Nachweis der GattungArmillaria (Fr.:Fr.) Staude undHeterobasidion annosum (Fr.) Bref. in Fichte (Picea abies [L.] Karst.) und Erfassung der klonalen Ausbreitung vonA. ostoyae-Genotypen unter Verwendung molekularer Methoden

  • S. Schulze
  • G. Bahnweg
Article

Zusammenfassung

Methoden zur Entwicklung molekularer Marker mit Hilfe der Polymerase-Kettenreaktion (PCR) werden beispielhaft an den beiden forstlichen PathogenenArmillaria spp. undH. annosum erläutert. Unter Verwendung der aus „internal transcribed spacer“ (ITS)-DNA-Sequenzen abgeleiteten Primerpaare ARM-1/ARM-2 bzw. HET-7/HET-8 konntenArmillaria spp.- bzw.H. annosum-spezifische DNA-Abschnitte mittels PCR amplifiziert werden. Diese PCR-Diagnosemethode erlaubt einen schnellen und effizienten Frühnachweis dieser beiden forstlich relevanten Wurzel- und Stockfäuleerreger in verschiedenen Substraten bzw. Pflanzengeweben. Die genetische Variabilität von 20A. ostoyae-Isolaten aus verschiedenen geographischen Herkunftsgebieten wurde untersucht. Die UPGMA-Clusteranalyse der unter Verwendung von 10 Decamer-Zufallsprimern (OPA 01-10) erhaltenen „random amplified polymorphic DNAs“ (RAPDs)-Muster gruppierte die Isolate in Untergruppen mit 40–96% Ähnlichkeit, was auf eine hohe intraspezifische genetische Variation hindeutet. Die potentielle Rolle der historischen und gegenwärtigen Ausbreitung von Fichtenpflanzen für die genetische Variation vonA. ostoyae in Europa wird diskutiert. Die aufgefundenen polymorphen DNA-Marker wurden für die Erfassung der Populationsstruktur bzw. -dynamik sowie der räumlichen Ausbreitung vonA. ostoyae-„genets“ in ausgewählten Befallsgebieten genutzt. Erste Untersuchungen haben unterschiedliche Ausbreitungsstrategien vonA. ostoyae in Abhängigkeit von Klima-, Standort- und Immissionsfaktoren aufgezeigt.

Schlüsselwörter

Armillaria spp. Heterobasidion annosum Diagnose ITS-PCR-Primer RAPDs Populationsstruktur 

Identification of the genusArmillaria (Fr.:Fr.) Staude andHeterobasidion annosum (Fr.) Bref. in Norway spruce (Picea abies [L.] Karst.) and determination of clonal distribution ofA. ostoyae-genotypes by molecular methods

Summary

Methods based on the polymerase chain reaction (PCR) for generating molecular markers are illustrated by two important forest pathogens,Armillaria spp. andH. annosum. Using the primer pairs ARM-1/ARM-2 and HET-7/HET-8, derived from sequences of the “internal transcribed spacer” (ITS) regions of the rDNA repeat,Armillaria spp.- andH. annosum-specific DNA fragments were amplified by PCR. This PCR-based detection method allows a rapid and definite diagnosis of both important root and butt rot pathogens in different substrates or plant tissues, especially in early stages. Genetic variability among 20A. ostoyae-isolates from different geographical origins was studied. UPGMA cluster analysis of random amplified polymorphic DNAs (RAPDs) profiles generated by 10 decamer random primers (OPA 01-10) grouped the isolates in subclusters at similarity levels between 40% and 96%, indicating high intraspecific genetic variation. The potential role of historical and current spread of spruce plants on the genetic variation ofA. ostoyae in Europe is discussed. The established polymorphic DNA markers were used to determine the population structure, dynamics and spatial distribution ofA. ostoyae-“genets” in areas colonized by the fungus. First investigations revealed different distribution strategies ofA. ostoyae, which may be mediated by climatic factors, location (e.g., soil characters), and pollutants.

Key words

Armillaria spp. Heterobasidion annosum diagnosis ITS-PCR-primer RAPDs population structure 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literatur

  1. Anderson, J. B.;Bailey, S. S.;Pukkila, P. J., 1989: Variation in ribosomal DNA among biological species ofArmillaria, a genus of root-infecting fungi. Evolution43, 1652–1662.CrossRefGoogle Scholar
  2. Anderson, J. B.;Petsche, D. M.;Smith, M. L., 1987: Restriction fragment polymorphisms in biological species ofArmillaria mellea. Mycologia79, 69–76.CrossRefGoogle Scholar
  3. Bachmann, K., 1994: Molecular markers in plant ecology. New Phytol.126, 403–418.CrossRefGoogle Scholar
  4. Bahnweg, G.; Schulze, S.; Möller, E. M.; Anegg, S.; Langebartels, Ch.; Sandermann, H. Jr., 1998: Detection and quantification of the root and Sutt rot fungus,Heterobasidion annosum (Fr.) Bref. in forest trees by PCR. Eur. J. For. Pathol., in press.Google Scholar
  5. Burdsall, H. H. Jr.;Banik, M.;Cook, M. E., 1990: Serological differentiation of three species ofArmillaria andLentinula edodes by enzyme-linked immunosorbent assay using immunized chikkens as a source of antibodies. Mycologia82, 415–423.CrossRefGoogle Scholar
  6. Chase, T. E.;Ullrich, R. C., 1988:Heterobasidion annosum, root- and butt-rot of trees. Adv. Plant Pathol.6, 501–510.CrossRefGoogle Scholar
  7. Delatour, C., 1980: LeFomes annosus (Fr.) Cke. en Europe de l’ouest: importance economique, orientation des recherches. In: 5th Int. Conf. on Problems of Root and Butt rots in Conifers (Kassel, Aug. 7.–12., 1978) Hess. Forstl. Versuchsanstalt, Hann. Münden, 9–18.Google Scholar
  8. Foster, L. M.;Kozak, K. R.;Loftus, M. G.;Stevens, J. J.;Ross, I. K., 1993: The polymerase chain reaction and its application to filamentous fungi. Mycol. Res.97(7), 769–781.CrossRefGoogle Scholar
  9. Garbelotto, M.;Ratcliff, A.;Bruns, T. D.;Cobb, F. W.;Otrosina, W. J., 1996: Use of taxon-specific competitive-priming PCR to study host specifity, hybridization, and intergroup gene flow in intersterility groups ofHeterobasidion annosum. Phytopathology86, 543–551.CrossRefGoogle Scholar
  10. Guillaumin, J. J.;Anderson, J. B.;Legrand, P.;Ghahari, S.;Berthelay, S., 1996: A comparison of different methods for the identification of genets ofArmillaria spp. New Phytol.133, 333–343.CrossRefGoogle Scholar
  11. Guillaumin, J. J.;Mohammed, C.;Anselmi, N.;Courtecuisse, R.;Gregory, S. C.;Holdenrieder, O.;Intini, M.;Lung, B.;Marxmüller, H.;Morrison, D.;Rishbeth, J.;Termorshuizen, A. J.;Tirro, A.;Van Dam, B., 1993: Geographical distribution and ecology of theArmillaria species in western Europe. Eur. J. For. Path.23, 321–341.CrossRefGoogle Scholar
  12. Harrington, T. C.;Worrall, J. J.;Baker, F. A., 1992:Armillaria. In: Methods for Research on Soil-borne Phytopathogenic Fungi. Ed. bySingleton, L. L.;Mihail, J. D.;Rush, C., APS Press, St. Paul, Minnesota, 81–85.Google Scholar
  13. Kato, F., 1969: Stammfäuleschäden derFichte. Ein Beitrag zur Schadensberechnung in den staatlichen Fichtenwäldern Niedersachsens. Forstarchiv40, 91–92.Google Scholar
  14. Keen, N. T., 1990: Gene-for-gene complementarity in plant-pathogen interactions. Annu. Rev. Genet.24, 447–463.CrossRefGoogle Scholar
  15. Kile, G. A., 1983: Identification of genotypes and the clonal development ofArmillaria luteobubalina Watling & Kile in eucalypt forests. Austral. J. Bot.31, 657–671.CrossRefGoogle Scholar
  16. Korhonen, K., 1978a: Interfertility and clonal size in theArmillariella mellea complex. Karstenia18, 31–42.CrossRefGoogle Scholar
  17. Korhonen, K., 1978b: Intersterility groups ofHeterobasidion annosum. Commun. Inst. For. Fenn.94(6), 1–25.Google Scholar
  18. Legrand, P.;Ghahari, S.;Guillaumin, J. J., 1996: Occurrence of genets ofArmillaria spp. in four mountain forests in Central France: the colonization strategy ofArmillaria ostoyae. New Phytol.133, 321–332.CrossRefGoogle Scholar
  19. Marxmüller, H., 1992: Some notes on the taxonomy and nomenclature of five EuropeanArmillaria species. MycotaxonXLIV, 267–274.Google Scholar
  20. Möller, E. M.;Bahnweg, G.;Sandermann, H. Jr.;Geiger, H. H., 1992: A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucl. Acids Res.20/22, 6115–6116.CrossRefGoogle Scholar
  21. Morrison, D. J.;Mallett, K., 1996: Silvicultural management ofArmillaria root disease in western Canadian forests. Can. J. of Plant Path.18, 194–199.CrossRefGoogle Scholar
  22. Otrosina, W. J.;Chase, T. E.;Cobb, F. W., Jr.;Korhonen, K., 1993: Population structure ofHeterobasidion annosum from North America and Europe. Can. J. Bot.71, 1064–1071.CrossRefGoogle Scholar
  23. Priestley, R.;Mohammed, C.;Dewey, F. M., 1994: The development of monoclonal antibody-based ELISA and dipstick assays for the detection and identification ofArmillaria species in infected wood. In: Modern Assays for Plant Pathogenic Fungi. Ed. bySchots, A.;Dewey, F. M.;Oliver, R. P., Oxford: CAB International, 149–156.Google Scholar
  24. Rayner, A. D. M., 1991: The challenge of the individualistic mycelium. Mycologia83, 48–71.CrossRefGoogle Scholar
  25. Rizzo, D. M.;Harrington, T. C., 1993: Delineation and biology of clones ofArmillaria ostoyae,A. gemina andA. calvescens. Mycologia85, 164–174.CrossRefGoogle Scholar
  26. Rohlf, F. J., 1992: NTSYS-pc, Numerical Taxonomy and Multivariate Analysis System. Setauket: Applied Biostatistics Inc.Google Scholar
  27. Roll-Hansen, F., 1985: TheArmillaria species in Europe. A literature review. Eur. J. For. Path.15, 22–31.CrossRefGoogle Scholar
  28. Sambrook, J., Fritsch, E. F.;Maniatis, T., 1989: Molecular Cloning. A laboratory manual., 2nd edn., Cold Spring Harbor: Cold Spring Harbor Laboratory.Google Scholar
  29. Schulze, S.;Bahnweg, G.;Möller, E. M.;Sandermann, H. Jr., 1997a: Identification of the genusArmillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation withinArmillaria ostoyae by rDNA-RFLP and RAPD analysis. Eur. J. For. Path.27, 225–239.CrossRefGoogle Scholar
  30. Schulze, S.;Bahnweg, G.;Tesche, M.;Sandermann, H. Jr., 1995: Identification of European-Armillaria species by restriction-fragment-length polymorphisms of ribosomal DNA. Eur. J. For. Path.25, 214–223.CrossRefGoogle Scholar
  31. Schulze, S.;Bahnweg, G.;Tesche, M.;Sandermann, H. Jr., 1997b: Identification techniques forArmillaria spp. andHeterobasidion annosum root and butt rot diseases. A critical review. J. Plant Dis. and Prot.104, 433–451.Google Scholar
  32. Shaw, C. G. III;Kile, G. A., 1991:Armillaria root disease. USDA Forest Service, Agriculture Handbook No. 691. Washington DC: USDA.Google Scholar
  33. Smith, M. L.;Anderson, J. B., 1989: Restriction fragment polymorphisms in mitochondrial DNAs ofArmillaria: identification of North American biological species. Mycol. Res.93, 247–256.CrossRefGoogle Scholar
  34. Smith, M. L.;Anderson, J. B., 1994: Mitochondrial DNAs of the fungusArmillaria ostoyae: restriction map and length variation. Curr. Genet.25, 545–553.CrossRefGoogle Scholar
  35. Smith, M. L.;Bruhn, J. N.;Anderson, J. B., 1994: Relatedness and spatial distribution ofArmillaria genets infecting red pine seedlings. Phytopathology84, 822–829.CrossRefGoogle Scholar
  36. Stalpers, J. A., 1978: Identification of wood-inhabiting Aphyllophorales in pure culture. Stud. Mycol.16, 248 pp.Google Scholar
  37. Williams, J. G. K.;Kubelik, A. R.;Livak, K. J.;Rafalski, J. A.;Tingey, S. V., 1990: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res.18, 6531–6535.CrossRefGoogle Scholar
  38. Worrall, J. J., 1994: Population structure ofArmillaria species in several forest types. Mycologia86, 401–407.CrossRefGoogle Scholar
  39. Worrall, J. J.;Harrington, T. C., 1992:Heterobasidion. In: Methods for Research on Soil-borne Phytopathogenic Fungi. Ed. bySingleton, L. L.;Mihail, J. D.;Rush, C. M., APS Press, St. Paul, Minnesota, 86–90.Google Scholar

Copyright information

© Blackwell Wissenschafts-Verlag 1998

Authors and Affiliations

  • S. Schulze
    • 1
  • G. Bahnweg
    • 2
  1. 1.Institut für Forstbotanik und ForstzoologieTU DresdenTharandt
  2. 2.GSF-Forschungszentrum für Umwelt und GesundheitInstitut für Biochemische PflanzenpathologieOberschleißheim

Personalised recommendations