Vineyard Development: Application of a Sophisticated Testing Technology to Detect Potential Disease

The fall season is the time that vineyard managers and winemakers should start to plan for new vineyard development.  It Is true, this is when you are the busiest with harvest and wine making activities.  However, this is the best time to spot the symptoms associated with most grapevine diseases.

My philosophy is if you wish to develop a healthy vineyard you need to plan ahead.  When planting a new vineyard, unless you are willing to take whatever is left at the nursery (not the best idea), you will need to place your order with a nursery at least one year ahead of the planting season.    With so many different diseases that are not regulated by certification programs, I recommend you hire a knowledgeable plant pathologist (consult with me!) to help you determine the best time to perform vineyard block and vine inspections as well and how and when to collect plant and soil samples for pathogen detection.

Diseases Originate in the Vineyard

Growers must be aware that many grapevine diseases can generate in the vineyard.  If a grower is replacing a vineyard, leaving the land fallow (with no vines) for a long period (2-3 years) may have advantages.  If vines infected with leafroll or red blotch virus (GRBV) were recently removed from the vineyard, it will be important to take some precautions. Some species of leafroll associated viruses (GLRaVs) are transmitted by mealybugs and GRBV is transmitted by the three-cornered alfalfa hopper. It is important to be careful when removing vines, as portions of infected roots that remain in the ground will be a source of reinfection.  Especially, if mealybugs are present in the vineyard block, these will be able to transmit the virus to the new vineyard.   In this situation, it would be impossible to determine if the symptoms in the vineyard a due to a newly vineyard planted with infected material or if it became infected by mealybugs that remained in the vineyard.

Agrobacterium vitis (the crown gall causal agent) and some fungi are soil borne pathogens and can be propagated in nursery material as well as field selections or be present in the soil prior to planting.   Agrobacterium vitis and a diverse group of fungal pathogens are present and sometimes are latent (no symptoms are visible) in vineyards.  For example, the crown gall disease agent can be present in certified planting material without showing symptoms until some stress factor (cracks due to freeze or physical damage) occurs. Obviously, the stress caused by the grafting process is enough to induce typical galling.  Grafted vines commonly display excess callus formation, enlarged graft unions, and galls.  Some symptoms are typical of crown gall disease while others could be difficult to diagnose visually.   To be safe this type of planting material should be tested as most vineyard managers are not able to distinguish between bacterial galling and callusing during the grafting process.

Traditional Diagnostic Methods May Fail to Detect Certain Pathogens

Testing the vines and soil before planting will give an indication of the type of fungal and bacterial organisms present.  Depending on the method used for testing, information of beneficial microorganisms and nematodes present in the vineyard soil could be obtained.

Traditional methods such as microbiological culture for the detection of Agrobacterium and fungal pathogens could fail to detect these pathogens in the laboratory.   While microbiological culture in plates with identification using microscope and/or further biochemical and molecular characterization are still being used, there are some important drawbacks to these methods. The plating of microbes is prone to competition between different fungal and bacterial species.   Generally, the microorganism that grows faster will be identified but may not necessarily be the cause of symptoms of disease.  Even when a more specific method is used for identification (i.e., polymerase chain reaction), the method may not be specific enough to characterize the fungi and bacteria.  For example, there are many Agrobacterium vitis stains that are non-pathogenic and do not cause crown gall disease.

Next Generation Sequencing as a Virus Discovery Tool

The next generation sequencing (NGS) technique also known as high throughput sequencing (HTS) or deep sequencing is able to determine the complete sequence of the genetic material in grapevines.  The data obtained is analyzed with software that is able to compare sequences available in a database and provides a list of the bacteria, fungi, or viruses present in a given sample.  The method can provide relative quantitative data (copy number) of the presence of each organism found.

Initially, the NGS method has been used as a tool to discover new plant viruses.  In 2011, NGS lead to the discovery of the first DNA virus to infect grapevines, Grapevine vein clearing virus a Badnavirus associated with severe vein-clearing and vine decline syndrome in the Midwest.  Subsequently, NGS has allowed the discovery of other DNA viruses: such as GRBV, Grapevine Geminivirus A, as well as many grapevine RNA associated viruses (e.g., Grapevine virus E, F, J).  The application of NGS will continue to allow for the discovery of new viruses.  However, it is important to understand that research must continue to characterize these newly discovered viruses to determine if they are harmful and/or if they pose a threat to vineyard health.

Next Generation Sequencing as a Diagnostic Tool

Recently at the 19th ICVG (International Council for the Study of Grapevine Viruses and Virus-Like Diseases) held in Santiago, Chile in April 2018, experts discussed the application of NGS technology for diagnostic purposes.  Comparative studies have suggested replacing the woody index technique with NGS studies certification and quarantine programs.  Presently, the NGS technology is already being applied for the verification of clean planting stock as well as exclusion of infected material in new variety introductions quarantine and certification programs in Italy and USA.

Commercial laboratories offer the testing of soil and plant tissue using NGS technology to detect bacterial and fungal pathogens in planting stock material and to determine the health status of established vineyards.

The NGS technology has become a powerful diagnostic tool but requires technical knowledge to interpret the complex results. Because of the complexity of the results, expertise is needed to determine which of the microorganisms present in the tested material are damaging to the vineyard health.  I have experienced receiving loads of data (enormous lists of fungi and bacteria) to sort out and determine the relevance of the findings.  The information has allowed me to help my clients make informed propagation, planting, and managing decisions.

Future research will allow us to answer what might be a pathogen copy number required to initiate infection and cause disease symptoms.  In my opinion, grapevine growers and winemakers will benefit when the NGS technology is widely applied to grapevine testing.  The application of new technologies will increase the health of planting material and subsequently decrease of presence of harmful pathogens in planted vineyards and ultimately increase wine quality.  I envision that in the near future the NGS technology will allow certification programs world-wide to exclude pathogenic bacterial, fungal, and viral species from their foundation blocks.

 Judit Monis, Ph.D.  is a California-based plant health consultant that provides support for grapevine disease management including pre-planting disease-test planning, in-field monitoring, and interpretation of laboratory testing results. She can be contacted at juditmonis@yahoo.com