By Judit Monis, Ph.D.
Presently there are many laboratories that provide testing services dedicated to the detection and diagnosis of plant pathogens. 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 in USA. 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. Generally speaking, choosing the lab because it offers the best prices or quicker turn-around-times might be a huge mistake.
This article will describe the different methods used for grapevine pathogen diagnostics and discuss the advantages and pitfalls of each of them. Ultimately, I will attempt to convince the reader that the standardization of the diagnostic methods used for the detection as well the development of an accreditation of testing laboratories should be a goal for the future.
Different testing Scenarios
In an ideal world, the nursery or grower is interested in learning that their propagation and planting material is free of important pathogens. But unfortunately, the grower may suspect disease in the vineyard due to specific symptoms. Or as you may have heard me say many times, diseased vines may not always be symptomatic. A knowledgeable plant pathologist will be able to help on statistical sampling as well as what type of laboratory is best suited for each case. Regardless of the purpose for testing, below I will describe the most common methods available for the detection of important bacterial, fungal, and viral infecting pathogens.
Microbiological Culture
Fungal and bacterial pathogens can be cultured and isolated in specialized media (see fig.1). However, microorganisms may compete among each other. Generally, the microbe(s) with the most competitive growth capacity will overshadow microbes that grow slower, making the diagnosis difficult or even impossible. In some cases, the diagnosis will be biased and a laboratory may not be able to report the disease causal agent unless sophisticated molecular methods are used (for details see NGS/HTS section). Generally, in the case of the diagnosis of a declining vine in the vineyard or nursery, the identification of the fungal family (i.e., Diatripaceae species are associated with cankers) or bacterial genus (Agrobacterium species causes crown 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
The Coronavirus pandemic has made some of the terminology that I use in my articles much easier to explain. The general media talks about antibody tests (ELISA is one) and PCR (this is a molecular test). ELISA is the abbreviation for “enzyme-linked immuno-sorbent assay, and consists of sticking the virus coat protein on a plastic test plate that was coated with specific antibodies (Fig.2 Shows the loading of an ELISA plate in the laboratory). 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. PCR, is the abbreviation for polymerase chain reaction. The technique allows the multiplication of viral nucleic acid from the initial titer (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 converting RNA into 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).
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 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 no ELISA has been developed as of yet, I recommend that PCR is performed to amplify at least two different portions of the viral genome.
Single Use Strips for “in house” detection
A molecular single use strip test has been developed for the detection of Grapevine red botch virus (GRBV) that claims it can be used for in-field testing. However, for 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 instructions must be carefully followed. The protocol includes many steps that are complicated and require measuring small quantities of reagents (microliters of components). In my opinion, 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. In the past, a kit was available for the “in house” detection of Grapevine leafroll associated -3 (GLRaV-3). However, many different leafroll viruses can cause leafroll disease and obtaining a negative result for GLRaV-3 would have given the false impression that the vineyard block or sample in question was not infected.
Next Generation or High Throughput Sequencing
The next generation sequencing (NGS) also known as high throughput sequencing (HTS) is a powerful method that allows the laboratory to detect any organism present in a sample.
When NGS or HTS is applied, the complete sequence of the genetic material or microbiome present in the tested plant material or soil can be 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 list the bacteria, fungi, virus, or other organisms (beneficial or pathogenic) 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. 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).
Need for Accreditation of Laboratories
As mentioned earlier, at the moment, there is no accreditation system for laboratories performing grapevine diagnostic testing. The closer we have gotten to these efforts is a ring (comparative) test run by the Lodi Wine Grape Commission. A ring test consists in providing laboratories with “blind” samples of known infection status to determine if the laboratory’s in-house procedures are able to detect the correct infection status in each sample. In the past, while affiliated to various laboratories I was a participant of such ring tests.
In the fall of 2018, the Lodi Wine Commission ran a ring test to evaluate the different labs that offer testing for the diagnostics of grapevine viruses. The laboratories received a large number of homogenized samples that were infected with various grapevine viruses. The results of each laboratory were shared privately with the participant laboratories. To the best of my knowledge no accreditation was granted. While it is a great first step to carry out a ring test with the laboratories, future tests could be improved by providing the laboratory with portions of grapevines rather than a homogenized powder. While it is understanding that homogenized samples may avoid the possibility of uneven distribution of viruses in the grapevine material, the capacity of the laboratory to process whole samples is important. The integrity of the samples would determine if the laboratory is proficient on processing each sample without cross contamination or degrading the potential viruses present.
Conclusions
The standardization of the diagnostic methods for the detection of grapevine pathogens should be a goal for the viticulture industry in the near future. The accreditation of laboratories is of upmost importance for evaluating the reliability of testing labs. Standardization of sampling and testing is common in other fields of food and plant biotechnology. It is puzzling that the grapevine industry has not adopted a system given the importance of this perennial crop. My philosophy is that a vineyard must be planted with the healthiest available material as vineyards must live a long healthy life. If a vineyard is planted with diseased material, its life expectancy is reduced (not to mention the possibility of perpetration and spread of pathogens in the vineyard and neighboring vineyards).
It is encouraging to know that new and more sensitive pathogen detection methods are being developed and applied for the diagnostic of grapevine pathogens. The next generation sequencing or HTS is becoming more affordable and available for the detection at the species level of microorganisms in plants and soil. It is expected that in the near future, these methods will be applied at the nursery and on new planting material to help develop healthy vineyards.
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.
