February 2008 Issue of Wines & Vines

Detecting Leafroll Disease

Strategies for testing, management and control

by Judit Monis, Ph.D.
Detecting Leafroll Disease
Leafroll disease comes in many varieties, all of them colorful, and none of them good for grapes or vines.

  • Plants infected with leafroll disease produce smaller grape clusters that ripen unevenly and have lower sugar content.
  • At least nine different virus species are associated with grapevine leafroll disease. Various types of mealybug are known to spread the disease.
  • Two types of laboratory genetic testing are recommended for detection of leaf-roll disease.
  • The control of leafroll spread in California will need to be based on a concerted effort among growers rather than on individual accomplishments.
Growers have accepted the presence of grapevine viruses, but have rarely considered their impact detrimental to the vineyard. This perception is being abandoned, due to the recent documented spread of leafroll disease in Napa Valley. However, the spread of certain virus species causing leafroll disease is neither new nor limited to California vineyards. Reports in Australia, Argentina, Europe and South Africa date as far back as 30 years.

It is known that different types of mealybugs transmit certain species of leafroll virus and other viruses, such as Grapevine Virus A and Grapevine Virus B. Therefore, the notion that viruses are spread solely by cuttings (e.g., vegetative propagation and grafting), is false. This article will focus on the management and control of leafroll disease spread in the vineyard by the application of strategic disease diagnostic methods.

Leafroll disease

Plants infected with leafroll disease produce smaller grape clusters that ripen unevenly and have lower sugar content. Foliar symptoms include downward rolling, reddening or yellowing of leaves. Other foliar colors associated with leafroll infection include purple and crimson-orange, and they vary depending on the grapevine variety. The leaf veins may remain green or take many other colors (yellow, purple, red, etc.).

At least nine different virus species are associated with grapevine leafroll disease. Although all leafroll-associated viruses belong to the Closteroviridae (Greek, clostero: thread-like) family, the only species known to be transmitted by mealybugs are classified within the Ampelovirus genera (Greek, ampelos: grapevine). Ampeloviruses include the majority of leafroll-associated viruses (GLRaV-1, -3, -4, -5, -6, -8, and 9). Recent research has shown that leafroll viruses are able to recombine in mixed infections, generating many variants of similar viruses. This phenomenon has serious implications on the diagnostics and detection of these viruses in the vineyard.

Ed Weber (UC Cooperative Extension, Napa County), Deborah Golino (UC Foundation Plant Services) and co-workers started mapping the spread of GLRaV-3 in a Napa vineyard in the fall of 2002. The assessment was done by visual observation (characteristic leaf roll disease symptoms) and initially confirmed the presence of GLRaV-3 in all symptomatic vines with a few exceptions (i.e., potential new infections). The incidence of leafroll symptoms in the vineyard was 23%, 41%, 46%, 59% and 66%, in 2002, 2003, 2004, 2005 and 2006 respectively. The study noted that grape mealybugs were present in the vineyard in populations too low to be considered to warrant treatment.

Work in the Golino lab has shown that the citrus (Planococcus citri), grape (Pseudococcus maritimus), long-tailed (Pseudococcus longispinus), obscure (Pseudococcus viburni) and vine (Planococcus ficus) mealybugs are able to transmit GLRaV-3. The same study showed that the long-tailed mealybug can transmit GLRaV-5, GVA (also transmitted by obscure, citrus and vine mealybug) and GVB (also transmitted by obscure and vine mealybug). Furthermore, work in South Africa has shown that a single individual of vine or long-tailed mealybug is capable of transmitting GLRaV-3 to healthy grapevines.

Disease testing in the lab

The correct identification of the disease causal agent is critical for devising a control strategy. Regular visual inspections and sampling of grapevines should be performed to monitor the disease status of a vineyard. It might not always be possible to correlate the presence of virus infection with symptoms, especially with new viral infections.

Complicating matters, other viruses and fungi cause similar symptoms in the vineyard. Worse, many scion-rootstock combinations may not develop symptoms at all. Symptoms may appear two or more years after top-working a vineyard with a new variety. Viruses associated with leafroll move slowly in the vine and may remain undetected by laboratory testing, unless sampling is done correctly. Collection of representative samples will allow the laboratory to detect the presence of viruses associated with leafroll. The season for testing is important. Samples can be collected from vines starting in the fall throughout dormancy. Testing both rootstock and scion plant material is recommended, especially if the vineyard was top worked or field budded recently.

In the laboratory, a battery of tests will be run. Two methods are standard for the detection of leafroll-associated viruses in grapevines: ELISA and RT-PCR. ELISA is an abbreviation for "enzyme-linked immuno-sorbent assay, and consists of trapping the virus protective protein (coat protein) on a test plate containing specific viral antibodies, and detection through a colorimetric enzyme reaction. Detection is limited to the amount of virus present in the sample.

RT-PCR, short for "reverse transcription-polymerase chain reaction, allows the amplification (i.e., making multiple copies) of viral RNA from the initial low concentration present in the vine. The process is specific, and utilizes tiny portions of the viral genome to start the amplification/copying process. The amplification is repeated many times, with each copy making more copies, so after the completion of an appropriate number of PCR cycles, more than a billion copies of the viral RNA can be produced. Therefore, RT-PCR is a sensitive technique for the detection of leafroll viruses.

The sensitivity and specificity (i.e., reacting to the exact virus genetic code) of the detection of viruses associated with leafroll is dependent on the method used for diagnostics, but is also influenced by the season and part of the vine from which samples are collected. While ELISA is generally thought to be less sensitive than RT-PCR, the ELISA has a broader spectrum of detection (i.e., it can detect a range of virus variants). On the other hand, RT-PCR could be too specific, and miss the detection of isolates of the same virus with minute changes due to recombination events (e.g., variant species).

We recommend using both ELISA and RT-PCR consecutively to reliably detect grapevine leafroll viruses, because both methods are designed to detect different portions of the virus. ELISA detects the coat protein, and RT-PCR detects the genomic RNA. Our experience is that the ELISA is more "forgiving" to small changes in the make-up of a virus, while RT-PCR is sensitive but will require the design of new primers to keep up with detection of virus variants. In summary, RT-PCR and ELISA complement each other on the detection of low virus concentrations, new infections and newly generated virus variants.

Detecting Leafroll Disease
A vineyard infected with leafroll disease contrasts sharply with neighboring healthy vines.
Disease management and control

Effective disease control requires above all a clean planting stock (i.e., certified disease-free tested status). However, to ensure that the vineyard remains disease free, the grower must be vigilant of virus infection in neighboring vineyards. The grower must devise procedures to protect the "clean vineyard" from potential external infection.

Mealybugs should be constantly monitored and controlled. Mealybugs are not always easy to observe in the vineyard. Special traps (including pheromone traps) are available to monitor the presence of mealybug infestation. For proper identification, samples should be collected and brought to a pest control advisor (PCA) or an extension office. The extension agent or PCA will recommend treatment based on specific mealybug identification.

The dispersal of mealybugs by field equipment, birds, workers or wind contribute to long distance spread of the virus. In South Africa, studies are in progress to determine the effect of sanitary practices on the spread of leafroll disease. These sanitary practices include fallow periods, sanitation of equipment and sanitation of workers' clothing. Another study involves the removal of infected vines and mealybugs (through use of systemic herbicide/insecticide) in order to treat virus-infected blocks. The results of these studies will provide guidelines for disease control in the near future.

Controlling the spread of harmful viruses calls for rigorous protocols while handling vines and performing cultural practices in the vineyard. Hot water treatment of vine cuttings has been shown to be an effective practice to control the movement of mealybugs from one site to another. Other suggestions include establishing wind traps, planting insecticidal cover or border crops, using site-dedicated clothing and shoes for workers, and avoiding the use of potentially contaminated equipment in the vineyard.

Related to clean stock, the California Department of Agriculture is working on revising the regulations for registration and certification of grapevines. The new regulations are expected to be strict about applying practices (i.e., site isolation, vector control) to keep the certified blocks free of disease infection.

UC Davis is organizing an all-day Leafroll Disease Symposium on June 10. Stay tuned for more details and information through the UC Davis Extension Services.

The control of leafroll spread in California will need to be based on a concerted effort among growers rather than on individual accomplishments. Creating a network of neighboring growers will allow open discussion, education and outreach. The application of cultural practices (especially sanitation and insect control applications) should be coordinated and scheduled by a local committee.

Judit Monis, Ph.D., is director of plant pathology at STA Laboratories in Gilroy, Calif. She has more than 15 years of experience in plant diagnostics and disease eradication, and was previously a supervisory plant pathologist-virologist at the USDA Center for Plant Health Science and Technology. To comment on this article, e-mail edit@winesandvines.com.
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