Crop Protection 127 (2020) 104947

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Crop Protection
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Control of Alternaria fruit rot in ’Pink Lady’ apples by fungicidal mixtures

Lior Gur a, b, Moshe Reuveni a, *, Yigal Cohen b
a
Shamir Research Institute, University of Haifa, P.O. Box 97, Katzrin, 12900, Israel
b
Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, 52900, Israel

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

Keywords: Fruit body rot and calyx rot caused by Alternaria alternata f.sp. mali recently appeared in Israel as a severe disease
Alternaria mali of apple, cv. Pink Lady. No such heavy rots of fruits were reported elsewhere, nor are there any known control
Alternaria alternata apple pathotype measures against the disease. We found that the disease may be successfully controlled by application of
Alternaria alternata forma specialis mali
fungicidal mixtures to the fruit at the proper phenological development stage. Ortiva-Top (azox­
Cripps pink
Disease control
ystrobinþdifenoconazole) and Azimut (azoxystrobinþtebuconazole) effectively inhibited spore germination,
Systemic fungicides mycelial growth, and rot development in detached fruits. Mixtures of Azimut or Ortiva-Top with Captan were
synergistically effective in controlling the disease in detached fruits. In seven field trials Ortiva-Top and Orpan
(tebuconazoleþcaptan) were the most effective, and reduced calyx and fruit body rot by 60–88 and 58–92%,
respectively, compared with untreated trees. Based on these findings a new spray program was adopted, in which
only four to six sprays of Ortiva-Top, Orpan or Azimut were employed, starting at about 115 days after petal fall;
it effectively controlled Alternaria fruit rot in ’Pink Lady’ apples in Israel.

1. Introduction
Alternaria leaf blotch and fruit spot of apple (Malus domestica Borkh),
caused by the fungus Alternaria alternata f. sp. mali (syn. Alternaria mali
Roberts, or Alternaria alternata apple pathotype) (Grove et al., 2003;
Harteveld et al., 2013; Woudenberg et al., 2015) is an important disease
of apple in many countries (Bulajic et al., 1996; Filajdic and Sutton,
1991, 1992a; Hanif et al., 2016; Harteveld et al., 2013; Marschall et al.,
2006; Ozgonen and Karaca, 2006; Sawamura, 1990; Soleimani and
Esmailzadeh, 2007; Toome-Heller et al., 2018), including Israel (Gur
et al., 2013, 2017). The disease affects cultivars such as ’Golden Deli­
cious’, ’Starking Delicious’, ’Indo’, ’Gala’, and ’Pink Lady’ (Ferree and
Warrington, 2003; Filajdic and Sutton, 1991; Gur et al., 2017; Rotondo
et al., 2012; Zhu et al., 2017). Leaf infection may cause 60–85% defo­
liation in susceptible cultivars (Filajdic and Sutton, 1992a; Jung, 2007).
Fruit symptoms are usually limited to small, dark, corky lesions, often
associated with the lenticels (Ferree and Warrington, 2003). The path­
ogen may cause soft rot, particularly when the skin was wounded by
mechanical damage or insects (Filajdic et al., 1995a, b; Jung, 2007), or
has cracks around the fruit calyx (Stern et al., 2013). Severe outbreaks of
Alternaria fruit spots have recently been observed on cv. Pink Lady in
northern Israel (Gur et al., 2017). Because of the large lesions and rots on
fruits in Israel, unlike the small spots seen in other countries, we termed
the disease Alternaria fruit rot (AFR) (Gur et al., 2018). Heavy infection
of fruits is common in Israel but rare in other countries (Jung, 2007; Li
et al., 2013). Lesions on the fruit body or adjacent to cracks around the
calyx may coalesce to produce large dark rotted areas, and such rots
have destroyed up to 80% of the fruits in some orchards in Israel (Gur
et al., 2017). Whereas leaf infection and defoliation are the main dam­
age caused by A. alternata f. sp. mali in most growing areas worldwide
(Jung, 2007; Li et al., 2013), fruit rot is the major damage inflicted by
this pathogen in Israel (Gur et al., 2017).
Attempts to control A. alternata f. sp. mali with foliar fungicidal
sprays were conducted worldwide during the past two decades, but most
trials in the USA, East Asia, and Australia aimed to control the foliar
form of the disease (Filajdic and Sutton, 1992b; Horlock, 2006; Jung,
2007; Lee and Kim, 1986). Iprodione was effective against leaf blotch,
and captan, mancozeb and their mixtures with benomyl were partially
effective. However, tolerance to iprodione was reported in mycelial
growth, spore formation and spore germination (Lee and Kim, 1986;
Filajdic and Sutton, 1992b). It was recommended to start applications of
protectant fungicides at 2–3 weeks after petal fall (Sawamura, 1990).
Fungicides such as thiram, oxine-copper, tebuconazole, chlorothalonil,
and polyoxin B were used against Alternaria leaf blotch and fruit spot of
apple (Jung, 2007). In Australia, dithianon (delan) and metiram (poly­
ram) were used, but efficacy was often erratic, and varied among regions

* Corresponding author.
E-mail address: mreuveni@research.haifa.ac.il (M. Reuveni).

https://doi.org/10.1016/j.cropro.2019.104947
Received 30 December 2018; Received in revised form 1 September 2019; Accepted 4 September 2019
Available online 5 September 2019
0261-2194/© 2019 Elsevier Ltd. All rights reserved.
L. Gur et al. Crop Protection 127 (2020) 104947

(Horlock, 2006). In most trials, solo fungicides were tested, therefore, Table 1
the risk of fungicide resistance development, and the putative advantage Fungicide concentrations (μg mL 1) used in laboratory experiments with
of synergistic effects of mixtures were ignored. In the present paper we A. alternata f. sp. mali.
present data on the ability of various fungicides, alone and in mixtures, Fungicide Type of experimenta
to control AFR in the orchard. We previously demonstrated that under Spore Mycelial growth Detached fruits
Israeli conditions fruits of ’Pink Lady’ acquired susceptibility to the germination
disease at about 115 days after petal fall (DAPF), when their diameter
Scoreb 1.25, 12.5, 50, 1.25, 12.5, 125 1.25, 12.5, 50
was �55 mm (Gur et al., 2018). 125
The specific objectives of the present study were: (i) to evaluate the Merpan 2.5, 25, 250 25, 250, 1250 250, 500, 625, 1250
inhibitory effects of fungicides, separately and in fungicide mixtures, on Ortiva- 1.625, 16.25, 1.625, 16.25, 81.25, 1.625, 8.125, 16.25,
development of A. alternata f. sp. mali, in vitro, and on rot development in Top 162.5 162.5 162.5
Azimut 0.96, 9.6, 96 0.96, 9.6, 96 0.96, 4.8, 9.6, 96, 160
detached apple fruits; and (ii) to evaluate the efficacy of selected fun­ Orpan 12, 120, 1200 12, 120, 1200 12, 120, 1200
gicides, separately and in mixtures, in controlling Alternaria fruit rot in
a
the orchard. Spore germination and mycelial growth were conducted in vitro. Rot
development assays were applied to detached fruits.
b
Score ¼ difenoconazole. Merpan ¼ captan. Ortiva-Top ¼ prepacked mixture
2. Materials and methods
of azoxystrobin þ difenoconazole. Azimut ¼ prepacked mixture of azoxystrobin
þ tebuconazole. Orpan ¼ prepacked mixture of captan þ tebuconazole.
2.1. Fungicides
and the EC50 and EC90 values (the concentrations of each fungicide
Two single fungicides and three fungicide mixtures were used. The
causing 50 and 90% inhibition, respectively) were calculated (Table 2).
single ones were Score 250SC (Syngenta, Basel, Switzerland), containing
The experiment was conducted twice.
difenoconazole, a sterol demethylation inhibitor (DMI, FRAC Code 3)
and Merpan 500 WP (Adama Makhteshim, Beer Sheva, Israel), which
contains captan, a protectant fungicide (FRAC Code M04). The pre­ 2.4. Inhibition of mycelial growth of A. alternata f. sp. mali in vitro
packed fungicide mixtures were: Ortiva-Top 325SC (Syngenta)
comprising azoxystrobin (FRAC Code 11) at 200 g/L and difenoconazole Five mm Agar disks, bearing A.alternata f. sp. mali were taken from
at 125 g/L; Orpan 400SC (Adama Makhteshim), comprising captan at the edge of a freshly growing colony and placed on PDA amended with
320 g/L and tebuconazole (FRAC Code 3), at 80 g/L; Azimut 320SC various concentrations of each fungicide in 9-cm Petri dishes, with two
(Adama Makhteshim), comprising azoxystrobin (FRAC Code 11) at discs in each dish. The fungicides used were: Merpan, Ortiva-Top and
120 g/L and tebuconazole (FRAC Code 3) at 200 g/L. All doses are given Azimut at the final concentrations shown in Table 1. Petri dishes con­
as grams of active ingredient (ai) per liter or per hectare. taining PDA and sterilized water were used as controls. Three dishes per
treatment were incubated at 25 � C in darkness for 6 days, and colony
2.2. Pathogen diameters were recorded at 2, 3 and 6 days. Dishes were placed in the
incubator in a completely randomized design. Percentage inhibition and
A single-spore culture of A. alternata f. sp. mali grown on potato the EC50 and EC90 values were calculated (Table 2). The experiment was
dextrose agar (PDA, Difco, 39 g in 1 L of distilled water) was maintained conducted twice.
at 25 � C in Petri dishes in the dark for 10–12 days until conidia were
produced. Conidia were harvested from PDA plates by adding a small 2.5. Bioassay to evaluate rot development in detached fruits
amount of sterile distilled water to each plate and gently rubbing the
sporulating mycelial mass with a bent glass rod. Spore concentration The detached fruits were inoculated according to Gur et al. (2017).
was adjusted with the aid of a hemocytometer, to obtain a suspension Briefly: mature fruits, cv. Pink Lady from untreated trees were brought
containing 5 � 105 spores mL 1 (Reuveni and Sheglov, 2002). We have to the laboratory, washed with soapy water, blotted with a paper towel,
previously isolated four isolates PL-EZ-B-2011-1, PL–SH–B-3011,
PL-OR- C, and PL-EZ-l and tested them for germination and mycelial
Table 2
growth inhibition by fungicides (Gur et al., 2017). Inhibition rates ob­
Efficacy values EC50 and EC90 (μg mL1, ai) of fungicides against spore germi­
tained by several fungicides were similar for all isolates. Therefore, the nation, mycelial growth and rot development of A. alternata f. sp. malia.
most virulent isolate, PL–SH–B-3011, isolated from body rot of a ’Pink
Fungicide Spore germination Mycelial growth Rot on detached
Lady’ fruit grown in Sha’al orchard (33� 070 43’’N, 35� 430 34’’E) in 2011,
fruit
was used in all subsequent experiments of this study.
EC50 EC90 EC50 EC90 EC50 EC90

2.3. Inhibition of spore germination of A. alternata f. sp. mali in vitro Score >125 >125 4.25 60.8 6.75 55.5
(�2.55e) (�46.1) (�0.4) (�6)
Merpan 1.5 16.2 >1250 >1250 732.5 >1250
Fresh conidial suspensions were kept in 2-mL tubes and mixed with (�0.15) (�5.75) (�22.5)
an aqueous suspension of each fungicide or mixture to achieve the final Ortiva- 2.4 39.8 24.6 >162.5 5.1 80.1
desired concentrations, with a uniform conidial concentration of Topb (�0.85) (�1.55) (�3.4) (�0.15) (�9.3)
2 � 104 mL 1. In light of the risk of reduced sensitivity and acquired Azimutc 5.8 35.5 >96 >96 4.55 88.8
(�3) (�26.4) (�0.35) (�17.8)
resistance of the fungus to fungicides like DMI (demethylation in­
Orpand 0.05 8 (�4) 50.7 1006.7 75.25 960.9
hibitors) or QoI (quinon outside inhibitors) a mixture of fungicides was (�0.04) (�1.8) (�52.7) (�21.9) (�132)
often used. The following fungicides or mixtures were used: Merpan,
In the absence of fungicides, conidial germination after 20 h was 95 � 4%, col­
Ortiva-Top, Azimut, and Orpan at the final concentrations shown in
ony diameter on PDA after 6 days was 45 � 1.5 mm and the average lesion
Table 1. Conidial suspension mixed with water served as control treat­
diameter in fruits after 13 days was 24 � 3 mm.
ment. A 70-μL sample of each mixture was pipetted into a depression on a
Values were calculated with probit analysis (SPSS).
a microscope slide. Three replicates were used for each mixture or for b
Ortiva-Top, a prepacked mixture containing azoxystrobin þ difenoconazole.
control, and each slide was placed on wet filter paper in three different c
Azimut, a prepacked mixture containing azoxystrobin þ tebuconazole.
Petri dishes and incubated at 25 � C for 20 h in darkness. The germinating d
Orpan, a prepacked mixture containing captan þ tebuconazole.
conidia were counted under a microscope and the percentage inhibition, e
Standard Error of the mean of two experiments.

2
L. Gur et al.
and allowed to dry at room temperature. Each fruit was wounded to a
depth of ~3 mm at six points on its surface, with a 2-mm-diameter
sterilized plastic tip. The wounded fruits then were inoculated by
pipetting 10 μL of conidial suspension containing 5 � 105 spores mL 1
into each wound. Following inoculation, the fruits were placed in trays
on wet filter paper, covered with plastic bags to maintain high humidity
and kept in a growth chamber at 25 � C in darkness for 13 days. Fruits
were placed in trays in a completely randomized design. Lesion diameter
at each inoculation site was measured every 2 or 3 days. This assay was
used to determine the activity of fungicdes against rot development.
2.6. Activity of fungicides against AFR in detached fruits
The efficacy of fungicides against rot development in detached fruits
was estimated as follows: an aqueous suspension of each fungicide was
mixed 1:1 (v:v) with a conidial suspension to obtain the desired final
concentration of each fungicide Merpan, Ortiva-Top, Azimut and Orpan
which were tested at the final concentrations shown in Table 1. Mixtures
of Score, Ortiva-Top or Azimut with Merpan, also were tested at reduced
doses (Table 1). Spore concentration in all treatments were kept uniform
at 5 � 105 spores mL 1. Conidial suspensions mixed with equal volume
of strerile water served as control. Three fruits were used for each
concentration of each fungicide or mixture. Each fruit was inoculated
and incubated as described above (paragraph 2.5). Following inocula­
tion, the diameter of the rot lesion produced at each inoculation site was
measured four times every 2 or 3 days. Each experiment was conducted
twice. Percentage inhibition and the EC50 and EC90 values were calcu­
lated (Table 2). Area Under Disease Progress Curves (AUDPC) were
calculated, based on rot lesion diameter (Shaner and Finney, 1977), 13
days after inoculation. A synergy factor (SF) based on Abbott (Gisi,
1996) was calculated for the fungicide mixtures by using the equation:
SF ¼ Cobs/Cexp in which Cobs is the experimentally observed efficacy of
the mixture, and Cexp is its expected efficacy. The value of Cexp was
calculated as Cexp ¼ A þ B – (AB/100), in which A and B are the control
levels obtained with the fungicides in the mixture when used separately.
2.7. Field experiments
Field experiments were conducted with apple trees cv. Pink Lady in
commercial orchards in the Golan and the Upper Galilee regions of Israel
during 2011 through 2014. Fertilization, irrigation and other cultural
practices were as recommended to commercial growers by the Extension
Service of the Israeli Ministry of Agriculture and Rural Development.
The annual rainfall in the region ranged between 800 and 900 mm, all
precipitated during the winter (October through April). In the summer,
average relative humidity (RH) and temperature at midday were
35–40% and 30 � C, respectively. The sky is cloudless during most sum­
mer days. Night temperatures occasionally fall to 14–20 � C between the
end of May and August, and dew accumulates on leaf surfaces during
75% of the nights.
2.8. Experimental design
Seven field experiments were conducted during 2011–2014, inten­
ded to determine fungicide efficacies in controlling fruit calyx and body
rot in ’Pink Lady’ apples. Following Gur et al. (2018), applications of
fungicides in these experiments started at 115–118 DAPF, i.e.,
mid-August, 2 weeks before calyx cracks appeared. In experiments 1–4,
in orchards in Ein-Zivan, Ortal, and Merom-Golan, fungicides were
sprayed to runoff with a Turbo 400 (100-L, 1,400 kPa) gun sprayer
(Degania Sprayers, Degania, Israel) at spray volumes of 800–1,
000 L ha 1, according to the size of the trees. Treatments within ex­
periments were arranged in a randomized complete block design. Each
replicate comprised two or three trees, and there were four replicates for
each treatment. Three large-scale demonstration experiments were
conducted in 2013 and 2014: one on a 0.5-ha plot in Alonei-Habashan
3
Crop Protection 127 (2020) 104947
orchard in the Golan region, and two experiments during 2013 and
2014 on a 0.3-ha plot in the Matityahu Experimental Station in the
Upper Galilee region. Fungicides were applied at 1,500 L ha 1, with a
tractor-mounted 2,000-L trailed turbo-blower sprayer (Degania
Sprayers, Israel).
2.9. Efficacy of fungicides against AFR in the field
2.9.1. Experiment 1–2011
In this experiment, in the Ortal orchard (33� 050 40’’N, 35� 460 32’’E),
the treatments were applied to 9-year-old ’Pink Lady’ trees grafted on
MM.106 rootstock, and spaced at (4 � 1.5) m. The efficacy against AFR
of two prepacked-mixtures Ortiva-Top and Orpan was evaluated, each in
two doses (Table 3). Untreated trees served as controls. Five foliar sprays
of each treatment were applied at 14-day intervals, starting at 116
DAPF, i.e. August 14, when fruits were about 55 mm in diameter. Each
replicate comprised two trees and there were four replicates per
treatment.
2.9.2. Experiment 2–2012
In this experiment the treatments were applied to 7-year-old ’Pink
Lady’ trees grafted on MM.106 rootstock in the Ein-Zivan orchard
(33� 060 58’’N, 35� 480 12’’E), and spaced at (4.5 � 1.8) m. The efficacy
against AFR of seven fungicides and mixtures was evaluated (Table 4).
Untreated trees served as controls. Six foliar sprays of each fungicide/
mixture were applied at 14-day intervals starting at 118 DAPF, i.e.
August 10, when fruits were about 55 mm in diameter. Each replicate
comprised two trees, and there were four replicates per treatment.
2.9.3. Experiment 3–2012
In this experiment the treatments were applied to 5-year-old ’Pink
Lady’ trees grafted on MM.106 rootstock, in the Merom-Golan orchard
(33� 070 41’’N, 35� 480 19’’E). Spacing between trees was (5 � 2) m. The
efficacy against AFR of eight fungicides and mixtures was evaluated
(Table 4). Untreated trees served as controls. Six foliar sprays of each
treatment were applied at 14-day intervals starting at 117 DAPF, i.e.
August 11, when fruits were about 55 mm in diameter. Each replicate
comprised two trees and there were four replicates per treatment.
2.9.4. Experiment 4–2012
The treatments were applied to 10-year-old ’Pink Lady’ trees grafted
on MM.106 rootstock in the Ortal orchard (33� 050 40’’N, 35� 460 32’’E)
and spaced at (4 � 1.5) m. The efficacy of Ortiva-Top against AFR and
Alternaria leaf blotch was evaluated. Untreated trees served as controls.
In each treatment six foliar sprays were applied at 14-day intervals
starting at 115 DAPF, i.e. August 16, when fruits were about 55 mm in
Table 3
Exp. 1–2011. Efficacy of fungicidal mixtures each applied at two doses in con­
trolling Alternaria calyx rot and fruit body rot in ’Pink Lady’ apple fruits.
Treatmenta Fruits with calyx rot (%) Fruits with body rot (%)
Control untreated 15.6 af 11.9 a
Ortiva-Topb 5b 5b
Ortiva-Top (1/2)c 3.1 b 6.2 ab
Orpand 2.5 b 5b
Orpan (1/2)e 6.9 ab 7.5 ab
a
Five foliar sprays were applied at 14-d intervals, starting in mid-August,
when fruits were about 55 mm in diameter.
b
Prepacked mixture of azoxystrobin (100 g ha 1) þ difenoconazole (62.5 g
1
ha ).
c
Prepacked mixture of azoxystrobin (50 g ha 1) þ difenoconazole (31.25 g
ha 1).
d
Prepacked mixture of captan (960 g ha 1) þ tebuconazole (240 g ha 1).
e
Prepacked mixture of captan (480 g ha 1) þ tebuconazole (120 g ha 1).
f
Means within columns followed by different letters are significantly different
(P < 0.05, Fisher’s LSD K-ratio t-test).
L. Gur et al. Crop Protection 127 (2020) 104947

Table 4 360 g ha 1); and (v) untreated trees as a control. All sprays were applied
Exp. 2 and Exp. 3–2012. Efficacy of various fungicides and their mixtures in at 14-day intervals starting when fruits were about 55–60 mm in
controlling Alternaria calyx rot and fruit body rot in ’Pink Lady’ apple fruits. diameter: either five sprays starting at 116 DAPF (i.e. August 21), or four
Treatmenta Modej Experiment 2 Experiment 3 sprays starting at 128 DAPF (i.e. September 3). The fungicides were
Fruits with Fruits with Fruits with Fruits with
applied with a tractor-mounted 2,000-L trailed turbo blower sprayer.
calyx rot body rot calyx rot body rot
(%) (%) (%) (%) 2.9.7. Experiment 7–2014 – large-scale demonstration experiment
Control NS k
22 a l
17.5 a 30 a 16 a This third large-scale experiment was conducted in 2014 to examine
untreated the efficacy of an alternating spray program. Each treatment comprised
Ortiva-Topb PM 5c 3.5 b 14 b 1.3 cd two rows of twenty 10-year-old ’Pink Lady’ trees, growing on Hashabi
Azimutc PM 9 bc 4b 22 ab 6b rootstock in the Matityahu Experimental Station orchard, on a 0.3-ha
Orpand PM 7 bc 1.5 b 16 b 3.3 bcd
Scoree Solo n.tm n.t 18 b 4 bcd
plot in which trees were spaced at (4 � 2) m. The experiment included
Merapnf Solo 9 bc 3b 21 ab 7.3 b three treatments: (i) four sprays of Ortiva-Top (rates as in experiment 5);
Score þ TM 6 bc 4.5 b 22 ab 6b (ii) four sprays alternating between Ortiva-Top and Orpan (rates as in
Merpang experiment 6); and (iii) untreated trees as control treatment. All sprays
Ortiva-Top þ TM 4.5 c 1.5 b 13 b 0d
were applied at 14-day intervals starting at 114 DAPF (i.e. August 3),
Merpanh
Azimut þ TM 12 b 2.5 b 14 b 4.6 bc when fruits were about 55 mm in diameter. The fungicides were applied
Merpani with a tractor-mounted 2,000-L trailed turbo blower sprayer.
a
Six foliar sprays were applied at 14-d intervals starting in mid-August, 118
DAPF (Exp 2), or 117 DAPF (Exp 3), when fruits were about 55 mm in diameter. 2.10. Assessment of infected fruit with rots and leaf blotch in the orchard
b
Prepacked mixture of azoxystrobin (100 g ha 1) þ difenoconazole (62.5 g
ha 1). A few days before comercial harvest, 50 fruits, growing at heights of
c
Prepacked mixture of azoxystrobin (360 g ha 1) þ tebuconazole (600 g 40–200 cm and comprising 25 from each side of the row, were randomly
ha 1). selected from each treatment and replicate, i.e. 200 fruits per treatment.
d
Prepacked mixture of captan (960 g ha 1) þ tebuconazole (240 g ha 1). For each fruit the presence of body or calyx rots was recorded and the
e
Difenoconazole (50 g ha 1). percentage of such fruits (incidence) was calculated for each treatment.
f
Captan (1250 g ha 1). Five shoots, each bearing five leaves, were randomly selected from each
g
Tank mixture of difenoconazole (50 g ha 1) þ captan (1250 g ha 1).
h
treatment and replicate, i.e. 100 leaves per treatment, and the per­
Tank mixture of Ortiva-Top (azoxystrobin þ difenoconazole at 162.5 g
centage of leaves with Alternaria spots (incidence), and the infected
ha 1) þ Merpan (captan at 1250 g ha 1).
i percentage of each leaf’s area (severity) were calculated for each
Tank mixture of Azimut (azoxystrobin þ tebuconazole at 960 g ha 1) þ
Merpan (captan at 1250 g ha 1). treatment.
j
Mode ¼ Mode of application.
k
NS ¼ not sprayed, PM ¼ Prepacked mixture, Solo ¼ Solo fungicide, 2.11. Data analysis
TM ¼ Tank mixture.
l
Means within columns followed by different letters are significantly different Each laboratory experiment was conducted at least twice. The data
(P < 0.05, Fisher’s LSD K-ratio t-test). from repeated experiments were combined for analysis because variance
m
n.t ¼ not tested. between experiments was homogeneous. The EC50 and EC90 values for
conidial germination, mycelial growth, or rot development in detached
diameter. Each replicate comprised one tree and there were seven rep­ fruits were calculated by means of probit analysis with SPSS software
licates per treatment. (IBM SPSS Statistics 20.0). In experiments on detached fruits, AUDPC
was calculated based on Shaner and Finney (1977).
2.9.5. Experiment 5–2013 – large-scale demonstration experiment All data were analysed with the GLM statistical software package
To further evaluate the potential of disease management by using version 9.2 or with the JMP statistics package version 12.1.0 (both from
effective fungicide mixtures against AFR, a large-scale demonstration SAS, Cary, NC, USA). For field experiments, analysis of variance
experiment was conducted in 2013. The experiment was conducted in (ANOVA) was applied to arcsin transformed data of the percentage of
the Matityahu Experimental Station orchard (33� 030 54’’N, 35� 260 19’’E) infected fruits, in order to achieve normal distribution. Data in tables is
in a 0.3-ha plot in which trees were spaced at (4 � 2) m. Each treatment reported in untransformed units. Fisher’s LSD K-ratio t-test was applied,
used two rows of twenty 9-year-old ’Pink Lady’ trees, grafted on a local to determine whether differences between treatments were significant at
Hashabi rootstock. Three treatments were tested: (i) two sprays during α ¼ 0.05.
the season — Ortiva-Top (azoxystrobin at 150 g ha 1 þ difenoconazole
at 93.75 g ha 1); (ii) four sprays as above, and (iii) untreated trees as 3. Results
controls. All sprays were applied at 14-day intervals starting when fruits
were about 55–58 mm in diameter, either at 115 DAPF (i.e. August 12) 3.1. Inhibition of spore germination of A. alternata f. sp. mali in vitro
and using four sprays, or starting at 132 DAPF (i.e. August 29) and using
two sprays. The fungicides were applied with a tractor-mounted 2,000-L In the absence of a fungicide, the average conidial germination rate
trailed turbo blower sprayer. was 95 � 4%. Orpan, Merpan, and Ortiva-Top were most inhibitory to
conidial germination, with EC90 of 0.05, 1.5, and 2.4 μg mL 1, respec­
2.9.6. Experiment 6–2013 – large-scale demonstration experiment tively (Table 2). Azimut was less effective, with EC50 and EC90 of 5.8 and
A second large-scale demonstration experiment was conducted in 35.5 μg mL 1, respectively. Score was ineffective, with EC50 of more
2013 in the Alonei-Habashan orchard (33� 170 63’’N, 35� 760 92’’E), on a than 125 μg mL 1 (Table 2).
0.5-ha plot in which trees were spaced at (4 � 2) m. Each treatment used
ninety 9-year-old ’Pink Lady’ trees, growing on M9 rootstock. The effi­ 3.2. Inhibition of mycelial growth of A. alternata f. sp. mali in vitro
cacy of four or five sprays during the season in controling AFR was
examined. The experiment included the following five treatments: (i þ In the absence of a fungicide, the average colony diameter on PDA
ii) four or five sprays of Ortiva-Top (rates as in experiment 5); (iii þ iv) after 6 days was 45 � 1.5 mm. Score, Ortiva-Top and Orpan were
four or five sprays of Orpan (captan at 1440 g ha 1 þ tebuconazole at effective against mycelial growth, with EC50 values of 4.25, 24.6, and

4
L. Gur et al.
50.7 μg mL 1, respectively (Table 2). Azimut was less effective, with
EC50 value of more than 96 μg mL 1. Merpan was ineffective, with EC50
and EC90 values > 1250 μg mL 1 (Table 2).
3.3. Activity of fungicides against AFR in detached fruits
In the absence of fungicides, the average diameter of lesions caused
by A. alternata f. sp. mali in fruits after 13 days was 24 � 3 mm. Azimut,
Ortiva-Top and Score were highly effective in suppressing AFR, with
EC50 values of 4.55, 5.1, and 6.75 μg mL 1, respectively, as compared
with 75.25 and 732.5 μg mL 1, respectively for Orpan and Merpan
(Table 2). Merpan was weakly active, with EC90 of >1250 μg mL 1,
compared with the recommended field-use concentration of
1250 μg mL 1.
The mixture of Score and Merpan at reduced concentrations of 12.5
and 625 μg mL 1, respectively, was not synergistic (Fig. 1a and b).
However, the mixture of Ortiva-Top plus Merpan, at reduced concen­
trations of 8.125 (for Ortiva-Top), and 625 μg mL 1 (for Merpan),
completely inhibited AFR development in inoculated fruits, exhibiting
synergistic action, with SF ¼ 1.43 (Fig. 2a and b). Although reduced
concentrations of Azimut (4.8 μg mL 1) or Merpan were not signifi­
cantly reduced fruit rot compared to control, the mixture of Azimut plus
Merpan, at reduced concentrations significantly inhibited AFR devel­
opment, compared to control, exhibiting synergistic action, with
SF ¼ 1.44 (Fig. 3a and b).
3.4. Efficacy of fungicides against fruit rot in the field
3.4.1. Experiment 1–2011
Both prepacked mixtures, Ortiva-Top and Orpan, at full doses of each
component were effective, providing 68–86% control of calyx rot, or
58% control of fruit body rot compared with control untreated fruits.
The prepacked mixture Orpan, with dosage reduced by half for each
component, was less effective than the full doses. However, the efficacy
of the prepacked mixture Ortiva-Top was similar at both doses in con­
trolling calyx rot, but not significantly different than the control in
reducing fruits with body rot (Table 3).
5
Crop Protection 127 (2020) 104947
3.4.2. Experiment 2–2012
Six applications of the prepacked fungicide mixtures Ortiva-Top or
Orpan, or the tank mixture of Score with Merpan reduced calyx-rotted
fruit numbers by 77, 68, and 73%, respectively, compared with un­
treated trees (Table 4). Applications of the fungicide Azimut or Merpan
provided only 59% control compared with untreated trees. Tank mix­
tures of Ortiva-Top with Merpan, or of Azimut with Merpan did not
improve efficacy compared with each fungicide alone. All treatments
significantly reduced percentages of body-rot-infected fruits by 75–90%
compared with untreated trees (Table 4).
3.4.3. Experiment 3–2012
Ortiva-Top and Orpan affectively reduced calyx rots by 47 and 54%,
respectively (Table 4), and adding Merpan to create a three-way tank
mixture with Ortiva-Top or Azimut slightly improved calyx rot control,
but Score, Merpan, or their mixture did not provide satisfactory control
of calyx rots (Table 4). All treatments reduced fruit body rot percentages
by 65–90% compared with untreated trees. The most effective treatment
was Ortiva-Top alone (with only 1.3% of the fruit bodies showing AFR
symptoms) or its tank mixture with Merpan (with no AFR symptoms
observed on the fruit body); the percentage on untreated trees was 16%
(Table 4).
3.4.4. Experiment 4–2012
Six applications of the prepacked Ortiva-Top reduced percentages of
fruits rotted on calyx and fruit body by 85 and 89%, respectively,
compared with untreated trees (Table 5). Ortiva-Top also reduced the
percentage of infected leaves by 51% and that of infected leaf area by
90% (Table 5).
3.4.5. Large-scale demonstration experiments – 2013
Two mixtures were selected for determination of the number of foliar
sprays required to achieve satisfactory control of AFR under commercial
conditions. With the first, the prepacked mixture Ortiva-Top was
sprayed either twice or four times. Fig. 4 shows that all fungicidal
treatments significantly reduced both calyx rot and fruit body rot
compared with control untreated fruits. Two foliar sprays were less
effective than four, and did not provide satisfactory results in reducing
fruit calyx rots (Fig. 4). This mixture together with Orpan was tested in
Fig. 1. a. Effects of difenoconazole (Score), captan
(Merpan), and their mixture on rot development in
detached apple fruits. Rot diameter at each inocu­
lation site was measured every 2 or 3 days. Three
fruits were used for each treatment. Concentrations
of fungicides are given as μg ai/mL. The Area Under
Disease Progress Curves (AUDPC) were calculated 13
days after inoculation. Mean values followed by
different letters are significantly (P < 0.05) different
according to Fisher’s LSD t-test; b. Effects of dife­
noconazole, captan, and their mixture on rot devel­
opment in detached apple fruits, 13 days after
inoculation. Each fruit was inoculated with an
aqueous suspension of each fungicide, mixed with a
conidial suspension. Fruits inoculated with conidial
suspension in water served as controls. Fungicide
concentrations are given as μg ai/mL.
L. Gur et al.
the second demonstration trial with four or five applications of each
mixture. The results showed no differences in calyx rot between four and
five sprays of Ortiva-Top (Fig. 5), but five sprays of Ortiva-Top improved
control of fruit body rot compared with four applications, reducing body
rots by 90% compared with untreated fruits. Although four sprays of
Orpan did not significantly reduced calyx rots compared with untreated
fruits, five sprays of Orpan significantly improved control of calyx rots,
reducing calyx rots by 88% compared with untreated fruits. No differ­
ences in fruit-body rot were seen between four and five applications of
6
Crop Protection 127 (2020) 104947
Fig. 2. a. Effects of Ortiva-Top, a prepacked-mixture
of azoxystrobin (200 g/L) plus difenoconazole
(125 g/L), captan (Merpan), and their 3-way mixture
on rot development in detached apple fruits. Rot
diameter at each inoculation site was measured
every 2 or 3 days. Three fruits were used per treat­
ment. Fungicide concentrations are given as μg ai/
mL. Area Under Disease Progress Curves (AUDPC) of
rot lesion diameter were calculated 13 days after
inoculation. Mean values followed by different let­
ters are significantly (P < 0.05) different according
to Fisher’s LSD t-test; b. Effects of a prepacked
mixture of azoxystrobin þ difenoconazole, captan,
and their three-way mixture on rot development in
detached apple fruit, 13 days after inoculation. The
fungicides were mixed with a conidial suspension
before inoculation. Fruits inoculated with conidial
suspension in water served as controls. Fungicide
concentrations are given as μg ai/mL.
Fig. 3. a. Effect of Azimut, a prepacked mixture of
azoxystrobin (120 g/L) plus tebuconazole (200 g/L),
captan (Merpan) and their 3-way mixture on rot
development in detached apple fruits. Rot lesion
diameter at each inoculation site was measured
every 2 or 3 days. Three fruits were used per treat­
ment. Fungicide concentrations are given as μg ai/
mL. Area Under Disease Progress Curves (AUDPC) of
rot lesion diameter were calculated 13 days after
inoculation. Mean values followed by different let­
ters are significantly (P < 0.05) different according
to Fisher’s LSD t-test; b. Effects of Azimut, a
prepacked-mixture of azoxystrobin þ tebuconazole,
captan and their three-way mixture on rot develop­
ment in detached apple fruits, 13 day after inocula­
tion. Fungicides were mixed with conidial
suspension before inoculation. Fruits inoculated with
conidial suspension in water served as control.
Fungicide concentrations are given as μg ai/mL.
Orpan (Fig. 5).
3.4.6. Large-scale demonstration experiment – 2014
The results of this experiment were similar to those achieved in 2013.
Four consecutive sprays of Ortiva-Top applied in a commercial manner
reduced fruit calyx rot and body rot by 69 and 81%, respectively,
compared with untreated fruits (Fig. 6). Four sprays of Ortiva-Top
alternated with Orpan were as effective as four sprays of Ortiva Top
(Fig. 6).
L. Gur et al. Crop Protection 127 (2020) 104947

Table 5
Exp. 5–2012. Efficacy of Ortiva-Top (azoxystrobin þ difenoconazole) in con­
trolling Alternaria calyx rot, fruit body rot and leaf blotch in ’Pink Lady’ apple
fruits.
Treatmenta Fruits with Fruits with Infected Infected leaf
calyx rot (%) body rot (%) leaves (%) area (%)

Control 51 ac 27 a 91 a 15 a
untreated
Ortiva-Topb 8b 3.5 b 45 b 1.5 b
a
Six foliar sprays were applied at 14-d intervals starting from mid-August,
115 DAPF when fruits were about 55 mm in diameter.
b
Prepacked mixture of azoxystrobin (100 g ha 1) þ difenoconazole (62.5 g
1
ha ).
c
Means within columns followed by different letters are significantly different
(P < 0.05, Fisher’s LSD K-ratio t-test).

Fig. 6. Large-scale demonstration experiment 7–2014. The efficacy of Ortiva-

Top (azoxystrobin þ difenoconazole), applied four times, or in alternation
with Orpan (captan þ tebuconazole) on Alternaria calyx rot and fruit body rot
in ’Pink Lady’ apple fruits. Untreated trees served as controls. Sprays were
applied at 14-d intervals starting at 114 DAPF (August 3) when fruits were
about 55 mm in diameter. Means above columns of the same color followed by
different letters are significantly (P < 0.05) different according to Fisher’s LSD
K-ratio t-test.

4. Discussion

Fig. 4. Large-scale demonstration experiment 5–2013. The efficacy of Ortiva-
Top (azoxystrobin þ difenoconazole) applied two or four times on Alternaria
calyx rot and fruit body rot in ’Pink Lady’ apple fruits. Untreated trees served as
controls. Four sprays were applied at 14-d intervals starting at 115 DAPF
(August 12), when fruits were about 55 mm in diameter, or two sprays, starting
at 132 DAPF (August 29), when fruits were about 58 mm in diameter. Means
above columns of the same color followed by different letters are significantly
(P < 0.0001) different according to Fisher’s LSD K-ratio t-test.
Fig. 5. Large-scale demonstration experiment 6–2013. The efficacy of Orpan
(captan þ tebuconazole), or Ortiva-Top (azoxystrobin þ difenoconazole)
applied four or five times on Alternaria calyx rot and fruit body rot in ’Pink
Lady’ apple fruits. Untreated trees served as controls. Sprays were applied at
14-d intervals: either five sprays starting at 116 DAPF (August 21) when fruits
were about 55 mm in diameter; or four sprays starting at 128 DAPF (September
3) when fruits were about 58 mm in diameter. Means above columns of the
same color followed by different letters are significantly (P < 0.05) different
according to Fisher’s LSD K-ratio t-test.
Alternaria Fruit Rot (AFR) is a new severe fruit rot in Pink Lady’
apple orchards in Israel caused by A. alternata f. sp. mali (Gur et al.,
2017, 2018). As no reports are available on control of AFR, this
encouraged us to conduct an intensive study, in the laboratory and in the
field, seeking effective fungicides and/or fungicidal mixtures to control
this severe disease of apple fruits.
We searched for chemicals that would inhibit conidial germination
and mycelial growth, and thereby reduce fruit rot from A. alternata f. sp.
mali infection. Of the tested chemicals, Merpan, Orpan, Azimut, and
Ortiva-Top were found the most effective in suppressing conidial
germination (Table 2).
Once infection has occurred, suppression of mycelial growth within
the fruit tissue becomes important for disease control. Mycelial growth
was suppressed by Score, Ortiva-Top and Azimut, but not by Merpan
(Table 2). These tested fungicides have differing modes of action:
azoxystrobin (a component of Ortiva-Top) is a Quinone outside Inhibitor
(QoI) that inhibits mitochondrial respiration by blocking electron
tranport at the cytochrome bc1 complex (complex III) (Anke, 1995;
Brandt et al., 1988). The QoIs control a range of fungal pathogens,
including Alternaria spp., by inhibiting spore germination and mycelial
growth (Anke, 1995; Bartlett et al., 2002). This accounts for the high in
vitro sensitivity of conidial germination and mycelial growth to
Ortiva-Top, which contains azoxystrobin.
Difenoconazole and tebuconazole are sterol biosynthesis inhibitors,
which inhibit C-14 demethylation of lanosterol or 24-methylenedihy­
drolanosterol, a biosynthesis step responsible for conversion of lano­
sterol to ergosterol, the final product of fungal cell membrane sterol
synthesis (Koller and Scheinpflug, 1987). This accounts for the in vitro
inhibition of mycelial growth of A. alternata f. sp. mali by Score (con­
taining difenoconazole) or Azimut (containing tebuconazole) (Table 2).
Merpan (containing captan) inhibits conidial germination by interupt­
ing the respiration that converts glucose to pyruvate (Hochstein and
Cox, 1956). This accounts for the high in vitro activity against conidial
germination, and the low sensitivity of mycelial growth to Merpan.
The efficacy of fungicides against rot development in detached fruits
was tested with a fruit-inoculation assay (Gur et al., 2017). Azimut,
Ortiva-Top and Score at full doses were highly effective against AFR
development in detached fruits (Table 2).
Evaluation of measures to reduce resistance, such as using fungicide
mixtures or alternation, was another motivation for the present study.

7
L. Gur et al.
Resistance of Alternaria alternata to QoI fungicides was reported in pis­
tachio (Ma et al., 2003) and tangerine (Vega and Dewdney, 2014), and
reduced sensitivity of A. alternata f. sp. mali to polyoxin and pyraclos­
trobin was reported in apples (Hwang and Yun, 1986; Lu et al., 2003).
The prepacked mixture Ortiva-Top contains difenoconazole, and Azimut
contains tebuconazole, both of which are sterol biosynthesis inhibitors
(SBI), which are considered medium-risk fungicides (FRAC Code List,
2018). These SBIs should provide background disease control in support
of the other component in these mixtures, i.e. azoxystrobin (a QoI
fungicide) which is considered a high-risk fungicide (FRAC Code List,
2018).
The high efficacy of the multisite fungicide Merpan against spore
germination, led us to combine it with reduced doses of Ortiva-Top,
Azimut, or Score. Merpan is regarded as a low-risk fungicide against
which no signs of resistance are known (FRAC Code List, 2018), there­
fore adding it, either as a part of a prepacked mixture or in a tank
mixture, while lowering the proportion of the at-risk fungicide reduces
the selection pressure for fungicide resistance, without compromising
effective disease control (Elderfield et al., 2018; van den Bosch et al.,
2014).
Fungicides are also combined in mixtures to expand their spectrum
of activity, to prolong their persistence, and to improve disease control
by exploiting synergistic interactions between their components (Gisi,
1996; van den Bosch et al., 2014). Synergy, which frequently occurs
between fungicides in mixtures, may involve antifungal compounds of
differing natures and sources, differing or identical modes of action, or
differing formulations (Gisi, 1996). The enhanced efficacy of Ortiva-Top
or Azimut when mixed with Merpan (Figs. 2 and 3) resulted from syn­
ergistic interactions (SF ¼ 1.43 and SF ¼ 1.44, respectively). Synergy
values larger than 1 indicate synergistic interaction between the fungi­
cides comprising a mixture (Gisi, 1996).
In laboratory experiments we showed that fungicides, both sepa­
rately and in mixtures, effectively controlled AFR in artificially inocu­
lated detached apple fruits. Field experiments verified the efficacy of
these products against the disease in the orchard. In light of our previous
findings (Gur et al., 2018) on the proneness of apple fruits to AFR, the
control strategy we adopted in the present experiments was to start
fungicide applications at 115–118 DAPF, i.e. about 15 days before fruit
cracking appeared, and when fruit diameter was about 55 mm. Unlike
our findings with detached fruits under laboratory conditions, the
addition of Merpan (captan) to a tank mixture with Ortiva-Top or Azi­
mut did not significantly improve disease control in the orchard
(Table 4). This may be related to the difference between the high spore
concentration (5 � 105 spores mL1) used for artificial inoculation in the
laboratory and the low spore concentration that occurs naturally on
fruits in the orchard. Against a low spore concentration each prepacked
mixture was effective, and the advantage gained by adding Merpan was
negligible. A tank mixture of difenoconazole and captan was not syn­
ergisic with detached fruits, and did not provide repeatable results in
differing field experiments. Application of Ortiva-Top was effective
against AFR, but also reduced the incidence and severity of leaf blotch
on apple leaves, similarly to other reported fungicides (Filajdic and
Sutton, 1992b; Horlock, 2006; Jung, 2007; Lee and Kim, 1986). Four or
five applications of Ortiva-Top and Orpan or their alternation, were
most effetive in controlling the disease in the field, also when applied in
a commercial manner by using a tractor and trailed turbo blower sprayer
(Figs. 4–6). Five applications seems to be more effective than four ap­
plications, probably because that in five applications the first spray was
applied in mid of August while in the case of four applications the first
spray was applied two weeks later. Our previous study (Gur et al., 2018)
demonstrated that the most susceptible stage of the fruit starts at 115
DAPF (mid of August) and fruit calyx cracking appears in nature towards
early September. Therefore, starting of foliar sprays is recommended
about 15 days before cracking appears. By that fruits will be protected
against the fungal infection through either cracks or wounds (Gur et al.,
2018). This emphasizes the importance of the first application two
8
Crop Protection 127 (2020) 104947
weeks before cracks appeared. Reducing the number of sprays below
four led to reduced efficacy.
It is important to note that A. alternata f. sp. mali is a wound pathogen
(Gur et al., 2017), and the naturally occurring calyx cracks on ’Pink
Lady’ fruits (Stern et al., 2013) are of major concern to growers because
they serve as a point of entry into the mesoderm, where the fungus
causes fruit rot. Reducing fruit calyx cracks by using growth regulators
such as cytokinins and gibberellins, as reported by Ginzberg et al.
(2014), may reduce the number of pentration sites available to the
fungus and thereby, together with fungicide applications, should reduce
calyx rots. Further study is required to evaluate this form of integrated
management.
Our present data clearly show that use of prepacked mixtures or tank
mixtures improved disease control in both detached fruits and in the
orchards. We urge the commercial use of Ortiva-Top and/or Orpan as
effective products for AFR disease control in Israel. Their efficacy was
enhanced by correctly timed application, i.e. when fruits become prone
to infection; this practice enabled effective control of Alternaria fruit rot
in apple by using a limited number of fungicidal sprays.
Acknowledgements
The authors thank Keren Levy, Suliman Farhat and the orchard teams
of Ein-Zivan, Ortal, Merom-Golan and Matityahu for their valuable
assistance.
This study was supported by research grant No. 38427 from the Is­
raeli Ministry of Science and Technology, and Adama Agricultural So­
lutions Ltd, Beer Sheva, Israel.
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