Doctor of Philosophy
Department of Biology
Hap, Gonzalo Hortelano, Studies on the nucleic acids of bacteria isolated from greening and dieback infected citrus, Doctor of Philosophy thesis, Department of Biology, University of Wollongong, 1991. http://ro.uow.edu.au/theses/1076
Greening disease is a severe disease of citrus affecting crops in Southern Africa and Asia. The disease is thought to be caused by bacteria seen in planta by electron microscopy. In Australia, Australian citrus dieback (ACD) has symptoms which closely resemble those of greening, although the aetiological agent requires confirmation. A number of bacterial isolates with an ultrastructure similar to that described for the putative causal agent of greening have been studied. The DNA from the different organisms was compared by pulsed field gel electrophoresis after bacterial restriction endonuclease digestion analysis (BRENDA) using a number of endonucleases, and restriction fragment length polymorphism (RFLP). The isolates studied were assigned to two groups. Group 1 contains several very homogeneous isolates, including an organism isolated from ACD infected material. Group 2 isolates are not as homogeneous and can be further divided into two subgroups. The chromosomal DNA of the Group 1 isolates was estimated to be 3,480 Kb after digestion of intact bacterial DNA with rare-cutter restriction endonucleases and separation of the resulting fragments by pulsed field electrophoresis. No extrachromosomal DNA was detected in any isolate. In an attempt to develop a detection system, a DNA library from a Group 1 isolate was constructed. The suitability of certain randomly-cloned fragments as probes to detect the organisms isolated from greening and dieback infected citrus was assessed. RFLP techniques were used to develop a signature pattern for the DNA of Group 1 isolates to allow direct comparison with other similar bacteria isolated from citrus. This technique was modified to detect the presence of Group 1 isolates in infected citrus samples, although the sensitivity of the system only allowed the positive identification of some samples. In order to taxonomically classify the bacterial isolates, the gene coding for the 16S fraction of the ribosomal RNA (rRNA) from Group 1 isolates was fully sequenced, both with reverse transcriptase and from PCR amplified 16S rRNA. The sequence was compared to other known sequences to generate a phylogenetic tree.