Abstract
Among the most devastating pests of Norway spruce (Picea abies) are the European spruce bark beetle (Ips typographus) and the associated pathogenic blue-stain fungus Ceratocystis polonica. Following attack and colonization, the beetle and the fungus must cope with induced host chemical defenses, such as monoterpenes that are generally thought to be toxic to both symbionts. The goal of this study was to better understand the response of Norway spruce following C. polonica inoculation at low density that does not overwhelm the tree and to identify monoterpenes mobilized toward the fungus. We inoculated healthy mature trees and monitored monoterpene profiles 2, 3, and 5 months post-inoculation. We also exposed three different C. polonica strains to the most abundant or significantly up-regulated monoterpenes to determine differences in monoterpene toxicity and resistance among strains. Total monoterpene levels, including limonene, were increased at 2 and 3 months after inoculation and had dropped after 5 months. In in vitro assays, all monoterpenes were inhibitory to C. polonica. Limonene and β-pinene were the most potent inhibitors of fungal growth. The extent of inhibition varied between the three strains tested. These results showed a defense response of Norway spruce to C. polonica, in which limonene may play a critical role in inhibiting the spread of the fungus. We also showed that differences between strains of C. polonica must be taken into account when assessing the role of the fungus in this bark beetle–symbiont system.
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References
Baier P, Führer E, Kirisits T, Rosner S (2002) Defence reactions of Norway spruce against bark beetles and the associated fungus Ceratocystis polonica in secondary pure and mixed species stands. For Ecol Manage 159:73–86
Bohlmann J (2012) Pine terpenoid defences in the mountain pine beetle epidemic and in other conifer pest interactions: specialized enemies are eating holes into a diverse, dynamic and durable defence system. Tree Physiol 32:943–945
Boone CK, Aukema BH, Bohlmann J, Carroll AL, Raffa KF (2011) Efficacy of tree defense physiology varies with bark beetle population density: a basis for positive feedback in eruptive species. Can J For Res 41:1174–1188
Christiansen E (1985) Ceratocysts polonica inoculated in Norway spruce: blue-staining in relation to inoculum density, resinosis and tree growth. For Pathol 15:160–167
Christiansen E, Bakke A (1988) The spruce bark beetle of Eurasia. In: Dynamics of forest insect populations: patterns, causes, implications. Plenum Press, New York
Croteau R, Gurkewitz S, Johnson MA, Fisk HJ (1987) Biochemistry of Oleoresinosis: monoterpene and diterpene biosynthesis in lodgepole pine saplings infected with Ceratocystis clavigera or treated with carbohydrate elicitors. Plant Physiol 85:1123–1128
DiGuistini S, Wang Y, Liao NY, Taylor G, Tanguay P, Feau N, Henrissat B, Chan SK, Hesse-Orce U, Alamouti SM, Tsui CKM, Docking RT, Levasseur A, Haridas S, Robertson G, Birol I, Holt RA, Marra MA, Hamelin RC, Hirst M, Jones SJM, Bohlmann J, Breuil C (2011) Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen. Proc Natl Acad Sci USA 108:2504–2509
Eckhardt LG, Menard RD, Gray ED (2009) Effects of oleoresins and monoterpenes on in vitro growth of fungi associated with pine decline in the Southern United States. For Pathol 39:157–167
Erbilgin N, Colgan LJ (2012) Differential effects of plant ontogeny and damage type on phloem and foliage monoterpenes in jack pine (Pinus banksiana). Tree Physiol 32:946–957
Erbilgin N, Krokene P, Christiansen E, Zeneli G, Gershenzon J (2006) Exogenous application of methyl jasmonate elicits defenses in Norway spruce (Picea abies) and reduces host colonization by the bark beetle Ips typographus. Oecologia 148:426–436
Evensen PC, Solheim H, Høiland K, Stenersen J (2000) Induced resistance of Norway spruce, variation of phenolic compounds and their effects on fungal pathogens. For Pathol 30:97–108
Faccoli M (2009) Effect of weather on Ips typographus (Coleoptera Curculionidae) phenology, voltinism, and associated spruce mortality in the southeastern Alps. Environ Entomol 38:307–316
Fäldt J, Solheim H (2006) Influence of fungal infection and wounding on contents and enantiomeric compositions of monoterpenes in phloem of Pinus sylvestris. J Chem Ecol 32:1779–1795
Franceschi VR, Krokene P, Christiansen E, Krekling T (2005) Anatomical and chemical defenses of conifer bark against bark beetles and other pests. New Phytol 167:353–375
Hall DE, Robert JA, Keeling CI, Domanski D, Quesada AL, Jancsik S, Kuzyk MA, Hamberger B, Borchers CH, Bohlmann J (2011) An integrated genomic, proteomic and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil. Plant J Cell Mol Biol 65:936–948
Hammerbacher A, Schmidt A, Wadke N, Wright LP, Schneider B, Bohlmann J, Brand WA, Fenning TM, Gershenzon J, Paetz C (2013) A common fungal associate of the spruce bark beetle metabolizes the stilbene defenses of Norway spruce. Plant Physiol 162(3):1324–1336
Horntvedt R, Christiansen E, Solheim H, Wang S (1983) Artificial inoculation with Ips typographus-associated blue-stain fungi can kill healthy Norway spruce trees (Ceratocystis polonica, Ceratocystis penicillata, water stress). Rep Nor For Res Inst 38:1–20
Jost R, Rice A (2008) Monoterpene emissions from lodgepole and jack pine bark inoculated with mountain pine beetle associated fungi. J Wood Chem 28:37–46
Keeling CI, Bohlmann J (2006a) Diterpene resin acids in conifers. Phytochemistry 67:2415–2423
Keeling CI, Bohlmann J (2006b) Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defence of conifers against insects and pathogens. New Phytol 170:657–675
Klepzig KD, Robison DJ, Fowler G, Minchin PR, Hain FP, Allen HL (2005) Effects of mass inoculation on induced oleoresin response in intensively managed loblolly pine. Tree Physiol 25:681–688
Klepzig KD, Adams AS, Handelsman J, Raffa KF (2009) Symbioses: a key driver of insect physiological processes, ecological interactions, evolutionary diversification, and impacts on humans. Environ Entomol 38:67–77
Klepzig KD, Smalley EB, Raffa KF (1996) Combined chemical defenses against an insect-fungal complex. J Chem Ecol 22:1367–1388
Krajnc AU (2009) A temporal analysis of antioxidative defense responses in the phloem of Picea abies after attack by Ips typographus. Tree Physiol 29:1059–1068
Krokene P, Solheim H (1996) Fungal associates of five bark beetle species colonizing Norway spruce. Can J For Res 2115–2122
Krokene P, Solheim H (1997) Growth of four bark-beetle-associated blue-stain fungi in relation to the induced wound response in Norway spruce. Can J Bot 75:618–625
Krokene P, Solheim H (1998) Pathogenicity of four blue-stain fungi associated with aggressive and nonaggressive bark beetles. Phytopathology 88:39–44
Krokene P, Solheim H (1999) What do low-density inoculations with fungus tell us about fungal virulence and tree resistance? In: Lieutier F, Mattson WJ, Wagner MR (eds) Physiology and genetics of tree-phytophage interactions INRA editions. Versailles, France, pp 353–362
Krokene P, Christiansen E, Solheim H, Franceschi V, Berryman A (1999) Induced resistance to pathogenic fungi in Norway spruce. Plant Physiol 121:565–570
Krokene P, Solheim H, Christiansen E (2001) Induction of disease resistance in Norway spruce (Picea abies) by necrotizing fungi. Plant Pathol 50:230–233
Krokene P, Nagy NE, Krekling T (2008) Traumatic resin ducts and polyphenolic parenchyma cells in conifers. In: Schaller A (ed) Induced plant resistance to herbivory. Springer, Netherlands, Dordrecht, pp 147–169
Krupa S, Fries N (1971) Studies on ectomycorrhizae of pine. Production of volatile organic compounds. Can J Bot 49:1425–1431
Lah L, Haridas S, Bohlmann J, Breuil C (2012a) The cytochromes P450 of Grosmannia clavigera: genome organization, phylogeny, and expression in response to pine host chemicals. Fungal Genet Biol 50:72–81
Lah L, Haridas S, Bohlmann J, Breuil C (2012) The cytochromes P450 of Grosmannia clavigera: genome organization, phylogeny, and expression in response to pine host chemicals. Fungal Genet Biol
Lewinsohn E, Savage T (1993) Simultaneous analysis of monoterpenes and diterpenoids of conifer oleoresin. Phytochem Anal 4:220–225
Li SH, Nagy NE, Hammerbacher A, Krokene P, Niu XM, Gershenzon J, Schneider B (2012) Localization of phenolics in phloem parenchyma cells of Norway spruce (Picea abies). Chembiochem: A Eur J Chem Biol 18:2707–2713
Lieutier F, Yart A, Salle A (2009) Stimulation of tree defenses by ophiostomatoid fungi can explain attack success of bark beetles on conifers. Ann For Sci 66:1–22
Linnakoski R, De Beer ZW, Niemelä P, Wingfield MJ (2012) Associations of conifer-infesting bark beetles and fungi in Fennoscandia. Insects 3:200–227
Lu M, Wingfield MJ, Gillette NE, Mori SR, Sun JH (2010a) Complex interactions among host pines and fungi vectored by an invasive bark beetle. New Phytol 187:8
Lu M, Wingfield MJ, Gillette NE, Mori SR, Sun J-H (2010b) Complex interactions among host pines and fungi vectored by an invasive bark beetle. New Phytol 187:859–866
Marei GIK, Abdel Rasoul MA, Abdelgaleil SAM (2012) Comparative antifungal activities and biochemical effects of monoterpenes on plant pathogenic fungi. Pestic Biochem Physiol 103:56–61
Martin D, Tholl D, Gershenzon J, Bohlmann J (2002) Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems. Plant Physiol 129:1003–1018
Paine TD, Raffa KF, Harrington TC (1997) Interactions among scolytid bark beetles, their associated fungi, and live host conifers. Annu Rev Entomol 42:179–206
Pasquier-Barre F, Palasse C, Goussard F, Auger-Rozenberg M-A, Géri C (2001) Relationship of Scots pine clone characteristics and water stress to hatching and larval performance of the sawfly Diprion pini (Hymenoptera: Diprionidae). Environ Entomol 30:1–6
Persson M, Sjödin K, Borg-Karlson A-K, Norin T, Ekberg I (1996) Relative amounts and enantiomeric compositions of monoterpene hydrocarbons in xylem and needles of Picea abies. Phytochemistry 42:1289–1297
Phillips MA, Croteau RB (1999) Resin-based defenses in conifers. Trends Plant Sci 4:184–190
Raffa KF, Berryman AA (1982) Accumulation of monoterpenes and associated volatiles following inoculation of grand fir with a fungus transmitted by the fir engraver, Scolytus ventralis (Coleoptera: Scolytidae). Can Entomol 114:797–810
Raffa KF, Berryman AA (1983a) Physiological aspects of lodgepole pine wound responses to a fungal symbiont of the mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Scolytidae). Can Entomol 115:723–734
Raffa KF, Berryman AA (1983b) The role of host plant resistance in the colonization behavior and ecology of bark beetles (Coleoptera: Scolytidae). Ecol Monogr 53:27
Raffa KF, Smalley EB (1995) Interaction of pre-attack and induced monoterpene concentrations in host conifer defense against bark beetle-fungal complexes. Oecologia 102:285–295
Raffa KF, Berryman AA, Simasko J, Teal W, Wong BL (1985) Effects of grand fir monoterpenes on the fir engraver, Scolytus ventralis (Coleoptera: Scolytidae), and its symbiotic fungus. Environ Entomol 14:5
Raffa KF, Aukema BH, Bentz BJ, Carroll AL, Hicke JA, Turner MG, Romme WH (2008) Cross-scale drivers of natural disturbances prone to anthropogenic amplification: the dynamics of bark beetle eruptions. Bioscience 58:501
Reed AN, Hanover JW, Furniss MM (1986) Douglas-fir and western larch: chemical and physical properties in relation to Douglas-fir bark beetle attack. Tree Physiol 1:277–287
Robert JA, Madilao LL, White R, Yanchuk A, King J, Bohlmann J (2010) Terpenoid metabolite profiling in Sitka spruce identifies association of dehydroabietic acid, (+)-3-carene, and terpinolene with resistance against white pine weevil. Botany 88:810–820
Rocchini LA, Lindgren BS, Bennett RG (2000) Effects of resin flow and monoterpene composition on susceptibility of lodgepole pine to attack by the Douglas-fir pitch moth, Synanthedon novaroensis (Lep., Sesiidae). J Appl Entomol 124:87–92
Sallé A, Monclus R, Yart A, Garcia J, Romary P, Lieutier F (2005) Fungal flora associated with Ips typographus: frequency, virulence, and ability to stimulate the host defence reaction in relation to insect population levels. Can J For Res 35:365–373
Schiebe C, Hammerbacher A, Birgersson G, Witzell J, Brodelius PE, Gershenzon J, Hansson BS, Krokene P, Schlyter F (2012) Inducibility of chemical defenses in Norway spruce bark is correlated with unsuccessful mass attacks by the spruce bark beetle. Oecologia 170:183–198
Silvestrini E, Michelozzi M, Skroppa T, Brancaleoni E, Ciccioli P (2004) Characterisation of different clones of Picea abies (L.) Karst using head-space sampling of cortical tissues combined with enantioselective capillary gas chromatography for the separation of chiral and non-chiral monoterpenes. J Chromatogr A 1034:183–189
Six DL, Wingfield MJ (2011) The role of phytopathogenicity in bark beetle-fungus symbioses: a challenge to the classic paradigm. Annu Rev Entomol 56:255–272
Sjödin K, Persson M, Faldt J, Ekberg I, Borg-Karlson A-K (2001) Occurrence and correlations of monoterpene hydrocarbon enantiomers in Pinus sylvestris and Picea abies. J Chem Ecol 26:1701–1720
Smith RH (1965) Effect of monoterpene vapors on the western pine beetle. J Econ Entomol 58:2
Solheim H (1986) Species of Ophiostomataceae isolated from Picea abies infested by the bark beetle Ips typographic. Nordic J Bot 6:199–207
Solheim H (1991) Oxygen deficiency and spruce resin inhibition of growth of blue stain fungi associated with Ips typographus. Mycol Res 95:1387–1392
Tausz M, Wonisch A, Grill D, Morales D, Jiménez MS (2003) Measuring antioxidants in tree species in the natural environment: from sampling to data evaluation. J Exp Bot 54:1505–1510
Viiri H, Annila E, Kitunen V, Niemelä P (2001) Induced responses in stilbenes and terpenes in fertilized Norway spruce after inoculation with blue-stain fungus, Ceratocystis polonica. Trees 15:112–122
Wallin KF, Raffa KF (1999) Altered constitutive and inducible phloem monoterpenes following natural defoliation of jack pine: implications to host mediated interguild interactions and plant defense theories. J Chem Ecol 25:861–880
Wang Y, Lim L, Diguistini S, Robertson G, Bohlmann J, Breuil C (2013) A specialized ABC efflux transporter GcABC-G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle-associated fungal pathogen of pine trees. New Phytol 197:886–898
Wermelinger B (2004) Ecology and management of the spruce bark beetle Ips typographus—a review of recent research. For Ecol Manage 202:67–82
Wright E (1933) A cork-borer method for inoculating trees. Phytopathology 23:487–488
Zamponi L, Michelozzi M, Capretti P (2007) Terpene response of Picea abies and Abies alba to infection with Heterobasidion sl. For Pathol 37:243–250
Zeneli G, Krokene P, Christiansen E, Krekling T, Gershenzon J (2006) Methyl jasmonate treatment of mature Norway spruce (Picea abies) trees increases the accumulation of terpenoid resin components and protects against infection by Ceratocystis polonica, a bark beetle-associated fungus. Tree Physiol 26:977–988
Zhao T, Krokene P, Björklund N, Långström B, Solheim H, Christiansen E, Borg-Karlson AK (2010) The influence of Ceratocystis polonica inoculation and methyl jasmonate application on terpene chemistry of Norway spruce, Picea abies. Phytochemistry 71:1332–1341
Zhao T, Krokene P, Hu J, Christiansen E, Björklund N, Långström B, Solheim H, Borg-Karlson AK (2011) Induced terpene accumulation in Norway spruce inhibits bark beetle colonization in a dose-dependent manner. PLoS ONE 6:e26649
Zulak KG, Bohlmann J (2010) Terpenoid biosynthesis and specialized vascular cells of conifer defense. J Integr Plant Biol 52:86–97
Zulak KG, Lippert DN, Kuzyk MA, Domanski D, Chou T, Borchers CH, Bohlmann J (2009) Targeted proteomics using selected reaction monitoring reveals the induction of specific terpene synthases in a multi-level study of methyl jasmonate-treated Norway spruce (Picea abies). Plant J 60:1015–1030
Acknowledgments
We thank Prof. Dr. Mario Lešnik from Faculty of Agriculture and Life Sciences, Peter Kramer from Maribor University Agriculture Centre, and Ciril Zupan from the Slovenian Forest Service for permission to use the experimental field in Pivola, Hoče, and support during the experimental work. We gratefully acknowledge support from Alfonso Lara Quesada for the assistance with data analysis and Justin G. A. Whitehill for help with the statistical analysis. We thank Mateja Felicijan for helping with the field sample collection and technical assistance with sample preparations. We thank Janez Gorenšek, Andreja Šober, Maja Svenšek, Peter Drevenšek, and Robi Gjergjek for excellent assistance in the field.
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Novak, M., Krajnc, A.U., Lah, L. et al. Low-density Ceratocystis polonica inoculation of Norway spruce (Picea abies) triggers accumulation of monoterpenes with antifungal properties. Eur J Forest Res 133, 573–583 (2014). https://doi.org/10.1007/s10342-013-0772-4
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DOI: https://doi.org/10.1007/s10342-013-0772-4