By Ed Weber, UC Cooperative
Deborah Golino, Director of the Foundation Plant Materials Service
& Adib Rowhani, Plant Pathologist, FPMS
Laboratory testing for grapevine diseases is useful for diagnosing
problems in existing vineyards. Identifying infected propagation
wood can help growers avoid spreading diseases to new vineyards.
In the past, detection of viruses in grapevines was largely accomplished
by laborious and slow biological tests. Today, many of the most
important grapevine viruses can be detected using fast laboratory
tests. Although this article will focus on viruses, similar testing
is available for diseases caused by fungi or bacteria.
Field diagnosis is challenging
Laboratory testing is a valuable tool when investigating vineyard
problems because field diagnosis of grapevine diseases can be difficult.
Symptoms displayed in the field are rarely unique to a particular
disease. In addition, some infected vines may not show any symptoms
indicative of their disease status.
In some cases, grapevine diseases produce distinct symptoms that
make them easy to identify in the field. Late spring foliage symptoms
of Eutypa, for example, are not likely to be confused with other
problems. In this case, laboratory testing would not be necessary
to identify the cause of the problem. With considerable experience,
several other diseases can be reliably diagnosed based on field
More often, a particular set of symptoms could result from a number
of causes. Red leaves, for example, could be an indication of a
virus disease, a fungal root disease, a nutritional deficiency,
physical damage, or feeding by mites, insects, or rodents. Similarly,
leaf scorch could be a symptom of Pierces disease, water stress,
sulfur burn, or spray damage. Disease testing is often used to help
sort out the true nature of the problem leading to symptom expression.
With most grapevine virus diseases, diagnostic symptoms only occur
during certain times of the year. Leafroll, for example, causes
leaves to redden in red-fruited varieties in the late summer and
fall. Examination of these vines in the spring would give no indication
of their disease status.
It is virtually impossible to diagnose grape virus diseases in the
field during the dormant season, a time when many growers and nurseries
cut wood for propagation.
In addition, grapevines infected with some disease agents (especially
viruses) may never show any obvious symptoms! The concentration
of disease-causing agents may be so low that no disease symptoms
develop, or the infection may be in a grape variety that is tolerant
to that particular disease.
Such latent infections can only be detected with reliable disease
testing methods. This is an important consideration when collecting
wood for propagating new vines, since virus diseases that may not
be evident on one rootstock, or in certain growing conditions, may
cause severe disease when infected wood is grafted to create new
Several methods of disease testing are available from commercial
plant pathology laboratories (see Table I). These include direct
culture of disease agents; serological tests, such as ELISA; and
molecular tests, such as Polymerase Chain Reaction (PCR). Other
types of tests, such as indexing with biological indicators, are
usually performed only at research institutions such as the University
culture of plant pathogens
Direct culture is the oldest form of laboratory testing and is still
commonly used to help identify many fungal and bacterial disease agents.
This method involves placing samples of diseased tissue onto selective
culture media and observing what organisms grow. Certain types of
selective media only allow fungi to grow, while others only allow
bacteria to grow.
Several media recipes have been designed so that only certain fungi
or bacteria will grow. The use of these selective media, along with
microscopic observation (or other diagnostic testing) of the organisms
that do grow, helps confirm the cause of many diseases.
In grapes, culture methods are commonly used for many fungal diseases,
such as Eutypa, Armillaria (oak root fungus), Phytophthora (crown
rot), Phomopsis, and Botrytis, as well as bacterial diseases, such
as crown gall and Pierces disease. Viruses cannot be detected
through culture methods.
In order to successfully culture a disease agent, it must be living
in the sample provided to the lab. Ideally, samples should include
active lesions or interfaces between live and dead tissue. Collecting
dry leaves or dead stems will be of no value towards culturing.
Culturing is a slow, labor-intensive practice. Also, considerable
expertise is needed to reliably identify organisms that grow on the
plates. Check with a plant pathology laboratory to see what types
of culture services they provide.
|Serological tests ELISA
Serological methods utilize antibody reactions with disease agents, usually
viruses or bacteria. Antiserum is produced by first injecting an animal
(typically a rabbit) with a purified preparation of a plant pathogen, such
as a virus. The animal reacts to this foreign material by producing antibodies.
These antibodies react specifically with the pathogen that was used to create
them. The antibodies are then purified from blood serum and the resulting
antiserum is used in diagnostic tests. The most commonly used serological
test is Enzyme-Linked Immunosorbent Assay (ELISA).
Many laboratories offer ELISA tests for grapevine diseases caused by viruses
and bacteria. ELISA tests are fairly simple to run and can provide results
in just one or two days. For ELISA to be successful, antiserum for each
disease agent must be available in the lab, and grapevine test samples must
come from the appropriate tissue, at the right time of year, and be in good
One limitation to ELISA testing is that antisera have not been produced
for all grapevine viruses. Therefore, a vine could be infected with a particular
virus, but if no antiserum against that virus exists, ELISA will be unable
to detect it. This is the situation with some of the leafroll viruses (see
Another issue with serological testing is the purity of the antiserum. In
some cases, an antiserum may react against more than one virus, or against
other components of plant sap that were present with the virus when the
antiserum was produced. This could lead to confusing results, or potentially
false positive results if the antiserum reacts to something other than the
virus. A good lab should use proper internal controls in each test and advise
clients when a particular ELISA test for a pathogen is prone to this problem.
Molecular tests PCR
Recently, molecular tests have been developed that directly target the genetic
material (genome) of plant pathogens. Rather than relying on antibody reactions,
they specifically test for molecular sequences that are unique to a particular
pathogen. One of the most sensitive molecular methods for pathogen detection
currently available is PCR.
PCR involves the selective amplification of a small part of a pathogens
genome. If the pathogen is present in a sample, even in very low amounts,
the amplification steps in PCR allow for its detection. It is this amplification
that makes PCR such a sensitive test.
PCR can be used for the detection of pathogens in grape because each virus,
bacteria, or fungus has its own unique genetic code. In the past decade,
molecular scientists have identified genetic markers for many of these pathogens.
Commercial PCR testing is currently available for many viral and bacterial
pathogens of grapevines. This is an active area of research and this list
is sure to expand.
Before reliable laboratory tests were available for grapevine viruses, biological
indexing (testing) was used to detect these pathogens. Herbaceous indexing
is performed in a greenhouse in the spring and involves rubbing an extract
from the test vine onto leaves of sensitive indicator plants. If certain
viruses were present in the test plant extract, the indicator plants will
develop diagnostic symptoms in several weeks.
Woody or field indexing requires two years to complete. Indicator grapevine
varieties that are especially sensitive to virus diseases are grafted with
buds from the vine being tested. They are planted in the field and observed
for two seasons for development of virus disease symptoms.
Although indexing tests are labor-intensive and time-consuming, they are
still very useful if the vines being tested are valuable and a high level
of confidence in the diagnosis is needed.
Reliability of laboratory testing
No diagnostic test is perfect. All the methods described above have the
potential to produce false positive or false negative results.
False positives occur when a plant was actually free of a particular disease,
but the test results indicate that it was present. False positives usually
occur as a result of contamination or mislabeling of samples. These errors
could occur in the field when samples are collected, or at the lab after
the samples arrive.
Contamination is of particular concern with PCR because of the sensitivity
of the test. Just a few bacteria or virus particles carried over from one
sample to another could lead to false positive results. With ELISA, false
positives can also occur if an antiserum reacts against plant constituents
in addition to the targeted pathogen.
False negative results are much more common than false positives. False
negatives occur when diseased vines are tested but the test results indicate
that no disease agent was present. Most false negatives occur because the
sample from the diseased vine did not have disease agents in it, or the
sample was mishandled and was not in good condition when it arrived at the
Virus infections are usually unevenly distributed in vineyards. Even within
a single vine, viruses may be present in some parts but not others. If the
tissue sent to the lab was from a part of a diseased vine that didnt
contain virus, the result will be negative, albeit a false one.
False negatives can also occur due to a variety of problems at the laboratory
that compromise the testing procedure. Good laboratories include controls
in their tests in hopes of identifying these types of problems so that the
test can be repeated.
|Sample selection and handling
Sample selection is important for minimizing the chances for false negative
results. Sampling particular parts of the vine at certain times of the year
can greatly increase the reliability of disease testing.
For example, to test vines for Grapevine Fanleaf Virus, shoot tips should
be collected for testing in the spring. This virus is heat-sensitive and
during summer, its concentration in vines becomes very low, making disease
testing more unreliable. See Table II for additional sampling recommendations.
Proper handling and shipping of samples is also important. In general, samples
should be delivered to the testing lab as soon as possible. If the samples
have been exposed to excessive heat or drying, or if they are stored for
too long, it will not be possible to get reliable results. Be sure to consult
with a lab for recommended delivery instructions.
Interpretation of results
In general, the larger the number of samples sent from a vineyard for testing,
the higher the confidence level in the results. Keep in mind that in a given
vineyard, more than one virus can infect individual vines, and it is possible
for vines in the same vineyard to be healthy, infected with only one virus,
or infected with more than one virus.
Positive results from a laboratory can generally be counted upon to be accurate.
False positives are not common unless there were significant problems at
the lab. Most labs run internal controls to check for these types of errors.
However, if you suspect a problem because every one of your samples comes
back positive, you might consider running them again. Including a healthy
sample along with your diseased ones is usually a good idea.
Negative results, on the other hand, are of limited value. Because of the
problems inherent in sampling, the uneven distribution of disease agents
in vines and concentration changes during the year, false negative results
occur with high frequency. Keep in mind that a negative test result does
not mean the vine is free of the disease being tested for.
If you suspect that a vine is diseased but the test results come back negative,
dont let this be the end of the story. Consider other evidence such
as vine performance and symptom expression, then do further testing until
you are satisfied.
If you are using laboratory tests to screen vines for propagation, extra
care is needed. Foundation vines in the California Registration and Certification
program are tested over a period of at least two years using a combination
of biological indicators, ELISA, and PCR tests. This provides a very high
level of confidence about the virus status of the selections.
Although practical considerations may require the use of wood from commercial
vineyards for propagation, particularly for field budding, it is highly
recommended that growers do everything possible to avoid spreading pathogens
with the wood.
Disease tests cannot be used to determine the general health
of a grapevine. Rather, they can help determine whether or not a vine is
infected with the particular pathogens being tested for. Because tests are
not available for all known grape diseases, no vine can ultimately be declared
disease-free. However, the new laboratory technology available
today makes the job of diagnosing vineyard diseases far more reliable than
it has ever been in the past.
Grapevine Virus Diseases
This virus group includes at least 13 different viruses that can cause
disease in grapevines. They share in common transmission by nematodes
and a polyhedral physical structure when purified and examined with an
electron microscope. This is the source of the name nepovirus:
ne for nematode, po for polyhedral. Fortunately,
only a few of these viruses are reported to be of importance in grapes
in the U.S.
Grapevine fanleaf virus GFLV
GFLV is perhaps the best characterized virus of grapevines, causing fanleaf
degeneration in affected plants. It is widely distributed throughout the
world. Fanleaf disease is a major viticultural problem in California,
causing reduced yields due to poor berry set. The reduction in yield can
be over 80% in some varieties. Symptoms include fan-like distortions of
leaves and chlorotic yellowing as ringspots, vein banding, and mottling
or mosaic patterns. The virus is transmitted by the nematode Xiphinema
index and can infect all Vitis species.
Yellow Vein Tomato ringspot virus ToRSV
ToRSV causes yellow vein disease. A similar disease is caused by tobacco
ringspot virus. These viruses are transmitted by several species of nematodes
including X. americanum, X. californicum and X. rivesi. Symptoms of both
diseases include shot berries, shoot stunting, and devigoration of the
vine. These diseases are common in vineyards in the eastern U.S. and in
fruit trees, but are rarely seen in California vineyards. The symptoms
of yellow vein resemble those described for fanleaf, and they can be easily
Arabis mosaic virus ArMV
This virus is widespread in grapevines in Europe. Although not found in
California vineyards, it has recently been reported as common in Missouri
and some infections have also been reported in Canada. Infected grapevines
show symptoms similar to those of fanleaf, and ArMV can be present in
a mixed infection with GFLV. Several nematode species can transmit ArMV
to grapevines, the most common being Xiphinema diversicaudatum.
There are at least seven distinct viruses reported to be associated with
leafroll disease. These viruses are collectively referred to as grapevine
leafroll-associated viruses (GLRaVs) and are designated GLRaV 1 through
GLRaV 7. ELISA tests are currently only available in commercial labs in
the U.S. for GLRaV 1-5.
Symptoms of leafroll disease may include downward rolling of leaves, leaf
reddening in the fall of red-fruited varieties, poor fruit color development,
and delayed fruit maturation. Yield losses of 10 to 20% may occur. In
cases of mixed infections with more than one virus, vines may be severely
weakened and vine death may occur.
RUGOSE WOOD COMPLEX
Diseases in the rugose wood complex are characterized by trunk and stem
disorders (pitting and grooving). Foliar symptoms similar to leafroll
may also occur. Diseases in this complex include corky bark, Kober stem
grooving and rupestris stem pitting. Their effects on grapevines vary
from mild to severe. Disease severity is compounded when multiple infections
of the rugose wood complex occur, or by the presence of other viruses
such as leafroll.
In recent years, individual viruses have been discovered and characterized
which has made the detection of these disease agents much easier. There
are still some rugose wood diseases for which the agent has not yet been
described, making it necessary to perform laborious and slow biological
Rupestris stem pitting-associated virus RSPaV
RSPaV is associated with rupestris stem pitting of grapevines. This disease
is usually of little consequence. Decline due to rupestris stem pitting
has been reported, but is not well-documented. RSPaV is widely distributed
and is not targeted for elimination in most certification programs.
Vitiviruses GVA, GVB, GVC, GVD
The vitiviruses are a group of viruses associated with the rugose wood
disease complex. Four vitiviruses have been discovered in grapevines:
grapevine vitivirus A (GVA), grapevine vitivirus B (GVB), grapevine vitivirus
C (GVC), and grapevine vitivirus D (GVD).
GVA is associated with Kober Stem Grooving. Affected vines may show swelling
at the graft union and fail to thrive. Ungrafted vines may be infected,
but usually do not show symptoms.
GVB is associated with corky bark disease. The disease affects only grafted
vines. The severity of corky bark is more pronounced in vines infected
with other rugose wood complex viruses.
Neither GVC nor GVD have been proven to cause disease in grapevine but
their structure and genetic profiles have shown that they belong to the
Grapevine fleck virus GFkV
GFkV is a graft-transmissible virus that causes symptoms of disease only
in V. rupestris. Other Vitis species can be infected but remain asymptomatic.
In infected V. rupestris, symptoms include localized clearings (flecks)
in the veinlets of young leaves. In older leaves, the symptoms diffuse
into a mosaic pattern and the leaves wrinkle and curl upward. Symptoms
persist during mild weather and disappear with the onset of hot temperatures.
Very little information is available about the economic importance of
Many other graft transmissible diseases, likely caused by viruses, can
infect grapevines. These include asteroid mosaic, enations, vein necrosis,
and vein mosaic, among others. These diseases have been studied to varying
degrees, but have never been demonstrated to be common or severe.
Occasionally, new diseases appear that are significant. Recently, a new
stem lesion virus disease was discovered in California (see California
Agriculture, July-August 2001). Also known as Redglobe virus, this disease
can kill vines on certain rootstocks. Continuing research is necessary
to identify important new diseases like this and to develop diagnostic
tools to help minimize their future impact.