TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.4 DETECTION/PSTVd

Section 2.4.1
Potato Spindle Tuber
Viroid(PSTVd)

Potato spindle tuber disease was first reported by Martin in 1922. The
term “spindle tuber” was used by South Jersey (USA) farmers to describe
the shape of affected tubers. This disease is caused by the potato
spindle tuber viroid (PSTVd) which is a small RNA molecule without a
protein coat. Unlike other potato viruses that thrive in temperate and cold
climates, PSTVd needs a tropical climate to multiply. Yield losses due to
this viroid depend on potato cultivar and viral strain. Severe strains can
cause losses of up to 65%.

Disease symptoms in potato also vary depending on the infected potato

cultivar and environmental conditions. In many cases, leaf symptoms are
difficult to detect.

Infected tubers are elongated and some cultivars develop pointed ends,
which are a typical characteristic of the disease. Other symptoms that
have been observed include deep eyes, necrotic spots around the
lenticels, surface cracks, and pulp necrosis. Neither all nor even some of
these symptoms are present in all tubers from infected plants.
 The mechanical transmission of PSTVd to a susceptible plant host is very
easy. Under field conditions, a sick plant may infect a nearby plant by
simple contact. The viroid can also be transmitted to other plants during
handling (i.e., by using contaminated tools) or through botanical seed.

Detection Methods

Serological detection of this viroid is not possible because it doesn’t have

a protein coat. Leaf symptoms are difficult to detect and, in many
varieties, the tuber does not show the characteristic spindle shape.
Thus, more sensitive techniques are required for detecting PSTVd. The
following detection methods are available.

1. Tomato test

a) Normal test

b) Yang and Hooker test

c) Challenge test

2. Scopolia sinensis test

3. Polyacrylamide gel electrophoresis (PAGE)

4. Nucleic acid spot hybridization (NASH)

1. Tomato Test

a) Normal test. This is a very simple test. Plant tissue to be tested is

macerated in distilled water and then mechanically inoculated onto
young tomato leaves (Lycopersicon esculentum cv. Rutgers).

The inoculated plants are placed in a growth chamber at 30°C until

symptoms appear. The infected tomatoes show initial epinasty,
rugosity of the leaves (which then roll), dwarfism, and initial vein
necrosis. Later, vein necrosis spreads to the whole leaf causing a
brown color. Severe PSTVd strains provoke clearly identifiable
symptoms. However, mild strains are difficult to detect because
symptoms are not clear and infected plants cannot be easily
differentiated from healthy controls.

b) Yang and Hooker test. This test is carried out as above, but the
inoculated plants are kept at 24–30 °C under continuous
fluorescent light (100 ft-c). The tomato plants infected with either
severe or mild PSTVd strains show albinism. In the case of a
severe strain, the plants also show vein necrosis. This method is
more sensitive than the normal test because it allows detection of
both viroid strains.

c) Challenge test. This is a modified normal test used to detect mild

PSTVd strains. Three tomato plants (A, B, and C) are used in
each test. First, tomato plants A and B are inoculated with sap of
the tested plant. If these plants show severe PSTVd symptoms,
the original potato plant is infected with the severe viroid strain. If

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 plants A and B do not show any symptoms, plants B and C are
inoculated with the severe PSTVd strain and kept at 30°C. After
two or three weeks, the symptoms in the three plants (A, B, and C)
are observed.

Plant C will show severe PSTVd symptoms. If plant B also shows

these symptoms, then the original plant was not infected with the
viroid. If plant B does not show symptoms, then the original plant
was infected with the mild PSTVd strain which prevented plant B
from becoming infected with the severe strain of the viroid due to
cross protection.

2. Scopolia sinensis test

Scopolia sinensis is a plant from the Solanaceae family. When

inoculated with PSTVd, it develops local necrotic lesions after
infection. The plants used in this test should be in a vigorous
growth condition and must be kept under mild lighting conditions
(300–400 ft-c) at 18–23 °C. Symptom development will take from 8
to 14 days. This test is not practical due to the strict lighting and
temperature requirements for plant growth, and difficulties in
producing seed of this plant.

3. Polyacrylamide gel electrophoresis (PAGE)

Until recently, electrophoretic separation of RNA of low molecular

weight isolated from plants in polyacrylamide gel was commonly
used at CIP for PSTVd detection. The test protocol is described in
a separate section. Electrophoresis is less sensitive than the
tomato test. Therefore, a combination of both tests is
recommended during routine detections to increase sensitivity.

The sap of test plants is inoculated to healthy nursery tomato

plants. After 2 to 3 weeks, low molecular weight RNA is extracted
from the inoculated tomato plants to carry out the PAGE test. The
advantage of this method is that, even with low viroid concentration
in the original plants, the concentration in inoculated tomatoes will
increase, thus allowing easy identification of the viroid through
symptom observation or using the PAGE test.

After gel staining, PSTVd RNA can be localized near the center of
the gel because PSTVd RNA migrates more slowly than 9S RNA.
In some cases, the 9S RNA band may not be clearly visible. Thus,
it is advisable to use infected (positive) and healthy controls in the
test run. The positive sample may be identified by comparing it to
the controls.

4. Nucleic acid hybridization (NASH)

Since 1983, CIP has used this technique for routine work due to its high
sensitivity, and because it is possible to simultaneously analyze a large
number of samples. Several conditions must be present to use this
technique:

a) Production of single strand cDNA complementary to the PSTVd

sequence (PSTVd-cDNA).

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 b) Availability of a large amount of PSTVd-cDNA. This is possible
through cloning in a vector (e.g., a plasmid) and introduction into
an E. coli bacterium where the plasmid multiplies. The M-13
bacteriphage also has been used as a vector.
c) Isolation of PSTVd-cDNA from the bacterium and labeling with a
radioactive isotope. Two companies have recently developed a
non-radioactive marker that can also be used.
The NASH test is based on the capacity of PSTVd-cDNA to form a
hybrid with the PSTVd molecule immobilized on a solid membrane.
Due to the presence of a radioactive marker in cDNA, this hybrid
can be detected through autoradiography on a photographic plate.
The presence of a radioactive hybrid indicates that the original
sample was infected by the viroid.

Experiments carried out at CIP show that this method is 10 times more
efficient than the PAGE test and can help to detect the viroid in 1 infected
seed out of 100.

At present, the use of radioactive test markers is a constraint. Tests with

commercial non-radioactive markers are underway at CIP to evaluate
their efficiency and adaptability for detecting PSTVd. Until the non-
radioactive method is available, CIP will analyze the samples from
national programs. However, this is only possible if the samples are
placed on a membrane and mailed to CIP where equipment is available.
If you are interested, contact Dr. Luis F. Salazar or Dr. M. Querci at CIP,
P.O. Box 1558, Lima 12, Peru. For additional information, please refer to
Section 3.4.3 on Nucleic Acid Hybridization.

Recommended Literature

Diener, T.O. and W.B. Raymer. 1967. Potato spindle tuber virus: Plant
viroid with properties of a free nucleic acid. Science 158:387.
Diener, T.O. 1979. Viroids and viroid diseases. John Wiley & Sons. New
York.
Sanger, H.L. 1982. Biology, structure, functions and possible origin of
viroids. In: Nucleic Acids and Proteins in Plants, Vol. 2. Springer-
Verlag. Berlin. 168 pp.

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