Category: Vineyard Info

By: Judit Monis, Ph.D. – Vineyard and Plant Health Consultant

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.

Virus Testing

  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 & 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 juditmonis@yahoo.com to request a consulting session at your vineyard.

By Igor Sill

Napa Valley is world renowned for its fine wines, and Napa’s wine appellation that surpasses all others is its famed Atlas Peak, Napa’s volcanic mountain wine growing region. Atlas Peak is the highest point in Napa at 2263′ above sea level. Our vineyards rest atop a plateau off of Atlas Peak Road, the main artery that leads you to stunning vistas with purity of air, serenity and diverse wildlife transporting one back in time.

The curvy, windy road remains lined with burnt oak trees and blackened rocks, a reminder of the 2017 Napa wildfires. In between the trees are modest looking estates and pristine vineyards spared from the fire’s devastation. Thankfully, vineyards act as a natural firebreak and prevented further destruction. Even though it’s just minutes from the hustle and bustle of tourist-rich Napa, Atlas Peak remains a quiet oasis, wine country’s hidden gem. Farming these soils is immensely challenging, but well worth the effort as vintners continue Atlas Peak’s reputation for consistently producing the finest wines since 1870.

We are now well into Napa’s deep winter season, a vine’s dormant period and a time when our mountain vineyards generally experiences that quiet tranquility of cold temperatures. This season’s snow and lots of it, froze Atlas Peak’s vineyard soils and halted all farming activity.

The late winter freeze swathed cover crop and vines with snow, six inches of it! With continued forecasts of more freezing rain in March, Atlas Peak is off to a prolonged series of winter storms that should continue to drench vineyards till Spring! All this much needed rain will also preclude irrigation until late summer, which is good for vineyards and good for our planet..

The goal of sustainably regenerative farming is to build healthy soils in harmony with a nature-based approach to decreasing carbon emissions and increasing carbon uptake and storage. This is accomplished primarily through the use of cover crops which can sequester up to two additional tons of carbon per acre while improving soil microbe diversity and increasing water absorption. Planting cover crops such as mustard, legumes, oats and clover significantly improve soil quality by enriching organic matter with nutrients, increasing microbial activity and attracting beneficial arthropods by providing a healthy ecosystem during the dormant season. The regenerative approach strengthens the health of vineyard soil, and yields tastier fruit while reversing the effects of climate change.

As Franklin Delano Roosevelt once noted, “The nation that destroys its soil, destroys itself.”

From the UC Davis CARC and the CITRIS Climate Initiative’s co-Director, Michele Barbato:

“Extreme weather events are increasing in frequency and intensity due to climate change, which impacts soil and growth conditions of vineyard crops. Regenerative agriculture looks to not only stop damaging our ecosystem but actually improves soil health by moving carbon from our atmosphere back into our soils using a variety of agricultural management practices that work in alignment with natural systems. I’m delighted to know that Napa is on the leading edge of environmental awareness and action.” says Professor Michele Barbato, co-director of the UC Davis Climate Adaptation Research Center (CARC) and director of the CITRIS Climate Initiative. “Regenerative farming is a vital solution to both mitigating the emissions of greenhouse gases from the agriculture industry and to limit the damaging impacts of climate change to crops and ensure a resilient and sustainable food system. The UC Davis Climate Adaptation Research Center is looking forward to working with local farmers to quantify the benefits of regenerative farming.” says Professor Erwan Monier, Co-Director of the UC Davis CARC.

In a natural process called “weathering”, Atlas Peak’s volcanic basalt rock terrain naturally absorbs carbon dioxide from the atmosphere.  Some Atlas Peak growers have added additional exposed volcanic basalt rock to this natural weathering process by relocating large amounts of stacked rocks where they surround vineyards and absorb carbon dioxide.

The 2017 Atlas Peak fires, and several more recent Napa and Sonoma fires, confirmed the need for reforestation to keep carbon absorbed and stored.  Post fires, many Atlas Peak growers planted new oak trees and carbon sequestering coyote shrubs in their vineyards to restore and capture carbon dioxide. Turns out that while Atlas Peak vines, trees, and shrubs capture carbon dioxide as they grow, they also provide a natural fuel source in a process called “bioenergy generation,” absorbing and holding carbon for a longer time.

It all starts with the burning of biomass, such as vine cuttings left over from pruning, to produce Biochar, a charcoal-like substance that stores carbon. Biochar is then raked into the soil to keep carbon out of the atmosphere for longer periods while it improves our soil’s health and sustainability. A recent Columbia Climate School scientific report highlighted both positive and negative impacts of climate change on plant growth. By proactively building soil health, growers are preparing their vineyards for changing climatic conditions, so that they can minimize negative impacts and capitalize on a potential CO2 fertilization effect.

Another component of responsible farming is that one honors farm workers fairly by paying a livable wage, which tends to be much higher than the national average, given Napa’s higher cost of living. Of course, nearly every aspect of this regenerative farming costs more than conventional farming and using organic compost preparations with natural nutrients versus chemical-based fertilizers, manual weed removal rather than spraying herbicide, all drive up the total costs. The economics matter, and Napa’s grape growers have had a tough time of it lately given Napa’s rigorously regulated agricultural compliance. Over these last year’s though, Napa growers have embraced their responsibility, and the fiscal requirements that come with it, to preserve their soil’s health for our generation and the many generations to come. The payoff of regenerative farming extends well beyond the benefits of establishing a climate-friendly farm to the rising awareness of our environment, protecting our water sources, storing carbon, reducing greenhouse gas emissions and maintaining soil health.

We all agree that we must dramatically cut our carbon dioxide emissions to limit global warming and avoid future climate catastrophes. The wonderful thing is that cutting carbon emissions and redirecting carbon into the soil significantly benefits the quality and health of Napa’s vines, ultimately the crafting of exquisite wines while continually adopting low-cost ways to operate more sustainably.

About the Author:

Igor Sill is living his dream pursuing his passion for regenerative farming on Atlas Peak Mountain in Napa. He’s a commonsense environmentalist, wine lover, winemaker, vintner, writer, Court of Master Sommeliers, attended UC Davis’ winemaking program, a Judge for the International Wine Challenge, London, UK; and holds his masters from Oxford University. Many thanks to Derek Irwin, Sill Family Vineyards’ Agronomist & Enologist, and UC Davis Climate Adaptation Research Center (CARC) and director of the CITRIS Climate Initiative, Michele Barbato.

By: Annie Klodd, University of Minnesota Extension Educator

Under-vine cover crops are managed plants grown underneath the rows of vines. Vineyard managers grow them on purpose to help achieve certain goals in the vineyard. I find them fascinating to talk about because their benefits and challenges are complex and never guaranteed.

  Grape growers working with overgrown vines might bring in competitive cover crops to slow their vigor. Organic-leaning vineyards often use cover crops to suppress weeds in place of herbicides. Those on steep hillsides should think about installing cover crops to stop erosion. If the cover crop is selected and managed well, vineyard managers should find success with these goals for at least several years.

  Other benefits are less straightforward. In some cases, cover crops might help increase soil organic matter and retain nitrogen, but the effects are often less that one might expect. Grasses tend to re-capture the nitrogen they put back into the soil after decomposition, so it may never reach the grapevines. Flowering cover crops will help attract pollinators like bees to the vineyard. This is wonderful as long as you are avoiding insecticides, which can kill those bees congregating around your flowering cover crop.

  Growers considering cover crops should first decide what goals you want to accomplish. Then, learn which types of cover crops to plant based on what will help achieve those goals. For example, a simple perennial fescue mix works wonders for most goals – vigorous fescues can easily outcompete most weeds, slow excess vine growth for several years, and support soil structure while requiring minimal mowing. Flowering species can be incorporated if desired. Vineyards hilling up graft unions each winter should consider annual cover crop species because the soil is frequently disturbed.

Slow Down Overly-ambitious Grapevines

  Is excessive vigor a problem in your vineyard? Are you constantly hedging and thinning to keep them under control? A strong, thick stand of under-vine cover crops might help slow the growth of overly-vigorous grapevines. Plants with dense root systems like perennial grasses compete with grapevines for water, nutrients, and space in the soil. When those roots get established, they tend to take up so much space in the soil that the grapevine roots avoid the top several inches of soil. The top few inches of soil are often the richest in nutrients and organic matter. So, when the grapevine roots are forced out of this area of soil, it can cause the vines to grow more slowly as they have limited access to the buffet of top soil resources.

  To use cover crops for vine vigor management, select a short, densely-growing perennial grass such as fine fescue. Wait to plant it until the vines are at least 3 years old, so that the grass competition does not stunt their early growth.

  There are many fine fescue species to choose from. The species that I have personally worked with is creeping red fescue. Fine fescues are short, only growing about 8 inches tall, and only need to be mowed 1-2 times per season. These grasses are also effective weed suppressors because they form a thick thatch of grass blades along the soil surface, blocking sunlight from hitting the soil surface. During my graduate research on fine fescues in vineyards, creeping red fescue formed a dense tangle of roots in the top 10 cm of soil, forcing the grapevine roots to avoid that area and grow deeper into the soil.

  To plant this cover crop, spread the seeds on lightly cultivated soil under the vine rows. Fescues are cool-season grasses that germinate best in cool, rainy weather. So, plant them in the spring after snow-melt for best results.

Say No to Weeds

  To suppress weeds, the stand of cover crops should be dense enough that it prevents direct sunlight from hitting the soil. Perennial fescues, described above, achieve this well. Clovers do a relatively poor job of densely covering the soil unless they are planted at very high seeding rates. If you want weed suppression but also insect attraction and nitrogen fixing, mix clover in with a fescue seed mix rather than planting it alone.

  Annual cover crops generally germinate and establish much faster than perennial species. They will do a better job of fighting weeds in the three months after planting. The obvious tradeoff is that they must be re-seeded each year. Perennial cover crops are less likely to provide effective weed management in the first year but will perform better long-term if managed well.

  Many annual species can be used for weed suppression if planted at high seeding rates. It is wise to include grasses in an annual cover crop mix if weed suppression is a goal, because they will help cover the soil more densely. Hairy vetch has also been shown to have good weed suppression but can become weedy. Whatever you do, just be sure to select low-growing species.

Cover Crops for Soil Health

  Cover crops improve healthy soil by adding organic matter, reducing erosion, and improving soil structure. Reducing erosion and improving soil structure are always good things in vineyards. But those considering cover crops for organic matter and nitrogen additions should ask themselves two questions first:  1) Will my cover crop add enough organic matter to make a difference? And 2) Does my vineyard actually need higher organic matter and nitrogen, or is my soil already ideal for grapevines?

  Sometimes certain cover crops can help retain or increase soil nitrogen, but this effect does not necessarily benefit the grapevines. If the soil and foliar tests indicate that the vines have sufficient nitrogen already, adding more is not beneficial. Most vineyards I know in the Midwest do not need to increase nitrogen.

  Secondly, even though grass cover crops put nitrogen back into the soil when they break down, their roots tend to take this nitrogen back up rather than releasing it into the soil for the grapevine roots to use. A 16-year study at a Cornell University vineyard found that bark mulch under the rows contributed more nitrogen to the soil than grass or herbicide treatments.

  This same 16-year study also found that bark mulch contributed more organic matter to the soil than cover crops, along with a 10-fold increase in phosphorous. While all plant matter contributes organic matter to the soil, the amount of matter added via cover crops is generally less than large outside inputs like mulch, compost, or manure.

  The ideal soil organic matter content for grapevines is 2-3%. Adding organic matter beyond that may not actually benefit your crop and can lead to excess vine vigor. Therefore, it is OK if a cover crop’s organic matter contribution is small.

  If, after reading this, you think that your vineyard’s soil could use more organic matter and nitrogen, consider cover crops, compost, or wood-based mulch accordingly.

Flower Power or Flower Downer?

  Flowering cover crops attract beneficial pollinators like bees. I am a full supporter of bees. That said, when deciding to add flowering cover crops to your vineyard, consider whether or not you plan to use insecticides going forward. Many broad-spectrum insecticides, including some organic ones, are highly toxic to bees.

  Because flowers attract these beneficial and sensitive insects to the vineyard, pollinator experts at Michigan State University and University of Minnesota have advised against spraying insecticides if flowers are in bloom. If you do use insecticides throughout the season, opt to plant flowering plants outside the vineyard rather than in the rows. Alternatively, you could try growing flowering plants and mow off the flowers before spraying insecticides.

Planting a Vineyard? Hold that Thought

  Just like weeds, thick stands of cover crops in the rows will slow the growth of newly planted vines, causing a delay in yield. In general, I advise waiting to plant cover crops under vine rows after Year 3, or until the grapevines have both cordons established (you should still plant grasses in the aisles during this time).

  The exception to this 3-year rule would be if the vines are excessively vigorous during establishment – in that case, a cover crop could help manage their rate of growth and suppress bull canes. I have seen this occur in a Minnesota vineyard with over 5% organic matter. Due to the extreme vine vigor during Years 2 and 3, I broke my rule and recommended planting an under-vine cover crop during vineyard establishment.

  If cover crops seem like the right choice for you after reading this far, give them a try! Reach out to your local seed representatives and ask for some samples to try in small areas of the vineyard. If it works for you, let your neighbors know!

Sources: 

•    turf.umn.edu

•    Atuche et al.  2011.  Long-term effects of four groundcover management systems in an apple orchard. 

•    HortScience 46(8)

•    Klodd et al. 2016 Coping with cover crop competition in mature grapevines.  Plant and Soil 400: 391-402

•   MSU webinar recording – April 8th, 2021

By: Judit Monis, Ph.D. – Vineyard and Plant Health Consultant

You probably know that there are many options when it comes to laboratory testing services.  It can be confusing to the grower, vineyard manager, and/or nursery staff to decide which laboratory to choose

  I am always asked by clients: “why the results of samples submitted from the same vineyard block yield different results at different laboratories?” There are many different reasons and I will try to clarify some aspects associated with laboratory issues (method/techniquess used) and sample collection that will affect the final disease diagnostic results.    Finally, I will introduce the concept of standardization of diagnostic methods used for the detection of grapevine pathogens. After reading this article, my hope is that you hire a knowledgeable plant pathologist who can determine your best options based on your needs and walks you through the process.

Description of Most Common Laboratory Techniques:

Microbiological Culture: Fungal and bacterial pathogens can be cultured and isolated in specialized media.  However, microorganisms could compete among each other.  Microbe(s) that grow faster will outcompete microbes that grow slower, making the diagnosis of certain bacterial or fungal pathogens difficult.  The diagnosis could be biased or the laboratory may not be able to report the disease causal agent unless sophisticated molecular methods are used in combination with culturing methods. However, in some cases, the identification of the fungal taxonomic family (i.e., species of the Diatripaceae or Botryosphaeriaceae family isolated from a canker) or bacterial genus (Agrobacterium species isolated from a typical gall) may be sufficient to decipher the cause of the problem.  Phytoplasmas (a special type of bacteria that lack cell walls) and viruses cannot be cultured and their identification must be carried out using molecular and serological methods.

  ELISA, PCR, RT-PCR, qPCR: ELISA is the abbreviation for “enzyme-linked immuno-sorbent assay, and consists the binding of a protein (coat protein, in the case of a virus) on a plastic test plate that was coated with specific antibodies.  A positive reaction is seen when there is a change of color in the wells of the test plate (colorimetric enzymatic reaction). ELISA detection is limited to the amount of virus present in the sample, therefore not prone to lab contamination.  During the Coronavirus pandemic you probably heard in the general media talk about antibody tests.  ELISA, although different from the rapid home COVID 19 tests based on immunochromatography, is an antibody test.  PCR, is the abbreviation for polymerase chain reaction (this is a molecular based test).  The technique allows the multiplication nucleic acid from the concentration of pathogen present in the vine. The process is specific, and utilizes copies of small portions of the pathogen’s genome (called primers) to start the 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 nucleic acid is produced. For RNA viruses the detection is done using RT-PCR (RT stands for reverse transcription, a molecular way of copying the RNA to produce DNA).  PCR and RT-PCR are sensitive techniques used for the detection of grapevine pathogens.  Quantitative or Real Time PCR is a modification of PCR that can provide the relative quantitation of the pathogen present in a sample (abbreviated as qPCR and qRT-PCR).

  The sensitivity and specificity of the detection of pathogens can be influenced by the season as well as the part of the vine from which samples are collected. While ELISA is generally thought to be less sensitive than RT-PCR, ELISA has a broader spectrum of detection and can detect a range of virus variants. On the other hand, PCR is very specific, this can be an advantage but also a disadvantage.  If the detection is too specific, it could miss the detection of isolates of the same virus even when small changes (mutations) are present.  This is even more true when TaqMan, a type of qPCR that in addition to specific primers uses a specific probe is applied for the detection of viruses in grapevine samples.   This is why running both ELISA and RT-PCR consecutively is recommended for the reliable detection of grapevine viruses, as each method is designed to detect different portions of a virus.   Since Grapevine red blotch virus is a DNA virus, and ELISA is not available, I recommend that PCR is performed to amplify at least two different locations of the viral genome.

Rapid Tests for the Detection of Grapevine red blotch and Grapevine Pinot Gris Viruses

  A single use strip test based on the recombinase polymerase amplification (RPA) assay has been developed for the detection of Grapevine red botch virus (GRBV) and Grapevine Pinot Gris virus (GPGV).  The manufacturer claims that these tests can be performed in the field. However, to obtain reliable results, the assays should be conducted by experienced technicians in a clean laboratory.  If a lay person were to attempt to run this type of assay, the assay instructions must be carefully followed.  The protocol includes many steps that require measuring small quantities of reagents (microliters).   Thus, it is worthwhile to have an experienced laboratory run these tests.  Laboratory personnel are used to running different protocols and are trained to keep the sample and other materials free of contamination.  Another drawback of these rapid tests is that these are only available for two grapevine viruses.  As I have noted in other articles, the symptoms caused by grapevine pathogens can confused.  For instance, a negative GRBV result, may give a false reassurance that the vines in the vineyard are healthy when they could otherwise be infected with leafroll (GLRaVs), Vitiviruses, a combination of these, and/ or bacterial or fungal pathogens.

Loop-Mediated Isothermal Amplification (LAMP)

  Like PCR, LAMP is a nucleotide amplification method that uses primers to initiate the copying process of the pathogen’s nucleic acid.  It differs however as the reaction often does not require the extraction of nucleic acid and is performed at a constant temperature (isothermal).  These LAMP assays have been developed in South Africa for the detection of GLRaV-3 and at Cornell University for the detection of GRBV.  Training of the methodology for the detection of GRBV was covered in various sessions by the Napa Valley Vineyard Technical Group.  The technique is claimed to be as or more sensitive than PCR.  Because LAMP assays are very sensitive, it is prone to contamination (i.e., yield false positives).  Like RPA, the operator will need to follow carefully a protocol that requires the measurement of very small volumes of reagents.  The technology also requires the initial investment of laboratory equipment and a clean area to perform the operations. 

Next Generation or High Throughput Sequencing:

The next generation sequencing (NGS) also known as high throughput sequencing (HTS) is a powerful method that allows a laboratory to detect any organism present in a sample.

  When NGS or HTS is applied, the complete sequence of the plant genetic material and its microbiome is obtained.  Generally, during the sample preparation, the pathogens specific sequences are enriched to increase the sensitivity of the assay (for example the lab may just amplify fungal sequences).  The data obtained is analyzed with sophisticated software that is able to analyze the bacterial, fungal, viral, or other microorganisms (beneficial or pathogenic) sequences present in the sample.  The method can provide relative quantitative data, generally expressed in percentages, of each organism found.   The NGS has been widely used in research and has allowed the discovery and characterization of important viruses such as Grapevine red blotch virus and many Vitiviruses. Presently, this technique is being applied commercially to test plant and soil samples for the detection of bacterial and fungal microorganisms.  It is recommended that a plant pathologist with expertise in bacterial, fungal, and/or viral taxonomy be available to associate the presence of the microorganisms found with disease symptoms (or potential disease development).

The Results Obtained from Different Laboratories can be Different

  Frequently, the cause of different testing results at different laboratories can be due to operational

errors.  In my career running plant diagnostic testing labs, I can confidently say that I have seen a lot of things that can go wrong during sample processing. It is not my intention to list them here. The key is that the laboratory has knowledgeable personnel capable of catching the mistake before the results go out to the client (i.e., something does not look right, lets repeat the test or investigate further). A false positive, is the reporting of a sample positive for certain pathogen(s) when it really is not infected.  This can be due to contamination in the lab but also due a mix-up in the field or in the laboratory during the process.  False negatives can also occur, and this can be due to the lack of sensitivity of the assay used, problems with sampling (either in the field or laboratory), and/or when the lab technician made a mistake.

  Let’s assume that everything goes well at the laboratory.  The operator uses the best technical skills and applies good quality assurance and control (QA/QC).  An important factor to take into consideration is the quality and type of sample collected in the vineyard submitted to the lab for testing.  It is known that even when sampling from different portions of the same vine, different results can be obtained. The failure to detect a pathogen that is present in low concentration is due to uneven distribution of certain pathogens in the vine   If your goal is to determine which is the best laboratory for pathogen detection, it is important to send samples  with known infection status to each laboratory.  Further, the collector must be able to prepare cuttings of the same vine material represented equally in samples submitted to each laboratory.

  At the time, there is no accreditation that is specific to grapevine diagnostic testing.  Therefore, each laboratory applies their own testing and sampling protocols. These methods were developed and optimized with positive (infected with the pathogen of choice) and negative controls (not infected with the pathogen of choice).  Additionally, a reputable laboratory should use specific internal controls to determine the quality of their processes. 

  The implementation of an accreditation and certification system for diagnostic laboratories would provide an unbiased evaluation of the laboratory processes.  Standardization of sampling and testing is common in other fields of food and plant biotechnology.  It is surprising that the grapevine industry has not adopted an accreditation system given the losses that pathogens cause to this perennial crop. The standardization of the diagnostic methods used for the detection of grapevine pathogens will provide reliable results to stakeholders.  The future goal of the viticulture industry should be to adopt the accreditation of grapevine diagnostic laboratories.

  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.  During the Coronavirus pandemic, you can also schedule virtual vineyard consultations.  Please visit juditmonis.com for information or contact juditmonis@yahoo.com to request a consulting session.