Date of Award

1985

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

Recombinant DNA technology was used to produce a probe for detection of Clavibacter xyli subsp. xyli, the sugarcane ratoon stunting disease agent. C. xyli, subsp. xyli chromosomal DNA was digested with the restriction enzyme Pst 1, inserted into the plasmid pUC 19, and transformed into Escherichia coli JM 83. Recombinant clones containing six different sizes of C. xyli subsp. xyli DNA sequences were obtained. Plasmid K3-5, with an insert of 4.9Kb, and C. xyli subsp. xyli chromosomal DNA, were radiolabelled by nick translation using dCTP or dATP (alpha-('32)P). The labelled DNAs were used to detect homologous DNA sequences present in DNA extracts from C. xyli subsp. xyli cells, C. xyli subsp. xyli cells and sap from C. xyli subsp. xyli infected sugarcane by dot blot hybridization. The technique proved to be rapid and sensitive for detecting C. xyli subsp. xyli in sugarcane sap. The degree of sensitivity varied, with the labelled C. xyli subsp. xyli chromosomal DNA probe being more sensitive than the cloned C. xyli subsp. xyli chromosomal DNA probe. Diluted sap from infected sugarcane plants gave positive hybridization signals whereas sap from unifected sugarcane plants failed to give hybridization signals.

Pages

43

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