ANR Coffee CupSome grapevine diseases can be identified easily in the field by signs of the pathogen, which include microscopic and macroscopic microbial cells, tissues, and structures. For example, the appearance of the powdery mildew fungus on the surface of infected leaves and fruit is quite characteristic (fig. 7.1). Some diseases, such as crown gall (fig. 7.2), are characterized by their symptoms, which are defined as abnormal plant growth caused by damage from the pathogen. In contrast, the stunted shoots, scorched leaves, or other nondescript symptoms associated with other diseases make it difficult to narrow the list of possible causes. (See section 5, “Diagnosing Vineyard Problems”). In addition, a few diseases are identifiable only through controlled inoculations of healthy plants. Many grapevine viruses, for example, can be identified only by using a combination of lengthy biological tests and laboratory tests. To address the fact that many grapevine diseases are difficult to diagnose simply by observation of symptoms, it may be necessary in some cases to submit samples to a diagnostic laboratory for identification of the disease agent.

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This excerpt from Grape Pest Management (Third Edition) was published with permission from UC ANR, and was written by Deborah A. Golino, Stephen J. Vasquez, George M. Leavitt, and Kendra Baumgartner.

GPM Cover Art - Hi Rez_color adjust

The Lodi Winegrape Commission and the University of California Division of Agriculture and Natural Resources (UC ANR) are partnering to provide Lodi winegrowers with the latest information about grape pest management. This is the first of several excerpts from the third edition of the Grape Pest Management book to be published here in the Coffee Shop.

In Grape Pest Management, more than 70 research scientists, cooperative extension advisors and specialists, growers, and pest control advisors have consolidated the latest scientific studies and research into one handy reference. The result is a comprehensive, easy-to-read pest management tool.

The new edition, the first to be published in over a decade, includes several new invasive species that are now major grape pests. It also reflects an improved understanding among researchers and growers about the biology of pests. With nine expansive chapters, helpful, colorful photos throughout.

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Figure 7.1: A diagnostic sign of the powdery mildew pathogen (Erysiphe necator) is the white, powdery appearance of the fungal tissue (hyphae) and spores that grow on the surfaces of fruit and leaves. Photo: L. J. Bettiga.

Figure 7.1: A diagnostic sign of the powdery mildew pathogen (Erysiphe necator) is the white, powdery appearance of the fungal tissue (hyphae) and spores that grow on the surfaces of fruit and leaves. Photo: L. J. Bettiga.

Laboratory technology for the detection of grapevine pathogens has undergone rapid development in the last decade. Reliable tests for numerous fungi, bacteria, and viruses have been developed in research labs. Many of these tests are currently offered by commercial and public laboratories, providing growers with the opportunity to submit samples from their vineyards for identification of grapevine diseases.

Several methods of disease testing are available from commercial plant pathology laboratories. These include direct culture of disease agents, serological tests such as ELISA, and nucleic acid sequence–based tests such as PCR. Other types of tests, such as indexing with biological indicators, are usually performed only at research institutions such as the University of California. (See “Management of Grape Virus Diseases” in section 24, “Grapevine Virus Diseases”).

Culturing Disease Agents

Many of the pathogens that cause fungal and bacterial diseases can be identified in the laboratory when isolated from a diseased grapevine and grown on an agar media. This technique, known as culturing, typically involves placing samples from diseased tissue onto agar media to detect the presence of characteristic microscopic structures or colony growth. Certain types of media are specifically formulated for fungi, while others promote growth of only bacteria. Media formulas that encourage the growth of certain families of fungi or bacteria are known as selective media. For pathogens that are too small for observation with a light microscope or for those that appear similar to nonpathogenic species, culturing is an important first step to nucleic acid sequence–based tests such as the polymerase chain reaction (PCR). The use of selective media, in conjunction with microscopic observations (or other diagnostic testing) of the pathogens, helps laboratories identify and often confirm the causal agents of many diseases.

Figure 7.2: A diagnostic symptom of crown gall is the abnormal tumorlike gall that sometimes forms on the vine trunk and canes in response to infection by the pathogen Agrobacterium vitis. Photo: S. J. Vasquez.

Figure 7.2: A diagnostic symptom of crown gall is the abnormal tumorlike gall that sometimes forms on the vine trunk and canes in response to infection by the pathogen Agrobacterium vitis. Photo: S. J. Vasquez.

In grapes, culture methods are commonly used to diagnose many fungal diseases, such as the following: canker diseases (Eutypa lata, Botryosphaeriaceae spp.); Armillaria root rot (Armillaria mellea, fig. 7.3); Phytophthora root rot (Phytophthora spp.); and esca (Phaeomoniella chlamydospora, Phaeoacremonium spp.). Culture methods are also important for diagnosis of bacterial diseases such as crown gall (Agrobacterium vitis) and Pierce’s disease (Xylella fastidiosa). This technique does not work for all grapevine diseases, though. For example, powdery mildew and all of the viruses are obligate parasites, which need living host material to survive and reproduce. There are also other fungi and bacteria that have not yet been successfully cultured.

In order to successfully culture an organism suspected of causing disease, it must be living in the sample provided to the lab. To ensure that the pathogens are viable, samples should include active lesions or interfaces between live and dead tissue (fig. 7.4). Collecting dead leaves, stems, or roots will be of no value toward culturing most pathogens.

Culturing is a slow, labor-intensive practice. Also, considerable expertise is needed to reliably identify organisms that grow on media. Check with your laboratory to see what types of culture services they provide. It may be necessary to consult several laboratories and a viticulturist to identify peculiar symptoms that may be caused by a pest, disease, or environmental condition.

Figure 7.3: The pathogen that causes Armillaria root rot, Armillaria mellea (oak root fungus), is easily cultured from symptomatic root wood or mycelial fans of the pathogen on a selective medium that excludes many soilborne fungi that contaminate petri plates and obscure or prevent growth of the pathogen. Photo: S. T. Koike.

Figure 7.3: The pathogen that causes Armillaria root rot, Armillaria mellea (oak root fungus), is easily cultured from symptomatic root wood or mycelial fans of the pathogen on a selective medium that excludes many soilborne fungi that contaminate petri plates and obscure or prevent growth of the pathogen. Photo: S. T. Koike.

Serological tests—ELISA

Serological methods of disease testing use 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’s immune system reacts to this foreign material by producing antibodies specific to the pathogen. 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. These tests are rarely offered commercially for fungal pathogens, as most fungi produce visible structures in culture or on infected plant tissue that are taxonomically informative, making the use of ELISA tests unnecessary (fig. 7.5). ELISA tests are fairly simple to run and can provide results in just 1 or 2 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, must be collected at the proper time of year for the pathogen life cycle, and must be fresh.

Figure 7.4: The dark brown, wedge-shaped canker in this cross-section of a live cordon is a suitable sample for diagnosis of infection by Eutypa lata or Botryosphaeria species. The tissue from which these organisms are cultured is the lighter-colored, healthy-looking wood at the margin of the canker, as this is most likely to be colonized by the pathogen. In contrast, the saprophytic fungi that follow the pathogen, as part of a normal succession of microbes, are likely to be cultured directly from the canker. Photo: L. J. Bettiga.

Figure 7.4: The dark brown, wedge-shaped canker in this cross-section of a live cordon is a suitable sample for diagnosis of infection by Eutypa lata or Botryosphaeria species. The tissue from which these organisms are cultured is the lighter-colored, healthy-looking wood at the margin of the canker, as this is most likely to be colonized by the pathogen. In contrast, the saprophytic fungi that follow the pathogen, as part of a normal succession of microbes, are likely to be cultured directly from the canker. Photo: L. J. Bettiga.

One limitation to ELISA testing for grapevine viruses is that antisera do not exist for all of the viruses that infect grapes. 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, where a negative result may not be meaningful if a leafroll is present in the plant for which no antiserum is available (see “Leafroll” in section 24, “Grapevine Virus Diseases”)

Another issue with serological testing is the purity of the antiserum. In some cases, an antiserum may react against more than one pathogen or against other components of plant sap. This could lead to confusing results or potentially false positive results if the antiserum reacts to something other than the pathogen. A good lab uses proper internal controls in each test and advises 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. They do not rely on antibody reactions, but rather look for nucleic acid sequences that are unique to a particular pathogen. One of the most sensitive molecular methods for pathogen detection currently available is polymerase chain reaction (PCR).

PCR involves the selective amplification (i.e., making many copies) of a small part of a pathogen’s 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 species of fungi, bacteria, or virus 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 16 bacterial and viral pathogens of grapevines. Additionally, researchers are evaluating the feasibility of detection of some of the more common fungal pathogens, such as Eutypa lata and Botrytis cinerea, in symptomless tissue. As this is an active area of research, the list of pathogens detectable in plant tissue by PCR is sure to expand, giving growers a higher level of confidence in matching visual symptoms with causal organisms.

Figure 7.5: This chasmothecium (arrow) is a microscopic reproductive structure produced by Erysiphe necator. As a biotrophic pathogen, E. necator is not culturable, but the common occurrence of numerous chasmothecia and white hyphae on the surfaces of leaves and fruit makes its presence suitable for confirmation of powdery mildew. Photo: L. J. Bettiga.

Figure 7.5: This chasmothecium (arrow) is a microscopic reproductive structure produced by Erysiphe necator. As a biotrophic pathogen, E. necator is not culturable, but the common occurrence of numerous chasmothecia and white hyphae on the surfaces of leaves and fruit makes its presence suitable for confirmation of powdery mildew. Photo: L. J. Bettiga.

Biological Indexing

Biological indexing (testing) is a valuable test for some pathogens, particularly viruses. Because these tests require greenhouses or field plantings, they are rarely offered by commercial labs and are most often used at research institutions. Although used most often to detect virus diseases, there are also biological indexing tests for bacteria, fungi, and herbicides.

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 virus will multiply in the plant, and within several weeks, diagnostic disease symptoms will appear on the herbaceous indicator plant. For grapevine viruses, common indicators include Chenopodium quinoa, C. amaranticolor, Nicotiana clevelandii, and Cucumis sativus. Although this test is very sensitive for some viruses (particularly the nepoviruses, including grapevine fanleaf virus), it works only for the limited number of grapevine viruses that can reproduce on herbaceous hosts. In addition, although it indicates that virus is present, it does not differentiate between viruses, as will an ELISA test or a PCR test.

Woody or field indexing requires 2 years to complete. Indicator grapevine cultivars that are especially sensitive to virus diseases are grafted with buds from the vine being tested. If virus is present, it will move from the buds to the indicator, causing infection and eventually diagnostic disease symptoms. Once the buds heal, the indicators are planted in the field and observed for two seasons for the development of virus disease symptoms. Although indexing tests are labor intensive and time consuming, they are very useful if the symptomatic grapevines are valuable (e.g., an imported cultivar or a new cultivar released by a grape breeder) and a high level of confidence in the diagnosis is needed. Biological indexing is a very sensitive test because it provides an opportunity for the pathogen to multiply from very low population levels that can readily be detected by the symptoms it causes. Grapevine field indexing programs generally are performed using the indicators Vitis rupestris St. George, LN33, Kober 5BB, and Cabernet franc.

Sample Selection and Handling

Samples must be selected and collected properly to minimize 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 its concentration in vines becomes very low during summer, making detection less reliable. Diagnostic laboratories should be able to provide useful guidance about optimal sampling times. This publication has additional information about seasonal aspects of pathogen detection in individual disease descriptions.

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 your lab for recommended delivery instructions.

Figure 7.6: Phytophthora cinnamomi decomposes the cambium of the root crown, resulting in a dark brown layer of dead tissue beneath the bark, as shown on the thick root in the foreground. Culture attempts from the margin of a lesion often yield the saprophytic secondary fungi that follow P. cinnamomi infection, primarily because the pathogen is quickly replaced by these ubiquitous soilborne fungi. To guard against detection of secondary fungi instead of the primary pathogen, request that only the margins of lesions are plated and that cultures obtained from the roots, root crown, and soil are compared before a diagnosis is made. Photo: K. Baumgartner.

Figure 7.6: Phytophthora cinnamomi decomposes the cambium of the root crown, resulting in a dark brown layer of dead tissue beneath the bark, as shown on the thick root in the foreground. Culture attempts from the margin of a lesion often yield the saprophytic secondary fungi that follow P. cinnamomi infection, primarily because the pathogen is quickly replaced by these ubiquitous soilborne fungi. To guard against detection of secondary fungi instead of the primary pathogen, request that only the margins of lesions are plated and that cultures obtained from the roots, root crown, and soil are compared before a diagnosis is made. Photo: K. Baumgartner.

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 test results indicate that a disease is present but the vines were actually free of the disease. 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 fungal cells 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 test results indicate that no disease is present but the vines were actually diseased. Most false negatives occur because the sample from the diseased vine did not have the suspect organism in it, or the sample was mishandled and was not in good condition when it arrived at the lab. Given that pathogens are unevenly distributed in vineyards and within an individual vine, it is important to collect multiple tissue samples from the same symptomatic vine or from different vines. If the tissue sent to a lab is from a part of a diseased vine that does not contain the pathogen, the result will be a false negative. False negatives can also occur due to a variety of problems at the laboratory that compromise the testing procedure. Careful laboratories include controls in their tests in hopes of identifying these types of problems so that if the quality of the test is not good, the test can be repeated. The addition of a control gives the researcher or lab staff a known result that can be compared with samples submitted for pathogen identification.

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 many microorganisms that can cause disease are present in vineyards much of the time. Your sample may test positive for fungi, bacteria, or a virus that may be present but is not likely to cause the problem of concern. To cite a good example, when grapevine roots are sampled and cultured, many fungi are frequently found that may or may not be the cause of a disease; the fungi are common but disease only results when additional factors contribute. Culture results can provide a good lead in determining the cause of a problem, and possible solutions should not be the only tool used in diagnosis. Sometimes test results include reports on the presence of rare pathogens or pathogens that are rarely aggressive in grapevines. Often, secondary organisms may exist in greater numbers only after the primary organism has caused infection first (fig. 7.6).

Figure 7.7: The nonuniform distribution of the Pierce’s disease pathogen, Xylella fastidiosa, in symptomatic vines can confound laboratory diagnosis of this disease. The pathogen may be absent from a symptomatic leaf such as this, because symptoms form in advance of the infection. To maximize the chance of a positive diagnosis, collect sections of the adjacent stems, where the pathogen is more likely to be detected. Also, collect multiple stems adjacent to leaves showing a range of symptoms, from moderate to severe. Photo: J. K. Clark.

Figure 7.7: The nonuniform distribution of the Pierce’s disease pathogen, Xylella fastidiosa, in symptomatic vines can confound laboratory diagnosis of this disease. The pathogen may be absent from a symptomatic leaf such as this, because symptoms form in advance of the infection. To maximize the chance of a positive diagnosis, collect sections of the adjacent stems, where the pathogen is more likely to be detected. Also, collect multiple stems adjacent to leaves showing a range of symptoms, from moderate to severe. Photo: J. K. Clark.

Positive results from a laboratory can generally be counted on as being 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, such as the uneven distribution of pathogens in the tissues of individual vines and changes in seasonal pathogen populations, false negative results are common (fig. 7.7). Keep in mind that a negative test result does not mean that the vine is free of the disease being tested for.

Test results for disease cannot be used to determine the general health of a grapevine. Rather, they can help determine whether 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, new technology is available today that makes the job of diagnosing vineyard diseases using laboratory tests far more reliable than it has ever been in the past. Guidelines for sampling and disease testing for the most common diseases found in California vineyards are organized in table 7.1. It is important to note that symptoms may be similar for many diseases, which might necessitate multiple diagnostic tests to properly identify the cause of disease.

Table 7.1. Guidelines for sampling and disease testing

Disease

Pathogen type

When to test

Tissue to sample

Diagnostic methods

Culturing

ELISA

PCR

Armillaria root rot

fungus

year-round

root crown and thick, woody roots

reliable

NA

reliable from pathogen culture

black foot disease

fungus

year-round

roots, root crown, base of trunk

reliable

NA

NA

Botryosphaeria dieback

fungus

spring through harvest

spurs, cordons, and trunk wood

reliable

NA

reliable from pathogen culture

Botrytis bunch rot

fungus

spring through harvest

fruit, shoots, and canes; scion/ rootstock of new plants

reliable

NA

reliable from pathogen culture

crown gall

bacterium

year-round

trunk wood, buds, galls from canes or trunks, and roots

reliable

reliable from pathogen culture

reliable from pathogen culture and only fresh galls on plant tissue

downy mildew

fungus

spring

leaves and shoots

NA

NA

reliable from plant tissue

esca

fungus

year-round

canes, cordon, and trunk

reliable

NA

NA

Eutypa dieback

fungus

spring through harvest

spurs, cordons, and trunk wood

reliable

NA

reliable from pathogen culture

fanleaf (and other nepoviruses)

virus

spring is best; fall and winter are okay

active shoot tips in spring; shoots/ canes for cambium scrapings in fall and winter

NA

reliable in spring

reliable in spring, less reliable in fall and winter

leafroll

virus

late summer, fall, and winter

petioles in late summer and fall; shoots/ canes for cambium scrapings in fall and winter

NA

Not available for all GLRaVs; reliable n late summer and fall

Not available for all GLRaVs; reliable in late summer, fall, and winter

Petri disease/vine decline

fungus

year-round

roots, root crown, base of trunk

reliable

NA

NA

Phomopsis cane and leaf spot

fungus

spring and winter

canes and shoots

reliable

NA

NA

Phytophthora crown and root rot

fungus

year-round

fine roots, root crown, and soil adhering to roots

reliable

NA

NA

Pierce’s disease

bacterium

late summer and fall

symptomatic leaves and adjacent stems

reliable

reliable from pathogen culture and plant tissue

reliable from pathogen culture and plant tissue

powdery mildew

fungus

spring through harvest

leaves, shoots, and fruit

NA

NA

reliable from pathogen scraped off plant tissue

rupestris stem pitting

virus

year-round

petioles, leaves, and cambial scrapings

NA

not generally available

reliable

Verticillium wilt

fungus

year-round

shoots

reliable

NA

NA

vitiviruses: GVA, GVB, and GVD

virus

spring, fall, and winter

petioles, leaves, or cambial scrapings

NA

available for GVA only; reliable in spring

available for GVA, GVB, and GVD