# Antimicrobial Volatiles of the Insect Pathogen _Metarhizium brunneum_
## 4 Discussion  
This study showed that _M. brunneum_ strains produced a wide array of VOCs with the profile (type and quantity of chemical) being influenced by the development stage of the fungus and cultural conditions. The EPF VOC profiles appear to vary with EPF species, isolate and culture conditions [22; 25; 30]. The unique blend of volatiles emitted by EPF is of significance from both ecological and biocontrol perspectives. It could explain why some EPF strains influence the behaviour of some invertebrates, while others do not [24; 26]. Compounds that attract invertebrates could help with spore dispersal or form part of the pathogens \"lure and kill\" strategy [24; 26]. Repellent compounds could reduce EPF efficacy. The implications for biocontrol are profound, as illustrated by studies conducted on sweet potato weevil and termites; these insects avoid virulent strains of _M. anisopliae_ with the response being linked to their VOCs [23; 30; 38]. A novel and salient feature of this study was that _M. brunneum_ produced VOCs with antimicrobial properties. The most potent antimicrobials were isovaleric acid and 1-octen-3-ol, which inhibited or killed bacteria, yeasts, filamentous fungi and the oomycete _P. ultimum_. In contrast, isoamyl formate, 3-octenone, cedrene and farnesene, although potent, were more restricted in their specificity. Some microbes were clearly more sensitive to _M. brunneum_ VOCs than others. In order of sensitivity, _F. graminearum_ was inhibited by far more compounds than any other test microbe, followed by _B. cinerea_, then _P. ultimum_. Of the test bacteria, _B. megaterium_ was the most sensitive, followed by _B. thuringiensis_. This differential sensitivity may reflect some evolutionary arms race between EPF and competing microbes, especially plant pathogens, occupying the same niche. Since EPF, such as _M. brunneum_, are highly successful at colonizing plant roots and existing as endophytes, it is tempting to speculate that the capacity to produce antifungal VOCs evolved to enable the fungus to inhibit/eliminate potential competitors or fungi that could harm the host plant. EPF were shown to be antagonistic to plant pathogenic fungi via the production of antifungal metabolites and induction of the host's defences [39]. However, the results of the current study suggested that _M. brunneum_ VOCs could play a role in plant disease suppression and concomitantly contribute to healthy plant growth. Many non-EPF, such as _Muscodor heveae, Xylaria_ sp., _Daldinia cf concentrica_ and _Saccharomyces cerevisiae_, also produce antifungal VOCs that inhibit the growth of a wide range of phytopathogens, including _Sclerotinia sclerotorium_, _Colletotrichum gloeosporoides_, _Botrytis cinerea_ and _Phyllosticita citricarpa_[40; 41; 42; 43; 44; 45; 46]. Interestingly, one of the most inhibitory compounds produced by these fungi was methyl-1-butanol (isoamyl alcohol), which, in the current study, showed moderate inhibition of _F. graminearum_ and _B. cinerea_ and weak inhibition of _P. ultimum_. Some antifungal VOCs appear to be unique to specific fungi. For  
Figure 7: Cryo-scanning electron micrographs of _Candidia glabrata_ in the (**a**,**b**) absence (**c**) and presence of isovaleric acid. example, 6-pentyl-\(\alpha\)-pyrone, trimethylamine and 3-methyl-acetate appear to be peculiar to _Trichoderma_ species, _Geotrichum candidium_ and _Muscodor albus_, respectively [47; 48; 49]. We identified several new antifungal VOCs, with the most potent being isovaleric acid, which also had strong antibacterial activity. Two compounds _M. brunneum_ has in common with other fungi are 1-octen-3-ol and 3-octanone [50], both of which have strong antifungal activity, with the former responsible for self-inhibition of conidia in several fungi [51; 52]. The current study showed that 1-octen-3-ol also had strong antibacterial properties. We are not aware of any study that explained the mechanism of inhibition.