Molecular characterisation of phytoplasmas in Australia
Creators: name: Padovan, Anna
Publisher: name: Charles Darwin University
Year: 1998

description: A phytoplasma was associated with sweet potato plants with little leaf disease occurring in northern Australia using classical approaches of symptomatology, dodder (Cuscuta australis) transmission to Ipomoea nil, fluorescence microscopy and electron microscopy. Sweet potato little leaf (SPLL) disease was then detected using the polymerase chain reaction (PCR) assay using universal primers targeting the 16S rRNA gene. An Australian phytoplasma survey was undertaken using universal primers in a PCR-based detection system. Phytoplasmas were detected in thirty two different plant species showing little leaf, proliferation, phyllody, virescence, and/or yellows symptoms. The genetic relatedness of these phytoplasmas was determined by restriction analysis of the ribosomal PCR product using a number of enzymes including Alu l, Rsa I, Mse I and Hpa II. All the phytoplasmas detected had similar restriction profiles to SPLL except for the Australian grapevine yellows (AGY) phytoplasma which produced a unique digestion pattern. The ribosomal PCR product of some of the phytoplasmas was cloned and the l6S/23S intergenic spacer region was sequenced to identify more subtle differences in the SPLL-type phytoplasmas. In some of the phytoplasmas some minor nucleotide changes were identified in the variable regions of the spacer region either side of the conserved tRNAIle. The entire SPLL 16S rRNA gene was sequenced to determine its taxonomic and phylogenetic position. Parsimony analysis showed that SPLL belonged to the faba bean phyllody strain cluster, and was closely related with tomato big bud (TBB) from Australia and to phytoplasmas occurring in southeast Asia, such as peanut witches' broom, sesame phyllody and sunhemp phyllody. Restriction fragment length polymorphisms (RFLP) analysis of 16S rDNA showed that AGY was different to other characterised phytoplasmas occurring in grapevine, and most closely resembled stolbur phytoplasmas associated with bois noir from France and Vergilbungskrankheit from Germany. Sequence analysis of 16S rDNA showed that AGY clustered with the aster yellows group and was most closely related to, but not the same as, the stolbur subgroup, forming a new subgroup. Polymorphisms identified in the amplified tuf gene coding for the elongation factor EF-Tu confirmed the uniqueness of AGY in comparison to stolbur phytoplasmas of grapevine. The phytoplasma associated with papaya dieback (PDB) from Australia gave the same restriction profile as AGY. Australian phytoplasmas were therefore found to belong to two phylogenetically distinct groups: the faba bean phyllody strain cluster and the aster yellows strain cluster (stolbur subgroup), with AGY forming a new subgroup within the AY group. To further understand the genetic relatedness of phytoplasmas, physical and gene maps of the SPLL phytoplasma chromosome were constructed. Pulsed field gel electrophoresis was used to determine the size of the SPLL-V 4 genome which was estimated to be 622 kb, the smallest phytoplasma genome recorded. A physical map was prepared using 2-dimensional reciprocal digestions of the restriction enzymes Bss HII, Sma I, Eag I and 1-Ceu I. Sixteen restriction sites were located on the map. Southern hybridisations of digested SPLL-V4 chromosomal DNA were done using random clones and PCR products as probes. This confirmed fragment positions and located the two ribosomal operons and the fus-tuf gene coding for elongation factors G and Tu on the physical map. Results confirmed the presence of two, non-linked 16S rRNA genes in phytoplasmas. Location of the fus-tuf gene represents the first mapping of a nonribosomal phytoplasma gene. Further hybridisations using the mycoplasma ribosomal probe pMC5 and digestions with the 23S ribosomal cutting enzyme I-Ceu I revealed an inversion of one of the ribosomal operons. Comparisons were made with the only other phytoplasma chromosome map published, and with other mollicute genome maps. Digestions of the TBB chromosome using Bss HII, Sma I, Eag I and 1-Ceu I revealed genome heterogeneity when compared to the closely related SPLL-V 4 , and a preliminary chromosome size of 662 kb was estimated. This mapping information has revealed that significant genome diversity exists within the phytoplasmas.