Bark beetles are destructive insect pests known to form symbioses with different fungal taxa, including yeasts. The aim of this study was to (1) determine the prevalence of the rare yeast Hyphopichia heimii in bark beetle frass from wild olive trees in South Africa and to (2) predict the potential interaction of this yeast with trees and bark beetles. Twenty-eight culturable yeast species were isolated from frass in 35 bark beetle galleries, including representatives of H. heimii from nine samples. Physiological characterization of H. heimii isolates revealed that none was able to degrade complex polymers present in hemicellulose; however, all were able to assimilate sucrose and cellobiose, sugars associated with an arboreal habitat. All isolates were able to produce the auxin indole acetic acid, indicative of a potential symbiosis with the tree. Sterol analysis revealed that the isolates possessed ergosterol quantities ranging from 3.644 ± 0.119 to 13.920 ± 1.230 mg/g dry cell weight, which suggested that H. heimii could serve as a source of sterols in bark beetle diets, as is known for other bark beetle-associated fungi. In addition, gas chromatography-mass spectrometry demonstrated that at least one of the isolates, Hyphopichia heimii CAB 1614, was able to convert the insect pheromone cis-verbenol to the anti-aggregation pheromone verbenone. This indicated that H. heimii could potentially influence beetle behaviour. These results support the contention of a tripartite symbiosis between H. heimii, olive trees, and bark beetles.

Department of Conservation Ecology and Entomology Stellenbosch University Stellenbosch 7602 South Africa

Department of Microbiology Stellenbosch University Private Bag X1 Stellenbosch 7602 South Africa

Zobrazit více v PubMed

Agnello AM, Combs DB, Filgueiras CC, Willett DS, Mafra-Neto A (2021) Reduced infestation by Xylosandrus germanus (Coleoptera: Curculionidae: Scolytinae) in apple trees treated with host plant defense compounds. J Econ Entomol 114:2162–2171. https://doi.org/10.1093/jee/toab153 PubMed DOI

Ali SS, Al-Tohamy R, Sun J, Wu J, Huizi L (2019) Screening and construction of a novel microbial consortium SSA-6 enriched from the gut symbionts of wood-feeding termite, Coptotermes formosanus and its biomass-based biorefineries. Fuel 236:1128–1145. https://doi.org/10.1016/j.fuel.2018.08.117 DOI

Anastasaki E, Psoma A, Partsinevelos G, Papachristos D, Milonas P (2021) Electrophysiological responses of Philaenus spumarius and Neophilaenus campestris females to plant volatiles. Phytochemistry 189:112848. https://doi.org/10.1016/j.phytochem.2021.112848 PubMed DOI

Arthington-Skaggs BA, Warnock DW, Morrison CJ (2000) Quantitation of Candida albicans ergosterol content improves the correlation between in vitro antifungal susceptibility test results and in vivo outcome after fluconazole treatment in a murine model of invasive candidiasis. Antimicrob Agents Chemother 44:2081–2085. https://doi.org/10.1128/AAC.44.8.2081-2085.2000 PubMed DOI PMC

Axelsson B-O, Saraf A, Larsson L (1995) Determination of ergosterol in organic dust by gas chromatography-mass spectrometry. J Chromatogr B Biomed Appl 666:77–84. https://doi.org/10.1016/0378-4347(94)00553-h PubMed DOI

Ayres MP, Wilkens RT, Ruel JJ, Lombardero MJ, Vallery E (2000) Nitrogen budgets of phloem-feeding bark beetles with and without symbiotic fungi. Ecology 81:2198–2210. https://doi.org/10.2307/177108 DOI

Behmer ST, Nes WD (2003) Insect sterol nutrition and physiology: a global overview. Adv Insect Physiol 31:1–72. https://doi.org/10.1016/S0065-2806(03)31001-X DOI

Behmer ST, Olszewski N, Sebastiani J, Palka S, Sparacino G, Sciarrno E, Grebenok RJ (2013) Plant phloem sterol content: forms, putative functions, and implications for phloem-feeding insects. Front Plant Sci 4:1–7. https://doi.org/10.3389/fpls.2013.00370 DOI

Bentz BJ, Six DL (2006) Ergosterol content of fungi associated with Dendroctonus ponderosae and Dendroctonus rufipennis (Coleoptera: Curculionidae, Scolytinae). Ann Entomol Soc Am 99:189–194. https://doi.org/10.1603/0013-8746(2006)099[0189:ecofaw]2.0.co;2 DOI

Blomquist GJ, Figueroa-Teran R, Aw M, Song M, Gorzalski A, Abbott NL, Chang E, Tittiger C (2010) Pheromone production in bark beetles. Insect Biochem Mol Biol 40:699–712. https://doi.org/10.1016/j.ibmb.2010.07.013

Bongi G (2002) Freezing avoidance in olive tree (Olea europaea L.): from proxies to targets of action. Adv Hort Sci 16:117–124. https://www.jstor.org/stable/42883314

Breivik ON, Owades JL (1957) Spectrophotometric semimicro determination of ergosterol in yeast. Agric Food Chem 5:360–363. https://doi.org/10.1021/jf60075a005

Callaham RZ, Shifrine M (1960) The yeasts associated with bark beetles. For Sci 6:146–154. https://doi.org/10.1093/forestscience/6.2.146 DOI

Christiansen E, Ericsson A (1986) Starch reserves in Picea abies in relation to defence reaction against a bark beetle transmitted blue-stain fungus, Ceratocystis polonica. Can J for Res 16:78–83. https://doi.org/10.1139/x86-013 DOI

Conde C, Delrot S, Gerós H (2008) Physiological, biochemical and molecular changes occurring during olive development and ripening. J Plant Physiol 165:1545–1562. https://doi.org/10.1016/j.jplph.2008.04.018 PubMed DOI

Davis TS, Hofstetter RW, Foster JT, Foote NE, Keim P (2011) Interactions between the yeast Ogataea pini and filamentous fungi associated with the western pine beetle. Microb Ecol 61:626–634. https://doi.org/10.1007/s00248-010-9773-8 PubMed DOI

Dowd PF, Shen SK (1990) The contribution of symbiotic yeast to toxin resistance of the cigarette beetle (Lasioderma serricorne). Entomol Exp Appl 56:241–248. https://doi.org/10.1111/j.1570-7458.1990.tb01402.x DOI

Durand A-A, Buffet J-P, Constant P, Déziel E, Guertin C (2018) Fungal communities associated with the eastern larch beetle: diversity and variation within developmental stages. bioRxiv 220780. https://doi.org/10.1101/220780

Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340 PubMed DOI PMC

Endoh R, Suzuki M, Benno Y, Futai K (2008) Candida kashinagacola sp. nov., C. pseudovanderkliftii sp. nov. and C. vanderkliftii sp. nov., three new yeasts from ambrosia beetle-associated sources. Antonie Van Leeuwenhoek 94:389–402. https://doi.org/10.1007/s10482-008-9256-9 PubMed DOI

Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 50:1351–1371. https://doi.org/10.1099/00207713-50-3-1351 PubMed DOI

Grieneisen ML (1994) Recent advances in our knowledge of ecdysteroid biosynthesis in insects and crustaceans. Insect Biochem Mol Biol 24:115–132. https://doi.org/10.1016/0965-1748(94)90078-7 DOI

Harman GE (2011) Multifunctional fungal plant symbionts: new tools to enhance plant growth and productivity. New Phytol 189:647–649. https://doi.org/10.1111/j.1469-8137.2010.03614.x PubMed DOI

Harrigan WF, McCance ME (1976) Statistical methods for the selection and examination of microbial colonies. In: Harrigan WF, McCance ME (eds) Laboratory methods in food and dairy microbiology. Academic Press, London, pp 47–49

Harrington T C (2005) Ecology and evolution of mycophagous bark beetles and their fungal partners. In: Vega F E, Blackwell M (eds), Ecological and evolutionary advances in insect-fungal associations. Oxford University Press, 257–291

Hernández-Martínez F, Briones-Roblero CI, Nelson DR, Rivera-Orduña FN, Zúñiga G (2016) Cytochrome P450 complement (CYPome) of Candida oregonensis, a gut-associated yeast of bark beetle, Dendroctonus rhizophagus. Fungal Biol 120:1077–1089.  https://doi.org/10.1016/j.funbio.2016.06.005

Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, Villari C (2020) Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81:101–113. https://doi.org/10.1007/s13199-020-00686-9 DOI

Hunt DWA, Borden JH (1990) Conversion of verbenols to verbenone by yeasts isolated from Dendroctonus ponderosae (Coleoptera: Scolytidae). J Chem Ecol 16:1385–1397. https://doi.org/10.1007/BF01021034 PubMed DOI

Jordal BH (2021) A phylogenetic and taxonomic assessment of Afrotropical Micracidini (Coleoptera, Scolytinae) reveals a strong diversifying role for Madagascar. Organisms Diversity & Evolution 21:245–278. https://doi.org/10.1007/s13127-021-00481-4

Jurišić Grubešić R, Nazlić M, Miletić T, Vuko E, Vuletić N, Ljubenkov I, Dunkić V (2021) Antioxidant capacity of free volatile compounds from Olea europaea L. cv. Oblica leaves depending on the vegetation stage. Antioxidants 10(11):1832. https://doi.org/10.3390/antiox10111832

Kasana RC, Salwan R, Dhar H, Dutt S, Gulati A (2008) A rapid and easy method for the detection of microbial cellulases on agar plates using Gram’s iodine. Curr Microbiol 57:503–507. https://doi.org/10.1007/s00284-008-9276-8 PubMed DOI

Klepzig KD, Six DL (2004) Bark beetle-fungal symbiosis: context dependency in complex associations. Symbiosis 37:189–205

Kok LT, Norris DM (1973) Comparative sterol compositions of adult female Xyleborus ferrugineus and its mutualistic fungal ectosymbionts. Comp Biochem Physiol 44:499–505. https://doi.org/10.1016/0305-0491(73)90024-2 DOI

Kreutz J, Zimmermann G, Vaupel O (2004) Horizontal transmission of the entomopathogenic fungus Beauveria bassiana among the spruce bark beetle, Ips typographus (Col., Scolytidae) in the laboratory and under field conditions. Biocontrol Sci Technol 14:837–848. https://doi.org/10.1080/788222844 DOI

Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096 PubMed DOI PMC

Kurtzman CP (1987) Two new species of Pichia from arboreal habitats. Mycologia 79:410–417. https://doi.org/10.2307/3807464 DOI

Kurtzman CP (2011a) Hyphopichia von Arx & van der Walt. In: Kurtzman CP, Fell JW, Boekhout T (eds) The yeasts: a taxonomic study, 5th edn. Elsevier, pp 435–438 DOI

Kurtzman CP, Fell JW, Boekhout T, Robert V (2011b) Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW, Boekhout T (eds) The yeasts: a taxonomic study, 5th edn. Elsevier, pp 87–110 DOI

Lachance M-A, Starmer WT, Rosa CA, Bowles JM, Barker JSF, Janzen DH (2001) Biogeography of the yeasts of ephemeral flowers and their insects. FEMS Yeast Res 1:1–8. https://doi.org/10.1016/S1567-1356(00)00003-9 PubMed DOI

Lam CK, Belanger FC, White JF Jr, Daie J (1995) Invertase activity in Epichloë/Acremonium fungal endophytes and its possible role in choke disease. Mycol Res 99:867–873. https://doi.org/10.1016/S0953-7562(09)80743-0 DOI

Leufvén A, Bergström G, Falsen E (1984) Interconversion of verbenols and verbenone by identified yeasts isolated from the spruce bark beetle Ips typographus. J Chem Ecol 10:1349–1361. https://doi.org/10.1007/BF00988116 PubMed DOI

Leufvén A, Nehls L (1986) Quantification of different yeasts associated with the bark beetle, Ips typographus, during its attack on a spruce tree. Microb Ecol 12:237–243. https://www.jstor.org/stable/4250882

Limtong S, Koowadjanakul N (2012) Yeasts from phylloplane and their capability to produce indole-3-acetic acid. World J Microbiol Biotechnol 28:3323–3335. https://doi.org/10.1007/s11274-012-1144-9 PubMed DOI

Limtong S, Kaewwichian R, Yongmanitchai W, Kawasaki H (2014) Diversity of culturable yeasts in phylloplane of sugarcane in Thailand and their capability to produce indole-3-acetic acid. World J Microbiol Biotechnol 30:1785–1796. https://doi.org/10.1007/s11274-014-1602-7 PubMed DOI

Linardi VR, Machado KMG (1990) Production of amylases by yeasts. Can J Microbiol 36:751–753. https://doi.org/10.1139/m90-129 PubMed DOI

Martini X, Sobel L, Conover D, Mafra-Neto A, Smith J (2020) Verbenone reduces landing of the redbay ambrosia beetle, vector of the laurel wilt pathogen, on live standing redbay trees. Agric for Entomol 22:83–91. https://doi.org/10.1111/afe.12364 DOI

Moller L, Kessler KD, Steyn A, Valentine AJ, Botha A (2016a) The role of Cryptococcus laurentii and mycorrhizal fungi in the nutritional physiology of Lupinus angustifolius L. hosting N DOI

Moller L, Lerm B, Botha A (2016b) Interactions of arboreal yeast endophytes: an unexplored discipline. Fungal Ecol 22:73–82. https://doi.org/10.1016/j.funeco.2016.03.003 DOI

Nes WR, Sekula BC, Nes WD, Adler JH (1978) The functional importance of structural features of ergosterol in yeast. J Biol Chem 253:6218–6225. https://doi.org/10.1016/S0021-9258(17)34602-1 PubMed DOI

Olatinwo R, Allison J, Meeker J, Johnson W, Streett D, Catherine Aime M, Carlton C (2013) Detection and identification of Amylostereum areolatum (Russulales: Amylostereaceae) in the mycangia of Sirex nigricornis (Hymenoptera: Siricidae) in Central Louisiana. Environ Entomol 42:1246–1256. https://doi.org/10.1603/EN13103 PubMed DOI

Pasanen A-L, Yli-Pietilä K, Pasanen P, Kalliokoski P, Tarhanen J (1999) Ergosterol content in various fungal species and biocontaminated building materials. Appl Environ Microbiol 65:138–142. https://doi.org/10.1128/aem.65.1.138-142.1999 PubMed DOI PMC

Pignal M (1970) A new species of yeast isolated from decaying insect-invaded wood. Antonie Van Leeuwenhoek 36:525–529. https://doi.org/10.1007/BF02069054 PubMed DOI

Postma F, Mesjasz-Przybyłowicz J, Przybyłowicz W, Stone W, Mouton M, Botha A (2012) Symbiotic interactions of culturable microbes with the nickel hyperaccumulator Berkheya coddii and the herbivorous insect Chrysolina clathrata. Symbiosis 58:209–220. https://doi.org/10.1007/s13199-012-0217-8 DOI

Raffa KF, Gregoire JC, Lindgren BS (2015) Natural history and ecology of bark beetles. In: Vega FE, Hofstetter RW (eds) Bark beetles. Academic Press, pp 1–40

Rodriguez RJ, White JF Jr, Arnold AE, Redman ARA (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330. https://doi.org/10.1111/j.1469-8137.2009.02773.x PubMed DOI

Rottava I, Cortina PF, Zanella CA, Cansian RL (2010) Microbial oxidation of ( - ) - α -pinene to verbenol production by newly isolated strains. Appl Biochem Biotechnol 162:2221–2231. https://doi.org/10.1007/s12010-010-8996-y PubMed DOI

Ruano F, Campos M, Sánchez-Raya AJ, Peña A (2010) Olive trees protected from the olive bark beetle, Phloeotribus scarabaeoides (Bernard 1788) (Coleoptera, Curculionidae, Scolytinae) with a pyrethroid insecticide: effect on the insect community of the olive grove. Chemosphere 80:35–40. https://doi.org/10.1016/j.chemosphere.2010.03.039 PubMed DOI

Scorzetti G, Petrescu I, Yarrow D, Fell JW (2000) Cryptococcus adeliensis sp. nov., a xylanase producing basidiomycetous yeast from Antarctica. Antonie Van Leeuwenhoek 77:153–157. https://doi.org/10.1023/a:1002124504936 PubMed DOI

Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A (2002) Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Res 2:495–517. https://doi.org/10.1016/S1567-1356(02)00128-9 PubMed DOI

Six DL (2013) The bark beetle holobiont: why microbes matter. J Chem Ecol 39:989–1002. https://doi.org/10.1007/s10886-013-0318-8 PubMed DOI

Six DL, Bentz BJ (2003) Fungi associated with the North American spruce beetle, Dendroctonus rufipennis. Can J for Res 33:1815–1820. https://doi.org/10.1139/x03-107 DOI

Staffan Lindgren B, Miller DR (2002) Effect of verbenone on five species of bark beetles (Coleoptera: Scolytidae) in lodgepole pine forests. Environmental Entomol 31:759–765. https://doi.org/10.1603/0046-225X-31.5.759

Sulman S, Rehman A (2013) Isolation and characterization of cellulose degrading Candida tropicalis W2 from environmental samples. Pakistan J Zool 45:809–816

Tiwari R, Köffel R, Schneiter R (2007) An acetylation/deacetylation cycle controls the export of sterols and steroids from S. cerevisiae. EMBO J 26:5109–5119. https://doi.org/10.1038/sj.emboj.7601924 PubMed DOI PMC

Valiev A, Ogel ZB, Klepzig KD (2009) Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi. Symbiosis 49:37–42. https://doi.org/10.1007/s13199-009-0007-0 DOI

van Heerden A, van Zyl WH, Cruywagen CW, Mouton M, Botha A (2011) The lignicolous fungus Coniochaeta pulveracea and its interactions with syntrophic yeasts from the woody phylloplane. Microb Ecol 62:609–619. https://doi.org/10.1007/s00248-011-9869-9 PubMed DOI

Vural N, Akay MA (2021) Chemical compounds, antioxidant properties and antimicrobial activity of olive leaves derived volatile oil in West Anatolia. J Turkish Chem Soc Section A: Chem 8(2):511–518. https://doi.org/10.18596/jotcsa.833139

Vreulink JM, Stone W, Botha A (2010) Effects of small increases in copper levels on culturable basidiomycetous yeasts in low-nutrient soils. J Appl Microbiol 109:1411–1421. https://doi.org/10.1111/j.1365-2672.2010.04770.x PubMed DOI

White RA, Agosin M, Franklin RT, Webb JW (1980) Bark beetle pheromones: evidence for physiological synthesis mechanisms and their ecological implications. Z Angew Ent 90:255–274. https://doi.org/10.1111/j.1439-0418.1980.tb03526.x

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–322

Wood SL, Bright DE (1992) Hosts of Scolytidae and Platypodidae. Great Basin Naturalist Memoirs 13:1241–1284

Worrall J (1991) Media for selective isolation of Hymenomycetes. Mycologia 83:296–302. https://doi.org/10.2307/3759989