Effects of temperature, pH and carbon and nitrogen sources on growth of in vitro cultures of ectomycorrhizal isolates from Pinus heldreichii forest

  • Jelena Lazarević University of Montenegro, Biotechnical Faculty, Podgorica.
  • Dragana Stojičić University of Niš, Faculty of Sciences and Mathematics, Niš.
  • Nenad Keča University of Belgrade, Faculty of Forestry, Belgrade.
Keywords: Lactarius, Montenegro, physiology, RFLP analysis, Russula, Suillus, Tricholoma


Aim of study: This study aims to provide basic information about physiological characteristics of isolates of Lactarius deliciosus (L.) Gray, Russula sanguinaria (Schumach.) Rauschert, Suillus collinitus (Fr) Kuntze, Suillus granulatus (L.) Rousell, Tricholoma batchii Gulden and Tricholoma imbricatum (Fr.) Kumm.

Area of study: The isolates are obtained from Pinus heldreichii H. Christ forest in the south-eastern part of Montenegro.

Material and methods: The isolates were molecularly characterised by internal transcribed spacer (ITS) sequencing and restriction fragment length polymorphism (RFLP) analysis. The effects of different temperatures (20, 22, 25°C), pHs (4, 4.5, 5.2, 5.8, 6.5, 7.5), and carbon (glucose, sucrose, dextrin, arabinose, xylose and starch) and nitrogen (NH4+, NO3- and protein) sources on their growth were examined under laboratory conditions.

Main results: The studied factors established significant differences in the development of isolates. Isolates of R. sanguinaria, L. deliciosus and both Suillus, were characterised by faster growth at 22°C, while Tricholoma isolates grew faster at 25°C. S. granulatus, S. collinitus and T. imbticatum isolates grew well at lower pH values (4 - 5.2), while L. deliciosus, R. sanguinaria and T. bachii exhibited faster growth at pHs between 5.8 and 6.5. The examined isolates were able to utilize various carbohydrates as carbon sources. The biggest mycelial growth was characterised for sucrose, then glucose, dextrin, arabinose, starch and xylose. They grew on all examined nitrogen sources, while the biggest mycelia growth was achieved on ammonium, followed by nitrate and protein. Those characteristics varied amongst the species.

Research highlights: Information about physiological characteristics of Tricholoma, Lactarius, Russula, as well as Suillus, are sparse. Hence, the data obtained in this study could contribute to the understanding of their function in ecosystems.



Download data is not yet available.

Author Biographies

Jelena Lazarević, University of Montenegro, Biotechnical Faculty, Podgorica.
Center for forestry
Dragana Stojičić, University of Niš, Faculty of Sciences and Mathematics, Niš.
Department of Biology and Ecology
Nenad Keča, University of Belgrade, Faculty of Forestry, Belgrade.
Department of forest protection



Abuzinadah RA, Read DJ, 1986. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants, Vol.I, Utilisation of peptides and proteins by ectomycorrhizal fungi. New Phytol 103: 481-493. http://dx.doi.org/10.1111/j.1469-8137.1986.tb02886.x

Allen EB, Allen MF, Helm DJ, Trappe JM, Molina M, Rincon E, 1995. Patterns and regulation of mycorrhizal plant and fungal communities. Plant Soil 170: 47-62. http://dx.doi.org/10.1007/BF02183054

Aguilera LM, Griffiths RP, Caldwell BA, 1993. Nitrogen in ectomycorrhizal mat and non-mat soils of different age Douglas-fir forests. Soil Biol Biochem 25:1015-1019. http://dx.doi.org/10.1016/0038-0717(93)90148-5

Azul AM, Nunes J, Ferreira I, Coelho AS, Verissino P, Trovao J, Campos A, Castro P, Freitas H, 2014. Valuing nature ectomycorrhizal fungi as a Mediterranean forestry component for sustainable and inovative solutions. Botany 92 (2): 161-171. http://dx.doi.org/10.1139/cjb-2013-0170

Basso MT, 1999. Lactarius Pers. in Fungi Europ., Alassio, Italy. 847 pp.

Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL, 2005. GenBank. Nucleic Acids Res 33: D34‐D38. http://dx.doi.org/10.1093/nar/gki063

Bowen GD, 1973. Mineral nutrition of ectomicorrhizae. In: Ectomycorrhiza, their ecology and physiology; Marx GC, Kozlowsky TT (eds). pp: 151-205.Academic Press, London, Great Britain.

Buscot F, Munch JC, Charcosset JY, Gardes M, Nehls U, Hampp R, 2000. Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems. FEMS Microbiol Rev 24: 601- 614. http://dx.doi.org/10.1111/j.1574-6976.2000.tb00561.x

Cairney JWG, 1999. Intraspecific physiological variation: implications for understanding functional diversity in ectomycorrhizal fungi. Mycorrhiza 9: 125-135. http://dx.doi.org/10.1007/s005720050297

Dahlberg A, Jonsson L, Nylund J-E, 1997. Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in oldgrowth Norway spruce forest in south Sweden. Can J Bot 75: 1323-1335. http://dx.doi.org/10.1139/b97-844

Dames JF, Starker CJ, Sholes MC, 1999. Ecological and anatomical characterisation of some Pinus patula ectomycorrhizas from Mpumalanga, South Africa. Mycorrhiza 9: 9-24. http://dx.doi.org/10.1007/s005720050258

Daza A, Manjon JL, Camacho M, Romero de la Osa L, Aguilar A, Santmaria C, 2006. Effect of carbon and nitrogen sources, pH and temperature on in vitro cultures of several isolates of Amanita caesarea (Scop.:Fr.) Pers. Mycorrhiza 16: 133-136. http://dx.doi.org/10.1007/s00572-005-0025-6

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 5: 197-204. http://dx.doi.org/10.1051/forest:2002006

Finlay RD, Forestegard A, Sonnerfeldt AM, 1992. Utilisation of organic nitrogen sources by ectomycorrhizal fungi in pure culture and in symbiosis with P. contorta Dough.ex Loud. New Phytol 120: 105-115. http://dx.doi.org/10.1111/j.1469-8137.1992.tb01063.x

Fuštić B., Đuretić G, 2000. Soils of Montenegro; University of Montenegro, Biotechnical institute, Podgorica, Montenegro 626 pp. (in Serbian)

Galli R, Mazza R, Consiglio G.1996. Le russule, Atlante pratco-monografico per la determinazione delle russule. Edinatura, Milano, Italy. 480 pp.

Gardes M, Bruns TD, 1993. ITS primers with enhanced specificity for basidiomycetes –application to the identification of mycorrhizae and rusts. Molec Ecol 2: 113-118. http://dx.doi.org/10.1111/j.1365-294X.1993.tb00005.x

Hampp R, Schaeffer C, 1999. Mycorrhiza carbohydrate and energy metabolism. In:Mycorrhizal structure, function,molecular biology and biotechnology; Varma A, Hock B (eds) . pp. 273-303. Springer-Verlag, Berlin, Germany. http://dx.doi.org/10.1007/978-3-662-03779-9_12

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.

Harley JL, Smith SE, 1983. Mycorrhizal symbiosis. Academic Press, London and New York. pp. 483.

Hydro-Meteorologic service of Montenegro 1995. Hydrometeorological base for spatial plan of Republic of Montenegro (report), Montenegro, Hydro-Meteorological service of Montenegro, Sector for meteorology, Podgorica. (in Serbian)

Hutchison LJ, 1990. Studies on the systematics of ectomycorrhizal fungi in axenic culture.II. The enzimatic degradation of selected carbon and nitrogen compounds. Can J Bot 68: 1522-1530. http://dx.doi.org/10.1139/b90-194

Janković M.M., 1960 . Betrachtungen uber gegenseitigen Bezichungen der Molika (Pinus peuce) und Panzerkiefer (Pinus heldreichii) sowie auch Uber ihre okologishen Eigenschaften, besonders in Bezug auf ihre geologishe Grundlage . Bull Inst Jardin Botaniq'Uni de Beograd 1(2):141-181 (in Serbian)

Keller G, 1996. Utilization of inorganic and organic nitrogen sources by high-subalpine ectomycorrhizal fungi of Pinus cembra in pure culture. Mycol Res 100 (8):989-998. http://dx.doi.org/10.1016/S0953-7562(96)80053-0

Kõljalg U, Larsson K-H, Kessy Abarenkov K, Nilsson HR, Ian J, Alexander IJ, Eberhardt U, Erland S, Høiland K, Kjøller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo L, Vrålstad T, Ursing MB, 2005. UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi, New Phytol 166:1063–1068. http://dx.doi.org/10.1111/j.1469-8137.2005.01376.x

Koide RT, Shumway DL, Xu B, Sharda JN. 2007. On temporal partitioning of a community of ectomycorrhizal fungi. New Phytol 174: 420–429. http://dx.doi.org/10.1111/j.1469-8137.2007.02000.x

Laperyrie F, Chilvers GA, Bhem CA, 1987. Oxalic acid synthesis by mycorrhoizal fungus Paxillus involvutus (Batch.ex. Fr.). New Phytol 106: 39-146.

Lazarević J, Perić B, Perić O, 2011. Ectomycorrhizal fungi in Montenegro- diversity and distribution, Mycol Monten XIV: 85-115.

Lilleskov EA, Hobbie EA, Fahey TJ, 2002. Ectomycorhizal fungal taxa differing in response to nitrogen deposition also differ in pure culture organic nitrogen use and natural abundance of nitrogen isotopes. New Phytol 154: 219-231 http://dx.doi.org/10.1046/j.1469-8137.2002.00367.x

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, Bryan WC, Davey CB, 1970. Influence of temperature on aseptic synthesis of ectomycorrhizae by Telephora terestis and Pisolithus tinctorius on loblolly pine. For Sci 16: 424-431.

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. http://dx.doi.org/10.1007/s00572-008-0207-0

Nygren C, 2008. Functional diversity in nutrient acquisition by ectomycorrhizal fungi. Doctoral thresis, Faculty of Natural Resources and Agricaltural Sciences, Swedish Univrsity of Agricultural Sciences, Uppsala, Sweden.

Read DJ, Perez-Moreno J, 2003. Mycorrhiza and nutrient cycling in ecosystem-a journey toward relewance? New Phytol 157: 475-492. http://dx.doi.org/10.1046/j.1469-8137.2003.00704.x

Rincon A, Alvarez I, Pera J, 1999. Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain. Mycorrhiza 8: 271-276. http://dx.doi.org/10.1007/s005720050245

Riva A, 2003. Trcholoma (Fr.) Staude. Eduzioni Candusso, Alasio, Italy. 620 pp.

Roux P, 2006. Mille et un champignons, Sainte-Sigolene, France.1224 pp.

Sanchez F, Honrubia M, Torres P, 2001. Effects of pH, water stress and temperature on in vitro growth of ectomycorrhizal fungi from Mediterranean forests. Cryptog Mycol 22(4): 243-258. http://dx.doi.org/10.1016/S0181-1584(01)01076-4

Smith S, Read DJ, 1997. Mycorrhizal symbiosys. Academic press, London UK, pp: 605.

Tedersoo L, May TW, Smith ME, 2010. Ectomycorrhizal lifestile in fungi: global diversity, distribution and evolution of phylogenetic lineages. Mycorrhiza 20: 217-263. http://dx.doi.org/10.1007/s00572-009-0274-x

Trojanowski J, Haider K, Huttermann A, 1984. Decomposition of 14C labelled lignin, holocellulose and lignocellulose by mycorrhizal fungi. Arch Mycrobiol 139: 202-206. http://dx.doi.org/10.1007/BF00402000

Zhang Z, Schwartz S, Wagner L, Miller W, 2000. A greedy algorithm for aligning DNA sequences. J Comput Biol 7 (1-2): 203-214. http://dx.doi.org/10.1089/10665270050081478

How to Cite
Lazarević, J., Stojičić, D., & Keča, N. (2016). Effects of temperature, pH and carbon and nitrogen sources on growth of in vitro cultures of ectomycorrhizal isolates from Pinus heldreichii forest. Forest Systems, 25(1), e048. https://doi.org/10.5424/fs/2016251-07036
Research Articles