Crop Protection 139 (2021) 105382

Contents lists available at ScienceDirect

Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Microencapsulation – An innovative technique to improve the fungicide

efficacy of copper against grapevine downy mildew
Karin Weitbrecht a, Stefan Schwab b, d, Christoph Rupp b, Evi Bieler c, Markus Dürrenberger c,
Gottfried Bleyer a, Stefan Schumacher a, Hanns-Heinz Kassemeyer a, René Fuchs a, *,
Eberhard Schlücker b
a
State Institute of Viticulture and Enology, Merzhauser Str. 119, 79100, Freiburg, Germany
b
Friedrich-Alexander-University Erlangen-Nuremberg, Institute of Process Machinery and Systems Engineering, Cauerstraße 4, 91058, Erlangen, Germany
c
University of Basel, Swiss Nanoscience Institute (SNI) – Nano Imaging Lab, Klingelbergstrasse 50/70, 4056, Basel, Switzerland
d
Agrolytix GmbH, Cauerstraße 4, 91058, Erlangen, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Copper based fungicides are plant protection products prevalently used in organic agriculture to control several
Copper fungicides plant diseases. Since copper can have various negative effects on the environment, the amount used should be
CuCaps reduced to the minimum necessary. However, this reduction should not lead to a loss of efficacy, as otherwise the
Grapevine
quality and quantity of the yield would be at risk. During this study, a copper sulfate/copper hydroxide mixture
Microparticles
Microencapsulation
was combined with an agent for better adhesion to grapevines. This was achieved by microencapsulation of the
Oomycetes active agents into a fat matrix which resulted in the so called CuCaps. Laboratory analysis of dispersibility and
Organic farming particle size proved that CuCaps form an applicable powder, suitable for use in commercial crop protection
Plasmopara viticola sprayers. Field experiments demonstrated that CuCaps have an equivalent effect compared to a commercially
Spray congealing available copper-based fungicide (CCF). Both, CuCaps and CCF were able to significantly (p < 0.05) reduce
Viticulture disease severity (DS) on leaves and bunches in three independent years compared to an untreated control (UTC).
Vitis vinifera This was even true if the applied amount of CuCaps was reduced by one third to 2 kg copper per year and hectare.
Additionally, in the particularly severe P. viticola epidemic of 2016, CuCaps showed a remarkably better effect on
flower clusters and bunches compared to the CCF. However, this effect was not statistically significant due to
strong variation within the repetitions. Taken together, this study shows that copper minimization by micro­
encapsulation is possible. Considering the restricted use of copper-based fungicides, CuCaps can be a valuable
option for organic farmers, especially in difficult years.

1. Introduction
One of the most devastating diseases in viticulture is grapevine
downy mildew caused by the biotrophic oomycete Plasmopara viticola
(Berk. & M.A. Curtis) Berl. & de Toni. Since P. viticola was introduced
into Europe in the late 19th century (Millardet, 1881) Vitis plants on this
continent were never challenged to develop effective defense mecha­
nisms against this pathogen during evolution. Consequently, all Vitis
vinifera cultivars are highly susceptible to this disease, whereas wild
species from North America such as V. rupestris, V. riparia or V. aestivalis,
show a distinct degree of resistance (Staudt and Kassemeyer, 1995; Boso
and Kassemeyer, 2008).
In warm and humid climates P. viticola is a threat to grapevine plants
throughout the whole production period. At the beginning of the season,
primary infections may occur as soon as soil temperatures exceed 11 ◦ C.
Oospores which were produced at the end of the last season and matured
in the soil over winter can now germinate by contact with water
whereby primary zoospores are released. These zoospores can be
transported to green plant parts by raindrop splashes. The motile zoo­
spores attach around the stomata and enter the grapevine through the
intercellular space by forming a penetration peg (Kiefer et al., 2002;
Riemann et al., 2002). In the sub stomatal cavity a vesicle develops and
within few hours a hypha with the first haustoria arises. At this time
P. viticola has established itself in the host tissue. In susceptible Vitis
genotypes colonization of the intercellular space and parasitation of host
cells by multiple haustoria begins (Unger et al., 2007; Boso and

* Corresponding author.
E-mail address: rene.fuchs@wbi.bwl.de (R. Fuchs).

https://doi.org/10.1016/j.cropro.2020.105382
Received 8 June 2020; Received in revised form 1 September 2020; Accepted 2 September 2020
Available online 6 September 2020
0261-2194/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
K. Weitbrecht et al.
Kassemeyer, 2008). After an incubation period of four to ten days under
favorable conditions such as high humidity and temperatures above
12 ◦ C, sporangiophores with sporangia can emerge from the stomata
(Rumbolz et al., 2002). During the season P. viticola reproduces asexu­
ally by abundant production of sporangia. These sporangia again release
heterokont flagellated zoospores at periods of sufficient leaf wetness.
P. viticola can consequently lead to epidemics with severe loss of quality
and yield and demands an enormous effort from the winemakers for its
control. In general, symptoms are usually more severe on young leaves,
inflorescences and young berry clusters since, as older leaves and berries
become increasingly resistant (ontogenetic resistance) (Kennelly et al.,
2005).
Although a large number of fungus-resistant grape varieties have
been developed in recent years, most of the vineyard area is still planted
with susceptible varieties (Pedneault and Provost, 2016). Infection of
the plants and an outbreak of the disease are therefor usually prevented
by the use of contact fungicides. While conventional viticulture can rely
on a wide range of substances, organic farming in Europe is almost
exclusively limited to copper-containing fungicides. The use of antimi­
crobial copper compounds used in agriculture has been reviewed
recently (Lamichhane et al., 2018; La Torre et al., 2018).
Copper-containing fungicides are used against a broad range of plant
diseases caused by bacteria, fungi and oomycetes. While these products
are popular due to their low costs and their multisite activity with a low
risk for resistance development on the part of the pathogens they also
possess several negative effects on the environment. Application rates up
to 30 kg/ha per year in viticulture led to high amounts of copper re­
siduals in vineyards all over the world (Kovacic et al., 2013; Lamichhane
et al., 2018). Negative effects of high copper amounts in the soil on the
microbial biomass and the diversity of microbial communities have
previously been shown (Kandeler et al., 1996). Large amounts of copper
in the ground have been also linked to a reduction of the biodiversity of
soil macroorganisms like earthworms, nematodes and snails (Buene­
mann et al., 2006). However, presently there are no approved alterna­
tives to copper containing fungicides available in organic agriculture
making its practicable reduction a priority objective in organic farming.
One tool to significantly reduce plant protection treatments are decision
support systems (DSS) like Agrometeo or VitiMeteo (Dubuis et al.,
2019). DSS allow winegrowers to precisely time their sprayings by the
use of weather forecast data for real time disease modelling and infec­
tion prediction. Over the last few years additional efforts have been
made to substitute or at least reduce copper application by the use of
new formulations (La Torre et al., 2011). A common problem, especially
with the application of water-soluble pesticides, is the rapid reduction of
a high initial concentration on the plant surface due to washing-off by
rain or other environmental effects (Markus and Linder, 2006). One
promising strategy to reduce this problem may be the encapsulation of
the water-soluble copper into a solid lipid microparticle. Lipophilic
microparticles may allow better attachment to the plant surface and a
controlled release of the active ingredient copper. The aim of this work
was to create such a product by the microencapsulation method. The
final product, the so-called CuCaps, was tested for their suitability as a
plant protection product used in commercially available sprayers by
analysis of technical parameters like suspensibility and particle size.
Consequently, wash-off resistance as well as biological activity of
CuCaps against P. viticola were tested in leaf disk assays and field trials in
the years 2013, 2014 and 2016.
2. Materials and methods
2.1. Development of CuCaps
2.1.1. Production
CuCaps production was carried out by spray congealing. The matrix
material of the particles, a hydrogenated rapeseed oil (melting point
70 ◦ C), was melted in a mixing tank at 80 ◦ C. After addition of benzoic
2
Crop Protection 139 (2021) 105382
acid (16.5%, w/w) and the emulsifier sorbitan fatty acid ester ethoxylate
(10.0%, w/w), the active ingredients, copper sulfate (18.84%, w/w) and
copper hydroxide (3.84%, w/w), were mixed to the blend by an ULTRA-
TURRAX® high-speed stirrer (IKA-Werke, Germany). The homogenized
dispersion was pumped into a heated two-substance nozzle (80 ◦ C).
Solidification was achieved by atomizing of the fluid by an approx. 80 ◦ C
hot air flow at 2 bar into a cooled spray tower. Separation of the solid
microparticles from the air flow was achieved by the help of a cyclone.
At the end of the production process the separated CuCaps, containing
10% copper (w/w), were collected in a plastic barrel.
2.1.2. Suspensibility test
To ensure that wettable powders (WP), such as CuCaps, can be used
well in field sprayers that are common in practice, their suspensibility in
water must be checked previously in the laboratory. A stable suspension
is necessary to avoid sedimentation in the sprayer, which can lead to
blockages in the sieves and nozzles, and inhomogeneous spray deposits
on the plant surface. Therefore, the CIPAC method MT 15 “Suspensi­
bility of wettable powders in water” (Dobrat and Martijn, 1995) with
some slight changes was applied. In the case of CuCaps, suspensibility
was defined as the amount of microcapsules suspended after a given
time in water. CuCaps were dispersed initially by weighting 1.172 g of
powder into a 100 ml beaker and adding water at 20 ◦ C to a total mass of
50 g. After mixing with a magnetic stirrer at 800 rpm for 1 min the
suspension was left to stand for 10 min in a 20 ◦ C incubator. The mixture
was transferred into a 250 ml mixing cylinder, which was filled with
water to a volume of 250 ml. For mixing the cylinder was inverted 30
times before being placed into the incubator again. After 30 min the top
90% of the volume in the cylinder were withdrawn by a peristaltic
pump. Finally, the dry mass of the remaining 10% was determined and
the suspensibility was calculated by equation (1) where c is the mass of
CuCaps in the initial suspension and Q is the dry mass of the bottom
10%.
10 100 (c − Q)
Suspensibility = × (1)
9 c
2.1.3. Particle size analysis
Particle size analysis provides additional information on the appli­
cability of WP formulations in the field sprayers. For optimum usability
no microcapsule agglomerates should form during dispersion in the tank
mix and the largest particles should be less than 100 μm in diameter.
Therefore, particle size was measured with a Mastersizer 2000 (Malvern
Panalytical, UK) equipped with the wet dispersion unit Hydro 2000S,
filled with bidistilled water. CuCaps were added into the dispersion unit
until an obscuration rate between 5 and 12% was reached. Possible
agglomerates were separated by using a stirrer or ultrasound. Initial
dispersion was conducted with a low-energy input by stirring at 1500
rpm. After five measurements (20 s per cycle) ultrasound was applied
during the following 10 measurements with an intensity of 10% to
determine the real particle size distribution. Finally, another five mea­
surements were carried out without ultrasound to check if re-
agglomeration takes place. For comparison values for Dv (10), Dv (50)
and Dv (90) were recorded, whereas Dv(x) is the particle size (D =
diameter), up to and including which x % of the total volume (v) of
particles in the sample is contained.
2.1.4. Microscopic analysis
Visualization of the three-dimensional structure of the capsules and
their attachment properties on the leaf surface was carried out with a
scanning-electron microscope (Philips XL30 ESEM) equipped with a
cryo preparation unit (Alto 2500, Gatan, UK). For this purpose, CuCaps
were applied to leaf discs as described in 2.1. Strips were taken imme­
diately after the application from which slices of approx. 3 mm × 3 mm
were excised with a scalpel. The slices were mounted with low tem­
perature glue on a specimen holder by carefully avoiding touching the
K. Weitbrecht et al. Crop Protection 139 (2021) 105382

leaf surface. Cryofixation was performed with nitrogen slush
(<− 185 ◦ C). The frozen samples were sputtered with 20 nm Au in a high
vacuum cryo preparation chamber and examined with a SE detector
operating with acceleration voltage of 5–10 kV at high vacuum and
− 150 ◦ C. In this way, the specimens were cryo-fixed, sputtered and
ready for analysis within 90 min after sampling. The surface structure of
each specimen was documented from at least three positions.
2.2. Characterization and efficacy tests of CuCaps
2.2.1. Leaf disc assays
To evaluate the efficacy of the different fungicides leaf disk assays
were carried out. For this purpose, the third to fifth fully developed
leaves from the top of Vitis vinifera cv. Mueller-Thurgau greenhouse
plants of at least 12 individuals were detached and shortly disinfected
with 70% ethanol. Afterwards leaf discs were punched out of each de­
tached leaf with a cork borer (Ø 15 mm) and evenly distributed over all
treatments. To avoid desiccation leaf discs were kept upside down on
plant-agar dishes (1%, w/v; 12*12 cm square petri dishes, Greiner,
Germany). One dish consisted of 36 leaf discs of at least 12 individuals.
These were then treated with the corresponding fungicides by means of
an automated application system (Schachtner, Germany). A commer­
cially available copper fungicide (CCF; Cuprozin progress ®, Spiess
Urania Chemicals) was used as a control at a concentration of 150 mg
(copper)/l. After air-drying leaf disks were infected with 80 μl of a
suspension containing 40.000 sporangia of P. viticola in distilled water.
Dishes were placed in the dark overnight. On the next morning sus­
pension drops were removed from the leaf disc surface and the dishes
were placed in a climate chamber at 24 ◦ C under long day conditions
(14 h day, 10 h night). After five to seven days disease severity (DS) was
scored via ImageJ (Wayne Rasband, National Institute of Health,
Bethesda, MD, USA) taking the area of sporulation as measurement.
These experiments were repeated four times.
2.2.2. Wash-off resistance assays
Leaves of six different V. vinifera cv. Mueller-Thurgau plants were
detached and treated as mentioned above. 16 leaf discs (Ø 45 mm) were
punched out and placed on water agar dishes with the same distribution
as in the assays without immersion. Afterwards dishes were treated with
concentrations corresponding to an application of 150 mg/l copper and
air dried. On the following day leaf discs were immersed in distilled
water for 2 s with forceps, placed back onto the agar dish and air dried
again. This procedure was repeated six to twelve times. Incubation and
evaluation of DS were carried out as mentioned above. These experi­
ments were repeated three times.
2.2.3. Field trials
Independent field trials were carried out in experimental vineyards
of the State Institute of Viticulture and Enology (WBI) in Ihringen and
Freiburg, Germany during the years 2013, 2014 and 2016 with two
different cultivars (V. vinifera cv. Scheurebe and V. vinifera cv. Pinot
noir). Each trial consisted of three randomized plots per treatment and
three randomized plots for an untreated control (Table 1). To ensure a
uniform and homogeneous infection pressure, artificial infections were
induced in May on a single leaf of a single shoot of every fourth plant.
For this purpose, a suspension containing 40.000 sporangia of P. viticola
from freshly infected greenhouse plants was used. Every second row
represented a buffer zone to avoid drift of the sprayed fungicides.
Application of fungicides was carried out with a standard sprayer for
viticulture. The CCF was applied as recommended by the manufacturer
in a basic amount of 0.4 l/ha (100 g copper/ha) in 400 l water and was
adjusted during the season according to the growing stage up to 1.6 l/ha
(400 g copper/ha) in 1.600 l water. Based on the copper contained,
CuCaps were applied in the same amounts. Fungicide applications were
timed according to the decision support system VitiMeteo (www.vitimet
eo.de; Bleyer et al., 2008) and the manufacturers’ recommendation of
the copper containing fungicides. This resulted in eight treatments in
2013, seven treatments in 2014 and ten treatments in 2016, due to the

Table 1
Field trial results of different copper-based fungicides for P. viticola control.Listed are disease incidence and severity of grapevine downy mildew at the end of the
season. The 2013 field trial was performed in a vineyard planted with V. vinifera cv. Scheurebe while field trials in the years 2014 and 2016 were performed in a
vineyard planted with V. vinifera cv. Pinot noir. CCF = commercially available copper fungicide (Cuprozin progress ®, Spiess Urania Chemicals); CuCaps reduced = 2/3
amount of copper compared to standard treatments with CCF and CuCaps. The statistical group was calculated by analysis of variance (ANOVA; p < 0.05).
Year Treatment Fungicide Leaves Bunches
amount
Disease Statistical Disease Statistical Disease Statistical Disease Statistical
aimed applied incidence group severity group incidence group severity group
(%) (incidence) (%) (severity) (%) (incidence) (%) (severity)

2013 CuCaps 2 kg 1.2 kg 50.07 ± A 8.59 ± a 20.67 ± ab 7.54 ± a

(reduced) 5.25 1.08 1.15 0.46
CuCaps 3 kg 1.8 kg 40.33 ± A 7.61 ± a 10.67 ± a 5.28 ± a
2.52 1.92 1.53 1.23
CCF 3 kg 1.8 kg 46.33 ± A 9.61 ± a 32.00 ± b 8.44 ± a
4.51 1.16 3.61 1.36
Untreated 0 0.2 kg 99.33 ± B 36.98 ± b 66.33 ± c 30.11 ± b
control 0.58 2.59 7.51 5.71

2014 CuCaps 2 kg 1.1 kg 96 ± 1.73 A 18.67 ± a 9.67 ± 3.21 a 1.44 ± a

(reduced) 4.09 1.14
CuCaps 3 kg 1.7 kg 96 ± 2.65 A 15.53 ± a 7.67 ± 4.16 a 0.71 ± a
1.55 0.60
CCF 3 kg 1.7 kg 91 ± 14.73 A 13.34 ± a 7.37 ± 3.56 a 0.76 ± a
4.92 0.61
Untreated 0 0 kg 100 ± 0 A 59.19 ± b 44.67 ± b 10.22 ± b
control 4.59 7.09 3.77

2016 CuCaps 2 kg 1.8 kg 49.33 ± A 5.32 ± a 61.67 ± a 31.40 ± a

(reduced) 7.37 1.16 12.66 10.65
CuCaps 3 kg 2.7 kg 39.33 ± A 5.06 ± a 36.67 ± 9.5 a 15.33 ± a
9.24 0.56 5.22
CCF 3 kg 2.7 kg 33.67 ± A 5.46 ± a 63.00 ± a 31.07 ± a
11.93 2.46 14.11 8.6
Untreated 0 0 96.33 ± B 34.33 ± b 100 ± 0 b 90.77 ± b
control 3.79 8.07 2.11

3
K. Weitbrecht et al.
different development of the grapevine downy mildew epidemic. Dis­
ease incidences (DI) were monitored weekly as soon as they reached
thirty percent, DI and DS on bunches and leaves were evaluated after
EPPO standards in three repetitions per treatment (3 × 100 leaves/­
grapes) for the whole field. At the beginning of berry ripening (BBCH 83)
a second detailed evaluation was carried out. To monitor the epidemic of
P. viticola DI and DS were additionally observed weekly in an untreated
vineyard (V. vinifera cv. Müller-Thurgau) according to EPPO standards
mentioned above.
2.2.4. Calculation of efficacies and significance
Efficacies were calculated for all laboratory experiments according
to Abbott WS (1925). An analysis of variance (ANOVA) was performed
on the data of the field trials with R (3.4.1) with a significance level of
alpha = 0.05.
3. Results and discussion
Microencapsulation of active compounds and additives may be
achieved by multiple technologies. However, for a plant protecting
agent only large-scale and cheap processes can be considered. Therefore,
the technology of choice is spray congealing (Gouin, 2004). During this
procedure copper is mixed into a hot melt of fats or waxes and atomized
by a spraying nozzle into a cooled tower. As the droplets cool beneath
their melting point, solidification takes place resulting in matrix-type
microparticles. Capsule production was realized in a pilot plant with a
throughput of roughly 15.6 kg/h, resulting in a CuCaps yield of 89%.
The loss of 11% may be explained by capsules sticking to the spraying
tower and incomplete separation from the airflow. Wettability and
consequently a measured suspensibility of 79% were achieved by inte­
gration of an emulsifier. Particle size analysis was carried out to deter­
mine the dispersibility of CuCaps in water without re-agglomeration by
use of a stirrer (Fig. 1).
The initial measurement showed a very high Dv (90) with 1120 μm
(out of scale) which was reduced to 75 μm within less than a minute and
remained constant until ultrasound was activated at minute four. Only a
slight change in particle size was observed upon activation of ultrasound
with a decrease of the Dv (90) to a stable value of 66 μm. This confirms
that the energy input of the stirrer alone can separate the agglomerates
almost completely. This is of importance because CuCaps need to be
applicable with the standard field sprayers which might be blocked if
particle size exceeds 100 μm. Separation of agglomeration is also crucial
for the best effect against P. viticola since CuCaps must form a homog­
enous layer of particles on wine leaves after spraying. After the deacti­
vation of ultrasound, no significant increase in particle size was
observed. This leads to the conclusion that the used emulsifier is capable
of stabilizing the dispersion. Taken together, particle and suspensibility
analysis proves that CuCaps form an applicable powder (Fig. 2A), suit­
able for further testing for biological activity.
Fig. 1. Particle size analysis of CuCaps in water. Shown are values for Dv (10), Dv (50) and Dv (90). Ultrasound treatment was added from minute four to minute
twelve. Dv(x) = particle size (D = diameter), up to and including which x % of the total volume (v) of particles in the sample is contained.
4
Crop Protection 139 (2021) 105382
For the best effect of fungicides, it is crucial that they not only form a
homogeneous layer on the leaves or inflorescences but also adhere to the
plant surface and have a high durability in rainfall. CuCaps combine a
lipid matrix with several additives to control the release of the included
copper salts, a mixture of copper sulfate and copper hydroxide, from the
particle. After application CuCaps are distributed over the waxy, hy­
drophobic cuticula of plants (Fig. 2B and C).
To characterize the efficacy of CuCaps in comparison to a commer­
cially available copper fungicide (CCF; Cuprozin progress ®, Spiess
Urania Chemicals) laboratory experiments were carried out (Fig. 3). The
dose-response curve shows a continuous increase in efficacy as a func­
tion of dose and a cutoff point between 200 mg and 250 mg of copper per
liter (Fig. 3). The CCF showed a similar efficacy as CuCaps. The control is
shown as a reference for the variability of the infection rate between the
different experiments.
Furthermore, CuCaps were tested for wash-off resistance (Fig. 4). To
assess a change in efficacy a copper concentration (150 mg/l) with a
medium effect on P. viticola growth was chosen intentionally. Compared
to a CCF, CuCaps behave similarly if being immersed in water. The ef­
ficacy of both products dropped equally after each immersion step.
However, soaking of leaf disks in water is a very artificial approach not
taking into regard the kinetic energy and size of the raindrop. Differ­
ences between CCF and CuCaps may be more obvious under field con­
ditions or in machines simulating natural rain. Nevertheless, the
advantage of the novel formulation of the CuCaps regarding their effi­
cacy in the control of grapevine downy mildew compared to the stan­
dard product could be demonstrated in three different field trials
(Table 1).
Field trails were carried out in 2013 (V. vinifera cv. Scheurebe), 2014
(V. vinifera cv. Pinot noir), and 2016 (V. vinifera cv. Pinot noir) to test the
effect of CuCaps against downy mildew. DI and DS varied considerably
from year to year and between leaves (UTC, 2013: DI = 99%, DS = 37%;
UTC, 2014: DI = 100%, DS = 59%; UTC, 2016: DI = 96%, DS = 34%)
and bunches (UTC, 2013: DI = 66%, DS = 30%; UTC, 2014: DI = 45%,
DS = 10%; UTC, 2016: DI = 100%, DS = 91%) (Table 1). Differences
between the three years can be partly explained by the different dates of
disease onset (Fig. 5) and consequently the strength of the epidemic
(Chen et al., 2020). In 2013, DI on leaves increased steadily from
mid-June, but could not exceed 80% by the end of June. The DI on
bunches also developed rather slowly and only increased rapidly from
BBCH 73 (berries groat-sized, bunches began to hang) onwards. The
infection pressure in 2014 was even lower. DI on leaves and bunches did
not rise above 20% before grape closure. However, onset of the disease
at the end of May in 2016 was unusually early which led to a particular
severe epidemic with a DI of almost 100% by the end of June. Due to
optimal weather conditions, e.g. high spring precipitation, for the
development of the disease, the year 2016 was especially well suited to
uncover the differences between the various treatments (Fig. 6). Be­
tween the first fungicide application in May and the last one in the
K. Weitbrecht et al.
Fig. 2. CryoSEM pictures of CuCaps. A: CuCaps of different particle size in the
finished product. B: CuCaps distributed on the lower surface of a leaf of
V. vinifera cv. Mueller-Thurgau. C: CuCap near a stoma of a V. vinifera cv.
Mueller-Thurgau leaf.
middle of August (August 12th, 2016), weekly applications of fungicides
were necessary, resulting in ten applications of copper containing fun­
gicides. CuCaps were applied in two amounts: 3 kg copper/hectar­
e/season representing the full amount and 2 kg of
copper/hectare/season representing a treatment with an amount
reduced by one third. As control only the full amount of Cuprozin
progress was applied due to space constraints in the field. On the leaves,
both fungicides, at both application rates, led to a significant (p < 0.05)
reduction of DI (CCF: 34%; CuCaps: 39%) and DS (CCF: 5%; CuCaps:
5%) compared to the untreated control (DI = 96%; DS = 34%) (Fig. 6).
Even the CuCaps application of the reduced amount of 2 kg copper did
not significantly differ from the two variants with the full amount of 3 kg
(CuCaps (reduced): DI = 49%, DS = 5%). Also on bunches CCF and
5
Crop Protection 139 (2021) 105382
Fig. 3. Efficacy of different doses of CuCaps against P. viticola in leaf disc as­
says. The graph shows the means and standard deviations of four independent
experiments. Each experiment consisted of 36 leaf discs of 12 individual
V. vinifera cv. Mueller-Thurgau plants for each different copper concentration
(mg (copper)/l). CCF = commercially available copper fungicide (Cuprozin
progress ®, Spiess Urania Chemicals), UTC = untreated control.
Fig. 4. CuCaps (CC) and the commercially available copper fungicide (CCF)
show a similar decrease in efficacy after repeated immersions in laboratory
experiments. Efficacy of 150 mg/l CC and a CCF without immersion and after
repeated immersions (I) in distilled water. The graph shows means and stan­
dard deviation of efficacies of all three independent experiments. In each
treatment a replicate consisted of 16 leaf discs from 8 different plants.
CuCaps significantly (p < 0.05) reduced DI and DS compared to the
untreated control (UTC: DI = 100%, DS = 91%). Although the results are
not significant due to high variability within the variants, CuCaps (DI =
37%, DS = 15%) seem to be better against grapevine downy mildew on
bunches than the commercially available copper fungicide (DI = 63%,
DS = 31%) when applied in the full application rate. Even if the amount
of CuCaps applied was reduced by one third the effect was still equally
good (DI = 62%, DS = 31%) as the effect of CCF in the full application
rate (DI = 63%, DS = 31%). This may be caused by a depot like char­
acteristic of the CuCaps. Experiments performed in the past showed that
the performance of a CCF against P. viticola on bunches is rather low
compared to its performance on leaves (Bleyer et al., 2020). While a CCF
K. Weitbrecht et al. Crop Protection 139 (2021) 105382

Fig. 5. Epidemic of grapevine downy mildew in 2016 developed particularly early and unusually severe.
Graphs show disease incidence of P. viticola on Vitis vinifera cv. Mueller-Thurgau in Freiburg, Germany in the years 2013 (A), 2014 (B) and 2016 (C). Gray line
indicates disease incidence on leaves, black line on bunches. Numbers in the graph indicate the developmental stage of the plants (BBCH).

is applied and distributed to the plant surface it is immobile and shows
no effect on new grown plant parts. Microencapsulated products may be
released from the matrix during rain and then distribute around the
capsule. However, the better performance of CuCaps on berries
compared to CCFs should be clarified in further experiments with a
corresponding reduction also in the CCF treatment. Nevertheless, the
results presented here lead to the conclusion that a reduction of copper
amounts could be possible with CuCaps. Since the amounts for copper
application are restricted to a maximum of 28 kg in 7 years (4 kg/ha/­
year) in the European Union and 17.5 kg in 5 years (3.5 kg/ha/year with
the possibility for 4 kg/ha/year in seasons with high infection pressure)
in Germany, CuCaps can be a valuable option for organic farmers in
particularly difficult years. However, the results of 2013 and 2014 also
show that copper reduction is already feasible by the flexible schedule of
spray treatments with the help of decision support systems like Viti­
Meteo (Bleyer et al., 2020). Taken together this study is a proof of
concept for microencapsulation of copper to increase its efficacy as a
fungicide.
4. Conclusions
Copper-based fungicides are the most effective plant protection
products against grapevine downy mildew that are approved in organic
farming at the moment. However, the maximum application rate is
limited and should be reduced to a necessary minimum from an
ecological point of view. Further reduction might be feasible with new
and improved formulations of copper salts. As the results of this study
have shown, microencapsulation of copper into a fatty matrix by the

6
K. Weitbrecht et al.
spray congealing method is possible and results in a wettable, dis­
persable powder with good suspensibility. The biological effectivity was
proven with leaf disc assays and field trials, where CuCaps showed
similar activity to a commercially available copper fungicide at even
lower amounts of copper used. In conclusion, the results presented here
show that on the one hand further copper minimization by microen­
capsulation is possible and on the other hand CuCaps can be a valuable
option for organic farmers in particularly difficult years with regard to
the quantitative limits.
Funding
This research was partly funded by a grant of the Bundesprogramm
Ökologischer Landbau und andere Formen nachhaltiger Landwirtschaft
(BÖLN; Grant. No: 512-06.01-2809OE057) and support money from
Baden-Württemberg State Ministry for Rural Areas, Nutrition and Con­
sumer Protection.
CRediT authorship contribution statement
Weitbrecht Karin: Data curation, Formal analysis, Investigation,
Methodology, Visualization, Roles, Writing - original draft. Schwab
Stefan: Data curation, Formal analysis, Writing - review & editing.
Rupp Christoph: Data curation, Formal analysis, Visualization. Bieler
Evi: Investigation, Methodology. Dürrenberger Markus: Resources.
Bleyer Gottfried: Investigation, Methodology, Writing - review &
editing. Schumacher Stefan: Data curation, Formal analysis, Visuali­
zation, Writing - review & editing. Kassemeyer Hanns-Heinz:
Conceptualization, Funding acquisition, Project administration, Writing
- review & editing. Fuchs René: Project administration, Resources,
Writing - review & editing. Schlücker Eberhard: Conceptualization,
Resources.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
7
Crop Protection 139 (2021) 105382
Fig. 6. Disease incidence and severity of
grapevine downy mildew in the field trial
performed during the year 2016 in a
V. vinifera cv. Pinot noir vineyard. The left
graph shows disease severity and incidence
on leaves, the right graph on bunches. Both
graphs show means and standard deviations
for the three plots at the beginning of berry
ripening (BBCH 83) of season 2016. CC
reduced = CuCaps (2/3 amount); CC =
CuCaps (full amount); CCF = commercially
available copper fungicide (Cuprozin prog­
ress ®, Spiess Urania Chemicals); UTC =
untreated control.
Acknowledgements
We thank Barbara Brengartner, Petra Erhardt and Mara Seger for
their support and continuous enthusiasm for this project. We are
furthermore grateful to Fedor Loesch, Martin Kury and Thomas Kal­
tenbach for excellent technical assistance.
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