Influence of temperature, water activity and incubation time on fungal growth and production of ochratoxin A and zearalenone by toxigenic Aspergillus tubingensis and Fusarium incarnatum isolates in sorghum seeds

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Highlights

  • Optimal growth conditions were 37 °C and 0.99 aw for the three A. tubingensis isolates.

  • Maximal OTA production was observed at 0.97 aw and 37 °C.

  • A. tubingensis isolates grew and produce OTA at 15 °C only at 0.97 and 0.99 aw.

  • Optimal growth conditions were 25 °C and 0.99 aw for F. incarnatum isolates.

  • There is no correlation between the growth and production of ZEA in the case of F. incarnatum.

Abstract

The major objective of this study was to describe the effect of water activity and temperature on radial growth and production of ochratoxin A (OTA) and zearalenone (ZEA) on sorghum grains of three Aspergillus tubingensis and three Fusarium incarnatum isolates. The water activity range was 0.91–0.99 aw for F. incarnatum isolates and 0.88–0.99 aw for A. tubingensis isolates. Temperatures of incubation were 15, 25 and 37 °C for both species. Mycotoxin production was determined after 7, 14, 21 and 28 days depending on the growth rate of the six isolates. Maximum growth rates (mm/day) were observed at 37 °C and 0.99 aw for A. tubingensis isolates and at 0.99 aw and 25 °C for F. incarnatum isolates. A. tubingensis was able to grow at 15 °C only at the highest aw levels (0.97 and 0.99 aw). However, at this temperature F. incarnatum grew at 0.94 aw. Optimum ochratoxin A production was observed at 0.97 aw × 37 °C whereas optimal conditions for ZEA production varied from one isolate to another. Moreover, isolates of F. incarnatum from Tunisia do not require high aw and temperature levels to yield maximum levels of ZEA. In general, our results showed that there is no correlation between the growth and production of ZEA in the case of F. incarnatum. This is the first study on the water activity and temperature effect on growth rate and ZEA production of F. incarnatum. Our results show that sorghum grains not only support growth but also OTA and ZEA production by A. tubingensis and F. incarnatum, respectively.

Introduction

Sorghum grains suffer from severe infection and colonization by several toxigenic fungi during panicle and grain developmental stages (Waliyar et al., 2008). Several species of Aspergillus, Alternaria, Fusarium, Cladosporium, Curvularia and Penicillium are among the prevalent grain mold pathogens in sorghum (Bandopadyay et al., 2000, Lahouar et al., 2015). Consequently, sorghum is usually contaminated by zearalenone, fumonisins, aflatoxins and ochratoxin A. In Tunisia, sorghum sold in the retail market is often found contaminated with ochratoxin A and zearalenone. Indeed, OTA was found in 51.0% of sorghum samples with an average of 5.4 ng/g (Ghali et al., 2009). However, Zaied et al. (2009) reported high levels of OTA in analyzed sorghum samples with an average of 117 μg/kg. ZEA has been also detected in sorghum seeds with an incidence of 23.5% (Ghali et al., 2008).

Aspergillus ochraceus and Penicillium verrucosum were initially believed to be the main OTA-producing species (Hesseltine et al., 1972, Pitt, 1987). Penicillium verrucosum is considered to be more frequently associated with cereals in temperate climates, however Aspergillus ochraceus is more commonly found in warm and tropical climates (Pitt and Hocking, 1997). Furthermore, the significance of black aspergilli as OTA-producing fungi has changed since the first description of OTA production by Aspergillus niger var. niger (Abarca et al., 1994) and Aspergillus carbonarius (Horie, 1995). It is now considered that in food commodities such as grapes, raisins and wine, OTA contamination is due mainly to A. carbonarius and some A. niger aggregate species, mainly A. niger and A. tubingensis (Abarca et al., 2001, Perrone et al., 2007, Perrone et al., 2008). Aspergillus section Nigri includes 19 species: four uniseriate (A. japonicus, A. uvarum, A. aculeatinus and A. aculeatus) and 15 biseriate species, e.g. A. brasiliensis, A. carbonarius, A. niger and A. tubingensis (Samson et al., 2007). The last two species are morphologically indistinguishable and are both commonly referred A. niger or A. niger aggregate. These two species have recently been distinguished by their ability to produce OTA (Medina et al., 2005, Perrone et al., 2006). The reported percentages of ochratoxigenic isolates belonging to the A. niger aggregate are lower than for A. carbonarius (Abarca et al., 2001, Medina et al., 2005, Chiotta et al., 2011). A. niger and other A. niger aggregate species were dominant in sorghum from Argentina, Thailand, Brazil and Tunisia (Pitt et al., 1994, Gonzalez et al., 1997, Alves dos Reis et al., 2010, Lahouar et al., 2015). However, A. carbonarius showed a higher distribution in Indian sorghum samples (Priyanka et al., 2014). This species was not detected in sorghum from Tunisia and all ochratoxigenic Aspergillus section Nigri isolates were Aspergillus niger aggregate species (Lahouar et al., 2015).

Fusarium incarnatum-equiseti species complex (FIESC) represents a complex of morphologically similar species. A multilocus sequence typing scheme analysis revealed that it comprises 30 phylogenetically distinct species (O'Donnell et al., 2009, O'Donnell et al., 2012), evenly divided among two clades designated Incarnatum and Equiseti. Fusarium equiseti is a cosmopolitan fungus (Burgess et al., 1994) distributed across regions with cool through to hot and arid climates (Leslie and Summerell, 2006). It behaves as a soil saprophyte associated with fruit rots and dead and other dying plant tissues but also may be a pathogen of a wide range of crops (Booth, 1971, Bosch and Mirocha, 1992). This species produces zearalenone (ZEA) (Kosiak et al., 2005, Leslie and Summerell, 2006). Fusarium incarnatum is extremely widespread and common in the tropics and subtropics but also found in the Mediterranean and occasionally in temperate regions (Leslie and Summerell, 2006). As suggested by Leslie and Summerell (2006), Fusarium incarnatum is synonymous with F. semitectum and F. pallidoroseum. Traditionally, F. semitectum has been considered to produce zearalenone (Schroeder and Hein, 1975, Aoyama et al., 2009). Fusarium incarnatum has been associated with sorghum seeds in India (Sharma et al., 2011, Divakara et al., 2013), Argentina (Gonzalez et al., 1997) and Tunisia (Lahouar et al., 2015). Aoyama et al. (2009) indicated that sorghum is a significant source of ZEA contamination and that the primary causative fungus is F. semitectum. Schroeder and Hein (1975)demonstrated ZEA contamination of sorghum in the United States with heavy infections by F. roseum ‘Gibbosum’ and F. roseum ‘Semitectum’. F. semitectum is frequently detected in sorghum grains (Lincy et al., 2011) and plant tissues from warmer regions (Leslie et al., 1990). However, Shotwell et al. (1980) reported that F. equiseti was present in sorghum samples contaminated with zearalenone and that isolates were able to produce this mycotoxin in cracked corn culture.

Knowledge of the effect of environmental conditions on mycotoxin production by toxigenic fungi may help prevent food contamination. Furthermore, current scientific literature on the effect of environmental factors on the level of OTA has been focused mainly on A. ochraceus (Pardo et al., 2004) and P. verrucosum (Lindblad et al., 2004, Cairns-Fuller et al., 2005, Czaban et al., 2006). Regarding black Aspergilli, studies have reported the influence of environmental conditions on OTA production by A. carbonarius in pistachios (Marín et al., 2008), coffee beans (Joosten et al., 2001) and synthetic grape-based medium (Marín et al., 2006, Lasram et al., 2010). However, Esteban et al., 2004, Esteban et al., 2006 have studied the effect of water activity on the OTA production by Aspergillus niger aggregate species in CYA and YES media. Studies on OTA production in natural substrates with A. niger species has been made in peanut and maize kernels (Astoreca et al., 2009a, Astoreca et al., 2009b, Alborch et al., 2011). A few studies have focused on the influence of abiotic factors on Aspergillus tubingensis growth in SNM medium (Selouane et al., 2009, García-Cela et al., 2014, Chiotta et al., 2015). Aspergillus tubingensis was detected in sorghum grains purchased from Tunisian market with an incidence of 23.05% (Lahouar et al., 2015), but the contribution of A. tubingensis to OTA contamination of sorghum seeds remains unknown. To date, all the research has been devoted to study the effect of environmental factors on fungal growth and mycotoxin production of several Fusarium species such as F. graminearum, F. verticillioides and F. proliferatum but no work has been done on F. incarnatum.

The aim of this work was to evaluate, under laboratory conditions, the effect of water activity (aw), temperature and incubation time on the fungal growth and production of zearalenone (ZEA) and ochratoxin A (OTA) in sorghum seeds inoculated with Fusarium incarnatum and Aspergillus tubingensis isolated from Tunisian sorghum.

Section snippets

Fungal strains

Three isolates of Fusarium incarnatum (F79, F86 and F115) and three isolates of Aspergillus tubingensis (AN1, AN28 and AN71) were used in this study. Strains were isolated from sorghum samples collected from the retail market of the region of the Sahel in Tunisia in 2011 and they are kept in the Food Technology department collection of the University of Lleida. Aspergillus tubingensis isolates were molecularly characterized using the primer pairs: NIG1, NIG2, AckS10R, AcKS10L and TUB1, TUB2 for

Growth assessment

Fig. 1 shows the interaction of the aw and temperature on the growth rate (mm/day) of three A. tubingensis and three F. incarnatum isolates inoculated on sorghum grain. It can be seen from Fig. 1 that the behavior of different isolates varies against aw and temperature.

Analysis of variance by the ANOVA test for A. tubingensis and F. incarnatum growth rates (Table 1) showed that the single factors: aw and temperature and the interactions: isolate × aw and aw × temperature had a significant effect on

Conclusion

This is the first study on the water activity and temperature effect on growth rate and ZEA production by Fusarium incarnatum. Previous studies have been conducted on other Fusarium species such as F. graminearum, F. oxysporum and F. culmorum. It can be concluded that there is no correlation between growth and ZEA production in the case of F. incarnatum. It is not possible to define the optimal conditions for ZEA production. Thus, to define safe conditions, studies in a wider range of

Acknowledgments

We thank Montse Prim for her encouragement and technical assistance. The authors are grateful to the European Union (MYCORED KBBE-2007-2-5-05 project) and Tunisian Government (U12E503) for the financial support.

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