Indicate (X) client(s) to whom this final report is submitted. Replace any of these with other relevant clients if required. FINAL REPORT FOR 2011

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1 CFPA Canning Fruit Producers Assoc. Submit to: Wiehahn Victor Tel: +27 (0) SAAPPA / SASPA / SAT Fruitgro Science Submit to: Louise Liebenberg Tel: +27 (0) /1 louise@fruitgro.co.za DFTS Dried Fruit Technical Services Submit to: Dappie Smit Tel: +27 (0) dappies@dtd.co.za Winetech Submit to: Jan Booysen Tel: +27 (0) booysenj@winetech.co.za Indicate (X) client(s) to whom this final report is submitted. Replace any of these with other relevant clients if required. FINAL REPORT FOR 2011 PROGRAMME & PROJECT LEADER INFORMATION Programme leader Title, initials, surname Prof. G. Pietersen Project leader Prof. G. Pietersen Present position Specialist Scientist/Extra-ordinary Specialist Scientist/Extraordinary Professor Professor Address ARC-PPRI c/o Dept. of ARC-PPRI c/o Dept. of Microbiology, Univ. of Pretoria Microbiology, Univ. of Pretoria Tel. / Cell no Fax gerhard.pietersen@up.ac.za gerhard.pietersen@up.ac.za PROJECT INFORMATION Project number WW 07/18 Project title Project Keywords Prevention of grapevine leafroll disease spread between successive vineyards at the same site Grapevine leafroll, fallow, control Industry programme CFPA Deciduous DFTS Winetech Other Vine improvement program Viruses of grapevines Fruit kind(s) Wine grapes Start date (dd/mm/yyyy) 01/04/2004 End date (dd/mm/yyyy) 31/03/2011 (Note: adjust footer insert the project number no, researcher and research institution)

2 Final report 2 FINAL REPORT (Completion of points 1-5 is compulsory) 1. Executive summary Give an executive summary of the total project in no more than 250 words In the South African Vine Certification Scheme field infection by grapevine leafroll disease of Vitis clones from which virus had been eliminated, is a major problem. A number of sources and means of spread have been identified in South Africa through spatio-temporal analyses of grapevine leafroll disease in foundation- and mother-blocks. At one site circumstantial evidence was obtained that leafroll infection of a newly established block occurred due to the presence of grapevine leafroll disease infection with attendant viruliferous mealybugs in the preceding vineyard on that site. Perpetuation of leafroll disease this way may be common, as producers have to utilise old vineyards immediately for new plantings for economic reasons. In this project we intend to confirm the spread of leafroll between two successive vineyard blocks and perform a field experiment to assess control methods such as fallow periods, and systemic insecticide use with or without herbicide to prevent the perpetuation of grapevine leafroll infection from a preceding vineyard. All replicated treatments (fallow, insecticide and herbicide) were conducted in an old, fully infected vineyard. Remaining vines in the old vineyard was removed and nuclear rootstock material established and the following season nuclear scion material was field grafted onto these. All vines were monitored on an individual bases for symptoms and representative vines monitored for mealybugs. All vines were tested by ELISA by personnel of Ernita Nurseries. To data 18 vines were found to display leafroll symptoms and test positive by ELISA. This confirms that and old infected vineyard can serve as the source for spread of leafroll to a new vineyard on that site. Of the infected vines 12 were distributed outside any of the treatments, within trial buffer zones. The numbers of vines in the new vineyard correlating with a specific treatment in the previous vineyard are too low currently to make any inferences regarding which treatments are most effective, and requires monitoring over a number of further seasons. 2. Problem identification and objectives State the problem being addressed and the ultimate aim of the project. The possibility exists that grapevine leafroll spreads between two successive vineyard blocks on the same site. The objective of this study is to confirm this means of spread and to evaluate the potential of control methods such as different length fallow periods between vineyards, systemic insecticide treatment of the vines being replaced and herbicide treatment of the old vineyard, to prevent the perpetuation of grapevine leafroll infection from a preceding vineyard. A replicated, controlled, field trial was conducted to address this. 3. Workplan (materials & methods) List trial sites, treatments, experimental layout and statistical detail, sampling detail, cold storage and examination stages and parameters. Trial site A commercial vineyard consisting of 6466 vines (2.3ha), planted in 1982 to Cabernet sauvignon Parker clone in the Paarl District (S33 o E19 o ) was used as the trial site. ELISA tests ELISA tests were conducted at ARC-PPRI on individual selected vines in the original vineyard. Samples were prepared by collecting six lower leaf petioles from vines to be tested in autumn. Pooled leaf petioles were cut and weighed and extracted in 5X (w/v) 0.1M Tris/HCl, ph 7.6 buffer with 0.01M MgSO 4, 4% polyvinyl-pyrrolidone (PVP-P) and 2% Triton

3 Final report 3 X-100 in filter separated plastic bags using a Homex 6 homogenizer (Bioreba AG, Switzerland). The ELISA utilized is a triple antibody sandwich type (TAS ELISA) capable of detecting grapevine leafroll associated virus type 1 (GLRaV-1), -2 (GLRaV-2) and GLRaV-3 separately or simultaneously (Goszczynski et al., 1997b). Virus specific antibodies were developed at the Plant Protection Research Institute, Pretoria (PPRI) from electrophoretically-separated coat proteins of the respective viruses (Goszczynski et al., 1997a). Commercial goat-anti-rabbit antibodies conjugated with alkaline phosphatase (Sigma, St. Louis, MO, USA) was used for sero-reaction detection. Vines were considered infected when they yielded an absorbance value (405nm) twice that of the healthy controls of a given microtiter plate. ELISA tests were conducted on all vines in the newly established vineyard by Distell annually in autumn by using pools of ten vines as an initial screen, followed by tests on individual vines in instances where a pool was positive. Monitoring of mealybug The spatial distribution of Planococcus ficus throughout the trials site and presence/absence was determined annually. This was done by visual monitoring of the mealybugs on systematically selected vines distributed throughout the trial. Monitoring was done by two persons per vine until live mealybugs were detected up to a maximum of 5 minutes. Presence of ants, honeydew, parasitized cadavers, egg sacs, and live mealybugs were recorded. The presence or absence of live mealybugs was plotted vs. the spatial position of the monitored vine to give a measure of distribution of mealybugs in the old vineyard. Once the new vineyard was established, 2009/2010, monitoring of Planococcus ficus, was also by P. ficus -specific pheromone capsules maintained in a yellow delta traps with replaceable sticky pads (Chempack, Paarl, South Africa) placed in the middle of the trial. Sticky traps were replaced biweekly. P. ficus male counts were made by the ARC- Infruitec- Nietvoorbij. Pheromone capsules were replaced every three months. Position of treatments The lat/long co-ordinates of the corners of all replicated blocks, pathways and surrounds of the trial site were determined using a Trimble GEO XH Hand held differential GPS with post processing was done in The position of leafroll infected vines within the newly established vineyard was recorded using the same GPS and correlated with the treatment applied in the preceding vineyard. Fallow period treatment Fallow treatments consisted of removal of vines from three hierarchical randomized replicated blocks of twenty plants by ten rows. Vines were mechanically removed in the subsequent winter following rains by a chain tied around the stem and ripping out the stem and roots using a tractor. The removed block was kept fallow for either, three, two or one growth season, during which time the position and numbers of volunteer plants were recorded and these plants removed by manual removal by digging out the plant. Herbicide treatment All vines in the replicated (n = 3) 10 rows by 20 vine blocks requiring herbicide treatment were treated with a 10% glyphosate (Roundup, Monsanto, USA) cut-stump application during spring 2007 (October, 2007). Herbicide treated vines were mechanically removed in the subsequent winter following rains by tractor as described above. Insecticide treatment Imidachloprid (Confidor 350SC, Bayer, Germany) was applied to all vines in spring 2007 (September, 2007) in the replicated (n = 3) blocks of 10 rows by 20 vines. Application was by

4 Final report 4 soil drench around the base of stems with 1.5ml product per vine in 100ml water followed by at least 4 litres of water. Establishment of a new vineyard Following removal of the last treatments, all vines were removed in autumn, 2008 (May, 2008) and the entire trial site was prepared for a new vineyard using standard industry practices. The site was ploughed and residual plant material removed and the new vine established directly thereafter in the summer of 2009 (December, 2009). Healthy nuclear US 8-7 which is a cross of Jacquez (Vitis aestivalis Mich. X V. cinereae Engel. X V. vinifera L.) and 99 Richter (V. berlandieri X V. Rupestris) rootstock material, which had been individually tested for GLRaV-1, -2 and -3, was established from rooted canes. Canes that did not take were placed within two months. Nuclear material of Cabernet franc 1J, individually testing negative for GLRaV-1, -2 and -3 were field grafted onto the rootstocks in late November, Monitoring for leafroll in the new vineyard. Vine for vine inspection was done annually in autumn since the establishment of the new vineyard on the trial site. All vines were also tested by ELISA for GLRaV-1, -2, and -3 by Ernita personnel, as described above. Prevention of primary virus spread from surrounding vineyards and secondary spread in the new vineyard Surrounding vineyards were removed shortly before or after removal of all the vines in the trial site and healthy vineyards established on these sites. The adjacent vineyard to the north was removed in 2006 and those to the south and east in 2010, resulting in the removal of all vineyards in the vicinity of the trial. Leafroll infected vines in the new vineyards on the trial site were treated by imidachloprid in spring of the season directly after they were observed and tested positive by ELISA in order to prevent them serving as sources of infection for secondary spread of leafroll. Volunteer vines in vineyards and feral vines growing in the proximity of the new vineyard were annually recorded and were removed following rain in the winter by digging them out 4. Results and discussion Various vineyards were assessed for their suitability for this trial. Criteria that the vineyard had to comply with was 1) had to be planted to a single black-berried cultivar clone/rootstock combination, 2) it had to be 100% grapevine leafroll disease infected, 3) it had to have a history of Planococcus ficus (Mealybug) infestation, 4) it had to have dimensions large enough that at least twenty treatment plots of about 200 vines each could be accommodated, 5) the producer had to agree not to apply any insecticides to the vineyard over the trial period, 6) the producer had to agree to a phased removal of some treatments over a four year period, 7) the producer had to agree to replace the vineyard directly after removal of the last treatment with a further black-berried cultivar, using only certified planting material, 8) the vineyard should be some distance from any adjoining vineyards, 9) the producer would have to agree to removing of treatments, maintenance and normal cultivation of vines on the trial site, and 10) removal of volunteer hosts where requested. During 2004 a vineyard was identified as a site suitable for this trail, by collaborators from Distell (Hannes vn Rensburg, Tossie and Marietta Louw). It consisted of 6466 vines (2.3ha), planted to Cabernet sauvignon Parker clone in the Paarl District (S33 o E19 o ) in It was confirmed that the site was suitable with regards leafroll infection having a 100% infection (visually assessed on a vine- for-vine basis). The virus present was shown to be GLRaV-3 by testing with IEM and ELISA on a sub-sample of vines from the vineyard. No insecticides had been applied in this vineyard since its establishment in 1982, and mealybug control was by natural predation and parasitism of mealybugs. Prior to the trial, high levels of

5 Final report 5 grape mealybug, distributed throughout the vineyard were typically observed annually until January/February after which the numbers of mealybugs observed dramatically declined. By harvest time mealybugs were seldom a problem (B. Faure, personal communication). As Distell magnanimously agreed to all the onerous requirements of this trial it was ideal for the trial. A spatial evaluation of mealybug presence/absence was done annually to confirm the presence and distribution of the vector throughout the life of the old vineyard. Mealybug was consistently found in the old vineyard on remaining vines (figure 1). Of some concern was that mealybugs were detected in the Imidachloprid-treated replicated blocks slightly more than 3 months after treatment. These vines were removed 6 months later, and while no mealybug monitoring was done it is anticipated that the treatment should have taken effect by then, with a total of nine months allowed for effective imidachloprid uptake.

6 Final report 6 Figure 1: Spatial representation of the presence (tall red bars) or absence (short green bars) of mealybug on the stems and foliage of individual remaining vines in the trial site. Monitored for live mealybugs was done for five minutes per vine. A = 2004/5; B =2005/6; C = 2006/7 and D = 2007/8.

7 Final report 7 Figure 2: Aerial image of position of grapevine leafroll infected vines obtained in 2011 in a newly established Cabernet franc vineyard in relation with the position of treatments performed on the preceding 100% infected leafroll infected vineyard. In order to ensure that planting material was leafroll free, planting material derived directly from nuclear material, which is tested annually and kept in an insect-free environment was used to establish the new vineyard. Unfortunately this meant that rootstocks were first established in the vineyard and a season later the scion grafted onto it. This is not normal industry practice were rooted grafted canes are planted directly. As mealybugs do not appear to thrive on rootstocks this deviation from industry norms may have reduced the potential of spread of leafroll from the old infected vineyard, and negatively influenced the trial. For two seasons following the establishment of the new vines no mealybugs were observed. It is not known if this was due to the vine consisting of only the rootstock in the initial season or if this is normal in newly established vineyards, were predation and parasitism is probably much more effective as the mealybugs are generally more exposed on the vines. In May, 2011, 18 leafroll infected plants were observed and also tested positive by ELISA amongst the vines in the newly established trial vineyard. A correlation of the position of the leafroll infected vines with that of treatments done in the preceding, 100% leafroll infected vineyard, revealed the following: One leafroll infected vine was found in one of the replicates which lay fallow for only 1 season. Similarly, a single leafroll infected vine was also found in one of the replicated blocks which had lain fallow for 2 seasons. However no infected vines were present in any of the replicates which had lain fallow for 3 seasons. No infected vines were present in positions that had been Herbicide treated while 2 leafroll infected vines were found in the

8 Final report 8 Imidachloprid-treated sites. In the replicates which had been re-planted without any treatment, 2 infected vines were observed, while in the buffer zones 12 infected vines were observed. The relatively low number of infected vines observed preclude any statistical tests for significant differences amongst treatments, but it is expected that within the next season or two, the number of infected plants may be such that such tests can be done. Should the number of infected vines not increase significantly the trial size will have been too small, and inferences regarding optimal control treatments cannot be made. The trial has achieved is first objective, in showing that spread of leafroll occurs from a preceding infected to a new one established at the same site. This was achieved by removing the possibility that infected vines, randomly spread throughout in the new vineyard was NOT introduced by planting material. The second objective, of obtaining a means of controlling this spread is not yet unequivocally determine. All the interventions, with the exception of Imidachloprid treatment have reduced infections relative to the untreated replicates. The low number of infected plants in the new vineyard however makes conclusive results impossible at this stage and the monitoring of leafroll infectiuon will continue for at least two more seasons. 5. Accumulated outputs Technology development, products and patents Confirmation of a previous vineyard as a source of grapevine leafroll infection of a new vineyard planted on the same site. Preliminary data suggests that all the treatments (various fallow periods, insecticide treatment and herbicide treatment reduce the spread of the disease from the old vineyards. Further monitoring of the trial site over the next few years may reveal which of these treatments is the most effective. The technology to control this means of spread has therefore been developed, but the most optimal method is not determined yet. Human resources development/training Student level (BSc, MSc, PhD, Post doc) Cost to project (R) 1. None None Publications (popular, press releases, semi-scientific, scientific) This project included as an aspect of the following: Popular publications PIETERSEN, G., Beheer van Wingerd rolblaarsiekte in Suid-Afrika. Winelands October, 2010, pg PIETERSEN, G., New Strategies to control leafroll. Australian Grapegrower and Winemaker, 570; June, Presentations/papers delivered This project included as an aspect within each of the following oral presentations:

9 Final report 9 PIETERSEN, G., Tackling the grapevine leafroll disease problem in South Africa. The Dr JE VanderPlanck Memorial Address. Proceedings of the 44rd Congress of the South African Society for Plant Pathology. Magalies Park Country Club January, 2006 PIETERSEN, G., Spatio-temporal distribution dynamics of grapevine leafroll disease in Western Cape vineyards. Pg Extended abstracts of the 15 th Meeting of the International Council for the Study of Virus and Virus-like diseases of the Grapevine (ICVG) 3-7 April, Stellenbosch, South Africa. PIETERSEN, G., Leafroll Control. Progress Report Presented to Winetech, 2 May, PIETERSEN, G., Virus Epidemiology. Winetech Virus Information Day., Stellenbosch, 4 May, PIETERSEN, G., Grapevine Leafroll disease in South Africa. Talk presented to Producers invited by New Zealand Wine, Martinborough, NZ, 6th March, PIETERSEN, G., Current Studies on leafroll control in South Africa. Talk presented to Producers invited by New Zealand Wine, Martinborough, NZ, 6th March, PIETERSEN, G., Grapevine Leafroll disease in South Africa. Talk presented to Producers invited by New Zealand Wine, Napier, NZ, 7th March, PIETERSEN, G., Current Studies on leafroll control in South Africa. Talk presented to Producers invited by New Zealand Wine, Napier, NZ, 7th March, PIETERSEN, G., Grapevine Leafroll disease in South Africa. Talk presented to Producers invited by New Zealand Wine, Gisborne, NZ, 10th March, PIETERSEN, G., Current Studies on leafroll control in South Africa. Talk presented to Producers invited by New Zealand Wine, Gisborne, NZ, 10th March, PIETERSEN, G., Grapevine Leafroll disease in South Africa. Talk presented to Producers invited by New Zealand Wine, Marlborough, NZ, 12th March, PIETERSEN, G., Current Studies on leafroll control in South Africa. Talk presented to Producers invited by New Zealand Wine, Marlborough, NZ, 12th March, PIETERSEN, G., Grapevine Leafroll disease in South Africa. Talk presented to Producers invited by New Zealand Wine, Nelson, NZ, 13th March, PIETERSEN, G., Current Studies on leafroll control in South Africa. Talk presented to Producers invited by New Zealand Wine, Nelson, NZ, 13th March, PIETERSEN, G., Current Studies on leafroll control in South Africa. Talk presented to Producers invited by Golan Heights Winery, Israel, 19th May, PIETERSEN, G., A South African experience in combating/controlling grapevine leafroll disease. Invited Plenary Session Paper for Romeo Bragato Conference, August, 2009, Napier, New Zealand.

10 Final report 10 PIETERSEN, G., Principles and problems related to rouging and replacement Strategies. Invited forum presentation Romeo Bragato Conference, August, 2009, Napier, New Zealand. PIETERSEN, G., Grapevine leafroll epidemiology and Control in South Africa. Invited forum presentation Romeo Bragato Conference, August, 2009, Napier, New Zealand. PIETERSEN G, 4 May, Vredendal, Western cape. Oral presentation at Vinpro/Winetech regional information day. Present two talks; 1) Review of Grapevine leafroll disease research and application and 2) Successful control of grapevine leafroll disease at Vergelegen Wine Estate. PIETERSEN G, 5 May, Malmesbury, Western cape. Oral presentation at Vinpro/Winetech regional information day. Present two talks; 1) Review of Grapevine leafroll disease research and application and 2) Successful control of grapevine leafroll disease at Vergelegen Wine Estate. PIETERSEN G, 6 May, Aan de doorns, Worcester, Western cape. Oral presentation at Vinpro/Winetech regional information day. Present two talks; 1) Review of Grapevine leafroll disease research and application and 2) Successful control of grapevine leafroll disease at Vergelegen Wine Estate. PIETERSEN G, 11 May, Robertson, Western cape. Oral presentation at Vinpro/Winetech regional information day. Present two talks; 1) Review of Grapevine leafroll disease research and application and 2) Successful control of grapevine leafroll disease at Vergelegen Wine Estate. PIETERSEN G, 12 May, Stellenbosch, Western cape. Oral presentation at Vinpro/Winetech regional information day. Present two talks; 1) Review of Grapevine leafroll disease research and application and 2) Successful control of grapevine leafroll disease at Vergelegen Wine Estate. PIETERSEN G, 26 May, Constantia, Cape Town, Western cape. Oral presentation at Constantia Interest Group Present two talks; 1) Principles of Grapevine leafroll disease control, and 2) Successful control of grapevine leafroll disease at Vergelegen Wine Estate. PIETERSEN G, 27 May, Domain Brahm, Windmeul, Wellington, Western cape. Oral presentation to Table Grape Producers. Present two talks; 1) Principles of Grapevine leafroll disease control, and 2) Successful control of grapevine leafroll disease at Vergelegen Wine Estate. PIETERSEN G, 2 June, Wellington, Western Cape. Oral presentation at Terrason Grapevine Training Program. The management of Grapevine leafroll disease. PIETERSEN, G Oral presentation, Control of grapevine leafroll in South Africa. Winetech Virus Day 24 th May, 2011, Stellenbosch. PIETERSEN, G., Oral Presentatiion; Progress on control of grapevine leafroll in South Africa. Uniwines Producers Study Group, Rawsonville, 21 st June, 2011.

11 Final report Total cost summary of project Year CFPA Deciduous DFTS Winetech THRIP ARC-PPRI TOTAL Total cost in real terms for year Total cost in real terms for year Total cost in real terms for year Total cost in real terms for year Total cost in real terms for year Total cost in real terms for year Total cost in real terms for year Total cost in real terms for year TOTAL