- 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.