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In vitro liquid culture and optimization of Steinernema jeffreyense using shake flasks

  • Murray D. Dunn
  • Prasanna D. Belur
  • Antoinette P. MalanEmail author
Article
  • 13 Downloads

Abstract

Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml−1 after 14 days, in comparison to 75% and 99,875 IJs ml−1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml−1), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.

Keywords

Entomopathogenic nematode In vitro culture Pathogenicity Shake flask Steinernema jeffreyense 

Notes

Acknowledgements

The authors would like to thank D.G. Nel, from the Centre for Statistical Consultation, Stellenbosch University, for assistance in statistical analysis. This work was supported by the South African Table Grape Industry (SATI), the South African/Indian Joint Science and Technology Research Collaboration (IND150923142961), the Technology and Human Resources for Industry Programme (THRIP: Grant Number: TP14062571871).

Funding

This study was funded by the South African Table Grape Industry (SATI), NemaBio (Pty) (Ltd) and National Research Foundation (THRIP-TP14062571871).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human and or/animal participants

The research does not involve human participant or animals.

References

  1. Akhurst RJ (1980) Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis. J Gen Microbiol 121:303–309Google Scholar
  2. Bedding RA (1981) Low cost in vitro mass production of Neoaplectana and Heterorhabditis species (Nematoda) for field control of insect pests. Nematologica 27:109–114CrossRefGoogle Scholar
  3. Bedding RA (1984) Large scale production, storage and transport of the insect-parasitic nematodes Neoaplectana spp. and Heterorhabditis spp. Appl Biol 104:117–120CrossRefGoogle Scholar
  4. Boemare N, Akhurst R, Mourant R (1993) DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. Int J Syst Bacteriol 43:249–255CrossRefGoogle Scholar
  5. Converse V, Miller RW (1999) Development of the one-on-one quality assessment assay for entomopathogenic nematodes. J Invertebr Pathol 74:143–148CrossRefGoogle Scholar
  6. De Waal JY, Malan AP, Addison MF (2011) Efficacy of entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae) against codling moth, Cydia pomonella (Lepidoptera: Tortricidae) in temperate regions. Biocontrol Sci Technol 20:489–502CrossRefGoogle Scholar
  7. Dreyer J, Malan AP, Dicks L (2017) Three novel Xenorhabdus-Steinernema associations and evidence of trains of X. khoisanae switching between different clades. Curr Microbiol 74:938–942CrossRefGoogle Scholar
  8. Ehlers RU (2001) Mass production of entomopathogenic nematodes for plant protection. Appl Microbiol Biotechnol 56:623–633CrossRefGoogle Scholar
  9. Ehlers RU, Hokkanen HMT (1996) Insect biocontrol with non-endemic entomopathogenic nematodes (Steinernema and Heterorhabditis spp.): conclusions and recommendations of a combined OECD and COST workshop on scientific and regulatory policy issues. Biocontrol Sci Technol 6:295–302CrossRefGoogle Scholar
  10. Ehlers RU, Wyss U, Stackebrandt E (1988) 16S rRNA cataloguing and the phylogenetic position of the genus Xenorhabdus. Syst Appl Microbiol 10:121–125CrossRefGoogle Scholar
  11. Ferreira T, Malan AP (2014) Xenorhabdus and Photorhabdus, bacterial symbionts of the entomopathogenic nematodes Steinernema and Heterorhabditis and their in vitro liquid mass culture: a review. Afr Entomol 22:1–14CrossRefGoogle Scholar
  12. Ferreira T, Van Reenen CA, Endo A, Spröer C, Malan AP, Dicks LMT (2013) Description of Xenorhabdus khoisanae sp. nov., a symbiont of the entomopathogenic nematode Steinernema khoisanae. Int J Syst Evol Microbiol 63:3220–3224CrossRefGoogle Scholar
  13. Ferreira T, Addison MF, Malan AP (2014) In vitro liquid culture of a South African isolate of Heterorhabditis zealandica for the control of insect pests. Afr Entomol 22:80–92CrossRefGoogle Scholar
  14. Ferreira T, Addison MF, Malan AP (2016) Development and population dynamics of Steinernema yirgalemense (Rhabditida: Steinernematidae) and growth characteristics of its associated Xenorhabdus indica symbiont in liquid culture. J Helminthol 90:364–371CrossRefGoogle Scholar
  15. Giese H, Azizan A, Kümmel A, Liaon A, Peter CP, Fonseca JA, Hermann R, Duarte TM, Buchs J (2014) Liquid films on shake flask walls explain increasing maximum oxygen transfer capacities with elevating viscosity. Biotechnol Bioeng 111:295–308CrossRefGoogle Scholar
  16. Gil G, Choo H, Gaugler R (2002) Enhancement of entomopathogenic nematode production in in vitro liquid culture of Heterorhabditis bacteriophora by fed-batch culture with glucose supplementation. Appl Microbiol Biotechnol 58:751–755CrossRefGoogle Scholar
  17. Glaser RW, Tenbroeck C (1940) The bacteria-free culture of a nematode parasite. Proc Soc Exp Biol Med 43:512–514CrossRefGoogle Scholar
  18. Golden JW, Riddle DL (1984) The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature. Dev Biol 102:368–378CrossRefPubMedPubMedCentralGoogle Scholar
  19. Grewal PS, Converse V, Georges R (1999) Influence of production and bioassay methods on infectivity of two ambush foragers (Nematoda: Steinernematidae). J Invertebr Pathol 73:40–44CrossRefPubMedPubMedCentralGoogle Scholar
  20. Han RC (1996) The effects of inoculum size on yield of Steinernema carpocapsae and Heterorhabditis bacteriophora in liquid culture. Nematologica 42:546–553CrossRefGoogle Scholar
  21. Hatting J, Malan AP (2017) Status of entomopathogenic nematodes in integrated pest management strategies in South Africa. In: Abd-Elgawad MMM, Askary TH, Coupland J (eds) Biocontrol agents: entomopathogenic and slug parasitic nematodes. CABI, Wallington, pp 409–428CrossRefGoogle Scholar
  22. Hirao A, Ehlers RU (2009) Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida). Appl Microbiol Biotechnol 84:77–85CrossRefPubMedPubMedCentralGoogle Scholar
  23. Hirao A, Ehlers RU (2010) Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida). Appl Microbiol Biotechnol 85:507–515CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hunt DJ, Nguyen KB (2016) Advances in entomopathogenic nematode taxonomy and phylogeny. Nematology monographs and perspectives. Brill, BostonGoogle Scholar
  25. Tachibana M, Uechi T, Suzuki N, Kawasuji, T (1995) Method of culturing nematodes. Int. Patent WO 95/02958Google Scholar
  26. Kaya H, Gaugler R (1993) Entomopathogenic nematodes. Annu Rev Entomol 38:181–206CrossRefGoogle Scholar
  27. Kaya H, Stock S (1997) Techniques in insect nematology. In: Lacey L (ed) Manual of techniques in insect pathology. Academic Press, San Diego, pp 281–324CrossRefGoogle Scholar
  28. Leite LG, Shapiro-Ilan DI, Hazir S, Jackson MA (2016) The effects of nutrient concentration, addition of thickeners, and agitation speed on liquid fermentation of Steinernema feltiae. J Nematol 48:126–133CrossRefPubMedPubMedCentralGoogle Scholar
  29. Leite LG, Shapiro-Ilan D, Hazir S, Jackson MA (2017) Effect of inoculum age and physical parameters on in vitro culture of the entomopathogenic nematode Steinernema feltiae. J Helminthol 2016:1–10Google Scholar
  30. Lunau S, Stoessel S, Schmidt-Peisker AJ, Ehlers RU (1993) Establishment of monoxenic inocula for scaling up in vitro cultures of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis. Nematologica 39:385–399CrossRefGoogle Scholar
  31. Maistrello L, Vaccari G, Sasanelli N (2010) Effect of chestnut tannins on the root-knot nematode Meloidogyne javanica. Helminthologia 47:48–57CrossRefGoogle Scholar
  32. Malan AP, Hatting J (2015) Entomopathogenic nematode exploitation: case studies in laboratory and field applications from South Africa. In: Campos-Herrera R (ed) Sustainability in plant and crop protection: ecology and applied technologies for sustainable plant and crop protection. Springer, Berne, pp 475–506Google Scholar
  33. Malan AP, Knoetze R, Tiedt LR (2016) Steinernema jeffreyense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from South Africa. J Helminthol 90:262–278CrossRefGoogle Scholar
  34. Mascarin GM, Jackson MA, Kobori NN, Behle RW, Dunlap CA, Delalibera JI (2015) Glucose concentration alters dissolved oxygen levels in liquid cultures of Beauveria bassiana and affects formation and bioefficacy of blastospores. Appl Microbiol Biotechnol 99:6653–6665CrossRefGoogle Scholar
  35. Neves JM, Teixeira JA, Simões N, Mota M (2001) Effect of airflow rate on yields of Steinernema carpocapse Az 20 in liquid culture in an external-loop airlift bioreactor. Biotechnol Bioeng 72:369–373CrossRefPubMedPubMedCentralGoogle Scholar
  36. Nguyen KB, Tesfamariam M, Gozel U, Gaugler R, Adams BJ (2004) Steinernema yirgalemense n. sp. (Rhabditida: Steinernematidae) from Ethiopia. Nematology 6:839–856CrossRefGoogle Scholar
  37. Poinar G, Thomas G (1996) Significance of Achromobacter nematophilus (Achromobacteraceae: Eubacteriales) in the development of the nematode. Parasitology 56:385–390CrossRefGoogle Scholar
  38. Ramakuwela T, Hatting J, Laing MD, Hazir S, Thiebaut N (2016) In vitro solid-state production of Steinernema innovationi with cost analysis. Biocontrol Sci Technol 26:792–808CrossRefGoogle Scholar
  39. Shapiro-Ilan DI, Gaugler R (2002) Production technology for entomopathogenic nematodes and their bacterial symbionts. J Ind Microbiol Biotechnol 28:137–146CrossRefPubMedPubMedCentralGoogle Scholar
  40. Shapiro-Ilan DI, Han R, Qui X (2014) Production of entomopathogenic nematodes. In: Morales-Ramos JA, Guadalupe Rojas M, Shapiro-Ilan D (eds) Mass production of beneficial organisms, invertebrates and entomopathogens. Elsevier Inc, Philadelphia, pp 321–355CrossRefGoogle Scholar
  41. Steyn VM, Malan AP, Addison P (2019) Controlling of false codling moth, Thaumatotibia leucotreta (Lepidoptera: Tortricidae), using in vitro-cultured Steinernema jeffreyense and S. yirgalemense. Biol Control (in press).  https://doi.org/10.1016/j.biocontrol.2019.104052 CrossRefGoogle Scholar
  42. Stoll NR (1953) Axenic cultivation of the parasitic nematode, Neoaplectana glaseri, in a fluid medium. J Parasitol 39:422–444CrossRefPubMedPubMedCentralGoogle Scholar
  43. Strauch O, Ehlers RU (1998) Food signal production of Photorhabdus luminescens inducing the recovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture. Appl Microbiol Biotechnol 50:369–374CrossRefGoogle Scholar
  44. Strauch O, Ehlers RU (2000) Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures. Appl Microbiol Biotechnol 54:9–13CrossRefPubMedPubMedCentralGoogle Scholar
  45. Strauch O, Stoessel S, Ehlers RU (1994) Culture conditions define automictic or amphimictic reproduction in entomopathogenic rhabditid nematodes of the genus Heterorhabditis. Fundam Appl Nematol 17:575–582Google Scholar
  46. Surrey MR, Davies RJ (1996) Pilot-scale liquid culture and harvesting of an entomopathogenic nematode, Heterorhabditis bacteriophora. J Invertebr Pathol 67:92–99CrossRefGoogle Scholar
  47. van Zyl C, Malan AP (2015) Cost effective culturing of Galleria mellonella and Tenebrio molitor and entomopathogenic nematode production in various hosts. Afr Entomol 23:361–375CrossRefGoogle Scholar

Copyright information

© International Organization for Biological Control (IOBC) 2019

Authors and Affiliations

  1. 1.Department of Conservation Ecology and Entomology, Faculty of AgriSciencesStellenbosch UniversityStellenboschSouth Africa
  2. 2.Department of Chemical EngineeringNational Institute of Technology Karnataka SurathkalMangaloreIndia

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