European Journal of Plant Pathology

, Volume 135, Issue 4, pp 817–829

Combining sanitation and disease modelling for control of grapevine powdery mildew

Authors

  • Tito Caffi
    • Istituto di Entomologia e Patologia vegetaleUniversità Cattolica del Sacro Cuore
  • Sara Elisabetta Legler
    • Istituto di Entomologia e Patologia vegetaleUniversità Cattolica del Sacro Cuore
  • Riccardo Bugiani
    • Plant Protection Service, Regione Emilia-Romagna
    • Istituto di Entomologia e Patologia vegetaleUniversità Cattolica del Sacro Cuore
Article

DOI: 10.1007/s10658-012-0124-0

Cite this article as:
Caffi, T., Legler, S.E., Bugiani, R. et al. Eur J Plant Pathol (2013) 135: 817. doi:10.1007/s10658-012-0124-0

Abstract

Chasmothecia of Erysiphe necator form in one season, survive winter and discharge ascospores that cause primary infections and trigger powdery mildew epidemics in the next season. A strategy for powdery mildew control was developed based on (i) the reduction in overwintering chasmothecia and on (ii) spring fungicide applications to control ascosporic infections timed based on estimate risk (two to five sprays per season). Several fungicides, the hyperparasite Ampelomyces quisqualis, and a mineral oil product were first tested as separate applications in a greenhouse and in vineyards. In the greenhouse, A. quisqualis suppressed chasmothecia formation by 41 %; fungicides and mineral oil suppressed chasmothecia formation by 63 % and ascospore viability by 71 %. In vineyards, application of boscalid + kresoxim-methyl or meptyldinocap once after harvest, as well as application of A. quisqualis pre- and post-harvest, delayed disease onset and epidemic development in the following season by 1 to 3 weeks and lowered disease severity (up to the pea-sized berry stage) by 56 to 63 %. Risk-based applications of sulphur and of synthetic fungicides provided the same control as the grower spray program but required fewer applications (average reduction of 47 %). Sanitation strategies were then tested by combining products and application times (late-season, and/or pre-bud break, and/or spring). Adequate disease control with a reduced number of sprays was achieved with the following combination: two applications of A. quisqualis (pre- and post-harvest), one application of mineral oil before bud break, and model-based applications of sulphur fungicides between bud break and fruit set.

Keywords

OverwinteringChasmotheciaAscosporic infectionFungicide schedulingBiocontrol

Introduction

Powdery mildew is a major grapevine disease throughout the world. The causal agent of the disease, Erysiphe necator Schwein. (syn. Uncinula necator (Schwein.) Burrill), is a biotrophic fungus that can survive the winter as ascospores in overwintering chasmothecia and as mycelium in infected buds (Pearson and Goheen 1988). The chasmothecia form and mature on powdery mildew colonies; when mature, they are dispersed by splashing rain (Gadoury and Pearson 1988). Chasmothecia overwinter on the bark of the vine trunk, on the soil surface, or on leaf litter. Chasmothecia survival, however, is consistently higher in exfoliating bark than on the other substrates (Gadoury and Pearson 1988). Ascospores are repeatedly released mainly between grapevine budbreak and bloom (Pearson and Gadoury 1987; Rossi et al. 2010) and cause primary infections on leaves. Once initial colonies are established, the fungus can asexually produce large numbers of conidia that disperse and infect additional leaves (Carisse et al. 2009). Conidia from leaf colonies serve as inoculum for infection of flowers and developing berries (Savary et al. 2009). Flag shoots that develop from infected dormant buds can also act as sources of primary inoculum, but they are usually absent in the commercial vineyards of North Italy and other viticultural regions (Pearson and Gadoury 1987; Cortesi et al. 1997; Legler et al. 2012).

Grape growers typically manage powdery mildew by applying foliar fungicides at fixed intervals through the growing season or according to grapevine growth stages (Dent 1995). Periodic fungicide applications at fixed intervals frequently lead to unneeded sprays, whereas applications based on growth stage do not account for current conditions and also lead to unjustified or poorly timed sprays. A common error in powdery mildew control is to delay fungicide application until the disease becomes evident in the vineyard (Magarey and Moyer 2010). Symptoms do not become evident until several weeks after the disease has been initiated, and at that time the epidemic is generally well established and difficult to control (Sozzani et al. 2010).

Control of powdery mildew could be improved and the number of fungicide applications reduced by reducing early season inoculum and subsequent infection so as to stop the epidemic before they reach the exponential phase (Magarey and Moyer 2010). This goal can be achieved by: (i) the reduction of the population of overwintering chasmothecia (Halleen and Holz 2001), and by (ii) the control of ascosporic infection until all the ascospores have been discharged (Hartman and Beale 2008).

Reduction of overwintering inoculum requires grower action late in the season. Growers frequently leave their vineyards unattended after harvest which can lead to severe infection of leaves and the production of high numbers of chasmothecia (Rossi et al. 2011) provided that environmental conditions are favourable (Legler et al. 2012). A strategy to reduce the production of chasmothecia may start by preventing high levels of leaf infection at the end of the season (Carisse et al. 2009), which may require management of mildew until veraison or beyond (Hed and Travis 2007) and which may also include the eradication of the produced chasmothecia. A method to eradicate overwintering chasmothecia on the bark of grapevines has been developed by Gadoury et al. (1994), who reported that aqueous solutions of lime sulphur applied as over-the-trellis sprays to dormant grapevines destroyed chasmothecia and provided good disease control. Other natural compounds and fungicides have also been tested (Schilder et al. 2008; D’Ascenzo and Corvi 2010). Application of Ampelomyces quisqualis, a hyperparasite of powdery mildews that is known to destroy chasmothecia ((Kiss et al. 2004; Angeli et al. 2009), reduced both the number of chasmothecia dispersed from leaves to the bark and their survival on bark (Falk et al. 1995; Legler et al. 2011).

For the control of ascosporic infection, a warning system was recently developed (Caffi et al. 2012) based on short-term weather forecasts, a model that simulates the severity of each E. necator ascosporic infection (Caffi et al. 2011). This warning system was evaluated in North Italy from 2006 to 2008, between bud break of vines and early berry development (most of berries touching, E-L stage 32, Coombe 1995). Use of the warning system reduced disease severity on leaves and bunches by >90 % compared to the unsprayed control and resulted in the same level of control of powdery mildew as the grower spray program but with 50 % less fungicide applications.

In previously cited works, efficacy of plant protection products in reducing the overwintering population of chasmothecia and of disease modelling for timing early season fungicide application have been tested separately but not in combination, i.e., not as part of an “combined-application” disease control strategy. The present work was conducted to develop and investigate a strategy for the control of grapevine powdery mildew based on reduction in the number of overwintering chasmothecia and targeted application of fungicides in spring against ascosporic infections.

Materials and methods

Several fungicides (Table 1), one biofungicide (A. quisqualis), and one mineral oil product were first tested as separate applications in greenhouse (to investigate their effect on chasmothecia production, ascospore maturation, and ascospore viability within the treated chasmothecia) and in vineyards (to investigate their effect in reducing disease severity in the next season). Afterwards, sanitation strategies were tested in vineyards; strategies were based on combinations of: (i) late-season application of the products to prevent chasmothecia formation, (ii) pre-bud break applications of the products to destroy overwintering chasmothecia, and (iii) spring application of fungicides based on a model that predicts ascosporic infection.
Table 1

List of products used in this study

Active ingredient or biocontrol agent

Commercial formulation

Manufacturer

Active compounds

Dose

(g l−1 except as noted)

(ml hl−1 except as noted)

Ampelomyces quisqualis

AQ10 WG

Intrachem

≥5 × 109 cfu g−1

20–30 (g ha−1)

Boscalid + kresoxim-methyl

Collis

Basf

200 + 100

30–40

Bupirimate

Nimrod

Makhteshim

250

80–100

Meptyldinocap

Karathane Star

Dow

350

40–60

Metrafenone

Vivando

Basf

500

20–25

Mineral oil

Polythiola

Cerexagri

400

5–6 (l hl−1)

Quinoxyfen + miclobutanyl

Arius System

Dow

45 + 45

100–125

Spiroxamine

Prosper 300 CS

Bayer

302.8

100–130

Tetraconazole

Domark 40 EW

Isagro

125

25

Sulphur

Heliosufre

Intrachem

700

150–300

Sulphur

Thiopron

Cerexagri

825

300–400

Sulphur

Thiovit Jet

Syngenta

80 %

200–400 (g hl−1)

aPolithiol is a preparation of highly refined paraffinic oil and co-formulants (including sulphur)

Greenhouse experiments

In September 2007, green cuttings of Vitis vinifera, Barbera, which is susceptible to E. necator (Rossi et al. 2006), were grown in a greenhouse (18 to 26 °C with a 12-h photoperiod provided by Philips Master Tl-D 90 Deluxe 18 W/950 lamps) in 10 × 10 cm pots containing a mixture of sand, peat, and soil. When plants were rooted and had reached the growth stage of ‘4–6 leaves unfolded’, the leaves were inoculated with a mixture of E. necator strains collected from different commercial vineyards of Emilia-Romagna (North Italy). For inoculation, a bulk conidial suspension was gently dusted on the adaxial surface of the grapevine leaves. After the inoculation, high humidity was provided by covering the plants with transparent plastic bags for 48 h.

Forty days after inoculation, the leaves were covered by powdery mildew colonies bearing chasmothecia at different stages of maturation. Absence of A. quisqualis natural parasitism was checked by microscopic observations of the colonies. The following active ingredients were applied: A. quisqualis, boscalid + kresoxim-methyl, bupirimate, meptyldinocap, mineral oil, and spiroxamine. Product characteristics are shown in Table 1. All products were applied with a hand sprayer that produced a fine mist to prevent washing off of mature chasmothecia. Viability of the A. quisqualis inoculum in the AQ10 product was confirmed before application with a spore germination assay. An untreated control was sprayed with water. For each treatment, four replicates were considered, arranged in a completely randomised design.

The greenhouse experiment performed in September 2007 was repeated in November 2007, October 2008, and November 2008 (greenhouse experiments 1 to 4, respectively). Because temperature influences the development of chasmothecia (Legler et al. 2012), the four experiments were performed at different temperatures to determine whether treatment effects were temperature dependent. Temperatures were registered by a data logger (Tinytag Plus 2, Gemini Data Loggers Ltd., Chichester, UK).

Before application of the products, a 2-cm-diameter circle was drawn on the adaxial surface of two leaves per cutting with a permanent felt marker. The leaf surface was then examined with a stereomicroscope (40× magnification), and all the chasmothecia in the circles were counted and classified as immature (i.e., yellow or brown and spherical) or mature (i.e., dark brown or black and concave–convex). Counts were repeated 3 weeks after fungicide application (when the experiments were terminated), and all chasmothecia counts were expressed as number per cm2 of leaf.

The maturity and viability of ascospores within the chasmothecia were also evaluated 3 weeks after product application. At least 30 visibly mature chasmothecia per treatment in each experiment were crushed on glass slides and examined microscopically. The ascospores were categorized as immature (i.e., with granular cytoplasm) or mature (i.e., with vacuolated cytoplasm) (Rossi et al. 2010). The percentage of mature ascospores was calculated. For assessment of ascospore viability, at least 30 chasmothecia per treatment in each experiment were crushed on glass slides, treated following the method of Cortesi et al. (1995), and observed with a fluorescence microscope. Ascospore viability (the number of viable ascospores expressed as a percentage of the total number of ascospores observed) was calculated.

Data analysis of greenhouse experiments

The numbers of chasmothecia (x) were transformed by the natural logarithm function, i.e., by ln(x + 1), to make variances uniform, and the transformed data were subjected to a factorial analysis of variance (ANOVA) in which experiment and product were factors. The Fisher Protected Least Square Difference (LSD) test was used at P = 0.05 to separate means. ANOVAs and the LSD tests were also applied to the data for percentages of mature and viable ascospores, which were arcsine transformed.

Field experiments

Two kinds of experiments were carried out, hereafter named as “single-application experiments” (six experiments in 2007/08 and 2008/09) and “combined-application experiments” (five experiments in 2009/10 and 2010/11), respectively. Experiments were conducted at six vineyards in Emilia-Romagna (North Italy): Marzeno, Modigliana, Castel S. Pietro, Fusignano, Conventello, and Tebano (Table 2). The four cultivars used in these experiments are highly susceptible to powdery mildew (Rossi et al. 2006). The vineyards were managed following standard practices except as indicated for fungicides. Downy mildew was controlled, when necessary, with fungicides that lack activity against E. necator.
Table 2

Characteristics of the experimental vineyards

Vineyard

Coordinates

Altitude (m)

Variety

Age in 2007 (years)

Training system

Within- × between-row spacing (m)

Marzeno

44°12′49″N

120

Pinot noir

 6

Guyot

1.5 × 2.5

11°49′01″E

Modigliana

44°11′51″N

100

Sangiovese

25

Guyot

1.5 × 2.5

11°49′47″E

Castel S. Pietro

44°24′29″N

 60

Sangiovese

12

permanent cordon spur pruning

1.5 × 4

11°33′15″E

Fusignano

44°30′37″N

  9

Pinot Blanc

 5

Casarsa

1.5 × 4

11°57′52″E

Conventello

44°29′45″N

  4

Chardonnay

 5

Casarsa

1.5 × 4

12°04′34″E

Tebano

44°17′29″N

 35

Pinot Noir

26

spurred cordon

1.5 × 4

11°47′20″E

In the single-application experiments, products were applied: (i) to prevent chasmothecia production, (ii) to destroy the overwintering chasmothecia, or (iii) to control ascosporic infection; and were compared with (iv) an unsprayed control, and (v) the grower spray program. To prevent chasmothecia production, products were applied before harvest (A. quisqualis) or post-harvest (A. quisqualis, boscalid + kresoxim-methyl, bupirimate, meptyldinocap, spiroxamine, and mineral oil). To destroy the overwintering chasmothecia, mineral oil was applied to the vine trunk before bud break. To control ascosporic infection, products (sulphur and a schedule of synthetic fungicides) were applied between bud break and fruit set (E-L stage 27 according to the modified Eichhorn-Lorenz system, Coombe 1995), scheduled according to a disease model (Caffi et al. 2011).

In the combined-application experiments, sanitation treatments based on: (i) pre- and post-harvest application of A. quisqualis, (ii) application of mineral oil before bud break, and (iii) application of both A. quisqualis and mineral oil were combined with model-based sulphur treatments between bud break and fruit set. These combinations of treatments were compared with the two model-based spray schedules that use: (iv) sulphur, (v) synthetic fungicides, (vi) the grower spray program, and (vii) an unsprayed control.

For operation of the model that estimates risk of powdery mildew, the weather data (air temperature, relative humidity, rainfall amount, and presence of wetness) were provided hourly by the Agro-meteorological Service of the Emilia-Romagna Region (North Italy) for the nodes (in a 5 × 5-km grid) corresponding to the geographical coordinates of the experimental vineyards. Weather forecasts for the following 72 h were also provided. The model (Caffi et al. 2011) predicts the occurrence of powdery mildew infection periods during the primary inoculum season, i.e., between bud break of vines and the time when the stock of ascospores is depleted. The model was operated daily using both measured and forecast data. Therefore, two kinds of model outputs were obtained daily: calculated infection for the previous day and predicted infection for the current and the following 2 days. Hence, two kinds of alarms were provided: one alarm indicated that an infection period had already occurred on the previous day, and the other predicted that an infection period was likely to occur on the current day or on the following 2 days. Possible secondary infections caused by the E. necator conidia were not considered for producing warnings. For either calculated or predicted infection periods, an SMS was sent to the mobile phone of the managers of the experimental vineyards, who applied fungicides accordingly. Dates when treatments were applied are shown in Table 3.
Table 3

Dates when treatments were applied in 11 vineyard experiments in North Italy

Vineyard

Dates when products were applied

Pre-harvest

Post-harvest

Before bud break

Bud break to fruit seta

Marzeno

16 August 2007

24 September 2007

19 March 2008

23 April; 16 May 2008 (5)

Modigliana

16 August 2007

24 September 2007

19 March 2008

23 April; 16 May 2008 (5)

Castel S. Pietro

16 August 2007

24 September 2007

25 March 2008

22 April; 19 May 2008 (7)

Fusignano

13 August 2008

17 September 2008

6 April 2009

21 April; 5, 29 May 2009 (5)

Castel S. Pietro

13 August 2008

17 September 2008

6 April 2009

21 April; 5, 29 May 2009 (7)

Conventello

28 August 2008

8 October 2008

3 April 2009

22 April; 6, 13, 26 May; 8 June 2009 (7)

Tebano

27 August 2009

6 October 2009

1 April 2010

22 April; 3, 14, 27 May 2010 (6)

Conventello

18 August 2009

23 September 2009

29 March 2010

22 April; 3, 14 May 2010 (5)

Castel S. Pietro

27 August 2009

6 October 2009

1 April 2010

22 April; 3, 14, 27 May 2010 (7)

Conventello

19 August 2010

20 September 2010

1 April 2011

12 April; 4, 16 May 2011 (5)

Castel S. Pietro

2 September 2010

5 October 2010

1 April 2011

13, 29 April; 17 May; 8 June 2011 (7)

aApplication dates were based on the advice provided by the disease model of Caffi et al. (2011); values in brackets are the number of fungicide sprays applied in the grower spray program. Fruit set is defined as the E-L stage 27 according to the modified Eichhorn-Lorenz system (Coombe 1995): fruit set is considered to have occurred when the diameter of young berries is >2 mm

For fungicide applications, vineyard managers selected among the products listed in Table 1. The active compounds in all of these products are considered effective for controlling powdery mildew and are included in the Annex I of the Regulation (EC) No. 396/2005. Products were used at the label dose (Table 1). The grower spray program was based on a fixed-interval schedule of the same fungicide list according to each grower’s schedule (Table 3). Plant protection products were applied with a knapsack air-blast sprayer SR 420 (Stihl Inc., Virginia Beach, VA, US) in such a way to ensure an even distribution on the whole plot with minimal drift and edge effects. Depending on the trellis system and growth stage of the vines, 4 to 8 hl of water with product was applied per ha.

Experiments were managed according to a randomized complete block design, with four replicate plots; each plot consisted of a single row, with at least six plants per row. For experiments conducted in the same vineyard, different subplots of vines were used in different years.

Chasmothecia collection and disease assessment in field experiments

The number of chasmothecia dispersed to the vine trunk in the vineyards was estimated with plastic trap funnels (13 cm diameter) as described by Gadoury and Pearson (1988). In mid-August of each year, five funnels were attached to each of three randomly selected vines per vineyard (15 funnels per vineyard) as follows: three funnels were secured to the trunk (at 1/3, 2/3, and 3/3 of the trunk height), and two funnels were secured to the permanent cordon or, for the Guyot-pruned vineyards, to the fruiting arm (at 1/3 and 2/3 of the cordon or arm length). The funnels were oriented so that the wide part was facing up. A disk of filter paper (15 cm diameter) was fixed in the mouth of each funnel by pushpins so that any material falling into the funnel top remained on the filter. In mid-August, E. necator colonies were present on leaves and/or on bunches but no chasmothecia had formed; microscopic observations of sample leaves and berries collected at the time of funnel installation confirmed the absence of visible fruiting bodies. Filters were replaced every 2 weeks until leaf fall was complete. Each filter was examined with a stereomicroscope (20× magnification), and chasmothecia were counted. The number of chasmothecia trapped per cm2 of trap surface was then calculated by dividing total counts by the total area of the funnel top opening. Numbers of chasmothecia trapped over time were expressed as a cumulative proportion of the total chasmothecia trapped in each vineyard.

Starting from bud break, the vineyards were inspected at least once per week to determine the prevalent growth stage of vines (i.e., the growth stage reached by >50 % of shoots in the vineyard) based on the modified Eichhorn-Lorenz system (Coombe 1995), and the time of appearance of the first disease symptoms, such as flag shoots or discrete pale spots on the abaxial surfaces of the basal leaves. Disease severity was assessed three times on a sample of 100 random leaves between beginning of flowering (E-L stage 19) and pea-sized berry stage (7 mm diameter) (E-L stage 31) and on 100 bunches per plot at pea-sized berry stage (EPPO 2002). Leaves and bunches were examined on both sides of the rows, limited to the central area of the plot to avoid interference between plots (EPPO 1999); leaves were not labelled and then they were not necessarily the same for the three assessment times. Leaves and bunches were carefully observed for powdery mildew colonies and classified as healthy or infected; disease severity was estimated visually and expressed as the percentage of the total leaf (or bunch) area with disease. Average disease severity of the sampled leaves (or bunches) was then calculated.

Data analysis of field experiments

Disease severity data were arcsine transformed to make variances uniform and were then subjected to an ANOVA for a randomized block design repeated over locations (i.e., vineyards), with four replicates. The two kinds of experiments (single- and combined-application) were analysed separately. The LSD test was used at P = 0.05 to separate means. Data from vineyards where the disease did not develop were not included in ANOVA.

Results

Greenhouse experiments

Depending on the greenhouse experiment, from 21.3 to 65.7 chasmothecia per cm2 of leaf were present when the products were applied; of these chasmothecia, 2 to 22 % were immature (Fig. 1). Three weeks later, the total number of chasmothecia on the untreated leaves increased by 5.0, 4.2, 1.6, and 1.4 times in experiment 1 to 4, respectively. Average temperature of the 3-week period was 21.7, 16.1, 17.0, and 25.0 °C in experiments 1 to 4, respectively.
https://static-content.springer.com/image/art%3A10.1007%2Fs10658-012-0124-0/MediaObjects/10658_2012_124_Fig1_HTML.gif
Fig. 1

Numbers of Erysiphe necator chasmothecia produced in powdery mildew colonies in greenhouse experiments 1 to 4. Chasmothecia were counted at the start of each experiment (day 0) on untreated leaves and after 3 weeks (day 21) at temperatures that differed for each experiment (see text). The proportion of immature chasmothecia, if any, is shown in black in each column. Whiskers indicate the standard error

All products significantly (P <0.001) reduced the total number of chasmothecia 3 weeks after application. Fungicides and mineral oil reduced the chasmothecia number by an average of 63.3 %, with no significant differences between products, while A. quisqualis reduced the chasmothecia by 40.9 % (Fig. 2a). No immature chasmothecia were present on the treated leaves 3 weeks after treatment, except with A. quisqualis and meptyldinocap (Fig. 2a). The interaction experiment × product (P = 0.021) accounted for only 13.5 % of the total variance. This interaction was mainly caused by differences between initial and final numbers of chasmothecia in the untreated control of the four experiments (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10658-012-0124-0/MediaObjects/10658_2012_124_Fig2_HTML.gif
Fig. 2

Effect of treatments on numbers of Erysiphe necator chasmothecia produced in powdery mildew colonies (a), ascospore maturity (b), and ascospore viability (c) in the greenhouse experiments. In (a), chasmothecia were counted at the start of the experiment (initial number) and 3 weeks later on untreated leaves and on leaves treated with the products listed in Table 1. In (b), ascospores were observed microscopically 3 weeks after treatment and categorized as immature (i.e., with granular cytoplasm) or mature (i.e., with vacuolated cytoplasm). In (c), viability was determined through fluorescence microscopy. In Fig. 2a, the proportion of immature chasmothecia is shown in black in each column. Whiskers indicate the standard error. In (a) and (c), bars with the same letter are not significantly different according to the LSD test (P = 0.05); in (b), significant differences between treatments were not detected

The treatments did not significantly influence (P = 0.35) the percentage of mature ascospores in chasmothecia 3 weeks after treatment (Fig. 2b). Fungicides and mineral oil significantly (P <0.001) reduced ascospore viability by 71 % in average (Fig. 2c).

Single-application field experiments

In the untreated plots, powdery mildew epidemics were severe at Castel S. Pietro and Modigliana in 2008 and at Castel S. Pietro in 2009, with 30 to 40 % disease severity on bunches at pea-sized berries (Fig. 3a). First disease symptoms were evident in early May at Castel S. Pietro and Modigliana in 2008, and in mid-June at Castel S. Pietro in 2009. At Conventello in 2009, first disease symptoms appeared in early June; disease severity on bunches was 6.3 % at pea-sized berry stage. At Marzeno in 2008 and Fusignano in 2009, first disease signs appeared in early to mid-July, and <1 % disease severity was observed on bunches at pea-sized berries. The number of chasmothecia trapped in the previous season was higher in the first three vineyards (2.7 to 6.4 chasmothecia per cm2 of trap surface) than in Conventello (1.0 chasmothecia per cm2 of trap surface); in the other two vineyards, no or few (< 0.01 chasmothecia per cm2 of trap surface) chasmothecia were caught (Fig. 3a). Flag shoots were not observed during the vineyard surveys. Data from Marzeno and Fusignano vineyards were not further analysed because no disease was present on leaves or bunches at pea-sized berry stage.
https://static-content.springer.com/image/art%3A10.1007%2Fs10658-012-0124-0/MediaObjects/10658_2012_124_Fig3_HTML.gif
Fig. 3

Powdery mildew severity (bars) in untreated bunches in field experiments in 2008 and 2009 (a), and in 2010 and 2011 (b). Numbers of Erysiphe necator chasmothecia (points) caught by trap funnels secured to the vine trunk between mid-August and complete leaf fall of the previous season are also indicated. Disease severity was estimated at the pea-size berry stage on a random sample of 100 bunches. Whiskers indicate the standard error

In treated plots, disease symptoms appeared 1 to 3 weeks later than in untreated ones, depending on vineyard and treatment. For instance, at Conventello in 2009, 2 % of leaves were infected in the untreated plots on 16 June, but no disease was observed in the plots treated with fungicides, only 0.5 and 1.0 % of leaves were infected in the plots treated with A. quisqualis (pre- and post-harvest) and mineral oil (before bud break), respectively. On 6 July, 18 % of the leaves were infected in untreated plots and a maximum of 9 % were infected in treated plots.

Sanitation treatments significantly influenced (P <0.001) disease severity on bunches at pea-sized berry stage, while the interaction of treatment × vineyard was not significant (P = 0.08). In particular, A. quisqualis applied at pre- and post-harvest and boscalid + kresoxim-methyl or meptyldinocap applied at post-harvest reduced the disease the following season by 62.7, 56.2, and 56.5 %, respectively, compared to the untreated control (Table 4). The best control of powdery mildew was obtained with the grower spray program (1.2 % disease severity at pea-sized berry stage), the model-based application of synthetic fungicides (1.5 % disease severity), and the model-based application of sulphur (5.7 % disease severity). For model-based applications, two to five sprays were applied between bud break and fruit set, depending on the vineyard, whereas five to seven sprays were applied in the grower spray program (Table 3). The three most effective sanitation treatments did not significantly differ from the two model-based and the grower spray schedules (Table 4). Disease severity did not significantly differ between untreated plots and plots treated with A. quisqualis and the other products (mineral oil, bupirimate, and spiroxamine) (Table 4). The treatments had the same effects on the disease on leaves and bunches at pea-sized berry stage, and the coefficient of correlation between disease severity on leaves and bunches was r = 0.786 (P <0.001, n = 60). In other words, whenever disease was reduced on bunches, it was also reduced on leaves.
Table 4

Severity of powdery mildew on bunches at the pea-sized berry stage in the single-application field experiments

Application schedulea

Disease severityb (% infected area)

Pre-harvest

Post-harvest

Before bud break

Bud break to fruit setc

A. quisqualis

-

-

-

17.9 ABCd

A. quisqualis

A. quisqualis

-

-

9.9 BCD

-

Mineral oil

-

-

20.4 AB

-

Boscalid + kresoxim-methyl

-

-

11.7 BCD

-

Bupirimate

-

-

21.5 AB

-

Meptyldinocap

-

-

11.6 BCD

-

Spiroxamine

-

-

15.3 ABC

-

-

Mineral oil

-

23.4 AB

-

-

-

Sulphure

5.7 CD

-

-

-

Fungicidese

1.5 D

-

-

-

Grower f

1.2 D

-

-

-

-g

26.7 A

aProducts were applied as indicated in Table 1 and 3

bAverage disease severity at Modigliana and Castel S. Pietro in 2008, and at Conventello and Castel S. Pietro in 2009; the disease did not develop at Marzeno in 2008 and at Fusignano in 2009. The interaction of product × vineyard was not significant (P = 0.08)

cFruit set is defined as the E-L stage 27 according to the modified Eichhorn-Lorenz system (Coombe 1995): fruit set is considered to have occurred when the diameter of young berries is >2 mm

dValues followed by the same letter are not significantly different according to the LSD test (P = 0.05)

eSulphur and synthetic fungicide applications were scheduled based on the disease model of Caffi et al. (2011) as indicated in Table 3; the products used are listed in Table 1

fFungicides listed in Table 1 were applied at fixed intervals according to each grower’s schedule, as indicated in Table 3

eUntreated control

Combined-application field experiments

In the untreated plots, powdery mildew caused severe epidemics at Castel S. Pietro in 2010 (51 % disease severity on bunches at pea sized berry stage), while disease severity on bunches ranged from 1.5 to 4.5 % at Conventello in 2010 and 2011 and at Castel S. Pietro in 2011. At Tebano in 2010, the disease did not develop (Fig. 3b), and these data were not further analysed. The number of chasmothecia trapped in the previous season was higher at Castel S. Pietro in 2010 (9.1 chasmothecia per cm2 of trap surface) than in the other vineyards (0.3 to 0.7 chasmothecia per cm2 of trap surface). Only few chasmothecia (< 0.01 chasmothecia per cm2 of trap surface) were trapped at Tebano in 2010 (Fig. 3b). Flag shoots were never observed during the vineyard surveys.

Compared to the untreated control, sanitation treatments with A. quisqualis and/or mineral oil combined with model-based sulphur sprays after bud break significantly (P <0.001) reduced disease severity on bunches at pea-sized berry stage and provided the same level of disease control as both the model-based fungicide program with synthetic fungicides and the grower spray schedule (Table 5). The model-based application of sulphur between bud break and fruit set provided less disease control without previous sanitation than with sanitation. The interaction of product × vineyard was not significant (P = 0.13).
Table 5

Severity of powdery mildew on bunches at the pea-sized berry stage in the combined-application field experiments

Application schedulea

Disease severityb (% infected area)

Pre-harvest

Post-harvest

Before bud break

Bud break to fruit setc

A. quisqualis

A. quisqualis

Mineral oil

Sulphur

0.8 Cd

A. quisqualis

A. quisqualis

-

Sulphur

0.4 C

-

-

Mineral oil

Sulphur

0.4 C

-

-

-

Sulphure

3.0 B

-

-

-

Fungicidese

0.1 C

-

-

-

Grower

0.1 C

-

-

-

-f

15.1 A

aProducts were applied as indicated in Table 1 and 3

bAverage disease severity at Conventello and Castel S. Pietro in 2010 and 2011; disease did not develop at Tebano in 2010. The interaction of product × vineyard was not significant (P = 0.13)

cFruit set is defined as the E-L stage 27 according to the modified Eichhorn-Lorenz system (Coombe 1995): fruit set is considered to have occurred when the diameter of young berries is >2 mm

dValues followed by the same letter are not significantly different according to the LSD test (P = 0.05)

eSulphur and synthetic fungicides were scheduled based on the disease model of Caffi et al. (2011) as indicated in Table 3; the products used are listed in Table 1

fFungicides listed in Table 1 were applied at fixed intervals according to each grower’s schedule, as indicated in Table 3

eUntreated control

Discussion

Chasmothecia develop in one season, survive the winter and trigger infection in the next season; the dose of chasmothecia within a vineyard is the main factor that influences the initial level of disease, which in turn can affect the difficulty in controlling powdery mildew in any season (Magarey and Moyer 2010). This was clearly supported by the results of the current study, in which there was a relationship between the number of chasmothecia trapped in the previous season and disease severity in the following season. Reduction of the primary inoculum formed in the first season is the first step in controlling powdery mildew epidemics in the second season (Magarey and Moyer 2010). Reduction of the primary inoculum of E. necator can be achieved by reducing chasmothecia production in the previous season and by eradicating chasmothecia that overwintered on vine bark. The disease control provided by these treatments is a form of sanitation considering that sanitation is a disease management approach aiming at reducing or destroying initial inoculum responsible for disease initiation (Van der Plank 1963).Both sanitation tactics were investigated in the present work.

In the greenhouse experiments, the chemical and biological products were applied to powdery mildew colonies that had produced some mature chasmothecia but were also still actively producing chasmothecia. The timing of application in the greenhouse experiments therefore corresponded to a late summer or autumn applications in the field. Fungicides and mineral oil reduced chasmothecia formation by 63 % in the greenhouse, in agreement with a previous work in which natural compounds (including calcium polysulfide, potassium bicarbonate, and organic acids) and tebuconazole applied in autumn reduced the total number of chasmothecia produced on powdery mildew colonies by 66 to 82 % (Schilder et al. 2008). In addition to reducing the total number of chasmothecia in the greenhouse experiments, some fungicides reduced the viability of ascospores within the treated chasmothecia. To date, only one work has been published showing the efficacy of fungicides in reducing ascospore viability within chasmothecia (Gadoury et al. 1994). In that study, 72 to 96 % of the ascospores were eliminated when chasmothecia were exposed to lime sulphur and copper compounds; dinocap reduced viability only with longer exposure time, while sulphur and triadimefon did not reduce ascospore viability. In addition to confirming the findings of Gadoury et al. (1994), the current study adds new elements to the list of products that are effective against ascospores and/or chasmothecia of E. necator. The biocontrol agent A. quisqualis reduced the number of chasmothecia by 41 % relative to the untreated control, but it did not reduce ascospore viability because this biocontrol agent destroys immature chasmothecia and, as a consequence, prevents ascospore production (Kiss et al. 2004).

Results from single-application experiments in vineyards showed that application of fungicides once after harvest, as well as application of A. quisqualis pre- and post-harvest, delayed disease onset and epidemic development in the following season and significantly lowered the disease severity until berries reached the pea-size stage. Although Gadoury et al. (1994) obtained similar results with an over-the-trellis application of lime sulphur on dormant vines, the required quantity of lime sulphur and volume of water were impractically high, and Gadoury et al. (1994) recognized the need for compounds more effective and economical than lime sulphur for reducing the primary inoculum of E. necator. Such compounds were identified in the present work which showed that disease control can be achieved at label rates and with normal volumes of water by applying fungicides once after harvest or A. quisqualis pre- and post-harvest. The implication in terms of fungicide resistance for those fungicides for which the intrinsic risk for resistance evolution is estimated to be low to high (spiroxamine, boscalid, bupirimate, and kresoxim-methyl) (FRAC 2009) should be investigated; application of these fungicides at the end of the season when not all the chasmothecia have been produced may stimulate resistance by selecting prior to mating resistant isolates.

In epidemiological terms, the disease progress curve of polycyclic diseases, such as grapevine powdery mildew, is driven by two main parameters: y0 (level of initial inoculum) and r (rate of disease development). Sanitation reduces y0, which delays the start of the epidemic and causes a consequent shift of the disease progress curve. The delay in time, or sanitation ratio, can be calculated as: ln(y0 / ys ) × 1/r, where ys is the percentage of initial inoculum remaining after sanitation (Nutter 2007). For instance, with ys = 36.7 ± 5.63 %, as it was for synthetic fungicides in our greenhouse experiments, and with r = 0.35 (maximum value of r in Sall 1980), the delay is 3 days with a 95 % confidence interval between 2 and 4 days. With r = 0.09 (i.e., one-fourth of the highest value), the delay is between 8 and 15 days. In this work, the appearance of first disease symptoms was delayed by 1 to 3 weeks in the treated plots; the consequent reduction in disease intensity at the berries pea-size stage was therefore consistent with these theoretical considerations.

In single-application field experiments, application of mineral oil over-the-trellis and before bud break had no significant effects on the disease. In previous studies, a winter spray of mineral oil reduced powdery mildew by up to 64 % on grape bunches over a 3-year period (D’Ascenzo and Corvi 2010). Because mineral oil is one of the products that can be used for controlling cochineals (Planococcus spp.) (Vandini et al. 2010), which are an increasing problem in North Italy (Scannavini et al. 2009), a possible parallel activity of mineral oil on E. necator chasmothecia overwintering on bark should be an added benefit.

The combined-application field experiments showed that two applications of A. quisqualis (pre- and post-harvest), as well an application of mineral oil before bud break, in combination with a model-based schedule of fungicide application between bud break and fruit set, may provide adequate control of powdery mildew until berries attained the pea-size stage with minimal input of sulphur and without synthetic fungicides, as is increasingly required by societal and political pressures in the European Union (Directive 2009/128/EU). This is important because berries rapidly acquire ontogenic resistance to powdery mildew infections after fruit set (Gadoury et al. 2003) thus it is essential to reach the pea-size stage with low disease severity.

In the current study, the disease did not develop in the vineyards with no or few chasmothecia trapped in the previous season, at least until berries attained the pea-size stage. Therefore, neither sanitation nor early fungicide application was justified in these vineyards. Assessment of the chasmothecia dose of the vineyard (Gadoury and Pearson 1988) in the late season may be a key component of decision-making for the control of powdery mildew in spring and early summer next year.

In addition to enhancing powdery mildew control in the following season, there are further reasons for introducing sanitation into disease control programs. By reducing disease severity in the prebloom stages of grapevine development, sanitation would also reduce bud infection by powdery mildew mycelium (Rademacher and Gubler 2002) and therefore reduce the possibility of flag shoot development in the following season. This is important in those grapevine growing regions where, in contrast to Northern Italy and other regions, flag shoots represent a major source of primary inoculum in spring. Sanitation should also contribute to the management of fungicide resistance which is increasingly difficult because of the reduction of the number of registered fungicides available for grapevine powdery mildew control due to regulatory actions (Directive 1991/414/CEE) in the EU. Reduction in the efficacy of demethylation inhibitors (DMIs) or mitochondrial inhibitors (QoIs) against E. necator has been reported worldwide (Debieu et al. 1995; Erickson and Wilcox 1997). It was shown that DMI resistance is transmitted over the seasons by overwintering ascospores (Gubler and Ypema 1996), thus the eradication of chasmothecia by A. quisqualis or mineral oil may reduce the perpetuation of resistance across seasons. Finally, sanitation reduces the number and size of powdery mildew colonies exposed to fungicides in spring and early summer and therefore reduces the probability of the development of fungicide resistance within the respective season (Brent and Hollomon 2007).

Acknowledgments

This research was funded by the Emilia-Romagna Region and the following companies: Intrachem Bio Italia, Basf, Makhteshim Agan Italia, Dow AgroSciences, Cerexagri, Bayer CropScience, and Isagro Italia. The authors thank G. Pradolesi and M. Scannavini for the management of the vineyards. S.E. Legler carried out this work within the Doctoral School on the Agro-Food System (Agrisystem) of the Università Cattolica del Sacro Cuore (Italy).

Copyright information

© KNPV 2012