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Culture age of Tulasnella affects symbiotic germination of the critically endangered Wyong sun orchid Thelymitra adorata (Orchidaceae)

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Abstract

Orchids (Orchidaceae) are dependent on mycorrhizal fungi for germination and to a varying extent as adult plants. We isolated fungi from wild plants of the critically endangered terrestrial orchid Thelymitra adorata and identified them using a multi-region barcoding approach as two undescribed Tulasnella species, one in each of phylogenetic group II and III (OTU1) of the Tulasnellaceae. Using symbiotic propagation methods, we investigated the role of Tulasnella identity (species and isolate) and age post isolation, on the fungus’s ability and efficacy in germinating T. adorata. The group II isolate did not support germination. Seed germination experiments were conducted using either (i) three different isolates of OTU1, (ii) 4- and 12-week-old fungal cultures (post isolation) of a single isolate of OTU1, and (iii) T. subasymmetrica which is widespread and known to associate with other species of Thelymitra. Culture age and fungal species significantly (P < 0.05) affected the time to germination and percentage of seed germination, with greater and faster germination with 4-week-old cultures. Tulasnella subasymmetrica was able to germinate T. adorata to leaf stage, although at slightly lower germination percentages than OTU1. The ability of T. adorata to germinate with T. subasymmetrica may allow for translocation sites to be considered outside of its native range. Our findings on the age of Tulasnella culture affecting germination may have applications for improving the symbiotic germination success of other orchids. Furthermore, storage of Tulasnella may need to take account of the culture age post-isolation, with storage at − 80 °C as soon as possible recommended, post isolation.

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Acknowledgements

We thank Antony Von Chrismar and Lucinda Ransom (NSW Department of Planning and Environment), Brian Towle (Ecoplanning Consultancy), Gavin Phillips and staff at the Australian PlantBank (Botanic Gardens of Sydney) for help with acquiring land access, collection, processing, storage and postage of Thelymitra adorata seed used in this study. We acknowledge the use of services and facilities of AGRF. We thank Dr Caroline Cristofolini for research assistance and Maja Zweck for technical assistance. We thank the volunteers at the RBGV Assoc Prof Charles Young, Eve Almond, Lynda Entwisle, Jenny Raven, Bryan Lawrence, Andrew Cosby and Neil Freestone for assistance in maintaining ex situ collections.

Funding

NSW Government’s Saving our Species (SoS) Program, NSW Department of Planning and Environment funding to NR and ARC Linkage Grant LP200200264 awarded to NR.

Author information

Authors and Affiliations

  1. Royal Botanic Gardens Victoria, Science Division, Corner of Ballarto Road and Botanic Drive, Cranbourne, VIC, 3977, Australia

    Noushka Reiter & Richard Dimon

  2. Ecology and Evolution, Research School of Biology, ANU College of Science, RN Robertson Building, 46 Sullivans Creek Road, The Australian National University, Canberra, ACT 2600, Australia

    Noushka Reiter, Arild Arifin & Celeste Linde

  3. Research Centre for Ecosystem Resilience, Botanic Gardens of Sydney, Mrs Macquaries Rd, Sydney, NSW 2000, Australia

    Richard Dimon

  4. Queensland Alliance of Agriculture and Food Innovation, University of Queensland, 306 Cermody Rd, St Lucia, QLD, Australia

    Richard Dimon

  5. Department of Plant Pathology, Washington State University Tree Fruit Research and Extension Center, Wenatchee, WA, 98801, USA

    Arild Arifin

Authors
  1. Noushka Reiter
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  2. Richard Dimon
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  3. Arild Arifin
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  4. Celeste Linde
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Contributions

N.R wrote the manuscript and took part in collecting and analysing the data. R.D took part in collecting and analysing the data. A.A and C.L undertook phylogenetic analysis. All authors reviewed the manuscript.

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Correspondence to Noushka Reiter.

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The authors declare no competing interests.

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Supplementary Information

Below is the link to the electronic supplementary material.

572_2023_1131_MOESM1_ESM.docx

Supplementary Table 1: Pairwise minimum and maximum ITS sequence divergences within and between Tulasnella (group III) dataset. (DOCX 14 KB)

Supplementary Table 2: Germination of Thelymitra adorata using one-week-old cultures. (DOCX 14 KB)

572_2023_1131_MOESM3_ESM.docx

Supplementary Table 3: Composition of terrestrial orchid soil mix for growing Thelymitra adorata to adult plants. (DOCX 14 KB)

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Reiter, N., Dimon, R., Arifin, A. et al. Culture age of Tulasnella affects symbiotic germination of the critically endangered Wyong sun orchid Thelymitra adorata (Orchidaceae). Mycorrhiza 33, 409–424 (2023). https://doi.org/10.1007/s00572-023-01131-7

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