Morphoanatomical and phylogenetic characterization of the ectomycorrhiza between Laccaria squarrosa with Pinus pseudostrobus and its relevance for reforestation programs

keywords: Ectomycorrhizal symbiosis, edible wild mushrooms, inoculants, Neotropics, pines


Background: Pinus (Coniferophyta) and Laccaria (Basidiomycota) establish ectomycorrhizal symbioses in natural forests. However, their detailed morphoanatomical and phylogenetic characterization have received little attention. Accurate identification of native host symbionts is of paramount relevance to the production of mycorrhized seedlings for successful reforestation programs.

Questions/Objective: We aimed to determine if L. squarrosa is able to establish ectomycorrhizal symbiosis with gymnosperms, thereby widening its host range and highlighting its relevance as a potential inoculant for pine seedlings. Currently, L. squarrosa is only known from its type collection associated with the angiosperm Fagus grandifolia var. mexicana.

Studied species: The fungus L. squarrosa and Pinus pseudostrobus, a tree endemic to Mexico.  

Study site and dates: A Pinus-Quercus forest in Piedra Canteada, Nanacamilpa, Tlaxcala; 2018-2020.

Methods: L. squarrosa basidiomata were identified and ectomycorrhizal roots were collected and morphoanatomically characterized. For molecular identification, DNA was extracted, PCR was performed targeting the nuclear ribosomal internal transcribed spacer region (nucrDNA ITS) for the mycobiont identification and the chloroplastic single-locus trnL region for the phytobiont.

Results: In the phylogenetic analyses, our sequences from basidiomata and ectomycorrhizae clustered together with L.squarrosa with high values of supporting identity. Meanwhile, P. pseudostrobus was molecularly identified as the phytobiont.

Conclusions: This is one of the few worldwide characterizations of Laccaria ectomycorrhiza under field conditions and contributes to the understanding of the ecology, distribution, and economic relevance of the symbiotic association. Our data suggest that L. squarrosa has potential for use as a native inoculant for P. pseudostrobus tree production.

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Author Biographies

Mariana Herrera, Germplasm Bank of Wild Species in Southwestern China, Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan

Postdoctoral Position

Fu-Qiang Yu, Germplasm Bank of Wild Species, Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan

Associate Professor 

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David Ramos-Rendón, Investigador independiente, Xalapa, Veracruz


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Magdalena Martínez-Reyes, Colegio de Postgraduados, Edafología, Campus Montecillo, Texcoco


Faustino Hernández-Santiago, Colegio de Postgraduados, Edafología, Campus Montecillo, Texcoco

Associate Professor

Jesus Perez-Moreno, Colegio de Postgraduados, Edafología, Campus Montecillo, Texcoco

Profesor Investigador Titular

Microbiologia, Edafologia

Campus Montecillo

Colegio de Postgraduados

Morphoanatomical and phylogenetic characterization of the ectomycorrhiza between <em>Laccaria squarrosa</em> with <em>Pinus pseudostrobus</em> and its relevance for reforestation programs


Agerer R. 1991. Characterization of ectomycorrhiza. Methods in Microbiology 23: 25-73. DOI:

Agerer R. 1987. Colour atlas of Ectomycorrhizae, Schwäbisch-Gmünd, Germany: Einhorn-Verlag Eduard Dietenberger. ISBN 3921703778

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 Research 25: 3389-3402. DOI:

Ashkannejhad S, Horton TR. 2005. Ectomycorrhizal ecology under primary succession on coastal sand dunes: interactions involving Pinus contorta, suilloid fungi and deer. New Phytologist 169: 345-354. DOI:

Carrasco-Hernández V, Pérez-Moreno J, Espinosa-Hernández V, Almaraz-Suárez JJ, Quintero-Lizaola R, Torres-Aquino M. 2010. Caracterización de micorrizas establecidas entre dos hongos comestibles silvestres y pinos nativos de México. Revista Mexicana de Ciencias Agrícolas 1: 567-577.

Chakravarty P, Hwang SF. 1991. Effect of an ectomycorrhizal fungus, Laccaria laccata, on Fusarium damping-off on Pinus banksiana seedlings. European Journal of Forest Pathology 21: 97-106. DOI:

Cho HJ, Park MS, Lee H, Oh S-Y, Wilson AW, Mueller GM, Lim YM. 2018. A systematic revision of the ectomycorrhizal genus Laccaria from Korea. Mycologia 110: 948-961. DOI:

Cuvelier JJ. 1991. Characterization of birch ectomycorrhizae (II): Laccaria amethystea and Russula ochroleuca. Belgian Journal of Botany 124: 195-203.

DEEMY. 2021. An information system for characterization and determination of ectomycorrhizae. Ludwig Maximilians, Universität München and Botanische Staatssammlung München. Munich. (accessed June 20, 2021).

Delgado P, Salas-Lizana R, Vázquez-Lobo A, Wegier A, Anzidei M, Alvarez-Buylla ER, Vendramin GG, Piñero D. 2007. Introgressive hybridization in Pinus montezumae Lamb and Pinus pseudostrobus Lindl. (Pinaceae): Morphological and molecular (cpSSR) evidence. International Journal of Plant Sciences 168: 861-875. DOI:

Duponnois R, Garbaye J. 1991. Mycorrhization helper bacteria associated with the Douglas fir- Laccaria laccata symbiosis: effect in aseptic and in glasshouse conditions. Annales des Sciences Forestières 48: 239-251. DOI:

Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792-1797. DOI:

Farfán-Heredia B, Casas A, Rangel-Landa S. 2018. Cultural, economic, and ecological factors influencing management of wild plants and mushrooms interchanged in Purépecha markets of Mexico. Journal of Ethnobiology and Ethnomedicine 14: 68. DOI:

Galindo-Flores G, Castillo-Guevara C, Campos-López A, Lara C. 2015. Caracterización de las ectomicorrizas formadas por Laccaria trichodermophora y Suillus tomentosus en Pinus montezumae. Botanical Sciences 93: 855-863. DOI:

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

Gardes M, Burns TD. 1996. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and below-ground views. Canadian Journal of Botany 74: 1572-1583. DOI:

Gómez-Romero M, Villegas J, Sáenz-Romero C, Lindig-Cisneros R. 2013. Efecto de la micorrización en el establecimiento de Pinus pseudostrobus en cárcavas. Madera y Bosque 19: 51-63. DOI:

INEGI [Instituto Nacional de Estadística y Geografía]. 1997. Anuario estadístico del estado de Tlaxcala. Instituto Nacional de Estadistica y Geografía, Aguascalientes, México.

Ingleby K, Mason PA, Last FT, Fleming LV. 1990. Identification of ectomycorrhizae. London, UK: HMSO Publisher. ISBN: 0117014613

Kalita K, Bezbaroa RN, Kumar R, Pandey S. 2016. Documentation of wild edible mushrooms from Meghalaya, Northeast India. Current Research in Environmental & Applied Mycology 6: 238-247. DOI:

Kang H, Chen X, Kemppainen M, Pardo AG, Veneault?Fourrey C, Kohler A, Martin FM. 2020. The small secreted effector protein MiSSP7.6 of Laccaria bicolor is required for the establishment of ectomycorrhizal symbiosis. Environmental Microbiology 22: 1435-1446. DOI:

Kropp BR, Mueller GM. 1999. Laccaria. In: Cairney JWG, Chambers SM, eds. Ectomycorrhizal Fungi Key Genera in Profile. Berlin, Germany: Springer, pp. 65-88. DOI:

Li Q, Yang L, Xiang D, Wanm Y, Qi W, Huangm W, Zhao G. 2020. The complete mitochondrial genomes of two model ectomycorrhizal fungi (Laccaria): features, intron dynamics and phylogenetic implications. International Journal of Biological Macromolecules 145: 974-984. DOI:

Martin F, Aerts A, Ahren D, Brun A, Danchin EGJ, Duchaussoy F, Gibon J, Kohler A, Lindquist E, Pereda V, Salamov A, Shapiro HJ, Wuyts J, Blaudez D, Buée M, Brokstein P, Canbäck B, Cohen D, Courty PE, Coutinho PM, Delaruelle C, Detter JC, Deveau A, DiFazio S, Duplessis S, Fraissinet-Tachet L, Lucic E, Frey-Klett P, Fourrey C, Feussner I, Gay G, Grimwood J, Hoegger PJ, Jain P, Kilaru S, Labbé J, Lin YC, Legué V, Le Tacon F, Marmeisse R, Melayah D, Montanini B, Muratet M, Nehls U, Niculita-Hirzel H, Oudot-Le Secq MP, Peter M, Quesneville H, Rajashekar B, Reich M, Rouhier N, Schmutz J, Yin T, Chalot M, Henrissat B, Kües U, Lucas S, Van de Peer Y, Podila GK, Polle A, Pukkila PJ, Richardson PM, Rouzé P, Sanders IR, Stajich JE, Tunlid A, Tuskan G, Grigoriev IV. 2008. The genome of Laccaria bicolor provides insights into ectomycorrhizal symbiosis. Nature 452: 88-93. DOI:

McKnight KH, Peterson RT, McKnight VB. 1998. A field guide to mushrooms: North America New York: Houghton Mifflin Harcourt. ISBN: 0-395-42101-2

Miller MA, Pfeiffer WT, Schwart ZT. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE10). New York: Association for Computing Machinery, pp. 1-8. DOI:

Mohan V, Natarajan K, Ingleby K. 1993. Anatomical studies on ectomycorrhizae. II. The ectomycorrhizae produced by Amanita muscaria, Laccaria laccata and Suillus brevipes on Pinus patula. Mycorrhiza 3: 43-49. DOI:

Molina R. 1980. Ectomycorrhizal inoculation of containerized western conifer seedlings. Oregon, Portland: United States Department of Agriculture Forest Service.

Molina R, Chamard J.1983. Use of the ectomycorrhizal fungus Laccaria laccata in forestry. II. Effects of fertilizer forms and levels on ectomycorrhizal development and growth of container-grown Douglar-fir and ponderosa pine seedlings. Canadian Journal of Forest Research 13: 89-95. DOI:

Montoya L, Bandala VM, Baroni T, Horton TR. 2015. A new species of Laccaria in a montane cloud forest from Eastern Mexico. Mycoscience 56: 597-605. DOI:

Mueller GM. 1984. New North American species of Laccaria (Agaricales). Mycotaxon 20: 1010-1116.

Mueller GM. 1992. Systematics of Laccaria (Agaricales) in the continental United States and Canada, with discussions on extralimital taxa and descriptions of extant types. Fieldiana Botany 30: 1-158. DOI:

Nara K. 2015. The role of the ectomycorrhizal networks in seedlings establishment and primary succession. In: Horton TR, ed. Mycorrhizal networks Dordrecht: Springer, pp. 117-201. ISBN 978-94-017-7395-9

Nara K, Nakaya H, Hogetsu T. 2003. Ectomycorrhizal sporocarps succession and production during the early primary succession on Mount Fuji. New Phytologist 158: 193-206. DOI:

Osmundson TW, Cripps CL, Mueller GM. 2005. Morphological and molecular systematics of Rocky Mountain alpine Laccaria. Mycologia 97: 949-972. DOI:

Pérez-Moreno J, Martínez-Reyes M, Yescas-Pérez A, Delgado-Alvarado A, Xoconostle-Cázares, B. 2008. Wild Mushroom Markets in Central Mexico and a Case Study at Ozumba. Economic Botany 62: 425-436. DOI:

Pérez-Moreno J, Martínez-Reyes M, Hernández-Santiago F, Ortiz-López I. 2020. Climate change, biotechnology, and Mexican Neotropical edible ectomycorrhizal mushrooms. In: Pérez-Moreno J, Guerin-Laguette A, Flores-Arzú R, Yu FQ, eds. Mushrooms, humans and nature in a changing world. Switzerland: Springer Nature, pp. 61- 100.

Perry JP. 1991. The pines of Mexico and Central America. Oregon, US: Timber Press. ISBN: 978-1604691108

Popa F, Rexer K H, Donges K, Yang ZL, Kost G. 2014. Three new Laccaria species from Southwest China (Yunnan). Mycological Progress 13: 1105-1117. DOI:

Popa F, Castillo-Jiménez SYC, Weisenborn J, Donges K, Rexer KH, Piepenbring M. 2016. A new Laccaria species from cloud forest of Fortuna, Panama. Mycological Progress 15: 1-8. DOI:

Ramos A, Bandala VM, Montoya L. 2017. A new species and a new record of Laccaria (Fungi, Basidiomycota) found in a relict forest of the endangered Fagus grandifolia var. mexicana. MycoKeys 27: 77-94. DOI:

Reverchon F, Ortega-Larrocea MP, Bonilla-Rosso G, Pérez-Moreno J. 2012. Structure and species composition of ectomycorrhizal fungal communities colonizing seedlings and adult trees of Pinus montezumae in Mexican neotropical forests. FEMS Microbiology Ecology 80: 479-487. DOI:

Rodríguez-Gutiérrez I, Ramírez-Martínez D, Garibay-Orijel R, Jacob-Cervantes V, Pérez-Moreno J, Ortega-Larrocea MP, Arellano-Torres E. 2019. Sympatric species develop more efficient ectomycorrhizae in the Pinus-Laccaria symbiosis. Revista Mexicana de Biodiversidad 90: e902868. DOI:

Santiago-Martínez G, Estrada-Torres A, Varela L, Herrera T. 2003. Crecimiento en siete medios nutritivos y síntesis in vitro de una cepa de Laccaria bicolor. Agrociencia 37: 575-584.

Sheedy E, Van de Wouw AP, Howlett BJ, May TW. 2013. Multigene sequence data reveal morphologically cryptic phylogenetic species within the genus Laccaria in southern Australia. Mycologia 105: 547-563. DOI:

Smith SE, Read DJ. 2008. Mycorrhizal Symbiosis. UK, London: Academic Press. ISBN: 978-0-12-370526-6

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6. Molecular Biology and Evolution 30: 2725-2729. DOI:

Tedersoo L, Jairus T, Horton BM, Abarenkov K, Suvi T, Saar I, Kõljalg U. 2008. Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers. New Phytologist 180: 479-490. DOI:

Trappe JM. 1962. Fungus associates of ectotrophic mycorrhizae. Botanical Review 28: 538-606. DOI:

Valencia AS, Flores-Franco G. 2006. Catálogo de Autoridad Taxonómica del género Quercus, Fagaceae en México. Base de Datos SNIB-CONABIO proyecto CS008. México: Facultad de Ciencias, Universidad Nacional Autónoma de México.

Vincenot L, Nara K, Sthultz C, Labbé J, Dubois MP, Tedersoo L, Martin F, Selosse MA. 2012. Extensive gene flow over Europe and possible speciation over Eurasia in the ectomyc¬orrhizal basidiomycete Laccaria amethystina complex. Molecular Ecology 21: 281-299. DOI:

Werner A, Zadworny M. 2003. In vitro evidence of mycoparasitism of the ectomycorrhizal fungus Laccaria laccata against Mucor hiemalis in the rhizosphere of Pinus sylvestris. Mycorrhiza 13: 41-47. DOI:

White TJ, Bruns TD, Lee SB, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JN, White TJ, eds. PCR protocols: a guide to methods and applications. New York: Academic Press, pp. 315-322. DOI:

Wilson AW, Hosaka K, Perry BA, Mueller GM. 2013. Laccaria (Agaricomycetes, Basidiomycota) from Tibet (Xizang Autonomous Region, China). Mycoscience 54: 406-419. DOI:

Wilson AW, Hosaka K, Mueller GM. 2017. Evolution of ectomycorrhizae as a driver of diversification and biogeographic patterns in the model mycorrhizal mushroom genus Laccaria. New Phytologist 213: 1862-1873.

Wu F, Zhou LW, Yang ZL, Bau T, Li TH, Dai YC. 2019. Resource diversity of Chinese marofungi: edible, medicinal, poisonous species. Fungal Diversity 98: 1-7. DOI:

How to Cite
Herrera, M., Yu, F.-Q., Ramos-Rendón, D., Martínez-Reyes, M., Hernández-Santiago, F., Chater, C. C., & Perez-Moreno, J. (2021). Morphoanatomical and phylogenetic characterization of the ectomycorrhiza between Laccaria squarrosa with Pinus pseudostrobus and its relevance for reforestation programs. Botanical Sciences, 100(2), 397-411.