# Antimicrobial Volatiles of the Insect Pathogen _Metarhizium brunneum_
## 3 Results
### VOC Antibacterial and Antifungal Activity  
_Metathizium anisopliae_ ARSEF 5469 radial growth was partially reduced by the 14 test compounds used (Supplementary Table S1), where isovaleric acid and 3-octanone caused the greatest reductions at 34 \(\pm\) 9.92% and 26 \(\pm\) 5.04% inhibition, respectively (Figure 2A-D). In contrast, the plant pathogenic fungi/oomycete were far more sensitive, being strongly inhibited (85-100%) by isovaleric acid and 1-octen-3-ol (Figure 2A-D). The yeasts, filamentous fungi and oomycete differed in their sensitivity to _M. brumeum_ VOCs. The yeasts, i.e., _C. albicans_ and _C. glabrata_, were only affected by isovaleric acid and 1-octen-3-ol, with the latter causing 100% inhibition (Figure 2D). The filamentous fungi _F. graminearum_ and _B. cinerea_ were inhibited by a wider range of VOCs than the other microbes (Figure 2A). _F. graminearum_ was sensitive to most (8/14) of the compounds tested, with inhibition ranging from 5 \(\pm\) 2% (farnesene) to 100 \(\pm\) 0.00% (isovaleric acid, 1-octen-3-ol). Inhibition of _B. cinerea_ ranged from 28 \(\pm\) 4% (methyl isovalerate) to 85 \(\pm\) 4.9% (1-octen-3-ol) (Figure 2A). The oomycete, i.e., _P. ultimum_, was highly sensitive to only four compounds, with inhibition ranging from 82 \(\pm\) 6.36% (isoamyl alcohol) to 100% (3-octanone, isovaleric acid, 1-octen-3-ol) (Figure 2A). Only one compound, isovaleric acid, inhibited all test bacteria, with inhibition ranging from 29 \(\pm\) 2.41% (_P. aeruginosa_) to 100% (_B. megaterium_) after 24 h (Figures 2B,C and 3). Isoamyl formate and farnesene totally inhibited _B. thuringiensis_ (Figures 2B and 3). 3-Octanone totally inhibited _E. coli_ without affecting any other test bacterium (Figures 2C and 3). Interestingly, _B. megaterium_ showed the greatest sensitivity to isovaleric acid, 1-octen-3-ol, cedrene and farnesene, which caused 100% inhibition. Cedrene caused 100% inhibition of _B. subtilis_ (Figures 2B and 3). Figure 2: Inhibition (%) of growth of **(a)** filamentous fungi, **(b)** G+ bacteria, (c) G\(-\) bacteria and **(d)** yeasts when exposed to VOCs. The bacteria and yeasts were examined 24 h post-exposure (to 10 \(\upmu\)L VOC) and the filamentous fungi 5 days post-exposure. Values represent mean (\(\pm\)SD) of two experiments, each with 4 technical replicates per treatment. Since the VOCs are ephemeral, the recovery of the bacteria and yeast was observed 48 h after treatment. The recovery depended on the compound and test organism. Whereas the majority of microbes recovered to various degrees, some failed to do so and were presumed dead. The yeast recovered totally once the VOC source was removed, rapidly colonising the clear (inhibition) zone (Figure 3). Full recovery was also recorded for _P. aeruginosa_,  
Figure 3: Growth inhibition (%) of bacteria and yeast exposed to volatile organic compounds. Microbial growth was recorded at 24 and 48 h post-exposure to 10 \(\mu\)L VOC. Values represent the mean (\(\pm\)SD) of two independent experiments, consisting of 4 technical replicates per treatment. An asterisk (*) denotes growth recovery. P. agglomerans, B. thuringiensis and S. aureus exposed to isovaleric acid, for P. agglomerans and B. thuringiensis exposed to 1-octen-3-ol and for B. subtilis exposed to cedrene (Figure 3). Partial recovery (55 \(\pm\) 7.4%) was observed for B. megaterium exposed to isovaleric acid. Full inhibition (100%) was recorded for E. coli exposed to 3-octanone 48 h post-treatment. Full inhibition was also recorded for B. thuringiensis exposed to isoamyl formate and farresene and for B. megaterium exposed to 1-octen-3-ol, cedrene and farresene (Figure 3). Where recovery was observed, bacterial and, to some extent, yeast colonies grew from the edge of the colony (interface between the surviving culture and zone of inhibition) towards the centre of the clear zone (Figure 4A).