WW0643 000302 Final report Halleen (1Sept2017) na Winetech · Aug 2015 Evaluate Oct to Dec 2015 Dec 2016 The effect of the time of clean pruning and treatment of wounds on the incidence

This document is confidential and any unauthorised disclosure is prohibited Consider all findings as preliminary Version 2015

SATI

CFPA

SAAPPA/SASPA

DFTS

Winetech

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___________________________________________________________________

FINAL REPORT

(2017)

1. PROGRAMME AND PROJECT LEADER INFORMATION

Research Organisation Programme leader

ARC Research Team Manager

Project leader

Title, initials, surname

Prof. B. Ndimba Roleen Carstens Dr Francois Halleen

Present position Senior Research Manager

Acting Research Team Manager: Plant Protection

Specialist Researcher

Organisation, department

ARC Infruitec-Nietvoorbij Private Bag X5026 Stellenbosch 7599



Tel. / Cell no. (021) 809 3000 (021) 809 3023 021 809 3040

E-mail [email protected] [email protected] [email protected]

2. PROJECT INFORMATION

Research Organisation Project number

302 (WW0643)

Project title Investigation into the cause of poor budburst and dying of single spurs in Sauvignon blanc and Cabernet Sauvignon

Short title Sauvignon blanc and Cabernet Sauvignon spur dieback

Fruit kind(s) Wine grapes

Start date (mm/yyyy) 01/04/2013

End date (mm/yyyy) 12/2016 (Original = 07/2016)

Key words Sauvignon blanc, Cabernet Sauvignon, spur dieback

Approved by Research Organisation Programme leader (tick box)

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Halleen (Spur dieback) 2 3. EXECUTIVE SUMMARY

Objectives & Rationale

The aim of the study is to investigate the cause of poor budburst and dying of single spurs in Sauvignon blanc (SB) and Cabernet Sauvignon (CS). Methods

Surveys of affected vineyards were conducted and ten dying spurs were collected from each vineyard to conduct fungal isolations in order to determine if specific trunk disease pathogens are associated with spurs that die. Incidence and severity of spur dieback was determined statistically for both cultivars. Field trials to determine the effect of the time of clean pruning on dieback of spurs, vine vigour and the incidence of trunk disease pathogens that infect wounds and the effect of wound protection during clean pruning were also conducted. Additionally, trials were conducted to determine whether newly discovered Diatrypaceae species (Eutypa-like pathogens) isolated from grapevine and other woody hosts were pathogenic on grapevine. During the 2015 season, shoots in the three vineyards used for the clean pruning trial, were also examined for the presence of the bud mite, Colomerus vitis. Key Results

Nineteen SB and 17 CS vineyards were surveyed for the presence of dead spurs during April - May 2013 and April - June 2014, respectively. Isolation results show that grapevine trunk disease pathogens could be implicated in the poor budburst and dieback of spurs. Most of the dying spurs collected were observed to be associated with wounds made during clean pruning. Pruning wounds are portals of entry of trunk disease pathogens. The surveys also identified the improper use of wires, plastic clips and elastic bands/tapes/rope, which are used to train young vines into the required shape (i.e. a vertical trunk and two horizontal cordons running alongside a wire) as a significant contributor to dieback. The time of clean pruning and protection of big wounds did not have an effect on the budding and bunch formation of individual SB and CS vineyards. The incidence of trunk disease pathogens remained similar between the treated and untreated wounds for each pruning time in both 2014 and 2015 seasons. Pathogenicity studies conducted with Diatrypaceae species (Eutypa-like pathogens studied), isolated during the grapevine surveys as well as isolates obtained from alternative hosts growing in close proximity to these vineyards, were pathogenic and individually capable of causing brown vascular discolorations when inoculated artificially into grapevine tissues. Examination of buds revealed differences in bud mite infestation among vineyards. High incidences of infestation by the bud mite (>30 % of buds in each treatment), which requires action to be taken, were found in the CS vineyard in Durbanville, followed by the SB vineyard in Constantia and very low infestation in the SB vineyard in Stellenbosch.

Conclusion/Discussion

A number of factors contributed to the dieback phenomenon observed in SB and CS spurs, including grapevine trunk disease pathogens, bud mites, as well as viticultural practices. Environmental factors did not influence the occurrence of dying spurs as similar percentages were observed between the cooler and warmer areas. The time of clean pruning and protection of big wounds did not have an effect on the budding and bunch formation of individual SB and CS vineyards. Treatment of wounds is still highly recommended.

Halleen (Spur dieback) 3 4. PROBLEM IDENTIFICATION AND OBJECTIVES

The problem was identified by the Western Cape Vineyard Discussion Group and was also identified as a priority within the grapevine industry by the Plant Protection Committee of Winetech. Sauvignon blanc and Cabernet Sauvignon spurs die prematurely and can already be seen on 4-year-old vines. Viticulturists noted that they observe this phenomenon especially in cooler, more humid areas of Grabouw, Constantia and certain areas within Stellenbosch. Some viticulturists are of the opinion that early clean pruning (“skoonsnoei”), as early as April, provided better results as they observed less dieback of spurs. However, during consultations in preparation for this proposal other viticulturists were of the opinion that all pruning actions must be delayed as long as possible. Various people were consulted and it is evident that there is no clear opinion on the cause of the problem, nor possible solutions. Although there are several opinions, nothing is based on scientific data. The objectives of this project:

1) To determine if specific trunk disease pathogens are associated with spurs that die by conducting surveys of affected vineyards.

2) To determine whether environmental factors, i.e. temperature, relative humidity and wind or viticultural practices play a role in the dieback of spurs.

3) To determine the effect of the time of clean pruning on dieback of spurs and vine vigour. 4) To determine the effect of the time of clean pruning on the incidence of trunk disease

pathogens that infect wounds. 5) To determine whether wound protection during clean pruning will affect dieback of spurs. 6) To conduct pathogenicity studies with newly found Diatrypaceae species from grapevine

as well as alternative hosts in close proximity to vineyards. 7) To evaluate the presence of the bud mite, Colomerus vitis, on shoots emerging from the

two buds left during clean pruning.

5. DETAILED REPORT

a) PERFORMANCE CHART (for the duration of the project)

Objectives Milestones (Significant event or stage in a project)

Original Target Date

as per application

Date achieved

To determine the cause of poor budburst and dying of single spurs in Sauvignon blanc and Cabernet Sauvignon

Surveys of affected vineyards: 1) Identify affected vineyards 2) Isolations from affected vines 3) Identify fungal pathogens 4) Isolations from affected vines 5) Identify fungal pathogens

Feb 2013 May 2013 Aug 2013 Feb 2014 March 2014

March 2013 June 2013 Oct 2013 June 2014 Oct 2014

The role of environmental factors and viticultural practices on dieback of spurs

March 2014 June 2014

The effect of the time of clean pruning on dieback of spurs and vine vigour general. Field trial (season 1)

Prune Mid-April to mid-Aug 2014 Evaluate Oct to Dec 2014

Dec 2014

The effect of the time of clean pruning and treatment of wounds on the incidence of trunk disease

May 2015 July 2014 (pathogen ID’s Oct 2015)

Halleen (Spur dieback) 4

pathogens that infect wounds (season 1).

The effect of the time of clean pruning on dieback of spurs and vine vigour. Field trial (season 2)

Prune Mid-April to mid-Aug 2015 Evaluate Oct to Dec 2015

Dec 2016

The effect of the time of clean pruning and treatment of wounds on the incidence of trunk disease pathogens that infect wounds (season 2).

1) Remove wounds 2) Fungal Isolations 3) Fungal ID’s 4) Statistical Analyses

May 2016 Aug 2016 Aug 2016 Sept/Oct 2016 Nov 2016

To determine the pathogen status of newly found Diatrypaceae species

Pathogenicity study July 2016

To determine the incidence of infestation of buds by the bud mite, Colomerus vitis

Evaluate buds for infestation by bud mites

Aug/Sept 2016

Final report July 2016 Aug 2017

Journal Publications Plant Disease (In Press) PDIS-05-17-0738-RE Diversity of Diatrypaceae species associated with dieback of grapevines in South Africa, with the description of Eutypa cremea sp. nov. P. Moyo, L. Mostert, C.F.J. Spies, U. Damm & F. Halleen

Aug 2017

b) WORK PLAN (MATERIALS AND METHODS)

Surveys of Cabernet Sauvignon and Sauvignon blanc vineyards affected by premature dieback of spurs:

Nineteen Sauvignon blanc (SB) vineyards (6 in Grabouw, 8 in Constantia and 5 in Stellenbosch) and 17 Cabernet Sauvignon (CS) vineyards (1 in Grabouw, 5 in Constantia, 2 in Durbanville, 6 in Stellenbosch and 3 in Robertson) between the ages of 4- and 8-years-old were surveyed for the presence of dead spurs during April - May 2013 and April – June 2014, respectively (Table 1). In each vineyard 100 vines were inspected and the number of dead/dying spurs recorded and subsequently, the average percentage of spur dieback calculated (Tables 2 & 3). The incidence and severity of spur dieback were then statistically determined. Incidence of spur dieback, in this case, refers to the percentage of vines affected by spur dieback whereas severity of spur dieback refers to the total number of dead/dying spurs in a vineyard. Ten dead/dying spurs were also collected from each vineyard in order to conduct fungal isolations. The incidence of spur infection, which refers to the percentage of spurs infected with trunk disease pathogens, was subsequently determined.

The collected spurs were immediately taken to the laboratory for investigation and isolations from symptomatic tissue. The spurs were split lengthwise to reveal possible infections. The two

Halleen (Spur dieback) 5 sections were triple sterilised by immersion into 70% ethanol for 30 s, 1 min in 0.35% NaOCl and again for 30 s in 70% ethanol. After sterilisation 12 pieces of tissue were removed aseptically from the edge of each symptom type and plated out onto potato dextrose agar (PDA, Biolab, Midrand, Johannesburg) amended with streptomycin sulphate (40 mg/L) to reduce bacterial growth. All isolations were done under sterile conditions in a laminar flow cabinet. After isolation, dishes were incubated at ± 25 °C for 4 weeks. Fungal cultures were identified based on morphological characteristics and sequencing where necessary.

Field trials to determine the effect of the time of clean pruning on dieback of spurs, vine vigour and the incidence of trunk disease pathogens that infect wounds and the effect of time of wound protection during clean pruning:

Four vineyards aged 4-years-old (1 SB and 1 CS in Constantia, 1 SB in Durbanville and 1 CS in Stellenbosch) were used for these trials during the first season (2014). Vines were clean-pruned every month from mid-May to mid-August 2014 (4 periods) and one half of the wounds made were treated with Trichoderma (Eco77, Plant Health Products) and the other half was left untreated. However, the bearer shoots of all the plants were left and only pruned to two buds in August 2014. The vines were evaluated in spring for the presence of dead or dying spurs. Each spur was evaluated to determine whether both buds emerged or not and the number of bunches formed was noted. The trials were performed in a complete block design with four blocks and eight treatments were applied in each block for each vineyard. Each treatment in each block was replicated 10 times. Percentages of budding (number of buds that emerged/20*100) and the average number of bunches (number of bunches/20) formed per treatment were ultimately determined and used for statistical analyses. Data were analysed using the General Linear Model one-way analysis of variance in SAS (version 9.2), with the budding percentage and average number of bunches as the dependant variables. This was followed by a least significant difference post hoc t test (α = 0.05) to compare means. The treated and untreated big wounds (clean prune wound) from the trial were removed and taken to the laboratory for investigation and isolations. Isolations were conducted to evaluate the incidence of trunk disease pathogens. Data were analysed by ARC Biometry Unit.

During the second season’s trials (2015), only three vineyards could be selected due to the absence of comparable vineyards in the various regions (i.e. clone differences, rootstock differences; based on data obtained from SAWIS), namely one 4-year-old CS vineyard in Durbanville, one SB in Stellenbosch and one SB in Constantia. Clean-pruning started mid-May and continued until mid-August 2015 (4 periods). One half of the wounds made were treated with Trichoderma (Eco77, Plant Health Products) and the other half was left untreated. The bearer shoots of all the plants were only pruned to two buds in August 2015. The treated and untreated clean pruned wounds were removed and subjected to fungal isolations and statistical analyses as described above. Pathogenicity studies conducted with Diatrypaceae species:

Seven Diatrypaceae species were isolated from grapevines during the surveys, five of which were not previously associated with grapevine in South Africa. Surveys conducted in other projects which aimed at identifying trunk disease pathogens on alternative hosts occurring in close proximity to vineyards resulted in seven additional Diatrypaceae species which are reported for the first time in South Africa. Six of the seven Diatrypaceae species found in the grapevine surveys were also found on alternative hosts. Hence, it was important to test the pathogenicity, on grapevine, of species found on alternative hosts to determine whether their existence in close proximity to vineyards poses a threat to grapevine production. To achieve this, three pathogenicity tests were conducted. Pathogenicity Trial 1 Pruning wounds of a 9-year-old Cabernet Sauvignon vineyard were inoculated with ascospores of 23 isolates of Diatrypaceae fungi belonging to eight species, in a statistically laid out trial. Shoots were pruned to two buds and 1000 ascospores applied directly onto the pruning wound.

Halleen (Spur dieback) 6 Each isolate application was replicated 10 times. Only four shoots per vine were inoculated, each with a different isolate. Ascospores were obtained by slicing open perithecia and adding a drop of water before picking up ascospores with a needle as described by Trouillas et al. (2010). Control shoots were inoculated with sterile water. After ten months, the canes were split longitudinally in half through the pruning wounds and the length of discouloration from the point of infection was measured and isolations conducted to determine re-isolation percentages. Pathogenicity Trial 2 One-year-old dormant canes of a 9-year-old Cabernet Sauvignon vineyard were inoculated with mycelia of 52 isolates of Diatrypaceae fungi belonging to 15 species, in a statistically laid out trial. Canes were wounded with a 4 mm cork borer between internodes 3 and 4 and agar plugs with fresh mycelium obtained from the margin of a 14-day-old colony were placed in each wound. Control shoots were inoculated with sterile PDA plugs and inoculated wounds were wrapped with Parafilm. Each isolate application was replicated 10 times. Only four shoots per vine were inoculated, each with a different isolate. Inoculated shoots were then pruned at internode 5. Ten months after inoculation, the inoculated shoots were excised for evaluation. The shoots were cut longitudinally through the inoculation point and the length of discolouration (lesion length) was measured, both upward and downward directions from point of inoculation, to determine the aggressiveness of each isolate and thereafter, isolations were made to determine the percentage recovery of the isolates. Pathogenicity Trial 3 Green shoots of a 9-year-old Cabernet Sauvignon and 11-year-old Sauvignon blanc vineyards were inoculated with mycelia of 52 isolates of Diatrypaceae fungi belonging to 15 species. The trial layout was similar to that mentioned in trial 2 above, except that evaluation was done five months post inoculation. Evaluation of pathogenicity trials Isolations were carried out by first surface sterilising the shoots in 70% ethanol for 30 s followed by 1 min in NAOCl and 30 s in 70% ethanol. There after 12 pieces (approximately 2 x 2 x 1mm) were cut from below the pruning wound (for ascospore inoculated shoots) or from both sides of the inoculation hole in the case of the agar plug inoculations. Four pieces of wood were plated on one plate of potato dextrose agar (PDA, Biolab, South Africa) amended with chloromycetin (250 mg/l) and all plates incubated at 24°C, exposed to approximately 12 h of daylight and 12 h of darkness, for 4 weeks. The Diatrypaceous fungi growing on plates were hyphal-tipped and transferred to new PDA plates for identification to verify if the inoculated strains could be re-isolated. Representative samples of each of the re-isolated species were subjected to molecular identification to confirm that the re-isolated fungus was the same as the one inoculated. Statistical analyses were conducted on the results to determine the extent of aggressiveness. Since Diatrypaceae species are difficult to distinguish morphologically and the co-occurrence of several species in diseased grapevine wood could result in misidentification of casual organisms, quantitative real-time polymerase chain reaction (qPCR) assays, targeting the β-tubulin gene, were developed for the detection and quantification of Cryptovalsa (C.) ampelina and Eutypa (E.) lata in grapevine wood. The developed qPCR assays were found to be reliable and specific when validated using grapevine wood samples artificially inoculated with the target species and the detection limit of both assays was 0.01 ng/µl of target DNA in grapevine wood (P. Moyo, PhD thesis, 2017). Examination of shoots for presence of bud mites:

Shoots emerging from one of the two buds left during clean pruning (in the 2015 season) were collected and taken to the laboratory to determine whether the buds on shoots were infested with mites. In each block, 10 shoots per treatment were examined for the presence of the bud mite, Colomerus vitis. Three buds were examined per shoot and percentage of buds infested by the mites was calculated for each treatment per block.


c) RESULTS AND DISCUSSION Surveys of Cabernet Sauvignon and Sauvignon blanc vineyards affected by premature dieback of spurs:

One-way analysis of variance using the General Linear Model in SAS (version 9.2) revealed that there were no significant differences [Cabernet Sauvignon (P = 0.43) & Sauvignon blanc (P = 0.066)] in incidence of spur dieback among vineyards of all ages and location. Overall, significant differences in spur dieback severity [Cabernet Sauvignon (P = 0.049) & Sauvignon blanc (P = 0.011)] and spur infection [Cabernet Sauvignon (P = 0.010) & Sauvignon blanc (P = 0.008)] between vineyards were found.

One would expect 8-year-old vineyards to be more affected by spur dieback and trunk disease infection compared to 4-year-old vineyards. However, there were no significant differences in spur dieback severity between 4-year-old and 8-year-old SB vineyards from the same location (Figure 1). Spur infection among vineyards of the same age from different locations also did not differ significantly. As expected, spurs of 4-year-old vines were less infected compared to 8-year-old vineyards from the same location with spur infection increasing with the age of vineyards. However, no significant differences in spur infection among vineyards aged 5 to 8 were observed (Figure 2). On the other hand, severity of spur dieback (Figure 3) and incidence of spur infection (Figure 4) were both not significantly different between 4-year-old and 8-year-old CS vineyards from the same area with exceptions being Stellenbosch and Robertson. Furthermore, these variables were not significantly different among same aged vineyards from different locations.

In total 274 trunk disease isolates were isolated from 190 dying and/or dead spurs collected from the 19 SB vineyards surveyed. Pathogens were successfully identified either by enzyme restriction profiles or sequencing. Species of the Botryosphaeriaceae were the most prevalent with 34 % of the isolates belonging to this group. The second most prevalent group was the Diatrypaceae (27 %), followed by the Diaporthales (23 %) and Petri disease fungi (16 %). Figure 5 shows the percentage of SB spurs infected with trunk disease pathogens among vineyards of different ages. Among the Botryosphaeriaceae, Neofusicoccum australe (57 %) and Diplodia seriata (28 %) were the most abundant followed by N. parvum (6 %), Botryosphaeria stevensii (5 %), Botryosphaeria luteum (2 %), N. protearum (1 %) and Lasidioplodia theobromae (1 %). Cryptovalsa ampelina was the most abundant (45 %) within the Diatrypaceae isolates, followed by Eutypa lata (29 %), Eutypella citricola (22 %) and an unidentified Eutypa species (4 %). The new Eutypa species was described as E. cremea (Moyo et al., 2017 Plant Disease, In Press). Diaporthe neotheicola made up the bulk of the Diaporthales isolates (64 %), followed by D. ampelina (14 %), D. ambigua (9 %), D. australafricana (7 %), D. cynaroidis (2 %), D. novem (2 %) and unidentified Phomopsis species (2 %). Phaeomoniella chlamydospora was the most prevalent species (74 %) of the Petri disease fungi with Phaeoacremonium aleophilum contributing 26 %.

A total of 170 dying spurs were collected from CS vineyards and 115 (68 %) of these were found to be infected with grapevine trunk disease pathogens. Similar to SB, most spurs were infected with pathogens belonging to the Botryosphaeriaceae (53 %), followed by the Diatrypaceae (41 %). The Diaporthales and Petri disease pathogens were isolated from 20 % and 29 % of the collected spurs, respectively. The percentages of spurs infected with different trunk pathogens in CS vineyards of different ages can be seen in Figure 6. The most abundant Diatrypaceae species was Cryptovalsa ampelina which was isolated from 23 % of dying Cabernet spurs, followed by Eutypella citricola (17 %), Eutypa lata (6 %), Eutypa cremea (2 %), Eutypella microtheca (1 %) and Cryptovalsa rabenhorstii (0.5 %). Phaeomoniella chlamydospora was the most prevalent species (21 %) of the Petri disease fungi with Phaeoacremonium spp. contributing 8 %. Diaporthe neotheicola was the most dominant species isolated within the Diaporthales (64 %), followed by D. ampelina (22 %), unidentified Phomopsis species (17 %), D. ambigua (7 %), Phomopsis sp. 5 (7 %), D. cynaroidis (3 %) and D. novem (3 %).

Halleen (Spur dieback) 8 This is the first report of Cryptovalsa rabenhorstii, Eutypella citricola, Eutypella microtheca and Eutypa cremea from grapevine in South Africa (Moyo et al., 2017 Plant Disease, In Press). Overall, many spurs were naturally infected with a number of important grapevine trunk disease pathogens and these pathogens could be implicated in the poor budburst and dieback of spurs in Cabernet Sauvignon and Sauvignon blanc. Most of the spurs collected were observed to be associated with large pruning wounds (Figures 7, 8, 9), which are portals of entry of trunk disease pathogens. On cutting open the spurs, dark brown lesions were observed internally (Figures 8, 9). These lesions could be a result of the vines responding to invasion of trunk pathogens. For example, the vines produce phenols and tyloses in response to pathogen invasion or wounding (Mundy & Manning, 2011) which may accumulate and block the vascular system resulting in dieback. Once grapevine trunk pathogens colonise the vine, they produce toxins which are translocated to shoots (Tey-Rulh et al. 1991; Tabacchi et al. 2000; Jim´enez-Teja et al. 2005). It is possible that new buds are susceptible to these toxins and hence the poor budburst of vines. Poor budburst in vines could also be a result of the diversion of the vine’s reserves (e.g. starch) to production of defence compounds which then leads to less resources being used in budding (Mundy & Manning, 2011). However, a common observation that was made among all the vineyards sampled was the improper use of wires (Figures 7, 10), plastic clips and elastic bands/tapes/rope (Figures 10, 11) which are used to train young vines into the required shape (i.e. a vertical trunk and two horizontal cordons running alongside a wire). If left on the vines for too long, they cut into the cordons and spurs, resulting in stressed vines. Any form of stress predisposes vines to pathogen attack and subsequently, lead to the scenario discussed above as a result of pathogen invasion. Both Sauvignon blanc and Cabernet Sauvignon are known as highly susceptible cultivars to trunk diseases and therefore it might be a combination of factors (susceptibility, big wounds, stress) that contribute to the high occurrence of dead spurs. Field trials to determine the effect of the time of clean pruning on dieback of spurs, vine vigour and the incidence of trunk disease pathogens that infect wounds and the effect of time of wound protection during clean pruning:

Statistical analysis revealed that the time of clean pruning and application of Eco77 on clean prune wounds had no significant effect on budding (P = 0.6200) and the average number of bunches (P = 0.067) in Sauvignon blanc vineyards in the first season. Comparing the effect of location on the measured variables in 2014, there was no difference in budding between the vineyards in Durbanville and Constantia (Figure 12) but the Durbanville vineyard had a higher average number of bunches compared to Constantia (Figure 13). There was also no significant difference (P = 0.6848, budding and P = 0.7797, bunch number) between treatments in the individual vineyards. The incidence of trunk disease pathogens from clean prune wounds was not significantly different among the different clean pruning times (P = 0.2204) but isolation of Trichoderma harzianum (Eco77) was significantly different among treatments (P < 0.001) in 2014, with higher isolation incidences of the biocontrol agent found on treated compared to untreated wounds in SB vineyards (Figure 14). Pathogens including species in the Botryosphaeriaceae, Diaporthales as well as Phaeomoniella chlamydospora were found throughout the pruning period in SB vineyards. Significant differences in the incidences of pathogens (P = 0.0001) and Trichoderma sp. (P = 0.0002) were, however, found between the two SB vineyards. Higher incidences of pathogens and Trichoderma were found in Durbanville and Constantia, respectively. Similar to the 2014 season, the time of clean pruning had no effect on the budding of spurs (P = 0.8306) as well as the average number of bunches formed (P = 0.9719) in the individual SB vineyards in the 2015 season. However, there was a difference in budding percentage (P = 0.0008) and bunch formation (P = 0.0132) between the two vineyards. To be precise, high percentage budding and high average bunch formation was recorded in treated spurs in May and July, respectively in Constantia in comparison to Stellenbosch.

Halleen (Spur dieback) 9 In 2015, significant differences in Trichoderma isolation were found between treatments (P < 0.0001) for SB, with treated wounds yielding more Trichoderma than untreated wounds. High Trichoderma incidences were found in Stellenbosch than in Constantia. Comparable to 2014, no significant differences in pathogen incidence between treated and untreated wounds made at the same time were observed. Differences in pathogen incidences were observed between the two vineyards (P < 0.0001), with the vineyard in Constantia yielding more isolates of trunk disease pathogens compare to the Stellenbosch vineyard. Results indicate that there was no significant difference (P = 0.5813) in budding between treatments in individual CS vineyards in 2014 but there was a slight difference (P = 0.310) when comparing different treatments between the two vineyards (Figure 15). Although the Stellenbosch vineyard had a high average number of bunches compared to the Constantia vineyard in 2014 (Figure 16), pruning time and treatment of wounds had no influence (P = 0.8308) on the average number of bunches in each location. Isolation results from wounds made in 2014 also showed that there were no significant differences in the incidence of pathogens among treatments (P = 0.2918) (Figure 17) as well as between the two CS vineyards (P = 0.8875). Pathogens including species in the Diatrypaceae, Botryosphaeriaceae, Diaporthales as well as Phaeomoniella chlamydospora were found throughout the pruning period in CS vineyards. As expected, the incidences of Trichoderma were higher in treated than untreated wounds (Figure 17).

In 2015, only one CS blanc vineyard was used in the trials and results from this vineyard showed that time of clean pruning had no effect on the budding of spurs (P = 0.2266) and bunch formation (P = 0.4070). Similar to 2014, significant differences in Trichoderma isolation were found between treatments (P < 0.0001), with higher incidences of Trichoderma found on treated than untreated wounds. Furthermore, there were no significant differences in the incidence of pathogens among treatments (P = 0.4340) and overall, no significant differences in pathogen incidence were found between treated and untreated wounds made at the same time.

Pathogenicity studies conducted with Diatrypaceae species:

Inoculations of grapevine pruning wounds with ascospores of various Diatrypaceae species (Trial 1) demonstrated the ability of these species to infect wounds shortly after pruning. All isolates tested caused lesions longer than those in control wounds and lesions were similar in length to those caused by isolates of Eutypa lata, with the exception of two isolates of Eutypa cremea (PMM 2408 and PMM 1439). Significant differences (P = 0.0011) in mean lesion lengths were observed among isolates of species. However, these differences were between treatments (isolates) and the control as well as between certain isolates of Cryptovalsa ampelina, Eutypa cremea and Eutypella citricola. Individual strains belonging to species of Eutypella australiensis, Eutypella microtheca, Eutypella leprosa, Eutypa lata and Eutypa consobrina behaved similarly (Table 4). Re-isolation percentages of inoculated isolates ranged from 60 to 100% and isolations from control shoots did not yield any Diatrypaceae species. All isolates inoculated in dormant shoots of CS (Trial 2) were pathogenic and produced brown lesions which extended in both directions from the point of inoculation after an incubation period of 10 months. These lesions were significantly longer than those of the controls. No differences in lesion lengths were observed among isolates of each species, with the exception of the isolate PMM 2647 (Eutypella citricola) which produced significantly shorter lesions compared to the Eu. citricola isolates PMM 2171 and PMM 2140. Although E. lata isolates produced the longest lesions on dormant canes, these lesions were not significantly different from those produced by isolates of C. ampelina, Eu. leprosa and Eu. microtheca (Table 5). All isolates were re-isolated from lesions but no Diatrypaceae species were isolated from control shoots. Re-isolation percentages of isolates ranged from 50 to 100%.

Halleen (Spur dieback) 10 Inoculation of green shoots (Trial 3) of CS and SB with isolates of Diatrypaceae species resulted in vascular discoloration (lesions) in the wood after five months of incubation in the field. Significant differences in mean lesions lengths within species was observed in both CS (P < 0.0001) and SB (P = 0.0005) vineyards. Mean lesion lengths differed among isolates of species including E. lata, C. ampelina, Eu. microtheca, Eu. citricola and Cryptosphaeria (Cr.) multicontinentalis in the CS vineyard. In the SB vineyard, differences in mean lesion lengths were observed among isolates of C. ampelina, Eu. microtheca, Eu. citricola and Eu. australiensis. Overall, isolates produced longer lesions in shoots of the CS vineyard compared to the SB vineyard. Mean lesion lengths ranged from 51.63 to 90.62 mm for CS and 45.82 to 80.07 mm for SB. All isolates produced lesions significantly longer than the control in CS but in the SB vineyard, all but two isolates (Cr. multicontinentalis PMM 2932 and Cr. multicontinentalis PMM 3063) produced lesions longer than those of the control (Table 5). All isolates inoculated in green shoots were re-isolated from the lesions. Re-isolations percentages ranged from 40 to 100% and 50 to 100% for CS and SB, respectively. No Diatrypaceae fungi were isolated from the control shoots. Examination of shoots for presence of bud mites:

Examination of buds revealed high incidences of infestation by the bud mite (>30 % of buds in each treatment) in the CS vineyard in Durbanville, followed by the Sauvignon blanc vineyard in Constantia. Low infestation was found in the SB vineyard in Stellenbosch, with many buds found to be either clean from bud mite or had less than 30 % bud mite infestation. In practice, bud mite control is recommended in blocks where >30 % of buds in the sample are infested.

d) CONCLUSIONS

Nineteen SB and 17 CS vineyards were surveyed for the presence of dead spurs during April - May 2013 and April - June 2014, respectively. Isolation results show that grapevine trunk disease pathogens could be implicated in the poor budburst and dieback of spurs. Most of the dying spurs collected were observed to be associated with wounds made during clean pruning. Pruning wounds are portals of entry of trunk disease pathogens. The surveys also identified the improper use of wires, plastic clips and elastic bands/tapes/rope, which are used to train young vines into the required shape (i.e. a vertical trunk and two horizontal cordons running alongside a wire) as a significant contributor to dieback. Results of the 2014 and 2015 field trials indicate that the time of pruning and application of a biological control agent on clean prune wounds did not have an effect on the budding and bunch formation of individual SB and CS vineyards. A study in Marlborough, New Zealand also found that pruning date (including pruning shortly after harvest) of 4-year-old SB vineyards resulted in minimal effect on carbohydrate reserves, bud break and bunch yield (Trought et al., 2011). The application of the biocontrol agent (Eco77) on clean pruned wounds in both 2014 and 2015 did not result in treated wounds having low incidences of trunk disease pathogens compared to the untreated wounds. Instead, in both cultivars, the incidence of isolation of trunk disease pathogens remained similar between the treated and untreated wounds for each pruning time. To give better protection of wounds against trunk pathogens, biological control agents need time to establish in the wounds before pathogens are introduced (John et al., 2005, 2008). The apparent inefficacy of the biocontrol agents to protect the treated wounds from infection by trunk disease pathogens, could be a result of natural infection of the 1-year-old shoots before pruning and application of the biological control agent and before the biological control agent was able to establish itself and increase its competitiveness in the wounds. Previous studies have shown a direct correlation between the extent of Trichoderma colonisation inside pruning wounds and its ability to reduce pathogen infections (Mutawila et al., 2011). Trichoderma colonisation in the current study was extremely low, namely 2-28% and 2-16% in 2014 and 2015, respectively. Additionally, clean pruning is made on older wood and the wound is usually big. All biological

Halleen (Spur dieback) 11 control efficacy trials thus far conducted in South Africa were conducted on 1-year-old wood (2-bud spur-pruned canes). Results further confirm findings of Van Niekerk et al. (2010) whose spore trapping experiments showed that inoculum of pathogens (Botryosphaeriaceae, Diatrypaceae, Diaporthales) was available throughout the period from June to September in Western Cape vineyards. Although there was no negative effect observed with regard to the time of clean pruning, there is enough evidence that inoculum exists throughout the winter and all wounds created should therefore be protected. Pathogenicity studies conducted with Diatrypaceae species showed that all taxa studied are pathogenic and individually capable of causing brown vascular discolorations when inoculated artificially into grapevine tissues. Although Diatrypaceae species demonstrated the ability to infect grapevine shoots in the inoculation studies, no cankers were observed on the inoculated shoots. It therefore remains unclear whether infections by these species would lead to foliar symptoms typical of Eutypa dieback. It usually takes 3-8 years post infection for Eutypa lata to cause visible symptoms of Eutypa dieback on grapevine. It is likely that a similar delay in symptom expression on grapevine occurs for the newly discovered Diatrypaceae species. Based on results of the inoculation of green shoots of Cabernet Sauvignon and Sauvignon blanc with Diatrypaceae species, it seems that Cabernet Sauvignon is more susceptible than Sauvignon blanc. Previous studies also reported on the fact that Cabernet Sauvignon was shown to have greater susceptibility to E. lata compared to Sauvignon blanc (Péros and Berger, 1994). Moreover, during surveys of vineyards affected by dying spurs, a high number of isolates and diversity of Diatrypaceae species was found in Cabernet Sauvignon compared to the Sauvignon blanc vineyards, despite a lower number of samples examined in the Cabernet Sauvignon vineyards. Nevertheless, species of Diatrypaceae can be considered as a significant threat to the sustainability of grapevine production. These pathogens colonise native and cultivated woody hosts and results of this study show that isolates of Diatrypaceae species collected from non-grapevine hosts (i.e. camphor trees, oaks, Peruvian pepper, seringa, blackwood, rose, willow, figs, persimmons, mulberry, apricots, plum, guava, quince, pomegranate, loquat, lemon, apple, etc.) are pathogenic to grapevine reinforcing a previous report that alternative hosts are sources of Diatrypaceae inoculum for grapevines (Trouillas and Gubler, 2010). Management practices for Eutypa dieback should therefore not only focus on vineyards but also consider non-grapevine hosts occurring in close proximity to vineyards. For instance, in the case of sanitation practices, i.e. removal of dead wood to reduce inoculum sources. Further strategies to reduce incidences of infection by Diatrypaceae species on grapevine should include protection of pruning wounds. A number of fungicides that are effective against E. lata were found to be effective against several Diatrypaceae species (Gramaje et al., 2012). The results of this study therefore provide a basis for future research aimed at managing Diatrypaceae species causing disease on grapevine in South Africa.

Halleen (Spur dieback) 12 Table 1: A summary of the Sauvignon blanc (SB) and Cabernet Sauvignon (CS) vineyards surveyed from April - May 2013 and April – June 2014, respectively.

Location Cultivar Plant date

2005 2006 2007 2008 2009 2010 Grabouw SB SB11O

SB11O

SB316

SB11O SB316G SB11

CS CS169B

Constantia SB SB316 SB11 SB11H

SB316

SB11O

SB11O

SB316C

SB11

CS CS338C CS23A CS169B CS169

CS23A

Stellenbosch SB SB11 SB242 SB11

SB316 SB11R

CS CS46C CS163 CS169B

CS163 CS169A CS46

Durbanville CS CS46 CS163

Robertson CS CS163 CS?

CS169B


Table 2: A summary of the Sauvignon blanc (SB) vineyards surveyed from April - May 2013, average percentages of vines with spur dieback and observations made (remarks) for each vineyard.

Area Cultivar Clone Year planted

Aver. % of vines with spur dieback

Remarks

Constantia SB SB 316C 2009 31 SB SB 11O 2009 78 SB SB11 2009 64 Shoots torn off probably by wind.

SB SB11O 2008 81 Most spurs were damaged by wind and/or suckering SB SB316 2008 48 SB SB11H 2008 75

SB SB11 2007 70 Trellising wires and rubber strings cutting into cordons and spurs

SB SB316 2005 93 Too many big wounds close to spurs. Spurs too long i.e. more than 2 buds

Grabouw SB SB11 2009 98 Typical spur dieback problem. Many spurs broken or damaged by wind and wires

SB SB316G 2008 41

SB SB11O 2007 43

SB SB11O 2005 100 SB SB316G 2005 100 SB SB11O 2005 95

Stellenbosch SB SB11 2009 92 Spurs were extremely short pruned, damaging buds SB SB11R 2009 72

SB SB242 2007 98 Wires cutting into cordons and spurs and some spurs were too long and many broken off

SB SB11 2005 97 SB SB316 2005 99 Spurs unusually long and dying spurs located where cordons are cut

into by trellising wires. Many shoots torn or broken off

Average % of spur dieback 78


Table 3. A summary of the Cabernet Sauvignon (CS) vineyards surveyed from April - June 2014, average percentages of vines with spur dieback and observations made (remarks) for each vineyard.

Area Cultivar Clone Year planted

Aver. % of vines with spur dieback

Remarks

Constantia CS CS169 2010 91 Severe wires and cardboard with wire cutting into arms of vines CS CS23A 2010 67 Many dying spurs observed closer to the mountain where it is windy.

Trellising wires cutting into arms of some vines

CS CS169B 2009 92 Trellising wires cutting into arms of vines

CS CS23A 2007 95

CS CS338C 2006 98 Trellising wires damaging arms and few shoots damaged by wind

Durbanville CS CS163 2010 82 Wire damage to arms/spurs CS CS46 2007 100 Trellising wires and ropes cutting into arms of vines and in most vines

with 2 spurs, often one is dead Grabouw CS CS169B 2007 100 Wires and trellising tape/rope cutting into arms and spurs. Some spurs

were too short Stellenbosch CS CS169B 2010 90 Plastic clips cutting into spurs/arms and shoots broken in a few vines CS CS46 2010 90 Plastic clips cutting into some spurs CS CS163 2008 80 Wires cutting into arms of vines CS CS169A 2008 82 Wire in arms of most vines and big wounds around dying spurs CS CS46C 2006 99 CS CS163 2006 100 Wire cutting into arms of vines Robertson CS CS 2010 85 Trellising wire and rope cutting into arms of vines CS CS169B 2010 60 Wire cutting into few spurs CS CS 2006 100 Trellising wire and rope cutting into arms of vines

Average % of spur dieback 89


Table 4. Mean lesion length caused by Diatrypaceae species from South Africa in Cabernet Sauvignon pruning wounds. Species Strain number Host Location Lesion length % re-isolation

Cryptovalsa ampelina PMM 1352 Vitis vinifera Darling 27.81 b-e 70

Cryptovalsa ampelina PMM 1040 Vitis vinifera Durbanville 36.70 a 100

Cryptovalsa ampelina PMM 1436 Ficus carica Stellenbosch 25.90 b-e 90

Cryptovalsa ampelina PMM 2637 Cinnamomum camphora Grabouw 31.35 a-c 80

Eutypa consobrina PMM 1961 Quercus sp. Durbanville 23.89 de 60

Eutypa consobrina PMM 2104 Blackwood Constantia 25.13 b-e 60

Eutypa lata PMM 1043 Vitis vinifera Durbanville 31.12 a-d 90

Eutypa lata PMM 2410 Vitis vinifera Bonnievale 26.76 b-e 80

Eutypa sp. PMM 1354 Vitis vinifera Darling 31.36 a-c 80

Eutypa sp. PMM 2408 Vitis vinifera Bonnievale 22.05 ef 100

Eutypa sp. PMM 3072 Morus sp. Franschhoek 27.92 b-e 90

Eutypa sp. PMM 1439 Quercus sp. Durbanville 21.30 ef 80

Eutypella australiensis PMM 2640 Unknown Hout Bay 30.21 a-d 60

Eutypella australiensis PMM 2641 Dalbergia sp. Constantia 27.48 b-e 70

Eutypella citricola PMM 1441 Vitis vinifera Durbanville 32.46 ab 80

Eutypella citricola PMM 2645 Vitis vinifera Durbanville 30.87 a-d 100

Eutypella citricola PMM 2411 Diospyros kaki Bonnievale 24.28 c-e 100

Eutypella citricola PMM 2107 Ficus carica Bonnievale 26.59 b-e 70

Eutypella leprosa PMM 2642 Ficus carica Hout Bay 29.64 a-d 70

Eutypella leprosa PMM 2102 Quercus sp. Constantia 30.17 a-d 80

Eutypella microtheca PMM 3084 Vitis vinifera Calitzdorp 24.96 c-e 70

Eutypella microtheca PMM 3082 Morus sp. Franschhoek 30.67 a-d 90

Eutypella microtheca PMM 2796 Melia azedarach Wellington 25.10 b-e 70

PDA plug - - - 14.74 f -

* Means followed by the same letter in the same column are not significantly different (P < 0.05).


Table 5. Mean lesion length caused by Diatrypaceae species from South Africa in green and dormant shoots of Cabernet Sauvignon (CS) and Sauvignon blanc (SB). Species Isolate Host origin Location Dormant shoots Green shoots

Cabernet Sauvignon Cabernet Sauvignon Sauvignon blanc

Lesion length % re-isolation



Cryptovalsa ampelina PMM 2646 Vitis vinifera Rawsonville 64.05 a-e 100 74.93 b-l 80 70.99 a-h 100

Cryptovalsa ampelina PMM 2116 Prunus armenica Bonnievale 66.94 ab 100 87.19 a-c 100 80.07 a 100

Cryptovalsa ampelina PMM 1038 Psidium guajava Klaver 66.48 a-c 90 79.61 a-j 100 68.81 a-j 100

Cryptovalsa ampelina PMM 1240 Punica granatum Bonnievale 63.45 a-e 80 82.77 a-h 90 61.96 b-n 100

Cryptovalsa ampelina PMM 2891 Schinus molle Durbanville 61.48 a-h 90 88.15 ab 100 76.70 ab 100

Cryptovalsa ampelina PMM 1239 Eriobotrya japonica Darling 59.13 a-k 90 83.87 a-f 100 65.03 a-m 80

Cryptovalsa ampelina PMM 1035 Rosa sp. Vredendal 61.98 a-g 70 71.57 e-l 90 68.33 a-k 100

Cryptovalsa rabenhorstii PMM 2125 Vitis vinifera Stellenbosch 60.31 a-i 70 69.15 g-m 60 65.91 a-m 70

Cryptosphaeria ligniota PMM 2929 Salix mucronata Stellenbosch 50 g-n 80 68.42 i-m 90 65.32 a-m 80

Cr. multicontinentalis PMM 2769 Salix mucronata Constantia 48.88 g-n 90 68.80 h-m 70 56.29 h-o 70

Cr. multicontinentalis PMM 3063 Salix mucronata Stellenbosch 53.91 d-n 80 76.62 a-l 90 48.63 n-p 80

Cr. multicontinentalis PMM 2932 Salix mucronata Stellenbosch 46.79 k-n 80 69.88 f-m 50 45.82 op 100

Cr. multicontinentalis PMM 2909 Salix mucronata Stellenbosch 57.23 a-n 90 65.63 j-n 90 53.71 k-o 70

Cr. multicontinentalis PMM 2787 Morus sp. Red Stone 45.71 n 80 51.63 n 70 51.85 l-o 60

Diatrypella vulgaris PMM 1356 Citrus limon Stellenbosch 53.79 d-n 100 80.74 a-i 80 58.94 e-o 100

Eutypa consobrina PMM 282 Vitis vinifera Grabouw 56.66 a-n 70 74.98 b-l 80 57.96 f-o 80

Eu. consobrina PMM 2919 Schinus molle Durbanville 46.61 l-n 80 75.29 b-l 80 61.02 c-n 70


Table 5 (continued) Species Isolate Host origin Location Dormant shoots Green shoots





Eu. lata PMM 3055 Vitis vinifera Hermanus 67.94 a 100 68.79 h-m 80 68.34 a-k 100

Eu. lata PMM 1476 Psidium guajava Constantia 68.40 a 100 90.62 a 100 75.41 a-c 100

Eutypa sp. PMM 2408 Vitis vinifera Bonnievale 57.06 a-n 90 81.87 a-i 90 55.02 j-o 100

Eutypa sp. PMM 2298 Prunus armenica Bonnievale 58.27 a-m 90 77.71 a-k 80 67.21 a-k 90

Eutypa sp. PMM 1450 Ficus carica Grabouw 53.31 d-n 80 81.20 a-i 90 67.49 a-k 90

Eutypa sp. PMM 2139 Malus domestica Bonnievale 53.89 d-n 10 80.39 a-i 50 59.63 d-o 60

Eutypa sp. PMM 2762 Salix mucronata Constantia 57.85 a-n 90 77.03 a-l 70 60.81 c-o 100

Eutypa sp. PMM 2884 Schinus molle Wellington 47.59 j-k 80 75.62 b-l 70 73.38 a-e 100

Eutypa sp. PMM 2750 Eriobotrya japonica Reebok 53.98 d-n 90 76.55 a-l 70 69.01 a-j 100

Eutypella australiensis CSN 869 Psidium guajava Stellenbosch 49.33 h-n 90 82.78 a-h 90 57.46 g-o 100

Eu. australiensis PMM 2641 Dalbergia sp. Constantia 52.66 e-n 80 83.68 a-f 70 56.73 g-o 60

Eu. australiensis PMM 2640 Unknown Hout Bay 47.35 k-n 80 88.30 ab 80 72.57 a-f 80

Eutypella citricola PMM 3056 Vitis vinifera Hermanus 55.37 b-n 100 75.04 b-l 90 54.41 j-o 100

Eu. citricola PMM 2111 Prunus armenica Bonnievale 48.58 i-n 90 81.13 a-i 80 71.51 a-g 90

Eu. citricola PMM 2171 Prunus salicina Bonnievale 59.84 a-i 100 86.32 a-d 80 56.84 g-o 100

Eu. citricola PMM 701 Psidium guajava Paarl 52.77 e-n 100 77.61 a-k 80 70.41 a-i 90

Eu. citricola PMM 1454 Eriobotrya japonica Grabouw 51.91 e-n 90 80.34 a-i 80 68.60 a-k 100

Eu. citricola PMM 2647 Melia azedarach Hout Bay 46.38 nm 80 71.05 e-l 100 65.27 a-m 100


Table 5 (continued) Species Isolate Host origin Location Dormant shoots Green shoots





Eu. citricola PMM 2140 Punica granatum Bonnievale 58.99 a-l 100 64.92 k-n 70 67.56 a-k 100

Eu. citricola PMM 2890 Schinus molle Durbanville 50.95 f-n 90 74.75 b-l 80 64.57 b-m 100

Eutypella leprosa PMM 2654 Ficus carica Hout Bay 56.19 a-n 70 88.60 ab 60 66.81 a-l 100

Eu. leprosa PMM 2656 Ficus carica Hout Bay 62.65 a-f 70 77.07 a-l 70 71.50 a-g 100

Eu. leprosa PMM 2648 Ficus carica Hout Bay 63.35 a-f 70 87.65 ab 60 62.62 b-n 100

Eutypella microtheca PMM 2134 Vitis vinfera Robertson 58.49 a-m 70 79.11 a-k 90 64.70 b-m 100

Eu. microtheca PMM 2723 Prunus armeniaca Calitzdorp 62.22 a-g 80 76.66 a-l 90 66.29 a-l 100

Eu. microtheca PMM 1037 Ficus carica Lutzville 57.57 a-n 70 80.90 a-i 100 50.95 m-o 100

Eu. microtheca PMM 1202 Morus sp. Franschhoek 66.75 a-c 70 83.22 a-g 100 58.23 f-o 100

Eu. microtheca PMM 2120 Cydonia oblonga Bonnievale 65.71 a-d 80 80.19 a-i 100 58.75 e-o 90

Eu. microtheca PMM 2728 Pyrus communis Calitzdorp 62.33 a-g 60 84.38 a-e 70 74.26 a-d 100

Eu. microtheca PMM 2724 Schinus molle Calitzdorp 58.14 a-n 70 86.58 a-d 90 58.17 f-o 100

Eu. microtheca PMM 540 Diospyros kaki Stellenbosch 61.64 a-h 70 62.88 k-n 90 59.42 d-o 90

Eutypella sp. 1 PMM 2789 Psidium guajava Calitzdorp 63.91 a-e 50 72.90 c-l 70 55.84 i-o 90

Eutypella sp. 2 PMM 1234 Diospyros kaki Bonnievale 48.34 i-n 70 56.38 nm 70 57.71 f-o 90

Eutypella sp. 3 PMM 2721 Schinus molle Outdshoorn 54.40 c-n 50 80.50 a-i 60 66.44 a-l 50

Eutypella sp. 3 PMM 2718 Schinus molle Outdshoorn 54.63 b-n 40 72.62 d-l 50 65.02 a-m 40

PDA plug 20.88 o - 23.78 o - 34.45 p -

* Means followed by the same letter in the same column are not significantly different (P < 0.05).


Figure 1. Severity of dieback in spurs of SB vineyards of different ages located in three areas. Severity is based on the total number of affected spurs as determined during a survey of 100 vines in each vineyard.

Figure 2. Incidence of spur infection in SB vineyards of different ages, located in different locations. Incidence is based on the number of spurs infected with trunk disease pathogens. In each vineyard, 10 spurs were collected for isolations.


Figure 3. Severity of spur dieback in CS vineyards of different ages and located in different locations. Severity is based on the total number of affected spurs as determined during a survey of 100 vines in each vineyard.

Figure 4. Incidence of spur infection in CS vineyards aged 4 to 8 years old and located in different locations. Incidence is based on the number of spurs infected with trunk disease pathogens. In each vineyard, 10 spurs were collected for isolations.


Accumulated outputs

Technology developed

None to date

Figure 5. Spur infection by different trunk disease pathogens in SB vineyards aged 4 to 8 years old. The infection is based on the isolations made from the 10 spurs collected in each vineyard.

Figure 6. Spur infection by different trunk disease pathogens in CS vineyards aged 4 to 8 years old. The infection is based on the isolations made from the 10 spurs collected in each vineyard.

0

10

20

30

40

50

60

70

80

90

100

4 5 6 7 8

% o

f in

fecte

d s

pu

rs

Vineyard age

Eutypa

dieback

Bot dieback

Phomopsis

dieback

Petri disease


Figure 7. Wire cutting directly into tappie and big pruning wounds close to two bud spur (left). Wire cutting into arm and several big pruning wounds close to two bud spur (right).

Figure 8. Several big wounds (left) close to two bud spur. Transverse cut through these big wounds reveal the extent of dieback and infections inside.

Figure 9. Big wounds close to two bud spurs (left and right). Transverse cut through these big wounds reveal the extent of dieback and infections inside.


Figure 10. Plastic tape cutting into tappie (left). Wire cutting into arm of 4-year-old vine and plastic clip cutting directly into spur (right).

Figure 11. Ropes strangling vine arms.


Figure 12. Effect of time of clean pruning and time of application of Eco77 on percentage budding of pruned spurs in SB vineyards located in Durbanville and Constantia in 2014.

a a a a a a

a a

a

a a

a a


Figure 13. Average number of bunches formed on treated and untreated spurs pruned during different times from May to August 2014 in SB.

a

e

abc

de

bcde

ab


Figure 14. Incidence of trunk disease pathogens (belonging to the Botryosphaeriaceae, Diaporthales, Diatrypaceae and Petri disease fungi) in clean prune wounds and incidence of Trichoderma (Eco77) in Sauvignon blanc wounds in the 2014 season.

Figure 15. Effect of time of clean pruning and time of application of Eco77 on percentage budding of pruned spurs in CS vineyards located in Stellenbosch and Constantia in 2014.

ab bc

ab c c

ab

c


Figure 16. Average number of bunches formed in treated and untreated spurs pruned during different times from May to August 2014 on CS.

Figure 17. Incidence of trunk disease pathogens (belonging to the Botryosphaeriaceae, Diaporthales, Diatrypaceae and Petri disease fungi) in clean prune wounds and incidence of Trichoderma (Eco77) in CS in the 2014 season.

Halleen (Spur dieback) 28 6. ACCUMULATED OUTPUTS

a) TECHNOLOGY DEVELOPED, PRODUCTS AND PATENTS

Quantitative real-time PCR (qPCR) assays were developed for the detection and quantification of E. lata and C. ampelina in grapevine wood. The qPCR assays were specific and successfully quantified target taxa in artificially inoculated wood samples.

b) SUGGESTIONS FOR TECHNOLOGY TRANSFER

A popular article will be prepared for publication in Winetech Tegnies.

c) HUMAN RESOURCES DEVELOPMENT/TRAINING

Student Name and Surname

Student Nationality Degree (e.g. MSc

Agric, MComm)

Level of studies in final year of

project

Total cost to industry

throughout the project

Honours students

Masters Students

Palesa Lesuthu SA MTech R0

PhD students

Providence Moyo (full-time student working on the project)

Zimbabwe PhD Agric R0

Postdocs

Dr. Chris Spies SA Postdoc R0

Support Personnel (not a requirement for HORTGRO Science)

PERSONS PARTICIPATING IN THE PROJECT (Excluding students)

Initials & Surname

Highest Qualification

Degree/ Diploma

registered for

Race (1)

Gender (2)

Institution & Department

Position (3)

Cost to Project

R

F. Halleen PhD W M ARC Infruitec-Nietvoorbij Plant Protection

PL

L. Mostert* PhD W F Plant Pathology, Stellenbosch University

Coll

C. Vermeulen Matric W F ARC Infruitec-Nietvoorbij Plant Protection

RA

J. Marais Matric W F ARC Infruitec-Nietvoorbij Plant Protection

RA

P. Lesuthu BTech MTech B F ARC Infruitec-Nietvoorbij Plant Protection

TA

D. Marais Martric W M ARC Infruitec-Nietvoorbij Plant Protection

RA

B. Sokwaliwa BTech B F ARC Infruitec-Nietvoorbij Plant Protection

RA

(1)Race B = African, Coloured or Indian W = White (2)Gender F = Female

Halleen (Spur dieback) 29 M = Male (3)Position Co = Co-worker ( other researcher at your institution) Coll = Collaborator ( participating researcher that does not receive funding for this project from industry) PF = Post-doctoral fellow PL = Project leader RA = Research assistant TA = Technical assistant/ technician

d) PUBLICATIONS (POPULAR, PRESS RELEASES, SEMI-SCIENTIFIC, SCIENTIFIC) Scientific Publications: P. Moyo, L. Mostert, C.F.J. Spies, U. Damm & F. Halleen (2017) Diversity of Diatrypaceae

species associated with dieback of grapevines in South Africa, with the description of Eutypa cremea sp. nov. Plant Disease (In Press) PDIS-05-17-0738-RE

PhD Thesis: P. Moyo. Identification and characterisation of Diatrypaceae species associated with declining grapevines and alternative hosts in South Africa. University of Stellenbosch, March 2017 e) PRESENTATIONS/PAPERS DELIVERED

Presentations/papers delivered Moyo P., Mostert L. and Halleen F. 2013. Investigation into the cause of poor budburst and

dying single spurs in Sauvignon blanc and Cabernet Sauvignon. 35th Congress of the South African Society for Enology and Viticulture, 13-15 November 2013, Simondium.

Moyo P., Mostert L. and Halleen F. 2014. Spur dieback in young Sauvignon blanc and Cabernet

Sauvignon vineyards: Results from recent surveys. 36th Congress of the South African Society for Enology and Viticulture, 12-14 November 2014, Somerset West.

Moyo P., Mostert L. and Halleen F. 2015. Diversity of Diatrypaceae associated with grapevine

and trees in the vicinity of vineyards in South Africa. US-PP Friday Forum, Stellenbosch, 26 June 2015.

Moyo P., Mostert L. and Halleen F. “Eutypa and its relatives: pathogens with many hosts”. 21

August 2015, Institute for Plant Protection in Fruit Crops and Viticulture of the Julius Kühn-Institute/Geilweilerhof, Germany. Invited lecture.

Moyo P., Mostert L., and Halleen F. Diversity of Diatrypaceae species from grapevines and

trees in the vicinity of vineyards in South Africa. XVIII. International Plant Protection Congress. 24-27 August 2015 Berlin, Germany (Poster presentation).

Moyo P., Mostert L., and Halleen. 2016. Diatrypaceae species associated with Eutypa dieback-

affected grapevines in South Africa: new insights into an old disease. Congress of the South African Society for Enology and Viticulture, 23-25 August 2016, Somerset West.

Spies CFJ, Moyo P., Bester MC, Lesuthu P, du Plessis IL, van Jaarsveld W.J., Mostert L., and

Halleen. 2017. Woody plants near vineyards harbour grapevine trunk pathogens. 50th SASPP Congress (15-19 January 2017, Drakensberg).

Moyo P., Mostert L., Spies, CFJ and Halleen. 2017. Diatrypaceae species associated with

dieback and cankers of grapevine and other woody hosts in South Africa. 50th SASPP Congress (15-19 January 2017, Drakensberg).

Halleen (Spur dieback) 30 Moyo P., Mostert L., and Halleen. 2017. Characterisation of Diatrypaceae species from

grapevine in South Africa. 10th International Workshop on Grapevine Trunk Diseases (4-7 July 2017, Reims, France)

Spies CFJ, Moyo P., Bester MC, Lesuthu P, du Plessis IL, van Jaarsveld W.J., Mostert L., and

Halleen.Diversity and host range of trunk disease pathogens in South Africa. INRA UMR SAVE (Bordeaux, France, 20 July 2017). Invited lecture.

7. BUDGET WW0643 (000302) TOTAL COST SUMMARY OF THE PROJECT

YEAR CFPA DFTS SAAPPA SASPA

SATI Winetech THRIP OTHER TOTAL

2013/2014 194090 202012 396102

2014/2015 209617 218390 428007

2015/2016 230578 240229 470807

2016/2017 144921 150836 295757

Halleen (Spur dieback) 31 EVALUATION BY INDUSTRY/PHI

This section is for office use only

Project number WW0643 (000302)

Project name Investigation into the cause of poor budburst and dying of single spurs in Sauvignon blanc and Cabernet Sauvignon

Name of Sub-Committee* Plant Protection

Comments on project

Committee’s recommendation (Review panel in the case of PHI)

• Accepted.

• Accepted provisionally if the sub-committee’s comments are also addressed. Resubmit this final report by___________________________________

• Unacceptable. Must resubmit final report. Chairperson__________________________________________ Date___________________

*SUB-COMMITTEES Winetech

Viticulture: Cultivation; Soil Science; Plant Biotechnology; Plant Protection; Plant Improvement; Oenology: Vinification Technology; Bottling, Packaging and Distribution; Environmental Impact; Brandy and Distilling; Microbiology Deciduous Fruit

Technical Advisory Committees: Post-Harvest; Crop Production; Crop Protection; Technology Transfer Peer Work Groups: Post-Harvest; Horticulture; Soil Science; Breeding and Evaluation; Pathology; Entomology

Documents

WW0643 000302 Final report Halleen (1Sept2017) na Winetech · Aug 2015 Evaluate Oct to Dec 2015 Dec 2016 The effect of the time of clean pruning and treatment of wounds on the incidence