Crop Protection 137 (2020) 105202
Contents lists available at ScienceDirect
Crop Protection
journal homepage: www.elsevier.com/locate/cropro
Integrated management of downy mildew of basil
Giovanna Gilardi a, *, Angelo Garibaldi a, Maria Lodovica Gullino a, b
a
b
Centre for Innovation in the Agro-Environmental Sector, AGROINNOVA, University of Torino, Largo Braccini 2, 10095, Grugliasco, TO, Italy
Department of Agricultural, Forest and Food Sciences (DISAFA), University of Torino, Largo Braccini 2, 10095, Grugliasco, TO, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ocimum basilicum
Peronospora belbahrii
Non –chemical products
Fungicides
Cultural practices
The downy mildew of basil, which is caused by Peronospora belbahrii, is one of the most devastating basil diseases
throughout the world, and management programmes with a limited use of fungicides are necessary to control it.
In order to develop efficient integrated disease management programmes, the efficacy of alternative products
(calcium oxide, zeolite, orange oil with ascorbic acid, potassium phosphites and bicarbonate), applied in
different spray programmes in trials carried out under artificial and natural infestations with the pathogen, has
been tested. Various combinations of the treatments were chosen, on the basis of the efficacy of the single
products, for further testing in experiments carried out in the presence of natural pathogen infestation and under
a reduced crop density. The effect of the tested programmes on downy mildew severity (% of affected leaf area),
disease development (AUDPC) and yield (fresh weight of plants) was evaluated. Calcium oxide, K-phosphite and
orange oil with ascorbic acid generally provided the most consistent disease control (58%, 40% and 26% effi­
cacy, respectively), when used alone under severe disease pressure, compared to the inoculated control. Spray
programmes based on calcium oxide, applied 2 to 4 times, in succession to one application of azoxystrobin, and
under a reduced crop density, were the most efficient treatment in the presence of a natural infestation by the
pathogen; this IPM programme reduced downy severity as much as 80%, providing the same effect as a con­
ventional chemical programme based on the alternation of azoxystrobin, fluopicolide þ propamocarb and
mandipropamid.
1. Introduction
Sweet basil (Ocimum basilicum L.) is an economically important herb
crop that is grown in several Mediterranean countries for fresh con­
sumption and for processing (as an ingredient in pesto sauce). The crop
is very important in Italy, and in some areas, such as the Liguria Region,
it represents a particular regional specialty crop protected by a desig­
nation of origin (Garibaldi et al., 1997), which endows the crop with a
very high cultural and economic value. Moreover, it is grown over
hundreds of hectares and involves hundreds of workers. It is also grown
in other regions, in open fields, for processing (frozen and pesto sauce)
and greenhouses for fresh consumption. However, Genovese types are
the only varieties used in Italian cooking, in order to avoid the mint
flavor that is common to other types of basil.
Because of its economic importance, both for fresh production and
for processing, basil is usually cultivated on specialized farms in either
greenhouses or open fields under monoculture conditions, both of which
can encourage epidemics of pests and diseases (Garibaldi et al., 1997).
At the beginning of 2000, downy mildew, caused by Peronospora bel­
bahrii (Belbahri et al., 2005; Thines et al., 2009), became the most
economically important basil disease throughout the world as it had
caused significant yield losses in several countries, including Italy
(Garibaldi et al., 2004a; Farr and Rossman, 2019), and affected the
production and long-term sustainability of the industry throughout the
world. The rapid spread of the pathogen to most basil growing areas was
probably favored by the fact that the pathogen is seed-transmitted
(Farahani-Kofoet et al., 2012; Garibaldi et al., 2004b). The threat of
production losses as a result of this new pathogen has stimulated a great
deal of research in many countries, which has led to a better under­
standing of the biology and epidemiology of the pathogen, and to the
development of cultural management strategies (Cohen et al., 2013;
Cohen et al., 2016; Cohen and Rubin, 2015a; Elad et al., 2016; Patel
et al., 2016). Most cultural practices, in the case of greenhouse-grown
basil, are based on climate-management techniques, which are aimed
at reducing the relative humidity and at manipulating the light and
temperature regimes. P. belbahrii can in fact start to infect basil over a
* Corresponding author.
E-mail address: giovanna.gilardi@unito.it (G. Gilardi).
https://doi.org/10.1016/j.cropro.2020.105202
Received 4 May 2019; Received in revised form 22 April 2020; Accepted 23 April 2020
Available online 29 April 2020
0261-2194/© 2020 Elsevier Ltd. All rights reserved.
G. Gilardi et al.
Crop Protection 137 (2020) 105202
wide range of temperatures, but it needs moisture on the leaf surface to
germinate and start a new infection (Elad et al., 2016; Cohen et al.,
2017; Garibaldi et al., 2007). Reducing the crop density is effective in
reducing the pressure of humidity-promoted foliar pathogens, such as
Botrytis cinerea on basil and chickpea, Puccinia arachidis on chickpea and
Peronospora sparsa on rose (Elad et al., 2014; O’Neill et al., 2002; Pande
et al., 1998; Pande and Rao, 2002), but further investigation is needed
for the downy mildew of basil. However, the current cultural practices
do not permit complete control of the pathogen in the presence of a high
disease pressure, and an integrated approach should be followed.
The use of fungicides on conventional farms is the preferred method
to manage basil downy mildew, since existing resistant cultivars have
not been developed commercially (Ben-Naim et al., 2015, 2018; Far­
ahani-Kofoet et al., 2012; Homa et al., 2016; Pyne et al., 2017; Wye­
nandt et al., 2010, 2018; Wyenandt et al., 2010). In practice, growers
rely on management schemes that rotate fungicides with different
modes of action, such as mefenoxam of the phenylamide inhibitors
(FRAC code 4), azoxystrobin of the quinone outside inhibitors (Qols,
FRAC code 11), mandipropamid and dimethomorph of the carboxylic
acid amide (CAA, FRAC code 40) group, and fluopicolide (belonging to
acylpicolides, FRAC code U), all of which are permitted for use on basil,
as well as on copper fungicides (Cohen et al., 2015 Cohen et al., 2015;
Gilardi et al., 2013, 2015 a,b; Gullino et al., 2009; Homa et al., 2014;
McGrath, 2015, 2016; Patel et al., 2014a b and 2015; Raid et al., 2011).
Management practices that limit the use of fungicides against downy
mildew in basil production are necessary due to the high risk of selecting
fungicide-resistant populations of the pathogen, but also to avoid
fungicide residues, and, more generally, to reduce the impact of fungi­
cides on the environment and on human health. However, after repeated
applications of mefenoxam, resistant populations of the pathogen have
been reported (Cohen et al., 2013b; Garibaldi et al., 2016; Pintore et al.,
2016), therefore making one of the most effective fungicides labeled for
this pathogen ineffective in some areas. Moreover, commercial fungi­
cides work best under low disease pressure in combination with other
management practices. If not properly applied, they leave unwanted
residues on the leaves; this is a peculiar problem due to the frequency of
harvests, especially in the case of basil grown for fresh consumption
(Gilardi et al., 2013; Homa et al., 2014).
The management of downy mildew on basil grown under organic
farming regulations is complicated by the limited effective options for
this kind of production. However, to this kind of production must be
given much attention, since it is very popular due to consumer demand.
Treatments allowed under organic crop management guidelines include
various salts (potassium bicarbonate, mono- and di-potassium salts of
phosphorous acid), biocontrol agents (Streptomyces lydicus, Bacillus
amyloliquefaciens strain D747, Bacillus subtilis), and plant extracts and
oils (i.e. from Reynoutria sachalinensis, Neem oil), although these have
not always provided consistent results when tested against basil downy
mildew under different condition and in different cultivation areas,
seem potentially as promising (Allen and Saska, 2013; Gilardi et al.,
2013, 2015 a, b; Homa et al., 2014; Mersha et al., 2011, 2012; McGrath,
2015; McGrath and Hunsberger, 2012; Patel et al., 2013, 2014 a; 2016).
The possibility of using products, acting as biostimulants and/or resis­
tance inducers, which is at present given much attention, is particularly
interesting for a crop such as basil. For instance, products based phos­
phates are intensively investigated against the disease (Gilardi et al.,
2013; Homa et al., 2014; Wyenandt et al., 2015; Patel et al., 2016).
Moreover, there is a lack of published literature in which the efficacy
of products alternative to synthetic fungicides such as salts, plant extract
and other natural sources, is compared with that of copper and other
fungicides, considering their possible application under Integrated Pest
Management (IPM) strategies.
The aim of this study was: i) to test products already known for their
activity against other downy mildews as possible alternatives to the
chemical fungicides against basil downy mildew in greenhouses under
controlled conditions; ii) to develop IPM protocols against basil downy
mildew, using different alternative products alone or combined with
conventional fungicides, considering different intervals between treat­
ments and different crop densities.
2. Materials and methods
2.1. Plant material and experimental design
Thirteen trials were carried out in 2017 and 2018 in the experimental
greenhouses of the Centre of Competence Agroinnova of the University
of Turin in Grugliasco (Turin, Italy). Trials 1 to 6 were conducted in
2017 and trials 7 to 13 in 2018. General information on each experiment
is given in Table 1.
Basil seeds (cv. Genovese selection ‘Italiano classico’, Pagano
Sementi Italy) were sown in plastic pots (12 L vol., 30 � 45 cm at 0.52 g/
pot - corresponding to 310–340 plants/pot, 1550–1700 plants/m2,
which is considered the standard density) in trials 1–9. Trials 1–9 were
carried out 3 times and conducted with 4–5 replicates per treatment
using one pot as experimental unit. The treated and untreated pots were
arranged on benches in a 64 m2 greenhouse compartment using a
completely randomised block design, with each pot representing one
experimental unit (Table 2). The distance among pots was about 1 m to
avoid the cross-contamination between different spray programmes
during the trials.
Trials 10 to 13 were carried out using the ‘Italiano classico’ basil
cultivar (Olter sementi, Italy) as a result of preliminary tests aimed to
select a seed lot naturally infested by the pathogen. Different plant
densities, namely 0.24 g/pot (corresponding to 144 plants/pot and
600–750 plants/m2) and 0.44 g/pot (corresponding to 240–270 plants/
pot and 1200–1350 plants/m2) were compared with the standard den­
sity (Table 2). In each of the trial 10–13, treatments were carried out at
the same time in three adjacent greenhouse compartments (64 m2) using
one crop density per greenhouse compartment. In each greenhouse
compartment the treated and untreated pots were arranged using a
completely randomised block design with one pot representing one
experimental unit with 4–5 replicates per treatment.
During all the trials, the environmental conditions were maintained
at a high relative humidity, close to 90%, and at temperatures ranging
between 24 and 27 � C, by using a program (software Magricomp Mul­
tilab, Agricontrol S.n.c., SV, Italy) that allows the same environmental
conditions set in the greenhouse to be obtained.
2.2. Products used in the trials
A list of the tested active ingredients, including the dose rates and
commercial formulations, is given in Table 3. The alternative products
to chemical fungicides (hereafter defined as alternative products) were
selected primarily on the basis of data from literature, because of their
efficacy against other downy mildews. Calcium oxide (Califol, Agri­
�, Spain),
NewTech, Italy), orange oil þ ascorbic acid (Snack, Masso
zeolite (Zem70, GreenLine, Italy), potassium bicarbonate (Armicarb 85,
�, Spain) were
SCAM, Italy) and potassium phosphite (Alexine, Masso
selected for the experiments.
The tested chemical fungicides, selected from among those regis­
tered for use on basil, were: azoxystrobin (Ortiva, 23.2% a. i., Syngenta
Crop Protection), mandipropamid (Pergado SC, 23.4% a. i., Syngenta
Crop Protection), dimethomorph þ pyraclostrobin (Cabrio Duo EC,
6.9% þ 3.8% a.i., BASF), fluopicolide þ propamocarb hydrochloride
(Volare, 5.53% þ 55.3% a.i., Bayer Crop Science) (Table 3) and copper
sulfite (Cuprotax SDI, 14.84% a.i. Sipcam, Italy).
The chemical fungicides and alternative products were prepared
according to the suppliers’ instructions and applied, as foliar sprays, to
basil plants when the first true leaves were fully expanded, i.e., 16–20
days after sowing, using a hand sprayer, according to the four protocols
(I to IV) described below and summarized in Table 2.
2
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Table 1
Inoculation and treatment dates for all trials performed in this study to determine the relative efficacy of fungicides and alternative products to control Peronospora
belbahrii on basil.
Operation
Sowing
Artificial
inoculation
1st* treatment
(To)
2nd* treatment
(T3)
3rd* treatment
(T6)
4th* treatment
(T9)
5th* treatment
(T12)
End of the trial
a
b
Protocol I
Protocol II
Protocol III
Protocol IV
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
Trial 6
Trial 7
Trial 8
Trial 9
Trial 10
Trial 11
Trial 12
Trial 13
29/06/
17
08/07/
17
07/07/
17
-b
07/07/
17
14/07/
17
13/07/
17
–
14/07/
17
25/07/
17
24/07/
17
–
25/07/
17
09/08/
17
05/08/
17
10/08/
17
15/01/
18
31/01/
18
30/01/
18
02/02/
18
05/02/
18
08/02/
18
12/02/
18
26/02/
18
29/01/
18
21/02/
18
20/02/
18
23/02/
18
26/02/
18
1/03/
18
5/03/
18
19/03/
18
23/08/
18
–
19/07/
17
26/07/
17
22/12/
17
10/01/
18
9/01/
18
12/01/
18
16/01/
18
19/01/
18
22/01/
18
2/02/
18
12/07/
18
–
30/07/
17
–
19/10/
17
01/11/
17
30/10/
17
6/11/
17
9/11/
17
12/11/
17
15/11/
17
24/11/
17
21/06/
18
–
19/07/
17
–
12/10/
17
25/10/
17
24/10/
17
27/10/
17
30/10/
17
02/11/
17
15/11/
17
17/11/
17
22/04/
18
-a
13/07/
17
-b
15/09/
17
4/10/
17
03/10/
17
06/10/
17
09/10/
17
13/10/
17
16/10/
17
23/10/
17
11/05/
18
14/05/
18
17/05/
18
21/05/
18
24/05/
18
03/06/
18
10/07/
18
13/07/
18
16/07/
18
19/07/
18
23/07/
18
30/07/
18
27/07/
18
30/07/
18
02/08/
18
06/08/
18
09/08/
18
17/08/
18
10/09/
18
13/09/
18
17/09/
18
20/09/
18
24/09/
18
02/10/
18
Natural downy mildew infestation by naturally infested seeds.
Not applied.
Table 2
Protocols adopted in the different trials.
Experimental
protocol (Trial
number, year)
Artificial
inoculation of the
basil plants
Fungicides
Alternative
products tested
Standard reference programme
Plant density
Spray programmes tested
I (1-2-3,
2017)
24 h after the first
treatments with 1
� 105 sporangia/ml
Pyraclostrobin þ
dimethomorph;
fluopicolide þ
propamocarb;
mandipropamid;
azoxystrobin
Copper Sulfite
Standard
(1550–1700
plants/m2)
Alternative products and
fungicides tested alone
with 3 sprays at 6-day
intervals
II (4-5-6, 2017;
2018)
24 h after the first
treatments with 1
� 105 sporangia/ml
Pyraclostrobin þ
dimethomorph followed by
fluopicolide þ propamocarb
and then by mandipropamid;
Standard
(1550–1700
plants/m2)
IPM: one fungicide spray
followed by 2 or 4
alternative product sprays
at 3 or 6-day intervals
III (7-8-9, 2018)
24 h after the first
treatments with 1
� 105 sporangia/ml
Potassium
phosphite and
calcium oxide
Azoxystrobin followed by
fluopicolide þ propamocarb
and then by mandipropamid;
Standard
(1550–1700
plants/m2)
IV (10-11-12- 13,
2018)
-a
Pyraclostrobin þ
dimethomorph;
fluopicolide þ
propamocarb;
mandipropamid
fluopicolide þ
propamocarb;
mandipropamid;
azoxystrobin
fluopicolide þ
propamocarb;
mandipropamid;
azoxystrobin
Orange oil þ
ascorbic acid
Potassium
bicarbonate
Calcium oxide
Potassium
phosphite
Zeolite
Potassium
phosphite and
calcium oxide
Orange oil þ
ascorbic acid
Calcium oxide
Potassium
phosphite
Azoxystrobin followed by
fluopicolide þ propamocarb
and then by mandipropamid;
Standard
(1550–1700
plants/m2)
Average
(1200–1350
plants/m2)
Reduced
(600–750
plants/m2)
IPM: one fungicide spray
followed by 2 or 4
alternative product sprays
at 3 or 6-day intervals
IPM: one fungicide spray
followed by 2 or 4
alternative product sprays
at 3 or 6-day intervals
a
Natural downy mildew infestation by naturally infested seeds.
2.3. Tested protocols
Protocols II and III were adopted in trials 4 to 9, which were con­
ducted in 2017–2018, to select the optimal frequency and application
timing, and the optimal combination of fungicides with the alternative
products, which were selected on the basis of their performance in trials
1–3.
The designed IPM consisted of one spray with a selected fungicide
(azoxystrobin, mandipropamid and fluopicolide þ propamocarb hy­
drochloride), followed by alternative products applied in succession at
intervals of 3 and 6 days in 2–4 applications (Table 2). The obtained
results were compared with those obtained from spray programmes
carried out by growers (hereafter defined as the standard commercial
treatment), corresponding to an alternation of pyraclostrobin þ dime­
thomorph, fluopicolide þ propamocarb hydrochloride and man­
dipropamid (Protocol II) or an alternation of azoxystrobin, fluopicolide
Each set of trials included one untreated and non-inoculated control,
used to provide evidence of possible natural infection by the pathogen
during the experiments, one untreated and inoculated control, and
different treatments with products tested alone or according to three
different designed spray programmes (Table 2).
In Protocol I the alternative products (calcium oxide, orange oil with
ascorbic acid, zeolite, potassium bicarbonate and potassium phosphite)
were tested in trials 1 to 3, which were conducted in 2017. Products
were applied three times alone at 6 day intervals. Copper sulfite was
selected as the standard reference, since it is permitted in organic
farming, while different fungicides that are registered for basil were
used as a chemical reference (Tables 2 and 3).
3
G. Gilardi et al.
Crop Protection 137 (2020) 105202
trials, 21–26% (average 23.5%) seedlings with sporulation of P. belbahrii
were observed (data not shown). Briefly, basil seeds of six commercially
available lots were planted in pot trays (15 � 15 cm) containing a steam
disinfested peat substrate (100 seeds/pot), and immediately closed in
transparent plastic bag to avoid cross contaminations between lots and
to provide a relative humidity up to 90%. Pot trays were placed in a
growth chamber at 23/19 � C for 16 h of light, under uniform light in­
tensity, since the stage of cotyledons (11–13 days after sowing), then
were maintained at dark for 24 h before the visual inspection for the
downy mildew sporulation.
Table 3
List of the alternative products and fungicides tested in the different trials with
the corresponding commercial formulations and the used dosages.
Product (acive
ingredient)
Commercial
formulation
(Company)
% active
ingredient
Dosage
(a. i) ml
or g/100
L
FRAC
code
Orange oil þ
ascorbic acid (C
organic þ
Norganic)
Potassium
bicarbonate
Calcium oxide
Snack (Mass�
o,
Spain)
10 þ 1.3
15 þ 2 ml
Not
classified
Armicarb 85
(SCAM, Italy)
Califol
(Agrinewtech,
Italy)
Zem70
(GreenLine, Italy)
Alexine (Mass�
o,
Spain)
85
255 g
22.1
110 ml
Not
classified
Not
classified
70
175 g
52 þ 42
130 þ
105 g
Not
classified
Not
classified
Cuprotax SDI
(Sipcam, Italy)
Cabrio Duo
(BASF, Italy)
Pergado SC
(Syngenta Crop
Protection, Italy)
Volare (Bayer
Crop Science,
Italy)
Ortiva (Syngenta
Crop Protection,
Italy)
14.84
59.4 ml
M1
6.9 þ 3.8
17.3 þ
9.5 ml
11.7 ml
40 þ 11
5.5 þ 55.3
8.8 þ
88.5 ml
U
23.2
18.6 ml
11
Zeolite
Mineral fertilizer P2O5 52%, K2O
42%
Copper sulfite
Dimethomorph þ
pyraclostrobin
Mandipropamid
Fluopicolide þ
propamocarb
hydrochloride
Azoxystrobin
23.4
2.5. Data collection and analysis
The trials were monitored daily and, starting from the appearance of
the first symptoms, disease severity (DS), corresponding to the leaf area
with signs of P. belbahrii as leaf chlorosis and sporulation, was visually
estimated on 100 leaves by individually rating a representative sub­
samples of about 50–60 plants. A 0–5 scale, corresponding to: 0 ¼ no
sign of pathogen infection (no sporulation or no yellow leaves), 1 ¼ from
1 to 10% (midpoint 5%) infected leaf area, 2 ¼ from 11 to 25%
(midpoint 18%) infected leaf area, 3 ¼ from 26 to 50% (midpoint 38%)
infected leaf area, 4 ¼ from 51 to 75% (midpoint 63%) infected leaf area
and 5 ¼ from 76 to 100% (midpoint 85%) infected leaf area, was used.
P
Disease severity was calculated using the formula: DS ¼ [ (number of
leaves � rating scale 0–5)/(total number of recorded leaves)]. The final
evaluation was carried out 7–10 days after the last foliar spray, that is,
27–41 days after sowing, because of the differences in plant growth
throughout the seasons.
The area under the disease-progress curve (AUDPC) was calculated
on the basis of three disease severity assessments, which were carried
out at 4–7 day intervals during three evaluations. The percentage data
were arcsine-transformed before further analysis.
The plant biomass, that is, the fresh weight of the leaves and stems of
all the plants in each experimental unit (pot), was measured at the end of
each trial. Both affected and healthy plants were cut, starting from 4 to 5
cm above the soil.
The disease severity (DS), the AUDPC, and the fresh weight of the
plant data were analyzed using SPSS software 25. The obtained data
were subjected to Levene’s test to determine the homogeneity of the
variance. One way-ANOVA was conducted using the General linear
model in SPSS to test for the effect of each factor (trial, treatments and
plant density), and their interactions on DS, AUDPC and the fresh weight
parameters. The means were separated by Tukey’s honestly significant
difference test at P � 0.05. The percentage of disease reduction was
calculated according to the formula: 100 – (100 � (disease severity
treatment/disease severitycontrol). Standard errors of the means were
calculated.
11
þ propamocarb hydrochloride and mandipropamid (Protocol III). The
alternative products and registered fungicides tested alone were
considered as the experimental reference (Tables 2 and 3).
Protocol IV was used in trials 10 to 13 in 2018 to evaluate the optimal
frequency and application timing, and the combination of fungicides
with alternative products (potassium phosphite, the biofertilizer based
calcium oxide and orange oil with ascorbic acid) (Tables 2 and 3),
considering three different crop densities. Protocol IV was applied, in a
full IPM strategy, to each crop density in three adjacent compartments of
the greenhouse under natural infestation, and different treatments,
based on alternative products (applied at 3 and 6 day intervals in 2–4
applications) applied in succession to one treatment with azoxystrobin,
were tested. The results were compared with those obtained from a
conventional fungicide program widely adopted by growers, which is
based on the alternation of azoxystrobin, fluopicolide þ propamocarb
hydrochloride and mandipropamid. The alternative products and
registered fungicides, were also tested alone at intervals of 3 and 6 days,
in 2–4 applications, as the experimental reference.
3. Results
2.4. The pathogen
3.1. Efficacy of the alternative products using protocol I
Trials 1–9 were carried out using an artificial inoculation of
P. belbahrii. One artificial inoculation with the pathogen was carried out
24 h after the first treatment in protocols I to III (Table 2). The
P. belbahrii population, which was collected in Piedmont (Northern
Italy) from diseased plants, was maintained on basil plants. The infected
leaves were shaken in 100 ml of sterile water containing 2 μl of Tween
20, and the obtained suspension was adjusted, with the aid of a hae­
mocytometer, to 1 � 105 sporangia/ml. The artificial inoculation was
carried out immediately after preparation through nebulization with a
laboratory spray bottle (10 ml capacity). One ml of suspension/pot per
replicate was used.
Trials 10–13 were carried out using naturally infested basil plants
obtained from a contaminated commercial seed sample (cv. Genovese
selection ‘Italiano classico’, Olter Sementi Italy), in which based four
According to one-way ANOVA, the data from trials 1–3 were com­
bined because no significant treatment � trial interactions was found for
DS (P ¼ 0.055), AUDPC ((P ¼ 0.065) or for the fresh weight (P ¼ 0.587).
The average severity of downy mildew reached at the end of the trials
was 61.4% in the untreated control, while a 17.6% of infected leaf area
was observed in the non-inoculated untreated control (Fig. 1A). The
calcium oxide, K-phosphite and orange oil with ascorbic acid treatments
resulted in significantly lower downy mildew severities of 30.0%, 39.1%
and 45.1%, respectively, than the inoculated untreated control. The
level of disease control provided by these products was significantly
higher (51%, 36% and 27% efficacy, respectively) than that provided by
copper (7% efficacy), potassium bicarbonate and zeolite, which did not
reduce the disease, compared to the untreated control. Three applica­
tions of dimethomorph þ pyraclostrobin, mandipropamid, fluopicolide
4
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Fig. 1. Effect of foliar sprays with fungicides and alternative products (three applications at 6 day intervals) against the P. belbahrii of basil ‘Italiano classico’ under
controlled conditions according to Protocol I. The data are expressed as the average value of three trials based on A) Downy mildew disease severity (DS) as % of the
infected leaf area B) the area under the disease-progress curve (AUDPC) and C) fresh weight FW (g/pot) at the end of trials 1–3. The data (means values of trials 1–3)
in each histogram with a different letter are significantly different at P � 0.05, according to the Tukey HSD test. Standard errors are also indicated.
þ propamocarb and azoxystrobin alone resulted in a disease control of
98.5%, 85.4%, 71.6% and 95.9%, respectively. In terms of disease
development (Fig. 1B), all the tested alternative products resulted in a
significant reduction in AUDPC, compared to the untreated control.
Potassium bicarbonate and zeolite resulted in a significantly similar
AUDPC to copper. A significant effect on disease development was
provided by orange oil with ascorbic acid and K-phosphite, while cal­
cium oxide had the lowest AUDPC values of all the other alternative
treatments. The lowest AUDPC values, compared to the non-treated
control, were provided by dimethomorph þ pyraclostrobin and man­
dipropamid, followed by fluopicolide þ propamocarb and azoxystrobin
(Fig. 1B). The fresh weigh was significantly higher in the pots treated
with dimethomorph þ pyraclostrobin than the other tested fungicides,
while only calcium oxide, orange oil with ascorbic acid and k-phosphite
significantly differed from the copper treatment and the other tested
products (Fig. 1C).
intervals, after one spray with pyraclostrobin þ dimethomorph, as
equivalent results to the application of the conventional standard
strategy were achieved. Generally, calcium oxide applied alone as a
foliar spray at 3-day intervals showed a lower disease severity and
AUDPC value than the foliar applications at 6-day intervals (Fig. 2 A, B,
C and D).
All the treatments, except copper sulfite, increased the fresh weight
of basil, compared to the inoculated untreated control. Among the tested
IPM strategies, pyraclostrobin þ dimethomorph, followed by the
application of calcium oxide at 6 day intervals, resulted in the greatest
fresh weight (Table 4).
Downy mildew was severe on the inoculated untreated basil plants in
trials 7–9, which showed mean values of 33.5, 40.4 and 50.0% of the leaf
surface covered with P. belbahrii, respectively and 1.6%, 4.7% and
18.8%, respectively in the non-inoculated and untreated control (Fig. 3).
Generally, in two out of three trials, spraying with azoxystrobin or
fluopicolide þ propamocarb, followed by calcium oxide or K-phosphite,
resulted in similar downy mildew severity to the standard chemical
control (Fig. 3). The efficacy shown by K-phosphite instead ranged from
25 to 83% compared to the inoculated and untreated control (Fig. 3).
Moreover, calcium oxide applied alone, at 3-day intervals, provided a
consistent downy mildew reduction of 70–74% and a lower AUDPC
value than applications at 6-day intervals (Figs. 3 and 4). Similarly, the
spray programme with azoxystrobin or fluopicolide þ propamocarb
applied once, followed by calcium oxide applied at 3-day intervals,
resulted in a consistent disease severity and AUDPC reduction in all the
trials (Figs. 3 and 4). The significant reduction in downy mildew
observed after foliar applications of azoxystrobin, followed by calcium
oxide, confirmed the greater fresh weight in trials 7–9, with similar re­
sults to those provided by the conventional standard strategy based on
the alternation of azoxystrobin, fluopicolide þ propamocarb and man­
dipropamid (Table 5).
3.2. Efficacy of the IPM strategy applied using protocols II and III
Trials 4–6 were combined for downy mildew severity only because
no significant treatment � trial interaction (P ¼ 0.196) was observed.
Downy mildew was moderately severe on all the untreated basil plants,
with mean values of 29% of the leaf surface covered with P. belbahrii in
the untreated pots (Fig. 2 A) and 7.4% leaf surface infested by the
pathogen in the non-inoculated and untreated control. Calcium oxide,
applied at three day intervals in combination with pyraclostrobin þ
dimethomorph, resulted in the highest downy mildew control (93%
efficacy), which was comparable with the standard chemical strategy
(94% efficacy) based on the alternation of pyraclostrobin þ dimetho­
morph, fluopicolide þ propamocarb and mandipropamid. Calcium
oxide, applied at 6 day intervals, provided a better control when used
after pyraclostrobin þ dimethomorph (88% efficacy) than the spray
program that included fluopicolide fluopicolide þ propamocarb and
mandipropamid (62% and 68% efficacy, respectively), with statistically
similar results to those obtained with potassium phosphite applied in
combination with fluopicolide þ propamocarb and mandipropamid and
with calcium oxide used alone at 3 day intervals (62–74% efficacy)
(Fig. 2A). Copper sulfite used at 6 day intervals was worse (30% effi­
cacy) than calcium oxide (48% efficacy) and K-phosphite (41% efficacy)
(Fig. 2A). Considering the disease development (AUDPC) achieved in
trials 4–6 (Fig. 2 B,C and D), the best level of disease control was pro­
vided by calcium oxide and K-phosphite, both applied at 3 and 6 day
3.3. Efficacy of IPM programmes according to protocol IV for a reduced
crop density
The data from each crop density tested in trials 10–13, carried out
under Protocol IV and under natural infestation, were analyzed sepa­
rately because the interaction of the crop density � treatment was sig­
nificant (P < 0.0001), then, were analyzed together for each crop
density because the interaction trial � treatment was not found to be
significant factor (P > 0.05) for DS, AUDPC or the fresh weight (Fig. 5).
5
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Fig. 2. Effect of foliar sprays with calcium oxide (CaO) and potassium phosphite (K-Phi) applied at 3 or 6 day-intervals alone or after pyraclostrobin þ dime­
thomorph (Py þ d), fluopicolide þ propamocarb (Fl) and mandipropamid (M), compared to fungicides used alone (Py þ d, Fl and M) or in alternation and copper
sulfite against the downy mildew of basil ‘Italiano classico’ according to protocol II. The number of applications of each product in the spay programme is reported in
brackets. The data are expressed as A) Downy mildew disease severity (DS, % of the infected leaf area) as the average value of trials 4, 5 and 6, and B, C, D) as
AUDPC at the end of trials 4, 5 and 6. The data in each histogram with a different letter are significantly different at P � 0.05, according to the Tukey HSD test.
Standard errors are also indicated.
6
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Table 4
Fresh weight of basil ‘Italiano classico’ harvested after foliar sprays based on calcium oxide (CaO) and K- phosphite applied alone or after fungicides according to
protocol II against the artificially inoculated Peronospora belbahrii compared to the copper sulphite and the standard spray programme. The data are expressed as mean
value (g/pot) at the end of trials 4, 5 and 6.
Fungicide (No. of sprays)
Alternative
products (No. of
sprays)
Interval
between sprays
(days)
Total
sprays
(No)
Fresh weigh (g/pot)
Trial 4
Trial 5
Inoculated untreated control
Pyraclostrobin þ dimethomorph (1)
–
CaO (4)
–
3
–
5
62.8
188.2
�12.0
�6.9
Fluopicolide þ propamocarb (1)
CaO (4)
3
5
222.4
�6.5
Mandipropamid (1)
CaO (4)
3
5
190.8
�9.5
Pyraclostrobin þ dimethomorph (1)
Fluopicolide þ propamocarb (1)
CaO (2)
CaO (2)
6
6
3
3
219.8
194.0
�5.1
�3.6
Mandipropamid (1)
CaO (2)
6
3
172.2
�10.2
Pyraclostrobin þ dimethomorph (1)
K-phosphite (2)
6
3
185.1
�11.4
Fluopicolide þ propamocarb (1)
K-phosphite (2)
6
3
137.7
Mandipropamid (1)
K-phosphite (2)
6
3
–
CaO (5)
3
–
CaO (3)
–
a
Trial 6
e
ac
a
83.8
216.1
�6.1
�23.0
246.1
�14.4
230.4
�12.8
251.4
226.8
�3.8
�9.5
216.5
�30.2
256.0
�22.6
ac
a
ac
ac
ac
cd
138.3
�13.9
5
134.6
6
3
K-phosphite (3)
6
Copper sulfite (3)
-b
Pyraclostrobin þ dimethomorph (1)
e
ac
a
85.3
185.0
�9.5
�11.2
165.5
�6.8
-
�17.1
ac
a
ac
ac
a
255.3
�27.6
cd
206.0
�16.0
�14.7
cd
222.3
�3.8
144.6
�22.4
204.9
�14.3
3
147.5
�10.2
189.1
�10.2
6
3
100.9
�8.3
bd
bd
de
161.3
�13.2
–
–b
1
193.5
�8.5
209.1
�11.0
Fluopicolide þ propamocarb (1)
–
–
1
157.8
�9.1
225.1
�11.6
Mandipropamid (1)
–
–
1
172.0
�27.5
150.5
�5.9
Pyraclostrobin þ dimethomorph (1);
fluopicolide þ propamocarb (1);
mandipropamid (1)c
Non-inoculated untreated control
–
6
3
206.4
�9.0
233.3
–
–
–
162.5
�6.6
121.3
ac
ad
ac
ab
ad
187.8
124.5
f
ac
ac
�7.7
�7.9
ab
df
135.8
�13.5
de
a
163.0
�8.1
–
164.8
�11.5
141.8
�5.5
124.5
�7.9
116.5
�6.5
ac
ce
df
ef
138.0
�5.9
de
152.0
�11.2
144.3
�8.6
�3.3
ad
ac
ad
ad
be
ad
ac
ce
ab
ac
195.5
�6.2
ae
be
a
�23.3
de
132.3
�7.3
de
–
a
The data in each column followed by a different letter are significantly different at P � 0.05, according to the Tukey HSD test. Standard errors are reported.
No treatments with alternative products or No intervals between sprays or No data available.
c
Standard spray programme based on the succession of fungicides with different mode of actions.
b
Average values of 44%, 55% and 70% of leaf surface covered with
P. belbahrii were observed at reduced (600–750 plants/m2) and average
(1200–1350 plants/m2) densities, respectively, compared to the stan­
dard density (1550–1700 plants/m2) (Fig. 5), and the progress of the
disease in the untreated controls was also lower, as indicated by the
lower AUDPC values for the untreated controls (Fig. 6).
Higher levels of disease control were observed for the treatments
with reduced densities, compared to the standard ones. The efficacy of
the strategies that included one spray with azoxystrobin, followed by 2
or 4 sprays with alternative products at a lower crop density, generally
increased ten-fold, compared to the level reached for a standard density
(Fig. 5). The best disease severity reduction (as much as 80% efficacy)
was provided by azoxystrobin, followed by calcium oxide at 3 dayintervals, and it was significantly comparable with the reduction ach­
ieved with the standard chemical program. Azoxystrobin, followed by
calcium oxide at 6 day-intervals, provided 63% downy mildew reduc­
tion for the standard crop density and 74–75% downy mildew reduction
for the reduced crop density (Fig. 5). When the alternative products
were tested alone under different crop densities, the best downy mildew
severity reduction (as much as 70% efficacy) was provided at the
reduced crop density by calcium oxide applied in the shortest spray
interval. Moreover, the spray program based calcium oxide at reduced
crop density provided significantly better downy mildew reduction than
K-phosphite (37.7–61.1% efficacy, respectively) and orange oil with
ascorbic acid (7.7–14.5% efficacy, respectively). All these products were
better that copper sulfite that reduced the downy mildew severity from
4.4% to 25%.
No significant difference in disease development was found when
considering the AUDPC values obtained for different spray programmes
for reduced and average crop densities (P ¼ 0.556) (Fig. 6). The impact
of calcium oxide on disease development was evident at both the
reduced and average crop density tested, which showed AUDPC values
from 63 to 190, compared to copper (253 and 300, respectively). The
lowest AUDPC values was found at the reduced and average crop density
tested using azoxystrobin, followed by calcium oxide at 3 or 6 dayintervals and it was significantly comparable to the standard fungi­
cides programme (Fig. 6). The fresh weight of the plants at the end of the
trials was generally higher for the reduced crop density (600–750
plants/m2) than for the 1200 to 1700 plants/m2 densities, with the
highest value measured in pots treated with azoxystrobin followed by
calcium oxide at 3 day intervals under a reduced crop density, which
resulted in significantly similar values to that obtained from the stan­
dard fungicide programme (Table 6).
4. Discussion
Research carried out in basil-growing areas affected by severe downy
mildew losses have shown that the pathogen is difficult to manage and
7
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Fig. 3. Effect of foliar sprays with calcium oxide (CaO) and potassium phosphite (K-Phi) applied at 3 or 6 day-intervals alone or after azoxystrobin (A), and
fluopicolide þ propamocarb (Fl), compared to fungicides used alone (A, Fl and mandipropamid M), to standard fungicides programme and copper sulfite against the
downy mildew of basil ‘Italiano classico’, according to protocol III. The number of applications of each product in the spay programme is reported in brackets. The
data are expressed as downy mildew disease severity (DS, % of the infected leaf area) at the end of trials 7, 8 and 9. The data in each histogram with a different letter
are significantly different at P � 0.05, according to the Tukey HSD test. Standard errors are also indicated.
Fig. 4. Effect of foliar sprays with calcium oxide (CaO) and potassium phosphite (K-Phi) applied at 3 or 6 day-intervals alone or after azoxystrobin (A) and
fluopicolide þ propamocarb (Fl), compared to fungicides used alone (A, Fl and mandipropamid M), to standard fungicides programme and copper sulfite against the
downy mildew of basil ‘Italiano classico’, according to protocol III. The number of applications of each product in the spay programme is reported in brackets. The
data are expressed as AUDPC at the end of trials 7, 8 and 9. The data in each histogram with a different letter are significantly different at P � 0.05, according to the
Tukey HSD test. Standard errors are also indicated.
that no single measure is capable of providing complete control (Elad
et al., 2016; Cohen et al., 2017; Gilardi et al., 2013, 2015 a,b; Gullino
et al., 2009; Homa et al., 2014; Patel et al., 2015, 2016). Different factors
influence the control of P. belbahrii, such as the biology and epidemi­
ology of the pathogen, as well as its seed-borne, wind-borne and poly­
cyclic nature (Cohen et al., 2017; Farahani-Kofoet et al., 2012; Garibaldi
et al., 2004a b; Garibaldi et al., 2007; Zhang et al., 2019), the utilized
cultural practices and the different modes of action of the available
fungicides, applied in different spray programmes. However, there is a
need to reduce the use of fungicides in general and, in this context, an
IPM approach is imperative to develop practical solutions to manage the
pathogen, to reduce disease pressure, to prevent the development of
resistance to pesticides and to ensure high quality basil, as requested by
the consumers.
In the present study, products already known for their activity
against other downy mildews have been tested as possible alternatives to
chemical fungicides. The alternative products that have been tested in
this study include phosphites, which have already been evaluated
extensively as a component of IPM for several crops, including basil,
against foliar and soilborne pathogens (Deliopoulos et al., 2010; Gilardi
8
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Table 5
Fresh weight of basil ‘Italiano classico’ harvested after foliar sprays based calcium oxide (CaO) and K-phosphite applied alone and after fungicides according to
protocol III against the artificially inoculated Peronospora belbahrii compared to the copper sulphite and the standard spray programme. The data are expressed as mean
value (g/pot) at the end of trials 7, 8 and 9.
Fungicide (No. of sprays)
Alternative products
(No. of sprays)
Interval between
sprays (days)
Total
sprays
(No.)
Fresh weigh (g/pot)
Trial 7
Trial 8
a
Trial 9
Inoculated untreated control
Azoxystrobin (1)
–
CaO (4)
–
3
–
5
111.7
210.3
�4.8
�13.7
c
a
164.8
314.8
�14.6
�30.0
Fluopicolide þ propamocarb (1)
CaO (4)
3
5
135.4
�4.3
bc
271.0
�8.6
Azoxystrobin (1)
Fluopicolide þ propamocarb (1)
CaO (2)
CaO (2)
6
6
3
3
165.9
140.4
�16.2
�8.1
ab
bc
339.0
197.0
�46.6
�17.9
Azoxystrobin (1)
Fluopicolide þ propamocarb (1)
K-phosphite (2)
K-phosphite (2)
6
6
3
3
161.3
139.9
�10.4
�6.0
b
bc
330.0
297.8
�52.7
�5.4
–
–
–
Copper sulfite (3)
Azoxystrobin (1)
CaO (5)
CaO (3)
K-phosphite (3)
-b
–
3
6
6
6
–b
5
3
3
3
1
160.7
169.2
144.4
146.7
138.9
�5.3
�10.2
�14.5
�9.6
�4.2
b
ab
bc
bc
bc
236.3
205.0
232.5
146.8
220.8
�8.0
�5.7
�14.7
�19.9
�6.1
Fluopicolide þ propamocarb (1)
–
–
1
146.9
�10.0
bc
271.8
�7.1
Mandipropamid (1)
–
–
1
180.9
�7.1
ab
283.5
�5.2
Azoxystrobin (1); fluopicolide þ
propamocarb (1); mandipropamid
(1)c
Non-inoculated untreated control
–
–
3
167.6
�3.8
ab
263.0
�14.2
–
–
–
180.3
�5.7
ab
221.3
�8.9
e-f
ac
ae
a
df
ab
ad
a-f
c-f
a-f
f
bf
ae
ad
ae
bf
30.5
247.0
�3.0
�7.8
f
a
130.8
�13.0
bc
245.5
146.5
�6.4
�3.6
a
bc
134.8
111.0
�9.7
�6.8
bc
cd
168.5
149.3
57.8
83.0
87.0
�6.4
�12.0
�5.7
�8.3
�1.9
b
bc
e
de
de
85.3
�5.7
de
120.3
�11.0
cd
220.5
�8.8
a
150.3
�14.5
bc
a
The data in each column followed by a different letter are significantly different at P � 0.05, according to the Tukey HSD test. Standard errors are reported.
No treatments with alternative products or No intervals between sprays.
c
Standard spray programme based the succession of fungicides with different modes of action.
b
Fig. 5. Effect of foliar sprays with calcium oxide (CaO), potassium phosphite (K-Phi) and orange oil (O-oil) applied at 3 or 6 day-intervals alone or after azoxystrobin
(A) compared to the standard fungicide programme (azoxystrobin A/fluopicolide þ propamogcarb Fl/mandipropamid M) and copper sulfite against the downy
mildew of ‘Italiano classico’ basil using different crop densities (1: reduced 600–750 plants/m2; 2: average 1200–1350 plants/m2; 3: standard 1550–1700 plants/m2).
The number of applications of each product in the spay programme is reported in brackets. The data are expressed as downy mildew disease severity (DS, % of the
infected leaf area) and were calculated as the average value of trials 10, 11, 12 and 13 for each crop density. The data in each histogram with a different letter are
significantly different at P � 0.05, according to the Tukey HSD test. Standard errors are also indicated.
et al., 2013, 2015 a,b; Liljerotha et al., 2016; Patel et al., 2016; Ramezani
et al., 2009) and a diversity of materials marketed as biostimulants or
fertilizers (Calvo et al., 2014; Demirci et al., 2014; Du Jardin, 2015;
Mersha et al., 2012; Ramesh et al., 2011).
In order to represents an ideal situation where a grower sprays pre­
ventatively, such products were applied immediately before the artifi­
cial inoculation with the pathogen, alone and combined with
conventional fungicides considering three different protocols. The arti­
ficial inoculation of basil plants provides a homogenous disease pres­
sure, and results in a higher disease pressure than a commercial
production system. Moreover, due to the polycyclic epidemics caused by
P. belbahrii, that is capable of several infection cycles a season, an un­
treated and non-inoculated control was used through the experiments.
So, the presented results should be considered in this perspective.
9
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Fig. 6. Effect of foliar sprays with calcium oxide (CaO), potassium phosphite (K-Phi), orange oil (O-oil) applied at 3 or 6 day-intervals alone or after azoxystrobin
(A) compared to the standard fungicide programme (azoxystrobin A/fluopicolide þ propamogcarb Fl/mandipropamid M) and copper sulfite against the downy
mildew of ‘Italiano classico’ basil using different crop densities (1: reduced 600–750 plants/m2; 2: average 1200–1350 plants/m2; 3: standard 1550–1700 plants/m2).
The number of application of each product in the spay programme is reported in brackets. The data are expressed as AUDPC value and were calculated as the average
value of trials 10, 11, 12 and 13 for each crop density. The data in each histogram with a different letter are significantly different at P � 0.05, according to the Tukey
HSD test. Standard errors are also indicated.
Table 6
Fresh weight of basil ‘Italiano classico’ harvested for three densities (1: 600–750; 2:1200-1350; 3, standard: 1550–1700 plants/m2), after foliar sprays based calcium
oxide (CaO), orange oil and K-phosphite applied alone or after fungicides according to protocol IV, against naturally infection by Peronospora belbahrii compared to the
copper sulphite and the standard spray programme. The data are expressed as mean value (g/pot) of trials 10–13 for each crop density.
Fungicide (No. of sprays)
Alternative products
(No. of sprays)
Interval between
sprays (days)
Total
sprays
(No.)
Reduced crop density
(1)
Untreated control
Azoxystrobin (1)
–
CaO (2)
–
6
–
3
83.6
202.3
�10.9
�11.3
Azoxystrobin (1)
K- phosphite (2)
6
3
155.1
�11.5
Azoxystrobin (1)
6
3
128.2
�10.5
–
Orange oil þ ascorbic
acid (2)
CaO (3)
6
3
125.7
�9.2
–
K- phosphite (3)
6
3
177.4
�11.3
–
Orange oil þ ascorbic
acid (3)
CaO (4)
K- phosphite (4)
6
3
80.9
3
3
5
5
Orange oil þ ascorbic
acid (4)
CaO (5)
3
Orange oil þ ascorbic
acid (5)
-b
–
Azoxystrobin (1)
Azoxystrobin (1)
Azoxystrobin (1)
–
–
Copper sulfite (3)
Azoxystrobin (1); Fluopicolide þ
propamocarb (1); mandipropamid
(1)c
Average crop density
(2)
Standard
Crop density (3)
ga
ac
c-f
30.9
175.2
�2.8
�18.9
e
ab
26.5
136.3
�5.1
�13.2
g
bc
106.4
�7.0
70.7
�7.8
73.8
�13.4
59.2
�6.9
53.4
�5.7
de
58.9
�8.8
161.6
�26.2
87.6
�7.3
46.6
�9.8
ac
de
df
eg
eg
de
�11.6
eg
eg
be
g
bd
de
35.6
�3.6
fg
229.8
162.1
�12.9
�12.9
ab
c-f
197.6
96.2
�16.9
�7.1
173.9
93.0
�11.3
�9.9
ab
de
5
114.3
�11.9
fg
77.7
�11.5
a
ce
de
67.8
�3.5
3
5
187.4
�14.9
205.6
�28.9
a
112.2
�8.0
3
5
87.4
�12.2
ad
g
eg
cd
47.8
�9.2
de
32.5
�2.8
fg
6
3
136.0
�16.9
66.6
�8.6
de
66.0
�11.2
6
3
244.7
�12.6
dg
a
176.6
�23.1
ab
179.0
�13.5
eg
a
a
The data in each column followed by a different letter are significantly different at P � 0.05, according to the Tukey HSD test. Standard errors are reported.
No treatments with alternative products or No intervals between sprays.
c
Standard spray programme based on the succession of fungicides with different modes of action.
b
Among the products tested in this study as possible alternatives to
the chemical fungicides, potassium bicarbonate and zeolite were
partially effective (7–10% efficacy), compared to the untreated control,
while orange oil with ascorbic acid used alone (26% efficacy) was found
to be much better than copper sulfite. Moreover, when orange oil was
applied in spray programmes after azoxystrobin, its efficacy was
improved by 40–45%. Potassium phosphite and calcium oxide provided
the most consistent disease control. The effect of the application of K-
phosphite confirmed previous results (Gilardi et al., 2013; Patel et al.,
2016). However, phosphites are not permitted as plant protection
products on basil in Europe, but may be used as plant strengthens. An­
alyses of the residue levels are needed to determine the amounts that can
be applied without exceeding the MRLs, which at present are the same as
those for phosethyl-Al (EFSA, 2018). Calcium oxide also results to be a
very good alternative, considering the intensive discussions about the
suitability of phosphite-based products and the aim of reducing the use
10
G. Gilardi et al.
Crop Protection 137 (2020) 105202
of copper salts. As it is classified as an agricultural and horticultural
fertilizer/biostimulant, there are no regulatory requirements for its use,
such as pre-harvest intervals or MRLs. To the best of our knowledge, this
is the first time that the effect of calcium oxide has been shown against
basil downy mildew. Calcium-based products are widely used as fertil­
izers/biostimulants in agriculture, but are also known for their disease
suppression properties. For instance, soil applied lime (calcium hy­
droxide) is known to suppress such Fusarium wilt agents as Fusarium
oxysporum f.sp. lycopersici on tomato (Edgerton, 1918; Woltz and Jones,
1973) and Fusarium oxysporum f.sp. spinaciae on spinach (Gatch and
duToit, 2017). Calcium oxide (CaO, also called burned lime) has been
tested against the oomycetes such as Phytophthora cinnamomi root rot of
Quercus (Serrano et al., 2012), Plasmodiophora brassicae on brassica
crops (Murakami et al., 2002; Tremblay et al., 2005) and Phytophthora
nicotianae on citrus (Campanella et al., 2002). Calcium-based sprays are
also used to reduce sprays of synthetic chemicals in orchards against
apple scab caused by Venturia inaequalis (Percival and Haynes, 2009)
and the powdery mildew of grapes (Pugliese et al., 2018).
In the present study, crop density was found to be a significant factor
in modulating the degree of efficacy of the different tested protocols. As
a cultural practice, a lower crop density has been shown to have a sig­
nificant impact on the success of IPM control measures on other crops.
For instance, Elad et al. (2014, 2015) reported that increased plant
spacing helped suppress both white mold, caused by Sclerotinia scle­
rotiorum, and gray mold, caused by Botrytis cinerea on basil. A positive
effect of a low crop density and/or plant spacing has also been reported
for other pathosystems, such as rose-Peronospora sparsa (O’Neill et al.,
2002; Salgado-Salazar et al., 2018), onion-Peronospora destructor
�lez et al., 2011), groundnut-Puccinia arachidis (Pande and Rao,
(Gonza
2002) and chickpea -Botrytis cinerea (Pande et al., 1998). The density at
which plants are grown may be expected to affect the relative humidity
of the leaf canopy and the rapidity at which leaves dry after a wetting
period (rain or irrigation), and may have a profound impact on the
development of foliar and soilborne diseases in different pathosystems
(Elad et al., 2014, 2016; Gamliel et al., 1996; O’Neill et al., 2002; Pande
et al., 1998; Pande and Rao, 2002). Indeed, it is well known that the
infection, sporulation and the duration of the latent period (from
infection to sporulation) of P. belbahrii depends to a great extent on the
occurrence of high levels of humidity (>85%), but also on the temper­
ature and light regime (Elad et al., 2016; Cohen et al., 2017; Garibaldi
et al., 2007).
In the present study, trials carried out under controlled conditions,
but with natural pathogen infestation, have permitted different crop
densities to be tested under closer conditions to those encountered in
commercial basil production. For instance, calcium oxide, applied after
azoxystrobin at a reduced crop density (600–750 plants/m2), signifi­
cantly delayed disease development, compared to the untreated control,
and improved its efficacy, compared to the same treatment applied
under standard density (1550–1700 plants/m2). Moreover, the results
were similar to those obtained with the standard chemical programme,
even in terms of the marketable yield of basil. These findings suggest the
possibility of reducing the number of fungicide applications in practice.
However, the alternation of fungicides with the different modes of ac­
tion used as the standard programme did not eradicate P. belbahrii for
the high crop density (73% efficacy), although the efficacy was
improved by 82–83%, while a significant effect was observed for the
reduced crop density. The effectiveness of a fungicide alternation pro­
gramme is therefore influenced by a number of factors, including such
properties of the fungicide as systematicity, translocation movement
within plants or the mode of action in the life cycle of the pathogen.
Growers are encouraged to alternate fungicides with different modes of
action in order to maintain their efficacy, according to Fungicide
Resistance Action Committee (https://www.frac.info/). The present
study has provided evidence that one application of azoxystrobin and
pyraclostrobin þ dimethomorph provided a longer protection against
basil downy mildew than the alternation of floupicolide þ propamocarb
with mandipropamid, thus indicating the importance of designing the
IPM programme and the interval between sprays according to the mode
of action of the used fungicides.
Although fungicide applications are currently necessary to achieve
an adequate disease control, alternative products such as biostimulant
‘that is any substance or microorganism applied to plants aimed to
enhance nutrition efficiency, abiotic stress tolerance and/or crop qual­
ity’ (Du Jardin, 2015) may be useful components of an IPM programme,
and may help develop an eco-friendly control strategy to manage basil
downy mildew. These products help reduce the risk of the development
of resistance to site-specific fungicides and may be used as a substitute
for copper, as required by the current European regulation (CE n.
354/2014). In the present study, a range of less toxic copper alternatives
such as calcium oxide and orange oil has been tested using artificial
inoculation with P. belbahrii and these alternatives have been shown to
be effective under severe disease pressure. Calcium oxide, K-phosphite
and orange oil generally provided the most consistent disease control
among the alternative compounds tested. For instance, orange oil with
ascorbic acid provided the same or a better effect than copper sulfite and
resulted in a better protection against basil downy mildew and an
improved yield, compared to copper sulfite. The calcium product used
here has a wider applicability than the orange oil products that are
registered as pesticides, but only in some EU countries. Thus,
calcium-based products should be compatible with organic farming
principles. Moreover, this study has shown that calcium oxide is able to
provide a significant control of downy mildew on basil when applied
alone or in several combinations with fungicides, and that it results in
the same protection as a typical fungicide rotation. It could also be used
as a component of more conventional IPM programmes that include a
limited application of traditional fungicides.
In the present study, the effect of products alternative to conven­
tional fungicides and copper salts such as calcium oxide, orange oil and
k-phosphite and their use in IPM programme has not be tested consid­
ering the situation in which some growers would begin spraying when
disease was first discovered. However to achieve the best results in
downy mildew control, both conventional and alternative products
based on salts and essential oil should be used in a preventative manner
to protect plants from infection. The curative effect of calcium oxide,
orange oil and potassium phosphite products and their possible use in
combination with fungicides should be further evaluated. Moreover, in
the future, basil varieties that are resistant to downy mildew and
commercially acceptable may become another component of IPM
programmes.
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.
CRediT authorship contribution statement
Giovanna Gilardi: Conceptualization, Investigation, Methodology,
Data curation, Writing - original draft, Writing - review & editing, Su­
pervision. Angelo Garibaldi: Conceptualization, Methodology, Writing
- review & editing, Supervision. Maria Lodovica Gullino: Conceptu­
alization, Funding acquisition, Project administration, Writing - original
draft, Writing - review & editing, Supervision.
Acknowledgements
This research has received funding from the European Union’s Ho­
rizon 2020 research and innovation programme under grant agreement
No. 634179. Effective Management of Pests and Harmful Alien Species Integrated Solutions^ (EMPHASIS). The authors would like to thank
Andrea China Gallo for her technical assistance, Marguerite Jones for
11
G. Gilardi et al.
Crop Protection 137 (2020) 105202
the language revision, the company AgriNewTech for providing Califol
and the anonymous referees for their valuable suggestions.
of resistance inducers, fungicides, biocontrol agents and natural products.
Phytoparasitica 41, 59–72.
Gilardi, G., Demarchi, S., Ramon, I., Gullino, M.L., Garibaldi, A., 2015a. Pre-planting
treatments with phosphite-based products against different foliar and soil-borne
pathogens of vegetable crops. Commun. Agric. Appl. Biol. Sci. 80, 445–451.
Gilardi, G., Pintore, I., Demarchi, S., Gullino, M.L., Garibaldi, A., 2015b. Seed dressing to
control downy mildew of basil. Phytoparasitica 44, 531–539.
Gonz�
alez, P.H., Colnago, P., Peluffo, S., Gonz�
alez, H., Zipitría, I.J., Galv�
an, G.A., 2011.
Quantitative studies on downy mildew (Peronospora destructor Berk. Casp.) affecting
onion seed production in southern Uruguay. Eur. J. Plant Pathol. 129, 303–314.
Gullino, M.L., Gilardi, G., Garibaldi, A., 2009. Chemical control of downy mildew on
lettuce and basil under greenhouse. Commun. Agric. Appl. Biol. Sci. 74, 933–940.
Homa, K., Barney, W.P., Ward, D.L., Wyenandt, C.A., Simon, J.E., 2014. Evaluation of
fungicides for the control of Peronospora belbahrii on sweet basil in New Jersey. Plant
Dis. 98, 1561–1566.
Homa, K., Barney, W.P., Ward, D.L., Wyenandt, C.A., Simon, J.E., 2016. Morphological
characteristics and susceptibility of basil species and cultivars to Peronospora
belbahrii. Hortscience 51, 1389–1396.
Liljerotha, E., Lankinen, A., Wiik, L., Dhar, Burra, D., Alexandersson, E., Andreassona, E.,
2016. Potassium phosphite combined with reduced doses of fungicides provides
efficient protection against potato late blight in large-scale field trials. Crop Protect.
86, 42–55.
McGrath, M., 2015. Evaluation of organic and conventional fungicide programs for
downy mildew in basil, 2014. Plant Dis. Manag. Rep. 9, V026.
McGrath, M.T., 2016. Efficacy of fungicides, biopesticides, and resistant varieties for
managing downy mildew in basil. Abstr.) Phytopathology 106 (Suppl. l), S4.
McGrath, M.T., Hunsberger, L.K., 2012. Evaluation of biopesticides for managing downy
mildew in basil, 2011. Plant Dis. Manag. Rep. 6, V099.
Mersha, Z., Zhang, S., Mo, X., 2011. Evaluation of biologicals and biorationals for control
of basil downy mildew under greenhouse conditions, 2010. Plant Dis. Manag. Rep. 6,
V059.
Mersha, Z., Zhang, S., Raid, R.N., 2012. Evaluation of systemic acquired resistance
inducers for control of downy mildew on basil. Crop Protect. 40, 83–90.
Murakami, H., Tsushima, S., Kuroyanagi, Y., Shishido, Y., 2002. Reduction of resting
spore density of Plasmodiophora brassicae and clubroot disease severity by liming.
Soil Sci. Plant Nutr. 48, 685–691.
O’Neill, T., Pye, D., Locke, T., 2002. The effect of fungicides, irrigation and plant density
on the development of Peronospora sparsa, the cause of downy mildew in rose and
blackberry. Ann. Appl. Biol. 140 (2), 207–214.
Pande, S., Rao, J.N., 2002. Effect of plant population densities on the severity of late leaf
spot and rust of groundnut. Plant Pathol. J. 18, 271–278.
Pande, S., Johansen, C., Narayana Rao, J., 1998. Management of botrytis gray mold of
chickpea. A review. In: Recent Advances in Research and Management of Botrytis
Gray Mold of Chickpea. Pp. 23-40. Summary Proceedings of the Fourth Working
Group Meeting to Discuss Collaborative Research on Botrytis Gray Mold of Chickpea,
23-26 February 1998, Joydebpur, Gazipur, Bangladesh.
Patel, J.S., Costa de Novaes, M.I., Barbier, M., Zhang, S., 2013. Evaluation of
biopesticides for managing downy mildew of basil. Plant Dis. Man. Rep. 7, V147.
Patel, J.S., Costa de Novaes, M.I., Zhang, S., 2015. Evaluation of the new compound
oxathiapiprolin for control of downy mildew in basil. Plant Health Prog. 16,
165–172.
Patel, S.J., Zhang, S., Zhang, S., McGrath, M.T., 2016. Red light increases suppression of
downy mildew in basil by chemical and organic products. J. Phytopathol. 164, 1–8.
Patel, J.S., Zhang, S., de Novaes, M.I.C., 2014a. Oxathiapiprolin: an effective new
chemistry for control of downy mildew of basil. Abstr.) Phytopathology 104 (Suppl.
l), S90.
Patel, J.S., Zhang, S.A., de Novaesi, M.I.C., 2014b. Effect of plant age and acibenzolar-Smethyl on development of downy mildew of basil. Hortscience 49, 1392–1396.
Percival, G.C., Haynes, I., 2009. The influence of calcium sprays to reduce fungicide
inputs against apple scab. Arboric. Urban For. 35, 263–270.
Pintore, I., Gilardi, G., Gullino, M.L., Garibaldi, A., 2016. Detection of mefenoxamresistant strains of Peronospora belbahrii, the causal agent of basil downy mildew,
transmitted through infected seeds. Phytoparasitica 44, 563–569.
Pugliese, M., Monchiero, M., Gullino, M.L., Garibaldi, A., 2018. Application of laminarin
and calcium oxide for the control of grape powdery mildew on Vitis vinifera cv.
Moscato.J. Plant Dis. Protect. 125, 477–482.
Pyne, R.M., Honig, J., Vaiciunas, J., Bonos, S., Wyenandt, A., Simon, J.E., 2017. A first
genetic map and downy mildew resistance QTL discovery for sweet basil (Ocimum
basilicum). PloS One 12, e0184319.
Raid, R.N., Sui, D., Zhang, S., 2011. Comparision of five fungicides of dissimilar modes of
action for control of downy mildew of basil, Spring 2010. Plant Dos. Manag.Rep. 5,
V047.
Ramesh, K., Reddy, D.D., Biswas, A.K., Subbarao, A., 2011. Potential uses of zeolite in
agriculture. Adv. Agron. 113, 219–241.
Ramezani, S., Rezaei, M.R., Sotoudehnia, P., 2009. Improved growth, yield and essential
oil content of basil grown under different levels of phosphorus sprays in the field.
J. Appl. Biol. Sci. 3, 96–101.
Salgado-Salazar, C., Shishkoff, N., Daughtrey, M.L., Palmer, C., Crouch, J.A., 2018.
Downy mildew: a serious disease threat to rose health worldwide. Plant Dis. https://
doi.org/10.1094/PDIS-12-17-1968-FE.
Serrano, M.S., De Vita, P., Fern�
andez-Rebollo, P., Hern�
andez, M.E.S., 2012. Calcium
fertilizers induce soil suppressiveness to Phytophthora cinnamomi root rot of Quercus
ilex. Eur. J. Plant Pathol. 132, 271–279.
Thines, M., Telle, S., Ploch, S., Runge, F., 2009. Identity of the downy mildew pathogens
of basil, coleus and sage with implications for quarantine measures. Mycol. Res. 113,
532–540.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cropro.2020.105202.
References
Allen, J.E., Saska, M., 2013. Basil downy mildew control using organic fungicides and
nitrogen fertilization rates. Plant Dis. Manag. Rep. 7, V045.
Belbahri, I., Calmin, G., Pawlowski, J., Lefort, F., 2005. Phylogenetic analysis and real
time PCR detection of a presumably undescribed Peronospora species on sweet basil
and sage. Mycol. Res. 109, 1276–1287.
Ben-Naim, Y., Falach, L., Cohen, Y., 2015. Resistance against basil downy mildew in
Ocimum Species. Phytopathology 105, 778–785.
Ben-Naim, Y., Falach, L., Cohen, Y., 2018. Transfer of downy mildew resistance from
wild basil (Ocimum americanum) to sweet basil (O. basilicum). Phytopathology 108,
114–123.
Calvo, P., Nelson, L., Kloepper, J.W., 2014. Agricultural uses of plant biostimulants. Plant
Soil 383, 3–41.
Campanella, V., Ippolito, A., Nigro, F., 2002. Activity of calcium salts in controlling
Phytophthora root rot of citrus. Crop Protect. 21, 751–756.
Cohen, Y., Vaknin, M., Ben Naim, Y., 2015b. Chemical control of downy mildew in basil
caused by Peronospora belbahrii. Phytoparasitica 43, 381.
Cohen, Y., Vaknin, M., Ben Naim, Y., 2015a. Chemical control of downy mildew in basil
caused by Peronospora belbahrii. Phytoparasitica 43, 381.
Cohen, Y., Rubin, A.E., 2015. Daytime solar heating controls downy mildew Peronospora
belbahrii in sweet basil. PLoS One 10, e0126103.
Cohen, Y., Vaknin, M., Ben-Naim, Y., Rubin, A.E., 2013a. Light suppresses sporulation
and epidemics of Peronospora belbahrii. PLoS One 8, e81282.
Cohen, Y., Vaknin, M., Ben-Naim, Y., Rubin, A.E., 2016. Nocturnal fanning suppresses
downy mildew epidemics in sweet basil. PLoS One 11, e0155330.
Cohen, Y., Vaknin, M., Ben-Naim, Y., Rubin, A.E., 2017. Epidemiology of basil downy
mildew. Phytopathology 107, 1149–1160.
Cohen, Y., Vaknin, M., Ben-Naim, Y., Rubin, A.E., Galperin, M., Silverman, D., Bitton, S.,
Adler, U., 2013b. First report of the occurrence and resistance to mefenoxam of
Peronospora belbahrii, causal agent of downy mildew of basil (Ocimum basilicum) in
Israel. Plant Dis. 97, 692.
Deliopoulos, T., Kettlewell, P.S., Hare, M.C., 2010. Fungal disease suppression by
inorganic salts: a review. Crop Protect. 29, 1059–1075.
Demirci, S., Ustao�
glu, Z., Yılmazer, G.A, Sahin, F., Baç, N., 2014. Antimicrobial
properties of zeolite-X and zeolite-A ion-exchanged with silver, copper and zinc
against a broad range of microorganisms. Appl. Biochem. Biotechnol. 172,
1652–1662.
Du Jardin, P., 2015. Plant biostimulants: definition, concept, main categories and
regulation. Sci. Hortic. 196, 3–14.
Edgerton, C.W., 1918. A study of wilt resistance in the seed bed. Phytopathology 8, 5–14.
EFSA, 2018. Peer review of the pesticide risk assessment of the active substance fosetyl.
EFSA J. 7, 307.
Elad, Y., Israeli, L., Fogel, M., David, D.R., Kenigsbuch, D., Chalupowicz, D., Maurer, D.,
Lichter, A., Silverman, D., Biton, S., Yitzhak, S., Harari, D., Maduel, A., Pivonia, S.,
Adler, U., 2014. Conditions influencing the development of sweet basil grey mould
and cultural measures for disease management. Crop Protect. 64, 67–77.
Elad, Y., Fogela, M., Silvermanb, D., Bitonb, S., Yitzhakc, S., Hararid, D., Adler, U., 2015.
White mould of sweet basil: conditions influencing its development in greenhouses
and cultural measures for disease management. Plant Pathol. 64, 951–960.
Elad, Y., Omer, C., Nisan, Z., Harari, D., Goren, H., Adler, U., Silverman, D., Biton, S.,
2016. Passive heat treatment of sweet basil crops suppresses Peronospora belbahrii
downy mildew. Ann. Appl. Biol. 168, 373–389.
Farahani-Kofoet, D.R., R€
omer, P., Grosch, R., 2012. Systemic spread of downy mildew in
basil plants and detection of the pathogen in seed and plant samples. Mycol. Prog.
11, 961–966.
Farr, D.F., Rossman, A.Y., 2019. Fungal databases U.S. National fungus collections, ARS.
USDA. http://nt.ars-grin.gov/fungaldatabases.
Gamliel, A., Katan, T., Yunis, H., Katan, I., 1996. Fusarium wilt and crown rot of sweet
basil: involvement of soilborne and airborne inoculum. Phytopathology 86, 56–62.
Garibaldi, A., Gullino, M.L., Minuto, G., 1997. Diseases of basil and their management.
Plant Dis. 81, 124–132.
Garibaldi, A., Minuto, A., Minuto, G., Gullino, M.L., 2004a. First report of downy mildew
of basil (Ocimum basilicum) in Italy. Plant Dis. 88, 312.
Garibaldi, A., Minuto, G., Bertetti, D., Gullino, M.L., 2004 b. Seed transmission of
Peronospora sp. of basil. J. Plant Dis. Prot. 111, 465–469.
Garibaldi, A., Pintore, I., Gilardi, G., Gullino, M.L., 2016. Resistenza a mefenoxam in
popolazioni di Peronospora belbahrii. Protezione delle Colture 9 (5), 13–16.
Garibaldi, A., Bertetti, D., Gullino, M.L., 2007. Effect of leaf wetness duration and
temperature on infection of downy mildew (Peronospora sp.) of basil. J. Plant Dis.
Prot. 114, 6–8.
Gatch, E.W., duToit, L.J., 2017. Limestone-mediated suppression of Fusarium wilt in
spinach seed crops. Plant Dis. 101, 81–94.
Gilardi, G., Demarchi, S., Garibaldi, A., Gullino, M.L., 2013. Management of downy
mildew of sweet basil (Ocimum basilicum) caused by Peronospora belbahrii by means
12
G. Gilardi et al.
Crop Protection 137 (2020) 105202
Tremblay, N., B�
elec, C., Coulombe, J., Godin, C., 2005. Evaluation of calcium cyanamide
and liming for control of clubroot disease in cauliflower. Crop Protect. 24, 798–803.
Woltz, S.S., Jones, J.P., 1973. Interactions in source of nitrogen fertilizer and liming
procedure in the control of Fusarium wilt of tomato. Hortscience 8, 137–138.
Wyenandt, C.A., Simon, J.E., Pyne, R.M., HomaK, McGrath, M.T., Zhang, S., Raid, R.N.,
Ma, L.J., Wick, R., Gou, L., Madeiras, A., 2015. Basil downy mildew (Peronospora
belbahrii): discoveries and challenges relative to its control. Phytopathology 105,
885–894.
Wyenandt, C.A., Maimone, L.R., Homa, K., Madeiras, A.M., Wick, R.L., Simon, J.E., 2018.
Detection of the downy mildew pathogen on seed of basil following field infection in
southern New Jersey. Hortecnology 28, 637–641.
Wyenandt, C.A., Simon, J.E., Mcgrath, M.T., Ward, D.L., 2010. Susceptibility of basil
cultivars and breeding lines to downy mildew (Peronospora belbahrii). Hortscience 45
(9), 1416–1419.
Zhang, G., Thompson, A., Schisler, D., Johnson, E.T., 2019. Characterization of the
infection process by Peronospora belbahrii on basil by scanning electron microscopy.
Heliyon 5. https://doi.org/10.1016/j.heliyon.2019.e011, 2019 e01117.
13