Effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain

Both water activity (aW) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 aW and 25 degrees C on both substrates. At 0.98 aW and 25 degrees C, a single colony of A. alternata grown on wheat extract agar produced 807 micrograms of AOH, 603 micrograms of AME, and 169 micrograms of AE ml in 30 days. However, production of all three mycotoxins at 0.95 aW was less than 40% of these amounts. Little toxin was produced at 0.90 aW. Changing temperature and aW altered the relative amounts of the different toxins produced on agar. At 15 degrees C and 0.98 aW, maxima of 52 micrograms of AOH and 25 micrograms of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 micrograms/ml after 40 days. At 15 degrees C and 0.95 aW, production was, respectively, 62, 10, and 5 micrograms/ml after 40 days. All three metabolites were produced at 5 degrees C and 0.98 to 0.95 aW and at 30 degrees C and 0.98 to 0.90 aW. On wheat grain at 25 degrees C and 0.98 to 0.95 aW, more AME was produced than AOH or AE, but at 15 degrees C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25 degrees C and 0.90 aW, but production of AME was inhibited at 30 degrees C and 0.95 aW or less.     

Nitrogen inhibition of mycotoxin production by Alternaria alternata.

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.     

Nitrogen inhibition of mycotoxin production by Alternaria alternata

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.     

Blue light inhibits mycotoxin production and increases total lipids and pigmentation in Alternaria alternata.

Light inhibits production of the mycotoxins alternariol and alternariol monomethyl ether, both polyketids produced by Alternaria alternata. This effect seems to be general because seven isolates of A. alternata with different alternariol- and alternariol monomethyl ether-producing abilities all respond to continuous light with reduced levels of alternariol and alternariol monomethyl ether when the mycotoxins were calculated on a microgram-per-milligram (dry weight) basis. Blue light inhibited alternariol and alternariol monomethyl ether production 69 and 77%, respectively. Red light gave no reduction of toxin levels. Total lipids were increased 25% when mycelium was grown in blue light as compared with red light or darkness. In white or blue light, but not in red light or darkness, a red-brown pigment accumulated by the mycelium.     

Effect of substrate on metabolite production of Alternaria alternata.

Alternariol and alternariol monomethyl ether are commonly associated with weathered grain sorghum. Production of these metabolites and altenuene by isolates of Alternaria alternata was evaluated on various sterile grain substrates. At 35% moisture content and 25 C, metabolite yields were highest on rice, intermediate on sorghums, and lowest on wheat and yellow corn. Fourteen-to 21-day cultures on milled rice were best in terms of ease of metabolite recovery, even though yields were higher on 28-day cultures of rough and brown rice. Metabolite production was reduced when rice was supplemented with yeast extract or yeast extract plus Czapek-Dox broth.     

Blue light inhibits mycotoxin production and increases total lipids and pigmentation in Alternaria alternata

Light inhibits production of the mycotoxins alternariol and alternariol monomethyl ether, both polyketids produced by Alternaria alternata. This effect seems to be general because seven isolates of A. alternata with different alternariol- and alternariol monomethyl ether-producing abilities all respond to continuous light with reduced levels of alternariol and alternariol monomethyl ether when the mycotoxins were calculated on a microgram-per-milligram (dry weight) basis. Blue light inhibited alternariol and alternariol monomethyl ether production 69 and 77%, respectively. Red light gave no reduction of toxin levels. Total lipids were increased 25% when mycelium was grown in blue light as compared with red light or darkness. In white or blue light, but not in red light or darkness, a red-brown pigment accumulated by the mycelium.     

Effect of water activity and temperature on Tenuazonic acid production byAlternaria alternata on sunflower seeds

Alternaria alternata is isolated in high frequency in sunflower seeds both in the field and storage. This species produces several toxic metabolites among them alternariol, alternariol monomethyl ether and tenuazonic acid. The accumulation of mycotoxins is regulated by physical, chemical and biological factors and for their production in many commodities nothing is known with regards to these conditions. In sunflower seeds the optimal conditions of temperature and water activity for tenuazonic acid production are unknown. The aim of this work was to investigate the effect of temperature and water activity on tenuazonic acid production byAlternaria alternata ITEM 539 in sunflower seeds. The temperature conditions evaluated were: 20, 25, and 30 °C and the water activities were 0.97, 0.90, 0.87, 0.80. The tenuazonic acid determinations were carried out during the incubation period at intervals of 7 days. Under conditions of constant temperature 25 °C) and variable water activities, 0.90 was the optimal value for tenuazonic acid production. At this water activity of the optimal temperature for tenuazonic acid production was 25 °C.     

Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton.

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.     

Influence of fungicides on mycotoxin production by Alternaria mali

Extracts of fungicide induced variants ofAlternaria mali were tested with mice and bacteria. Both the living fungi and their crude chloroform extracts inhibited growth ofStaphylococcus aureaus, Sarcina lutea, Bacillus mycoides, andB. subtilis. B. megaterium was not sensitive to most of the extracts and was only slightly so to the remainder. The LD₅₀ in mice when injected intraperitoneally ranged from 300 mg/kg to 2400 mg/kg; however, in some cases there were no lethal effects. The toxicity of the wild type was greatly reduced when grown in the presence of fungicide decomposition products. Altenuene, alternariol, and alternariol monomethyl ether were not found in any of the extracts.     

Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.     

Effect of environmental factors on tenuazonic acid production by Alternaria alternata on soybean-based media

Aims: To determine the effects of water activity (a_W; 0·995–0·90), temperature (5, 18, 25 and 30°C), time of incubation (7–35 days) and their interactions on tenuazonic acid (TA) production on 2% soybean‐based agar by two Alternaria alternata strains isolated from soybean in Argentina. Methods and Results: TA production by two isolates of A. alternata was examined under interacting conditions of a_W, temperature and time of incubation on 2% soybean‐based agar. Maximum TA production was obtained for both strains at 0·98 a_W, but at 30 and 25°C for the strains for RC 21and RC 39, respectively. The toxin concentration varied considerably depending on a_W, temperature, incubation time and strain interactions. TA was produced over the temperature range from 5 to 30°C and a_W range from 0·92 to 0·995, however at 5 and 18°C little TA was produced at a_W below 0·94. Contour maps were developed from these data to identify areas where conditions indicate a significant risk for TA accumulation. Conclusions: The optimum and marginal conditions for TA production by A. alternata on soybean‐based agar were identified. The results indicated that TA production by A. alternata is favoured by different temperatures in different strains. Significance and Impact of the Study: Data obtained provide very useful information for predicting the possible risk factors for TA contamination of soybean as the a_W and temperature range used in this study simulate those occurring during grain ripening. The knowledge of TA production under marginal or sub‐optimal temperature and a_W conditions for growth are relevant as improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality.     

Incidence and mycotoxin production by Alternaria tenuis in decayed apples

Alternaria tenuis was the main species of Alternaria which produced post-harvest decay in apples. Alternariol (AOH) and alternariol monomethyl ether (AME) were the major mycotoxins produced in Alternaria -decayed apples at 25°C and 2°C. Only the strain Alternaria tenuis CMTA 65 at 25°C produced tenuazonic acid (TA). Altertoxin-I (ATX-I) and altertoxin-II (ATX-II) were not detected in any of the apple samples.
A large percentage of strains of A. tenuis studied produced TA (97%) and AT-I (82·3%) when grown in a yeast extract sucrose medium (YES) at 25°C. A much smaller percentage of strains produced AOH and AME and none were found to produce detectable levels of ATX-II.     

Simultaneous production of glucose oxidase and catalase by Alternaria alternata

Summary A number of factors affecting simultaneous production of cell-bound glucose oxidase and catalase by the fungus Alternaria alternata have been investigated. Consecutive optimization of the type and concentration of nitrogen and carbon source, the initial pH and growth temperature resulted in a simultaneous increase in glucose oxidase and catalase by 780% and 68% respectively. Two second-order equations, describing the combined effect of pH and temperature on the activity of each enzyme, revealed that glucose oxidase had its optima at pH 7.9 and 32.3°C and catalase at pH 8.5 and 18.1°C. Under certain growth conditions, yields as high as 23.5 and 18,100 units/g carbon source for glucose oxidase and catalase, respectively, were simultaneously obtained.Offprint requests to: B. J. Macris     

Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro

Previously, Alternaria extract and metabolite mutagenicities+/-nitrosylation were characterized using Ames Salmonella strains TA98 and TA100, which are both reverted at GC sites. To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined+/-nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ATX I was also assessed for mammalian mutagenicity at the Hprt gene locus in Chinese hamster V79 lung fibroblasts and rat hepatoma H4IIE cells. When tested from 1 to 100 microg/plate without nitrosylation, ATX I was mutagenic in TA102+/-rat liver S9 for activation and weakly mutagenic in TA104+/-S9, demonstrating direct-acting AT base pair mutagenicity. AOH was also directly mutagenic at AT sites in TA102+/-S9 while AME was weakly mutagenic in TA102+/-S9 and TA104+S9. Nitrosylation of ATX I enhanced mutagenicity at AT sites in TA104+/-S9 but produced little change in TA102+/-S9 compared to native ATX I. However, nitrosylated ATX I generated a potent direct-acting frameshift mutagen at C sites in TA97+/-S9. While ATX I was not mutagenic in either V79 cells or H4IIE cells, 5 and 10 microg/ml nitrosylated ATX I produced a doubling of 6-thioguanine resistant V79 colonies and 0.5 and 1 microg/ml were mutagenic to H4IIE cells, becoming toxic at higher concentrations. These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.     

Two cases of cutaneous phaeohyphomycosis by Alternaria alternata and Alternaria tenuissima

Two cases of cutaneous phaeohyphomycosis, one with a nodular appearance and the other with an erythematous infiltrating patch, are reported in immunocompromised patients. Diagnosis was based on histological examination, which revealed hyphae and round-shaped fungal cells in a granulomatous dermal infiltrate, and on identification of the moulds when biopsy fragments were cultured on Sabouraud-dextrose agar without cycloheximide. The pathogens were Alternaria tenuissima in the first case and A. alternata in the second. The fungi were examined by scanning electron microscopy. The patients were checked for bone and lung involvement and were then treated with surgical excision and itraconazole, and itraconazole only, respectively, with clinical and mycological resolution. This revised version was published online in August 2006 with corrections to the Cover Date.     

Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

Characterization of epoxide hydrolase activity in shape Alternaria alternata f. sp. shape lycopersici. Possible involvement in toxin production

Using trans-diphenylpropane oxide (tDPPO) as a substrate, we measured epoxide hydrolase (EH) activity in subcellular fractions of Alternaria alternata f. sp. lycopersici (Aal), a fungus that produces host-specific toxins. The activity was mainly (>99.5%) located in the soluble fraction (100,000 × g supernatant) with the optimum pH at 7.4. An increase of toxin production between days 3 and 9 found in a Aal liquid culture over a 15 days period was concomitant with a period of high EH activity. EH activity remained constant during the same period in an Alternaria alternata culture, a fungus which does not produce toxin. In vivo treatment of Aal culture with the peroxisome proliferator clofibrate stimulated EH activity by 83% and enhanced toxin production 6.3 fold. Both 4-fluorochalcone oxide (4-FCO) and (2S,3S)-(-)-3-(4-nitrophenyl)-glycidol (SS-NPG) inhibited EH activity in vitro with a IM₅₀f 23 ± 1 μM and 72 ± 19 μM, respectively. The possible physiological substrate 9,10-epoxystearic acid was hydrolyzed more efficiently by Aal sEH than the model substrates trans- and cis-stilbene oxide (TSO and CSO) and trans- and cis-diphenylpropane oxide (tDPPO and cDPPO). This revised version was published online in June 2006 with corrections to the Cover Date.