Controlling Cucumber Powdery Mildew using Cow Milk and Whey under Greenhouse Conditions

Full text

Egyptian Journal of Phytopathology, Vol. 48 (2020) 58-70.
DOI: 10.21608/ejp.2020.45238.1009
ORIGNAL PAPER
Controlling Cucumber Powdery Mildew using Cow Milk and Whey under
Greenhouse Conditions
Kamel*, S.M. and Afifi, M.M.I.
Received: 5 October 2020 / Accepted: 10 December 2020 / Published online: 16 December 2020.
© Egyptian Phytopathological Society 2020
ABSTRACT
The role of raw cow's milk and whey (40 and 50% dilution in water) and number of sprays against cucumber powdery
mildew caused by Sphaerotheca fuliginea was studied in greenhouse experiments during 2017/18 and 2018/19. As a
result of number of sprays, the disease severity percentage and area under the disease progress curve (AUDPC) were
affected by the number of sprays with raw milk and whey. The number of sprays played an important role in
controlling powdery mildew, and significant differences have occurred among all applied sprays. Four sprays with
intervals of one week was the effective treatment in reducing disease severity, AUDPC values, compared to other
treatments, i.e., two and three sprays in comparison to the fungicide as well as a control treatment. Additionally,
treated plants showed a significant increase in plant growth and yield parameters in case of spraying four times. Also,
the activity of defense-related enzymes (catalase, peroxidase, and polyphenoloxidase) and amino acid proline was
high in response to increasing the number of sprays at four times compared with untreated plants. Light and scanning
electron microscopic observations showed a clear change in the morphology of S. fuliginea spores, in addition
to hyphae degradation and loss the vitality of conidia as well as rupturing the content of conidia. The results obtained
also indicated that cow's milk and whey dilution effectively reduced the powdery mildew signs and symptoms on
infected cucumber plants at 50% with the four sprays treatment being the most effective treatment.
Key words: Cucumber, Cucumis sativus, enzyme, greenhouse, Cow milk, Sphaerotheca fuliginea, Whey
*Correspondence: Kamel, S.M.
said_kamel88@yahoo.com
Said M. Kamel
https://orcid.org/0000-0001-7243-3276
Plant Pathology Research Institute, Agricultural
Research Center, 12619, Giza, Egypt.
Mohamed M.I. Afifi
https://orcid.org/0000-0002-0345-4011
Soils, Water and Environment Research Institute,
Agricultural Research Center, 12619, Giza, Egypt.
INTRODUCTION
Worldwide, the cultivated cucurbits under
greenhouse conditions are mainly infected and
damaged by powdery mildew pathogen
Sphaerotheca fuliginea, which attacks leaves, stems,
and even fruits. The chemical fungicides are used as
the main strategy to control the fungal diseases
affecting cucurbits production under commercial
conditions (Kimati et al., 1997). Usage of chemical
fungicides especially repeating the application
times contributes to environmental pollutions and
resulted in resistant populations of S. fuliginea to
the used fungicides (McGrath, 1996). So, searching
for alternatives to chemical fungicides or reducing
their application times is the urgent target. In this
respect, Bettiol et al. (1999) used milk, an eco-
friendly product to control squash powdery mildew
caused by S. fuliginea. So, they suggested that foliar
sprays of aqueous dilutions of fresh, non-
pasteurized cow’s milk can be quite effective in
controlling the powdery mildew on zucchini where
the weekly foliar sprays using an aqueous solution
of 40% milk reduced the powdery mildew disease
severity by 85-90%. Also, using milk with different
concentrations gave a good reduction in powdery
mildew disease severity and this may be attributed
to its contents of fatty acids, proteins, sugars, salts,
enzymes, minerals, and vitamins. In this regard,
Crisp et al. (2006) suggested that management of
powdery mildew on grapevine could be done
through potential alternatives to synthetic
fungicides such as whey, milk individually or
mixed with oils or bicarbonate. Whey of raw cow
milk was also used to control the powdery mildew
of squash and cucumber (Bettiol et al., 2008). On
the other hand, Smith et al. (2003) mentioned that
the milk-based foliar sprays of pumpkin infected
naturally with Sphaerotheca fuliginea, muskmelon
infected with S. fuliginea and tomato infected with
Leveillula taurica reduced the powdery mildew
signs and symptoms under open field conditions.

Kamel and Afifi 59
Also, powdery mildew disease severity on pumpkin
plants was reduced by 85-90% when 40% of milk
was applied as foliar sprays (Ferrandino and Smith
2007). Recently, Kamel et al. (2017) reported that
whey dilutions (50 and 40%) followed by milk
dilutions (50 and 40%) were significantly effective
in reducing the foliar symptoms of cucumber
powdery mildew disease severity by 43-74%.
An efficient anti-oxidative system comprising of
the enzymatic, as well as non-enzymatic
antioxidants, is involved in scavenging or
detoxification of excess ROS (Noctor et al., 2007).
As for the role of antioxidant enzymatic
components, i.e. catalase (CAT), peroxidase
(POX), and polyphenoloxidase (PPO) play a vital
role during high ROS levels, thereby and protect
plants from pathogen attack (Hafez et al., 2016).
Whereas non-enzymatic antioxidants include
phenolics, ascorbate, glutathione, carotenoids, and
tocopherols (Gill and Tuteja, 2010)
The present work was conducted to study the
role of raw cow milk and whey 40 and 50%
dilutions and the number of sprays against
cucumber powdery mildew caused by S. fuliginea
under greenhouse conditions.
MATERIALS AND METHODS
Plant source:
The efficacy of cow milk and whey with two
dilutions (40 and 50%) was evaluated under
greenhouse conditions to determine the best
number of sprays against S. fuliginea the causal
agent of cucumber powdery mildew compared to
fungicide Topas 100 EC (Penconazole 10.2%
W/W, Syngenta Company). In this respect, seeds
of cucumber (Cucumis sativus L., Barracuda F1)
batch No. 0161531598 (Seminis Company) were
obtained kindly from the Horticultural Research
Institute, Agricultural Research Center (ARC).
Rectangular plastic trays (5×5×7 cm) were filled
with autoclaved commercial potting medium
(peat-based container medium, containing peat
moss and vermiculite). Seedlings 30-days old of
cucumber cv. Barracuda were transplanted at the
rate of 10 plants per 5 m in a row in the
greenhouse located in Sakha, Kafr El-Sheikh
governorate, Egypt. Recommendations of the
Ministry of Agriculture for irrigation,
fertilization and weed control were applied
precisely.
Milk and whey preparation:
Cow’s raw fresh milk, unpasteurized and
unhomogenized was obtained from Animal
Research Station located in Sakha, Kafr El-Sheikh
governorate, Egypt. The whey was prepared from
cow’s fresh milk by heating the milk at 72°C in a
water bath for 20 sec then transferred immediately
to water bath heated at 42°C for 1 hr. For obtaining
the gelatinous materials of the curd, rennin enzyme
was added 1ml/L milk and then incubated at 42°C
for 3 hr. For obtaining the whey, cheesecloth was
used for the filtration process of the gelatinous
materials of the curd.
Experimental design:
This experiment was conducted during
2017/2018 and 2018/2019 as complete randomized
blocks design. The treatments were spraying fresh
milk and whey, each with two dilutions (40 and
50%) compared to the fungicide Topas-100 EC at
the ratio 0.25ml/L and untreated control, in a
greenhouse (9 x 40 m). To determine the best
number of sprays on controlling cucumber powdery
mildew, three replicates were sprayed with each
dilution of each tested substance, viz. two sprays,
three sprays and four sprays treatments with one
week interval. Foliar spray was carried out when the
natural infection by powdery mildew was appeared
on the first five true expanded leaves. Control
treatment and fungicide Topas 100 EC at ratio 0.25
ml/L. were used for comparison. For studying the
effect of number of sprays on disease development,
the final disease severity percentage, efficacy %,
area under disease progress curve (AUDPC) and r-
value were estimated.
Disease assessment:
Seven days after the appearance of powdery
mildew symptoms, disease severity was assessed
five times during the experiment, zero time, 7, 14,
21 and 28 days. The disease severity (percentage of
the leaf surface covered with powdery mildew
symptoms) was evaluated based on the scale of 0 to
6 according to Yan, et al., 2006, and calculated
according to the equation described by Descalzo et
al. (1990) as follow:
R = [∑ (a × b)/N × K] × 100.
Where: R =disease severity %, a =number of
infected leaves rated, b = numerical value of
each grade, N =total number of examined plants,
K = the highest degree of infection in the scale.

Egyptian Journal of Phytopathology, Vol. 48 (2020) 60
The efficacy of the treatments was calculated
according to the following formula:
Efficacy % =
DS% control – DS% treatment
× 100
DS% control
The method described by Pandey et al. (1989) was
used for calculation area under disease progress
curve (AUDPC) as follow:
AUDPC =
D [1/2 (Y1 +YK) +Y2 + Y3 +….+Y (K-1)]
Where: D = days between each successive two
readings, Y1 = first disease record. Yk = last
disease record.
r-value =
1
(loge
X2
- Loge
X1
)
t2 - t1
1 - X2
1 - X1
Where:
X1 = Disease severity (%) at t1 date.
X2 = Disease severity (%) at t2 date.
t2-t1= Time interval in days between two
observations.
Growth and yield parameters:
Plant height, chlorophyll content, fresh and dry
weights were also estimated (Data are the average
of 2017/2018 and 2018/2019). Plant height was
taken from the base of cucumber plants to the top
by centimeter unit, while chlorophyll content was
measured by portable leaf chlorophyll meter
(Minolta SPAD-502, Japan) to determine the
greenness or relative content of leaves to analyze
the photosynthetic pigments according to the
method of Torres-Netto et al. (2005) in treated and
untreated fully expanded cucumber leaves. Yield
parameters such as number of fruits/plant, mean
weight fruit (g), weight of fruits/plant (kg) and
mean product/ greenhouse (ton) were investigated,
(Data are the average of 2017/2018 and 2018/2019).
Enzymes activity and proline:
For the three enzyme assays and amino acid
proline, 0.5 g of fresh treated cucumber leaf was
homogenized at 0–4ºC in 3 mL of 50mM TRIS
buffer (pH 7.8), containing 1mM EDTA-Na2 and
7.5% polyvinylpyrrolidone. The homogenates were
centrifuged (12,000 rpm, 20 min, 4ºC), the enzymes
activity was determined spectrophotometrically.
The measurements were carried out at 25ºC, using
the model UV-160A spectrophotometer. The
activity of catalase (CAT) was measured according
to Aebi (1984). Peroxidase (POX) activity was
measured as described by Hammerschmidt et al.
(1982). Polyphenoloxidase (PPO) activity was
measured according to Malik and Singh (1980).
Proline was measured as described by Bates et al.
(1973).
Microscopic observations:
Light microscope (Leica DM1000) examination
was used to study the mode of milk and whey action
on conidiophores and conidia of S. fuliginea on
naturally infected cucumber leaves with powdery
mildew sampled at 24 h after the tested treating with
substances every week (5 times). Photographing by
microscope was done at the Agricultural Botany
Dept., Faculty of Agriculture, Kafrelsheikh
University, Egypt.
For scanning electron microscope (SEM),
cucumber leaves bearing lesions of powdery
mildew (with and without treatment) were
processed according to Harley and Fergusen
(1990). Tissue pieces of ~4 mm2 were fixed in 3%
glutaraldehyde in 0.2 M phosphate buffer (pH 7.2)
for 24 h at 4°C, followed by exposure to osmium
tetraoxide (1% OsO4) for 1 h at 25°C. Samples were
dehydrated by passing through ascending
concentrations of acetone and dried till the critical
point. Samples were sputter-coated with gold. The
examination and photographing were done using a
Jeol Scanning Electron Microscope (JSM-T.330 A)
in the Central Laboratory, Faculty of Agriculture,
Mansoura University, Egypt.
Statistical analysis:
All experiments were designed with complete
randomized block design. WASP software (Web
Agriculture Stat Package) was used for analysis of
variance (ANOVA). The comparisons of means
were determined by Duncan’s multiple range tests,
at P≤0.05 (Gomez and Gomez, 1984).
RESULTS
Nowadays, the major ways of modern, safe and
sustainable in agriculture depend on eco-friendly
disease management strategies. Naturally infected
cucumber plants by powdery mildew were foliar
sprayed two, three and four times with natural
compounds such as raw milk and whey in dilution
of 40 and 50 % compared to fungicide and control
treatment. The obtained results indicated that whey
and milk could be used as a curative treatment, as a
result of their antifungal activities observed in these
products.

Kamel and Afifi 61
1- Effect of whey on controlling cucumber
powdery mildew:
The present results show that 50% dilution of
whey was very effective than 40% in reducing the
disease severity percentages of cucumber powdery
mildew during 2017/2018 (Fig. 1). The number of
sprays was a very effective factor, as shown in Fig.
(1) which indicates that spraying cucumber plants
for four times with 50% whey was more effective
than 2 and 3 sprays, where the recorded disease
severity values were 21.7, 14.3 and 7.0 % for the
tested treatments, i.e., 2, 3 and 4 sprays,
respectively compared to fungicide treatment which
recorded 17.7, 11.3 and 4.3 %, respectively.
The experiment was repeated in 2018/2019 and
the obtained results confirmed that diluted whey at
50% was more effective than diluted whey at 40%
in reducing disease severity percentages (Fig. 2). In
this respect, the number of sprays was a very
effective factor in reducing infection with powdery
mildew as shown in Fig. (2) where, four sprays
treatment in case of diluted whey at 50% was
effective in reducing disease severity% than
treatments of two and three sprays, being 19.3, 13.3
and 7.3 %, respectively, compared to fungicide
treatment which recorded 16.3, 11.0 and 5.3 %,
respectively.
Figure (1): Effect of diluted whey at 40 and 50 % and number of sprays (2, 3 and 4) on disease
severity percentages during 2017/2018.
Figure (2): Effect of diluted whey at 40 and 50 % and number of sprays (2, 3 and 4) on disease severity
percentages during 2018/2019.
The effect of diluted whey at 40 and 50 % with
2, 3 and 4 sprays treatments against powdery
mildew pathogen was evaluated. In this respect, the
number of sprays affected positively the final
disease severity percentage, efficacy %, area under
disease progress curve (AUDPC) and r-value
(Table, 1). The recorded final disease severity
percentages due to using the effectively diluted
whey at 50 % with two, three and four sprays were
21.7, 14.3 and 7.0 %, respectively, compared to the
fungicide treatment which recorded 17.7, 11.3 and
4.3 %, respectively, at the same conditions during

Egyptian Journal of Phytopathology, Vol. 48 (2020) 62
2017/2018. Similar results were obtained during
2018/2019 (Table 1). Consequently, the recorded
efficacy percentages were significantly higher with
using diluted whey (50 %) for 4 sprays treatment.
Also, AUDPC values and r-value were
significantly reduced by spraying cucumber plants
four times with whey 50% (Table 1).
Table (1): Effect of whey on some disease parameters of cucumber powdery mildew caused by S.
fuliginea during 2017/18 and 2018/19.
Treatment
No. of
sprays
Final disease
severity (%)
Efficacy (%)
AUDPC
r-value
2017/18
2018/19
2017/18
2018/19
2017/18
2018/19
2017/18
2018/19
Control
-
45.3 a
41.3 a
-
-
1023.a
933.1a
0.022a
0.021a
Whey
40%
2
21.3 b
20.3 b
52.9
50.8
612.2b
604.5b
-0.015b
-0.015b
3
16.7 c
14.7 cd
63.1
64.4
578.2bc
543.9c
-0.023c
-0.029de
4
9.4 f
8.3 f
79.2
79.9
510.7de
479.9de
-0.043e
-0.046g
Whey
50%
2
21.7 b
19.3 b
52.1
53.3
616.7b
604.5b
-0.010b
-0.019bc
3
14.3 d
13.3 d
68.4
67.8
540.1cd
546.0c
-0.031d
-0.031e
4
7.0 g
7.3 f
84.5
82.3
474.3e
490.7d
-0.057f
-0.052h
Topas 100
fungicide
2
17.7 c
16.3 c
60.9
60.5
554.1cd
530.6c
-0.022c
-0.024cd
3
11.3 e
11.0 e
75.1
73.4
472.2e
484.1d
-0.038e
-0.037f
4
4.3 h
5.3 g
90.5
87.2
425.9f
448.7e
-0.075g
-0.064i
Values in the same column followed by the same letter are not significantly differed at P<0.05 level.
2- Effect of cow milk on controlling cucumber
powdery mildew:
Data in Fig. (3) indicate that diluted cow milk
at 40, 50% was more effective in inhibiting and
reducing the severity of cucumber powdery
mildew when the plants were sprayed weekly as
mentioned before. In this respect, four sprays
treatment with cow milk 50% achieved the lowest
disease severity (5.7%) when it was applied every
week followed by treatments sprayed 3 and 2
times, which recorded 11.7 and 17.3% disease
severity, respectively. Topas fungicide treatment
recorded the lowest percentage of disease severity,
where it recorded 4.3%. On the other hand, the
recorded disease severity % in control treatment
was 43.7% at 28 days of application.
Also, results indicate that cow milk 50% was
very effective than 40% in reducing the disease
severity percentages of cucumber powdery mildew
during 2017/2018 (Fig. 3).
It is clear from the obtained results (Fig, 3) that
the number of sprays was a very effective factor in
controlling powdery mildew on cucumber plants
where spraying plants 4 times with cow milk 50%
and fungicide treatment were more effective than
two and three sprays treatments in reducing disease
severity percentage compared to control treatment.
The trial was repeated during 2018/2019 and
the obtained results showed the same trend
obtained during 2017/2018 to confirm that milk in
dilution 50% was very effective than 40% in
reducing the disease severity percentages (Fig. 4).
Also, the number of sprays was a highly effective
factor and four sprays with cow milk 50% and
fungicide treatment were more effective than 2 and
3 sprays in reducing the cucumber powdery
mildew disease severity compared to control
treatment.
Data presented in Table (2) show the effect of
number of sprays, two, three and four using cow
milk 40 and 50 % against cucumber powdery
mildew. In this respect, all values of final disease
severity percentage, efficacy %, area under disease
progress curve (AUDPC) and r-value were affected
by the number of sprays. However, all the three
tested sprays treatments reduced final disease
severity, area under the disease progress curve
(AUDPC) and r-value of cucumber powdery
mildew compared to control treatment. In contrast,
four sprays treatment achieved the highest
efficacy % of controlling the disease on sprayed
cucumber plants with cow milk 40 and 50%. Also,
spraying cucumber plants with cow milk 40 and
50% showed the highest reduction of AUDPC
(499.3) and (460.9) during 2017/2018 and
2018/2019 and (432.7) and (427.0) AUDPC,
during 2017/ 2018 and 2018/2019, respectively,
followed by using three and two sprays treatment,
compared to control treatment which recorded
1011.8 and 932.1 for AUDPC, respectively, during
2017/2018 and 2018/2019. However, Topas
fungicide recorded the highest reduction of

Kamel and Afifi 63
AUDPC (409.2 and 420 AUDPC, respectively,
during 2017/2018 and 2018/2019) when the plants
were sprayed four times. Also, the highest
reduction of r-value was recorded when cucumber
plants were sprayed 4 times.
Figure (3): Effect of diluted cow milk at 40 and 50 % and number of sprays (2, 3 and 4) on disease
severity percentages at zero time, 7, 14, 21 and 28 days during 2017/2018.
Figure (4). Effect of diluted cow milk at 40 and 50 % and number of sprays (2, 3 and 4) on disease
severity percentages at zero time, 7, 14, 21 and 28 days during 2018/2019.
Table (2): Effect of cow milk on some disease parameters of cucumber powdery mildew caused by S.
fuliginea during 2017/18 and 2018/19.
Treatment
No. of
sprays
Final disease
severity (%)
Efficacy (%)
AUDPC
r-value
2017/18
2018/19
2017/18
2018/19
2017/18
2018/19
2017/18
2018/19
Control
-
43.7 a
40.0 a
-
-
1011.8a
932.1a
0.019a
0.017a
Cow milk
40%
2
18.5 b
19.7 b
57.7
50.8
574.3b
568.4b
-0.017b
-0.014b
3
12.7 c
14.0 d
71.2
65.0
516.9cd
512.4d
-0.036de
-0.030cd
4
9.3 d
8.2 f
78.7
79.5
499.3de
460.9e
-0.050f
-0.049f
Cow milk
50%
2
17.3 b
16.0 c
60.4
60.0
567.8b
527.8c
-0.026c
-0.027c
3
11.7 c
12.7 d
73.2
68.3
525.0 c
513.1d
-0.043ef
-0.034de
4
5.7 e
5.0 g
86.9
87.5
432.7f
427.0e
-0.063g
-0.064g
Topas 100
fungicide
2
13.3 c
14.0 d
69.6
65.0
485.4e
521.5cd
-0.034d
-0.030cd
3
9.4 d
11.0 e
78.5
72.5
442.4f
473.5 e
-0.045f
-0.038e
4
4.3 e
4.0 h
90.2
90.0
409.2g
420.4f
-0.075h
-0.076h
- Values in the same column followed by the same letter are not significantly differed at P<0.5 level.

Egyptian Journal of Phytopathology, Vol. 48 (2020) 64
3- Effect of cow milk and whey on some growth
parameters of treated cucumber plants:
All of plant height, chlorophyll content, fresh
and dry weights were significantly affected as a
result of treating cucumber plants with cow milk
and whey 40 and 50 % three times compared to the
chemical fungicide and control treatment (Table
3). Plant height of cucumber was significantly
increased when cow milk and whey 40 and 50%
were sprayed four times comparing with control
treatment followed by those sprayed two and three
sprays (Table 3). Interestingly, spraying cow milk
and whey at 50% four times was the best treatment
in increasing chlorophyll content, fresh and dry
weights to be similar in their effects with those
treated with chemical fungicide followed by those
treated two and three sprays at the same dilution.
Table (3): Effect of cow milk and whey applications on some growth parameters of treated cucumber
plants, data are the average of 2017/18 and 2018/19.
Treatment
No. of
sprays
Plant height (cm)
Chlorophyll
(SPAD)
Fresh weight (g)
Dry weight (g)
Cow
milk
Whey
Cow
milk
Whey
Cow
milk
Whey
Cow
milk
Whey
Control
-
174.7f
170.3f
21.3e
19.8f
202.1g
199.3i
16.6g
15.9g
Dilution
40%
2
234.3e
227.4e
33.5d
31.9e
224.0f
221.9h
19.2f
18.7f
3
240.2de
235.5d
34.3cd
31.7e
229.8d
228.1f
21.8de
20.2e
4
250.1bc
242.3bc
36.2ab
33.5bcd
233.5c
230.7e
24.8c
22.9d
Dilution
50%
2
236.8e
226.5e
34.2cd
32.7cde
226.4e
223.8g
21.1e
19.4ef
3
244.9cd
236.3d
35.4bc
33.1bcd
229.9d
227.2f
22.7d
20.0e
4
256.0ab
245.5b
37.4a
34.3ab
238.4a
234.4c
27.1b
26.7b
Topas 100
fungicide
2
236.0e
237.1d
34.3cd
32.0de
231.7cd
231.7d
23.9c
24.3c
3
245.0cd
241.4c
35.4bc
33.7bc
235.3b
236.2b
26.7b
26.8b
4
259.0a
257.8a
35.5bc
35.7a
239.2a
240.1a
30.6a
31.2a
- Values in the same column followed by the same letter are not significantly differed at P<0.05 level.
4- Effect of cow milk and whey on some yield
parameters of treated cucumber plants:
Data presented in Table (4) show that all yield
parameters such as number of fruits/plant, mean
weight fruit (g), the weight of fruits/plant (kg) and
mean product/ greenhouse (ton) were investigated.
Cow milk, whey at 40 or 50% dilutions and
fungicide using any number of sprays significantly
affected the cucumber yield components compared
to control treatment. No significant differences
were found between the chemical fungicide and
cow milk at 50% when sprayed four times on mean
product/ greenhouse (ton), being 2.89 and 2.78 ton,
respectively, followed by whey at the same
dilution.
Table (4): Effect of cow milk and whey on some yield parameters of treated cucumber plants, data
are the average of 2017/18 and 2018/19.
Treatment
No. of
sprays
No. of fruit/plant
Mean weight fruit
(g)
Weight of fruit/
plant (kg)
Mean product/
greenhouse
(ton)
Cow
milk
Whey
Cow
milk
Whey
Cow
milk
Whey
Cow
milk
Whey
Control
-
21.3f
20.8f
77.3c
77.4c
1.65h
1.61f
1.32f
1.29e
Dilution
40%
2
32.7e
30.3e
80.2b
80.2b
2.62g
2.43e
2.10de
1.98d
3
36.3d
34.4d
80.3ab
81.4ab
2.91e
2.80d
2.33de
2.24c
4
42.3b
39.7bc
81.2ab
81,2ab
3.43b
3.22bc
2.74ab
2.58b
Dilution
50%
2
33.7e
31.2e
81.7ab
80.9ab
2.75f
2.52e
2.20e
2.02d
3
37.3d
34.3d
80.7ab
82.0a
3.01d
2.81d
2.41cd
2.24c
4
42.7ab
40.0b
81.2ab
81.5ab
3.47b
3.26b
2.78a
2.61b
Topas 100
fungicide
2
34.3e
34.2d
80.2b
80.3b
2.75f
2.75d
2.20e
2.20c
3
39.2c
38.2c
81.5ab
81.4ab
3.20c
3.11c
2.56bc
2.49b
4
44.3a
43.8a
81.8a
82.0a
3.62a
3.59a
2.89a
2.87a
- Values in the same column followed by the same letter are not significantly differed at P<0.05 level.

Kamel and Afifi 65
5- Effect of cow milk and whey on activities of
defense related enzymes in treated cucumber
plants:
Powdery mildew is one of the biotic stresses
affecting plant parameters and plant productivity,
where, it causes an increase of free radicals in a
plant which cause plant acquisition self-defence
through antioxidant enzymes (CAT, POX. and
PPO). Data presented in Fig. (5) show that cow
milk and whey have a vital role in controlling
powdery mildew where they activated main plant
self-defenses through antioxidant enzymes.
Dilutions of both cow milk and whey 50% and
using them four times recorded the highest
activities of defence-related enzymes, i.e., catalase,
peroxidase and polyphenoloxidase in addition to
the amino acid proline. In this respect, spraying
cow milk at 50% four times caused the highest
activities of catalase enzyme (32.9 mMH2O2 g-1
FW Min-1) comparing with control and other
treatments. Also, application whey 50% four times
was more effective in increasing the activities of
catalase to be 28.8 mM H2O2 g-1 FW Min-1 than
other treatments and control. Also, cow milk and
whey at 50% increased the activities of peroxidase
more than other treatments and control, being
1.202 and 1.121 mMH2O2 g-1 FW Min-1,
respectively, when cucumber plants were sprayed
four times. Also, spraying 50% dilution of cow
milk or whey was more effective dilution in
increasing the activities of polyphenoloxidase
enzyme than the other dilution (40%) and other
treatments, being 0.109 and 0.100 µ mol/min-1 g-1
(FW) with milk and whey, respectively.
On the other hand, there was a clear increment
in determining activities of proline as a related
defense amino acid as a result of spraying
cucumber plants with cow milk or whey 50%. The
recorded activity values were 0.173 and 0.167 µ
mol/min-1 g-1 (FW), respectively.
The lowest activity of the CAT, POX and PPO
enzymes as well as proline amino acid was
obtained when the fungicide Topas was applied in
compare with the other tested treatments.
6- Microscopic observations on S. fuliginea,
the causal agent of cucumber powdery mildew:
Light microscope examinations show that cow
milk and whey at 50% affected the size and shape as
well as caused shrinking of treated hyphae,
conidiophores and conidia of S. fuliginea on naturally
infected cucumber leaves with powdery mildew at 24
h post-treatment with four sprays (Fig. 6). Also,
scanning electron microscope examination of the
fungal structures taken from cucumber leaf surfaces
treated with milk as well as whey at 50 % dilution
showed abnormalities and alterations in the conidia,
conidiophores and hyphae of S. fuliginea compared
to control treatment (Fig. 7). Moreover, twisting and
plasmolysis of hyphae and conidiophores, conidial
shrinking and collapsing were also observed (Fig.7.
B and C).
DISCUSSION
Through the dairy industry, elimination of whey
which is a by-product of fresh milk processing is
very necessary because it could be an
environmental problem. Control of powdery
mildew diseases infecting various crops through
cheaper and safe alternatives to fungicides such as
whey of fresh milk was initiated in 1999 (Bettiol,
2001). So, the main target of this research is to test
both cow milk and whey by two dilutions (40 and
50%) with two, three and four sprays treatments for
controlling powdery mildew in cucumber plants.
The obtained results indicated that spraying
cucumber plants infected with powdery mildew,
with cow milk and whey dilutions 40 and 50% four
times were able to reduce disease severity
percentage. Also, different dilutions of cow milk,
as well as whey, gave a good reduction in powdery
mildew area under disease progress curve, efficacy
percentage and r-value through 2017/2018 and
2018/2019. These results are in agreement with
Bettiol (2001) who reported that powdery mildew
of squash could be controlled through the
suppression of disease by using whey dilutions.
Also, Crisp et al. (2005 and 2006) found that
disease severity of powdery mildew of grapevine
could be reduced through whey application. This
may be due to its contents which consist of oil, fatty
acids, proteins, sugar, salts, enzymes, vitamins and
minerals (Stadnik and Bettiol, 2001). Also,
Medeiros et al. (2006) reported that application of
milk as foliar spray was reported to be participated
with increasing of beneficial bacterial populations
which may help in suppression process of the
pathogen causing powdery mildew on cucumber
and zucchini. In addition, Modler et al. (1998) and
Ravensberg et al. (2006) observed that bovine milk
was very effective in the control process of
powdery mildew in tomato, rose, sweet pepper and
cucumber because of lactoperoxidase, which
known as an important factor in the anti-microbial
system.

Egyptian Journal of Phytopathology, Vol. 48 (2020) 66
Fig. (5): Effect of cow milk and whey on activities of defense related enzymes in treated and untreated
cucumber plants, A= catalase, B= Peroxidase and C= Polyphenoloxidase activity and D=
Amino acid proline.

Kamel and Afifi 67
Figure (6): Light microscope observations of hyphae, conidiophores and conidia of S. fuliginea
collected from cucumber leaf surface sprayed with cow milk or whey 50 % showing, A:
Untreated control with normal conidia and conidiophores (black arrows). B: cow milk 50
%and C: whey 50 %, showing twisting of hyphae and conidiophores, conidial shrinking and
collapsing (black arrows), (400X).
Figure (7): Scanning electron microscope observations of hyphae, conidiophores and conidia of S.
fuliginea taken from cucumber leaf surface sprayed with cow milk or whey 50 % showing,
A: Untreated control with normal conidia and conidiophores (white arrows). B: cow milk
50 %and C: whey 50 %, showing twisting of hyphae and conidiophores, conidial shrinking
and collapsing (white arrows).
Another suggested active component of milk
and whey is lactoferrin which is responsible for
damage and collapse of U. necator conidia at 48 h
than at 24 h after exposure the majority of the
damage caused to hyphae after treatment with
milk, whey occurred within 24 h of application.
Also, lactoferrin, iron binding glycoprotein, binds
to the membranes of various bacteria and fungi,
causing damage to membranes and loss of
cytoplasmic fluids (Samaranayake et al., 2001).
Possibly, components of milk other than lactoferrin
also caused damage to the hyphae, or the
interaction with other components of milk that
increased the activity of lactoferrin against hyphae
(Crisp et al., 2003). Reduction of disease severity
of powdery mildew in grapevines was reported by
Tzeng and DeVay (1989) to be associated with the
production of free radicals when sulfur-rich amino
acids, riboflavin and methionine exposure to light.
The same action could be happened with milk or
whey solids when influenced by solar radiations
including UV and result in the production of free
radicals. So the timing of application (on bright,
warm days) is important factor to maximize the
production of free radicals than an application on
cooler and cloudy days. Crisp et al. (2006)
explained that milk and its components could
produce oxygen free radicals (ROS), which attacks

Egyptian Journal of Phytopathology, Vol. 48 (2020) 68
the pathogen directly in the infection sites during
natural sunlight. Production of ROS early during
the infection process could be used by plants as
weapons in defense responsive system, while
continuous production might lead to susceptibility
reaction and initiate of programming cell death
(Ketta, 2015). the different metabolic and
developmental processes that involve the
interaction between ROS and hormones when high
proportions ROS in plants include closing the
stomata (Neill et al., 2008), programmed cell death
(Bethke and Jones, 2001), control of apical root
organization (Jiang and Feldman, 2003) and
gaining tolerance to both biotic and abiotic stresses
(Miller et al., 2008). ROS is constantly
reduced/erased by plant-based antioxidant defence
systems that maintain it at certain stable levels
under stressful conditions (Tuteja et al., 2012).
The results indicated that the 50% dilution of
both cow milk and whey, when used four times,
recorded the highest activities of defence-related
antioxidant enzymes, i.e., catalase, peroxidase and
polyphenoloxidase which play an important role of
increasing defence of plant to infection by powdery
mildew. Various enzymatic antioxidants comprise
of superoxide dismutase, catalase, peroxidase and
polyphenoloxidase (Foyer and Noctor, 2003 and
Sarhan et al., 2020). Increased activities of many
antioxidant enzymes have been observed in plants
to combat oxidative stress induced by various
stresses and also to maintain cellular homeostasis
(Almeselmani et al., 2006). Maintenance of a high
antioxidant capacity to scavenge the toxic ROS has
been linked to an increase in tolerance of plants to
these diseases stress (Hasanuzzaman et al., 2013).
Also, our results showed that amino acid proline
showed noticeable increasing with four sprays of
cow milk and whey at a dilution 50%. This may be
attributed to milk and its derivatives content of
high proline which has a role in low deleterious on
plant. Proline is considered one of the self-defense
tools for plant when plant is under stress. Scano et
al. (2014) reported that the chemical composition
analysis done by GC-MS of cow milk revealed that
the aqueous fraction obtained by the extraction
procedure was rich in short-chain hydroxylated
carboxylic acids, such as lactic acid, succinic acid,
fumaric acid, malic acid, 2-hydroxyglutaric acid,
and gluconic acid; long-chain stearic and palmitic
acids were also found. Among free amino acids,
proline, serine, valine, glycine, alanine and
glutamine were detected.
Light and scanning electron microscope
examinations showed that cow milk and whey at
50% affected the size and shape as well as caused
shrinking of treated hyphae, conidiophores and
conidia of S. fuliginea collected from naturally
infected cucumber leaves with powdery mildew at
24 h post-treatment. These results are in harmony
with Kamel et al. (2017) who mentioned that
microscopic observations showed abnormal shape,
shrinking and collapsing of conidia with twisting
and plasmolysis of conidiophores and hyphae of
Sphaerotheca fuliginea when they were treated
with many dilutions of both raw milk and whey.
Also, Crisp et al. (2006) found that application of
fresh milk or whey on Uncinula necator, which
causes the powdery mildew of grapevine, caused
collapse of hyphae and conidia within 24 h post-
treatment. They hypothesized that the observed
collapse of hyphae and conidia was related to
increased production of free radicals. This may be
due to several milk properties such as chemical
and/or physical barrier could be responsible for
reducing the attachment of the fungus to the
cucumber leaf surfaces in addition to direct
suppression of the pathogen. Slightly basic pH of
milk could be related to the development
disturbance of the fungus.
CONCLUSION
The authors recommend using cow milk and
whey dilutions 40 and 50% under four sprays could
be promising tools as potential alternatives to
chemical fungicides against cucumber powdery
mildew because of their efficacy, safety and
environment eco-friendly in addition to, whey is
less expensive because it considered as a waste of
industrial milk process. In addition, they have a
vital role and significantly effective against
cucumber powdery mildew. So, we need more
studies to better understand the mechanisms of cow
milk and whey in controlling plant diseases.
ACKNOWLEDGEMENTS
The authors would like to thank Prof. Dr.
Hammad Ketta, Agric. Botany Dept., Plant
Pathology Branch, Faculty of Agriculture,
Kafrelsheikh University, for his critical review of
the manuscript.
COMPETING INTERESTS
The authors declare that they have no
competing interests.

Kamel and Afifi 69
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