Skip to main content
Log in

Isolation and characterisation of the entomopathogenic bacterium, Photorhabdus temperata producing a heat stable insecticidal toxin

  • Published:
Journal of Plant Diseases and Protection Aims and scope Submit manuscript

Abstract

Entomopathogenic nematodes symbiotically associated with Photorhabdus spp. invade the larvae of susceptible insects and Photorhabdus bacteria are released into the insect hemolymph, which produce toxins and kill the insect larvae. In this study, five symbiotic bacteria isolates from soil nematodes, Heterorhabditis spp., have been reported. The bacteria were screened for insecticidal toxicity against larvae of Galleria mellonella. All were identified as different strains of Photorhabdus temperata via 16 s rDNA sequencing. The insecticidal activity was highest after 3–4 days of pure culture and the level of toxicity was higher in culture supernatant than in the cell pellet. The heat stability of the insecticidal activity was tested by heat treatments for 30 min at the range of 25°C to 100°C. Out of all isolated strains, P. temperata strains J4 and J5 produced heat-stable toxins. The supernatants extracted from the culture of these strains preserved up to 95% of insecticidal activity after heat treatment for 30 min at 80°C. The insecticidal activities of culture supernatants of all the selected strains were mostly maintained after proteinase K treatment. Based upon these findings, it can be inferred that the insecticidal toxins produced by Photorhabdus spp. were not pure protein substances.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • 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, 303–309.

    Google Scholar 

  • Akhurst RJ, 1982. Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae. J Gen Microbiol 128, 3061–3065.

    CAS  PubMed  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ, 1990. Basic local alignment search tool. J Mol Biol 215, 403–410.

    Article  CAS  PubMed  Google Scholar 

  • Bedding RA & Akhurst RJ, 1975. A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21, 109–110.

    Article  Google Scholar 

  • Blackburn MB, Domek JM, Gelman DB & Hu JS, 2005. The broadly insecticidal Photorhabdus luminescens toxin complex a (Tca): activity against the Colorado potato beetle, Leptinotarsa decemlineata, and sweet potato whitefly, Bemisia tabaci. J Insect Sci 5, 32.

    Article  PubMed  PubMed Central  Google Scholar 

  • Boemare NE & Akhurst RJ, 1988. Biochemical and physiological characterization of colony form variants in Xenorhabdus spp. J Gen Microbiol 134, 751–761.

    CAS  Google Scholar 

  • Boemare NE, Akhurst RJ & Mourant RG, 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, 249–255.

    Article  CAS  Google Scholar 

  • Bowen DJ & Ensign JC, 1998. Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl Environ Microbiol 64, 3029–3035.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.

    Article  CAS  PubMed  Google Scholar 

  • Brillard J, Duchaud E, Boemare N, Kunst F & Givaudan A, 2002. The PhlA hemolysin from the entomopathogenic bacterium Photorhabdus luminescens belongs to the two-partner secretion family of hemolysins. J Bacteriol 184, 3871–3878.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chiche TA, Blackburn M, Carney JR & Ensign JC, 2003. Photobactin: a catechol siderophore produced by Photorhabdus luminescens an entomopathogen mutually associated with Heterorhabditis bacteriophora NC1 nematodes. Appl Environ Microbiol 69, 4706–4713.

    Article  Google Scholar 

  • Cho S & Kim YG, 2004. Hemocyte apoptosis induced by entomopathogenic bacteria, Xenorhabdus and Photorhabdus, in Bombyx mori. J Asia-Pacific Entomol 7, 195–200.

    Article  Google Scholar 

  • Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, Taourit S, Bocs S, Boursaux-Eude C, Chandler M, Charles JF, Dassa E, Derose R, Derzelle S, Freyssinet G, Gaudriault S, Médigue C, Lanois A, Powell K, Siguier P, Vincent R, Wingate V, Zouine M, Glaser P, Boemare N, Danchin A & Kunst F, 2003. The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens. Nat Biotechnol 21, 1307–1313.

    Article  CAS  PubMed  Google Scholar 

  • Forst S, Dowds B, Boemare N & Stackebrandt E, 1997. Xenorhabdus and Photorhabdus spp.: Bugs that kill bugs. Ann Rev Microbiol 51, 47–72.

    Article  CAS  Google Scholar 

  • ffrench-Constant RH & Bowen DJ, 2000. Novel insecticidal toxins from nematode-symbiotic bacteria. Cell Mol Life Sci 57, 828–833.

    Article  CAS  PubMed  Google Scholar 

  • Fischer-Le Saux M VV, Brunel B, Normand P & Boemare NE, 1999. Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov. Int J Syst Bacteriol 49, 1645–1656.

    Google Scholar 

  • Gouge DH & Snyder JL, 2006. Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria. J Invert Pathol 91, 147–157.

    Article  Google Scholar 

  • Guo L, Fatig ROI, Orr GL, Schafer BW, Strickland JA, Sukhapinda K, Woodsworth AT & Petell JK, 1999. Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. J Biol Chem 274, 9836–9842.

    Article  CAS  PubMed  Google Scholar 

  • Hu KJ, Li JX, Li B, Webster JM & Chen GH, 2006. A novel antimicrobial epoxide isolated from larval Galleria mellonella infected by the nematode symbiont, Photorhabdus luminescens (Enterobacteriaceae). Bioorg Med Chem 14, 4677–4681.

    Article  CAS  PubMed  Google Scholar 

  • Hall TA, 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95–98.

    CAS  Google Scholar 

  • Ji D & Kim Y, 2004. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua. J Insect Physiol 50, 489–496.

    Article  CAS  PubMed  Google Scholar 

  • Kaya HK & Gaugler R, 1993. Entomopathogenic nematodes. Annu Rev Entomol 38, 181–206.

    Article  Google Scholar 

  • Kim JY & Park SH, 2002. Characterization of symbiotic bacteria from entomopathogenic nematode. Kor J Biotech Bioeng 17, 276–282.

    Google Scholar 

  • Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  CAS  PubMed  Google Scholar 

  • Lane DJ, 1991. 16S/23S rRNA sequencing. London, John Wiley & Sons Ltd.

    Google Scholar 

  • Li J, Chen G & Webster JM, 1997. Nematophin, a novel antimicrobial substance produced by Xenorhabdus nematophilus (Enterobacteraceae). Can J Microbiol 43, 770–773.

    Article  CAS  PubMed  Google Scholar 

  • Lysenko O & Weiser J, 1974. Bacteria associated with the nematode Neoaplectana carpocapsae and the pathogenicity of this complex for Galleria mellonella larvae. J Invert Pathol 24, 332–336.

    Article  CAS  Google Scholar 

  • Morgan JAW, Sergeant M, Ellis D, Ousley M & Jarrett P, 2001. Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296. Appl Environ Microbiol 67, 2062–2069.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park Y & Kim Y, 2000. Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the entomopathogenic nematode Steinernema carpocapsae. J Insect Physiol 46, 1469–1476.

    Article  CAS  PubMed  Google Scholar 

  • Park Y & Kim Y, 2003. Xenorhabdus nematophilus inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua. Arch Insect Biochem Physiol 54, 134–142.

    Article  CAS  PubMed  Google Scholar 

  • Park Y, Kim Y, Tunaz H & Stanley DW, 2004. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocytic phospholipase A2(PLA2) in tobacco hornworms Manduca sexta. J Invertebr Pathol 86, 65–71.

    Article  CAS  PubMed  Google Scholar 

  • Peel MM, Alfredson DA, Gerrard JG, Davis JM, Robson JM, McDougall RJ, Scullie BL & Akhurst RJ, 1999. Isolation, identification, and molecular characterization of strains of Photorhabdus luminescens from infected humans in Australia. J Clin Microbiol 37, 3647–3653.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Saitou N & Nei M, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.

    CAS  PubMed  Google Scholar 

  • Stanley DW, 2000. Eicosanoids in invertebrate signal transduction systems. New Jersey, Princeton University Press.

    Google Scholar 

  • Weiser J, 1963. Diseases of insects of medical importance in Europe. Bull World Health Org 28, 121–127.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wessel DFU, 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138, 141–143.

    Article  CAS  PubMed  Google Scholar 

  • Yu YS & Park SH, 2000. Optimal culture conditions for the production of insecticidal toxin by Xenorhabdus nematophilus isolated from Steinernema carpocapsae. Kor J Biotech Bioeng 5, 100–105.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jae-Ho Shin.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jang, EK., Ullah, I., Kim, MS. et al. Isolation and characterisation of the entomopathogenic bacterium, Photorhabdus temperata producing a heat stable insecticidal toxin. J Plant Dis Prot 118, 178–184 (2011). https://doi.org/10.1007/BF03356401

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03356401

Key words

Navigation