South African photorhabdus spp. : genetic and antibiotic diversity.
Van Wyngaard, Matthew George Dennis.
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As antibiotic producing symbionts of entomopathogenic nematodes, members of the genus Photorhabdus are candidates for biological control of insects and phytopathogenic microorganisms. The aim of this project was to establish the levels of genetic and antibiotic diversity amongst twenty Photorhabdus sp. isolates acquired from the South African Small Grain Institute, Agricultural Research Council (ARC) culture collection. These isolates represent a subset of Photorhabdus sp. isolates obtained from the Freestate and Western Cape provinces of South Africa. Bacterial strain diversity was investigated using several techniques; which included phenotypic testing, genomic and proteomic fingerprinting approaches. Phenotypic characteristics were assayed using API 20E biochemical test strips in conjunction with several supplementary tests. Genomic fingerprinting involved 16S rRNA gene PCR-RFLP and RAPD-PCR. Phylogenetic relationships between the isolates were determined using partial 16S rRNA gene sequence analysis. MALDI-TOF-MS was used to generate proteomic profiles for isolate identification and mass spectra comparison. Species-level differentiation between isolates and reference strains was achieved using 16S rRNA gene PCR-RFLP and partial 16S rRNA gene sequence analyses. Higher levels of strain differentiation between isolates were detected by RAPD-PCR and with MALDI-TOF-MS. In comparison to the high resolution of isolate diversity achieved by DNA-based analysis methods, the phenotypic characteristics proved to be of limited value. All methods evaluated suggest that the isolates are closely-related strains of P. luminescens. Two isolates were found to be non-Photorhabdus and identified as strains of Xenorhabdus sp. and Pseudomonas sp.. Antimicrobial activity amongst the isolates was screened for using disc-diffusion bioassays against Escherichia coli, Micrococcus luteus, Bacillus subtilis, Rhizoctonia solani and Botrytis cinerea. All isolates showed bioactivity against the Gram positive test organisms with weaker and variable antagonism against both fungal species. Three representative isolates were taken forward for further analysis of bioactive compounds produced. Antibiotic extraction and purification was attempted using several techniques including liquid-liquid extraction of broth supernatant using ethyl acetate, methanol extraction of cell pellet material and methanol extraction of lyophilised broth. Analysis of crude and partially purified antibiotic extracts was performed using C18 Sep-Pak clean-up, TLC, UPLC-ESI-TOF-MS and GC-MS. All antimicrobial extraction methods yielded bioactive fractions. UPLC-ESI-TOF-MS proved the most valuable analytical method with data obtained suggesting that each isolate produced identical compounds. The dominant UPLC peak for all isolates and extraction procedures displayed an ESI-TOF-MS mass spectrum consistent with the antibiotic 3,5-dihydroxy-4-isopropyistilbene. These results show that very limited genetic and phenotypic diversity existed between isolates. All isolates were identified as members of P. luminescens but did not cluster closely with any previously described subspecies on the basis of 16S rRNA partial gene sequence analysis. Likewise antimicrobial compound diversity between the three isolates assessed was found to be low, with the major bioactive compound identified as one previously described from Photorhabdus spp..