Grapevine leafroll, a serious disease of grapevines, has a number of viruses associated with it, with grapevine leafroll associated virus type 3 (GLRaV-3), and its predominant vector, the vine mealybug Planococcus ficus, being the most important in South Africa.

Grapevine leafroll, a serious disease of grapevines, has a number of viruses associated with it, with grapevine leafroll associated virus type 3 (GLRaV-3), and its predominant vector, the vine mealybug Planococcus ficus, (Figure 1) being the most important in South Africa. Within the South African Wine Grape Certification Scheme these viruses are successfully eliminated from nuclear planting material by heat treatment and meristem tip culture or somatic embryogenesis. This virus-free material remains susceptible to viruses and become infected again once propagated in the field. Over time this generally negates the advances achieved by planting certified material.Previous studies, during 2000 to 2007, on grapevine leafroll disease spread within 70 vine mother blocks in the Western Cape, South Africa by the author with funding from Winetech, revealed four major spread patterns occurring predominantly (See Wynboer, Technical Yearbook 2004/5, Pages 44 to 47).

These are:

Significant occurrences of directly adjacent infected vines, mainly next to each other in the rows, increasing in number from season to season and then also expanding to vines in adjoining rows (Figure 2). This is indicative of secondary spread (spread within a field) and constitutes the most common and important pattern of leafroll spread in local vineyards. These patterns are caused by the acquisition of virus by mealybugs from infected vines and dissemination over short distances to adjacent or closely adjoining vines of the virus-carrying (viruliferous) mealybug vectors on labourers, on implements or through their own motility.
Random early occurrence of infected vines in newly planted vineyards often associated with specific planting material sources. This spatial pattern being indicative of planting infected propagation material, with virus present originally in either the parent rootstocks or parent scion vine from which the material was derived.

Wynboer - October 2010 - Grapevine leafroll disease control in South Africa Wynboer - October 2010 - Grapevine leafroll disease control in South Africa Wynboer - October 2010 - Grapevine leafroll disease control in South Africa Wynboer - October 2010 - Grapevine leafroll disease control in South Africa Wynboer - October 2010 - Grapevine leafroll disease control in South Africa Wynboer - October 2010 - Grapevine leafroll disease control in South Africa Wynboer - October 2010 - Grapevine leafroll disease control in South Africa Wynboer - October 2010 - Grapevine leafroll disease control in South Africa

The occurrence of leafroll infected vines in newly established vineyards spatially correlated with a preceding infected vineyard at the same site. This pattern is probably caused by mealybug transmission of the virus from infected original host material, not properly removed resulting in volunteer plants (Figure 3), or by survival of viruliferous mealybugs on remnant roots in the period between the removal of the old leafroll infected vineyard and into the lifetime of the newly planted one. This is due to the economically induced imperative of short intervals between removing old vineyards and planting new vineyards, and
Gradients of leafroll infected plants decreasing from edges to the middle of vineyards or across whole vineyards, probably caused by various combinations of spread of the virus in mealybugs disseminated on labourers, implements, birds, wind or through their own motility from adjoining infected vineyards or spatially distant infected vineyards which share labourers and or implements.

While many of the underlying causes of the spread patterns still have to be proven, and a huge amount of quantitative information must still be acquired through scientific studies, these will take many years to complete. In the meantime it is imperative that steps be taken to curb the spread of the disease. By making the assumption that the underlying causes of the spread pattern are indeed those postulated, a number of control strategies can be formulated, and tested, as done in South Africa on Vergelegen Wine Estate. These strategies are based on existing knowledge of the epidemiology of GLRaV-3, the recorded disease spatial distribution patterns observed in South Africa, and the potential methods of dissemination leading to the various spatial patterns.

To control the spread of the disease, each of these potential individual spread mechanisms must be taken into account. It must however be kept in mind that multiple sources of virus and spread mechanisms could occur concurrently, complicating disease spatial distribution patterns observed. Furthermore additional unidentified virus spread or vector dispersal methods potentially exist. These may induce unique spatial patterns that would not have been observed in the earlier studies due to masking by the predominant spatial patterns. These further dissemination mechanisms will only become apparent once the predominant or obvious means of spread have been removed. Combinations of all, or some, of the following control procedures will generally be required to control the disease. Each estate will probably have to emphasize different aspects of the control methods to suit their particular situation. Ideally the control strategies should be applied area-wide where vineyards are in close proximity albeit on different estates, or where implements or labourers are shared amongst estates.

In essence control strategies fall into two broad categories;

reducing/eliminating the leafroll inoculums and
control/reduction of mealybug (mainly P. ficus) numbers and dissemination.

Chemical control of the vine mealybug and other mealybugs and soft-scale insects, known to be vectors of GLRaV-3 is difficult as these insects often have a number of hosts other than Vitis, they tend to be difficult to reach with contact insecticides as they most commonly occur on the lower surface of leaves, under the bark, in crevices, and on the roots of vines, and infestations are often composed of overlapping generations each preferring different parts of the vine, making the timing and targeting of pesticide application difficult. Furthermore, limited options exist with regards systemic insecticides containing different active ingredients. The excessive use of these similar-acting insecticides raises the spectre of a build up of insecticide-resistance. To add to this, Dr. K. Kruger (University of Pretoria, South Africa) has demonstrated in laboratory tests, that a single mealybug individual may transmit the virus to a previously healthy vine (Kruger per. comm.) and therefore control of the viruliferous mealybug must be to extremely low levels, requiring intensive spray programs, with obvious disadvantages to the environment as well as of unacceptable pesticide residues in the berries.

In contrast, the only known hosts of GLRaV-3 are members of the Vitis sp., and consequently the only source from where GLRaV-3 can be acquired and spread is from infected Vitis plants. It is therefore anticipated that a greater impact in disease control will be achieved by reducing virus inoculum (GLRaV-3 infected vines), than to control the vector and its dissemination. Having said this however it is important that concomitant to infected vine removal, control of viruliferous mealybugs must also take place, until disease infection levels are very low or completely removed.

Producers aiming at controlling the disease have to accept that the only long term solution to control is to remove virus-infected vines. This is simpler to perform in practice with red wine cultivars where symptoms are obvious in autumn (Figure 4, 5), than on white wine cultivars where diseased vines are often not discernable from healthy vines visually, with laboratory tests required to identify infected vines. Means of preventing the various methods of spread are discussed below.

To prevent secondary spread from infected vines to adjoining vines the infected plants must be removed in toto (rouged) as soon as the disease or virus presence is determined. Every effort must be made to remove as much of the infected vine as possible, and not allow any new sprouts (Figure 6), from which mealybugs may acquire virus, to shoot. In the case of red wine cultivars visual inspection in autumn is adequate, but in the white wine cultivars laboratory-based tests like enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) must be used. When this is done early enough in the lifetime of a new vineyard, when only single infected plants are present, only the clearly infected vine need to be removed. In older vineyards where some secondary spread has already occurred, as seen by infected plants clustered in close proximity (usually within the row), some surrounding plants may already be infected but do not show symptoms or have sub-detectable levels of the virus. This lack of symptom manifestation and lower virus levels in these vines are due to the fact that they were more recently infected than the initial infected vine from which they became infected, and the virus has had less time to replicate.

It is often not possible to differentiate the latent infected plants from healthy ones, visually or even by laboratory tests. It does not make sense to remove all the vines adjacent to an infected one as a number of them may be healthy. However it is recommended that the plants surrounding clearly infected clusters be treated with a long-acting systemic insecticide according to the manufacturers recommendations eg. imidachlorprid which is efficacious for up to 18 months . This allows the virus in any infected plants to replicate to detectable levels and the disease to manifest itself, when the vine must be removed. Because of the systemic insecticide treatment, the infected vines will not support mealybugs, or scale insects, which could transfer the virus to additional plants by further secondary spread. The number of latent infected surrounding vines requiring treatment with systemic insecticide is positively correlated with the amount of secondary spread that has already occurred. While an exact mathematical relationship has not been determined thus far, it is known that in South Africa the average number of additional infected plants annually per infection foci more than doubles. The minimum number of plants that need to be treated surrounding the infection foci must therefore be at least the number already infected. As spread is predominantly along vineyard rows greater emphasis must be placed on treating the vines adjacent the infection focus within the same row.

The most important method to prevent leafroll spread by infected planting material is to have a efficient certification scheme whereby virus is removed from valuable clones, tested for virus, and virus-free material propagated for as many generations as possible under conditions to ensure they remain virus-free. Producers must be advised to plant only certified material. Unfortunately in South Africa, pre-2002, the majority of such material was propagated in foundation and mother-blocks in grape-growing areas, where they were subjected to leafroll disease pressure from neighbouring infected vineyards. Therefore some vines, previously healthy, became infected again. While obviously symptomatic vines were marked and not collected after the annual inspections as prescribed by the certification scheme at the time, infected white cultivar vines and newly leafroll-infected black-berried cultivars, not yet showing any symptoms, would not have been detected. This resulted in virus infected cuttings being harvested from these plants and the infection spread to newly planted vineyards with this planting material. The infected vines were generally also not removed in mother blocks.

The risk of obtaining infected planting material via the South African Certification scheme has been dramatically reduced over the past seven or eight years by improvements introduced by the largest Plant Improvement Organization, supplying up to 80% of the industries planting material. They have established foundation blocks in new non-traditional grapevine production areas where virus-free vines are not subjected to leafroll disease pressure, have increased virus testing with sensitive detection methods, rigorously remove infected plants and their immediately adjacent neighbours and apply greater mealybug control. Planting material with lower risk is classified as three star materials within the newly introduced star rating of certification material and is more expensive, but represents a sound investment. This planting material is now freely available in South Africa for the most common clones, but is more scarce for the lesser known cultivars, where most vines still being planted in mother-blocks in traditional vine growing areas, albeit with greater emphasis on removal of infected vines, vector control and more tests for viruses.

It is therefore still recommended that, with the exception of 3-star status certified material, all newly planted vines be treated with a systemic insecticide eg. imidachlorprid, from 1 to 6 weeks after establishment. As in the case of preventing secondary spread, the theory behind this being that potentially infected new vines derived from infected parent material would be toxic to mealybugs and therefore could not serve as reservoirs from which the virus could be spread to adjoining plants. Such infected plants would then be removed as soon as they displayed symptoms or tested positive in annual ELISA tests for grapevine-leafroll associated viruses 1, 2, and 3. Use of these insecticides, which commonly have a long withholding period, at this early stage in the vineyards development should not be a problem with regards residues as the vineyards will not be in production yet. Experiments are currently being performed in South Africa to determine whether vines can be protected against mealybugs by treated in the nurseries with the systemic insecticide in advance of being planted in the new vineyard.

Infection in a vineyard caused by infected planting material is difficult to prove when the vineyard is established on a site previously planted to vines as it manifests itself in a similar fashion to transmission from the old infected vineyard (early random infection foci). Usually it can only be inferred when two or more sources/types of planting material is established and the infection is correlated with a specific source or clon

The extremely high cost of establishing vineyards and the relatively long period of time before they become productive often result in old vineyards being replaced by new vineyards without an alternate crop or fallow period. Also total removal of all the root material of the old vines is difficult and expensive and is seldom properly done, resulting in volunteer plants. Under these conditions the new vineyard may rapidly become infected by leafroll disease, due either to the leafroll infected volunteer vines hosting viruliferous mealybugs, or viruliferous mealybugs surviving the relatively short period between old and new vines on residual vine root debris. This spread mechanism would manifest itself as the appearance of single randomly infected vines fairly early in the lifetime of the vineyard, and is difficult to differentiate from disease spread due to infected planting material. It can be differentiated from this in instances where parts of the new vineyard do not overlap with the old vineyard and correlations of the new infections can be made with the old vineyard. This is possibly a common means of perpetuating leafroll disease in South Africa in spite of the use of certified planting material. To prevent this means of spread the producer must remove the old vines as thoroughly as possible (Figure 7). The potential use of systemic herbicides to kill the roots totally before vine removal is currently being investigated, but does not look very promising.

The site must then be left fallow or planted to another crop for one or two seasons before planting certified vines. During this period all volunteer vines must be removed completely. The rational of this strategy is to remove all sources of the virus and hence prevent acquisition of virus by mealybugs. In theory, once infected Vitis plants are removed only a single generation of mealybugs needs to pass away before all mealybugs from the site would be non-viruliferous, as transovarial (from mother to progeny) transmission of GLRaV-3 is not known, and GLRaV-3 is not known to affect host other than Vitis plants. Because of the scarcity of land available for grapevine production in South Africa it is generally not possible for a producer to afford fallow fields on their estates. Under these conditions, the following alternative strategy has been proposed.

The producer must apply intensive chemical control of mealybug in the vineyard designated for removal. Systemic insecticide treatment of infected vines must be implemented directly after the last harvest of that vineyard, and a few months before removal of the vines. This counter-intuitive financial outlay in an old vineyard destined for removal must be viewed as an essential investment in the new vineyard to be planted. Vines must then be removed as thoroughly as possible (the possible use of herbicides are still being investigated) and soil prepared for the new vines. Once planted these vines must also be subjected to treatment with systemic insecticide. Volunteer plants must be diligently removed as they emerge and all new vines showing leafroll symptoms too. The rationale here is to provide a mealybug-free period between re-plantings in instances where a Vitis-free period cannot be achieved. Studies to assess this control strategy as well as to determine the effect of different fallow periods are currently underway.

In order to prevent primary spread (coming from external sources to the vineyard) of grapevine leafroll disease, the various means by which viruliferous mealybugs are disseminated from surrounding infected vineyards to healthy or relatively uninfected vineyards must be controlled. The actual dissemination methods have yet to be proven in South Africa, but it is likely that spread is on implements, on labourers, by wind, and possibly even by birds over both long and short distances. Where vineyards are closely adjoined, the mealybugs may also be disseminated by their own motility.

Older, severely infected vineyards in close proximity to new ones are those that pose the greatest risk for the new vineyard, and mealybugs should be extremely well controlled on such infected vineyards until their productivity declines to the extent that they need to be removed. This may be done through a combination of systemic insecticides as well as release of biological control agents. Where possible distances between adjacent vineyards where the row orientation is parallel may be increased through removal of a row of two of vines. The exact number of rows is not known, but the relatively slow secondary spread of leafroll across rows suggests that the spread would be dramatically reduced with every row removed. Experiments to confirm this are in the planning stages.

To prevent labourers and implements disseminating the disease, vineyards on an estate need to be classified with regards leafroll infection levels. Work should always commence in the healthy vineyards first and then only can it move to infected vineyards. If it is unavoidable to move from infected vineyards, which potentially contain high numbers of viruliferous mealybugs, to less infected or healthy vineyards, methods to remove the mealybugs from clothing or implements must be used. Workers should have a change of clothes, or at least of their overalls. Implements must be washed down with a weak detergent solution or high-pressure water. Alternatively the estate should be partitioned into two, where teams of labourers and implements work 1) either only in healthy vineyards or solely in infected vineyards, 2) or they work on infected or healthy vineyards on different days with changes of clothes and a washing down of implements between the days. While these sanitation strategies are very difficult to apply in commercial estates they should be thought of as interim measures while a large disparity exists between different vineyards with regards the level of leafroll infection. Producers must actively plan replacement of highly infected vineyards with new healthy vines, and removal of individual infected plants in less infected vineyards. Once most vineyards on the farm are leafroll free these sanitary interventions are no longer necessary.

Control of wind dissemination of viruliferous mealybugs, as demonstrated recently in New Zealand, must be with physical barriers, for example natural or artificial windbreaks, where the wind-borne mealybugs can be trapped. Much information is required on this potential mode of dissemination of mealybugs.

Much remains to be done in order to quantify various aspects of the means of disease spread as well as to optimise and test the efficacy of the above control strategies in scientific experiments. The heavy reliance on insecticides, especially the few systemic insecticides available, is not ideal and should only be used as an interim measure while local virus-reservoir levels, and consequently viruliferous mealybugs are still relatively high. Once virus reservoirs numbers are reduced or eliminated and the majority of mealybugs found in the particular vineyards are no longer viruliferous, it is desirable to convert to biological control of mealybugs, where control of populations no longer needs to be absolute. Application of the control strategies proposed above has been done at Vergelegen Wine Estate, Somerset West with great success (Figure 8) and will be discussed in a later article. A number of studies on the use of biological control for vine mealybug and its attendant ant populations have been done and are currently being performed under the auspices of Winetech funding. See for more information on these.

Gerhard Pietersen.
Agricultural Research Council- Plant Protection Research Institute and Department of Microbiology and Plant Pathology, University of Pretoria, 0002, South Africa.

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