By: Judit Monis, Ph.D.
You probably know that I worked and developed several grapevine disease detection labs in the past. Now I am on the other side and am able to choose the lab that is able to fir with the needs of the specific project I am working on and evaluate their practices.
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 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 for grapevine diagnostic laboratories in USA. Therefore, each laboratory is free to develop their own testing and sampling methodologies.
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 accreditation of testing laboratories should be adopted by the grapevine industry
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, many times, the grower may suspect disease in the vineyard due to specific symptoms. 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. However, microorganisms may compete among each other. Generally, the microbe(s) with the most competitive growth capacity will overshadow slow microbes that grow slower, making the diagnosis difficult or even sometimes 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 (see NGS/HTS section). However, 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, and RT-PCR
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. The detection can be 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 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 = a copy of the viral RNA via reverse transcription) before PCR can start. PCR and RT-PCR are sensitive techniques used for the detection of grapevine pathogens.
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, the ELISA has a broader spectrum of detection but is available only for grapevine viruses and can detect a range of virus variants. On the other hand, PCR can be too specific, and miss the detection of isolates of the same virus even when slightly different. Using both ELISA and RT-PCR consecutively is recommended to reliably detect grapevine leafroll viruses, as each method is designed to detect different portions of a virus.
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 can be used for in-field testing. Although, this test is marketed for use in the field, for reliable results, the assays should be conducted by experienced technicians in a clean laboratory. If a lay person were to attempt to run the assay, they the instructions must be carefully followed, as the steps are complicated and require measuring small quantities of material (microliters of components). In my opinion, it is worthwhile to have an experienced laboratory run these tests. It is expected that laboratory personnel 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 associated virus. Presently, this technique is being applied for the commercial testing of plant and soil samples to detect bacterial and fungal microorganisms. A plant pathologist with expertise in bacterial, fungal, and viral taxonomy is able 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 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 pathogens. 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, the 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 on new planting material and 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. Please visit juditmonis.com for information or contact juditmonis@yahoo.com to request a consulting session at your vineyard.
