To investigate the aetiology of mealybug wilt disease (MWD) in Australian pineapples, a virus
diversity study was completed, in particular to determine what mealybug-transmitted viruses are
present in pineapple.
Pineapple mealybug wilt-associated virus 1 (PMWaV-1), -2 (PMWaV-2) and -3 (PMWaV-3) were
all detected in Australian commercial pineapple crops and shown to be transmitted by Australian
colonies of the mealybug species Dysmicoccus brevipes. A further previously undescribed
ampelovirus, for which the name Pineapple mealybug wilt-associated virus 5 (PMWaV-5) is
proposed was also identified in Australian pineapples. In surveys of mealybug wilt disease (MWD)-
affected crops, none of these four viruses was clearly associated with the disease at all survey sites.
At one site, PMWaV-2 was associated with disease symptoms and PMWaV-1 and -3 equally
distributed between symptomless and MWD-affected plants. However, at the remaining three
survey sites PMWaV-2 was at a low incidence and instead either PMWaV-1 and/or -3 were
associated with MWD. PMWaV-5 was only occasionally found at all sites.
The previously published partial sequence for Pineapple bacilliform virus was shown to be not from
a badnavirus as previously thought, but from a new Ty3-gypsy retrotransposon for which the name
Ananas metavirus (AMtV) is proposed. Two unique caulimovirid sequences isolated from
pineapple were associated with bacilliform virions, were transmitted by mealybugs and
phylogenetic analyses indicates that they are probably new species of the Badnavirus genus. The
names Pineapple bacilliform comosus virus (PBCoV) and Pineapple bacilliform erectifolius virus
(PBErV) are proposed for these viruses. Both viruses were transmitted by D. brevipes, and PBCoV
also by Planococcus citri. Neither virus was associated with MWD at any of the four disease survey
sites. Furthermore, PBErV was only detected in a few commercial field plants whereas the
incidence of PBCoV varied from uniform to only rarely found depending on the site surveyed.
Evidence suggests that a third caulimovirid sequence was not derived from encapsidated DNA but
more likely present as an endogenous form and as such the name endogenous Pineapple
pararetrovirus-1 (ePPRV-1) is proposed. In phylogenetic analyses the ePPRV-1 sequence clusters
with caulimovirids, but doesn’t appear to belong to any of the existing genera within the
Caulimoviridae family. A preliminary investigation indicates that MWD is not associated with the
presence of ePPRV-1-like RNA transcripts. It is not known if the RNA transcripts detected in
pineapple were derived from activation of ePPRV-1 or from episomal forms of the same virus.
However, no virions or DNA was detected from a sample which was positive for ePPRV-1-like
RNA transcripts, suggesting that for at least this single sample, the RNA was derived from ePPRV-
1 DNA present within the host chromosomes.
This study also highlighted the need for diagnostic assays which effectively discriminate episomal
caulimovirid from endogenous infections of the same virus. To this end, a number of PCR-based
assays were evaluated using the well described endogenous and episomal badnaviruses associated
with banana streak mosaic disease, as a model system. Most notably, a new approach for
immunocapture PCR was developed using this model system which not only discriminated the two
viral forms but also improved the efficiency of virus detection through the reduction of incubation
times. A multiplex diagnostic assay for the detection of pineapple-infecting ampleoviruses was also
developed during the study and used to screen MWD-affected crops. This assay allowed
simultaneous detection of four unique virus species and helped to minimise sample processing
times. The new approach for immunocapture was then used to develop a triplex RT-PCR assay for
the detection of the three ampeloviruses shown to be associated with MWD in Australia.
Finally, the results from this study are used to propose a hypothesis to explain the cycling of
symptoms in individual MWD-affected plants.