Development of a DNA hybridisation method for the identification of Rhizobium and Bradyrhizobium: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in microbiology at Massey University, New Zealand

Abstract

The potential of a DNA hybridisation method, utilising a biotin-labelling system with a streptavidin/alkaline phosphatase detection system (ENZO Biochem), was investigated as an identification method for Rhizobium species and Bradyrhizobium sp. (Lotus) strains using nodule, colony and pure DNA. The method used for extracting DNA from colonies and crushed nodules and binding it to nitrocellulose did not purify the DNA sufficiently to stop non-specific binding occurring between the streptavidin-alkaline phosphatase conjugate and the sample. An alternative method of colony hybridisation that removed more of the cellular constituents was required. Only pure DNA could be used. The method was altered as follows: i) Tris/EDTA buffer was used to terminate the colour reaction in place of allowing the membrane to air dry; ii) 5% milk powder was used in place of 10% bovine serum albumin in the blocking buffer, complex detection buffer and washing buffer used in the detection of hybridised biotin-labelled DNA; iii) 5% dextran sulphate was included in the hybridisation buffer to decrease the minimum hybridisation time from 6hr to 3hr. Investigation of the effect of variable conditions on the intensity of colour produced showed that: i) the incubation of alkaline phosphatase with its substrate at room temperature resulted in fluctuation of the development time as the enzyme reaction rate is sensitive over this range of temperature (approximately 1s0 c to 30°C); ii) increasing the concentration of labelled DNA in the hybridisation buffer increased the intensity of colour produced, the minimum concentration that could be used without lowering the detection limit was 200 ng/ml; iii) continued incubation of alkaline phosphatase with its substrate after colour development in the negative control had begun gave an increased colour intensity in the sample but since this increase was not proportional to that of the negative control the net response (sample minus control) decreased. When genomic probes were hybridised with slot-blots containing homologous DNA the detection limit was between 63 and 125 ng of DNA. Both 32P-labelled and biotin­ labelled genomic Rhizobium leguminosarum biovar trifolii DNA probes were able to distinguish between Rhizobium leguminosarum and other Rhizobium species but not between the biovars of R.leguminosarum. To distinguish between closely related species or strains when using 32P-labelled or biotin-labelled probes a specific DNA sequence was required for use as the probe. Two distinct DNA homology groups have been described in Bradyrhizobium sp. (Lotus). From a gene library of Bradyrhizobium sp. (Lotus) strain cc814S (homology group I) 8 clones were isolated that contained sequences that distinguish a representative of homology group I (strain cc814S) from a representative of homology group II (strain NZP2076). This was achieved by hybridising total genomic DNA from strain cc814S with total genomic DNA from strain NZP2076 and removing the single stranded specific sequences with hydroxylapatite. The specific DNA was used to probe the gene library. Increased selection for group-specific sequences by substituting another homology group I strain (NZP2021) for strain cc814S and subcloning one of the clones isolated gave inconclusive results but indicated that a group specific sequence could be derived in this way.