Characterisation of symbionts of entomopathogenic nematodes by universally primed-PCR (UP-PCR) and UP-PCR product cross-hybridisation.
This work introduces and demonstrates the applicability of universally primed-PCR (UP-PCR) for differentiating bacterial symbionts of entomopathogenic nematodes. Furthermore, typing by UP-PCR product cross-hybridisation was successfully introduced to cluster the bacterial strains. The work was initiated by isolating 10 isolates of Photorhabdus temperata (S172) from the nematode host Heterorhabditis sp. (DK172) and 12 isolates of Xenorhabdus bovienii (S1) from the nematode Steinernema feltiae (DK1). The isolates were compared by UP-PCR using different primers. The two bacterial species (P. temperata and X. bovienii) could be distinguished on the basis of the banding pattern whereas isolates isolated from the same nematode host had identical banding patterns. Three isolates obtained from DK172 and DK1, respectively, were then selected along with a number of reference strains (Hb, HP88, C1, K122, HSH2, HL81, T228, 61, AN6, Q58) and further characterised by UP-PCR product cross-hybridisation. The Xenorhabdus strains (Q58, AN6, 61, T228, S1) represented three species and these species were separated by the hybridisation technique. The Photorhabdus strains (Hb, HP88, C1, K122, HSH2, HL81, S172) represented two species and were also separated according to this in the cross-hybridisation. Within each species of Photorhabdus, two subgroups were formed as a result of intensity of the hybridisation signals. This grouping was in agreement with previous studies in other laboratories.
Nielsen
O
O
Department of Ecology, Zoology Section, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 3, 1871, Frederiksberg C, Denmark. on@kvl.dk
Lübeck
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PS
D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005838:Genotype; D009348:Nematoda / Q000382:microbiology*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020637:Photorhabdus / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D013559:Symbiosis; D020593:Xenorhabdus / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial