DNA extracted from three papaya (Carica papaya L.) plants, individually affected by dieback, yellow crinkle or mosaic diseases, was subjected to PCR using phytoplasma-specific primers to amplify the 165 rRNA gene plus 165-235 rRNA intergenic spacer region. Near-complete DNA sequences obtained for the three PCR amplimers were subjected to phylogenetic analyses and direct sequence comparison with other phytoplasma 165 rDNA and 165-235 spacer region DNA sequences. The papaya yellow crinkle (PpYC) and papaya mosaic (PpM) sequences were identical to each other, but distinctly different from the papaya dieback (PpDB) sequence, showing 9003% identity in the 165 rDNA and 8708% identity in the 165-235 spacer region DNA sequences. A phylogenetic tree based on 165 rDNA sequences was calculated, in which PpYC and PpM are most closely related to the tomato big bud phytoplasma (TBB; 99.7% 165 rDNA sequence identity) from Australia, within subclade iii. This subclade consists of strains only reported occurring in the Southern Asian region and Australia, which indicates an Asian/Australasian origin. PpDB is most closely related to the Phormium yellow leaf phytoplasma from New Zealand (PYL; 99.9% identity) and the Australian grapevine yellows phytoplasma (AGY; 99.7 YO identity). These three phytoplasma strains form a distinct clade within subclade xii, which also includes the European strains STOL and VK as another distinct clade. The origin of the closely related but geographically separated AGY-like strains and STOL-like strains of subclade xii is unclear. It is proposed that phytoplasma strains PpDB, PYL and AGY be included in the previously described taxon Candidatus Phytoplasma australiense ' 8 and that PpYc, PpM and TBB be assigned to a new taxon, Candidatus Phytoplasma australasia I .

Dieback-affected papaya plants were characterized by a discoloration of the contents of laticifers, while the anatomy of sieve elements was healthy in appearance until the necrotic stages of the disorder were reached. Laticifer discoloration was not always associated with the presence of phytoplasma in affected tissue, as judged by polymerase chain reaction (PCR) using primers based on the 16S rRNA gene and 16S-23S intergenic spacer region. Phytoplasma DNA was detected in a range of plant tissues, including roots, but not in mature leaves which would act as photoassimilate sources. As plants recovered from a dieback period, the extent of the distribution of both laticifer discoloration and phytoplasma DNA decreased. Phytoplasma cells were not observed in transmission electron microscopy studies of mature sieve elements of dieback-affected leaf, stem, or fruit tissue from plants at various stages of symptom expression, although PCR tests indicated the presence of phytoplasma DNA. Membrane-bound structures, similar in shape and size to phytoplasma cells but interpreted as autophagic vesicles or latex vesicles in immature laticifers, were observed within vacuoles of cells in phloem tissue in leaves displaying tissue breakdown in the form of a water-soaked appearance to veins (“X-Y” patterning). In contrast, phytoplasmas were readily observed in papaya leaves displaying symptoms of yellow crinkle. We conclude that phytoplasma cells are present in very low titer in dieback-affected tissues and that, while the plant appears to limit proliferation of the dieback-associated pathogen, this defense strategy is ultimately unsuccessful because it is associated with a rapid decline of the papaya plant.

Three phytoplasma-related diseases of papaya (Carica papaya), dieback, yellow crinkle, and mosaic, are recognized within Australia. Immature leaf material was sampled every week for 8 months from a cohort of 60 female plants, located within a commercial papaya plantation, to determine the minimum time between infection and symptom expression. Phytoplasma DNA was detected using the polymerase chain reaction (PCR) with primers specific for phytoplasmas in general, and for the stolbur group of phytoplasmas. The dieback-associated phytoplasma was detected 1 week prior to (four cases) or the same week (nine cases) as symptom expression, while phytoplasma DNA was detected between 3 and 11 weeks prior to expression of mosaic symptom (six cases). Lateral shoot regrowth on the lower stem of plants which had suffered dieback disease failed to generate stolbur-specific PCR products in 15 cases. A dual infection with dieback and yellow crinkle or mosaic was diagnosed in a further two cases, using restriction fragment length polymorphism digests, and both cases were interpreted as secondary infections by the dieback-associated phytoplasma. Regrowth in three of seven cases of yellow crinkle- and three of nine cases of mosaic-affected plants tested positive for phytoplasma-specific DNA. Ratooning of dieback-affected plants and removal of yellow crinkle- or mosaic-affected plants is suggested for the management of these diseases.

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