## 3 Results and Discussion
### Following the A. niger Metabolites Biomarkers Pattern over 7 Days
#### 3.2.2 By Direct Analysis of Contaminated Table Grapes--In Situ SPME  
The followed volatile metabolite profile of the disease was evaluated through 7 days of growth by using 42 metabolites associated with _A. niger_ metabolism, because in this study only 42 of the total set of metabolites were identified in grapes. By employing this strategy, 2-methyl-6-phenyl-1,6-heptadiene and methyl 2-methylropenoate were not found. Although there is no clear indication regarding their absence, the differences in the substrate can slightly modify metabolite production, indicating the importance of in situ studies. An in situ SPME strategy was performed using a non-invasive headspace approach. Data were equally subjected to statistical analysis using MetaboAnalyst 4.0 tool to identify possible groupings of the investigated samples. Figure 5 shows that there was a clear separation between the samples, associated with a variation in the concentration of metabolites over the days of fungal growth. In vivo SPME studies have been widely applied in food analysis, since coated surfaces are biocompatible and also provide higher sensitivity and reduced analysis time [66; 67; 68]. PLS-DA scores plot (Figure 5) revealed the dispersion of the samples according to main factors; time of growth and variety. The latent variable 1 (LV1) is mainly responsible for explaining the data variability (41.1%), which showed dispersion of the samples according to the growth time, from 1 day (LV1 negative) to 7 days (LV1 positive). Along LV2, which explained 13.1% of the data variability, the samples are organized according to the variety, with Do mainly in the LV2 positive and the RG in the LV2 negative. Figure 4: PLS-DA biplot of samples (LV1\(\times\)LV2) using the set of 44 metabolites listed in Table 1, which shows that the grapes contaminated with _A. niger_ presents a metabolomics pattern like the pure cultures. The areas were previously standardized by CFU mL\({}^{-1}\) (Table S1). Results obtained by performing the pure culture growth in solid YGC\({}_{\text{A}}\) culture medium and by contaminated grapes (Red Globe and Dominga) with _A. niger_. In addition, from these data it was also possible to infer that red variety (RG) was more resistant to contamination than white one (Do), as for RG after 4 days of contamination the metabolite profile was still quite similar to that of the first day. This fact can be explained by the higher concentration of phenolic and antioxidant compounds in general present in this type of grape, ensuring a higher resistance to contamination. A recent study demonstrated the antagonistic activity of essential oils of eight plants against strains of _A. niger_, and one of the factors highlighted by the authors was the presence of antioxidant compounds that helped to control the disease in stored wheat grains [69; 70]. [PERSON] et al. demonstrated that fresh grapes are rich in bioactive phenolic compounds, and the concentration depends heavily on the grape varieties. The Mascot Kyoho variety (red skin) had the highest concentration of antioxidant compounds such as (+)-catechin, (\(-\))-epicatechin, rutin, isoquercitrin, and kaempfer. The phenolic compounds of grapes are mainly in their skins, and this corroborates the results shown above, where the high content of antioxidant compounds promotes resistance to the growth of the pathogen. The effect of inhibiting the growth of _A. niger_ against antioxidant enzymes has been previously studied and possibly suggests the induction of antioxidant defense response by Trichoderma bio-controller to combat oxidative bursts produced by invading pathogens [70; 71]. Additionally, the dendrogram and heatmap (Figure S1) illustrate the formation of two main clusters related to the two grape varieties, within each one there is organization of the samples as a function of growth time. The biplot (Figure 5) and heatmaps (Figure S1) demonstrate that the most important compounds directly related to _A.