A PCR based method to detect Russula spp. in soil samples and Limodorum abortivum roots in Mediterranean environments

  • Eduardo Larriba Multidisciplinary Institute for Environmental Studies (MIES) ‘‘Ramón Margalef”, Department of Marine Sciences and Applied Biology. University of Alicante. Alicante.
  • Antonio Belda Department of Environment and Earth Sciences, University of Alicante. Alicante.
  • Luis Vicente López-Llorca Multidisciplinary Institute for Environmental Studies (MIES) ‘‘Ramón Margalef”, Department of Marine Sciences and Applied Biology. University of Alicante. Alicante.


Aim of study. Orchidaceae has the largest number of species of any family in the plant kingdom. This family is subject to a high risk of extinction in natural environments, such as natural parks and protected areas. Recent studies have shown the prevalence of many species of orchids to be linked to fungal soil diversity, due to their myco-heterotrophic behaviour. Plant communities determine fungal soil diversity, and both generate optimal conditions for orchid development.

Area of study. The work was carried out in n the two most important natural parks in Alicante (Font Roja and Sierra Mariola), in South-eastern of Spain.

Material and Methods. We designed a molecular tool to monitor the presence of Russula spp. in soil and orchids roots, combined with phytosociological methods.

Main results. Using a PCR-based method, we detected the presence in the soil and Limodorum abortivum orchid roots of the mycorrhizal fungi Russula spp. The species with highest coverage was Quercus rotundifolia in areas where the orchid was present.

Research highlights. We present a useful tool based on PCR to detect the presence of Russula spp. in a natural environment.  These results are consistent with those obtained in different studies that linked the presence of the mycorrhizal fungi Russula spp. in roots of the species Limodorum and the interaction between these fungal species and Quercus ilex trees in Mediterranean forest environments.

Key words: Detection; GIS; Russula spp.; Limodorum abortivum; PCR.


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Belda A, Arques J, Martínez JE, Peiró V, Seva E, 2009. Análisis de la biodiversidad de fauna vertebrada en el Parque Natural de la Sierra de Mariola mediante fototrampeo. Mediter 20: 7-34.

Braun-Blanquet J, 1965. Plant sociology: the study of plant communities. Translated, revised and edited by Fuller GD, Conard HS, Hafner Publishing Co, New York and London. 439 pp.

Fu Q, Ruegger P, Bent E, Chrobak M, Borneman J, 2008. PRISE (PRImerSElector): Software for designing sequence-selective PCR primers. J Microbiol Meth 72: 263-267. http://dx.doi.org/10.1016/j.mimet.2007.12.004

Girlanda M, Selosse MA, Cafasso D, Brilli F, Delfine S, Fabbian R, Ghignone S, Pinelli P, Segreto R, Loreto F, Cozzolino S, Perotto S, 2006. Inefficient photosynthesis in the Mediterranean orchid Limodorum abortivum is mirrored by specific association to ectomycorrhizal Russulaceae. Mol Ecol 15: 491-504. http://dx.doi.org/10.1111/j.1365-294X.2005.02770.x

Grant SA, 1981. Sward components. In: Sward measurement handbook (Hodgson J, Baker RD, Davies A, Laidlaw AS, Leaver JD, eds). British Grassland Society, Hurley, Maidenhead, Berkshire, UK. pp: 71-92.

Hutchings MJ, 2010. The population biology of the early spider orchid Ophrys sphegodes Mill. III. Demography over three decades. J Ecol. 98: 867-878. http://dx.doi.org/10.1111/j.1365-2745.2010.01661.x

Kindlmann P, Willems JH, Whigham DF, 2002. Trends and fluctuations and underlying mechanisms in terrestrial orchid populations. Backhuys Publishers, Leiden, The Netherlands. 254 pp.

Leake JR, Cameron DD, 2012. Untangling above- and belowground mycorrhizal fungal networks in tropical orchids. Mol Ecol 20: 4921-4924. http://dx.doi.org/10.1111/j.1365-294X.2012.05718.x

Nicol F, Brzosko E, Till-Bottraud I, 2005. Population viability analysis of Cypripedium calceolus in a protected area: longevity, stability and persistence. J Ecol 93: 716-726. http://dx.doi.org/10.1111/j.1365-2745.2005.01010.x

Richard F, Roy M, Shahin O, Sthultz C, Duchemin M, Joffre R, Selosse MA, 2011. Ectomycorrhizal communities in a Mediterranean forest ecosystem dominated by Quercus ilex: Seasonal dynamics and response to drought in the surface organic horizon. Ann For Sci 68: 57–68. http://dx.doi.org/10.1007/s13595-010-0007-5

Selosse MA, Martos F, Perry BA, Padamsee M, Roy M, Pailler T, 2010. Saprotrophic fungal mycorrhizal symbionts in achlorophyllous orchids: finding treasures among the 'molecular scraps'? Plant Signal Behav 5: 349-353. http://dx.doi.org/10.4161/psb.5.4.10791

Selosse MA, Rousset F, 2011. Evolution. The plant-fungal marketplace. Science 333: 828-829. http://dx.doi.org/10.1126/science.1210722

Sun X, Guo LD, 2012. Endophytic fungal diversity: review of traditional and molecular techniques. Mycology 3: 65-76.

Tĕšitelová T, Tĕšitel J, Jersáková J, RÍhová G, Selosse MA, 2012. Symbiotic germination capability of four Epipactis species (Orchidaceae) is broader than expected from adult ecology. Am J Bot 99: 1020-32. http://dx.doi.org/10.3732/ajb.1100503

White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols, a guide to methods and applications. (Innis MA, Gelfand DH, Sninsky JJ, White TJ, Eds), Academic Press, San Diego, USA. pp: 315-322. http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1

How to Cite
LarribaE., BeldaA., & López-LlorcaL. V. (2015). A PCR based method to detect Russula spp. in soil samples and Limodorum abortivum roots in Mediterranean environments. Forest Systems, 24(1), e019. https://doi.org/10.5424/fs/2015241-06249
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