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Genetic stability of ectomycorrhizal fungi is not affected by cryopreservation at −130 °C or cold storage with repeated sub-cultivations over a period of 2 years

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Abstract

Cryopreservation is considered the most reliable method for storage of filamentous fungi including ectomycorrhizal (ECM) fungi. A number of studies, however, have reported genetic changes in fungus cultures following cryopreservation. In the present study, the genetic stability of six ECM fungus isolates was analyzed using amplified fragment length polymorphism (AFLP). The isolates were preserved for 2 years either by cryopreservation (at −130 °C) or by storage at 4 °C with regular sub-cultivation. A third preservation treatment consisting of isolates maintained on Petri dishes at 22–23 °C for 2 years (i.e., without any sub-cultivation) was included and used as a control. The differences observed in AFLP patterns between the three preservation methods remained within the range of the total error generated by the AFLP procedure (6.85%). Therefore, cryopreservation at −130 °C and cold storage with regular sub-cultivation did not affect the genetic stability of the ECM fungus isolates, and both methods can be used for the routine storage of ECM fungus isolates over a period of 2 years.

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References

  • Amalfi M (2016) Fomitiporia (Basidiomycota) revisited. Species concept, phylogenetic structure and biogeographical patterns. PhD thesis, Université catholique de Louvain, Louvain-la-Neuve, Belgium.

  • Bonin A, Bellemain E, Eidesen PB, Pompanon F, Brochmann C, Taberlet P (2004) How to track and assess genotyping errors in population genetics studies. Mol Ecol 13:3261–3273

    Article  CAS  PubMed  Google Scholar 

  • Broughton R, Buddie A, Smith D, Ryan M (2012) The effect of cryopreservation on genomic stability in strains of the fungus Trichoderma. CryoLetters 33:299–226

    CAS  PubMed  Google Scholar 

  • Brundrett MC, Bougher N, Dell B, Grove T, Malajczuk N (1996) Working with mycorrhizas in forestry and agriculture. Australian Centre for International Agricultural Research, Canberra

    Google Scholar 

  • Butt TM, Wang C, Shah FA, Hall R (2006) Degeneration of entomogenous fungi. In: Eilenberg J, Hokkanen H (eds) An ecological and societal approach to biological control, progress in biological control vol 2. Springer, Dordrecht, pp 213–226

    Chapter  Google Scholar 

  • Colpaert JV, Vandenkoornhuyse P, Adriaensen K, Vangronsveld J (2000) Genetic variation and heavy metal tolerance in the ectomycorrhizal basidiomycete Suillus luteus. New Phytol 147:367–379

    Article  CAS  Google Scholar 

  • Crahay C, Declerck S, Colpaert JV, Pigeon M, Munaut F (2013a) Viability of ectomycorrhizal fungi following cryopreservation. Fungal Biol 117:103–111

    Article  CAS  PubMed  Google Scholar 

  • Crahay C, Wevers J, Munaut F, Colpaert JV, Declerck S (2013b) Cryopreservation of ectomycorrhizal fungi has minor effects on root colonization of Pinus sylvestris plantlets and their subsequent nutrient uptake capacity. Mycorrhiza 23:463–471

    Article  CAS  PubMed  Google Scholar 

  • Crawford LA, Koscinski D, Keyghobadi N (2012) A call for more transparent reporting of error rates: the quality of AFLP data in ecological and evolutionary research. Mol Ecol 21:5911–5917

    Article  PubMed  Google Scholar 

  • Duponnois R, Bâ A, Mousain D, Galiana A, Baudoin E, Dreyfus B, Prin Y (2011) Biotechnological processes used in controlled ectomycorrhizal practices. In: Rai M, Varma A (eds) Diversity and biotechnology of Ectomycorrhizae, soil biology vol 25. Springer, Berlin Heidelberg, pp 143–155

    Google Scholar 

  • Hajek AE, Humber R, Griggs M (1990) Decline in virulence of Entomophaga maimaiga (Zygomycetes: Entomophthorales) with repeated in vitro subculture. J Invertebr Pathol 56:91–97

    Article  Google Scholar 

  • Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. CryoLetters 25:3–22

    PubMed  Google Scholar 

  • Homolka L (2014) Preservation of live cultures of basidiomycetes—recent methods. Fungal Biol 118:107–125

    Article  PubMed  Google Scholar 

  • Homolka L, Lisá L, Eichlerová I, Valášková V, Baldrian P (2010) Effect of long-term preservation of basidiomycetes on perlite in liquid nitrogen on their growth, morphological, enzymatic and genetic characteristics. Fungal Biol 114:929–935

    Article  CAS  PubMed  Google Scholar 

  • Hubálek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46:205–229

    Article  PubMed  Google Scholar 

  • Johnston JW, Benson EE, Harding K (2009) Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm. Plant Physiol Biochem 47:123–131

    Article  CAS  PubMed  Google Scholar 

  • Kaity A, Ashmore SE, Drew RA, Dulloo ME (2008) Assessment of genetic and epigenetic changes following cryopreservation in papaya. Plant Cell Rep 27:1529–1539

    Article  CAS  PubMed  Google Scholar 

  • Karwa A, Varma A, Rai M (2011) Edible ectomycorrhizal fungi: cultivation, conservation and challenges. In: Rai M, Varma A (eds) Diversity and biotechnology of Ectomycorrhizae, soil biology vol 25. Springer, Berlin Heidelberg, pp 459–453

    Google Scholar 

  • Keirle MR, Avis PG, Hemmes DE, Mueller GM (2014) Testing the “one-log-one-genet” hypothesis: methodological challenges of population sampling for the Hawaiian wood-decay fungus Rhodocollybia laulaha. Mycologia 106:896–903

    Article  PubMed  Google Scholar 

  • Kuek C (1994) Issues concerning the production and use of inocula of ectomycorrhizal fungi in increasing the economic productivity of plantations. In: Robson A, Abbott L, Malajczuk N (eds) Management of mycorrhizas in agriculture, horticulture and forestry. Kluwer Academic Publishers, Dordrecht, pp 221–230

    Google Scholar 

  • Kumar S, Styanarayana T (2002) Isolation of ectomycorrhizal fungi: methods and techniques. In: Mukerji KG, Manoharachary C, Chamola BP (eds.), Techniques in Mycorrhizal Studies. Kluwer Academic Publishers, Dordrecht, pp 143–166

  • Laiho O (1970) Paxillus involutus as a mycorrhizal symbiont of forest trees. Acta Forestalia Fennica 106:1–73

  • Lalaymia I, Declerck S, Cranenbrouck S (2013) Cryopreservation of in vitro-produced Rhizophagus species has minor effects on their morphology, physiology, and genetic stability. Mycorrhiza 23:675–682

    Article  PubMed  Google Scholar 

  • Lalaymia I, Cranenbrouck S, Declerck S (2014) Maintenance and preservation of ectomycorrhizal and arbuscular mycorrhizal fungi. Mycorrhiza 24:323–337

    Article  PubMed  Google Scholar 

  • Liu J (2005) N-containing compounds of Macromycetes. Chem Rev 105:2723–2744

    Article  CAS  PubMed  Google Scholar 

  • Martin F, Aerts A, Ahrén D et al (2008) The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452:88–92

    Article  CAS  PubMed  Google Scholar 

  • Marx DH (1981) Variability in ectomycorrhizal development and growth among isolates of Pisolithus tinctorius as affected by source, age, and reisolation. Can J For Res 11:168–169

    Article  Google Scholar 

  • Marx DH, Daniel WJ (1976) Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage. Can J Microbiol 22:338–341

    Article  CAS  PubMed  Google Scholar 

  • Mikulášková E, Fér T, Kučabová V (2012) The effect of different DNA isolation protocols and AFLP fingerprinting optimizations on error rate estimates in the bryophyte Campylopus introflexus. Lindbergia 35:7–17

    Google Scholar 

  • Paterson RRM, Lima N (2013) Biochemical mutagens affect the preservation of fungi and biodiversity estimations. Appl Microbiol Biotechnol 97:77–85

    Article  CAS  PubMed  Google Scholar 

  • Peredo EL, Arroyo-García R, Reed BM, Revilla MÁ (2008) Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.) Cryobiology 57:234–241

    Article  CAS  PubMed  Google Scholar 

  • Peredo EL, Arroyo-García R, Reed BM, Revilla MÁ (2009) Genetic stability of in vitro conserved germplasm of Humulus lupulus L. Agric Food Sci 18:144–151

    Article  CAS  Google Scholar 

  • Prakash O, Nimonkar Y, Shouche YS (2013) Practice and prospects of microbial preservation. FEMS Microbiol Lett 339:1–9

    Article  CAS  PubMed  Google Scholar 

  • Repáč I (2011) Ectomycorrhizal inoculum and inoculation techniques. In: Ray M, Varma A (eds.) Diversity and biotechnology of ectomycorrhizae, Soil biology vol 25. Springer, Berlin Heidelberg, pp. 43–63

  • Richter DL (2008) Revival of saprotrophic and mycorrhizal basidiomycete cultures after 20 years in cold storage in sterile water. Can J Microbiol 54:595–599

    Article  CAS  PubMed  Google Scholar 

  • Richter DL, Dixon TG, Smith JK (2016) Revival of saprotrophic and mycorrhizal basidiomycete cultures after 30 years in cold storage in sterile water. Can J Microbiol 62:932–937

    Article  CAS  PubMed  Google Scholar 

  • Ryan MJ, Jeffries P, Bridge PD, Smith D (2001) Developing cryopreservation protocols to secure fungal gene function. CryoLettersetters 22:115–124

    CAS  Google Scholar 

  • Ryan MJ, Kasulyte-Creasey D, Kermode A, San SP, Buddie AG (2014) Controlled rate cooling of fungi using a stirling cycle freezer. CryoLetters 35:63–69

    CAS  PubMed  Google Scholar 

  • Siddiqui ZA, Kataoka R (2011). Mycorrhizal inoculants: progress in inoculant production technology. In: Ahmad I, Ahmad F, Pitchel J (eds.), Microbes and Microbial Technology: Agricultural and Environmental Applications. Springer, New York, pp. 489–506

  • Singh SK, Upadhyay RC, Kamal S, Tiwari M (2004) Mushroom cryopreservation and its effect on survival, yield and genetic stability. CryoLetters 25:23–32

    CAS  PubMed  Google Scholar 

  • Smith D (1998) The use of cryopreservation in the ex-situ conservation of fungi. CryoLetters 19:79–90

    Google Scholar 

  • Smith D, Onions A (1994) Preservation and maintenance of living fungi, 2nd edn. CAB International, Wallingford

    Google Scholar 

  • Smith SE, Read D (2008) Mycorrhizal Symbiosis, 3rd edn. Academic Press, London

    Google Scholar 

  • Smith D, Ryan MJ (2012) Implementing best practices and validation of cryopreservation techniques for microorganisms. Sci World J 2012:805659. doi:10.1100/2012/805659

    Article  Google Scholar 

  • Smith JE, McKay D, Molina R (1994) Survival of mycorrhizal fungal isolates stored in sterile water at two temperatures and retrieved on solid and liquid nutrient media. Can J Microbiol 40:736–742

    Article  Google Scholar 

  • Sterner O, Bergman R, Kesler E, Magnusson G, Nilsson L, Wickberg B, Zimerson E, Zetterberg G (1982) Mutagens in larger fungi. I. Forty-eight species screened for mutagenic activity in the salmonella/microsome assay. Mutat Res 101:269–281

    Article  CAS  PubMed  Google Scholar 

  • Thomson BD, Malajczuk N, Grove TS (1993) Improving the colonization capacity and effectiveness of ectomycorrhizal fungal cultures by association with a host plant and re-isolation. Mycol Res 97:839–844

    Article  Google Scholar 

  • Voyron S, Roussel S, Munaut F, Varese GC, Ginepro M, Declerck S, Filipello Marchisio V (2009) Vitality and genetic fidelity of white-rot fungi mycelia following different methods of preservation. Mycol Res 113:1027–1038

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project). The authors wish to thank Mario Amalfi for providing the CTAB DNA extraction protocol slightly modified in this study.

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Authors and Affiliations

  1. Earth and Life Institute, Applied Microbiology, Mycology, Université catholique de Louvain, Croix du Sud 2, bte L7.05.06, 1348, Louvain-la-Neuve, Belgium

    Charlotte Crahay & Stéphane Declerck

  2. Earth and Life Institute, Applied Microbiology, Phytopathology, Université catholique de Louvain, Croix du Sud 2, bte L7.05.03, 1348, Louvain-la-Neuve, Belgium

    Françoise Munaut

  3. Center for Environmental Sciences, Environmental Biology Group, Universiteit Hasselt, Agoralaan, Gebouw D, 3590, Diepenbeek, Belgium

    Jan V. Colpaert

  4. Earth and Life Institute, Applied Microbiology, Mycology, Université catholique de Louvain, Mycothèque de l’Université catholique de Louvain (MUCL), Croix du Sud 2, box L7.05.06, 1348, Louvain-la-Neuve, Belgium

    Stéphanie Huret

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  1. Charlotte Crahay
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  2. Françoise Munaut
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  5. Stéphane Declerck
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Correspondence to Stéphane Declerck.

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Crahay, C., Munaut, F., Colpaert, J.V. et al. Genetic stability of ectomycorrhizal fungi is not affected by cryopreservation at −130 °C or cold storage with repeated sub-cultivations over a period of 2 years. Mycorrhiza 27, 595–601 (2017). https://doi.org/10.1007/s00572-017-0770-3

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