Mycorrhization of containerized Pinus nigra seedlings with Suillus granulatus under open field conditions

  • J. S. Lazarevic University of Montenegro Biotechnical faculty. Podgorica, Montenegro
  • N. D. Keca University of Belgrade. Faculty of Forestry. Belgrade, Serbia
  • A. B. Martinovic University of Montenegro Biotechnical faculty. Podgorica, Montenegro


Seedling mycorrhization acts as an efficient tool for improving the quality of seedlings. In this study, the effectiveness of Suillus granulatus, originating from Pinus heldreichii forests (Montenegro), to produce containerized ectomycorrhizal seedlings of autochthonous Pinus nigra in open field conditions was investigated. Spore (106, 107, 108) and vegetative (1:16, 1:8, 1:4) inoculation on ectomycorrhizal formation and seedling growth were tested. Spore and vegetative inoculums of autochthonous Pisolithus arhizus were used in the same trial as additional control treatments. The utilization of vegetative and spore inoculums of autochthonous S. granulatus has proven to be an effective method of obtaining containerized ectomycorrhizal P. nigra seedlings under open field conditions after 11 months. S. granulatus spore inoculations resulted in well developed ectomycorrhiza, decreasing the growth of the P. nigra seedlings in the first growing season. Mycelial inoculations resulted in slightly developed S. granulatus ectomycorrhiza, which increased the growth of the seedlings. Therefore, it would be feasible to use spore inocula of S. granulatus, with 106 spores per plant, to produce ectomycorrhizal P. nigra plants on a large scale. Controlled mycorrhizal inoculation of seedlings is not a common practice in Montenegrin and Serbian nurseries; as such, the obtained results will contribute to the enhancement of nursery production of Pinus nigra and other conifers. This also could be assumed as a starting point for many further efforts and investigations with autochthonous fungal and plant material in this region.


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Author Biographies

J. S. Lazarevic, University of Montenegro Biotechnical faculty. Podgorica, Montenegro
Department for Forestry
N. D. Keca, University of Belgrade. Faculty of Forestry. Belgrade, Serbia
Department for forest protection


Agerer R. 2001. Exploration types of ectomycorrhizae. Mycorrhiza 11, 107-114.

Anonymous 2008. National Forest Policy of Montenegro, Ministry of Agriculture, Forestry and Water Management of Montenegro, document adopted from Montenegrin Government no. 03-3982, April 24th.68 pp.

Argyrous G. 2005. Statistics for Research: With a Guide to SPSS, Sage, London. 401pp.

Berman JT, Bledsoe CS. 1998. Soil transfers from valley oak (Quercus lobata Nee) stands increase ectomycorrhizal diversity and alter root and shoot growth on valley oak seedlings. Mycorrhiza 7, 223-235.

Burić D, Ivanović R, Mitrović L. 2007. The Climate of Podgorica. Hydrological and Meteorological Service of Montenegro, Podgorica. 106 pp.

Castellano MA. 1996. Outplanting performance of mycorrhizal inoculated seedlings. In: Concepts in mycorrhizal research (Mukerji KG, Ed.). Kluwer Academic Publishers, Dordrecht, The Netherlands. pp. 223-291.

Castellano MA. 1994. Current status of outplanting studies using ectomycorrhiza-inoculated forest trees. In: Mycorrhiza and plant health (Pflenger F.L., Linderman R.G. eds.). APS press, USA. pp. 261-281.

Castellano MA, Molina R. 1989. Mycorrhizae. In: The Container Tree Nursery Manual, Vol 5. Agric. Handbk. 674. (Landis, TD., Tinus RW., McDonald SE., Barnett JP.eds.). U.S. Department of Agriculture, Forest Service, Washington DC, USA. pp: 101-167.

Cairney JWG, Chambers S M. 1997. Interactions between Pisolithus tinctorius and its hosts: a review of current knowledge. Mycorrhiza 7, 117-131.

Cairney JWG. 1999. Intraspecific physiological variation: implications for understanding functional diversity in ectomycorrhizal fungi. Mycorrhiza 9, 125-135.

Cline M, Reid P. 1982. Seed source and mycorrhizal fungus effects on growth of containerized Pinus contorta and Pinus ponderosa seedlings. For. Sci. 28/2, 237-250.

Dunabeitia MK, Hormilla S, Garcia-Plazola JI, Txarterina K, Arteche U, Becerril JM. 2004. Differential response of three fungal species to enviromental factors and their role in the mycorrhization of Pinus radiata D.Don. Mycorrhiza 14, 11-18. PMid:14513336

El Karkouri K, Martin F, Mousain D. 2002. Dominance of the mycorrhizal fungus Rhizopogon rubescens in a plantation of Pinus pinea seedlings inoculated with Suillus collinitus. Ann. For. Sci. 59, 197-204.

El Karkouri K, Selosse M-A, Mousain D. 2006. Molecular markers detecting an ectomycorrhiyal Suillus collinitus strain on Pinus halepensis roots suggest successful inoculation and persistence in Mediterranean nursery and plantation. FEMS Microbiology Ecology 55, 146-158. PMid:16420623

Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes — application to the identification of mycorrhizae and rusts. Molecular Ecology 2, 113-118. PMid:8180733

Gomes EA, de Barros EG, Kasuya MCM, Araujo EF. 1999. Molecular characterization of Pisolithus spp. isolate by r DNA PCR-RFLP. Mycorrhiza 8, 197-202.

Hacskaylo E. 1973. Carbohydrate physiology of ectomzcorrhizae. In: Ectomycorrhizae, their ecology and function (Marks GC, Kozloeski TT, eds) Academic Press, London, Great Britain. pp. 207-230.

Hall TA. 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series 41, 95-98.

Jovanović B. 2007. Dendrology, University in Belgrade, Faculty of Forestry, Belgrade. 536 pp.

Karen O, Hogberg N, Dahlberg A, Jonsson L, Nylund J.-E. 1997. Inter-and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytologist 136, 313-325.

Knežević M, Košanin O, Milošević R. 2011. Assessment of production potential of podzolised and typical acid brown soils in some forest types in the area of Veliki Jastrebac, Bulletin of the Faculty of Forestry 103, University of Belgrade-Faculty of Forestry, Belgrade (57-72), DOI:10.2298/GSF1103057K.

Landis DH. 1989. Disease and Pest Management, In: The Container Tree Nursery Manual, Vol 5. Agric. Handbook. 674. (Landis, TD, Tinus RW., McDonald SE, Barnett JP, eds.). U.S. Department of Agriculture, Forest Service, Washington DC, USA. pp: 1-99.

Lazarević J. 2010. Mycorrhization of containerized Pinus nigra seedlings with autochthonous Pisolithus arhizus. Proceedings First Serbian Forestry Congress "Future with Forests", Faculty of Forestry University of Belgrade, Belgrade (Serbia) November 11-13. pp. 348-354.

Manian S, Sreenivasaprasad S, Bending GD, Mills PR. 2001. Genetic diversity and interrelationships among common European Suillus species based on ribosomal DNA sequences. FEMS Microbiology Letters 204, 117-121. PMid:11682189

Marx DH, 1969. The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic onfections. I. Antagonism of ectomycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59, 153-163.

Marx DH, Ruehle JL, Kenney DS, Cordell CE, Riffle J.W, Molina R.J, et al. (1982): Commercial vegetative inoculum of Pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on container-grown tree seedlings. For Sci 28/2, 373-400.

Molina R. 1979. Ectomycorrhizal inoculation of containerized Douglas-fir and lodgepole pine seedlings with six isolates of Pisolithus tinctorius. For Sci 25, 585-590.

Munnoz JA. 2005. Boletus s. l. In: Fungi Europaei Vol 1 Edizioni Candusso. Alassio. 951 pp.

Nieto MP, Carbone SS, 2009. Characterisation of juvenile maritime pine (Pinus pinaster Ait) ectomycorrhiyal fungal community using morphotyping, direct sequencing and fruitbodies sampling. Mycorrhiza 19, 91-98. PMid:18972139

Parlade J, Pera J, Alvarez IF. 1996. Inoculation of conteinerized Pseudotsuga menziesii and Pinus pinaster seedlings with spores of five species of ectomycorrhizal fungi. Mycorrhiza 6, 237-245.

Perić B, Perić O. 2004. The Provisory red list of endangered macromycetes of Montenegro. Mycologia Montenegrina 7, 7-33.

Perić B, Perić O. 2006. Boletus s.l. in Montenegro (Contribution to the study of Macromycetes of Montenegro 51°). Mycologia Montenegrina 9, 35-54.

Pera J, Parlade J. 2005. Inoculacion controlada con hongos ectomicorricicos en la production de planta destinada a repoblaciones forestales:estado actual en Espana. Invest Agrar Sist Recur For 14, 419-433.

Reddy MS, Natarajn K. 1997. Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery, Mycorrhiza 7, 133-138.

Riffle JW, Tinus RW. 1982. Ectomycorrhizal characteristics, growth, and survival of artificially inoculated ponderosa and scots pine in a greenhouse and plantation, For. Sci. 28, 646-660.

Rincon A, Alvarez I, Pera J. 1999. Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain. Mycorrhiza 8, 271-276.

Rincon A, Alvarez I, Pera J.2001. Inoculation of containerized Pinus pinea L. seedlings with seven ectomycorrhizal fungi. Mycorrhiza 11, 265-271.

Rincon A, Ruiz-Diez B, Garcia-Fraile S, Lucas-Garcia JA, Fernandez-Pascual M, Pueyo JJ, de Felipe MR. 2005. Colonization of Pinus halepensis roots by Pseudomonas fluorescens and interaction with the ectomycorrhizal fungus Suillus granulatus. FEMS Microbiology Ecology 51, 303-311. PMid:16329878

Rincon A, de Felipe MR, Fernandez-Pascual M. 2007. Inoculation of Pinus halepensis Mill. with selected ectomycorrhizal fungi improves seedling establishment 2 years after planting in degraded gypsum soil. Mycorrhiza 18, 23-32. PMid:17874144

Ruiz-Diez B, Rincon AM, de Felipe MR, Fernandey-Pascual M. 2006. Molecular characterisation end evaluation of mycorrhiyal capacity of Suillus isolates from Central Spain for the selection of fungal inoculants. Mycorrhiza 16, 465-474. PMid:16896798

Sarasini M. 2005. Gasteromycetes Astromiceti epigei A.M.B. Centro Studi Micologici. Trento. 406 pp. PMCid:1169090

Sanchez F, Honrubia M, Torres P. 2001. Effects of pH, water stress and temperature on in vitro cultures of ectomycorrhizal fungi from Mediterranean forests. Cryptogamie Mycol 22, 243-258.

Stilinović S, 1990. Eco-technological approach in choice of type of planting material for afforestation. Proceedings "Contemporary methods of afforestation, maintenance and protection in conservation and enlargement of Serbian forest area", Arandjelovac (Serbia), March15-19th. pp. 341-354.

Šijačić-Nikolić M, Vilotić D, Milovanović J, Veselinović M, Stanković D. 2010. Application of superabsorbent polymers in the production of Scotch pine (Pinus sylvestris L.) and Austrian pine (Pinus nigra Arn.) seedlings. Fresenius Environmental Bulletin 19, 1180-1185.

Torres P, Honrubia M. 1994. Inoculation of containerized Pinus halepensis (Miller) seedlings with basidiospores of Pisolithus arhizus (Pers) Rauschert, Rhizopogon roseolus (Corda) Th. M. Fr. and Suillus collinitus (Fr.) O Knutze. Ann Sci For 51, 521-528.

Wallander H, Mahmood S, Hagerberg D, Johansson L, Pallon J. 2003. Elemental composition of ectomycorrhizal mycelia identified by PCR-RFLP analysis and grown in contact with apatite or wood ash in forest soil, FEMS Microbiology Ecology 44, 57-65. PMid:19719651

Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm for aligning DNA sequences. J. Comput Biol. 7 (1-2), 203-214. PMid:10890397

How to Cite
Lazarevic, J. S., Keca, N. D., & Martinovic, A. B. (2012). Mycorrhization of containerized Pinus nigra seedlings with Suillus granulatus under open field conditions. Forest Systems, 21(3), 498-507.
Research Articles