By: Judit Monis, Ph.D., Plant Health and Vineyard Consultant
Presently there are many laboratories that provide testing services for the detection and diagnosis of plant pathogens (disease causing agents). It can be confusing to the grower, vineyard manager, and/or nursery staff to decide which laboratory to choose. My recommendation is to work with a plant pathologist who can provide guidelines towards the best option. At the time, there is no accreditation that is specific for grapevine diagnostic laboratories. Therefore, each laboratory is free to develop their own testing and sampling methodologies.
My expertise in grapevine disease diagnostics and my past work on developing state-of-the-art testing laboratories puts me in a position to evaluate the different choices for my clients. The short answer is that there is no “one lab that fits all”. In my experience, it is best to choose a lab based on the knowledge and capabilities specific to the needs of the project.
This article will describe the different methods used for grapevine pathogen diagnostics and discuss how they are best applied depending on needs. In some cases, you may have to submit samples to different laboratories to test their proficiency. However, different labs may offer different services or may be more knowledgeable in the detection of certain bacterial, fungal, or viral infecting pathogens. Therefore, a lab may be a better fit depending on the testing needs.
Different Testing Scenarios
Just as there are different laboratories that offer testing services, there are different reasons for submitting samples to a testing laboratory. Generally, I am called to a vineyard to determine the cause of a problem after the vineyard was planted. This is not an ideal situation, as I have to “play” detective to determine if the disease originated at the nursery (i.e., plants were infected before planting) or at the vineyard (i.e., transmission occurred after planting). Therefore, planning before planting is of upmost importance. It is best to learn that the propagation or planting material is free of important pathogens before planting either at a vineyard or the nursery.
It is possible that specific symptoms of disease are present and can help decide which tests to run. But it is also possible that infected vines will not show any symptoms at all. Depending on these different scenarios, I am able to help devise the best sampling strategy as well as what laboratory would be best suited for each situation.
Description of Common Laboratory Methods
Microbiological Culture: Fungal and bacterial pathogens can be cultured and isolated in specialized (selective or semi-selective) media. However, microorganisms often compete with each other. For example, saprophytic (those that feed in dead material) bacteria and fungi grow much faster than pathogenic ones. Consequently, the faster organicism’s often outcompete the slow growing ones, making the diagnosis of certain bacterial or fungal pathogens difficult. Here is where laboratory expertise is important to avoid a biased diagnosis. Often times the laboratory may not be able to report the disease causal agent by using a traditional microbiological culture, but could apply sophisticated molecular methods in combination with culturing methods.
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, it is rare for contaminations when this method is applied in the lab. ELISA is an antibody-based test, although it is different from the rapid home tests that detect the flu and COVID 19. 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. TaqMan, is a special type of qPCR that in addition to specific primers uses a specific probe to increase the sensitivity of the test. Because the test is so specific, it is known to miss detection of viruses prone to being mutated 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 & 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 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 be interested in amplifying only fungal sequences). The data obtained will be analyzed using a software that compares the bacterial, fungal, viral, or other microorganisms (beneficial or pathogenic) sequences present in the sample with those available in a database (known sequences that are published). 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).
Why Results Can Vary When Submitting Samples
Throughout my career I have heard the complaint of many clients about obtaining different testing results when samples are submitted to different laboratories. This can be due to operational errors by the lab personnel but can also could be due to submitting the wrong sample type or its labeling. A false positive, is the reporting of a positive result when the sample 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. There are many reasons for the reporting of a false negative: a lack of sensitivity of the assay used, the pathogen(s) are present in low concentrations, uneven distribution of certain pathogens in the vine. Furthermore, submitting the wrong sample will not yield trust wordy results. For example, once we received foliar samples to determine the cause of vine decline suspected to be caused by viruses. However, the panel we ran showed no evidence of infection of the viruses associated with these symptoms.
When I visited the vineyard, during vine inspection I noted that every plant had huge galls caused by Agrobacterium vitis (now called Allorhizobium vitis). Had the correct sample been submitted to the lab we would have been able to provide the correct diagnosis to the grower.
To determine which laboratory is best suited for client’s testing needs, I frequently help perform inter-laboratory comparative tests. This allows my clients to find a laboratory of choice to submit samples regularly. The process involves submitting samples to different laboratories, requesting the same panel or tests and compare the results with what is expected. For the process to work, it is important to collect and submit samples with known infection status. Further, the collector must be able to prepare homogeneous samples collected from the same vine material represented equally in each of the samples submitted to each laboratory. My involvement in the process includes sample collection, interviewing the laboratory manager to obtain more information on the personnel’s knowledge and capabilities, and analyses of results.
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. You can also schedule virtual vineyard consultations. Please visit juditmonis.com for information or contact juditmonis@yahoo.com to request a consulting session.
