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Abstract
In grade to obtain high quality and safety food along with the environment protection from pesticides hazards,
four bio agents were used to control fruit rot disease caused by Botrytis cinerea in strawberry fruits. Chlorella
vulgris, Spirulina platensis, Azotobacter chroococcum, Trichoderma harizianum and their combinations (T1, T2, T3,
T4, T12) were used as spray treatment in the open field and on strawberry after picking. The individual bio agents
treatments decreased liner growth and spore production of B. cinerea in comparing with mixed bio agents. Also, all
the treatments decreased Disease Severity% (DS%). The treatments of T1, T2, T3 and T4 caused 13.9, 22.3, 22.3
and 24.1 DS% during 2014; 12.26, 15.5, 17.0 and 21.86 DS% during 2015 in comparing with the mixed bio agents
(T12) which caused 29.3 and 29.03 DS% during 2014 and 2015, respect., after three sprayings in the field. Also,
the treatments of T1, T2, T3 and T4 caused 0.00 DS% after 2nd spray through the storage at 5C. The treatment of T12
caused the highest increase in the total sugar content in strawberry fruits which infected naturally with B. cinerea
and stored at 5C for three weeks increased with T12. In addition, the treatment of T12 caused the highest increase in
polyphenol oxidase (PPO) activity and peroxidase (PO) which were 0.459 and 0.360 U/mg fresh weight respect., in
comparing with T1, T2, T3 and T4 which caused 0.278, 0.287, 0.298, 0.313 and 0.139, 0.202, 0.276, 0.302 U/mg fresh
weight respect., after storage for 6 weeks. The treatment of T1 gave the best result in K% in leaves (1.82). T5 gave
the best value of N% in soil (72.2%), T3 gave the best one in P% in soil (36.0%) and T4 caused the best amount of
K% (16.0) in the soil.
Red and brown algae are mainly used as human healthy food
sources, due to their high concentration in polysaccharides, natural
richness in minerals, polyunsaturated fatty acids and vitamins.
Macroalgae (seaweeds) are rich sources of structurally new and
biologically active metabolites. In recent years, there have been many
reports of macro algae derived compounds that have a broad range of
biological activities, such as antibacterial, antiviral, antioxidant, antiinflammatory, cytotoxic and antimitotic activities Special attention has
been reported for antiviral, antibacterial and/or antifungal activities
against human pathogens and biostimulant properties of seaweeds are
explored for use in agriculture [4-10]. Biochemical resistance through
the accumulation of various phenolic compounds and phytoalexins
and the activation of peroxidases, polyphenol oxidases and key
enzymes in the phenylpropanoid and isoflavonoid pathways may play
a crucial role in the biological control of and resistance to pathogenic
attack in plants. The defense strategy of plants consists of two stages.
The first stage is assumed to involve the rapid accumulation of phenols
at the infection site, which function to slow the growth rate of the
pathogen and to allow for the activation of secondary strategies that
will more thoroughly restrict the pathogen. The secondary responses
involve the activation of specific defense mechanisms, such as the
synthesis of molecules related to pathogen stress [11]. However, it is
still not easy and costly in application. It can serve as the best control
Citation: El-ghanam AA, Farfour SA, Ragab SS (2015) Bio-Suppression of Strawberry Fruit Rot Disease Caused by Botrytis cinerea. J Plant Pathol
Microbiol S3: 005. doi:10.4172/2157-7471.S3-005
Page 2 of 7
Where:
dc-dt
100
dc
Treatments
Four bio agents i.e., Chlorella vulgris, Spirulina platensis, Azotobacter
chroococcum, Trichoderma harizianum and their combinations were
applied in 12 treatments (from T1 to T2) in comparing with chemical
control (T13) and untreated control (T14) as shown in Table 1.
Effect of tested bio agents on Botrytis cinerea spore production:
After determining linear growth of the pathogen, spores were collected
and transferred to sterilized water (50 ml/treatment). Spores were then
counted by using haemocytometer and spore concentration/ml was
determined according to the following equation:
Spore conc.=total spores count four squares 2500 dilution factor.
Filed experiment: Experiment was carried out at El-Tahreer city
regions. Healthy strawberry transplants cv. Festival were used for fresh
planting system. Treatments were mentioned in Table 1: (T1, T2, T3,
T4, T5, T6 T7, T8, T9, T10, T11, T12 T13, T14) and sprayed in January, 2014
and 2015 three times a week interval with four replicates; each replicate
contained 50 plants. The percentage of fruit rot severity caused by B.
cinera was estimated by the equation adopted by Hanounik.
Disease severity ( DS) %=
Where:
(NPCCR) 100
NIPMSC
Citation: El-ghanam AA, Farfour SA, Ragab SS (2015) Bio-Suppression of Strawberry Fruit Rot Disease Caused by Botrytis cinerea. J Plant Pathol
Microbiol S3: 005. doi:10.4172/2157-7471.S3-005
Page 3 of 7
Treatment
Codeessese
Chlorella vulgris
T1
Spirulina platensis
T2
Azotobacter chroococcum
T3
Trichoderma harizianum
T4
T5
Trichoderma harizianum+Spirulinaplatensis
T6
T7
T8
Azotobacter chroococcum+Spirulinaplatensis
T9
Azotobacterchroococcum+Chlorellavulgris+Trichoderma
harizianum
T10
T11
Azotobacter chroococcum+Chlorellavulgris+Spirulina
platensis+Trichodermaharizianum
T12
T13
Untreated control
T14
Table 1: The experimental treatments codes.
with all the tested bio agents (Chlorella vulgaris, Spirulina platensis,
Azotobacter chroococcum and Trichoderma harizianum) alone or
as mixture. Regardless of the sequence of application rates differed
according to the potency of the tested bio agents and application
sequence.
The highest reduction in sporulation of B. cinerea were obtained
with T1 treatment (2.2%), compared with those of the other tested bio
agents, followed by T3 and T4 (3.3%).
In this respect, cyanobacteria (blue-green algae) and eukaryotic
algae produce biologically active compounds that have antifungal
activity antibiotic and toxic activity ( Bonjouklian and Kiviranta
[27,28]. Also Hussien [29] evaluated the effect of culture filtrates of
nine algal strains at concentrations of 10, 20, 30, and 40% on mycelium
growth and spore production of Cercospora beticola causing leaf spot
disease in sugar beet. Generally, they found that all the algal culture
filtrates reduced the fungal mycelium growth, but the best results
were obtained by Spirulina platensis, Oscillatoria sp. and Nostoc
muscorum; the highest fungal mycelium growth inhibition percentage
was achieved by the concentrations of 30% (100, 100, and 82%,
respectively.) and at 40% (100, 100, and 100%, respectively). Fungal
spore production (number of spores) was completely inhibited by the
previous three algal cultural filtrates particularly at the concentration
of 40%. They conclude that the antifungal activity of the algal culture
filtrates has been attributed to the presence of bioactive compounds,
that is, total phenolic compounds, total saponins, and alkaloids in the
algal culture filtrates. Also, several workers reported that the extracts
of Nostoc muscorum significantly inhibited the growth of Candida
albicans and Sclerotinia sclerotiorum and [30] aBis (2, 3-dibromo-4,
5-dihydroxybenzyl) ether (BDDE) is a bromophenol isolated from
marine algae which possesses cytotoxic and antibacterial activity.
BDDE inhibits the growth of B. cinerea cultured on a solid medium
Bio agents
N%
P%
K%
Chlorella vulgaris
1.62
0.39
3.96
Spirulina platensis
1.17
0.19
2.09
Azotobacter chroococcum
3.2
0.28
2.25
Spores production
x103
T1
58.3
37.5
2.2
T2
50.1
45.0
4.5
T3
61.1
35.0
3.3
T4
55.5
40.0
3.3
T5
25.0
67.5
5.5
T6
23.3
72.5
5.5
T7
22.2
70.0
6.1
T8
20.5
71.5
6.2
T9
25.0
67.5
5.5
T10
30.5
62.5
13.6
T11
23.3
72.5
14.0
T12
19.4
74.0
14.9
Switch (2200
ppm)
100.0
0.0
0.0
Untreated
Control t
0.0
90.0
100
L.S.D at 5%
0.23
0.60
Table 3: The effect of different bio agents on the liner growth and spores production
of B. cinerea.
Citation: El-ghanam AA, Farfour SA, Ragab SS (2015) Bio-Suppression of Strawberry Fruit Rot Disease Caused by Botrytis cinerea. J Plant Pathol
Microbiol S3: 005. doi:10.4172/2157-7471.S3-005
Page 4 of 7
Treatment
1st spray*
2nd spray**
3rd spray**
4th spray**
T1
0.3
0.0
0.0
0.0
T2
0.6
0.0
0.0
0.0
T3
0.3
0.0
0.0
0.0
T4
1.6
0.0
0.0
0.0
T5
24.0
20.0
9.0
3.0
T6
22.3
14.3
8.6
2.6
T7
27.3
13.0
7.6
3.3
T8
19.9
12.6
9.0
6.6
T9
19.3
13.6
6.7
2.6
T10
36.6
20.8
13.0
8.2
T11
33.6
19.7
13.5
7.0
T12
40.3
25.0
20
9.0
Switch (T13)
0.0
0.0
0.0
0.0
Untreated control
(T14)
100
100
100
100
L.S.D at 5%
0.48
0.47
0.47
0.46
* In the Lab.
**In the field.
Table 4: Effect of some bio agents on disease severity percentage of strawberry
fruits after storage for six weeks at 5C.
Disease severity
Treatment
2014
2015
1st spray
2nd
spray
3rd
1st
2nd
3rd
Mean
spray
spray spray Spray
T1
19.6
13.3
9.0
13.9
17.5
12.3
7.0
12.2
T2
21.0
18.0
11.6
22.3
20.0
17.0
9.6
15.5
T3
32.0
19.0
16.0
22.3
19.0
18.0
14.0
17.0
T4
35.3
20.3
17.0
24.1
30.3
20.3
15.0
21.8
T5
38.3
23.6
20.0
27.3
38.3
22.6
19.0
26.6
T6
28.3
32.6
16.3
25.7
26.3
30.6
15.3
24.6
T7
23.8
23.0
15.3
20.0
22.6
20.0
14.3
18.9
T8
29.0
27.0
20.3
25.4
28.0
26.6
19.3
24.0
T9
23.3
22.0
17.3
20.8
22.6
21.0
16.3
19.9
T10
35.3
23.6
20.6
26.4
18.3
15.6
12.6
15.4
T11
40.3
29.0
20.6
29.9
39.6
28.0
18.3
28.6
T12
41.6
27.0
19.3
29.3
40.0
26.6
20.5
29.0
Switch (T13)
13.6
8.6
5.3
9.16
13.6
7.6
4.3
8.5
Untreated
control (T14)
55.7
53.3
54.0
54.3
56.o
53.3
50.0
53.1
L.S.D at 5%
0.58
0.92
1.29
0.60
0.95
1.30
Mean
Citation: El-ghanam AA, Farfour SA, Ragab SS (2015) Bio-Suppression of Strawberry Fruit Rot Disease Caused by Botrytis cinerea. J Plant Pathol
Microbiol S3: 005. doi:10.4172/2157-7471.S3-005
Page 5 of 7
Sugar content (mg/g fresh weight of fruit)
Treatment
1 week
2 weeks
3 weeks
NR
NR
NR
T1
73.1
11.6
84.7
83.5
11.7
95.2
93.2
11.7
210.2
T2
88.3
11.7
100
93.5
21.4
114.9
93.6
26.6
120.2
T3
86.3
12.1
98.4
92.5
20.4
112.9
92.6
25.5
118.1
T4
86.3
12.1
98.4
92.3
27.3
119.6
104.2
22.5
126.4
T5
93.2
22.2
115.4
98.4
27.3
125.7
100.1
27.3
127.4
T6
93.1
21.3
114.4
98.1
31.2
129.3
104
27.2
131.2
T7
94.4
22.5
116.9
99.3
31.2
130.5
108
32.4
140.4
T8
98.2
27.0
125.2
102.1
46.5
148.6
109
46.4
155.4
T9
99.3
27.6
126.9
104.0
51.5
155.5
118.5
47.2
165.7
T10
102.1
31.2
133.3
116.4
67.4
183.4
128.1
67.4
195.5
T11
109.8
50.4
160.2
114.9
77.3
192.2
134.8
82.6
217.4
T12
118.7
62.3
181
120.2
83.2
203.4
138.7
95.6
234.3
Switch(T13)
73.1
11.6
84.7
78.7
21.2
99.9
93.1
11.7
104.8
Untreated control(T14)
128.1
64.3
192.4
130.3
85.2
215.5
140.3
97.5
237.8
L.S.D at 5%
0.49
0.54
0.60
0.49
0.59`
0.44
0.57
0.4
0.59
2nd spray
3rd spray
1st spray
2nd spray
3rd spray
T1
0. 278
0.309
0.320
0. 139
0. 231
0. 267
T2
0. 287
0.310
0.325
0. 202
0. 305
0. 325
T3
0. 298
0.314
0.330
0. 276
0. 312
0.368
T4
0. 313
0.330
0.350
0. 302
0. 313
0.370
T5
0. 325
0.452
0.520
0. 304
0. 345
0.375
T6
0.367
0.509
0.624
0.314
0. 391
0.400
T7
0. 375
0.596
0.600
0. 326
0.401
0.435
T8
0.329
0.620
0.630
0. 335
0. 423
0.450
T9
0. 363
0.724
0.730
0. 345
0. 444
0.455
T10
0.379
0.858
0.860
0.350
0. 445
0.456
T11
0. 380
0.870
0.880
0. 358
0.488
0.470
T12
0. 459
0.897
0.910
0.360
0.490
0.475
Switch(T13)
0. 220
0.310
0.310
0.150
0. 228
0.270
Untreated control(T14)l
0. 410
0.911
0.920
0.360
0.495
0.500
L.S.D at 5%
0.012
0.003
0.82
0.45
0.04
0.29
Table 7: Effect of different bio agents on fruits Polyphenol oxidase and Peroxidase activities in treated strawberry fruits after storage six weeks at 5C.
bio agents treatment (T12) after storage periods 1, 2 and 3 weeks with
averages 118.7, 123.0 and 138.7 mg/g fresh weight of fruit, respectively.
Generally, reducing, non-reducing and total sugars increased sharply in
case of treatments T10, T11 and T12 compared with the other treatments
but the treatment (T14) had the highest values.
Early studies have reported that germination in most B. cinerea
isolates is dependend on the presence of nutrients, mainly sugars, and
that this process can be stimulated by exudates from mature fruits
and [39-41]. Fouri and Holzm showed that in B. cinerea fructose at
micro molar concentration was a more potent germination inducer
than glucose. Glucose, fructose and sucrose were the better inducers
of germination.
Data presented in Table 7 indicate that the enzyme activities
increased with treatments of mixed bio agents. The least activities of
Polyphenol oxidase and Peroxidase cleared with T1, T2, T3 and T4 after
spraying three times with different bio agents and stored for six weeks
under 5C. At the same time treatment with the fungicide (switch) led
to decrease in the enzymes activity, in contrast control treatment had
the highest values. Generally, mixing the bio agents resulted excess in
the activity for both enzymes compared to the treatment separately.
J Plant Pathol Microbiol
Citation: El-ghanam AA, Farfour SA, Ragab SS (2015) Bio-Suppression of Strawberry Fruit Rot Disease Caused by Botrytis cinerea. J Plant Pathol
Microbiol S3: 005. doi:10.4172/2157-7471.S3-005
Page 6 of 7
In Leaves of strawberry
In soil
N%
P%
K%
N%
P%
K%
T1
1.21
0.34
1. 82
27.7
15.0
112.0
T2
1.31
0.32
1. 79
37.8
20.0
120.0
T3
1.41
026
1. 77
36.1
36.0
120.0
T4
1.33
0.29
1. 54
34.4
10.0
160.0
T5
1.32
0.32
1. 51
72.2
8.0
128.0
T6
1.24
0.32
1.44
47.0
8.0
72.0
T7
1.17
0.30
1.69
42.0
10.0
88.0
T8
1.23
0.31
1. 62
61.3
9.0
96.0
T9
1.24
0.28
1.45
56.3
8.0
80.0
T10
1.30
0.35
1. 46
52.1
8.0
80.0
T11
1.32
0.29
1.47
60.5
9.0
88.0
T12
1.33
0.35
1.43
50.0
10.0
80.0
Switch(T13)
1.41
0.27
1.62
27.7
15.0
112.0
Untreated
control(T14)l
1.28
0.38
1.54
37.8
20.0
120.0
L.S.D at 5%
0.49
0.01
0.54
0.63
0.57
0.54
Citation: El-ghanam AA, Farfour SA, Ragab SS (2015) Bio-Suppression of Strawberry Fruit Rot Disease Caused by Botrytis cinerea. J Plant Pathol
Microbiol S3: 005. doi:10.4172/2157-7471.S3-005
Page 7 of 7
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