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.
