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

Abstract

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 °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 28 days of incubation at 21 °C. A temperature of 21 °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 °C. The optimum conditions for TA accumulation were 0.982 a_w and 21 °C. At the 0.904 a_w no growth or germination was registered at 6 °C and 15 °C over the whole incubation period. At 21 °C and 35 °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 °C after the whole incubation period. A storage temperature of 6 °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.

Introduction

The genus Alternaria includes both plant-pathogenic and saprophytic species which may affect crops in the field or cause harvest and post harvest decay of plant products (Logrieco et al., 2009). As is common for many soft-skinned vegetables and fruits, tomatoes are especially susceptible to fungal invasion. Alternaria spp. have been reported to be the most frequent fungal species invading tomatoes (Barkai-Golan and Paster, 2008).

Species of Alternaria are known to produce many metabolites which play an important role in plant pathogenesis. Certain species produce several mycotoxins in the infected plants (Logrieco et al., 2009). Alternaria spp. can produce a wide variety of metabolites belonging to three different structural groups: (i) the dibenzopyrone derivatives, alternariol (AOH), alternariol monomethyl ether (AME), and altenuene (ALT); (ii) the perylene derivatives altertoxins (ATX-I and II); and (iii) the tetramic acid derivative, tenuazonic acid (TA). TA, AOH, AME and ATX-I are the main Alternaria mycotoxins that can be found as contaminants of food commodities. They are produced by Alternaria alternata which is considered the most common Alternaria species in harvested fruits and vegetables (Barkai-Golan and Paster, 2008).

The chemical and toxicological aspects of the Alternaria toxins have been recently reviewed (Logrieco et al., 2009, Ostry, 2008, Scott, 2001). Of particular health concern is the association found between A. alternata contamination in cereal grains and the high levels of human esophageal cancer in China (Liu et al., 1991). The toxicity of TA has been reported in plants, in chick embryos and several animal species, including guinea pigs, mice, rabbits, dogs, and rhesus monkeys (Solfrizzo et al., 2005). In dogs, it caused haemorrhages in several organs and in chickens sub-acute toxicity was observed. Precancerous changes were observed in esophageal mucosa of mice. The possible involvement of TA in the etiology of inlay, a human hematological disorder occurring in Africa, has been suggested (Scott, 2004). AME and AOH were mutagenic in microbial and mammalian cell systems (An et al., 1989, Liu et al., 1992, Scott and Stolz, 1980). There is also some evidence of carcinogenic properties: squamous cell carcinoma were induced in mice subcutaneously inoculated with human embryo esophageal tissue that had been treated with alternariol; and NIH/3 T3 cells transformed by AME were subcutaneously tumorigenic in mice (Scott, 2004). Lehmann et al. (2006) have recently reported on the estrogenic potential of AOH, its inhibitory effects on cell proliferation, and its genotoxic effect in cultured mammalian cells. Altertoxin I and related compounds may cause acute toxicity in mice, and were reported to be more potent mutagens than AOH and AME (Ostry, 2008).

Natural occurrence of AOH and AME has been reported in various fruits, including tomatoes, olives, mandarines, melons, peppers, apples and raspberries, and also in grains, sunflower seeds, oilseed rape and pecans. The occurrence of low levels of AOH in processed fruit products – apple juice, processed tomato products, grape juice, red wine, cranberry nectar, prune nectar and raspberry juice – is possibly of human health interest; in apple juice, prune nectar and tomato products, AME has also been detected (Scott, 2004, Terminiello et al., 2006). TA has been shown to occur in several Alternaria infected fruits and vegetables, and in other foodstuffs, such as grains and seeds (Azcarate et al., 2008, Scott, 2001). It has also been found in tomato products and spoiled tomatoes in Canada and the US (Scott and Kanhere, 1980, Stack et al., 1985), in Brazilian tomato products (da Motta and Valente Soares, 2001) and in Argentinean tomato puree (Terminiello et al., 2006). Few studies on the stability and reactions have been carried out on Alternaria mycotoxins although in common with other mycotoxins they are probably quite stable. A major proportion of the toxins survived the autoclaving of tomatoes in producing tomato paste. Alternariol, alternariol monomethyl ether, and altertoxin I were stable in fruit juices and wine over 20 days or at 80 °C after 20 min (Lawley, 2010).

Mycotoxin production depends on the fungal strain, the substrate on which it grows and the environmental growth conditions. The two most important environmental factors on the development of fungi and mycotoxin production are water activity (a_w) and temperature (Magan et al., 1984). Alternaria species grow best at room temperature but also are capable of growing at low temperatures. For this reason they are often involved in spoilage of fruit and vegetables during refrigerated transport and storage (Barkai-Golan and Paster, 2008). Knowledge of the influence of environmental factors on growth and mycotoxin formation can be an important aid in predicting mycotoxin contamination of food. Furthermore, the a_w and temperature limits for growth and mycotoxin production are sometimes markedly different (Magan et al., 1984). Growth of mycotoxigenic Alternaria species and its relation with these factors have been described in different substrates (Lacey, 1992, Pose et al., 2009). However, no studies have been carried out on mycotoxin production by Alternaria strains isolated from tomatoes or on tomato products. Such information is important in developing realistic forecasting systems for predicting risk of mycotoxin contamination of fruits or processed products.

Due to the high incidence of Alternaria and its mycotoxins in commodities and food products in Argentina, the objective of this work was to study the effects of a_w and temperature on the production of AOH, AME and TA by strains of A. alternata isolated from tomato fruits affected by Blackmould on a synthetic tomato medium.