V. Fernández Pinto

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.

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 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.

A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds

Thirty-five samples of poultry feeds and corresponding raw materials (maize, soybean and meat meal) from a processing plant were analyzed to evaluate the distribution and toxigenicity of Aspergillus section Flavi isolates. Mycological analysis of the samples indicated the presence of five fungal genera (Aspergillus, Penicillium, Fusarium, Cladosporium, and Eurotium). Aspergillus flavus was the predominant species being present in 48.5% of the analyzed samples. Ninety-one isolates belonging to Aspergillus section Flavi were isolated; ninety were identified as A. flavus and only one as A. parasiticus. Fifty-seven isolates were capable of producing sclerotia, 41 were identified as L-type strains and 16 as type S. Fifty-seven percent of the isolates produced AFB₁ levels ranging from 0.05 μg/kg to 27.7 μg/kg whereas 86.8% produced CPA from 1.5 μg/kg to 137.8 μg/kg. L-strains produced from 0.05 to 14.8 μg/kg of aflatoxin and type S produced levels from 0.05 to 1.65 μg/kg. No significant differences in CPA production among S- and L-strains were observed. Sclerotial isolates produced AFB₁ levels ranging between 0.05 and 27.7 μg/kg and CPA levels from 3.8 to 47.3 μg/kg. More than half of the A. flavus isolates were able to produce AFB and CPA simultaneously. Twenty percent of the 35 samples were contaminated with aflatoxin B₁ whereas 34.3% were contaminated with CPA. The high rate of CPA producing isolates represents a potential risk of contamination with this toxin in poultry feeds.

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.

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.

Temperature and water stress impacts on growth and production of altertoxin-II by strains of Alternaria tenuissima from Argentinean wheat

Alternaria tenuissima is commonly isolated from wheat in Argentina. The objective of this study was to examine the effects of temperature (15-34 °C) and water activity (0.98, 0.95 aw) on growth and temporal altertoxin II (ALTX-II) production by two strains over 14-21 days on a milled wheat agar. It was shown that growth occurred over the whole temperature range tested and was optimum at 25-30 °C and 0.98 aw, and 30 °C at 0.95 aw. The incubation time did not show any significant effect on ALTX-II accumulation. The optimum conditions for ALTX-II production were 0.98 aw and 30 °C for both strains. The strains also accumulated significant amounts of this toxin at 34 °C. This is the first study to evaluate the ecology of growth and production of ALTX-II by strains of A. tenuissima.