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• W65684055 abstract "Mealybug wilt disease (MBW) remains the most significant field disease of pineapples today. Currently two different virus types have been found to infect pineapples in Australia (Wakman et al., 1995) and Hawaii (Gunasinghe and German, 1989; Hu et al., 1996), Pineapple bacilliform virus (PBV) and pineapple closterovirus(es) (PCV). The work undertaken in this thesis focuses on the development of a detection system for pineapple closterovirus(es) and the elimination of both viruses from infected pineapple material. An effective purification system which would significantly Increase the proportion of PCV particles, and removing PBV particles from the final preparation was not developed in this study. A substantial Improvement of the previous technique used by Wakman (1994; Wakman et al., 1995) was achieved by optimising each step in the purification process, but still did not yield preparations of very high purity. Such preparations still contained PBV particles and significant amounts of host plant contamination. A variety of different techniques were used in attempts to transmit PCV and PBV to alternative plant hosts. It was hoped that a particular host plant species or cultivar may be infected by only one of the viruses, or may contain higher concentrations of virus particles, or might lead to less host plant contamination. Inoculation by mechanical methods, graft or wound contact techniques and insect vectors failed to transit either virus to new plant species or even other pineapples. Serological studies involved the improvement of an immunosorbent electron microscope (ISEM) technique developed by Wakman (1994; Wakman et al., 1995). ISEM became more effective at detecting PBV particles after the addition of Sugarcane bacilliform virus antiserum (provided by B.E. Lockhart) to the trapping antibody. Although initially promising, a polyclonal antiserum produced by Wakman (1994; Wakman et al., 1995) was found to be unsuitable for use in ELISA. Attempts made by the author to produce PCV-specific monoclonal antibodies were unsuccessful due to a combination of impure virus preparations (Chapter 3) and an inferior adjuvant (Chapter 4). Extraction of dsRNA from MBW affected pineapples gave inconsistent results. The method of Gonsalves (1993) for PCV worked on one occasion, but was not reliable. A number of different methods were also attempted, but all failed to yield dsRNA. Cloning of cDNA from MBW affected pineapples provided by John Hu (University of Hawaii) produced two plasmid clones with inserts, which have only recently been confirmed to contain PCV-specific sequence. These two clones contain sequences with similarity to the helicase and replicase genes of Pineapple mealybug-wilt associated virus 1 (PMWaVl) and PMWaV2 respectively (personal communication John Hu; Melzer et al., 2001). A specific RT-PCR assay was developed from the sequence of the replicase clone, but was not sensitive enough for diagnostic use. The usefulness of this test remains unclear as it only detects a subset of the PCV particles observed by ISEM. Elimination of virus(es) from planting material, ie pineapple crowns, was attempted using hot water treatment (HWT) and plant tissue culture. Contrary to the findings of Ullman et al. (1991; Ullman et al., 1993) HWT was not able to eliminate either PCV or PBV from any crown. Meristem tip culture also failed to eliminate the viruses, even when combined with in vitro heat treatments." @default.
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• W65684055 date "2004-01-01" @default.
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• W65684055 title "Detection and elimination of mealybug wilt-associated viruses in pineapple" @default.
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