Fungal diversity, pest damage and biocontrol of aflatoxins in GM and conventional Brazilian maize cultivars under existing and future climate change scenarios.

This study (a) evaluated the fungal biodiversity, toxigenic mycobiota and mycotoxin profiles associated with conventional (non-GM) and genetically modified (GM) isogenic maize cultivars (cvs) from Brazil, (b) studied the ecology of the isolated strains of toxigenic Aspergillus flavus using non-GM and the isogenic GM cv as substrates under different water activity(aw) and temperature interactions in vitro, (c) screened mycobiota for potential biocontrol agents (BCAs) and compared the interactions between atoxigenic (AFL⁻) and toxigenic (AFL⁺ ) A. flavus strains and other antagonistic species for in vitro control of aflatoxins (AFs) using different spore inoculum ratios, (d) examined the best potential BCAs to apply in situ in stored GM and non-GM isogenic maize cvs on AFs production and related expression of structural (aflD) and regulatory (aflR) toxin biosynthetic genes, and (e) examined the resilience of the biocontrol efficacy under simulated pest damage and climate change (CC) scenarios. The majority of the GM and isogenic non-GM cvs analysed (20 samples; 10 each type) had moisture content (%MC) and aw levels within the safety range for safe storage (<0.70 aw). Fusarium and Penicillium spp were the predominant genera identified with a low percentage of isolation of A. flavus strains in the maize cultivars examined. There was no significant difference (p<0.05) in the frequency of isolation between non-GM and GM cvs. A total of 22 A. flavus strains were isolated, of which 15 were non-aflatoxin producers, and 7 were aflatoxin B₁ (AFB₁) producers. Six of these strains were from non-GM maize cvs. Six pairs of isogenic GM- and non-GM cvs (n=12) out of the 20 used in this work were selected and analysed in more detail using LC-MS/MS. The mycotoxin profiles showed 29 compounds present, with higher amounts of Fusarium toxins than any other, which paralleled the high isolation frequency of Fusarium spp. AFs were not detected, while Fumonisins (B₁ or B₂) were present in 10 out of the 12 cvs, with only 2 non-GM cultivars having contamination levels above the EU legislative limits (4000 µg kg⁻1). The distribution of the mycotoxins indicated differences between the non-GM and GM cvs (p<0.05) with the latter having lower overall concentrations of mycotoxins. Subsequently, from the 22 isolated strains of A. flavus 4 were selected (3 toxigenic and 1 atoxigenic) for ecological studies using 3 pairs of GM- and non- GM maize cvs as substrate. The strains were able to colonize and grow on maize- based nutritional matrices from both GM (two pesticide and one herbicide + pesticide resistant) and non-GM cvs. The type of cvs did not have a significant effect on the growth of A. flavus, however temperature and aw had a significant effect (p<0.05) on the fungal development. The optimal conditions for growth were slightly different from those for AFB₁ production. Optimal growth occurred at 30-35ᴼ C and 0.99 aw, whereas AFB₁ production was optimal at 25-35ᴼ C and 0.99 aw. Each strain showed a different pattern of AFB₁ production and there was a shift in the optimal conditions depending on the combination of aw × To C × maize cv. In vitro a total of 8 atoxigenic (AFL⁻) and 8 other strains from different genera were tested as BCAs against 5 toxigenic strains (AFL⁺ ). This showed that A. flavus was highly dominant in vitro. One yeast strain (Y6) was able to compete against A. flavus on malt extract agar (MEA) at 0.98 aw but when it was co- cultivated in milled-maize agar (MMA) against the toxigenic AFL⁺ strain resulted in an increase in AFB₁ when compared to the control. The interaction of the toxigenic AFL⁺ × atoxigenic AFL⁻ strains were mutual intermingling on both MEA and MMA. On MMA for co-cultivation of different inoculum ratios the screening was only done against 3 AFL⁺ toxigenic strains to examine effects on AFB₁ control. The overall control of AFB₁ ranged from 29 to 100%. The most effective ratio of spores of the atoxigenic vs toxigenic strains was found to be a mixture of 50:50 mixed conidial inoculum of each strain. Based on the in vitro screening for potential BCAs, the atoxigenic A. flavus strains were examined to determine whether they had a deletion in biosynthetic genes involved in AFs and cyclopiazonic acid (CPA) production using multiplex PCR. Five atoxigenic strains (AFL⁻) were found to have large deletions of genes in the AFs cluster. While 3 atoxigenic strains amplified most of the markers in the AF cluster, however they were still unable to produce AFs. The strain selected for in situ biocontrol studies (Af53H – AFL4⁻ ) had a large deletion of AF markers but had all the CPA markers. The AFL4⁻ was able to significantly reduce AFB₁ when paired with toxigenic strains in a 50:50 spore ratio in stored GM and non-GM maize cvs. The relative gene expression of aflD and aflR in one of the toxigenic strains (AFLb⁺ ) used as pathogen was significantly inhibited by the chosen BCA. The correlation of gene expression ´ AFB₁ was positive indicating that suppression in the gene expression pathway contributed to the lower toxin levels. The overall biocontrol action seems to have been most effective when used in stored GM maize cultivars. Different levels of simulated pest damage (0, 5 and 15%) showed that AFB₁ production did not increase with a higher level of damage regardless of whether pesticide resistance or herbicide + pesticide resistance cvs were compared with non-GM isogenic ones. The toxin production in 15% damaged maize grain was lower or equal to that with no or 5% damage. The gene expression of aflR and aflD involved in AFs biosynthesis showed differences between the maize cvs. However, the correlation of gene expression × AFB₁ was not significantly positive. The BCA showed resilience under TᴼC × CO₂ × aw × simulated pest damage conditions with similar control levels of AFB₁ which was achieved under existing environmental conditions. The use of a GM cvs showed better results for biocontrol under water stress (0.95 aw) and elevated CO₂ at 35ᴼ C when the kernels were undamaged. However, biocontrol in conventional maize was better when there were damaged kernels at 0.95 aw × 35ᴼ C ×1000 ppm CO₂.