# Antimicrobial Volatiles of the Insect Pathogen _Metarhizium brunneum_
## 3 Results
### VOC Antibacterial and Antifungal Activity
In some instances, growth at the interface was extremely vigorous, resulting in a pronounced collar (Figure 4A), but in other instances, growth was faint, resulting in a halo effect (Figure 4B). Occasionally, small colonies arose within the clear zone presumably from cells protected by other cells (Figure 4C). Figure 4: Appearance of bacterial cultures when exposed to selected volatile organic compounds. (**a**) _M. luteus_ NCIB 8553 culture exposed to 10 uL isovaleric acid. Note the dense growth at the edge of the inhibition zone. (**b**) _E. coli_ NCIB 8277 culture exposed to 10 uL isovaleric acid. Note the dense growth in the uninhibited zone and moderate growth in the recovery zone, with a clear zone at the centre where the concentration of the chemical was the highest. (**c**) _B. subtilis_ NCIP 3610 culture exposed to 10 uL cedrene. Note that the culture was mostly inhibited, except for a few colonies when exposed 24 h to this VOC, but total recovery occurred once the culture was ventilated and allowed to incubate for a further 24 h.  
The pattern of regrowth in filamentous fungi (_F. graminearrum_) differed markedly, with growth being stunted partially or totally depending on the VOC (Figure 5A,B). Closer examination of the bacterial and yeast cells using conventional SEM and Cryo-SEM showed that cells had lysed (Figures 6 and 7). Lysis was often preceded by unusual deposits appearing on the bacterial surface, followed by loss of form, then complete disappearance of the bacteria, leaving behind a \"debris-free\" clear zone (Figure 6). This pattern appeared to be true for both rod and spherical bacteria (Figure 6A,B). In some instances, micro-colonies and remnants of an extracellular matrix were observed at the interface of the surviving culture and clear zone (Figure 6A-C). The micro-colonies were made of several layers, with the surface cells clearly losing form when exposed to inhibitory VOCs (Figure 6C). No gross morphological changes were observed in yeast exposed to inhibitory VOCs, but cells had clearly disappeared in the clear (inhibition) zone, with the absence of any wall residue suggesting total digestion of the cell (Figure 7). Figure 5: Inhibition of fungal cultures exposed to selected volatile organic compounds. (**a**) _Candida albicans_ NCYC3778 exposed to 10 \(\mu\)L 1-octene-3-ol. Substantial inhibition was achieved within 24 h, but the cells appeared to recover after ventilation and a further 24 h incubation (i.e., 48 h). (**b**) Five-day-old cultures of _F. graminearrum_ in the (**i**) absence and presence (15 \(\mu\)L) of (**ii**) 3-octanone, (**iii**) isoamyl alcohol, (**iv**) isoamyl formate, (**v**) 1-octene-3-ol and (**vi**) isovaleric acid. Figure 6: Scanning electron micrographs of bacteria exposed to selected volatile organic compounds. (**a**) _E. coli_ in the (**i**) absence and (**ii**,**iii**) presence of isovaleric acid. (**b**) _M. luteus_ in the (**i**) absence and (**ii**,**iii**) presence of isovaleric acid. Cell surface blebbing is visible, with an altered shape and evidence of autolysis. (**c**) Cryo-SEM of _M. luteus_ in the absence (**i**) and presence of isovaleric acid: (**ii**) edge of the zone of inhibition showing healthy cells alongside lysed cells, (**iii**) zone of inhibition showing islands of intact and unhealthy cells, (**iv**) details of healthy and dying cells, (**v**) micro-colony of healthy cells in the zone of inhibition and (**vi**) micro-colony of dying cells.