# Antimicrobial Volatiles of the Insect Pathogen _Metarhizium brunneum_
## 3 Results  
### VOC Profiles Were Influenced by Strain and Culture Medium  
A wide range (>40) of VOCs were produced by _M. brunneum_ (ARSEF4556, ARSEF3297, V275), with some compounds being produced more consistently than others (Supplementary Table S3). The relative quantities of each VOC varied between strains, development stage (young mycelial versus older sporulating cultures) and culture conditions (Figure 1). For convenience, the compounds were expressed as a percentage relative to the compound with the largest peak (100%) in the GC-MS chromatogram. VOCs of all three _M. brunneum_ strains included a variety of saturated and unsaturated hydrocarbons, alcohols, acids, ketones, esters, terpenes and other assorted compounds (Supplementary Table S3). Figure 1: Profiles of volatile organic compounds produced by _M. brunneum_ ARSEF4556, V275 and ARSEF3297 on different substrates. The heatmap represents values that were proportional to the compound with the highest peak (100%) in the chromatogram. \(n\) = 5. OSM, osmotic stress medium; HCN, high C:N medium; ICN, intermediate C:N medium; LCN, low C:N medium. Note: no conidia were present in the ARSEF 4556 at 7 days. The VOC profiles of 7-day-old non-sporulating cultures differed from the 14-day-old cultures that consisted of mycelium, conidiophores and conidia (Supplementary Table S4). Some compounds were produced by all three strains but not necessarily on all media tested (e.g., acetic acid, isoamyl alcohol, 1-octene, 1,3-octadiene, isovaleric acid, methyl 2-ethylhexanoate, cedrene). Methyl 2-ethylhexanoate and cedrene were the most observed compounds (Figure 1). Acetic acid and isovaleric acid were produced only during the mycelial phase of growth, while pentane, heptane, octane, 1,3-octadiene, 1-octen-3-ol, 3-octanone, 4,4-dimethyl-2-neopentyl-1-pentene, 1-methyl-2-pentylcyclohexane and 4,4,7-trimethyl-2,4,5,6,7,7-a-hexahhydro-1H-inden-1-one were only produced during the sporulation phase (Figure 1; Supplementary Table S4). Farnesene was specific to ARSEF 4556 and produced during the mycelial and sporulation phases on all media tested (Figure 1, Supplementary Table S4); it was particularly abundant during the sporulation phase. Cedrene was emitted by V275 and ARSEF 3297 at almost all development stages, but it was only observed in ARSEF 4556 during sporulation. There was a noticeable difference in the class of VOCs produced by each strain, for example, ARSEF 4556 tended to produce alkanes (e.g., pentane, heptane, and octane) and terpenoids (cedrene, bergamotene, farnesene). Strain V275 tended to produce alcohols, esters and ketones, while ARSEF 3297 produced a much smaller range of volatiles. _Metathizium_ VOC profiles were influenced by the culture medium (OSM, HCN, ICN and LCN), independent of fungal strain (Figure 1, Supplementary Table S4). Variations in the VOC profiles were evident in OSM, but similar patterns were observed on the other media, albeit in different quantities. In 7-day-old mycelial cultures of _M. brunneum_ ARSEF 4556, isoamyl alcohol was more predominant than acetic acid on OSM, but the opposite was observed for cultures grown on HCN, ICN and LCN (Figure 1, Supplementary Table S4). V275 produced fewer VOCs on OSM than on the other media, with the most notable absences being that of ethyl acetate, 3-methyl-2-butanone, 2,3-butanediol and isoamyl acetate. For _M. brunneum_ ARSEF 3297, isoamyl alcohol was only produced by 7-day-old cultures grown on OSM. However, 14-day-old cultures produced 4,4-dimethyl-2-neopentyl-1-pentene and 1-methyl-2-pentylcyclohexane on all media except OSM (Figure 1, Supplementary Table S4). The 7-day-old non-sporulating cultures of _M. brunneum_ differed in the VOC profile from that of the 14-day-old sporulating cultures (Figure 1, Supplementary Table S4). Collectively, the aliphatic C8 compounds, i.e., 1-octene, octane, 1,3-octadiene, 1-octen-3-ol and 3-octanone, were found in older cultures, whereas acetic acid and isoamyl alcohol were exclusively produced by the young non-sporulating cultures.