# Endophytic Yeasts for the Biocontrol of _Phycetema vagabunda_ in Apples
## 4 Discussion  
In this study, we screened endophytic yeasts isolated from organic 'Cripps Pink' apples for the control of _P. vagabunda_. The most effective yeast that significantly decreased the incidence of bull's-eye rot in apples was the EPL29.5 strain which was identified as _Vishniacozyma victoriae_. Previously, the yeast _V. victoriae_ had been reported to be an effective biocontrol agent for _Botrytis cinerea_ and _Penicillium expansum_ on pears [47] and _Penicillium crustosum_ and _Mucor piriformis_ on cherries [48]. To the best of our knowledge, no study on _P. vagabunda_ has yet been recorded, making this the first report of a yeast controlling bull's-eye rot in apples. _P. vagabunda_ fruit infection occurs in the orchard [2], so the presence of the antagonist prior to or during the initial phases of the disease cycle is of crucial importance, considering the difficulties in controlling previously established infections [48]. We tested that the application of _V. victoriae_ EPL29.5 24 h before the pathogen reduces the incidence of _P. vtagabunda_ by 58.3%. The pre-harvest application of antagonistic yeasts could protect fruits against pathogen infection in the orchard, but for the biological control to be successful, the yeast needs to possess effective mechanisms to cope with the abiotic stresses to which they are exposed [49]. In this sense, endophytic yeasts could be a very promising new source of biological control agents because they can grow and develop inside plants, thus avoiding the negative influences of environmental factors such as solar radiation and desiccation [38]. It has been suggested that biological control agents utilize different strategies depending on the pathogen, host, and environment [50]. Understanding these diverse mechanisms is essential to determine how a combination of different yeasts affects pathogen control in order to take advantage of their multiple means of action [51]. This is also an important topic in the development process of bio fungicide formulations because it permits an increase in the performance of biocontrol agents [52]. In this study, we evaluated different mechanisms of action of _V. victoriae_ (EPL4.5 and EPL29.5), including antibiosis, pathogen hyphal adhesion, and siderophore production (Figures S1-S3 Supplementary Material), biofilm-forming capacity and production of volatile organic compounds. Only _V. victoriae_ EPL29.5 formed biofilms in sterile apple juice at 20 \({}^{\circ}\)C. This mechanism has been demonstrated by [PERSON] et al. [53] at \(0\pm 1\)\({}^{\circ}\)C in pear juice with glucose peptone yeasts extract as a culture medium. Biofilms are a network of cells and extracellular polysaccharides that form a gel that holds microorganisms together [54], creating a mechanical barrier between the wound and the pathogen surface [55], thereby preventing the onset of the infection process. Several studies have shown that the production of VOCs by yeasts has a significant role in their antagonistic activities [56; 57; 58]. In this study, VOCs emitted by the _V. victoriae_ strains EPL4.5 and EPL29.5 reduced colony-forming unit (CFU) and mycelial growth of _P. vagabunda_. Strain EPL4.5 reduced CFU by 54.8% and mycelial growth by 69.1% when grown in a YPD medium, while strain EPL29.5 reduced CFU by 50.63% and mycelial growth by 66.1%. The analyses of the VOC profiles produced by the evaluated yeast strains were conducted with HS-SMPE coupled with GC-MS, which indicated that the VOCs were mainly alcohols, with the main components being 1-Butanol, 2-methyl-, (S)- in the AJA medium and 1-Butanol in the YPD medium (Table 3). These compounds have previously been observed in the yeasts _Aureobasidium pullulans_ and _Meyerozyma caribbica_ when grown on a Nutrient Broth, Yeast Extract, Dextrose Agar, and PDA [32, 43]. [PERSON] et al. [56] found that the main VOCs produced by _C.