Ethanol-Enriched Substrate Facilitates Ambrosia Beetle Fungi, but Inhibits Their Pathogens and Fungal Symbionts of Bark Beetles

Abstract

Bark beetles (sensu lato) colonize woody tissues like phloem or xylem and are associated with a broad range of micro-organisms. Specific fungi in the ascomycete orders Hypocreales, Microascales and Ophistomatales as well as the basidiomycete Russulales have been found to be of high importance for successful tree colonization and reproduction in many species. While fungal mutualisms are facultative for most phloem-colonizing bark beetles (sensu stricto), xylem-colonizing ambrosia beetles are long known to obligatorily depend on mutualistic fungi for nutrition of adults and larvae. Recently, a defensive role of fungal mutualists for their ambrosia beetle hosts was revealed: Few tested mutualists outcompeted other beetle-antagonistic fungi by their ability to produce, detoxify and metabolize ethanol, which is naturally occurring in stressed and/or dying trees that many ambrosia beetle species preferentially colonize. Here, we aim to test (i) how widespread beneficial effects of ethanol are among the independently evolved lineages of ambrosia beetle fungal mutualists and (ii) whether it is also present in common fungal symbionts of two bark beetle species (Ips typographus, Dendroctonus ponderosae) and some general fungal antagonists of bark and ambrosia beetle species. The majority of mutualistic ambrosia beetle fungi tested benefited (or at least were not harmed) by the presence of ethanol in terms of growth parameters (e.g., biomass), whereas fungal antagonists were inhibited. This confirms the competitive advantage of nutritional mutualists in the beetle's preferred, ethanol-containing host material. Even though most bark beetle fungi are found in the same phylogenetic lineages and ancestral to the ambrosia beetle (sensu stricto) fungi, most of them were highly negatively affected by ethanol and only a nutritional mutualist of Dendroctonus ponderosae benefited, however. This suggests that ethanol tolerance is a derived trait in nutritional fungal mutualists, particularly in ambrosia beetles that show cooperative farming of their fungi.

Keywords: Ips typographus; ambrosia fungi; bark and ambrosia beetles; detoxification; ethanol; symbiont selection.

FIGURE 1

Phylogenetic placement based on 49 fungal LSU sequences. Black-colored species were examined within this study (N = 12), while species in gray act as a fungal outgroup (N = 37). The LSU region of the following fungi was sequenced by us and used for the phylogenetic analysis (strain IDs in brackets): Ophiostoma bicolor (P22), Grosmannia penicillata (2), Endoconidiophora polonica (F5), Beauveria bassiana (P13), Entomocorticium sp. (P8), Entomocorticium dendroctoni (P163), and Fusarium euwallaceae (P170). Sequences from all other species were obtained from NCBI GenBank. For accession numbers and further information of each individual strain see Supplementary Tables 1, 2. Ophiostomatales were divided into their four sub-clades that are currently hypothesized to have evolved independently as nutritional mutualists of ambrosia beetles (Biedermann and Vega, 2020). All examined species were classified into (i) ambrosia beetle fungi, (ii) bark beetle fungi, and (iii) free-living fungi. Triangles denote significant effects on fungal biomass (decrease, increase or not significant; p < 0.05) between the 0 and 1% EtOH treatments. Nutritional mutualists of bark and ambrosia beetles are underlined. Due to its abundant sporulation we could not receive comparable data from Beauveria bassiana (indicated with *) and excluded it from all further analyses.

FIGURE 2

Representative images of seven studied fungi growing on MEA treated with five investigated ethanol concentrations (0, 1, 2, 3, and 5% vol/vol). (a): ambrosia beetle fungi = Ambrosiella hartigii, Raffaelea sulphurea; (b): bark beetle fungi = Grosmannia penicillata, Endoconidiophora polonica; (c): pathogens/free-living = Chaetomium globosum, Esteya vermicola, Entomocorticium sp. For incubation times and further information of each individual strain see Supplementary Table 3. The five remaining fungal species are displayed in Supplemantary Figure 1.

FIGURE 3

Fungal biomass in mg (dry-weight) as a function of the EtOH (% v/v) treatment (0, 1, 2, 3, and 5% vol/vol). Fungi were classified into (A) positively affected (Ambrosiella hartigii, Entomocorticium dendroctoni, Esteya vermicola) and not affected (Raffaelea canadensis, R. sulphurea Fusarium euwallaceae) by the 1% EtOH treatment or (B) negatively affected (Chaetomium globosum, Endoconidiophora polonica, Entomocorticium sp. (E.sp.), Grosmannia penicillata, Ophiostoma bicolor) by the 1% EtOH treatment. With one exception (E. vermicola) all species in the first group are nutritional mutualists of bark and ambrosia beetles, whereas all species in the second group are either plant-defenses-detoxifying mutualists of bark beetles, free-living or antagonistic species. (AB) denotes ambrosia beetle associates and (BB) bark beetle associates. Means and standard deviations are given for each EtOH treatment. Significant differences between the 0% and 1% and 2% EtOH treatments are given: *p < 0.05, **p < 0.01, ***p < 0.001. Additionally, we added (+) and (–) to indicate a significant increase, respectively, decrease of fungal biomass. N = 8 replicates per fungal species.