# The Biology of _Pichia membranifaciens_ Killer Toxins
## 6 Applications, Conclusions and Future Perspectives of _P. membranarificiens_ Killer Toxins  
Although killer toxins were originally described and studied for their ecological function in the early 1960s [1], and they promptly received much attention from geneticists for the understanding of their genetic basis at the end of this decade [86; 136], their biocidal nature has an inherent applicability in clinical, food and biocontrol fields that was not systematically explored until the 1990s (for medicine: [137; 138; 139]; for food: [140; 141; 142; 143]; for biocontrol: [144; 145; 146]). As mentioned at the beginning, _P. membranarificiens_ killer yeasts exhibit a broad action spectrum including yeasts of biotechnological concern (_Brettanomyces/Dekkena_, _Candida boidiniii_, _Zygosaccharomyces_) and phytopatogenic fungi (_Fusarium proliferatum_, _Botrytis cinerea_), as well as other killer yeasts [21; 104]. To a large extent, the potential applications that have been proposed for yeast killer toxins have been conditioned by the aforementioned recognized liability of such toxins. The proposed applications are largely restricted to those that provide physical chemical conditions compatible with the stability of killer toxins, that is, those that can be performed at relatively low pH and low temperature values (Table 3). Although the majority of killer toxins (_W. mrakii_ var. _mrakii_ HM-1, _W. saturnus_ toxin, _P. anomalous_ PaKT and _Z. bailii_ zygocin) have shown activity against pathogenic yeasts, such as _Candida albicans_, _Cryptococcus neoformans_, etc., they have been suggested to be potentially useful as potential antifungal agents for infections caused by fungi, their direct application is of limited real importance because of their instability at physiological temperatures and pH values and their antigenicity [39; 104; 146; 147; 148; 149; 150; 151]. Once again, only the killer toxins that display a remarkable stability (e.g., the _Williopsis_ toxins) could be therapeutically used, but restricted to topical applications such as on superficial skin infections [151]. In this context, the role and use of _P. membranarificiens_ and its killer toxins to control human or animal infections and other clinical diseases is still uncertain. Since the use of _P. membranarificiens_ killer toxins seems to be not really useful at clinical stages, their use for biocontrol purposes has a great interest in agro-food industry. The applications of the biocide activity of _P. membranarificiens_ were initially unveiled by [PERSON] et al. (2001), observing that the mycelium of the _B. cinerea_ treated with _P. membranarificiens_ failed to develop the characteristic grey mould symptoms when it was re-inoculated onto grapevine [152]. This destructive process was suggested to be mediated by the production of exo- and endo-\(\beta\)-1,3-glucanases, as was also demonstrated by [PERSON] et al. (2002) [153]. However, as described by [PERSON] and colleagues in both grapevine and apple matrixes, this antagonistic phenomenon could also be mediated by killer toxin (PMKT) activity [19; 154]. The co-existence of different mechanisms, including hydrolytic enzymes and killer toxin production, is one of the reasons that justifies the interest and versatility of _P. membranarificiens_ as a biocontrol agent against _B. cinerea_, since it was also proved effective in postharvest peers [155]. The existence of similar and additional antagonistic mechanisms in other yeasts and their use to control _B. cinerea_ spoilage have also been reported. For example, the production of exo-glucanase toxins in _W. anomalus_ was reported by [PERSON] et al. (2016) [156], but the production of certain volatile organic compounds (i.e., 2-phenylethanol) has also been suggested as determinant for the biocontrol activity against _B. cinerea_ found in this species and in others such as _Candida intermedia_, _Sporidiobulus pararoseus_, _Starmerella bacilaris_ and _S. cerevisiae_[157; 158; 159; 160; 161; 162]. In a similar context, _P.