Optimization of copper applications using Laminarine for the sustainable control of grape downy mildew

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*Corresponding author: francesco.cavazza@astrainnovazione.it
Optimization of copper applications using Laminarine for the sustainable control of grape
downy mildew
Bergamaschi1, A., R., Cavazza2, F., Bugiani3, R.
1 UPL Italia – Cesena, Italy
2 Astra Innovazione e Sviluppo – Via Tebano, 45, 48018 Faenza, Italy
3 Servizio Fitosanitario – Regione Emilia-Romagna - Via A. da Formigine, 3, 40129 Bologna, Italy
______________________________________________________________________________
1 Introduction
Grapevine Downy mildew (GDM) is caused by the oomycete
Plasmopara viticola, affecting the leaves and berries under
warm and humid environmental conditions. P. viticola may
reduce grape yield and affects the aroma and flavours in wine,
with organoleptic impacts on berry quality and vinification.
GDM is mainly controlled using chemical fungicides in
conventional vineyards, where problems with pesticide
residues and resistance to active ingredients are increasing,
while copper and sulphur are used in organic farms (Gessler
et al., 2011; Gadoury et al., 2012). Due to the high number of
treatments carried out in viticulture restrictions have been
applied by the European Commission on the number of
pesticide treatments (Directive 2009/128/EC) and on the
maximum quantity per year of copper fungicides (Regulation
2002/473/EC). Recently, copper, which is still considered the
most important antifungal product in organic viticulture, has
been added to the list of candidates for substitution (European
Commission Implementing Regulation 2018/84) and limited
to 4 kg per hectare/year spread over 7 years (European
Commission Implementing Regulation 2018/1981 of 13
December 2018). Therefore, new strategies have been
developed to prevent grapevine downy mildew (GDM) in a
more sustainable way. Algae extracts are known among the
resistance inducers of natural origin (Vera et al., 2011), and
among these the laminarine, a glucanoligosaccharide
extracted from the brown alga Laminaria digitata, is well
known as an elicitor of the defense mechanisms of plants
(Labarre and Orieux, 2010; Bernardon Mery and Joubert,
2012), including grape (Aziz et al., 2003; Trouvelot et al.,
2008) on which the laminarin sulphate is active (Ménard et
al., 2004). Such activity has often been tested on grape against
Plasmopara viticola and Botrytis cinerea (Aziz et al., 2003;
Gauthier et al., 2014; Chalal et al., 2015; Romanazzi et al.,
2016). In the present work semi-field and field trials were
carried out to evaluate the efficacy of laminarine in
combination to a reduced amount of copper-based product to
contain the GDM in a sustainable viticulture.
2 Materials and Methods
In 2019 and 2020 3 efficacy trials were carried out as
following with the aim to evaluate the efficacy of laminarine
in combination with a reduced amount of copper-based
product. The characteristics of employed products are shown
in table 1.
Product
a.i.
Conc.
% or
g/L
Formulation
Poltiglia
Dissperss®
Copper -
Bordeaux
mixture
20
WG
Vacciplant®
Laminarine
45
SL
Table 1 Characteristics of employed products
Field Trials. In 2019, field efficacy trial was accrued out on
Cv. Trebbiano Romagnolo on Kober 5BB rootstock planted
in 2003, trained as “Casarsa” and spaced at 1.5 x 3.5 m. In
2020, the trial was carried out Cv. Sangiovese on Kober
5BB rootstock planted in 1999, trained as spour cordon and
spaced at 1.2 x 3.0 m. Both trials were experimentally
designed as Randomized Complete Block (RCB) with 4
replications in 2019 and 5 replications in 2020. Sprays and
volumes were applied as shown in table 1 & 2.
Semi-field trial. Potted grapevine plants) cv Pinot Grigio,
were previously moved inside the greenhouse for
acclimation. After the experimental application with the test
products (performed outside the greenhouse) the plants were
kept inside the climatic chamber to avoid any runoff due to
rain events. The artificial infection was performed outside the
greenhouse and then the plants were kept in the climatic
chamber as previously described. A total of 1 litre per
treatment, corresponding to 0.33 litres per plant, was applied
in a single experimental application. The plants were brought
outside the greenhouse only for the fungicide application:
they were moved just before the spray and were brought back
inside the greenhouse after the complete dry-off of the
application (when the canopy was dried). Application was
carried out 2 days before the artificial inoculum at three
different dosages. Artificial inoculation was carried out with
a hand-pump using a water suspension containing 5 x 104
sporangium/mL of Plasmopara viticola. A total of 2.5 litres
of solution was prepared and inoculated (having on average
30-32 mL of inoculum per plant). The selected pathogen
strain was characterized by complete sensitivity to the
fungicides included in the trial.
In field trials, disease evaluation was performed by visual
observation of 100 leaves and 50 bunches per plot estimating
disease incidence (percentage of affected leaves or bunches)
and severity (percentage of injured leaf or bunch area) using
the following score: 0 = absence of symptoms; 1 = 0.1 ÷ 5.0%
of surface with damage; 2 = 5.1 ÷ 15.0% of surface with
damage; 3 = 15.1 ÷ 40.0% of surface with damage; 4 = 40.1
÷ 70.0% of surface with damage; 5 = 70.1 ÷ 100.0% of
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 50, 03005 (2022) https://doi.org/10.1051/bioconf/20225003005
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*

surface with damage. For the trial in semi field condition
visual observation of 100 leaves, with the same assessment
procedure above described, was performed. Statistical
analysis Analysis of Variance (ANOVA) and Duncan's New
MRT (p ≤ 0.05) was used in 2019 trail, while SNK test (p ≤
0.05) and Abbott’s formula were used to calculate the
efficacy in 2020 trials
2019 Efficacy trial open filed
The final scope in the trail was to evaluate the combined
efficacy of Vacciplan (laminarine 45 g/L) at the dosage of 1,5
L/ha with a copper compound at half dosage, to obtain an
annual copper amount distributed within the European limit.
The sequence of 4 or 3 applications (tank mix) were carried
out in a specific growth period as reported in table 2: at the
beginning ABCD corresponding from 3 leaves developed to
pre flowering; in the core EFGH from flowering to berry set
up; at the end of growth cycle from berry pea size to bunch
closing ILM. The standard was a full copper dosage rate for
the whole cycle and compared with a total amount of copper
per hectar respect the EU rules (4 kg Cu/ha). Spray interval
was fixed at 7 days.
The trial was carried out in the countryside of Ravenna,
Emilia-Romagna, a region in the northeast of Italy, in a
typical wine production area, where Downy mildew
(Plasmopara viticola - PLASVI) often causes heavy yield
losses.
The trial was made in a commercial vineyard of Trebbiano
Romagnolo on Kober 5BB rootstock planted in 2003, trained
as “Casarsa” and spaced at 1.5 x 3.5 m. The five-vine plots
were arranged in four blocks using a randomized complete
block design. An untreated plot per block was present.
Trt
No.
Treatment Name
Rate kg/hl
Application
Code
Spray
Volume
L/ha
1
Untreated Check
2
Poltiglia Disperss
0,4
ABCD
400
Poltiglia Disperss
0,4
EFGH
600
Poltigia Disperss
0,4
ILM
1000
3
Poltiglia Disperss +
Vacciplant
0,2 + 1,5
L/ha
ABCD
400
Poltiglia Disperss
0,4
EFGH
600
Poltiglia Disperss
0,4
ILM
1000
4
Poltiglia Disperss
0,4
ABCD
400
Poltiglia Disperss +
Vacciplant
0,2 + 1,5
L/ha
EFGH
600
Poltiglia Disperss
0,4
ILM
1000
5
Poltiglia Disperss
0,4
ABCD
400
Poltiglia Disperss
0,4
EFGH
600
Poltiglia Disperss+
Vacciplant
0,2 +
1,5L/ha
ILM
1000
6
Poltiglia Disperss
0.8 kg/ha
AB
400
Poltiglia Disperss
1.2 kg/ha
CD
400
Poltiglia Disperss
1.6 kg/ha
EFGH
600
Poltiglia Disperss
2.4 kg/ha
ILM
1000
Table 2 Treatments detail
Application code
A = BBCH13 19th April 2019; B = BBCH14 25th April 2019;
C = BBCH16 2nd May 2019; D = BBCH16 8th May 2019; E
= BBCH53 15th May 2019; F = BBCH55 22nd May 2019; G
= BBCH57 28th May 2019; H = BBCH60 5th Jun 2019; I =
BBCH71 13th June 2019; L = BBCH73 21st June 2019; M =
BBCH77 28th June 2019
Assessments:
BBCH57 27th May 2019 Leaves (evaluation efficacy ABCD
applications); BBCH57 10th June 2019 leaves and bunches
(evaluation efficacy EFGH applications); BBCH77 3rd July
2019 leaves and bunches (evaluation efficacy ILM
applications)
2020 efficacy trial open filed
The trial site was selected in Ravenna province (Emilia
Romagna region); it was located in the area near Faenza, an
area particularly suitable to vine growing and downy mildew
infections. The trial was made in a commercial vineyard of
Cv. Sangiovese on Kober 5BB rootstock planted in 1999,
trained as spour cordon and spaced at 1.2 x 3.0 m. The trial
has been set up as randomized Complete Block (RCB), with
5 replications, single control randomized in each block. The
treatments details are reported in table 3. Spray interval was
fixed at 7 days.
Trt
No.
Treatment Name
Rate
kg/hl
Application
Code
Spray
Volume
L/ha
1
Untreated Check
2
Poltiglia Disperss
0,4
AB
555
Poltiglia Disperss +
Vacciplant
0,2 +
1,5 L/ha
CD
667
Poltiglia Disperss
0,2 +
1,5 L/ha
E
889
Poltiglia Disperss
0,4
F
889
Poltiglia Disperss +
Vacciplant
0,2 +
1,5 L/ha
GHI
1000
Poltiglia Disperss
0,4
LM
1000
3
Poltiglia Disperss
0,4
AB
555
Poltiglia Disperss +
Vacciplant
0,2 +
1,5 L/ha
CD
667
Poltiglia Disperss +
Vacciplant
0,2 +
1,5 L/ha
E
889
Poltiglia Disperss
0,4
F
889
Poltiglia Disperss
0,4
G
1000
Poltiglia Disperss+
Vacciplant
0,2 +
1,5 L/ha
HIL
1000
Poltiglia Disperss
0,4
M
1000
4
Poltiglia Disperss
0,4
AB
555
Poltiglia Disperss
0,4
CD
667
Poltiglia Disperss
0,4
EF
889
Poltiglia Disperss
0,4
GHILM
1000
Table 3 Treatments detail
Application code
BBCH53 27th April 2020; BBCH57 6th May 2020; BBCH60
13th May 2020; BBCH61 20th May 2020; BBCH69 26th
May 2020; BBCH71 3rd June 2020; BBCH73 9th June 2020;
BBCH75 16th June 2020; BBCH75/77 23rd June 2020;
BBCH77 1st July 2020; BBCH79 8th July 2020
Assessments:
BBCH75/77 22 June 2020 Leaves and bunches; BBCH79
16th July 2020 leaves and bunches.
In figure 6 – 7 – 8- 9 thesis 2 and 3 are respectively reported
as thesis 2 = Modality 1 and thesis 3 = Modality 2
2
BIO Web of Conferences 50, 03005 (2022) https://doi.org/10.1051/bioconf/20225003005
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2020 efficacy semi field trial
The trial was performed in semi-field conditions in the
greenhouse. The trial detail is reported in table 3. Potted
grapevine plants, cv Pinot Grigio, were previously moved
inside greenhouse for acclimation. After the experimental
application with the test product (performed outside the
greenhouse) the plants were kept inside the climatic chamber
to avoid any runoff due to rain events. A total of 1 litre per
treatment, corresponding to 0.33 litres per plant, was applied
in a single experimental application. The plants were brought
outside the greenhouse only for the fungicide application:
they were moved just before the spray and were brought back
inside the greenhouse after the complete dry-off of the
application (when the canopy was dried). Treatment was
performed on May 2nd 2020 using a backpack sprayer Stihl
SR 430. The artificial infection was performed 2 days after,
on May 4th 2020. Artificial inoculation was performed with a
solution containing 5 x 104 sporangium/mL of Plasmopara
viticola A total of 2.5 litres of solution was prepared and
inoculated (having on average 30-32 mL of inoculum per
plant). The selected pathogen strain was characterized by
complete sensitivity to the fungicides included in the trial.
The artificial infection was performed with a hand-pump on
May 4th on all the plants. Plants were then closed in a plastic
bag, kept in the climatic chamber and incubated for 24 hours.
Evaluation of the disease was performed 1 week after
symptom occurrence.
Trt
No.
Treatment Name
Rate
ml/hl
Application
Code
1
Untreated Check
2
Vacciplant
100
A
3
Vacciplant
150
A
4
Vacciplant
200
A
Table 4 Protocol trial
A = 2 days before the artificial inoculum
Assessment 15th May
3 Results and discussion
Efficacy trials open field 2019
3 assessments were carried out, to evaluate the activity of
Vacciplant in tank mix with a half dosage rate of copper
(Politglia Disperss) compared with a full dose rate. Treatment
6 follow the rules about EU copper limitation at 4 kgCu/ha.
To focus the attention for each block of application the table
below reported the follow treatments: Poltiglia disperss full
dosage rate; Poltiglia disperss half dosage rate + Vacciplant;
Poltiglia disperss complaining to 4 kg Cu/ha.
Figure 1: Evaluation of Vacciplant applications in ABCD
Assessment BBCH57 27th May 2019. Disease Incidence and
severity on leaves (bars) and tested products efficacy (lines)
%
Figure 2: Evaluation of Vacciplant applications in EFGH
Assessment BBCH57 10th June 2019. Disease Incidence and
severity on bunches (bars) and tested products % efficacy
(lines).
Figure 3: Evaluation Vacciplant applications ILM
Assessment BBCH77 3rd July 2019. Disease Incidence and
severity on leaves (histograms) and tested products efficacy
(lines) %
.
3
BIO Web of Conferences 50, 03005 (2022) https://doi.org/10.1051/bioconf/20225003005
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Figure 4: Evaluation Vacciplant applications at ILM
Assessment BBCH77 3rd July 2019. Disease Incidence and
severity on bunches (histograms) and tested products efficacy
(lines) %
Efficacy trials open field 2020
The experimental design adopted was the randomized block
with 5 replications and each plot consisted of 5 contiguous
plants on the rows. The applications were performed with a
pneumatic shoulder-supported nebuliser (mod. Stihl SR 430)
distributing a variable volume of water between about 555
and 1000 L/Ha, depending on the phenological stage of the
plant. The assessment to evaluate the efficacy of the products,
the incidence (% affected leaves and bunches) and the
severity (% affected area) of the disease was performed
observing 100 leaves and 50 bunches per plot.
In the vineyard where the trial plots were located, the first
symptoms of the disease were observed on the leaves on May
26th as consequence of the rains occurred on May 17th and
May 19th and of the first overcanopy irrigation executed on
May 16th.
The disease appeared slight late in the season with a natural
infection. Weather in the spring 2020 was not favourable for
downy mildew characterized by a drought climate with a
significantly low rainfall. From mid-May to June, canopy
irrigations were done twice a week to create favourable
conditions to the disease. A total of 13 artificial overcanopy
irrigations were carried out (15 mm each). Irrigations were
done utilizing micro sprinkler that works at 2,2 bar. During
the trial, the crop condition was normal in accordance with
local practices for commercial production purposes and
allowed to evaluate the activity of plant protection products
under test. The assessment performed on June 22nd (figure 6)
showed in the untreated check an incidence of 20.4% on
leaves with a severity of 3.40%. All strategies applied
demonstrated to obtain, on leaves very good results with a
control ranging from 97% to 100%. No statistical differences
among the different strategies were detected.
Figure 5: Assessment on leaves 22nd June Disease Incidence
and severity on leaves (histograms) and tested products
efficacy (lines) %
Figure 6: Assessment on bunches 22nd June Disease Incidence
and severity on bunches (histograms) and tested products
efficacy (lines) %
Figure 7: Assessment on leaves 16th July Disease Incidence
and severity on leaves (histograms) and tested products
efficacy (lines) %
4
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Figure 8: Assessment on bunches 16th July.
Disease Incidence and severity on bunches (histograms) and
tested products efficacy (lines) %
The assessment on bunches (figure 7) showed the untreated
check an incidence of 10.0% with a severity of 1.75%. As
seen previously, all strategies obtained an excellent efficacy
around 100% of control for disease incidence and severity.
The last assessment, was carried out on July 16th (figure 8 and
9) on leaves and bunches, showed in the untreated check an
incidence on leaves of 64.6% with a severity of 31.85% and
on bunches of 45.6% with a severity of 9.52%. The trial
highlighted as the strategies in which Poltiglia disperss was
used at low dosage (200 g/hL) and in tank mixtures with
Laminarine (in phase B and D) obtained similar results (60-
87% of efficacy on disease incidence and severity on leaves
respectively and 90-95% of disease control on bunches)
respect to that achieved by the Poltiglia disperss used at
higher dosage (400 g/hL) during all the season (57-85% of
efficacy on disease incidence and severity on leaves
respectively and 95-99% of disease control on bunches).
No symptoms of phytotoxicity occurred during the trials.
Efficacy trials semi field 2020
The trial was conducted adopting the experimental design,
with 3 repetitions/treatment. Every repetition was based on
one single potted grapevine plant. The experimental
application was performed with a backpacked nebulizer
sprayer Mod Stihl SR430. A solution containing 5 x 104
sporangium/mL of Plasmopara viticola was sprayed on the
plants treated 2 days before the artificial infection. The results
were evaluated 11 days from the infection in terms of disease
incidence and severity on the leaves of the potted grapevine
plants. The assessment performed on May 15th (at 11 days
after the application of the inoculum – Figure 10) showed
how in the untreated check the inoculum originated infections
on 54,7% of the leaves present, affecting a leaf area estimated
close to 5%. The results of the trial showed that Vacciplant
obtained a good control of the disease (82,6-95,3% of
efficacy respectively on the incidence and the severity of the
disease). Vacciplant when used at 200 mL/100L increasing
efficacy although not statistically evident. Trial demonstrated
that two days between application and the artificial inoculum
were enough to induce an elicitor effect (self-plants
protection). The selectivity assessments were performed just
before the artificial infection, to be sure that the plants were
all asymptomatic and no problem of phytotoxicity could
interfere with the experiment. No phytotoxicity symptom
occurred during the experimental application.
Figure 9: Efficacy of Laminarine application at 2 days
before the artificial inoculum
4 Conclusions
In 2 years, experimentation trials indoor and outdoor, it was
demonstrated that laminarine based product (Vacciplant), can
be effectively included in a strategy for the control of
grapevine downy mildew in tank mix with copper to optimize
the copper rate to be complaint on EU rules. The elicitor
laminarine activity express its activity within one day of the
application and maintain a good activity until 9 – 10 days
(Taibi O. et al.). The laminarine behavioir and activity could
be synchronized with the copper contact product in a
sustainable spray interval, with low dosage rate of copper and
a very good bunches protection.
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