Graduate Thesis Or Dissertation
 

Tobacco rattle virus : weed hosts, diagnosis, characterization, purification, and interaction with potato virus X

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  • A series of studies was conducted to determine the presence of tobacco rattle virus (TRV), potato virus X (PVX), and other viruses in wild plants and to evaluate the efficacy of the detection methods; to characterize and purify an isolate of TRV; and to study the interaction between TRV and PVX. Symptoms on Nicotiana tabacum L. cv. 'Samsun NN' and Phaseolus vulgaris L. cv. 'Bountiful' permitted detection of TRV, PVX, and other viruses. Identification was confirmed by comparison of these symptoms with those from standard cultures of TRV and PVX, by electron microscopy, and by serology. Portulaca oleracea Solanum sarrachoides Sendt., and Amaranthus retroflexus L. were carriers of TRV; the latter two also carried PVX. Most TRV carriers were found adjacent to infected potato tubers. Naturally infected S. sarrachoides contained TRV in the roots at relatively high concentration. Mechanical inoculation of TRV to A. retroflexus and S. sarrachoides produced variable symptoms. These included red spots on the former and chlorosis and stunting on the latter. TRV moved systemically in both species. Methods of detection were adequate but electron microscopy and serology required increased TRV concentration to confirm identification. The isolate from S. sarrachoides was characterized by symptoms, serology, and electron microscopy. To relate TRV concentration to infectivity, several purification methods were carried out. The isolate behaved similarly to other TRV strains in symptomatology and serology. Most purification procedures provided good virus yield and/or infectivity. Purity (260/280 nm) was higher when butanol was used. Precipitation with PEG provided good purity (260/280 = 1.17-1.18) typical of low RNA rod-shaped viruses. Sucrose ratezonal density gradient reduced yields and provided imperfect separation of modal lengths. Particle breakage was excessive when using butanol, freezing, and thioglycolate, and was reduced somewhat by using only chloroform. However, particle breakage was mainly an artifact of staining and thus not affecting the correlation virus concentration-infectivity. Fixation with glutaraldehyde reduced breakage and provided two modal lengths comprising particles; 85.4 and 206.7 nm for long and short particles, respectively. Histograms differed from those reported in the literature. The proportion of long to short particles, the width of particles, and the length of short particles were within values reported in literature. In addition, long particles were longer than previously reported. The interaction between TRV and PVX on 'Samsun NN' tobacco plants was studied using the half-leaf method on 'Bountiful' bean plants. Eleven-day-old bean plants which were 1 cm long from primary leaves to tip of trifoliate leaf were the most sensitive. Evaluation was done by comparing extraction from singly (TRV=A) and doubly (TRV + PVX=B) inoculated tobacco plants on opposite halves of bean leaves, or by comparing A or B against a standard TRV preparation on opposite halves of bean leaves. When the standard was used, concentration was estimated by referring to a regression equation relating concentration to infectivity. Using this method, a consis tent depressive effect of PVX on TRV was measured which reached the maximum at 96 hrs of incubation. At this time, TRV concentration in extracts from A was about 3 times as great as that from B. The depression was somewhat independent of the time lapse between the entry of one virus and the other. It was higher when TRV was inoculated first than when PVX preceded TRV. However, this depression was not larger than that obtained by simultaneous inoculation at 96 hrs of incubation. Symptoms caused by double inoculation on tobacco were remarkably different and developed faster than those induced by single inoculation with TRV or PVX. Symptoms from double inoculation were sunken lesions and, in some instances, severe stunting. TRV-inoculated tobaccos often were symptomless but their extracts were more infective than those from doubly inoculated plants. TRV replication in symptomless plants, increased severity of symptoms due to simultaneous presence of TRV and PVX, and depression of TRV triggered by PVX could explain some of the problems encountered when detection of TRV is attempted in potato tubers and potato plants showing conspicuous symptoms. If concentration of TRV in the field is lower than in plants used in this thesis, the model we used might predict an even higher depression of TRV under natural conditions in potatoes.
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