4.1. In Vitro Effects of Climate-Related Abiotic Factors on Growth and OTA Production by Strains of A. westerdijkiae
This study has examined the resilience of strains of A. westerdijkiae in relation to two-way interacting abiotic factors (aw × temperature) and three-way interacting climate-related factors (aw × temperature × CO2) on growth and OTA production in vitro on coffee extract-based matrices and stored coffee. Interestingly, while growth was similar on both green coffee extract and roasted coffee extract media, OTA production on the roasted coffee-based medium was practically undetectable. Certainly the strains were able to grow well over a wide range of temperatures, with reduced growth at 0.90 aw. This suggests that rapid colonization of drying green coffee would occur in the range of 25–35 °C under 0.90–0.98 aw conditions and result in a higher risk of OTA contamination. The two strains chosen for further studies did produce significant amounts of OTA, and were thus used for more detailed studies on the impact of three-way interacting climate-related abiotic factors.
Previous studies by Taniwaki et al. [
17,
18] reported that
A. ochraceus (=
A. westerdijkiae) was primarily responsible for the OTA contamination of green coffee, suggesting a minimum a
w for growth of about 0.85. Pardo et al. [
19] suggested that in vitro growth on green coffee-based media was optimal for
A. ochraceus (=
A. westerdijkiae) strains at 0.95–0.99 a
w and 20–30 °C. They suggested the a
w minimum for germination was 0.80, and for mycelial growth was 0.85 a
w. Maximum OTA production for
A. ochraceus occurred at 0.99 a
w with no toxin produced at 0.80 a
w. Previous studies suggest that optimum OTA production by
A. ochraceus (=
A. westerdijkiae) was at 0.95 a
w, with no production at 0.90 a
w. The maximum growth for the type strains of
A. westerdijkiae on YES media occurred at 0.95 a
w and 30 °C. Optimum OTA production was at 25 °C [
1]. Akbar et al. [
13] used a 20% coffee-extract medium that influenced relative growth rates and OTA production although optimum conditions, especially for growth, were relatively similar. With the exception of the work of Pardo et al. [
19,
20] and Akbar et al. [
13], most of the other studies were carried out have used defined laboratory media instead of a heterogenous coffee-based medium. The latter medium may provide more useful data and may more accurately simulate what may occur in situ.
This study has shown the resilience of the strains of
A. westerdijkiae to two-way interacting climatic conditions that form a platform for examining in more detail the effects of three-way interacting climate-related abiotic factors by including exposure to existing and elevated CO
2 levels (400 vs. 1000 ppm) [
3]. Coffee beans during the drying and storage phase may be exposed to these three-way interacting conditions, which will influence both the dominance of toxigenic fungal groups including those from the
Aspergillus section
Circumdati and the section
Nigri species [
13,
21,
22]. The present study has focused on one of the key species from the section
Circumdati only. Studies have shown, however, that slightly elevated CO
2 levels combined with drought stress and increased temperature may enhance OTA production by strains of
A. westerdijkiae on green coffee-based media. Interestingly, the impact on OTA production appeared to be more pronounced than the effects on colonization rates. In addition, elevated CO
2 (1000 ppm) + elevated temperature (35 °C) increased OTA production when compared with 30 °C for one of the
A. westerdijkiae strains (B 2). Both strains had optimum growth at 0.95 a
w and 35 °C, while at 30 °C, the optimum was at 0.98 a
w.
Most previous studies that have examined the impact of a
w × elevated CO
2 have focused on modified atmosphere storage to try and control OTA production post-harvest in both coffee and other commodities. Cairns-Fuller et al. [
23] suggested that 50% CO
2 was required to inhibit growth and OTA production by
P. verrucosum in moist grain by >75% at 0.90–0.995 a
w. Paster et al. [
24] reported that no growth of
A. ochraceus (=
A. westerdijkiae) occurred at 80 or 100% CO
2, and that growth was partially inhibited by 60% CO
2. Similarly, Pateraki et al. [
25] found that 50% CO
2 inhibited
A. carbonarius growth after five days. Some studies have reported a reduction in growth rate at 5–10% of CO
2 for some species at low a
w levels, including an increase in the lag phases prior to growth. Although at ≥15% CO
2 most strains showed growth inhibition, especially under water stress [
26]. Valero et al. [
27] found that there was a significant reduction in growth and OTA production by
Aspergillus section
Nigri species such as
A. carbonarius and
A. niger at 1% O
2 when combined with 15% CO
2.
Medina et al. [
3] suggested that often while exposure of mycotoxigenic fungi such as
A. flavus to interacting climate-related abiotic factors has little effect on growth, this does however result in a significant stimulation of aflatoxin B
1 (AFB
1) production on milled maize-based media and in stored maize. Indeed, recent studies have shown that the kinetics of AFB
1 production change over time when this species is exposed to climate-related abiotic factors [
28]. This is supported by relative expression of key structural and regulatory genes involved in the biosynthesis of aflatoxins. Mycotoxigenic fungi show significant plasticity in physiologically responding to stress factors, and thus three-way interacting stress factors may have a more significant impact on the colonization of different commodities and production of mycotoxins and other secondary metabolites by these toxigenic species.
4.2. In Situ Effect of Three-Way Interacting Climate-Related Abiotic Factors on OTA Production in Stored Green Coffee
This study examined the effects of three-way climate-related abiotic factors on contamination of stored green Arabica coffee beans with OTA when inoculated with individual strains of ochratoxigenic Aspergillus section Circumdati species. Based on the water adsorption curve, the range of aw levels used represent a moisture content (m.c.) of between approx. 40% and 20–22%, which is equivalent to the range of 0.90–0.97 aw. This study has shown that there was a high OTA contamination of stored green coffee when colonized by A. westerdijkiae strains, especially at 0.95 and 0.97 aw and 30 °C, regardless of CO2 levels. At 35 °C, the OTA contamination levels were significantly lower than at 30 °C regardless of other abiotic treatments. However, at 35 °C there was higher OTA contamination levels in these two A. westerdijkiae strains at 0.95 and 0.90 aw and 1000 ppm CO2. This suggests that the stress of interacting abiotic factors results in a stimulation of OTA production by this species.
Previously, Palacios-Cabrera et al. [
15] found high amounts of OTA produced by
A. carbonarius in irradiated raw coffee beans when stored at 100% Equilibrium Relative Humidity (=1.00 a
w). Similarly, maximum growth rates of
A. ochraceus on irradiated green coffee beans was found to occur at 30 °C and 0.95–0.99 a
w, which was similar to the in vitro studies in the present work. Other studies on irradiated coffee beans have shown OTA production between 40–17,000 ng g
−1 over this a
w range at 30 °C [
20]. This study also showed that limited OTA was produced at 10 °C and 0.80 a
w. It has been specified that during post-fermentation and drying, the final safe storage m.c. for green coffee beans should be in the range of 10–12% (approx. 0.65–0.70 a
w [
29]). However, uneven drying can result in pockets of wetter coffee beans that are conducive to colonization by ochratoxigenic species.
Prior to the reclassification of the
Aspergillus section
Circumdati, Taniwaki et al. [
18] found that there was very low accumulation of OTA by
A. ochraceus (=
A. westerdijkiae) at 0.80 a
w and 25 °C, with an increased contamination at 0.95 a
w after three weeks storage. Bucheli et al. [
22] also found the lowest a
w levels for OTA contamination of green coffee by
A. ochraceus (=
A. westerdijkiae) were 0.85 and 20–30 °C. However, these earlier studies did not examine the effects of >30 °C on interactions with other abiotic factors, especially water stress and elevated CO
2. Akbar et al. [
7] found that there were differential effects of three-way interacting climate-related abiotic factors on species of the section
Circumdati and section
Nigri. The species from the latter group (
A. carbonarius,
A. niger) appeared to be less affected by such abiotic changes and thus had a better resilience to such conditions. This may of course have implications, such as the fungal community structure and dominance of different toxigenic species colonizing the ripening coffee cherries pre-harvest, and post-harvest drying and processing may change under climate-related conditions and influence both colonization and mycotoxin contamination in the coffee production chain [
3,
5].
In the in situ study only Arabica coffee beans were used. There are significant and inherent differences in the caffeine concentration between this type (0.6%) and Robusta (4%) [
30]. Caffeine has been shown to have some anti-fungal inhibitory effects and can inhibit both growth and mycotoxin production significantly. For example, Akbar et al. [
21] showed that 0.5–1.0% caffeine inhibited growth of toxigenic species from both Section
Circumdati and
Nigri groups on a conducive defined medium. Thus, it may be important to compare the effect of climate-related abiotic factors on the colonization of both Arabica and Robusta coffee beans to evaluate and compare the impacts on OTA production. Inherent caffeine concentrations may inhibit colonization by some toxigenic species and perhaps influence the levels of OTA contamination that may occur. Robusta coffee beans are mainly grown at lower altitudes, and may have better tolerance to climate-related abiotic factors than Arabica, which is grown at higher altitudes and may be more sensitive to climate-related agronomic factors [
31]. In addition, changes in diversity of pests could lead to increased damage to ripening coffee beans, and such damage has been shown to have an impact on infection by toxigenic fungi and perhaps also toxin contamination levels post-harvest [
32].
Other important abiotic factors have not been considered such as UV radiation and fluctuations in temperature × water availability and exposure to elevated CO
2 conditions [
32,
33]. Some of these factors have been shown to impact growth and OTA production by strains of
A. carbonarius isolated from grapes [
33]. The impact of acclimatization of strains of species from both sections
Circumdati and
Niger have not been addressed. Studies by Vary et al. [
34] on
Fusarium graminearum and by Medina et al. [
3] on
A. flavus have shown that exposing these species to elevated CO
2 conditions for 10–20 generations can result in enhanced tolerance and pathogenicity and lead to increased mycotoxin production.