AbstractIn Australia the strawberry diseases, lethal yellows and green petal, are associated with Candidatus Phytoplasma australiense (Ca. P. australiense). In addition, a rickettsia-like-organism (RLO) is associated with Australian strawberry lethal yellows (SLY) disease. Ca. P. australiense is also associated with the plant diseases, papaya dieback (PDB), Australian grapevine yellows (AGY) and Phormium yellow leaf (PYL; New Zealand). The tomato big bud (TBB) phytoplasma is associated with a wider range of plant diseases throughout Australia. In contrast, the RLO has only been identified in association with SLY disease, and Ca. P. australiense has only been detected in a limited number of plant host species. Diversity studies of phytoplasmas have shown that the conserved 16S rRNA gene may not distinguish closely related phytoplasmas within the Ca. P. australiense group. Identification of more variable genes may facilitate the differentiation of these closely related phytoplasmas.
The aims of this study were to; 1) develop a peR based diagnostic test for the SLY RLO, 2) use this and existing tests to examine the phloem limited organisms associated with strawberry green petal (SGP) and lethal yellows diseases, 3) measure the level of association between the RLOs and phytoplasmas, and strawberry diseases, 4) identify plant species that are possible reservoirs for the phytoplasmas and RLOs associated with SLY and SOP diseases, 5) identify new genes that may be suitable for differentiating closely related phytoplasmas, 6) use these genes and previously identified phytoplasma genes to investigate genetic variation among Ca. P. australiense strains and determine whether there is a relationship between strains and a particular host plant species.
Two plants with SLY disease were subjected to PCR using papaya bunchy top (PBT) RLO primers and the amplicons were sequenced. Both amplicons shared 96% sequence identity with the sdhA gene of the PBT RLO and this provided the first molecular evidence that an RLO is associated with strawberry lethal yellows. Only two plants with strawberry green petal disease were observed and collected between January 2000 and October 2002, compared with 363 plants with SLY disease symptoms. Of the 363 SLY samples, 117 tested positive for the RLO, 67 tested positive for Ca. P. australiense AGY strain and 11 plants tested positive for Ca. P. australiense Phormium yellow leaf variant strain. On runner production farms at Stanthorpe, the RLO was detected in SLY diseased plants more frequently than phytoplasmas. On fruit production farms at the Sunshine Coast, Queensland, Ca. P. australiense was detected in SLY disease plants more frequently than the RLO. Thirty one other plant species from south-east Queensland were observed with disease between 2001 and 2003 and of these, 18 species tested positive using phytoplasma specific primers. The RLO was detected III diseased lacksonia scoparia and Modiola caroliniana samples collected at Stanthorpe. The TBB phytoplasma was detected in 16 different plant species and Ca. P. australiense AGY strain was detected in six species. The TBB phytoplasma was detected in plants collected at Nambour, Stanthorpe, Warwick and Brisbane. Ca. P. australiense was detected in plants collected at Nambour, Stanthorpe, Gatton and AHora.
A TBB phytoplasma random clone genomic library was analysed as part of a search for new genes that could be used to distinguish closely related phytoplasmas. Twenty clones were analysed and these contained 26 genes comprising 19.0kbp of TBB phytoplasma genomic DNA. Half of the TBB phytoplasma genes identified were involved in DNA replication, transcription and translation. The remaining TBB phytoplasma genes were involved in protein secretion, cellular processes, transport and energy metabolism. Most primers designed based on the newly identified phytoplasma genes amplified a product from TBB and sweet potato little leaf strain V4 (SPLL-V4) phytoplasma samples. When amplified products were subjected to restriction fragment length polymorphism (RFLP) analysis, the restriction patterns were the same as the respective clones. These primers did not amplify Ca. P. australiense DNA but sequence analysis of the tuf gene and rp gene operon showed that Ca. P. australiense strains could be differentiated into four subgroups. These subgroups were named 16SrXII-BUuf-Australia I)(rp-A), 16SrXII-BCtuf-New Zealand I; rp-B), 16SrXII-B(tuf-New Zealand II) and 16SrXII-B (rp-C). While no relationship was observed between these phytoplasma subgroups and collection date, location or host plant, the existence of subgroups indicated distinct inoculation events and possibly different alternative hosts and different vectors.
PCR results and sequence analysis indicated the PBT RLO PCR primers are suitable for screening SLY disease samples for the presence of an RLO. peR screening of diseased strawberry plants showed RLOs and phytoplasmas are equally associated with SLY disease and the RLO is more prevalent at runner production areas while Ca. P. australiense is more frequently detected at fruit production farms. These findings suggest that RLO SLY disease management is required in the runner production areas while Ca. P. australiense control strategies should be implemented in fruit production areas. The TBB phytoplasma was most frequently detected in the other plant species collected during the study so there is a plentiful supply of TBB phytoplasma inoculum. Despite this, the TBB phytoplasma was infrequently detected in diseased strawberry plants. Six plant species were identified as possible sources of Ca. P. australiense inoculum for SLY disease while only two species are possible reservoirs for the SLY RLO. For SLY disease control strategies to be effective they will have to address the supply of inoculum in the areas surrounding strawberry farms.
|Date of Award
|Karen Gibb (Supervisor)