High-Throughput Volatilome Fingerprint Using PTR-ToF-MS Shows Species-Specific Patterns in Mortierella and Closely Related Genera

Abstract

In ecology, Volatile Organic Compounds (VOCs) have a high bioactive and signaling potential. VOCs are not only metabolic products, but are also relevant in microbial cross talk and plant interaction. Here, we report the first large-scale VOC study of 13 different species of Mortierella sensu lato (s. l.) isolated from a range of different alpine environments. Proton Transfer Reaction-Time-of-Flight Mass Spectrometry (PTR-ToF-MS) was applied for a rapid, high-throughput and non-invasive VOC fingerprinting of 72 Mortierella s. l. isolates growing under standardized conditions. Overall, we detected 139 mass peaks in the headspaces of all 13 Mortierella s. l. species studied here. Thus, Mortierella s. l. species generally produce a high number of different VOCs. Mortierella species could clearly be discriminated based on their volatilomes, even if only high-concentration mass peaks were considered. The volatilomes were partially phylogenetically conserved. There were no VOCs produced by only one species, but the relative concentrations of VOCs differed between species. From a univariate perspective, we detected mass peaks with distinctively high concentrations in single species. Here, we provide initial evidence that VOCs may provide a competitive advantage and modulate Mortierella s. l. species distribution on a global scale.

Keywords: Entomortierella; Linnemannia; Mortierellomycotina; PTR–ToF–MS; Podila; fungal volatiles; volatilomes.

Figure 1

Phylogeny of the Mortierella s. l. species included in the VOC analysis. The 72 pure culture strains fall into 13 well-supported clades. Maximum Likelihood phylogram (log likelihood—1974.28) based on ITS sequences. Branch support (Bayesian posterior probabilities/Parsimony Bootstrap support ≥ 70) is shown above the respective branches. Sequences generated from type specimen are highlighted in red.

Figure 2

Overview of Mortierella s. l. isolates obtained from different locations. The percentages of different isolates (biological replicates) found in a certain sampling site are illustrated. The width of each bar corresponds to the relative sample size. Please note that the total numbers of isolates isolated from sampling sites cannot be inferred from the figure. ALGL = M. alpina/globalpina, AN = M. angusta, BA = M. bainieri, EX = L. exigua, GA = L. gamsii, GE = M. gemmifera, HO = P. horticola, HUVE = P. humilis/verticillata, HY = L. hyalina, PA = E. parvispora, PZ = M. pseudozygospora, SO = M. solitaria, ZO = M. zonata.

Figure 3

Dendrogram clusters of Mortierella s. l. species based on volatilomes (left) and ITS sequences (right). Clusters were calculated on an average cluster algorithm using median distances of biological replicates. ALGL = M. alpina/globalpina, AN = M. angusta, BA = M. bainieri, EX = L. exigua, GA = L. gamsii, GE = M. gemmifera, HO = P. horticola, HUVE = P. humilis/verticillata, HY = L. hyalina, PA = E. parvispora, PZ = M. pseudozygospora, SO = M. solitaria, ZO = M. zonata. Different colours (blue and pale orange) highlight identical clustering of species, the red rectangles show species with different clustering.

Figure 4

Relative concentrations of 118 mass peaks among 13 Mortierella s. l. species observed in this study. All mass peak concentrations illustrated differ significantly between species (p_Kruskal < 0.05). For illustration, the concentrations were centered and scaled by columns. Consequently, color shades can be compared between species but not between mass peaks. Darker colors indicate higher and lighter colors indicate lower concentrations. ALGL = M. alpina/globalpina, AN = M. angusta, BA = M. bainieri, EX = L. exigua, GA = L. gamsii, GE = M. gemmifera, HO = P. horticola, HUVE = P. humilis/verticillata, HY = L. hyalina, PA = E. parvispora, PZ = M. pseudozygospora, SO = M. solitaria, ZO = M. zonata.

Figure 5

Mass peaks enriched in certain Mortierella s. l. species. Species differ significantly with regards to relative concentrations of these mass peaks according to Kruskal test and ANOVA (p < 0.05). ALGL = M. alpina/globalpina, AN = M. angusta, BA = M. bainieri, EX = L. exigua, GA = L. gamsii, GE = M. gemmifera, HO = P. horticola, HUVE = P. humilis/verticillata, HY = P. hyalina, PA = E. parvispora, PZ = M. pseudozygospora, SO = M. solitaria, ZO = M. zonata.