Effect of Some Algal Filtrates and Chemical Inducers On Root-Rot Incidence of Faba Bean

Research Article
Agri Res Tech Open Access J
Volume 1 Issue 1 - 2015
Copyright All rights are reserved by Abeer M Mousa
Effect of Some Algal Filtrates and

Chemical Inducers on Root-Rot
Incidence of faba Bean
Sahar A EL-sayed1 and Abeer M Mousa2*
1
Plant pathology institution, Agricultural Research Center, Egypt
Soil and Water Research Department, Nuclear Research Center, Atomic Energy Authority, Cairo, Egypt
Submission: September 17, 2015; Published: September 25, 2015

*Corresponding author: Abeer M Mousa, Lecturer, Department of Soil and Water Research, Nuclear Research Center, Atomic Energy Authority,
Egypt, Tel: +201001958616; Email:
Abstract
Some algal filtrates namely Sargassum, Fucus and Nostoc spp., and chemical inducers namely Dipotassium Phosphate (K2HPo4), Salicylic
acid and Ascorbic acid to investigate their effect on the control of faba bean root-rot caused by Rhizocptina solani, Fusarium solani and
Fusarium oxysporum in the laboratory and green house using the faba bean (Giza3 Mohassan). Results of in vitro studies showed that algal
filtrates or chemical inducers significantly inhibited the mycelial growth of the pathogen, when compared to the untreated control. Under
greenhouse conditions, all isolated fungi proved to be pathogenic and caused pre- and post emergence damping-off. Considerable increases
in activity of oxidative reductive enzymes (peroxidase and polyphenol oxidase) and chitinase enzyme were recorded in plants grown from
treated faba bean seeds.
Keywords: Algal filtrates; Chemical inducers; Root-rot; Chitinase; Peroxidase; Polyphenol oxidase
Introduction
Faba bean (Vicia faba L.) is a legume crop with high
nutritional value. It contains about 18.5 and 37.8 % protein [1].
Faba bean plants are reacted with many fungal pathogens which
cause considerable yield losses [2]. Fungi belonging to various
genera were isolated from infected plants. Rhizoctonia solani
was the most pathogen causing root-rot diseases [3-5] isolated
Rhizoctonia solani and Fusarium oxysporum from wilted and
rotten roots of faba bean in different parts of the world. AbdelKader [6] several root rot and wilt pathogen such as Rhizoctonia
solani, Fusarium oxysporum and Macrophomina phaseolina are
reported to attack faba bean roots and stem base causing serious
losses in seed germination and plant stand as well. However,
algal filtrates have been record as active inhibitors to the in vitro
fungal growth [7,8] found that all the tested algal filtrates except
Anabaena decreases the mycelial growth of S. Sclerotiorum as
compared with control. It may be possible to utilize a scheme
to inducible plant defenses which provides protection against a
broad spectrum of diseases causing organisms. Among synthetic
inducer salicylic acid (S.A.) and hydrogen peroxides (H2O2) have
been found to be active as antimicrobial agents in various trials
as diseases resistance inducers. Theses have been reported for
inducing resistance agents several plant pathogens such as F.
oxysporum in tomato [9]. El-ghanam [10] reported that, the
Agri Res Tech Open Access J 1(1): ARTOAJ.MS.ID.555552 (2015)
percentage of diseases severity of fruits rot decreased by spraying

strawberry fruits with all different algae in field and laboratory.
Abdel-Monaim [9] found that S.A. follow by H2O2 recorded the

lowest reduction of growth in all tested fungi. Several investigators
studied the effectiveness of these chemical inducers on root
rot disease [11]. The treatment sensitizes the plant to respond
rapidly after treatment. These responses include phytoalexin an
accumulation, phenol, lignifications and activation of peroxidase,
polyphenol oxidase and chitinase.
Therefore, the objectives of the present study were to study
the effect of algal filtrates and chemical inducer on root rot
diseases of faba bean, in order to increase the number of survival
plants.
Material and Methods
The present study dealt with the management of root rot

disease of faba bean under laboratory experiments and green
house conditions.
Source of inocula
The concerned disease caused by Rhizocptina solani,

Fusarium solani and Fusarium oxysporum pathogenic fungi
were obtained from infected roots of faba bean collected from
001
Agricultural Research & Technology: Open Access Journal

different locations throughout Egypt. The used isolates were
purified and identified following the methods adapted by Sneh et
al. [12]. Different experiments were carried out in a randomized
complete plot design with three replicates for each treatment
using the cv.Qiz93 Mohassan were obtained from the Field Crop
Institute, Agricultural Research Center, Ministry of Agriculture,
Egypt.
Laboratory experiments
Algal materials and filtrate: two seaweeds were collected

from Suez Canal (Egypt) at Depressor during June 2014. The
algae were collected and transported to the laboratory within 8
hours in a tank with sea water. The algal species were identified
based on the schemes reported in the literature [13-20].
Algal cultures were grown in enriched artificial or natural

seawater. Artificial seawater was based on the recipe by
Harrison et al. [21] as modified by Thompson et al. [22]. Natural
seawater was treated with activated charcoal (to remove organic
compounds), gravity filtrated through a Whatman 1 filter paper,
re filtrated through 0.8 m Millipore filter autoclaved and then
enriched with ES levels of nutrients trace metals and vitamins
[21]. Cultures were exposed to continuous light (provided by
Vita-lite fluorescent tubes). At 19 C and 35 mol photo ns-2S-1.
Two weeks later, the developed algal growth was filtrated under
aseptic conditions to obtain culture filtrates. Nostoc sp. was
kindly supplied by Botany and Microbiology Department-Faculty
of science (for girls) Al-Azhar University Cairo- Egypt.
Nostoc sp. were diluted with sterilized distilled water to make
1/10 dilution series. Ten serial dilutions were then carried out
with sterilized distilled water one ml of 10-2 to 10-6 dilutions were
poured on the surface of the agar medium and spread with glass
spatula, then incubated in inverted position at room temperature
for 3 weeks. Isolation was carried out using dissociating
microscopic and Nostoc colony was picked out using inoculation
needle for propagation, purification and identification. Slant agar
refrigerated Nostoc (at 5 C) was Exposed to light (500 Lux) for
2 days then inoculated to a liquid medium. Nostoc grew in 500
ml conical flasks containing 100 ml sterilized BGII medium
(MgSO47H2O, K2HPO43H2O, Cacl2 2H2O, Na2CO3, Citric acid) and
incubated under initiation (3000 Lux) at temperature 28-32
C. Two weeks later, the developed Nostoc growth was filtrated
under aseptic conditions to obtain culture filtrates.
Effect of some algal filtrates on mycelial growth of

pathogenic fungi
The crude filtrate of each alga, prepared as mentioned before

was individually mixed with PDA medium at the rate of 1 ml/gmL
media at 45 C and poured into plates [23]. Plates centers were
incubated at 20 C for 5 days. Algal filtrate free PDA plates were
used as control.
The effect of some chemical inducers on mycelial

growth of pathogenic fungi in vitro
The effect of different concentrations of three chemical

inducers, K2HPO4 (10, 25, and 50 mM), Salicylic acid (1, 2, 10
mM) and Ascorbic acid (1, 2, 10 mM) were added to PDA medium
002
and then poured in Petri dishes (Dimensions: (DxH) 94mm x

16mm) and inoculated in center with plates were incubated at 27
C for 7 days. The diameter of pathogen radial was calculated as
mentioned previously.
Green House Experiment
Effect of soil treatment with some algal filtrates on soil

treatment with some algal filtrates on damping-off of faba bean: -
Crude algal filtrates are individually added to the soil infested

with R. solani, F. solani and F. oxysporum isolates (30 ml/ Kg soil)
at the rate of 90 ml/pot (20-cm-diam). Seeds of faba bean were
sown at rate of faba bean were sown at the rate of faba bean were
sown at the rate of 10 seeds/pot (Giza 3 Mohassan). Soil treated
with water only was served as control. Three replicates were
used for each treatment.
Diseases assessment
Percentage of pre- and post emergence damping-off as well as

healthy survival plants in each treatment were determined15 and
30 days after sowing respectively using the formula according to
[24]
Pre emergence (%) =
Number of non germinated seeds 100

Total number of sown seeds
Post emergence (%) =
Survival plant (%) =
Number of dead seedling 100

Number of survival plant 100

The plant height of faba bean plants and disease severity

were also recorded after 35 days from sowing.
Effect of soaking faba bean seeds in some chemical

inducers agents on controlling root rot under green
house conditions
Faba bean seeds were soaked in the solution of each tested
chemical for 2.5 hrs [25] before planting. Each particular
treatment consisted of one concentration K2HPO4 (50 mM),
Salicylicacid10 mM and Ascorbic acid 10 mM. The welted seeds
were spread in the thin layer and left about 24 hours, then sown
in the infested potted soil with the virulent R. solani, F. solani and
F. oxysporum isolates. Seeds of Giza 3 Mohassan were soaked in
tap water and were sown in pots to serve as control. Three pots
for each treatment were used as replicates. Pre-post emergence
root rot and survival plant incidence were recorded after 15 and
30 days of sowing. The height of faba bean plants and disease
severity were also recorded after 35 days from sowing.
Fresh samples were taken from plants grown from previously
treated and untreated faba bean seeds and extracts were used for
assaying biochemical change associated with tested treatments
of algal and chemical inducers on the activities of peroxidase
enzyme [26] and polyphenol oxidase enzyme [27] and chitinase
enzyme [28] were determined.
Statistical analysis
The obtained data were statistically treated by analysis of
How to cite this article: EL-syed SA, Mousa AM. Production, Effect of Some Algal Filtrates and Chemical Inducers on Root-Rot Incidence of
faba Bean. Agri Res Tech Open Access J. 2015;1(1): 555552. DOI: 10.19080/ARTOAJ.2015.01.555552

Effect of some chemical inducers on mycelia growth of
fungi under laboratory condition
variance (ANOVA) using the fisher L.S.D. method. Means were

separated by Fishers protected least significant differences [29].
Results
The effect of the tested chemicals on mycelia growth of R.

solani, F. solani and F. oxysporum are shown in Table 2. Data
indicate that low concentrations of Salicylic acid (S.A.), Ascorbic
acid (A.A.) and K2HPO4 slightly reduced the linear growth of fungi.
The highest reduction in the linear growth was achieved when
All the tested algal filtrates significantly reduced the mycelia

growth of R. solani, F. solani and F. oxysporum as compared with
the control Table 1 filtrates of Sargassum, Fucus and Nostoc spp.
completely inhibited the mycelia growth of the fungi.
Table 1: Effect of some algal filtrates on mycelia growth of fungi.
Fungi
Algae
Rhizoctonia solani
Fusarium solani
Fusarium oxysporum
Radial growth
(mm) %
Reduction %
Radial growth (mm)

%
Reduction %
Radial growth (mm)

%
Reduction %
35.00
61.11
20.00
77.78
24.43
62.85
0.0
100.0
Sargassum
Fucus
0.0
Nostoc
Control
100.0
90.0
0.0
100.00
90.0
0.0
0.0
0.0
L.S.D. Data 0.01 %=treatment =0.20; Concentration = 0.26; T X C= 0.46
100.0
0.0
100.0
90.0
0.0
0.0
Table 2: Effect of three selected chemical on linear growth of pathogenic fungi grown on PDA medium.
Rhizoctonia solani
Fungi
K2HPO4
Inhibition %
Linear growth
(mm)
Inhibition %
Linear
growth (mm)
Inhibition %
10 mM
25 mM
50 mM
40.00
25.00
0.0
45.55
62.22
100.0
35.00
20.0
0.0
61.11
77.77
100.0
50.00
35.00
20.00
44.44
61.11
33.33
-------------
90.0
0.0
90.0
0.0
90.0
0.0
1 mM
5 mM
10 mM
1 mM
5 mM
10 mM
S.A.
A.A.
Control
(pathogen)
Fusarium oxysporum
Linear
growth
(mm)
Concentration
Treatment
Fusarium solani
100.0
50.00
20.60
0.0
60.00
40.00
20.00
44.44
77.11
100.00
33.33
55.55
77.77
30.0
0.0
0.0
40.0
20.0
0.0
66.67
100.0
100.0
55.55
77.77
100.0
60.0
30.0
10.0
66.00
40.0
20.0
77.77
66.67
88.89
26.67
55.55
77.77
Table 3: Effect of soaking seeds in some algal filtrates for controlling root-rot disease of faba bean seedling under green house conditions
Fungi
Rhizoctonia solani
Pre
%
Algal
Post
%
Fusarium solani
Survival
plant
Plant
height
Diseases
severity
Pre
%
Post
%
Survival Plant
plant height
Fusarium oxysporum
Diseases
severity
Pre
%
Post
%
Survival
plant
Plant
height
Diseases
severity
Fucus
0.0
10.0
90.0
35.50
6.66
0.0
0.0
100.0
32.0
6.66
10.0
10.0
80.0
30.0
3.33
Nostoc
0.0
0.0
100
37.00
6.66
0.0
0.0
100.0
35.0
6.66
10.0
10.0
80.0
35.0
3.33
Control
(pathogen)
40
40
25
23.33
30
10
60.0
26.0
20.0
30.0
20.0
20.0
25.0
18.88
Sargassum
0.0
20.0
20
80.0
35.00
8.88
0.0
10.0

003
90.0
34.0
9.99
20.0
10.0
70.0
33.0
4.44

Table 4: Effect of chemical inducers on root rot incidence under greenhouse conditions
Fungi
Rhizoctonia solani
Pre
%
Chemical
inducers
K2HPO4
S. A.
A. A.
Control
(pathogen)
Post
%
Fusarium solani
Survival Plant Diseases

plant
height severity
Pre
%
Post
%
Fusarium oxysporum

plant
height severity
Pre
%
Post
%

plant
height severity
10.0 10.0
80.0
33.00
11.11
10.0
0.0
90.0
30.0
13.33
20.0
10.0
70.0
30.0
11.11
0.0
90.0
33.00
11.11
10.0
10.0
80.0
31.0
11.11
10.0
10.0
80.0
30.0
6.66
10.0
10.0 20.0
40.0 20.0
70.0
40.0
30.00
25.0
12.22
23.33
10.0
30.0
20.0
10.0
70.0
28.0
60.0
26.0
13.33
20.0
20.0
30.0
20.0
60.0
30.0
50.0
25.0
12.22
25.0
18.88
Table 5: Determination of peroxidase activity (activity/min) in treated faba bean grown from seeds and planted in soil infested with fungi under
greenhouse conditions.
Fungi
Treatment
Fucus
Nostoc
Sargassum
K2HPO4
Rhizoctonia solani
Concentration
% to control
Peroxidase
% to control
peroxidase
% to control
90 ml/pot
4.00
261
3.90
202
4.00
228
90 ml/pot
3.00
196
3.12
161
3.34
190
90 ml/pot
4.00
261
3.95
204
4.20
240
50mM
3.70
241
3.75
194
3.80
217
10mM
3.30
215
3.20
165
3.50
200
10 mM
Control
(pathogen)
-------------
3.80
1.53
248
-------
Salicylic acid and Ascorbic acid followed by K2HPO4 (Dipotassium

phosphate).
Effect of soaking seeds in some algal filtrates on root

incidence under green house conditions
Data in Table 3 indicate that the tested algae significantly

reduced pre and post emergence damping-off caused by of
R. solani, F. solani and F. oxysporum compared to the control.
However, Nostoc spp. was the most effective tested algal filtrates
on decreasing the percentage of pre- and post emergence rootrot followed by Fucus and sargassum. However all treatment
significantly reduced the disease severity root-rot symptoms
caused by R. solani, F. solani and F. oxysporum.
Effect of the tested chemical inducers on root rot

incidence under green house condition
Data in Table 4 show significant effects of salicylic acid,

Ascorbic acid and Dipotassium Phosphate (K2HPO4) on reducing
the percentages of pre- and post emergence damping off
004
Fusarium oxysporum
peroxidase
S.A.
A. A.
Fusarium solani
3.90
1.93
202
-------
4.00
1.75
228
-------
caused by R. solani, F. solani and F. oxysporum compared to the

control. However, Salicylic acid was the most effective tested
chemical inducers on decreasing the percentage of pre- and
post emergence root rot. However, all treatments significantly
reduced the disease severity of root rot symptoms caused by R.
solani, F. solani and F. oxysporum.
Effect of soaking faba bean seeds in some algal filtrates and

solution of some chemical inducers on the biochemical changes
of faba bean plant in soil infected with R. solani, F. solani and F.
oxysporum.
Effect on peroxidase and polyphenol oxidase activity
Data presented in Table 5 & 6 indicate that faba bean plants

grown from seeds soaked in some algal filtrates and solutions of
some chemical inducers resulted in an increase of peroxidase and
polyphenol oxidase activity compared to the untreated control.
Fucus and Nostoc gave the highest increase in peroxidase
activity followed by Salicylic acid. Meanwhile Ascorbic acid

Table 6: Determination of polyphenol oxidase activity (activity/min) in faba bean plants grown from treated seeds and planted in soil infested with
fungi under greenhouse conditions.
Fungi
Treatment
Rhizoctonia soloni
Fusariumsoloni
Fusariumoxysporum
Concentration
Polyphenol
oxidase
% to control
Polyphenol
oxidase
% to control
Polyphenol
oxidase
% to control
90 ml/pot
2.00
181
2.50
166
2.30
164
90 ml/pot
1.81
164
2.00
133
1.95
139
10 mM
2.00
181
2.30
153
2.10
Fucus
Nostoc
90 ml/pot
Sargassum
K2HPO4
50 mM
S.A.
A. A.
10 mM
Control
(pathogen)
-------------
2.20
1.90
1.80
1.10
200
172
164
-------
2.60
173
2.20
146
2.10
140
1.50
-------
2.40
171
2.00
142
150
190
135
1.40
-------
Table 7: Determination of chitinase activity (activity/min) in faba bean plants grown from seeds soaked in the solution of some treatments and
planted in soil infested with pathogenic fungi under greenhouse conditions.
Fungi
Treatment
Fucus
Nostoc
Sargassum
K2HPO4
Concentration
90 ml/pot
Fusarium solani
Fusarium oxysporum
6.43
6.00
5.95
5.18
5.57
5.40
90 ml/pot
6.50
50mM
4.92
4.70
4.95
10mM
4.80
4.60
4.90
90 ml/pot
S.A.
10 mM
Control
(pathogen)
-------------
A. A.
Rhizoctonia solani
5.90
2.55
gave the least activity compared to control (check) treatment.

Furthermore, Nostoc gave the highest increasing in polyphenol
oxidase activity followed by Fucus and Salicylic acid followed by
K2HPO4 respectively. Meanwhile Ascorbic acid gave the lowest
value compared to control (check) treatment.
Chitinase enzyme activity
Data in Table 7 recorded that treatment of faba bean seeds

with some algal filtrates and chemical inducers resulted in
increase chitinase activity compared to the untreated control
(check). Nostoc gave the highest increasing followed by Fucus
while Ascorbic acid gave the least compared to other treatments.
Discussion
Faba bean (Vicia faba L.) is legume crop with high nutritional
value. Soil borne diseases including root-rot cause considerable
yield losses. The surveys showed differences in the frequency
of the isolated fungi. Similar results were early reported by Elsayed [1], [2,5,30,31]. Testing the inhibitory activities of algal
005
6.17
5.85
2.00
6.12
5.75
2.30
filtrates on mycelial growth of R. solani, F. solani and F. oxysporum

demonstrated that Fucus and Nostoc spp was the most effective
antifungal agent against R. solani, F. solani and F. oxysporum. This
activity might indicate the ability of algae to produce bioactive
secondary compounds, secreted into the surrounding medium.
These bioactive ingredients seem to hinder growth of the
isolates of the tested fungi (Fucus and Nostoc spp.) as they have
the ability to produce variety of lethal toxins [32]. These results
are in agreement with Caccamese et al. [33] who reported that
cyanobacteria are probably best known for production of toxins
by certain species that live in both fresh and salt water. These
results are somewhat similar to those obtained by Biondi et al.
[23] who mentioned that adding culture filtrate of Nostoc strain
to soil resulted in complete inhibition of fungi
The effectiveness of soaking seed in algae filtrates might be
due to the absorption of active substances which prevented the
infection and disease development. As for soil treatment Biondi
et al. [23] stated that the efficacy of irrigated soil with culture

filtrates may be due to the capability of antifungal like substances
to penetrate into the fungal cell, consequently causing alterations
in fungal metabolism. Furthermore, induced systemic resistance
of plants against pathogens in wide spread phenomenon that
has bean investigated with respect to the underling signaling
pathways as well as its potential use in plant protection [34].
The tested chemical inducers might stimulate some defense
mechanisms such as phenolic compounds, oxidative enzymes
and some metabolites.
The present work demonstrated that application of algae

and chemical inducer resulted in plant health. These treatments
caused a significant reduction in root rot incidence of faba bean.
In addition, the activity of peroxidase and polyphenol oxidase
and chitinase enzymes were obviously higher in plants grown
from treated seed compared to the untreated. In general, salicylic
acid, Nostoc and Fucus were the most effective for stimulating
these defense mechanisms. The present results are in agreement
with those recorded by Ibrahim [35]. It may be concluded that
application of algae filtrate is considered an applicable, safa and
cost-effective method for controlling such soil borne disease.
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Effect of Some Algal Filtrates and Chemical Inducers On Root-Rot Incidence of Faba Bean

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Effect of Some Algal Filtrates and Chemical Inducers On Root-Rot Incidence of Faba Bean

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Research Article

Agri Res Tech Open Access J

Volume 1 Issue 1 - 2015

Copyright All rights are reserved by Abeer M Mousa

Effect of Some Algal Filtrates and

Plant pathology institution, Agricultural Research Center, Egypt

Submission: September 17, 2015; Published: September 25, 2015

percentage of diseases severity of fruits rot decreased by spraying

Abdel-Monaim [9] found that S.A. follow by H2O2 recorded the

Material and Methods

The present study dealt with the management of root rot

The concerned disease caused by Rhizocptina solani,

Agricultural Research & Technology: Open Access Journal

Algal materials and filtrate: two seaweeds were collected

Algal cultures were grown in enriched artificial or natural

Effect of some algal filtrates on mycelial growth of

The crude filtrate of each alga, prepared as mentioned before

The effect of some chemical inducers on mycelial

The effect of different concentrations of three chemical

and then poured in Petri dishes (Dimensions: (DxH) 94mm x

Green House Experiment

Effect of soil treatment with some algal filtrates on soil

Crude algal filtrates are individually added to the soil infested

Percentage of pre- and post emergence damping-off as well as

Number of non germinated seeds 100

Post emergence (%) =

Survival plant (%) =

Number of dead seedling 100

Number of survival plant 100

The plant height of faba bean plants and disease severity

Effect of soaking faba bean seeds in some chemical

The obtained data were statistically treated by analysis of

Agricultural Research & Technology: Open Access Journal

variance (ANOVA) using the fisher L.S.D. method. Means were

The effect of the tested chemicals on mycelia growth of R.

All the tested algal filtrates significantly reduced the mycelia

Radial growth (mm)

Radial growth (mm)

L.S.D. Data 0.01 %=treatment =0.20; Concentration = 0.26; T X C= 0.46

L.S.D. Data 0.01 %=treatment =0.27; Concentration = 0.31; T X C= 0.59

L.S.D. Data 0.05 %=treatment =2.28; Concentration = 2.02; T X C= 4.50

Agricultural Research & Technology: Open Access Journal

Survival Plant Diseases

Survival Plant Diseases

Survival Plant Diseases

L.S.D. Data 0.05 %=treatment =1.18; Concentration = 2.03; T X C= 4.34

Salicylic acid and Ascorbic acid followed by K2HPO4 (Dipotassium

Effect of soaking seeds in some algal filtrates on root

Data in Table 3 indicate that the tested algae significantly

Effect of the tested chemical inducers on root rot

Data in Table 4 show significant effects of salicylic acid,

caused by R. solani, F. solani and F. oxysporum compared to the

Effect of soaking faba bean seeds in some algal filtrates and

Effect on peroxidase and polyphenol oxidase activity

Data presented in Table 5 & 6 indicate that faba bean plants

Agricultural Research & Technology: Open Access Journal

gave the least activity compared to control (check) treatment.

Chitinase enzyme activity

Data in Table 7 recorded that treatment of faba bean seeds

filtrates on mycelial growth of R. solani, F. solani and F. oxysporum

Agricultural Research & Technology: Open Access Journal

The present work demonstrated that application of algae

1. El-sayed F, Nakoul H, Williams P (1982) Distribution of protein