the effect of water activity (a(w), 0.904, 0.922, 0.954, and 0.982) and temperature (6, 15, 21 and 35 degrees C) on mycotoxin production on a synthetic tomato medium of a cocktail inoculum of five strains of A. alternata isolated from tomato fruits affected by Blackmould. The optimum AOH production occurred at 0.954 a(w) after 28days of incubation at 21 degrees C. A temperature of 21 degrees C was the most favourable for AOH synthesis at all a(w) levels. The maximum concentration of AME was determined at 0.954 a(w) and 35 degrees C. The optimum conditions for TA accumulation were 0.982 a(w) and 21 degrees C. At the 0.904 a(w) no growth or germination was registered at 6 degrees C and 15 degrees C over the whole incubation period. At 21 degrees C and 35 degrees C growth occurred slowly but none of the toxins were detected at this a(w) level. In general, high a(w) levels were favourable for mycotoxin production. None of the other toxins was detected at quantifiable levels at 6 degrees C after the whole incubation period. A storage temperature of 6 degrees C or below could be considered as safe for tomato fruits and high moisture tomato products (a(w)>0.95), in relation with Alternaria toxins. The results obtained here could be extrapolated to evaluate the risk of spoilage in tomato fruits and tomato products caused by this pathogen.

Effect of water activity and temperature on growth of Alternaria alternata on a synthetic tomato medium.

Alternaria alternata is a toxigenic fungus, predominantly responsible for Blackmould of ripe tomato fruits, a disease frequently causing substantial losses of tomatoes, especially those used for canning. The objective of this study was to determine the effect of water activity (a(w), 0.904, 0.922, 0.954, 0.982) and temperature (6, 15, 21 and 35 degrees C) on germination and radial growth rate on a synthetic tomato medium of a cocktail inoculum of five strains of A. alternata isolated from tomato fruits affected by Blackmould. The shortest germination time (1.5 days) was observed at 0.982 a(w), both at 21 degrees C and 35 degrees C. The germination time increased with a reduction on a(w). The fastest growth rate was registered at 0.982 a(w) and 21 degrees C (8.31 mm/day). Growth rates were higher when a(w) increased. No growth or germination was observed at the lowest a(w) level evaluated (0.904) after 100 days of incubation at 6 degrees C and 15 degrees C. A temperature of 6 degrees C caused a significant reduction in growth rates, even at the optimum a(w) level. The knowledge on the ecophysiology of the fungus in this substrate is necessary to elaborate future strategies to prevent its development and evaluate the consumer health risk.

Alternaria toxins in wheat during the 2004 to 2005 Argentinean harvest.

The natural occurrence of Alternaria mycotoxins in Argentinean wheat from the zone 5 South during the 2004 to 2005 harvest was investigated in 64 wheat samples. All samples were highly contaminated with a wide range of fungal species. Alternaria was found as the main component of the mycota, with an infection percentage of 100%. Three mycotoxins produced by species of Alternaria were determined in wheat: alternariol, alternariol monomethyl ether, and tenuazonic acid. Alternariol was detected in 4 (6%) of 64 samples, with a range of 645 to 1,388 microg/kg (mean of 1,054 microg/kg); alternariol monomethyl ether, with a range of 566 to 7,451 microg/kg (mean of 2,118 microg/kg) in 15 (23%) of 64 samples; and tenuazonic acid in 12 (19%) of 64 samples, with a range of 1,001 to 8,814 microg/kg (mean, 2,313 microg/kg). Alternariol monomethyl ether was the predominant toxin, but tenuazonic acid was detected in higher concentrations. Alternariol was present in fewer samples and in lower levels than were the other toxins. Tenuazonic acid and alternariol monomethyl ether occurred together in four samples, while tenuazonic acid and alternariol co-occurred in one sample. This the first report of the natural occurrence of Alternaria mycotoxins in Argentinean wheat. Toxin levels were high, probably due to the heavy infection with Alternaria species found in the samples.

Mycotoxin production by Alternaria strains isolated from Argentinean wheat.

The toxigenic potential of Alternaria strains isolated from Argentinean wheat was investigated. A total of 123 strains were assayed for the production of tenuazonic acid (TA), alternariol (AOH) and alternariol monomethyl ether (AME). All but one of the isolates were able to produce at least one of the three mycotoxins. TA was produced by 72% of the strains (1-14782 mg/kg), AOH by 87% (4-622 mg/kg) and AME by 91% (7-2625 mg/kg). The average level of TA detected for all strains (1757 mg/kg) was higher than the average level of both alternariols (162 mg/kg for AOH and 620 mg/kg for AME). TA was the toxin produced at the highest concentration but in lower frequency. Most of the strains were able to synthesize more than one toxin: 74 isolates (60%) were positive for all three toxins, 30 (24%) for both AOH and AME, 5 (4%) for both TA and AME, and 2 (2%) for TA and AOH. The widespread occurrence of Alternaria in wheat and its ability to produce mycotoxins suggests the possible occurrence of its toxins in wheat naturally infected with this fungus.

Effect of water activity and temperature on growth of ochratoxigenic strains of Aspergillus carbonarius isolated from Argentinean dried vine fruits.

Aspergillus carbonarius is an ochratoxin A (OTA) producing fungus, predominantly responsible for the production of this mycotoxin in grapes, wine and dried vine fruits. The objective of this study was to determine the in vitro effects of water activity (a(w), 0.80-0.95) and temperature (15-35 degrees C) on lag phase extension and radial growth rate of a cocktail inoculum of four strains of A. carbonarius. The maximum growth rate was observed at 0.95 a(w) and 30 degrees C (17.46 mm day(-1)). In general, growth rates increased with the increment of a(w). No growth was observed at a(w) below 0.85. For all a(w) levels tested the highest growth rate was detected at 30 degrees C. At 15 degrees C growth only occurred at the higher a(w) levels evaluated (0.925 and 0.95) at a growth rate of 3.82 and 5.57 mm day(-1) respectively. The shortest lag phase (0.26 days) was found at 0.925 a(w) and 35 degrees C. At marginal conditions of a(w) and temperature the lag phases increased, being the highest registered at 20 degrees C and 0.89 a(w) (33.7 days). The pattern of effects of environmental factors on growth was similar among Argentinean A. carbonarius strains and those from several European countries, Israel and Australia.

Occurrence of alternariol, alternariol monomethyl ether and tenuazonic acid in Argentinean tomato puree.

The occurrence ofAlternaria mycotoxins was investigated in 80 samples of tomato puree processed and sold in Argentina. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) were searched for by liquid chromatography. Thirty-nine of the 80 samples showed mycotoxin contamination. TA was found in 23 samples (39-4021 µg/kg), AOH in 5 samples (187-8756 µg/kg), and AME in 21 samples (84-1734 µg/kg). Co-occurrence of two of these toxins was detected in 10 samples. This is the first report of natural occurrence of AOH, AME and TA in tomato products in Argentina.

Toxigenic fungi isolated from dried vine fruits in Argentina.

To evaluate the potential for mycotoxin production by fungi in dried vine fruits, the mycobiota was determined both before and after surface disinfection. Predominant genera were Aspergillus (50.2%), Eurotium (21.4%) and Penicillium (13.5%). Aspergillus section Nigri ("black aspergilli") were isolated with relatively high frequency. Aspergillus niger was the most common species but only 3 of 293 isolates screened were ochratoxin A (OTA) producers. Aspergillus carbonarius was less common but 96% of 48 strains screened were ochratoxigenic. OTA was not produced by A. japonicus. Other toxigenic fungi detected were A. ochraceus (3 strains produced OTA), Aspergillus flavus (5 strains produced cyclopiazonic acid but not aflatoxins), P. citrinum (19 strains were strong citrinin producers) and Alternaria alternata (15 strains were producers of tenuazonic acid, alternariol and alternariol methyl ether). In spite of the high incidence of A. carbonarius capable of producing OTA, low levels of this toxin were detected in the samples analysed.

Mycotoxin production byAlternaria strains isolated from tomatoes affected by Blackmold in Argentina.

A survey on 28 samples of Blackmold" affected tomatoes cultivated in Argentina was made.A. alternata was the most frequently occurring species.A. tenuissima, A. longipes yUdocladium spp. were also isolated. All theAlternaria strains (25) were able to produce mycotoxins, including Alternariol, Alternariol Monomethyl Ether and Tenuazonic Acid, on autoclaved rice (at 25°C and 8°C) and in fresh surface disinfected tomatoes at 25°C.

Natural co-occurrence of aflatoxin and cyclopiazonic acid in peanuts grown in Argentina.

Natural occurrence of aflatoxins and cyclopiazonic acid (CPA) contamination in peanuts was investigated. Co-occurrence of CPA and aflatoxins was detected in two of 50 samples analysed. The levels of these toxins found in positive samples were 4300 and 493 microg kg(-1) for CPA, 625 and 435 microg kg(-1) for aflatoxin B1 (AFB1), and 625 and 83 microg kg(-1) for aflatoxin G1 (AFG1), respectively. Levels of CPA contamination in the positive samples were similar to those registered in other substrates. This is the first report of natural co-occurrence of CPA and aflatoxins in Argentina.

Influence of water activity and temperature on the growth of Wallemia sebi: application of a predictive model.

Germination and growth of Wallemia sebi were examined on media of a(w) adjusted with glycerol in the range of 0.96-0.77, at 25 degrees C and 30 degrees C. The effect of temperature on the germination time was significant except between 0.95 and 0.88 a(w). At low a(w) levels as well as above 0.95, the increase of temperature produced an increment in the germination time. The minimum a(w) for germination was also affected by temperature, being lower at 25 degrees C (0.80 a(w)) than at 30 degrees C (0.82 a(w)). Radial growth rates at 25 degrees C were higher than at 30 degrees C. The optimum a(w) value for growth of W. sebi was 0.94 at both temperatures. The minimum a(w) for growth was higher than minimum for germination and was also dependent on temperature (0.84 at 25 degrees C and 0.86 at 30 degrees C). An empirical mathematical model was fitted to the measured growth data, providing a good approach to the description of the effect of a(w) on the radial growth rate of W. sebi.

Influence of water activity on deoxynivalenol accumulation in wheat.

The influence of water activity (aw) on deoxynivalenol accumulation in wheat at 25°C was studied. Gnotobiotic grains were conditioned at different aw levels, inoculated with a toxigenic Fusarium graminearum strain, and incubated for ten weeks. The highest accumulation of deoxynivalenol (1130 ug/kg) was detected at aw 0.980. At aw 0.945 and 0.925 the maximum quantities of toxin accumulated were 113 ug/kg and 93 ug/kg respectively. Deoxynivalenol was not detected in the substrate at aw 0.900. Results suggest that intermediate aw levels (0.97 - 0.92) are particularly critical at post harvest time because those are conditions under which deoxynivalenol production is most likely to be initiated naturally.

Influence of water activity on Penicillium citrinum growth and kinetics of citrinin accumulation in wheat.

The influence of water activity (aw) on both Penicillium citrinum growth and citrinin accumulation in wheat was studied. Wheat conditioned at different levels of aw and inoculated with a citrinin producer strain was incubated at 30 degrees C for 2 months. Fungal growth was assessed by microscopic examination. P. citrinum grew down to aw 0.775. Citrinin was not detected in the substrate at aw 0.800 and lower. As aw increased the toxin was detected earlier and the maximum accumulation increased markedly (65 microg/kg at aw 0.810, 460 microg/kg at aw 0.825 and 22 mg/kg at aw 0.885). Citrinin concentration declines rapidly after reaching the maximum at each aw level.

Amaranth grain as substrate for aflatoxin and zearalenone production at different water activity levels.

The influence of water activity (a(w)) on the kinetics of aflatoxin and zearalenone production in amaranth grains at 25 degrees C was studied. Minimum a(w) for aflatoxin production in this substrate was 0.825. Accumulation of the four aflatoxins (B1, B2, G1 and G2) was similar at a(w) 0.825 (maximum 81.2 microg/kg after 42 days) and 0.868 (maximum 109.6 microg/kg after 49 days). Maximum accumulation of total aflatoxins at a(w) 0.902 (260.4 microg/kg) was detected after 21 days, with an appreciable increment in the concentration of aflatoxins B1 and G1. These quantities were lower than those reported for aflatoxin production on other cereals and legumes, indicating that amaranth is not a good substrate for aflatoxin production. Zearalenone was not detected at a(w) 0.902. Maximum accumulation of zearalenone was 1.5 microg/g after 35 days at a(w) 0.925 and 11.1 microg/g after 49 days at a(w) 0.950.

[Mycotoxin-producing fungi in foods]. / Hongos productores de micotoxinas en alimentos.

This is an overview of the most important toxicogenic species of molds that contaminate human food and animal feed. Effects of fungal secondary metabolites are described. Attention is focused on toxicogenic species of Aspergillus, Penicillium and Fusarium. Species of other genera reported as toxicogenic are also considered.

[Effect of water activity on the growth of the xerophilic mold Eurotium herbariorum]. / Influencia de la actividad de agua en el crecimiento del hongo xerofílico Eurotium herbariorum.

The influence of water activity (aw) on growth of a xerophilic mold isolated from dried figs and identified as Eurotium herbariorum was studied on culture media of aw adjusted with sucrose or glycerol. Rate of radial growth (kr) and lag period were the kinetic parameters analyzed. Fungal growth was inhibited at aw > 0.97. In the presence of sucrose, optimum growth was found in M6OY agar (malt agar with yeast extract and 60% W/V of sucrose, aw = 0.95). On glycerol (aw ranging from 0.65 to 0.90) the fungus did not grow at aw < 0.80. Sucrose supported better growth than glycerol at aw 0.90.

Influencia de la actividad de agua en el crecimiento del hongo xerofílico Eurotium herbariorum / [Effect of water activity on the growth of the xerophilic mold Eurotium herbariorum]

The influence of water activity (aw) on growth of a xerophilic mold isolated from dried figs and identified as Eurotium herbariorum was studied on culture media of aw adjusted with sucrose or glycerol. Rate of radial growth (kr) and lag period were the kinetic parameters analyzed. Fungal growth was inhibited at aw > 0.97. In the presence of sucrose, optimum growth was found in M6OY agar (malt agar with yeast extract and 60

W/V of sucrose, aw = 0.95). On glycerol (aw ranging from 0.65 to 0.90) the fungus did not grow at aw < 0.80. Sucrose supported better growth than glycerol at aw 0.90.

Influencia de la actividad de agua en el crecimiento del hongo xerofílico Eurotium herbariorum. / [Effect of water activity on the growth of the xerophilic mold Eurotium herbariorum]

The influence of water activity (aw) on growth of a xerophilic mold isolated from dried figs and identified as Eurotium herbariorum was studied on culture media of aw adjusted with sucrose or glycerol. Rate of radial growth (kr) and lag period were the kinetic parameters analyzed. Fungal growth was inhibited at aw > 0.97. In the presence of sucrose, optimum growth was found in M6OY agar (malt agar with yeast extract and 60

W/V of sucrose, aw = 0.95). On glycerol (aw ranging from 0.65 to 0.90) the fungus did not grow at aw < 0.80. Sucrose supported better growth than glycerol at aw 0.90.