## 4 Discussion  
One of the major challenges in developing microbial biological control agents against pathogens is related to the method used for their screening [32]. The selection of antagonists often relies on in vitro dual culture assays in an artificial medium; however, the antagonism observed in this approach is not always reflected in in vivo assays [33]. Indeed, the in vitro screening method is only based on inhibition of pathogen growth due to antimicrobial metabolites [32]. However, other modes of action of the antagonist can contribute in planta to a better protection of plants from damage by diseases [32]. Screening methods for microbial biological control agents may therefore rely on assays that take into account the interaction between the pathogen, host plant and antagonist [32; 33]. In our work, the antagonistic effect of _A. pullulans_ and _S. summerbelli_ against the causal agent of olive anthracose, _C. acutatum_, was assessed based on this approach by using both in vitro and in vivo assays. In the first assay, dual cultures were established in a medium containing macerated olives, in an attempt to mimic the natural habitat under which antagonists should be active, while in vivo assays were performed in detached fruits. Similar results were obtained in both assays, by showing the greatest efficacy of _A. pullulans_ in controlling _C. acutatum_. This endophyte significantly reduced the development of disease, as well as the growth, sporulation and germination of _C. acutatum_. _A. pullulans_ is a well-known biocontrol agent against several pathogenic fungi, especially postharvest pathogens, but also pathogens present in the field [14; 18; 19]. For instance, _A. pullulans_ was shown to suppress the mycelium growth and/or conidia germination of _Botrytis cinerea_[18; 19], _Alternaria alternata_[19], _Colletotrichum acutatum_[15], _Penicillium expansum_, _Penicillium digitatum_ and _Penicillium italicum_[14], under in vitro and/or in vivo conditions. Its potential in the biocontrol of olive anthracose was previously reported as well. In field trays, this yeast was shown to significantly reduce anthracose severity by 40% and latent infection by 14% [17]. Despite the inability of _S. summerbelli_ to suppress _C. acutatum_ in the present study, some endophytic species of the genus _Sarocladium_ have already been reported to display significant antifungal activity against a broad spectrum of pathogens, including the genus _Colletotrichum_[16]. The in vitro inhibitory effect displayed by _A. pullulans_ against _C. acutatum_ in the present study started to occur prior to physical contact among the microbes, suggesting that the inhibition was caused by diffusible and/or volatile compounds produced by the endophyte. Therefore, changes in the VOC emission among olives inoculated with fungi (i.e., _A. pullulans_, _C. acutatum_ or _A. pullulans + C. acutatum_) and Tween (control) were evaluated after 2, 8 and 16 days post-inoculation. Overall, the VOCs detected were mainly alcohols, aldehydes and ketones, with alcohols being the most abundant and diversified compounds. This result is consistent with previous works that identified these three chemical classes among the most abundant volatile compounds produced by fungi [34]. These classes encompass VOCs with recognized antimicrobial effects against several plant pathogens [14; 35]. In the present study, the composition of the VOC blend was revealed to be different among treatments, with 2-undecanone being exclusive to olives inoculated only with _C. acutatum_. This result suggests that this compound may play an important role in the development of anthracose, probably being required for the pathogenicity of _C. acutatum_, as previously suggested for other pathosystems [36]. Alternatively, this VOC may be produced by olives to prevent _C. acutatum_ infection, as plants are known to protect themselves from pathogens by synthesizing diversified metabolites, including VOCs [37].