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Fungi in Roots of Nursery Grown Pinus sylvestris: Ectomycorrhizal Colonisation, Genetic Diversity and Spatial Distribution

  • Fungal Microbiology
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Abstract

The aims of this study were to investigate patterns of ectomycorrhizal (ECM) colonisation and community structure on nursery grown seedlings of Pinus sylvestris, spatial distribution of ECMs in the nursery plot and genetic diversity of commonly isolated ECM basidiomycete Hebeloma cavipes. One hundred seedlings were sampled in 225 m2 area using a systematic grid design. For each seedling, 20 individual root tips were randomly collected, morphotyped, and surface sterilised for fungal isolation in pure culture. Results showed that ECM community was comprised of nine distinct morphotypes among which Thelephora terrestris (39.7%), Hebeloma sp. (17.8%) and Suillus luteus (6.1%) were the most abundant. Spatial distribution of ECMs in the nursery plot was determined by their relative abundance: even in common ECMs and random in rare ones. Fungal isolation yielded 606 pure cultures, representing 71 distinct taxa. The most commonly isolated fungi were the ascomycetes Neonectria macrodidyma (20.3%), Phialocephala fortinii (13.5%), Neonectria radicicola (6.3%) and the ECM basidiomycete H. cavipes (4.5%). Intraspecific genetic diversity within 27 H. cavipes isolates was studied using two methods: restriction digestion of the amplified intergenic spacer of nuclear ribosomal DNA and genealogical concordance of five genetic markers. Five and eight genotypes were revealed by each respective method, but both of those were largely consistent, in particular, in determining the largest genotype (A) composed of 18 isolates. Mapping positions for each H. cavipes isolate and genotype in the field showed that isolates of the A genotype covered a large part of the nursery plot. This suggests that H. cavipes is largely disseminated by vegetative means of local genotypes and that nursery cultivation practices are likely to contribute to the dissemination of this species in the forest nursery soils.

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References

  1. Agerer R (1986–2006) Colour atlas of ectomycorrhizae. Einhorn, München

    Google Scholar 

  2. Alaniz S, León M, Vicent A, García-Jiménez J, Abad-Campos P, Armengol J (2007) Characterization of Cylindrocarpon species associated with black foot disease of grapevine in Spain. Plant Dis 91s:1187–1193

    Article  Google Scholar 

  3. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed  Google Scholar 

  4. Arnaud J, Mélanie M, Blanca-Rosa R-P, Bernard B, Bruno A, Anne-Marie M, Annick B, Michel C (2003) Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum. Mol Microbiol 47:411–430

    Article  Google Scholar 

  5. Auger J, Esterio M, Pérez I (2007) First report of black foot disease of grapevine caused by Cylindrocarpon macrodidymum in Chile. Plant Dis 91:470–470

    Article  Google Scholar 

  6. Brody JR, Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36:214–216

    CAS  PubMed  Google Scholar 

  7. Chakravarty P, Unestam T (1985) Role of mycorrhizal fungi in protecting damping-off of Pinus sylvestris L. seedlings. In: Gianinazzi-Pearson V, Gianinazzi S (eds.) Physiological and genetical aspects of mycorrhizae. Institut national de le recherche agronomique, Dijon, France, Proceedings of the 1st European Symposium on Mycorrhizae, pp. 811–814

  8. Clark PJ, Evans FC (1954) Distance to nearest neighbor as a measure of spatial relationships in populations. Ecology 35:445–453

    Article  Google Scholar 

  9. Colpaert JV, Vanassche JA (1992) Zinc toxicity in ectomycorrhizal Pinus sylvestris. Plant Soil 143:201–211

    Article  CAS  Google Scholar 

  10. Cram MM, Melax JG, Souter R (1999) Successful reforestation of South Carolina sandhills is not influenced by seedling inoculation with Pisolithus tinctorius in the nursery. South J Appl For 23:46–52

    Google Scholar 

  11. Dahlberg A (2002) Effects of fire on ectomycorrhizal fungi in Fennoscandian boreal forests. Silva Fenn 36:69–80

    Google Scholar 

  12. Dunabeitia M, Rodriguez N, Salcedo I, Sarrionandia E (2004) Field mycorrhization and its influence on the establishment and development of the seedlings in a broadleaf plantation in the Basque Country. Forest Ecol Manag 195:129–139

    Article  Google Scholar 

  13. Garbaye J, Churin JL (1997) Growth stimulation of young oak plantations inoculated with the ectomycorrhizal fungus Paxillus involutus with special reference to summer drought. Forest Ecol Manag 98:221–228

    Article  Google Scholar 

  14. Gardes M, Bruns T (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118

    Article  CAS  PubMed  Google Scholar 

  15. Guidot A, Lumini E, Debaud J-C, Marmeisse R (1999) The nuclear ribosomal DNA intergenic spacer as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum directly on Pinus root systems. Appl Environ Microb 65:903–909

    CAS  Google Scholar 

  16. Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acid S 41:95–98

    CAS  Google Scholar 

  17. Halleen F, Schroers HJ, Groenewald JZ, Crous PW (2004) Novel species of Cylindrocarpon (Neonectria) and Campylocarpon gen. nov associated with black foot disease of grapevines (Vitis spp.). Stud Mycol 50:431–455

    Google Scholar 

  18. Hasegawa M, Kishino H, Yano T (1985) Dating the human ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol 22:160–174

    Article  CAS  PubMed  Google Scholar 

  19. Haselwandter K, Bowen GD (1996) Mycorrhizal relations in trees for agroforestry and land rehabilitation. Forest Ecol Manag 81:1–17

    Article  Google Scholar 

  20. Ivory M, Munga F (1983) Growth and survival of container-grown Pinus caribaea infected with various ectomycorrhizal fungi. Plant Soil 71:339–344

    Article  Google Scholar 

  21. Iwanski M, Rudawska M (2007) Ectomycorrhizal colonization of naturally regenerating Pinus sylvestris L. seedlings growing in different micro-habitats in boreal forest. Mycorrhiza 17:461–467

    Article  PubMed  Google Scholar 

  22. Iwanski M, Rudawska M, Leski T (2006) Mycorrhizal associations of nursery grown Scots pine (Pinus sylvestris L.) seedlings in Poland. Ann For Sci 63:715–723

    Article  Google Scholar 

  23. Jonsson L, Dahlberg A, Nilsson MC, Karen O, Zackrisson O (1999) Continuity of ectomycorrhizal fungi in self-regenerating boreal Pinus sylvestris forests studied by comparing mycobiont diversity on seedlings and mature trees. New Phytol 142:151–162

    Article  Google Scholar 

  24. Jumpponen A (2001) Dark septate endophytes—are they mycorrhizal? Mycorrhiza 11:207–211

    Article  Google Scholar 

  25. Kernaghan G, Sigler L, Khasa D (2003) Mycorrhizal and root endophytic fungi of containerized Picea glauca seedlings assessed by rDNA sequence analysis. Microb Ecol 45:128–136

    Article  CAS  PubMed  Google Scholar 

  26. Koljalg U, Larsson KH, Abarenkov K, Nilsson RH, Alexander IJ, Eberhardt U, Erland S, Hoiland K, Kjoller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo L, Vralstad T, Ursing BM (2005) UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi. New Phytol 166:1063–1068

    Article  CAS  PubMed  Google Scholar 

  27. Koufopanou V, Burt A, Taylor JW (1997) Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis. P Natl Acad Sci USA 94:5478–5482

    Article  CAS  Google Scholar 

  28. Kropp BR, Langlois CG (1990) Ectomycorrhizae in reforestation. Can J For Res 20:438–451

    Article  Google Scholar 

  29. Laatikainen T, Heinonen-Tanski H (2002) Mycorrhizal growth in pure cultures in the presence of pesticides. Microbiol Res 157:127–137

    Article  CAS  PubMed  Google Scholar 

  30. Le Tacon F, Alvarez IF, Bouchard D, Henrion B, Jackson MR, Luff S, Parlade JI, Pera J, Stenström E, Villeneuve N, Walker C (1994) Variations in field response of forest trees to nursery ectomycorrhizal inoculation in Europe. In: Read DJ, Lewis DH, Fitter AH, Alexander IJ (eds) Mycorrhizas in ecosystems. CAB International, Wallingford, pp 119–134

    Google Scholar 

  31. Lee KJ (1992) A ten-year result of artificial inoculation of pines with ectomycorrhizal fungi, Pisolithus tinctorius and Thelephora terrestris. J Kor Forest Soc 81:156–163

    Google Scholar 

  32. Marx DH (1969) The influence of ectotrophic ectomycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to pathogenic fungi and soil bacteria. Phytopathology 59:153–163

    Google Scholar 

  33. Marx DH, Bryan WC, Grand LF (1970) Colonization, isolation, and cultural descriptions of Thelephora terrestris and other ectomycorrhizal fungi of shortleaf pine seedlings grown in fumigated soil. Can J Bot 48:207–211

    Article  Google Scholar 

  34. Marx DH, Cordell CE, Kenney DS, Mexal JG, Artman JD, Riffle JW, Molina R (1984) Commercial vegetative inoculum of Pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on bare root tree seedlings. Forest Sci Monograph 25:101

    Google Scholar 

  35. Marx DH, Hedin A, Toe SFP (1985) Field performance of Pinus caribaea var. hondurensis seedlings with specific ectomycorrhizae and fertilizer after three years on a savanna site in Liberia. Forest Ecol Manag 13:1–25

    Article  Google Scholar 

  36. Menkis A, Vasiliauskas R, Taylor AFS, Stenlid J, Finlay R (2005) Fungal communities in mycorrhizal roots of conifer seedlings in forest nurseries under different cultivation systems, assessed by morphotyping, direct sequencing and mycelial isolation. Mycorrhiza 16:33–41

    Article  PubMed  Google Scholar 

  37. Menkis A, Vasiliauskas R, Taylor AFS, Stenlid J, Finlay R (2007) Afforestation of abandoned farmland with conifer seedlings inoculated with three ectomycorrhizal fungi - impact on plant performance and ectomycorrhizal community. Mycorrhiza 17:337–348

    Article  CAS  PubMed  Google Scholar 

  38. Menkis A, Vasiliauskas R, Taylor AFS, Stenström E, Stenlid J, Finlay R (2006) Fungi in decayed roots of conifer seedlings from forest nurseries, afforested clearcuts and abandoned farmland. Plant Pathol 55:117–129

    Article  CAS  Google Scholar 

  39. Minitab® Inc (2003) Minitab statistical software. Release 15.1. Minitab Inc, Pennsyllvania

    Google Scholar 

  40. Morin C, Samson J, Dessureault M (1999) Protection of black spruce seedlings against Cylindrocladium root rot with ectomycorrhizal fungi. Can J Bot 77:169–174

    Article  Google Scholar 

  41. O'Neill JJM, Mitchell DT (2000) Effects of benomyl and captan on growth and mycorrhizal colonization of Sitka-spruce (Picea sitchensis) and ash (Fraxinus excelsior) in Irish nursery soil. For Pathol 30:165–174

    Google Scholar 

  42. Ortega U, Dunabeitia M, Menendez S, Gonzalez-Murua C, Majada J (2004) Effectiveness of mycorrhizal inoculation in the nursery on growth and water relations of Pinus radiata in different water regimes. Tree Physiol 24:65–73

    CAS  PubMed  Google Scholar 

  43. Pera J, Alvarez IF, Rincon A, Parlade J (1999) Field performance in northern Spain of Douglas-fir seedlings inoculated with ectomycorrhizal fungi. Mycorrhiza 9:77–84

    Google Scholar 

  44. Reddy MS, Natarajan K (1997) Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery. Mycorrhiza 7:133–138

    Article  Google Scholar 

  45. Rosling A, Landeweert R, Lindahl BD, Larsson KH, Kuyper TW, Taylor AFS, Finlay RD (2003) Vertical distribution of ectomycorrhizal fungal taxa in a podzol soil profile. New Phytol 159:775–783

    Article  CAS  Google Scholar 

  46. Rudawska M, Leski T, Trocha LK, Gornowicz R (2006) Ectomycorrhizal status of Norway spruce seedlings from bare-root forest nurseries. Forest Ecol Manag 236:375–384

    Article  Google Scholar 

  47. Shaw CG, Kile GA (1991) Armillaria root disease. Agricultural Handbook 691. USDA Forest Service, Washington

    Google Scholar 

  48. Smith SE, Read DJ (1997) Mycorrhizal symbiosis. Academic, London

    Google Scholar 

  49. Soll D (2000) The ins and outs of DNA fingerprinting the infectious fungi. Clin Microbiol Rev 13:332–370

    Article  CAS  PubMed  Google Scholar 

  50. Stenström E, Damm E, Unestam T (1997) Le role des mycorhizes dans la protection des arbres forestiers contre les agents pathogenes du sol. Revue forestière française XLIX-no sp.: 121–128

  51. Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland

    Google Scholar 

  52. Taylor AFS, Martin F, Read DJ (2000) Fungal diversity in ectomycorrhizal communities of Norway spruce (Picea abies (L.) Karst.) and beech (Fagus sylvatica L.) along north-south transects in Europe. In: Schulze ED (ed) Carbon and nitrogen cycling in European forest ecosystems. Springer Verlag, Heidelberg, pp 343–365

    Google Scholar 

  53. Thorn RG, Reddy CA, Harris D, Paul EA (1996) Isolation of saprophytic basidiomycetes from soil. Appl Environ Microb 62:4288–4292

    CAS  Google Scholar 

  54. Trocha LK, Rudawska M, Leski T, Dabert M (2006) Genetic diversity of naturally established ectomycorrhizal fungi on Norway spruce seedlings under nursery conditions. Microb Ecol 52:418–425

    Article  CAS  PubMed  Google Scholar 

  55. van Aarle I, Viennois G, Amenc L, Tatry M-V, Luu D, Plassard C (2007) Fluorescent in situ RT-PCR to visualise the expression of a phosphate transporter gene from an ectomycorrhizal fungus. Mycorrhiza 17:487–494

    Article  PubMed  Google Scholar 

  56. Van der Heijden EW, Kuyper TW (2001) Does origin of mycorrhizal fungus or mycorrhizal plant influence effectiveness of the mycorrhizal symbiosis? Plant Soil 230:161–174

    Article  Google Scholar 

  57. Van Tichelen KK, Colpaert JV, Vangronsveld J (2001) Ectomycorrhizal protection of Pinus sylvestris against copper toxicity. New Phytol 150:203–213

    Article  Google Scholar 

  58. Väre H (1990) Effects of soil fertility on root colonization and plant growth of Pinus sylvestris nursery seedlings inoculated with different ectomycorrhizal fungi. Scand J Forest Res 5:493–499

    Article  Google Scholar 

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

    Google Scholar 

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Acknowledgements

We thank Karelyn Cruz for linguistic corrections. Financial support is greatly acknowledged to The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and European Commission Marie Curie Transfer of Knowledge Project No. 042622 LITCOAST.

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  1. Uppsala BioCenter, Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, P.O. Box 7026, SE-75007, Uppsala, Sweden

    Audrius Menkis & Rimvydas Vasaitis

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  1. Audrius Menkis
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  2. Rimvydas Vasaitis
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Correspondence to Audrius Menkis.

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Menkis, A., Vasaitis, R. Fungi in Roots of Nursery Grown Pinus sylvestris: Ectomycorrhizal Colonisation, Genetic Diversity and Spatial Distribution. Microb Ecol 61, 52–63 (2011). https://doi.org/10.1007/s00248-010-9676-8

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