By: Judit Monis, Ph.D., Plant Health Consultant
After writing about the presentations at the Unified Symposium last January I promised I would write an article that focuses on the control of the spread of leafroll viruses in the vineyard. Transmission and spread of leafroll viruses have been documented in all grape growing areas worldwide. Specifically, leafroll disease has been reported spread in Australia, Argentina, Europe, New Zealand, South Africa, USA, and other important viticulture areas in the world. Different types of mealybugs and soft scale insects can transmit some species of leafroll virus and Vitiviruses. However, long distance dispersion of viruses (as well as other pathogens) is most effective by producing cuttings and grafting. If you follow my work, you know that I recommend that you plant healthy vines to prevent virus infection in the vineyard.
Grapevine Leafroll Disease
The most important effect of leafroll disease is the production of small grape clusters with uneven ripeness. The grapes have lower sugar content (reduced brix values). Foliar symptoms include downward rolling, reddening or yellowing of leaves and vary depending on the grapevine variety or winegrowing area. The leaf veins may remain green or take many other colors (yellow, purple, red, etc.). Sadly, some wine and tourist magazines use photos of infected vineyards to promote their wine regions. Although all leafroll associated viruses belong to the Closteroviridae (Greek, clostero: thread-like) family, the only species known to be transmitted by insects are found in the Ampelovirus genus (Greek, ampelos: grapevine). Ampeloviruses include the majority of Grapevine leafroll associated viruses (GLRaV-1, -3, and -4). So far, no vector has been reported for GLRaV -2 or GLRaV-7 (but as other viruses are propagated by cuttings). Research has shown that leafroll viruses are able to recombine in mixed infections, generating many variants of similar viruses that scientists define as a quasispecies (i.e., almost a species). These genomic changes have serious implications on virus detection as standard methods may miss infection. Transmission by mealybug and soft scale insects has been reported for GLRaV-1, -3, and -4. Different mealibugs such as the grape (Pseudococcus maritimus), citrus (Planococcus citri), long-tailed (Pseudococcus longispinus), obscure (Pseudococcus viburni) and vine (Planococcus ficus) are able to transmit Ampeloviruses and Vitiviruses non-specifically. Furthermore, work in South Africa has shown that a single individual of vine or long-tailed mealybug is capable of starting a GLRaV-3 infection.
Sampling in The Vineyard:
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, fungi, or nutritional deficiencies can cause similar symptoms in grapevines. Furthermore, vines planted on their own roots do not develop typical symptoms. 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 and samples should be collected from vines late in the summer throughout dormancy.
Two methods can be used for the detection of leafroll-associated viruses: ELISA and RT-PCR. Each method is designed to detect different portions of the virus. ELISA detects the capsid protein (coat or protective cover), and RT-PCR detects the viral genomic RNA (genetic information). Therefore, ELISA and RT-PCR complement each other on the detection of virus and virus variants. ELISA stands for enzyme-linked immuno-sorbent assay, and consists of trapping the virus protective protein on a plastic test plate containing specific viral antibodies. The detection is done through a colorimetric enzymatic reaction (positive samples yield a yellow color). The method is limited to the amount of virus present in the sample (i.e., there is not amplification or danger of laboratory contamination). RT-PCR, is the abbreviation for reverse transcription-polymerase chain reaction. The method involves the amplification or multiplication of viral RNA present in the vine. The process is specific, and utilizes a couple of primers to start the amplification process. Primers are essentially artificial copies of a portion of the viral genome. The amplification is repeated many times, each step or cycle continues to makes more copies of the viral segment. Consequently, RT-PCR is a sensitive technique for the detection of plant viruses.
As mentioned above, the sensitivity and specificity of the detection of viruses associated with leafroll is dependent on the method used for diagnostics. However, detection it is also influenced by the season and part of the vine from which samples were collected as well as the quality of reagents used. While ELISA is known to be less sensitive than RT-PCR, the ELISA has a broader spectrum of detection (i.e., it detects virus variants). On the other hand, RT-PCR (especially Taqman PCR) may be too specific, and could miss the detection of a virus with small changes due to mutation or recombination (e.g., variant species). Lately, fewer quality ELISA reagents are being developed as designing PCR primers is less time consuming. My recommendation is to use ELISA initially (commercial reagents work well for GLRaV-1 to -4) and RT-PCR to confirm infection (or lack of infection). When mapping infection in a white fruited grape block such as Chardonnay or Sauvignon Blanc, ELISA may be the only economical way of determining the leafroll disease status of specific vines as many samples must be tested in a yearly basis.
Leafroll Disease Management and Control Strategies
To manage leafroll disease, it is important to develop a map of virus infected vines. The map will provide information of virus spread and determine the pattern of infection. A random distribution of symptomatic vines would generally be associated with planting infected vines. While symptomatic vines aggregated or clustered in one area of the vineyard block is an indication of virus spread. The initial assessment must be done using a testing lab to determine the presence of a virus species capable of being transmitted. Once the vineyard manager is familiar with symptoms, the mapping could be done by visual observation. It is much easier to determine the characteristic leaf roll disease symptoms in red fruited varieties. In white varieties it may be possible to train eyes to determine the presence of infection but likely will rely on the confirmation of the presence of virus by testing vines.
Effective disease control requires the availability of clean planting stock (i.e., certified disease-free tested). However, to ensure that the vineyard remains disease free, the grower must be aware of the presence of virus infection in neighboring vineyards. It is important to devise procedures to protect a healthy vineyard from potential disease introduction. The presence of insect vectors and ants should be routinely monitored and controlled. Unfortunately, mealybugs are not always easy to observe in the vineyard, however the presence of ants are usually associated with the presence of mealybugs. Special traps (including pheromone traps) are available to monitor the presence of mealybug infestation. The dispersal of mealybugs by birds, wind, field equipment, and/or workers are responsible for long distance spread of virus. Sanitary practices such as fallow periods, sanitation of equipment, and sanitation of field worker’s clothing are recommended to avoid moving disease vectors from one vineyard block to another. I always recommend to start work in the non-infected blocks and move workers to infected blocks at the later part of the day.
Controlling the spread of viruses requires strict protocols for handling vines and performing cultural practices in the vineyard and nursery. Hot water treatment of vine cuttings and grafted vines are effective controlling the movement of mealybugs from one site to another. Other recommended practices include establishing wind traps, planting insecticidal cover or border crops, using site dedicated clothing and/or shoes for workers, and avoiding the use of potentially contaminated equipment in the vineyard.
Ultimately, the removal of infected vines or entire blocks will be key to reducing the source of infection. Guidelines call for rouging (removal of individual infected vines) if there is less than 25% disease incidence and entire blocks (greater than 25% disease incidence). A common mistake is the removal of a portion of the infected vineyard block (see photo 2). This decision is made to avoid production losses at the vineyard. However, in no time, the newly planted vines will become infected by the same virus present in the other portion of the vineyard.
The control of leafroll spread needs to be based on a concerted effort among growers. In California the development of a network of neighboring growers has allowed open discussion of infection status of blocks and applied control measures. The use of cultural practices (especially sanitation and insect control applications) should be coordinated and scheduled to include area-wide treatments as grapevine viruses and their vectors do not know or respect neighboring borders.
Judit Monis, Ph.D. provides specialized services to help growers, vineyard managers, and nursery personnel avoid the propagation and transmission of disease caused by bacteria, fungi, and viruses in their vineyard blocks. Judit (based in California) is fluent in Spanish and is available to consult in all wine grape growing regions of the word. Please visit juditmonis.com for information or contact firstname.lastname@example.org to request a consulting session at your vineyard.