Journal of Phytopathology and Pest Management 5(2): 48-62, 2018
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
A.A.M. Ali,
E-mail: abdallahali461@gmail.com
48
Copyright © 2018
Efficiency of some organic acids as safe
control mean against root and stem rot
disease of
Coleus forskohlii
A.E.A. Halawa
1
, A.A.M. Ali*
2
, M.M.H. Hassanin
1
1
Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt
2
Plant Pathology Department, Faculty of Agriculture and Natural Resources, Aswan
University, Aswan, Egypt
Abstract
Keywords: Coleus forskohlii, root and stem rot, ascorbic acid, oxalic acid, salicylic acid.
Coleus (Coleus forskohlii) is an important medicinal crop containing forskolin in
their roots. Root rot is considered the most important disease in coleus, causing
serious losses affecting growth and yield. Infected coleus plants showing root and
stem rots were collected from Giza gardens and nurseries. The pathogenicity tests of
the isolated fungi (Fusarium oxysporum, F. roseum, F. semitectum, F. solani,
Macrophomina phaseolina, Pythium splendens and Rhizoctonia solani) revealed the
ability of these fungi to cause infection. Application of three organic acids (Ascorbic,
oxalic and salicylic acid) each at 250 and 500 ppm, against R. solani, F. semitectum,
F. oxysporum and F. solani was performed in the greenhouse. All the tested organic
acids reduced significantly disease incidence when used as dipping unrooted
cuttings and/or spray plants 30 days after planting or soil drenching under
greenhouse conditions particularly at 500 ppm compared with untreated plants. In
general, oxalic acid at 500 ppm was more efficient in reducing infection with root
and stem rot diseases. As for plant growth parameters, soil drenching with oxalic
acid was the best treatment in increasing plant height and branch number
compared with the other treatments. However, ascorbic acid was the least effective
treatment compared with the other treatments. On the other hand, dual
combination of three organic acids was superior on the other treatments and also
recorded superiority in peroxidase and polyphenol oxidase enzyme activities.
Halawa et al., 2018
49
Introduction
Coleus forskohlii
Briq. belonging to
family lamiaceae is one of the
commercial ornamental and medicinal
plants grown extensively in Egypt; it is
used as a bedding plant for public
gardens.
C. forskohlii
is cultivated mainly
for its medicinal uses in some countries.
Forskolin, a labdane diterpene compound,
originates from coleus roots, has
vasodilatory properties and considered a
potent hypotensive agent (Shah &
Kalakoti, 1991; Seamon, 1984). Its
ethnomedicinal uses for a remedy for
cough, eczema, epidermal infections,
tumors and furuncles have been recorded
(Rupp et al
.,
1986).
C. forskohlii
is
susceptible to many diseases, of which
root rot disease is a major constraint
throughout the world (Srivastava et al.,
2001; Mihail, 1992).The disease is caused
by soil-borne pathogens; it infects plant
through root causing discoloration and
foliar chlorosis and the disease cause
huge loss (Kamalakannan et al., 2006).
Regarding management of medicinal
plant diseases, organic acids are being
considered because fungicides can result
in the accumulation of harmful residues
which may lead to health risks and
serious ecological effects. The use of
organic acids (antioxidants) to control the
fungal diseases was reported by several
researchers (Zaky & Mohamed, 2009;
Khan et al., 2001; Prusky et al., 1995).
The main objective of this study was to
evaluate some organic acids (Ascorbic,
oxalic and salicylic acid) for controlling
root and stem rot of
C. forskohlii.
Materials and methods
Isolation and identification of fungi:
Samples of
C. forskohlii
plants showing
root and stem rot disease symptoms were
collected from different locations of Giza
governorate, Egypt during 2017. Infected
tissues were washed in tap water, and cut
into sections with sterilized scalpel. The
sections were surface sterilized in 1%
sodium hypochlorite for 2 minutes and
rinsed with sterilized distilled water. The
sterilized tissues were then transferred
into plates containing potato dextrose
agar (PDA) medium. The plated petri
dishes were incubated at 27
o
C for seven
days and checked daily for fungal
development. The growing fungi were
purified and identified according to the
description given by Gilman (1957),
Barnett & Hunter (1972) and Nelson et
al
.
(1983). The identification was also
kindly confirmed by department of
mycology and plant disease survey
research, Plant Pathology Research
Institute., ARC, Giza, Egypt. Isolation
frequency of the developing colonies of
each fungus was calculated as percentage
of the total developing colonies.
Inoculum preparation:
The isolated
fungi were cultured on PDA medium and
incubated at 27°C. Inoculum of each
fungus (
Fusarium oxysporum, F. roseum,
F. semitectum, F. solani, Macrophomina
phaseolina, Pythium splendens
and
Rhizoctonia solani
) was grown on maize
meal-sand medium in 500 ml glass
bottles. Bottles were incubated at 27°C
for 20 days.
Pathogenicity tests:
The experiment of
pathogenicity tests was carried out under
greenhouse conditions. Formalin
sterilized pots (20-cm diameter) packed
with sterilized clay sand soil (1:1 w/w)
were infested with each fungal inoculum
at the rates of 0.5 and 1% (w/w). Each
Halawa et al., 2018
50
pot was planted with four unrooted stem
tip cuttings. Three replicates were used
for each treatment. Results were recorded
as percentage of dead plants 30 and 60
days after sowing as follows:
Dead plants (% )=
No. of dead plants
×100
Total No. of plants
Effect of three organic acids on the
percentage of disease incidence under
greenhouse conditions:
Ascorbic acid
(AA), oxalic acid (OA) and salicylic acid
(SA) and dual combination of them, were
tested for controlling
C. forskohlii
root
and stem rot disease caused by
Fusarium
oxysporum, F. semitectum, F. solani
and
Rhizoctonia solani
at two concentrations
(250 and 500 ppm) in pot experiments
compared with treatment of carbendazim
50% WP (2g/l) [Common name:
Carbendazim, Chemical composition: 2-
(Methoxycarbomylsmino-benzimidazole)
and Manufacture: Agriphar S.A.,
Ballgium.] and untreated plants.
Formalin sterilized pots (20-cm diam.)
packed with sterilized clay sand soil (1:1
w/w) were infested separately with each
fungal inoculum at 1% (w/w). The
infested soils were watered for 7 days to
improve distribution and development of
the fungal inoculum. The organic acids
were applied by different methods as
follows: (1) Dipping unrooted cuttings:
Healthy unrooted cuttings were dipped in
the tested solutions for 15 min. before
planting (96 unrooted cuttings per 1000
ml tested solution), then four cuttings
were planted in each pot (20 cm in
diameters). Three pots for each test were
used as replicates. The Percentages of
dead plants were recorded 60 days after
planting. The plant height and number of
branches per plant were also recorded at
the end of the experiment. (2) Dipping
unrooted cuttings before planting and
then spraying with different treatments
30 days after planting: Healthy unrooted
cuttings were dipped in the tested
solutions for 15 minutes before planting
(96 unrooted cuttings per 1000 ml tested
solution), then four cuttings were planted
in each pot (20 cm in diameters), and
they were spread 30 days after planting
date with the tested solutions. Three pots
were used as replicates for each test.
Percent of dead plants incidence and
growth parameters were recorded as
mentioned before. (3) Soil drenching:
Cuttings unrooted and non-treated were
planted in pots (four per pot). Pots were
irrigated (50 ml/pot) with (AA), (OA)
and (SA) solutions at two concentrations
(250, 500 ppm) when they were planted.
Three replicates were used for each test.
Percent of dead plants incidence and
growth parameters were recorded as
mentioned before.
Effect of three organic acids and
Carbendazim 50% WP as dipping
unrooted cuttings of coleus on some
defense-related enzymes:
Coleus leaves
samples (about 10 gm/treatment) were
obtained from each greenhouse treatment
and control to determine peroxidase and
polyphenol oxidase enzymes in plants72
h after each treatment. Using a mortar
and pestle, the samples were grinded in
10 ml of 0.1 M sodium phosphate buffer
(pH 6.8). The samples were then strained
through four layers of cheese-cloth and
the filtrates were centrifuged at 3000 rpm
for 20 min. at 6
o
C (Aina
et al
.,
2012).
The supernatant was then taken and used
for enzyme assay.
Peroxidase:
The activity of peroxidase
enzyme was determined by measuring
Halawa et al., 2018
51
the oxidation of pyrogallol to
purpurogallin in the presence of H
2
O
2
at
425 nm. The reaction mixture contained
0.3 ml 0.05 M pyrogallol, 0.5 ml of 0.1
M sodium phosphate buffer solution (pH
7.0), 0.1 ml of 1.0 % H
2
O
2
and 0.3 ml
sample extract, then completed with
distilled water up to 3 ml. The activity
was expressed as absorbance change per
minute (Abs/min.) (Aina
et al
.,
2012).
Polyphenol oxidase:
Polyphenol oxidase
enzyme activity was determined
colorimetrically by the method of (Quiles
et al
.,
2005). The reaction mixture
contained 1.0 ml of 10
3
N catechol, 1.0
ml of 0.2 M sodium phosphate buffer
(pH 7.0) and 1.0 ml sample extract, then
completed to a final volume of 6.0 ml
with distilled water. The polyphenol
oxidase activity was expressed as the
change in absorbance of the reaction
mixture per min. at 495 nm.
Statistical analysis:
The plan of this trial
as designed factorial experiment in a total
randomized design with three replicates
(Snedecor & Cochran, 1980). This
statistical analysis was performed by
using the computer program MSTAT-C
statistical package version (4) using
Least Significant Difference (L.S.D) test
at 0.05.
Results
Frequency of the isolated fungi:
Seven
fungi (
Fusarium oxysporum
Schlecht
., F.
roseum
Lk. Emend. Snyd. & Hans
., F.
semitectum
Berk. & Rav.
, F. solani
(Mart) Sacc.,
Macrophomina phaseolina
(Tassi) Goid
., Pythium splendens
Braun
and
Rhizoctonia solani
Kuehn) were
isolated from
C. forskohlii
diseased
plants collected from Giza governorate,
Egypt (Table 1 and Figure 1). Data in
Table (1) indicated that
R. solani
was the
most frequently isolated fungus (27.01%)
followed by
F. oxysporum
(21.33%),
F.
semitectum
(14.69%) and
Pythium
splendens
(14.22%), while
F. roseum
(4.74%),
M. phaseolina,
(7.58%) and
F.
solani
(10.43%) showed the lowest
isolation frequency.
Table 1: Frequency of fungi isolated from C. forskohlii diseased plants,
collected from gardens and nurseries of Giza governorate, Egypt.
Isolated fungi
Frequency (%)
Fusarium oxysporum
21.33
F. roseum
4.74
F. semitectum
14.69
F. solani
10.43
Macrophomina phaseolina
7.58
Pythium sp.
14.22
Rhizoctonia solani
27.01
Total
100
Halawa et al., 2018
52
Figure 1: Coleus plants naturally infected by root and stem rot disease: A (Healthy control) and B, C, D, E
(Diseased plants).
Pathogenicity tests:
All fungi (Table 2)
were able to infect the roots and stems of
C. forskohlii
plants, grown in artificially
infested soil. The infected plant organs
usually rotted, therefore stunting and/or
complete death appeared on these plants.
However, percentages of infection were
positively correlated with the inoculum
rate (0.5% and 1%) and by the time
elapse from 30 to 60 days.
R. solani
was
the most virulent fungi resulted in (75.0%
and 91.7%), followed by
F. oxysporum
(66.7% and 83.3%),
F. solani
(66.7% and
83.3%) and
F. semitectum
(50.0% and
75.0%), at the inoculum rates (0.5% and
1%), respectively. In contrast,
Pythium
splendens
,
F. roseum
and
M. phaseolina
were the least pathogenic fungi, causing
(41.7%) at the inoculum rate (0.5%),
while,
M. phaseolina
and
F. roseum
were
the least pathogenic fungi, causing
(50.0%) at the inoculum rate (1%).
Symptoms of artificially infection by the
pathogenic fungi on
C. forskohlii
plants,
however, appear in Figure (2).
Effect of three organic acids as dipping
unrooted cuttings on dead plants:
The
efficacy of organic acids AA, OA and
SA at two concentrations (250, 500 ppm)
was determined as dipping unrooted
cuttings treatment, on percentages of
dead plants after 30 and 60 days of
planting in soil artificially infested with
R. solani
,
F. semitectum
,
F. oxysporum
and
F. solani
. Dipping unrooted cuttings
with organic acids solutions and
carbendazim 50%WP resulted in an
increase
of coleus plants resistance
against infection with the tested fungi
Halawa et al., 2018
53
(Table 3). Data in Table (3) indicate that
SA was superior treatment than OA and
AA in decreasing dead plants. SA gave
the highest decrease percentage (100%)
for
F. oxysporum
,
F. semitectum
and
F.
solani
at concentration (500 ppm).
However, OA and AA gave the highest
decrease percentages (100%) for (
F.
semitectum
and
F. solani
) and (
F.
oxysporum
,
F. semitectum
), respectively.
Carbendazim 50%WP gave the highest
decrease percentages (100%) for (
F.
semitectum
and
R. solani)
after 30 and 60
days from planting. Dual combinations
of the three organic acids gave the
highest decreasing disease incidence
with all tested fungi. The highest
reduction was obtained by using the
combined treatments between AA+OA,
AA+SA and OA+SA at a concentration
of 500 ppm, which reduced the disease
incidence by 100% after 30 and 60 days
from planting. However, the decrease in
disease incidence with all tested fungi
was not significant with any treatment
after 60 days from planting.
Table 2: Percentages of dead coleus plants, 30 and 60 days after planting with unrooted stem tip cuttings
in infested soil under greenhouse conditions.
Fungi
Inoculum rate (0.5%)
Inoculum rate (1%)
Dead plants (%) after
Survivals
(%)
Dead plants (%) after
Survivals
(%)
30 days
60 days
30 days
60 days
Fusarium oxysporum
41.7
66.7
33.3
58.3
83.3
16.7
F. roseum
25.0
41.7
58.3
33.3
50.0
50.0
F. semitectum
33.3
50.0
50.0
50.0
75.0
25.0
F. solani
41.7
66.7
33.3
50.0
83.3
16.7
Macrophomina phaseolina
25.0
41.7
58.3
33.3
50.0
50.0
Pythium splendens
25.0
41.7
58.3
41.7
66.7
33.3
Rhizoctonia solani
25.0
75.0
25.0
25.0
91.7
8.3
Control (without fungus)
0.00
0.00
100.00
0.00
0.00
100.00
L.S.D. at 5%
0.4
0.5
-
2.0
0.7
-
Figure 2: Coleus plants grown in soil infested with: F. oxysporum (B), F. solani, (C), F. semitectum (D), P.
splendens (E) F. roseum (F) M. phaseolina (G) R. solani (H) showing root and stem rot disease symptoms on
plant growth compared with the healthy plants (A).
Halawa et al., 2018
54
Table 3: Effect of three organic acids as dipping unrooted cuttings treatment on incidence (%) of root and stem rot
disease of coleus plants under greenhouse conditions..
Treatments
Conc.
F. oxysporum
F. semitectum
F. solani
R. solani
Disease
incidence (%)
Disease
incidence (%)
Disease
incidence (%)
Disease
incidence (%)
30
days
60
days
30
days
60
Days
30
days
60
days
30
days
60
days
AA
250 ppm
8.3
8.3
0.0
8.3
8.3
25.0
16.7
25.0
500 ppm
0.0
0.0
0.0
0.0
0.0
16.7
8.3
16.7
OA
250 ppm
33.3
33.3
16.7
25.0
0.0
16.7
16.7
25.0
500 ppm
8.3
8.3
0.0
0.0
0.0
0.0
8.3
16.7
SA
250 ppm
16.7
33.3
16.7
25.0
0.0
8.3
41.7
50.0
500 ppm
0.0
0.0
0.0
0.0
0.0
0.0
25.0
33.3
AA + OA
250 ppm
0.0
8.3
0.0
0.0
0.0
8.3
8.3
8.3
500 ppm
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
AA + SA
250 ppm
8.3
8.3
8.3
8.3
0.0
0.0
16.7
16.7
500 ppm
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
OA + SA
250 ppm
8.3
8.3
8.3
8.3
0.0
0.0
16.7
16.7
500 ppm
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Carbendazim 50% WP
2 g/l
0.0
8.3
0.0
0.0
8.3
8.3
0.0
0.0
Control
-
66.7
83.3
58.3
75.0
50.0
75.0
50.0
50.0
L.S.D. at 5%
-
0.5
0.3
1.3
0.2
0.8
0.2
1.0
0.4
On the other hand, dipping unrooted
cuttings in organic acids increased plant
height and no. of branch/plant compared
to untreated plants under infection with
tested fungi. Plant height and branch
number increase by increasing of organic
acid concentration. Carbendazim
50%WP and dual combination between
OA+SA at 500 ppm recorded superiority
in increasing plant height and No. of
branches per plant, followed by OA at
500 ppm compared to any of the other
samples which underwent treatment with
all tested fungi (Table 4).
Table 4: Effect of three organic acids as dipping unrooted cuttings treatment on some plant growth parameters of
coleus plants in soil infested with each of four pathogenic fungi under greenhouse conditions.
Treatments
Conc.
F. oxysporum
F. semitectum
F. solani
R. solani
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
AA
250 ppm
10.0
4.1
11.8
4.2
10.4
5.0
11.3
4.1
500 ppm
10.8
4.2
12.1
4.9
11.6
5.4
12.0
4.8
OA
250 ppm
12.7
5.4
13.3
5.4
15.0
6.3
14.6
6.3
500 ppm
13.3
5.9
14.4
6.3
16.4
6.7
15.3
7.0
SA
250 ppm
10.9
4.8
12.0
5.4
12.1
5.4
12.9
5.0
500 ppm
11.8
5.0
13.1
5.9
12.7
5.9
13.1
5.7
AA + OA
250 ppm
12.0
4.1
12.0
4.7
10.9
5.3
13.3
4.7
500 ppm
13.0
5.2
14.4
6.7
16.1
6.3
15.1
6.7
AA + SA
250 ppm
10.9
4.7
12.7
5.4
10.8
4.2
13.1
5.7
500 ppm
12.0
5.4
13.3
5.9
11.8
5.0
15.3
6.7
OA + SA
250 ppm
12.4
5.0
12.9
4.7
12.0
6.3
13.3
5.4
500 ppm
13.4
5.7
14.6
6.7
16.4
7.0
16.1
7.0
Carbendazim 50% WP
2 g/l
13.5
5.9
14.7
7.0
17.8
7.3
18.1
7.3
Control
-
8.7
2.7
10.7
2.9
10.2
2.6
10.1
2.9
L.S.D. at 5%
-
0.6
0.1
0.4
0.1
0.7
0.1
0.3
0.5
Halawa et al., 2018
55
Effect of three organic acids as dipping
unrooted cuttings and spraying plants
30 days after planting date on the
disease incidence:
Dipping unrooted
cuttings and spraying plants with organic
acids solutions and carbendazim 50%
WP fungicide resulted in an increase of
C. forskohlii
resistance against to
infection with the tested fungi (Table 5).
The resistance of coleus was enhanced
by increasing organic acid concentration.
Carbendazim 50% WP and OA gave
significant decreases in disease incidence
than the controls with all fungi under
study, followed by SA at the highest
concentration. However, AA was the
least effective treatment
.
On the other
hand, dual combination of organic acids
gave the highest decreasing disease
incidence with all tested fungi. The
highest reduction was obtained by using
the combined treatments between OA +
SA at concentrations of 250 and 500 ppm
and AA + SA at concentration 500 ppm,
which reduced the disease incidence by
100% after 30 and 60 days from
planting. . However, the decrease in
disease incidence with all tested fungi
was not significant with any treatment
after 60 days from planting. Dipping
unrooted cuttings and spraying plants 30
days after planting with organic acids
and carbendazim 50% WP fungicide
significantly increase all parameters
compared to control under infection with
tested fungi. Plant height and branch
number increase by increasing of
organic acid concentration. Dual
combination between AA + OA at 500
ppm recorded superiority in increasing
plant height and No. of branches per
plant, followed by OA + SA at the same
concentration compared to any of the
other samples which underwent
treatment with all tested fungi (Table 6).
Table 5: Effect of three organic acids as dipping unrooted cuttings before planting then spraying with them 30 days
after planting on incidence (%) of root and stem rot disease of coleus plants under greenhouse conditions.
Treatments
Conc.
F. oxysporum
F. semitectum
F. solani
R. solani
Disease incidence
(%) after
Disease incidence
(%) after
Disease incidence
(%) after
Disease incidence
(%) after
30
days
60
days
30
days
60
Days
30
days
60
days
30
days
60
days
AA
250 ppm
33.3
33.3
33.3
41.7
33.3
41.7
41.7
41.7
500 ppm
25.0
25.0
8.3
16.7
8.3
16.7
16.7
25.0
OA
250 ppm
8.3
16.7
0.0
16.7
16.7
25.0
8.3
16.7
500 ppm
0.0
0.0
0.0
8.3
0.0
8.3
0.0
8.3
SA
250 ppm
25.0
33.3
33.3
41.7
8.3
16.7
33.3
33.3
500 ppm
8.3
16.7
16.7
25.0
0.0
0.0
8.3
8.3
AA + OA
250 ppm
8.3
16.7
16.7
25.0
16.7
25.0
8.3
8.3
500 ppm
8.3
8.3
8.3
8.3
8.3
8.3
0.0
0.0
AA + SA
250 ppm
16.7
16.7
8.3
8.3
8.3
8.3
8.3
16.7
500 ppm
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
OA + SA
250 ppm
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
500 ppm
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Carbendazim 50% WP
2 g/l
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Control
-
66.7
83.3
58.3
60.0
50.0
75.0
50.0
66.7
L.S.D. at 5%
-
0.8
0.1
0.5
0.3
0.8
0.6
0.5
0.6
Halawa et al., 2018
56
Table 6: Effect of three organic acids as dipping unrooted cuttings before planting then spraying with them 30
days after planting on some plant growth parameters of coleus plants in soil infested with each of four pathogenic
fungi under greenhouse conditions.
Treatments
Conc.
F. oxysporum
F. semitectum
F. solani
R. solani
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
AA
250 ppm
12.1
4.8
14.6
5.0
11.0
5.1
14.0
4.2
500 ppm
13.4
5.2
15.2
5.2
13.4
5.5
15.7
5.0
OA
250 ppm
15.0
7.3
16.1
7.6
13.9
7.4
16.2
7.6
500 ppm
16.2
8.0
17.0
8.3
16.6
8.2
18.3
8.2
SA
250 ppm
14.4
7.0
14.4
5.4
13.1
6.3
13.3
6.0
500 ppm
14.8
7.6
15.4
6.0
15.0
6.8
16.8
6.9
AA + OA
250 ppm
18.8
8.3
17.9
9.4
19.5
9.3
19.8
8.7
500 ppm
22.7
10.3
21.4
11.0
21.7
12.3
22.9
12.7
AA + SA
250 ppm
16.3
7.7
16.4
7.3
18.0
7.7
17.9
7.3
500 ppm
17.9
9.4
18.0
7.3
19.8
8.7
20.4
11.6
OA + SA
250 ppm
19.3
8.8
18.0
7.0
18.0
7.3
20.0
11.0
500 ppm
21.5
9.7
20.7
10.0
20.0
9.4
21.8
8.8
Carbendazim 50% WP
2 g/l
17.7
9.3
18.9
9.7
17.8
10.3
18.1
10.0
Control
-
8.4
2.7
10.3
2.4
10.9
3.0
10.2
3.1
L.S.D. at 5%
-
1.7
1.1
0.5
1.3
0.3
0.9
0.1
1.2
Effect of three organic acids applied as
soil drenching treatment on the disease
incidence:
Drenching the soil with
organic acid solutions resulted in an
increase of coleus resistance against to
infection with the tested fungi (Table 7).
The results revealed that all organic acids
reduced disease incidence by increasing
concentration. OA gave significant
decreases in disease incidence than the
control with all fungi under study,
followed by SA at the highest
concentration. In contrast, AA was the
least effective treatments in decreasing
disease incidence (%), except for
F.
solani
, since it gave the highest
decreases percentage at concentration
(500 ppm).
Table 7: Effect of three organic acids as soil drenching treatment on incidence (%) of root and stem rot disease of
coleus plants, under greenhouse conditions.
Treatments
Conc.
F. oxysporum
F. semitectum
F. solani
R. solani
Disease incidence
(%)
Disease incidence
(%)
Disease incidence
(%)
Disease incidence
(%)
30
days
60
days
30
days
60
Days
30
days
60
days
30
days
60
days
AA
250 ppm
33.3
41.7
41.7
41.7
8.3
16.7
41.7
41.7
500 ppm
25.0
25.0
25.0
33.3
0.0
0.0
25.0
33.3
OA
250 ppm
33.3
41.7
0.0
0.0
8.3
16.7
8.3
16.7
500 ppm
8.3
16.7
0.0
0.0
0.0
0.0
0.0
8.3
SA
250 ppm
33.3
41.7
41.7
41.7
25.0
25.0
41.7
41.7
500 ppm
16.7
25.0
33.3
33.3
8.3
16.7
16.7
25.0
AA + OA
250 ppm
16.7
16.7
8.3
16.7
8.3
16.7
16.7
16.7
500 ppm
8.3
8.3
0.0
0.0
0.0
0.0
0.0
0.0
AA + SA
250 ppm
8.3
16.7
16.7
25.0
8.3
8.3
16.7
25.0
500 ppm
8.3
8.3
8.3
8.3
0.0
0.0
8.3
8.3
OA + SA
250 ppm
8.3
8.3
8.3
16.7
8.3
8.3
16.7
16.7
500 ppm
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Carbendazim 50% WP
2 g/l
16.0
16.7
8.3
8.3
8.3
8.3
8.3
16.7
Control
-
66.7
83.3
58.3
75.0
50.0
75.0
50.0
66.7
L.S.D. at 5%
-
1.0
0.9
1.2
0.4
0.8
0.2
1.0
0.1
Halawa et al., 2018
57
On the other hand, dual combinations of
organic acids gave the highest decreasing
disease incidence with all tested fungi.
The highest reduction was obtained by
using the combined treatments between
OA+SA at concentration of 500 ppm
which reduced the disease incidence by
100% after 30 and 60 days from
planting.
Drenching the soil with organic
acids significantly increased all
parameters compared to control. Soil
drenching with OA single gave the
highest results in all parameters (plant
height and branch number) with all fungi
compared to untreated plants, while,
using the combined treatments between
OA+SA was superiorly compared to any
other treatment with all tested fungi
(Table 8). On the other hand, dual
combinations of the three organic acids
was superiorly in both applied methods
(dipping unrooted cuttings or dipping
unrooted cuttings + spraying plants 30
days after planting or soil drenching).
Table 8: Effect of three organic acids as dipping unrooted cuttings treatment on some plant growth parameters of
coleus plants in soil infested with each of four pathogenic fungi under greenhouse conditions.
Treatments
Conc.
F. oxysporum
F. semitectum
F. solani
R. solani
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
Plant
Height
(cm)
No. of
branches/
plant
AA
250 ppm
14.6
5.2
14.3
6.0
15.0
6.3
15.3
7.3
500 ppm
15.3
6.3
15.6
7.3
15.8
7.2
16.0
8.1
OA
250 ppm
20.0
8.0
20.3
8.7
20.1
7.0
19.8
8.3
500 ppm
21.5
9.4
21.1
10.0
21.8
8.8
21.7
10.0
SA
250 ppm
15.3
6.8
16.0
7.3
16.2
7.0
17.1
7.0
500 ppm
16.9
7.8
16.9
8.8
17.1
7.6
18.0
8.1
AA + OA
250 ppm
14.6
5.0
14.0
4.2
15.7
5.0
16.8
6.9
500 ppm
16.2
8.3
16.1
7.6
17.0
8.3
18.3
8.2
AA + SA
250 ppm
15.0
7.3
16.1
7.6
15.0
6.8
16.2
7.6
500 ppm
16.2
8.0
17.0
8.3
16.6
8.2
18.3
8.2
OA + SA
250 ppm
18.8
8.3
17.9
9.3
19.5
9.3
19.8
8.7
500 ppm
22.7
10.3
20.4
11.0
21.7
12.3
22.9
12.7
Carbendazim 50% WP
2 g/l
17.7
9.3
18.9
9.7
17.8
10.3
18.1
10.0
Control
-
8.8
2.8
10.2
2.3
10.2
2.3
10.7
2.1
L.S.D. at 5%
-
3.2
1.9
2.3
2.1
1.1
0.2
0.7
0.8
Effect of three organic acids and the
fungicide Carbendazim 50%WP as
dipping unrooted cuttings of coleus on
defense-related enzymes:
Data in table
(9) showed the oxidative enzyme
activities (peroxidase and polyphenol
oxidase) due to treating coleus cuttings
unrooted with three organic acids (alone
or dual combinations) and carbendazim.
Dual combination between OA+SA
recorded superiority in peroxidase and
polyphenol oxidase activities with
F.
oxysporum, F. semitectum
and
R. solani.
While, AA+SA recorded superiority in
peroxidase and polyphenol oxidase
activities with
F. solani
. In contrast, OA
exhibited lower peroxidase and
polyphenol oxidase activities.
Halawa et al., 2018
58
Table 9: Activity of enzymes in coleus plants treated with different treatments as dipping unrooted cuttings.
Treatments
Concentrations
Enzymes
F. oxysporum
F. semitectum
F. solani
R. solani
AA
500 ppm
Peroxidase
1.81
1.38
1.90
1.90
Polyphenol oxidase
0.70
0.48
0.76
0.74
OA
Peroxidase
1.31
1.20
1.38
1.48
Polyphenol oxidase
0.23
0.20
0.25
0.46
SA
Peroxidase
1.38
1.80
1.75
1.49
Polyphenol oxidase
0.25
0.71
0.76
0.53
AA + OA
Peroxidase
1.90
1.50
1.92
1.62
Polyphenol oxidase
0.67
0.29
0.77
0.50
AA + SA
Peroxidase
1.83
1.49
1.93
1.91
Polyphenol oxidase
0.70
0.31
0.78
0.78
OA + SA
Peroxidase
1.92
1.81
1.80
1.95
Polyphenol oxidase
0.78
0.75
0.75
0.79
Carbendazim 50% WP
2 g/l
Peroxidase
1.83
1.77
1.80
1.56
Polyphenol oxidase
0.71
0.50
0.76
0.59
Control
-
Peroxidase
0.76
0.65
0.55
0.68
Polyphenol oxidase
0.13
0.12
0.14
0.12
Correlation between disease incidence
and each of peroxidase and polyphenol
oxidase:
Table (10) showed that there
was a significant or highly significant
correlation between the activities of
peroxidase and polyphenol oxidase in
tissues infected with all fungi. The
correlation between disease incidence
and each of peroxidase and polyphenol
oxidase was negative for
F. oxysporum
and
R. solani
, however the correlation
was nonsignificant in both cases.
Table 10: Correlation between disease incidence and each of peroxidase and polyphenol oxidase.
Fungi
Variable
Variable
PO (X1)
PPO (X2)
F. oxysporum
Peroxidase (X1)
Polyphenol oxidase (X2)
0.986
a
(0.000)
b
(%) Disease incidence after 30 days(X3)
- 0.686
a
(0.132)
b
- 0.645
a
(0.167)
b
F. semitectum
Peroxidase (X1)
Polyphenol oxidase (X2)
0.881
a
(0.020)
b
(%) Disease incidence after 30 days (X3)
.
c
.
c
F. solani
Peroxidase (X1)
Polyphenol oxidase (X2)
0.950
a
(0.004)
b
(%) Disease incidence after 30 days (X3)
.
c
.
c
R. solani
Peroxidase (X1)
Polyphenol oxidase (X2)
0.966
a
(0.002)
b
(%) Disease incidence after 30 days (X3)
- 0.603
a
(0.205)
b
- 0.423
a
(0.403)
b
a
Linear correlation coefficient, n= 6,
b
Probability level,
c
correlation cannot be computed because DI
was constant.
Discussion
Coleus (
Coleus forskohlii
Briq.) is grown
for use as a bedding plant for public
gardens. Many soil-borne fungi are
capable of causing stem and root rot of
plants. Species of
Fusarium,
Macrophomina, Pythium
and
Rhizoctonia
were among the most
dominant fungi in isolation trials from
diseased plants. The causative agents of
stem and root rot as a result of isolation
trials were somewhat similar to those
reported by many authors (Pulla et al
.
,
Halawa et al., 2018
59
2013; Boby & Bagyaraj, 2003;
Srivastava et al., 2001; Mihail, 1992).
Results of the present study showed that
isolated fungi were able to infect roots
and stems of
C. forskohlii
plants, grown
in artificially infested soil.
R. solani
was
the most virulent fungi, followed by
F.
oxysporum
,
F. solani
and
F. semitectum
,
respectively. Similar results were
reported by Shivkumar et al. (2006),
Singh et al. (2009) and more recently by
Pulla et al
.
(2013). Testing three organic
acids (ascorbic acid, oxalic acid and
salicylic acid) against stem and root rot
diseases gave satisfactory effectiveness
in most cases. Dipping unrooted cuttings
with organic acids testes was of great
value in decreasing disease incidence and
plant growth parameters such as plant
height and no. of branch/plant were
greatly improved. SA was superior
treatment than OA and AA in decreasing
disease incidence. However, OA was
significantly superior in plant growth
parameters. On the other hand, dual
combination of the three organic acids
was superior to single treatment. The
highest reduction was obtained by using
the combined treatments of AA+OA,
AA+SA and OA+SA. Organic acids have
the properties of antioxidants which are
considered to be an important mechanism
of membrane deterioration during ageing
of tissues (Droillard et al., 1987; Paulin et
al., 1986; Mayak et al., 1983; Dhindsa et
al., 198; Kellog & Fridovich, 1975). The
two applications (Dipping + spraying) of
plant with organic acids were of great
value in decreasing dead plants and plant
growth parameters. Carbendazim and OA
gave significant decreases in dead plants
and plant growth parameters than the
controls with all fungi under study. On
the other hand, dual combination of the
three organic acids was superior to the
single treatment. Variability in the effect
of organic acids (Antioxidants) can be
attributed to differences in their activity
or variation in the responses dictated by
the host due to the phytotoxicity of the
compounds or to the ability of the
compounds to penetrate their tissues
(Edlich et al
.
, 1989). Also, some studies
have been shown that oxidants may
enhance host resistance to fungal
infection (Prusky et al., 1995; Elad,
1992; Prusky, 1988). Drenching the soil
with organic acids was of great value in
decreasing disease incidence and plant
growth parameters. OA gave significant
decreases in disease incidence and plant
growth parameters than the control with
all fungi under study. On the other hand,
dual combination of the three organic
acids was superior on single treatment.
These results were in agreement with the
results of Galal and Abdou (1996) who
found that soil drench application of
salicylic or ascorbic acid was better than
foliar application to control fusarial
diseases of cowpea. In the same sense,
Mostafa (2006) reported that soaking
cumin seeds or soil drenching with
antioxidant solutions (salicylic, ascorbic,
coumaric and benzoic acids) before
planting resulted in resistant cumin
seedlings against infection with
F.
oxysporum cumini
and
Acremonium
egypticum
. Treating coleus cuttings
unrooted with three organic acids and the
fungicide carbendazim increased in the
oxidative enzyme activities (peroxidase
and polyphenol oxidase). Combination
between OA+SA recorded superiority in
peroxidase and polyphenol oxidase
activities with
F. oxysporum, F.
semitectum
and
R. solani,
while, AA+SA
recorded superiority in peroxidase and
Halawa et al., 2018
60
polyphenol oxidase activities with
F.
solani
. The antioxidant mode of action
was reported in many interactions of
host-pathogen i.e. many oxidative
enzymes such as peroxidase, catalase,
ascorbate oxidase and polyphenol
oxidase were detected as a result of
infection with many pathogens (Clark
et
al.,
2002) or as a result of treatments with
various antioxidants (Abdel-Monaim et
al., 2011; Ragab et al., 2009; El-Khallal,
2007; Takahama & Oniki, 1994). There
was a significant or highly significant
correlation between the activities of
peroxidase and polyphenol oxidase in
tissues infected with all fungi. This
correlation indicated that it is not
necessary to determine the activity of the
two enzymes, because any of them is
indicator for the other one. The
correlation between disease incidence
and each of peroxidase and polyphenol
oxidase was negative for
F. oxysporum
and
R. solani
, however the correlation
was nonsignificant in both cases. This
nonsignificant could be due to the limited
sample size (n=6). In conclusion, the
present study indicated that each of
peroxidase or polyphenol oxidase is not
correlated with disease incidence.
Acknowledgements
The authors express their appreciation to
Professor A.A. Aly, Cotton Disease
Research Department, Plant Pathology
Research Institute, Agricultural Research
Center, Giza, Egypt, for his
encouragement and his advices and
revise this article.
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