The path towards reversing the red: The mycorrhizal fungus Tulasnella amonilioides highly contributes to the Cattleya intermedia (Orchidaceae) translocation success

Delio Endres Júnior; Márcio Hisayuki Sasamori; Genivaldo Alves-Silva; Rosa Mara Borges da Silveira; Annette Droste

doi:10.5327/Z2176-94782698

Authors

Delio Endres Júnior Universidade Feevale - Brazil https://orcid.org/0000-0002-1067-4362
Márcio Hisayuki Sasamori Universidade Feevale - Brazil https://orcid.org/0000-0003-4381-6301
Genivaldo Alves-Silva Universidade Federal do Rio Grande do Sul (UFRGS) - Brazil https://orcid.org/0000-0002-8142-6665
Rosa Mara Borges da Silveira Universidade Federal do Rio Grande do Sul (UFRGS) - Brazil https://orcid.org/0000-0003-1578-5034
Annette Droste Universidade Feevale - Brazil https://orcid.org/0000-0001-8866-1599

DOI:

https://doi.org/10.5327/Z2176-94782698

Keywords:

conservation; mycorrhiza; Orchidaceae; ex situ propagation; forest regeneration.

Abstract

Orchids are a key functional group in forest environments that require obligate symbiosis with mycorrhizal fungi at least during the early stages of development. The present study investigated whether Cattleya intermedia derived from asymbiotic in vitro propagation establishes symbiosis with mycorrhizal fungi after translocation to the wild, and whether this relationship varies along the vertical gradient of phorophytes. Fungi were isolated from plant roots four years after translocation to the trunk and canopy of phorophytes in a subtropical forest in Brazil. The orchid–fungus specificity pattern observed in translocated seedlings and adult plants resembled those reported in the literature for wild seedlings and adult individuals of species in the same environment, revealing the presence of C. intermedia mycorrhizal fungi (CiMF) and non-CiMF endophytes of various morphotypes. Isolated CiMF promoted seed germination and in vitro seedling development, with 14 isolates obtained from orchids translocated to the canopy and eight from those on the trunk. Plants in symbiosis with canopyderived CiMF produced a greater number of leaves compared to those associated with trunk-derived CiMF. Phylogenetic analysis of ITS (Internal Transcribed Spacer) region sequences indicated that CiMF belong to Tulasnellaceae, with 21 isolates identified as Tulasnella amonilioides, a species also found in symbiosis with wild C. intermedia in this environment. The prospecting of compatible mycorrhizal fungi enabled the isolation of new strains, effective for the symbiotic propagation of orchids and contributing to the conservation of C. intermedia.

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References

Airin, A.A.; Arafat, M.I.; Begum, R.A.; Islam, M.R.; Seraj, Z.I., 2023. Plant growth-promoting endophytic fungi of the wild halophytic rice Oryza coarctata. Annals of Microbiology, v. 73, Article 36. https://doi.org/10.1186/s13213-023-01738-3.

Almeida, C.E.A.; Ruppenthal, C.D.; Boniek, D.; Mendes, R.S.M.; Scotti, M.R., 2025. Tripartite Orchid–OMF–AMF symbioses in the Brazilian campo rupestre: implications for plant and soil nitrogen dynamics. Rhizosphere, v. 36, Article 101169. https://doi.org/10.1016/j.rhisph.2025.101169.

Almeida, P.R.M.; Van den Berg, C.; Goes-Neto, A., 2014. Epulorhiza amonilioides sp. nov.: A new anamorphic species of orchid mycorrhiza from Brazil. Neodiversity, v. 7, (1), 1-10. https://doi.org/10.13102/neod.71.1.

Alvares, C.A.; Stape, J.L.; Sentelhas, P.C.; Gonçalves, J.L.M.; Sparovek, G., 2013. Köppen’s climate classification map for Brazil. Meteorologische Zeitschrift, v. 22, (6), 711-728. https://doi.org/10.1127/0941-2948/2013/0507.

Arifin, R.R.; May, T.W.; Linde, C.C., 2021. New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae. Mycologia, v. 113, (1), 212-230. https://doi.org/10.1080/00275514.2020.1813473.

Arifin, R.R.; Reiter, N.H.; May, T.W.; Linde, C.C., 2022. New species of Tulasnella associated with Australian terrestrial orchids in the subtribes Megastylidinae and Thelymitrinae. Mycologia, v. 114, (2), 388-412. https://doi.org/10.1080/00275514.2021.2019547.

Attri, L.K., 2023. Studies on mycorrhizal associations in an orchid. Asian Journal of Biological and Life Sciences, v. 12, (1), 179-186. https://doi.org/10.5530/ajbls.2023.12.24.

Batty, A.L.; Dixon, K.W.; Brundrett, M.; Sivasithamparam, K., 2001. Constraints to symbiotic germination of terrestrial orchid seed in a mediterranean bushland. New Phytologist, v. 152, (3), 511-520. https://doi.org/10.1046/j.0028-646X.2001.00277.x.

Benzing, D.H., 1990. Vascular epiphytes: general biology and related biota. Cambridge University Press, Cambridge, 376 p.

Chauhan, P.; Attri, L.K., 2024. Mycorrhizal associations in oOrchids: A review. Asian Journal of Biological and Life Science, v. 13, (3), 278-286. https://doi.org/10.5530/ajbls.2024.13.36.

Crous, P.W.; Costa, M.M.; Kandemir, H.; Vermaas, M.; Vu, D. et al., 2023. Fungal Planet description sheets: 1550-1613. Persoonia, v. 51, 280-417. https://doi.org/10.3767/persoonia.2023.51.08.

Dentinger, B.T.M.; Margaritescu, S.; Moncalvo, J.-M.M., 2010. Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms. Molecular Ecology Resources, v. 10, (4), 628-633. https://doi.org/10.1111/j.1755-0998.2009.02825.x.

Dixon, K.W., 1987. Raising terrestrial orchids from seed. In: Harris, W.K. (Ed.), Modern orchid growing for pleasure and profit. Orchid Club of South Australia, Adelaide, pp. 47-100.

Eaton, D.A.R., 2020. Toytree: A minimalist tree visualization and manipulation library for Python. Methods in Ecology and Evolution, v. 11, (1), 187-191. https://doi.org/10.1111/2041-210X.13313.

Endres Júnior, D.; Alves-Silva, G.; Sasamori, M.H.; Siveira, R.M.B.; Droste, A., 2023. Successful Tulasnella amonilioides isolation from wild Cattleya intermedia and effectiveness of the mycobiont on in vitro propagation of this threatened Orchidaceae. Journal of Environmental Analysis and Progress, v. 8, (1), 9-29. https://doi.org/10.24221/jeap.8.1.2023.5160.009-029.

Endres Júnior, D.; Alves-Silva, G.; Silveira, R.M.B.; Droste, A., 2025. Data for "Towards reversing the red: Tulasnella amonilioides highly contributes to the Cattleya intermedia translocation success". Harvard Dataverse. https://doi.org/10.7910/DVN/ZBYVEZ.

Endres Júnior, D.; Sasamori, M.H.; Petry, C.T.; Droste, A., 2024. Responses of translocated Cattleya intermedia (Orchidaceae) to environmental key features in a vertical gradient of subtropical forest, Brazil. Revista Brasileira de Geografia Física, v. 17, (1), 668-688. https://doi.org/10.26848/rbgf.v17.1.p668-688.

Endres Júnior, D.; Sasamori, M.H.; Petry, C.T.; Santos, M.S.; Droste, A., 2019. Host tree bark traits and development of reintroduced Cattleya intermedia (Orchidaceae) plants in Southern Brazil. Rodriguésia, v. 70, Article e03092017. https://doi.org/10.1590/2175-7860201970046.

Endres Júnior, D.; Sasamori, M.H.; Silveira, T.; Schmitt, J.L.; Droste, A., 2015. Reintrodução de Cattleya intermedia Graham (Orchidaceae) em borda e interior de um fragmento de Floresta Estacional Semidecidual no sul do Brasil. Revista Brasileira de Biociências, v. 13, (1), 33-40.

Fernández, M.; Kaur, J.; Sharma, J., 2023. Co-occurring epiphytic orchids have specialized mycorrhizal fungal niches that are also linked to ontogeny. Mycorrhiza, v. 33, (1-2), 87-105. https://doi.org/10.1007/s00572-022-01099-w.

Freestone, M.; Reiter, N.; Swarts, N.D.; Linde, C.C., 2024. Temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal communities with ontogenetic and phenological development in Prasophyllum (Orchidaceae). Annals of Botany, v. 134, (6), 933-948. https://doi.org/10.1093/aob/mcae089.

Freitas, E.F.S.; Silva, M.; Cruz, E.S.; Mangaravite, E.; Bocayuva, M.F. et al., 2020. Diversity of mycorrhizal Tulasnella associated with epiphytic and rupicolous orchids from the Brazilian Atlantic Forest, including four new species. Scientific Reports, v. 10, Article 7069. https://doi.org/10.1038/s41598-020-63885-w.

Fujimori, S.; Abe, J.P.; Okane, I.; Yamaoka, Y., 2019. Three new species in the genus Tulasnella isolated from orchid mycorrhiza of Spiranthes sinensis var. amoena (Orchidaceae). Mycoscience, v. 60, (1), 71-81. https://doi.org/10.1016/j.myc.2018.09.003.

Gale, S.W.; Fischer, G.A.; Cribb, P.J.; Fay, M.F., 2018. Orchid conservation: bridging the gap between science and practice. Botanical Journal of the Linnean Society, v. 186, (4), 425-434. https://doi.org/10.1093/botlinnean/boy003.

Gao, X.; Wang, Y.; Deng, D.; Luo, Y.; Shao, S. et al., 2023. Morphogenesis changes in protocorm development during symbiotic seed germination of Dendrobium chrysotoxum (Orchidaceae) with its mycobiont, Tulasnella sp. Horticulturae, v. 9, (5), Article 531. https://doi.org/10.3390/horticulturae9050531.

Góes-Neto, A.; Loguercio-Leite, C.; Guerrero, R.T., 2005. DNA extraction from frozen field-collected and dehydrated herbarium fungal basidiomata: performance of SDS and CTAB based methods. Biotemas, v. 18, (2), 19-32.

Gonçalves, C.N.; Waechter, J.L., 2003. Aspectos florísticos e ecológicos de epífitos vasculares sobre figueiras isoladas no norte da planície costeira do Rio Grande do Sul. Acta Botanica Brasilica, v. 17, (1), 89-100. https://doi.org/10.1590/S0102-33062003000100007.

Herrera, H.; Valadares, R.; Contreras, D.; Bashan, Y.; Arriagada, C., 2017. Mycorrhizal compatibility and symbiotic seed germination of orchids from the Coastal Range and Andes in south centra Chile. Mycorrhiza, v. 27, 175-188. https://doi.org/10.1007/s00572-016-0733-0.

Hsu, R. C.C.; Chen, Y.C.; Lin, C., 2024. The impact of changing climate on an endangered epiphytic orchid (Pleione formosana) in a montane cloud forest and the conservation challenge ahead. Plants, v. 13, (17), Article 2414. https://doi.org/10.3390/plants13172414.

Instituto Brasileiro de Geografia e Estatística (IBGE), 2012. Manual técnico da vegetação brasileira. IBGE, Rio de Janeiro, 272 p.

International Union for Conservation of Nature, Species Survival Commission (IUCN/SSC), 2013. Guidelines for reintroductions and other conservation translocations, version 1.0. IUCN Species Survival Commission, Gland, 57 p. (Accessed October 10, 2024) at:. https://portals.iucn.org/library/efiles/documents/2013-009.pdf.

Jersáková, J.; Malinová, T., 2007. Spatial aspects of seed dispersal and seedling recruitment in orchids. New Phytologist, v. 176, (2), 237-241. https://doi.org/10.1111/j.1469-8137.2007.02223.x.

Johnson, L.J.A.N.; Kane, M.E.; Zettler, L.W.; Mueller, G.M., 2023. Diversity and specificity of orchid mycorrhizal fungi in a leafless epiphytic orchid, Dendrophylax lindenii and the potential role of fungi in shaping its fine-scale distribution. Frontiers in Ecology and Evolution, v. 11, Article 1057940. https://doi.org/10.3389/fevo.2023.1057940.

Kartzinel, T.R.; Trapnell, D.W.; Shefferson, R.P., 2013. Critical importance of large native trees for conservation of a rare Neotropical epiphyte. Journal of Ecology, v. 101, (6), 1429-1438. https://doi.org/10.1111/1365-2745.12145.

Kersten, R.A., 2010. Epífitas vasculares - histórico, participação taxonômica e aspectos relevantes, com ênfase na Mata Atlântica. Hoehnea, v. 37, (1), 9-38. https://doi.org/10.1590/S2236-89062010000100001.

Lee, Y.I.; Yeung, E.C., 2023. The orchid seed coat: a developmental and functional perspective. Botanical Studies, v. 64, (1), Article 27. https://doi.org/10.1186/s40529-023-00400-0.

Li, T.; Wu, S.; Yang, W.; Selosse, M.-A.; Gao, J., 2021. How mycorrhizal associations influence orchid distribution and population dynamics. Frontiers in Plant Science, v. 12, Article 647114. https://doi.org/10.3389/fpls.2021.647114.

Menini Neto, L.; Barros, F.; Vinhos, F.; Furtado, S.G.; Judice, D.M. et al., 2013. Orchidaceae. In: Martinelli, G.; Moraes, M.A. (Eds.), Livro vermelho da Flora do Brasil. Jardim Botânico do Rio de Janeiro, Rio de Janeiro, pp. 749-818. (Accessed September 11, 2024) at:. https://dspace.jbrj.gov.br/jspui/handle/doc/26.

Murashige, T.; Skoog, F.A., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, v. 15, (3), 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Nogueira, P.T.S.; Freitas, E.F.S.; Silva, J.A.R.; Kasuya, M.C.M.; Pereira, O.L., 2024. Efficiency of mycorrhizal fungi for seed germination and protocorms development of commercial Cattleya species (Orchidaceae). Brazilian Journal of Microbiology, v. 56, 589-599. https://doi.org/10.1007/s42770-024-01597-3.

Novo Hamburgo, 1999. Lei Complementar nº 167, de 1 de março de 1999. Aprova o Plano de Manejo do Parque Municipal Henrique Luís Roessler, estabelece as normas de uso e ocupação da área e dá outras providências (Accessed October 15, 2024) at:. https://leismunicipais.com.br/a/rs/n/novo-hamburgo/lei-complementar/1999/17/167/lei-complementar-n-167-1999-aprova-o-plano-de-manejo-do-parque-municipal-henrique-luis-roessler-estabelece-as-normas-de-uso-e-ocupacao-da-area-e-da-outras-providencias.

Oberwinkler, F.; Cruz, D.; Suárez, J.P., 2017. Biogeography and ecology of Tulasnellaceae. In: Tedersoo, L. (Ed.), Ecological Studies. Springer, Cham, v. 230, pp. 237-271. https://doi.org/10.1007/978-3-319-56363-3_12.

Pecoraro, L.; Rasmussen, H. N.; Gomes, S. I. F.; Wang, X.; Merckx, V. S. F. T. et al., 2021. Fungal diversity driven by bark features affects phorophyte preference in epiphytic orchids from southern China. Scientific Reports, v. 11, (1), Article 11287. https://doi.org/10.1038/s41598‑021‑90877‑1.

Petrolli, R.; Vieira, C.A.; Jakalski, M.; Bocayuva, M.F.; Vallé, C. et al., 2021. A fine-scale spatial analysis of fungal communities on tropical tree bark unveils the epiphytic rhizosphere in orchids. New Phytologist, v. 231, (5), 2002-2014. https://doi.org/10.1111/nph.17459.

Phillips, R.D.; Reiter, N.; Peakall, R., 2020. Orchid Conservation: From theory to practice. Annals of Botany, v. 126, (3), 345-362. https://doi.org/10.1093/aob/mcaa093.

Qin, J.; Feng, J.-Q.; Zhang, W.; Zhang, S.-B., 2021. Mycorrhizal fungal partners remain constant during a root lifecycle of pleione bulbocodioides (Orchidaceae). Journal of Fungi, v. 7, (11), 994. https://doi.org/10.3390/jof7110994.

Ramsay, M.M.; Dixon, K.W., 2003. Propagation science, recovery and translocation of terrestrial orchids. In: Dixon, K.W.; Kell, S.P.; Barrett, R.L.; Cribb, P.J. (Eds.), Orchid conservation. Natural History Publications, Kota Kinabalu, pp. 259-288.

Rasmussen, H.N.; Dixon, K.W.; Jersáková, J.; Těšitelová, T., 2015. Germination and seedling establishment in orchids: a complex of requirements. Annals of Botany, v. 116, (3), 391-402. https://doi.org/10.1093/aob/mcv087.

Rasmussen, H.N.; Rasmussen, F.N., 2009. Orchid mycorrhiza: implications of a mycophagous life style. Oikos, v. 118, 334-345. https://doi.org/10.1111/j.1600-0706.2008.17116.x.

Rasmussen, H.N.; Rasmussen, F.N., 2014. Seedling mycorrhiza: A discussion of origin and evolution in Orchidaceae. Botanical Journal of the Linnean Society, v. 175, (3), 313-327. https://doi.org/10.1111/boj.12170.

Reiter, N.; Menz, M.H.M., 2022. Optimizing conservation translocations of threatened Caladenia (Orchidaceae) by identifying adult microsite and germination niche. Australian Journal of Botany, v. 70, (3), 231-247. https://doi.org/10.1071/BT21132.

Reiter, N.; Whitfield, J.; Pollard, G.; Bedggood, W.; Argall, M. et al., 2016. Orchid re-introductions: an evaluation of success and ecological considerations using key comparative studies from Australia. Plant Ecology, v. 217, 81-95. https://doi.org/10.1007/s11258-015-0561-x.

Rio Grande do Sul, 2014. Decreto nº 52.109, de 1 de dezembro de 2014. Declara as espécies da flora nativa ameaçadas de extinção no Estado do Rio Grande do Sul. Lex-Diário Oficial do Rio Grande do Sul, ano LXXII, (233), 2-11.

Sasamori, M.H.; Endres Júnior, D.; Droste, A., 2015. Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Botanica Brasilica, v. 29, (3), 292-298. https://doi.org/10.1590/0102-33062014abb0054.

Selosse, M.A.; Alaux, P.L.; Deloche, L.; Delannoy, E.; Minasiewicz, J. et al., 2025. Mixotrophy in orchids: facts, questions, and perspectives. New Phytologist, v. 246, (5), 1912-1921. https://doi.org/10.1111/nph.70106.

Shao, S.C.; Wang, Q.X.; Beng, K.C.; Zhao, D.K.; Jacquemyn, H., 2020. Fungi isolated from host protocorms accelerate symbiotic seed germination in an endangered orchid species (Dendrobium chrysotoxum) from southern China. Mycorrhiza, v. 30, 529-539. https://doi.org/10.1007/s00572-020-00964-w.

Tikendra, L.; Singh, A. R.; Vendrame, W.; Nongdam, P., 2025. In vitro propagation of endangered Vanda coerulea Griff. ex Lindl.: asymbiotic seed germination, genetic homogeneity assessment, and micro‑morpho‑anatomical analysis for effective conservation. Agronomy, v. 15, (5), Article 1195. https://doi.org/10.3390/agronomy15051195.

Van den Berg, C., 2024. Cattleya in Flora e Funga do Brasil. Jardim Botânico do Rio de Janeiro (Accessed October 15, 2024) at:. https://floradobrasil.jbrj.gov.br/FB11338.

Wu, K.; Zeng, S.; Lin, D.; Silva, J.A.T.; Bu, Z. et al., 2014. In vitro propagation and reintroduction of the endangered Renanthera imschootiana Rolfe. PLoS One, v. 9, (10), Article e110033. https://doi.org/10.1371/journal.pone.0110033.

Yeh, C.M.; Chung, K.; Liang, C.K.; Tsai, W.C., 2019. New insights into the symbiotic relationship between orchids and fungi. Applied Sciences, v. 9, (3), Article 585. https://doi.org/10.3390/app9030585.

Zahn, F.E.; Söll, E.; Chapin, T.K.; Wang, D.; Gomes, S.I.F. et al., 2023. Novel insights into orchid mycorrhiza functioning from stable isotope signatures of fungal pelotons. New Phytologist, v. 239, (4), 1449-1463. https://doi.org/10.1111/nph.18991.

Zhao, D-K.; Mou, Z-M.; Ruan, Y-L., 2024. Orchids acquire fungal carbon for seed germination: pathways and players. Trends in Plant Science, v. 29, (7), 733-741. https://doi.org/10.1016/j.tplants.2024.02.001.

Zhao, D-K.; Selosse, M-A.; Wu, L.; Luo, Y.; Shao, S-C. et al., 2021. Orchid reintroduction based on seed germination-promoting mycorrhizal fungi derived from protocorms or seedlings. Frontiers in Plant Science, v. 12, Article 701152. https://doi.org/10.3389/fpls.2021.701152.