Selective isolation, characterisation and identification of Streptosporangia

PhD ThesisLarge numbers of actinomycetes were isolated from composite soil samples using procedures considered to be selective for the isolation of streptosporangia and related sporoactinomycetes from environmental samples. The highest streptosporangial counts were obtained when suspensions of air-dried soil were heated in the presence of yeast extract for 20 minutes at 4000 then plated onto humic acid vitamins agar supplemented with actidione (50mg/1) and nalidixic acid (30mg/1) and incubated for 4 weeks at 30°C. The highest count, 7.94 ± 1.19 x 104 colony forming units per gram dry soil, were obtained from samples of Ginseng field soil. Representative strains had morphological and chemical properties consistent with their classification in the genus Streptosporangium. Representativeis olatesa nd marker strainso f the genusS treptosporangium were examined for diagnostic features recommended for computer-assisted identification of unknown streptosporangia. Stringent criteria were adopted for positive identifications of both known and unknown strains following a critical evaluation of identification scores obtained for the marker cultures. Sixty-five of the seventy marker strains and twelve of the hundred and thirty six unknown streptosporangia were identified to known streptosporangial taxa. A further nineteen of the isolates were assigned to known taxa using less stringent cut-off points for positive identifications. 5S ribosomalR NA sequencews ere determinedf or nine representativeos f the genus Streptosporangium including centrotype strains of two taxa, clusters 1 and 2, circumscribed in a recent numerical phenetic survey and two Ginseng field soil isolates. The primary and secondary structure of the resultant sequences were of the type characteristic of Gram-positive bacteria with DNA rich in guanine and cytosine. It was evident from the phylogenetic tree that the genus Streptosporangium is heterogeneous as the type strains of Streptosporangium albidum and Streptosporangium viridogriseum subspecies viridogriseum were sharply separated from the remaining test strains; a result in good agreement with current trends in streptosporangial systematics. Pilot experiments were designed to determine the potential of Curie point pyrolysis mass spectrometry and rapid fluorogenic enzyme tests in the classification and identification of streptosporangia. The pyrolysis mass spectral data supported the taxonomic integrity of clusters 1 and 2 and showed that Streptosporangium viridogriseum subspecies viridogriseum had little in common with bona fide members of the genus Streptosporangium. Pyrolysis data also supported the results of the computer-assisted identification exercise as ten isolates assigned to cluster 1 using stringent cut-off criteria were found to be closely related to representatives of cluster 1. There was evidence that some of the conjugated substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone have potential as taxonomic markers for the classification of streptosporangia and related actinomycetes