Characterisation of Phormium yellow leaf phytoplasma

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dc.contributor.advisor Forster, Richard en
dc.contributor.advisor Gardner, Richard en
dc.contributor.advisor Pearson, Mike en Liefting, Lia en 2007-07-09T12:48:18Z en 2007-07-09T12:48:18Z en 1997 en
dc.identifier THESIS 98-080 en
dc.identifier.citation Thesis (PhD--Plant Science (Biological Sciences))--University of Auckland, 1997 en
dc.identifier.uri en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract Phormium yellow leaf (PYL) is a lethal disease of the monocotyledons, New Zealand flax (Phormium tenax) and mountain flax (P. cookianum). The disease was first discovered in 1908 and its phytoplasma etiology was determined in 1969. However, no further characterisation had occurred since that time, in part due to the lack of suitable scientific methods. The recent application of DNA based approaches to the study of phytoplasmas has provided the opportunity for a re-appraisal of this pathogen. During my study, PYL phytoplasma was transmitted from diseased to healthy flax by the native planthopper, Oliarus atkinsoni (Homoptera: Cixiidae). Successful transmission of PYL phytoplasma from flax to flax by O. atkinsoni was demonstrated by symptomatology and by PCR of the test plants using phytoplasma-specific primers to the l6S rRNA genes. When the salivary glands and the remaining body of the planthoppers used in the transmission studies were tested separately by PCR for the presence of phytoplasma, PYL phytoplasma was detected in 100% of both the salivary glands and the bodies of pre-transmission O. atkinsoni, and in 44% of the salivary glands and in 67% of the bodies from the planthoppers remaining alive after the transmission experiments. In contrast, transmission of PYL phytoplasma between flax plants was not effected by the introduced leafhopper, the passionvine hopper, Scolypopa australis (Homoptera: Ricaniidae), and the phytoplasma was not detected by PCR in the whole bodies of hoppers of S. australis. PYL phytoplasma was also unable to be transmitted from flax by the parasitic plant, dodder (Cuscuta campestris), due to the failure of dodder to establish on flax. O. atkinsoni was not able to transmit PYL phytoplasma from infected flax to periwinkle (Catharanthus roseus). The l6S rRNA gene and l6S/23S spacer region of PYL phytoplasma were amplified from infected flax by PCR, cloned and the nucleotide sequences determined. DNA sequencing and Southern hybridisation analysis of genomic DNA indicated the presence of two copies of the l6S rRNA gene. The two l6S rRNA genes exhibited sequence heterogeneity in four nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. Phylogenetic trees based on 16S rRNA gene and l6S/23S spacer region sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas. These phytoplasmas previously formed the stolbur subgroup of the aster yellows strain cluster but have now formed a phylogenetic group distinct from the aster yellows subclade, designated subclade xii. Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the l6S/23S spacer regions of PYL phytoplasma with the phytoplasmas associated with two diseases in Australia, Australian grapevine yellows (AGY) and papaya dieback (PDB), revealed that they are virtually identical and can be considered as the same phytoplasma species. The species name, Candidatus Phytoplasma australiense, has been proposed for AGY and this name will therefore also apply to the naming of PDB and PYL phytoplasmas at the species level. In addition to the characterisation of PYL phytoplasma, two further aspects of the phytoplasma genome were studied using tomato big bud (TBB) phytoplasma as a model system. Three attempts to obtain amino acid sequence of the major, immunodominant membrane protein of TBB phytoplasma were unsuccessful. However, the development of an affinity purification procedure using protein A-agarose beads cross-linked with the phytoplasma antibody, combined with 16-BAC/SDS-PAGE two-dimensional gel electrophoresis, showed potential for further work on this protein. Preliminary work aimed at the construction of a physical map was initiated for TBB phytoplasma. Small amounts of TBB genomic DNA were obtained, and a range of restriction enzymes were screened for their ability to cleave the TBB phytoplasma chromosome into suitable size fragments. Digests identified six single-cutter enzymes and three enzymes which cleaved the chromosome three to seven times. The genome size of TBB phytoplasma was estimated at 610 kb. en
dc.language.iso en en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA9969922614002091 en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland. en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights.uri en
dc.title Characterisation of Phormium yellow leaf phytoplasma en
dc.type Thesis en Plant Science en The University of Auckland en Doctoral en PhD en
dc.rights.holder Copyright: The author en

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