Main Article Content
Abstract
Conservation of orchids can be possible with effective seed germination and seedling growth methods. In this context, ex vitro symbiotic seed germination and seedling growth of orchid seeds may be convenient and advantageous. In this study, both the diversity of the root endophytic fungi in Serapias vomeracea (Burm.f.) Briq. and the ex vitro effects of these fungi on seed germination, seedling development and tuber formation were revealed. The fungi were isolated monthly for two years from S. vomeracea roots and the isolates were identified based on morphological characters and internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) sequences. All of the Rhizoctonia-like isolates that joined the mycorrhizal association were closely related to Tulasnella calospora (thirty isolates). Non-Rhizoctonia isolates are closely related to Fusarium tricinctum (two isolates), Aspergillus spelaeus (one isolate) and Talaromyces pinophilus (Pezizales) (one isolate). The viability rate of the seeds was 90.32%. The seed packs were placed in soils containing fungus and the germination process was followed. All isolates associated with Tulasnella calospora promoted germination and seedling development. Isolate Svl 21 (Tulasnella sp.) was found to have the highest germination rate (98%) but isolate Svl 4 developed seedlings with advanced leaves (stage 4 (S4): seedlings with advanced leaves and/or rooted, 13.67%). All seedlings at S4 were transferred to the natural environment; the first tubers were observed seven months after. In this study, for the first time, a tuberous European orchid, S. vomeracea developed from seed to adult plant in a natural environment.
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Copyright (c) 2022 Yasemin Ozdener Kompe
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References
- Acemi, A., Özen, F., 2019: Optimization of in vitro asymbiotic seed germination protocol for Serapias vomeracea. The EuroBiotech Journal 3, 143–151.
- Aciego Pietri, J. C., Brookes, P.C., 2008: Relationship between soil pH and microbial properties in a UK arable soil. Soil Biology and Biochemistry 40(7), 1856–1861.
- Aewsakul, N., Maneesorn, D., Serivichyaswat, P., Taluengiit, A.T., Nontachaiyapoom, S., 2013: Ex vitro symbiotic seed germination of Spathoglottis plicata Blume on common orchid cultivation substrates. Scientia Horticulturae 160, 238–242.
- Bailarote, B.C., Lievens, B., Jacquemyn, H., 2012: Does mycorrhizal specificity affect orchid decline and rarity? American Journal of Botany 99, 1655–1665.
- Bayman, P., Otero, J. T., 2006: Microbial Endophytes of Orchid Roots. In: Schulz B.J.E., Boyle, C.J.C., Sieber, T.N. (eds.), Microbial root endophytes, 153–177. Springer-Verlag, Berlin Heidelberg.
- Bandoni, R.J., 1979: Safranin O as a rapid nuclear stain for fungi. Mycologia 11, 873–874.
- Bowman, G.M., Hutka J., 2002: Particle size analysis. In: McKenzie, N.J., Cresswell, H.P., Coughlan, K.J. (eds.), Soil physical measurement and interpretation for land evaluation, 224–239. CSIRO Publishing: Collingwood.
- Clements, M.A., Muir, H., Cribb, P.J., 1986: A Preliminary report on the symbiotic Germination of European terrestrial orchids. Kew Bulletin 41, 437–445.
- Ding, R., Chen, X.H., Zhang, L.J., Yu, X.D., Qu, B., Duan, R., Xu, Y.F., 2014: Identity and specificity of Rhizoctonia-like fungi from different populations of Liparis japonica (Orchidaceae) in Northeast China. Plos One 9, e105573.
- Fracchia, S., Silvani, V., Flachsland, E., Terada, G., Sede, S., 2014: Symbiotic seed germination and protocorm development of Aa achalensis Schltr., a terrestrial orchid endemic from Argentina. Mycorrhiza 24, 35–43.
- Ghorbani, A., Gravendee, l.B., Naghibi, F, de Boer, H., 2014: Wild orchid tuber collection in Iran: a wake-up call for conservation. Biodiversity and Conservation 23, 2749–2760.
- Girlanda, M., Segreto, R., Cafasso, D., Liebel, H.T., Rodda, M., Ercole, E., Cozzolino, S., Gebauer, G., Perotto, S., 2011: Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations. American Journal of Botany 98, 1148–1163.
- Guevara-Suarez, M., Sutton, D.A., Cano-Lira, J.F, Garcia, D., Martin-Vicente, A., Wiederhold, N., Guarro, J., Gene, J., 2016: Identification and antifungal susceptibility of penicillium- like fungi from clinical samples in the United States. Journal of Clinical Microbiology 54, 2155–2161.
- Hall, T.A., 1999: BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/. NT Nucleic Acids Symposium Series 41, 95–98.
- Jacquemyn, H., Deja, A., De Hert, K., Bailatore, B.C., Lievens, B., 2012: Variation in mycorrhizal associations with Tulasnelloid fungi among populations of five Dactylorhiza species. PLos ONE 7, e42212.
- Kreziou, A., De Boer, H., Gravendell, B., 2016: Harvesting of salep orchids in North-western Greece continues to threaten natural populations. Oryx 50, 393–396.
- Kömpe, Y.O., Mutlu, V.A., Ozkoc, I., 2020: The viability of Anacamptis laxiflora (Orchidaceae) seeds and the symbiotic germination. Plant Biosystems 154, 800–805.
- Kömpe, Y.O., Mutlu, V.A., 2017: Mycorrhizal diversity in some species of Dactylorhiza genus (Orchidaceae), Biological Diversity and Conservation 10, 55–64.
- McCormick, M.K., Parker, K.L., Szlavecz, K., Whigham, D.F., 2013: Native and exotic earthworms affect orchid seed loss. AoB Plants 5, plt018.
- Mehlich, M., 1984: Mehlich 3 soil test extractant: a modification of the Mehlich 2 extractant. Communications in Soils Science and Plant Analysis 15,1409–1416.
- Moore, R.T., 1988: The genera of Rhizoctonia-like fungi: Asorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. nov., Moniliopsis and Rhizoctonia. Mycotaxon 29, 91–99.
- Murphy, J., Ridly, J.P., 1962: A modified single solution method for determination of phosphate innatural water. Analytica Chimica Acta 27, 31–36.
- Mutlu, V.A., Kömpe, Y.O., 2020: Mycorrhizal fungi of some orchis species of Turkey. Pakistan Journal of Botany 52, 687–695.
- Nelson, D.W., Sommers, L.E., 1982: Total carbon, organic carbon, and organic matter. In: Page, A.L. (ed.), Methods of soil analysis, Part 2, Chemical and microbiological properties, 539–579. Soil Science Society of America Inc, Madison, Wisconsin, USA.
- Ozkoç, I., Dalcı, M., 1993: Germination of the seeds Serapias vomeracea (Burm fil.) Briq. subsp. laxiflora (Soo’) Gölz et. Reinhard (Orchidaceae) through asymbiotic culture techniques. Turkish Journal of Biology 17, 5–11.
- Pascual, C.B., Toda, T., Raymondo, A.D., Hyakumachi, M., 2000: Characterization by conventional techniques and PCR of Rhizoctonia solani isolates causing banded leaf sheath blight in maize. Plant Pathology 49, 108–118.
- Paul, S., Kumaria, S., Tandon, P., 2012: An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast. AoB Plants plr 032, 1–6.
- Quay, L., McComb, J.A., Dixon, K.W., 1995: Methods for ex vitro germination of Australian terrestrial orchids. Hortscience 30, 1445–1446.
- Rasmussen, H.N., 2002: Recent developments in the study of orchid mycorrhiza. Plant and Soil 244, 149–163.
- Rhoades, J.D., 1996: Salinity: Electrical conductivity and total dissolved solids. In: Sparks, D.L. (ed.), Methods of soil analysis, Part 3, Chemical methods, 417–435. Soil Science Society of America Inc, Madison, Wisconsin.
- Rozas, J., Sanchez-Del Barrio, J.C., Messeguer, X., Rozas, R., 2017: DnaSP 6: DNA sequence polymorphism analysis of large datasets. Molecular Biology and Evolution 34, 3299–3302.
- Salazar, O., Schneider, J.H.M., Julian, M.C., Keije, J., Rubio, V., 1999: Phylogenetic subgrouping of Rhizoctonia solani AG2 isolates based on ribosomal ITS sequences. Mycologia 91, 459–467.
- Sezik, E., 2002: Turkish orchids and salep. Acta Pharmaceutica Turcica 44, 151–157.
- Stark, C., Babık, W., Durka, W., 2009: Fungi from the roots of the common terrestrial orchid Gymnadenia conopsea. Mycological Research 113, 952–959.
- Tamura, K., Stecher, G., Peterson, D., Filipsk., A., Kumar, S., 2013: MEGA6: Molecular evolutionary genetics analysis, version 6.0. Molecular Biology and Evolution 30, 2725–2729.
- Tĕšitelová, T., Jersáková, J., Roy, M., Kubátová, B., Tĕšitel, J., Urfus, T., Trávniček, P., Suda, J., 2013: Ploidy-specific symbiotic interactions: divergence of mycorrhizal fungi between cytotypes of the Gymnadenia conopsea group (Orchidaceae). New Phytologist 199, 1022–1033.
- Tondello, A., Vendramin, E., Vilani, M., Baldan, B., Squartini, A., 2012: Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall. Fungal Biology 116, 543–549.
- Van Waes, J.M., Deberg, P.C., 1986: In vitro germination of some Western European orchids. Physiologia Plantarum 67, 253–261.
- Vujanovic, V., Arnaud, S.M., Barabe, D., Thibeault, G., 2000: Viability testing of orchid seed and the promotion of colouration and germination. Annuals of Botany 86, 79–86.
- White, T. J., Bruns, T. D., Lee, S., Taylor, J. W., 1990: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Gelfand, D.H., Sninsky, J., White, T.J. (eds.), PCR Protocols: a Guide to Methods and Applications, 315–322. AcademicPress, San Diego.
References
Acemi, A., Özen, F., 2019: Optimization of in vitro asymbiotic seed germination protocol for Serapias vomeracea. The EuroBiotech Journal 3, 143–151.
Aciego Pietri, J. C., Brookes, P.C., 2008: Relationship between soil pH and microbial properties in a UK arable soil. Soil Biology and Biochemistry 40(7), 1856–1861.
Aewsakul, N., Maneesorn, D., Serivichyaswat, P., Taluengiit, A.T., Nontachaiyapoom, S., 2013: Ex vitro symbiotic seed germination of Spathoglottis plicata Blume on common orchid cultivation substrates. Scientia Horticulturae 160, 238–242.
Bailarote, B.C., Lievens, B., Jacquemyn, H., 2012: Does mycorrhizal specificity affect orchid decline and rarity? American Journal of Botany 99, 1655–1665.
Bayman, P., Otero, J. T., 2006: Microbial Endophytes of Orchid Roots. In: Schulz B.J.E., Boyle, C.J.C., Sieber, T.N. (eds.), Microbial root endophytes, 153–177. Springer-Verlag, Berlin Heidelberg.
Bandoni, R.J., 1979: Safranin O as a rapid nuclear stain for fungi. Mycologia 11, 873–874.
Bowman, G.M., Hutka J., 2002: Particle size analysis. In: McKenzie, N.J., Cresswell, H.P., Coughlan, K.J. (eds.), Soil physical measurement and interpretation for land evaluation, 224–239. CSIRO Publishing: Collingwood.
Clements, M.A., Muir, H., Cribb, P.J., 1986: A Preliminary report on the symbiotic Germination of European terrestrial orchids. Kew Bulletin 41, 437–445.
Ding, R., Chen, X.H., Zhang, L.J., Yu, X.D., Qu, B., Duan, R., Xu, Y.F., 2014: Identity and specificity of Rhizoctonia-like fungi from different populations of Liparis japonica (Orchidaceae) in Northeast China. Plos One 9, e105573.
Fracchia, S., Silvani, V., Flachsland, E., Terada, G., Sede, S., 2014: Symbiotic seed germination and protocorm development of Aa achalensis Schltr., a terrestrial orchid endemic from Argentina. Mycorrhiza 24, 35–43.
Ghorbani, A., Gravendee, l.B., Naghibi, F, de Boer, H., 2014: Wild orchid tuber collection in Iran: a wake-up call for conservation. Biodiversity and Conservation 23, 2749–2760.
Girlanda, M., Segreto, R., Cafasso, D., Liebel, H.T., Rodda, M., Ercole, E., Cozzolino, S., Gebauer, G., Perotto, S., 2011: Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations. American Journal of Botany 98, 1148–1163.
Guevara-Suarez, M., Sutton, D.A., Cano-Lira, J.F, Garcia, D., Martin-Vicente, A., Wiederhold, N., Guarro, J., Gene, J., 2016: Identification and antifungal susceptibility of penicillium- like fungi from clinical samples in the United States. Journal of Clinical Microbiology 54, 2155–2161.
Hall, T.A., 1999: BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/. NT Nucleic Acids Symposium Series 41, 95–98.
Jacquemyn, H., Deja, A., De Hert, K., Bailatore, B.C., Lievens, B., 2012: Variation in mycorrhizal associations with Tulasnelloid fungi among populations of five Dactylorhiza species. PLos ONE 7, e42212.
Kreziou, A., De Boer, H., Gravendell, B., 2016: Harvesting of salep orchids in North-western Greece continues to threaten natural populations. Oryx 50, 393–396.
Kömpe, Y.O., Mutlu, V.A., Ozkoc, I., 2020: The viability of Anacamptis laxiflora (Orchidaceae) seeds and the symbiotic germination. Plant Biosystems 154, 800–805.
Kömpe, Y.O., Mutlu, V.A., 2017: Mycorrhizal diversity in some species of Dactylorhiza genus (Orchidaceae), Biological Diversity and Conservation 10, 55–64.
McCormick, M.K., Parker, K.L., Szlavecz, K., Whigham, D.F., 2013: Native and exotic earthworms affect orchid seed loss. AoB Plants 5, plt018.
Mehlich, M., 1984: Mehlich 3 soil test extractant: a modification of the Mehlich 2 extractant. Communications in Soils Science and Plant Analysis 15,1409–1416.
Moore, R.T., 1988: The genera of Rhizoctonia-like fungi: Asorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. nov., Moniliopsis and Rhizoctonia. Mycotaxon 29, 91–99.
Murphy, J., Ridly, J.P., 1962: A modified single solution method for determination of phosphate innatural water. Analytica Chimica Acta 27, 31–36.
Mutlu, V.A., Kömpe, Y.O., 2020: Mycorrhizal fungi of some orchis species of Turkey. Pakistan Journal of Botany 52, 687–695.
Nelson, D.W., Sommers, L.E., 1982: Total carbon, organic carbon, and organic matter. In: Page, A.L. (ed.), Methods of soil analysis, Part 2, Chemical and microbiological properties, 539–579. Soil Science Society of America Inc, Madison, Wisconsin, USA.
Ozkoç, I., Dalcı, M., 1993: Germination of the seeds Serapias vomeracea (Burm fil.) Briq. subsp. laxiflora (Soo’) Gölz et. Reinhard (Orchidaceae) through asymbiotic culture techniques. Turkish Journal of Biology 17, 5–11.
Pascual, C.B., Toda, T., Raymondo, A.D., Hyakumachi, M., 2000: Characterization by conventional techniques and PCR of Rhizoctonia solani isolates causing banded leaf sheath blight in maize. Plant Pathology 49, 108–118.
Paul, S., Kumaria, S., Tandon, P., 2012: An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast. AoB Plants plr 032, 1–6.
Quay, L., McComb, J.A., Dixon, K.W., 1995: Methods for ex vitro germination of Australian terrestrial orchids. Hortscience 30, 1445–1446.
Rasmussen, H.N., 2002: Recent developments in the study of orchid mycorrhiza. Plant and Soil 244, 149–163.
Rhoades, J.D., 1996: Salinity: Electrical conductivity and total dissolved solids. In: Sparks, D.L. (ed.), Methods of soil analysis, Part 3, Chemical methods, 417–435. Soil Science Society of America Inc, Madison, Wisconsin.
Rozas, J., Sanchez-Del Barrio, J.C., Messeguer, X., Rozas, R., 2017: DnaSP 6: DNA sequence polymorphism analysis of large datasets. Molecular Biology and Evolution 34, 3299–3302.
Salazar, O., Schneider, J.H.M., Julian, M.C., Keije, J., Rubio, V., 1999: Phylogenetic subgrouping of Rhizoctonia solani AG2 isolates based on ribosomal ITS sequences. Mycologia 91, 459–467.
Sezik, E., 2002: Turkish orchids and salep. Acta Pharmaceutica Turcica 44, 151–157.
Stark, C., Babık, W., Durka, W., 2009: Fungi from the roots of the common terrestrial orchid Gymnadenia conopsea. Mycological Research 113, 952–959.
Tamura, K., Stecher, G., Peterson, D., Filipsk., A., Kumar, S., 2013: MEGA6: Molecular evolutionary genetics analysis, version 6.0. Molecular Biology and Evolution 30, 2725–2729.
Tĕšitelová, T., Jersáková, J., Roy, M., Kubátová, B., Tĕšitel, J., Urfus, T., Trávniček, P., Suda, J., 2013: Ploidy-specific symbiotic interactions: divergence of mycorrhizal fungi between cytotypes of the Gymnadenia conopsea group (Orchidaceae). New Phytologist 199, 1022–1033.
Tondello, A., Vendramin, E., Vilani, M., Baldan, B., Squartini, A., 2012: Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall. Fungal Biology 116, 543–549.
Van Waes, J.M., Deberg, P.C., 1986: In vitro germination of some Western European orchids. Physiologia Plantarum 67, 253–261.
Vujanovic, V., Arnaud, S.M., Barabe, D., Thibeault, G., 2000: Viability testing of orchid seed and the promotion of colouration and germination. Annuals of Botany 86, 79–86.
White, T. J., Bruns, T. D., Lee, S., Taylor, J. W., 1990: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Gelfand, D.H., Sninsky, J., White, T.J. (eds.), PCR Protocols: a Guide to Methods and Applications, 315–322. AcademicPress, San Diego.