Impacts of Climate Change Interacting Abiotic Factors on Growth, aflD and aflR Gene Expression and Aflatoxin B1 Production by Aspergillus flavus Strains In Vitro and on Pistachio Nuts

Abstract

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus, mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B₁ (AFB₁). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO₂ levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO₂ (400 vs. 1000 ppm), and water stress (0.98-0.93 water activity, a_w) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB₁ production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB₁ production was significantly stimulated (p < 0.05), especially when exposed to 1000 ppm CO₂ at 0.98-0.95 a_w and 35 °C, and sometimes in the 37 °C treatment group at 0.98 a_w. The relative expression of the structural aflD gene involved in AFB₁ biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the a_w level examined. For the regulatory aflR gene expression, there was a significant (p < 0.05) increase in 1000 ppm CO₂ and 37 °C for both strains, especially at 0.95 a_w. The in situ colonization of pistachio nuts resulted in a significant (p < 0.05) stimulation of AFB₁ production at 35 °C and 1000 ppm CO₂ for both strains, especially at 0.98 a_w. At 37 °C, AFB₁ production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO₂. This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO₂ and the level of water stress on AFB₁ production.

Keywords: Aspergillus flavus; aflD; aflR; aflatoxins; climate change; interacting abiotic factors; pistachios.