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Spread of Grapevine Leafroll Disease in South Africa - a difficult, but not insurmountable problem


Prof Gerhard Pietersen, Citrus Research International, Pretoria

Summary

Grapevine leafroll disease (Leafroll) is an important disease of grapevines affecting both yield and quality of grapes. It is the most important disease of grapevines in South Africa where spread of the disease is common. While a number of viruses are associated with this disease, Grapevine Leafroll-associated Virus type 3 (GLRaV-3) is the main causative agent in South Africa. Control of this disease has relied mainly on virus elimination within the S.A. Certification scheme for Wine grapes. This Scheme has been very effective in virus-elimination, but, as these virus-free vines are still susceptible to virus, they become infected again once planted in the field. This is because the vine mealybug, a very efficient vector of GLRaV-3, occurs commonly in South African vineyards. A control strategy to prevent the spread of Grapevine Leafroll disease to virus-free material is therefore required. This is most effectively achieved by preventing, or reducing, the most prevalent means of dispersal of virus-carrying mealybugs to healthy vines. To determine what these are, a survey has been conducted locally where Leafroll spread spatial patterns and dynamics have been monitored and analyzed. This showed that secondary spread from infected vines to adjoining vines along a row is the most prevalent means of spread. Control of Leafroll foci by rouging and the use of systemic insecticides are recommended under specific conditions. The second most common means of Leafroll spread appears to be from adjacent infected vineyards, and a number of recommendations to reduce this are made. In the survey, one vineyard showed a spatial pattern suggestive of infection by Leafroll-infected planting material while another one yielded a spread pattern suggestive of Leafroll-infection from a preceding vineyard at that site. While the relative importance of the last two means of spread could not be ascertained, recommendations to prevent spread by these means are discussed. The study has illustrated the complexity of spread of the disease, but has also highlighted a number of means to reduce or prevent this.

The problem

Grapevine Leafroll Disease is an important disease of grapevines worldwide, and causes reduction in yield as well as a deterioration of quality in both wine and table grapes. Symptoms are most obvious in autumn on red wine cultivars, where leaves become red while the midrib and main veins remain green or turn yellow. Leaf margins will often also roll downwards, hence the name of the disease (Figure 1). In white wine cultivars the symptoms are not as obvious as the leaves do not turn red, and the extent of leaf roll varies from severe curling in some cultivars such as Chardonnay, to very mild curling in some other green-berried cultivars such as Chenin blanc. Rootstocks do not display any apparent Leafroll symptoms. The symptoms are caused by a degeneration of the vascular system of the vine, resulting in reduced berry yields, delayed maturity, and poor pigmentation of the berries. All these have negative influences on the wine produced.


Fig 1:

Grapevine Leafroll Disease (hereafter referred to as Leafroll) is the most important disease of grapevines in South Africa and is widespread in all the grape-producing areas of the country. Spread to healthy vines is common (Figure 2). A number of phloem-limited viruses called Grapevine Leafroll-associated Virus types -1 to -9 (GLRaV-1 to GLRaV-9) have been associated with the disease. A recent survey by the ARC-Plant Protection Research Institute (Pietersen and Kasdorf, unpublished) in the Western Cape, South Africa, showed that GLRaV-3 (Figure 3) was present in all surveyed vines whenever Leafroll symptoms were observed. In about 25% of vines with Leafroll symptoms, GLRaV-3 was accompanied by grapevine virus A (a virus not normally associated with Leafroll), or, more rarely, by GLRaV-1, GLRaV-2, or by unidentified spherical viruses. GLRaV-3, present whenever Leafroll symptoms are present, is therefore the most important virus associated with Leafroll locally. Most efforts should therefore focus on the control of spread of this virus, as the other associated viruses are rarely found


Fig 2:

GLRaV-3 is not known to infect any plant genus other than Vitis, and spread is always considered to have occurred from infected Vitis plants. The virus itself cannot be transmitted by mechanical means, for example in the sap of an infected plant during pruning. However, the virus can be spread by grafting infected plant material or by vegetative propagation of infected planting material. The virus can also be transmitted from vine to vine by a number of mealybug and scale insect species. The most common one of these in South Africa is the vine mealybug, Planococcus ficus, a very efficient vector of GLRaV-3 (Figure 4).


Fig 3:

Control strategies for Leafroll

To control plant diseases, some strategy targeted at one or more components of the disease triangle consisting of the plant, the vector and the virus, is usually applied. These interventions, in relation to Leafroll are discussed below.

The plant:
To use changes to the plant as a means of controlling Leafroll is hampered by the fact that natural resistance to GLRaV-3 in Vitis vinifera has not yet been demonstrated. Various transgenic approaches are therefore currently being assessed worldwide and also in South Africa (Prof. J. Burger, Stellenbosch University). These are unlikely to be available for field use within the near future though, due to a number of technical hurdles still to cross, long efficiency-assessment times, as well as the low acceptance of this technology by the public.


Fig 4:

The virus:
Unlike for bacteria or fungi, there are no agro-chemicals applicable to the field situation that can kill the virus selectively within an infected plant. The virus can however be eliminated from plants on laboratory-scale by means of specialized techniques. Valuable clone material is subjected to either heat therapy and meristem tip culture or somatic embryogenesis to eliminate Leafroll-associated and other viruses. This takes place under the auspices of the SA Certification Scheme for Wine Grapes, in accordance with the Plant Improvement Act (Act no. 53 of 1976). Vines derived by this process, the so-called nuclear material, are still susceptible to virus infection. They are therefore maintained under strict vector-free conditions and regularly screened using the most effective techniques available to detect specific viruses. Virus elimination, isolation, and screening of nuclear material have been very successful in eliminating GLRaV-3. No examples have been found of vines either being infected with any of the Leafroll-associated viruses, or showing symptoms of Leafroll while kept in the Nuclear-blocks of the two major plant improvement organizations, which supplied more than 90% of certified material in South Africa during the last decade. However, the small amount of planting material obtainable from Nuclear-block vines is insufficient to meet the needs of the industry. Therefore, mass production of material from these vines is done within the Certification Scheme, first in Foundation-blocks and then in Mother-blocks, from where Certified material is purchased by commercial nurseries. Any one, or all of these phases are conducted in the field to gain sufficient amounts of planting material. The majority of Mother-blocks and Nurseries are currently within commercial production areas where certified vines often become infected with GLRaV-3, in spite of control measure requirements such as isolation distances and virus indexing requirements. Material from Foundation-blocks are screened for GLRaV-3 by virologists using sensitive virus-specific detection methods (ELISA or PCR), whereas Mother-blocks and Nurseries are visually screened for Leafroll symptoms by well-trained, experienced industry inspectors. A number of infected plants however escape detection due to a lag period that exists between the time when a virus infects a vine, and when the virus becomes detectable by virus detection techniques. This situation is not unique to grapevines. Even an instance such as HIV/AIDS, where PCR is also used, being the most sensitive virus detection technique available to virologists, such a lag period exists. Certified material from the Scheme does therefore not guarantee that all material is virus-free. It does however ensure that the planting material is substantially healthy, and represents the best planting material available to producers. Various improvements to the standard operating procedures of the Scheme, aimed at greater Leafroll control in Foundation- and Mother-blocks, are discussed, evaluated and implemented annually as new information on the epidemiology of the disease emerges.

The vector:
Given the limitations of the control strategies based on the plant and the virus discussed above, it is clear that the Certification Scheme must be supplemented by effective control of the vector(s) of GLRaV-3, both within the scheme itself as well as by producers using certified vines. To gain more insight into aspects of virus spread by the vectors, the following studies, all funded by Winetech, have been or are currently being conducted:

  1. Epidemiology of Grapevine Leafroll Disease (Prof. G. Pietersen, Citrus Research International, formerly at ARC-PPRI),
  2. Studies on the biology of Pl. ficus, (Dr. V.Walton, Stellenbosch University, formerly ARC-Infruitec/Nietvoorbij).
  3. Biological control of Pl. ficus (Dr. V.Walton, Stellenbosch University),
  4. Control of Pl ficus by degree-day and pheromone trap monitoring (Dr. V.Walton, Stellenbosch University),.
  5. Transmission properties of GLRaV-3 by Pl. ficus (Dr. K. Krüger, University of Pretoria, formerly ARC-PPRI),
  6. Identification of local vectors of GLRaV-3 (Dr. K. Krüger, University of Pretoria), and
  7. Control of ants in vineyards (Me. P. Addisson, ARC- Infruitec/Nietvoorbij).
  8. Detection of grapevine leafroll in rootstocks (Mr. G.G.F. Kasdorf, ARC-PPRI)

The adult vine mealybug stage is relatively sessile and even the mobile crawlers (first instars) can only move relatively short distances using their own motility. However, viruliferous (virus-carrying) mealybugs may be dispersed over longer distances by wind, implements, laborers, birds and ants tending them. While the relative importance of the different dispersal mechanisms in transmitting Leafroll disease is not known, some studies have yielded indirect evidence for some dispersal mechanisms in vineyards in Spain, Australia, New Zealand and the USA. This is crucial information necessary to control the disease, as different dispersal mechanisms require different methods of control. For example, if dispersal of viruliferous mealybugs by wind is important, then establishing effective wind barriers between infected and healthy vineyards will have a significant effect in preventing the spread of the disease.

Mechanisms of Leafroll spread in South Africa

In order to address the mechanisms whereby Leafroll spreads the ARC-Plant Protection Research Institute, initiated, and has been conducting a research project, funded by Winetech, on the spatial distribution and spatial dynamics (changes in the patterns) of Leafroll disease within Mother-blocks of the South African Certification Scheme for the past three years (2001 to 2003). Initially studies were performed to confirm the correlation between Leafroll disease symptoms and the presence of GLRaV-3 in selected red wine cultivars. Then seventy Mother-blocks between 4 and 7 years old in the Stellenbosch-, Paarl-, Wellington-, Worcester-, and Somerset West grape production areas were selected for analysis. During autumn of each year the relative position of vines showing Leafroll symptoms were recorded and plotted in a XY matrix using the row number and vine position as co-ordinates. To assess the spatial pattern of infected plants, data was analyzed statistically (ordinary runs analysis) to test the significance of infected plants being adjacent to each other within individual rows and vine positions over rows, as well as over the whole vineyard. Where the number of infected plants was sufficiently high mathematical models were used to describe the distribution and to determine the presence of gradients. Randomness, regularity or clumping of infected plants was also tested using statistical techniques widely used by epidemiologists (e.g. Morisita's Index of dispersal).

This study has not yet been concluded, and some results need further analysis and verification before being reported. However, in view of the need to implement control strategies as rapidly as possible to control Leafroll spread in South Africa, preliminary data on the most common spatial distribution patterns of Leafroll, and their potential underlying mechanisms of spread, are presented here, along with some possible means of control. Spatial patterns were interpreted based on current understandings of the transmission of Leafroll and the dispersal of mealybugs, and may need to be modified and refined as more epidemiological information is generated.


Fig 5:

The most common pattern observed, in the majority of the vineyards analyzed, is the statistically significant occurrence of two or more Leafroll infected vines directly adjacent to each other in rows (Figure 5). Newly infected vines typically spread from these vines along the rows in either direction, before spreading to adjacent plants across rows. This means of spread from an infection focus in a vineyard, is termed secondary spread by epidemiologists as it takes place within a vineyard. It may be caused by non-vector spread, for example by adjacent root graft union, but is more likely due to the movement of the virus-carrying (viruliferous) mealybug crawlers 1) by their own motility, 2) on implements, 3) on laborers moving along rows, or 4) by various combinations of these. Experiments to identify the precise methods of secondary spread are being planned. The spread from these foci is relatively slow (in plant pathology terms) and is clearly the consequence of having an initial infected plant at a specific position in the vineyard. Removal of infected vines (rouging) is therefore likely to be a successful means of control of secondary spread, irrespective of the mechanism of spread, as this removes the source of the virus. This control method has been attempted in four Mother-blocks where infected vines were removed annually after a positive ELISA test for GLRaV-1, or -2 or -3 (by KWV-Vititec). In these blocks the number of new vines which became infected was dramatically reduced compared to Mother-blocks in which rouging was not applied. Some new infections in the rouged blocks did occur however, and were often one or two vines away from where infected vines had formerly been. This suggests that, in this example, although the rouging dramatically reduced the secondary spread of Leafroll it did not completely eliminate it. Spread may have occurred 1) from the infected vines shortly before the virus testing was done, resulting in still sub-detectable concentrations of virus in these newly infected vines, or 2) by viruliferous mealybugs left behind after removal of the infected vine that moved to adjoining vines where they transmitted the disease, or 3) because the vine was not completely removed, and viruliferous mealybugs survived on remaining roots and then moved to adjacent vines which were then subsequently infected.

Rouging is not expected to be effective under all conditions. It is only feasible to apply rouging in vineyards where primary infection (that which brings the disease into the vineyard) is low, where the incidence of (secondarily) infected plants is relatively low, and where latent infections (no symptoms visible or negative virus test results but plants are infected already) are not excessive. Rouging may also not be effective in cultivars with long latent infection periods (e.g. green-berried cultivars if using visual assessments). Under these conditions rouging may need to be supplemented by application of a systemic insecticide to make it more effective. Treatment of infected vines with a systemic insecticide prior to removing them will kill viruliferous mealybugs still feeding on the vine and prevent their movement to adjacent plants. Latent infections have been shown during a survey by ARC-PPRI, to occur mainly at vines directly adjacent to infected vines in rows. Therefore, treating potentially infected plants adjoining infected vines with a systemic insecticide at the time of rouging, will allow any virus present in them to replicate to detectable amounts without the vine serving as a source of further infection, as mealybugs will not survive on them. Subsequent removal of such vines can then be done to eradicate the virus from those foci. If total physical removal of vines is not possible, the use of a systemic herbicide to kill all parts of the infected vine may be required, as re-growth of the infected vine (after the effective insecticide period has passed) can serve as a source from where virus can again be acquired and spread by mealybugs. The number of adjoining vines that need to be treated with the systemic insecticide can be gauged by 1) the amount of secondary spread (clumps of infected plants) already detectable, 2) the age of the vines (older vines are likely to have a longer latent period, allowing a longer period in which sub-detectable spread of the virus can occur) and 3) by the numbers of mealybugs present.

A second common spatial distribution pattern observed is a preponderance of Leafroll infected vines at the edges of a vineyard with a diminishing gradient of infected plants towards the middle or other side of the vineyard (Figure 6). This spatial pattern suggests that the Leafroll has been introduced to the vineyard after its establishment from a source external to it and from a specific direction. In most instances the number of infected vines in these gradients increase in the direction of infected, older proximal vineyards. These probably serve as the sources of Leafroll from where mealybugs acquire the virus and spread to neighboring vineyards by their own motility, by wind, by laborers or implements, or once again, by various combinations of these. It is envisaged that studies on the epidemiology of Leafroll will be expanded to correlate these gradients with the potential mechanisms through which they arise (e.g. wind dispersal of mealybugs, dispersal of mealybugs on implements, own motility of mealybugs etc). The possibility also exists that gradients and edge effects detected are not solely due to viruliferous mealybugs being introduced from external sources but that non-viruliferous mealybugs may be introduced or present at higher levels at the edges of vineyards, from where spread of existing Leafroll disease foci would be more rapid. In the absence of direct evidence for the causes of the gradients found some control strategies are proposed:

  1. Plant as far away from old, infected vineyards as practical,
  2. Plant upwind (prevailing summer winds) of old infected vineyards where possible (wind dispersal of vector not quantified on vines),
  3. Establish large blocks (as close to a square shape as possible) rather than small blocks with high edge/inside vine ratios,
  4. Consider wind-rows between vineyards, and
  5. Ensure that all activities involving laborers and implements are done in the youngest, least infected blocks first, and then only in older, infected blocks (especially during summer months, when mealybug crawlers are present)
Failing this, take steps to remove viruliferous mealybugs on laborers and implements between working in infected vineyards and moving to healthy vineyards must be taken such as change of clothing and washing down with detergents.

In applying the above strategies, make the assumption that older green-berried cultivar vineyards are Leafroll infected (since one cannot easily distinguish between Infected/healthy vines based on symptoms only).

A third spatial pattern observed during the study is the presence of randomly distributed Leafroll infected vines (or foci of vines) within young vineyards. This suggests primary spread due to the establishment infected planting material. In view of the lag phase between virus infection and the ability to detect the virus it can be expected that, while Leafroll infection occurs at Foundation-, Mother-block, and Nursery stages, some plant material from the Certification Scheme will be infected. However, primary infection can only be ascribed with certainty to infected planting material when found in newly established vineyards in instances where these vineyards were established on formerly virgin soils and where the presence of these random foci of infection can be correlated with a specific clone/rootstock combination at that site (i.e. One has to discount the possibility of the infection being due to an external factor at the site). Only one Mother-block analyzed during the survey met these criteria (Figure 7). Therefore, while showing that this mechanism of spread of Leafroll does exists, its relative importance in South Africa remains unknown. In order to control this method of spread in red cultivars it is important that nurseries selling vines also improve their control of Leafroll spread at the nursery. It is recommended that only certified planting material be purchased from reputable nurseries, and that this planting material be pre-treated at the nursery with a systemic insecticide effective against mealybugs. Failing this, newly established vineyards should be treated with such systemic insecticides at the time of planting. The systemic insecticide may need to be re-applied in such a manner that it protects the young vine for at least the first two seasons. In this way, if such a vine was latently infected with GLRaV-3, time is given for either the virus concentration to rise to detectable levels or for symptoms to develop, without serving as a source for secondary spread by mealybugs to adjoining healthy vines. Diseased vines must then be rouged during the first two or three seasons.


Fig 7:

An unusual distribution of Leafroll infected vines detected at one vineyard is suggestive of a further means of spread of Leafroll. This vineyard planted in 2000 to a homogeneous source of Cabernet Sauvignon on a 101-14 rootstock, was found to have significantly (P <0.01) greater number of Leafroll infected vines, randomly distributed, in one half of a young vineyard (Figure 8). No obvious adjoining source of infection could be identified and the distribution within the affected half did not have a significant disease gradient. However, the distribution correlated spatially with an earlier vineyard that had a high incidence of Leafroll infected plants, whereas the less affected half was spatially correlated with an area that had lain fallow for a season. This provides some circumstantial evidence that the new vineyard may have become infected with Leafroll from the previously highly infected vineyard at that site. This has major implications for the South African Wine industry, as vineyards are regularly re-established at sites previously planted to grapevines. However the relative importance of this mode of spread in the South African context could also not be ascertained from the survey as this can only be concluded at sites where a new vineyard planted to a single, homogeneous clone/rootstock only partially overlaps a preceding infected vineyard, and only one vineyard met these criteria during the survey. This mode of spread could occur due to the survival of viruliferous mealybugs between removal of the old vineyard and re-establishment of the new one at that site, possibly on some residual vine roots. Trials to confirm this means of Leafroll spread and an assessment of control methods to reduce this have recently been initiated. Theoretically, this mode of Leafroll spread could be controlled 1) by eliminating mealybugs (by treating vines with a systemic insecticide) in the old vineyard prior to removal, 2) lengthening the period that the site lies fallow between plantings, 3) the very thorough removal of old, infected vines and their roots, or herbicide treatment of them, also killing and removing volunteer vines, 4) and then establishing the new block with certified planting material treated with a systemic insecticide followed by rouging for the first two or three seasons (as for the control of spread of infected planting material).


Fig 8:

Conclusions

The information obtained from the survey of spatial patterns induced by Leafroll spread has confirmed the complexity of spread of Leafroll disease. While a number of spread patterns now found confirmed spread patterns detected in other grape- producing countries, circumstantial evidence of additional means of spread of GLRaV-3 viruliferous mealybugs have been obtained. These insights allow for more efficient control of this disease. Producers are however urged to analyze the local spread patterns occurring in their vineyards, incidence of disease, and risk of further infection from surrounding vineyards before applying either mass rouging (which will not always effectively control spread), or large-scale systemic insecticide applications. This, if used injudiciously, may lead to a build-up of resistance of the mealybugs to the insecticide which will severely hamper control strategies in future.

For more information contact:
Prof. Gerhard Pietersen
Citrus Research International/University of Pretoria
gp@cri.co.za

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