Journal of Phytopathology and Pest Management 5(3): 77-84, 2018
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Kamal A.M. Abo-Elyousr,
E-mail: elyousr@aun.edu.eg, ka@kau.edu.eg
77
Copyright © 2018
Control of tomato bacterial wilt using
certain of plant ethanol extracts
Kamal A.M. Abo-Elyousr
1,2*
, Hadeel M.M.K. Bagy
1
1
Plant Pathology Department, Faculty of Agriculture, Assiut University, 71526 Assiut, Egypt
2
Department of Arid Land Agriculture, Faculty of Meteorology, Environment and Arid Land
Agriculture, King Abdulaziz University, 80208 Jeddah, Saudi Arabia
Abstract
Keywords: Ralstonia solanacearum, plant extracts, bacterial wilt, tomato, disease control.
78
Introduction
Tomatoes (
Solanum lycopersicum
L.) are
considered the maximum global
significant vegetable crops after potato
(Khoso, 1994). Yield loss of tomato plant
10–20% annually caused by soil borne
diseases.
Ralstonia solanacearum
is an
important pathogen of many crops
(Yabuuchi
et al
., 1995). Also, it is the
greatest eradicative soil-borne pathogen
that impacts tomatoes causing bacterial
wilt disease in all over the world,
especially in temperate, subtropical and
tropical regions (Yuliar
et al.,
2015;
Champoiseau
et al.,
2009). Bacteria wilt
is a hard control disease. Chemical
control of tomato bacterial wilt is
dangerous to environment, human and
animal as well as raising the pathogens of
chemical resistant. So, there is a critical
requirement to substitute or at minimize
the use of chemicals for reducing of the
environment pollution. Besides, the use
of chemicals has not been very effective
in the control of such disease because the
causal pathogen is a soil borne and is
systemic in its nature. Antibiotics showed
low effect and resistant varieties occurred
due to the strain diversity and latent
infection of the pathogen (Farag
et al.,
1982). Use of crop rotation with non-host
crops can decrease the populations of
pathogen in the soil (Ahmed
et al
., 2000).
The use of copper-based bactericides and
antibiotics seldom gave satisfactory
control. Use of plant extracts could be
useful in control of this disease because
of their natural origin are biodegradable
and they do not leave any toxic residues
or by products to accumulate in the
environment (Abo-Elyousr & Asran,
2009). Therefore, under this scenario,
botanical pesticides seem to be ideal
candidate to be exploited in the tomato
bacterial wilt control in view of the
safety, renewable nature, cost effective
and high target specificity. Hence an
investigation was conducted to study the
effect of extracts from a few important
medicinal plants against
R.
solanacearum
. Plant products are
considered as significant sources for
control of certain plant diseases (Abdel-
Monaim
et. al.
, 2011; Hassan
et. al.
,
2009; Ji
et. al.
, 2005; Regnault
et. al.
2005; Kagale
et. al.
, 2004). Plant extracts
are actually safe alternatives for
environment and it can be included in the
disease control programs. Hassan
et. al.
(2009) used the ethanol extracts of
Eucalyptus globules
Punica granatum
and
Hibiscus sabdariffa,
against the
potato bacterial wilt disease. This study
was planned to study the efficiency of
Citrus sinensis
,
Citrus reticulate
,
Punica
granatum
and
Cinnamomum camphorm
extracts against the tomato bacterial wilt
causal pathogen, and to examine the
efficiency of time of applications on the
disease severity
.
Materials and methods
Source of the pathogen and inoculum
production:
A virulent isolate of
Ralstonia solanacearum
obtained from
previous study (Abo-Elyousr & Asran
2009) was used. The pathogen isolate
was grown on nutrient agar medium for
48 h at 28 °C. Pathogen cells were
immersed with sterilized distilled water
then scraped off the nutrient agar plate
using a sterile cotton swab to make a
suspension (Wai
et al
., 2013). The
bacterial suspension concentration was
adjusted using spectrophotometer (OD
600
= 0.3) to 1.5 × 10
8
CFU/mL (Lin
et al
.,
2014; Hindi, 2013) and they utilized in
the following experiments.
Plants collection:
Four local plants
namely,
Citrus sinensis
,
Citrus
79
reticulate
,
Punica granatum
and
Cinnamomum camphorm
were collected
from markets in Assiut, Egypt were used
in the current study. The name of plant
and its parts were used in this study are
presented in Table (1).
Table 1:
Plants sampled and parts used.
Botanical name
English Name
Part of plant used
Citrus sinensis
Orange
peel
Citrus reticulate
Tangerine
peel
Punica granatum
Pomegranate
peel
Cinnamomum camphora
Camphor
leaf
Plant extracts preparation:
Fresh plant
parts of
C. camphorm
and peel of
C.
sinensis
,
C. reticulate
and
P. granatum
were carefully washed with tap water to
remove the dust and other unwanted
materials accumulated on the leaves and
peel. The samples were dried under
laboratory conditions for 20 days. Then,
the dried leaves and peel were powdered
using an electric blender. Finally, the
powdered materials either leaves or peel
were sieved through the strainer and the
fine powder was collected and used for
the extraction process. Ten grams of the
weighed plant materials powder was
soaked in 100 ml of ethanol into 200 ml
conical flask plugged with sterile cotton
and covered with aluminium foil and kept
in a reciprocating shaker at 200 rpm for
24 h. Then, the extract was filtered
through muslin cloth cloth and this
repeated three times, then through
Whatman no 1 filter paper. The solvent
from the extracts was removed by using
rotary vacuum evaporator. As a final
point, the residues were collected, and the
concentrations of 15 and 20% were
prepared and used in the next
experiments.
Efficacy of plant extracts against the
pathogen
in vitro
:
Antibacterial
activities of the tested concentrations (15
and 20%) of the ethanol extracts plants
were studied
in vitro
using the agar well
diffusion method. The NA plate surface
is inoculated by spreading a 100 µl of the
pathogen suspension (10
8
CFU/ml) over
the entire surface (Balestra
et al
., 2009).
After that, a hole with a 9 mm diameter
is aseptically bored with a sterilized cork
borer. Hundred µl of test extracts was
added in each well. Water was used as a
control. After overnight incubation at
27˚C, inhibition zones were measured by
a transparent ruler. The experiment was
repeated twice.
Effect of plant extracts on seed
germination under laboratory
conditions:
Tomato seeds cv. Super
Marmande were soaked in each tested
concentration of plant extracts and then
treated with the pathogen. Seeds were
carefully shaken for 1 h then, left to dry,
and finally plated on wet blotters. Seeds
treated only with the pathogen were used
as control and the germination was
measured according to the filter paper
method (Abo-Elyousr & El-Handawy
Hoda, 2008).
Effect of plant extracts on the disease
severity under greenhouse conditions:
Tomato plants cv. Super Marmande were
grown in 20 cm sterilized pots filled with
sterilized clay and sand mixture (2:1
w/w) under greenhouse conditions and
80
watered regularly. The plants were
fertilized once with 100 mL/plant of
micronutrients hydrosol fertilizer one
week after transplanting. Plants were
inoculated by clipping the lower leaf with
scissors dipped in a fresh bacterial
suspension of 10
8
CFU/mL (Hindi, 2013).
The potted plants were arranged in two
groups, the plants in the first group were
inoculated with the pathogen two days
after application with plant extract (40 ml
of each extract) and the plants in the
second group were inoculated with the
pathogen two days before the plant
extract application. Plants within each
group were treated only with the
pathogen were used as control. Each
group was divided into two
concentrations 15% and 20%. The
experiment was repeated twice. Disease
severity percentages were calculated four
weeks after the application according to
Wai
et al
. (2013). The 1-5 disease
severity scale was used where, 1 = no
visible symptoms, 2 = one leaf to half of
the foliage wilting, 3 = nearly all of the
foliage wilting, 4 = the whole plant
wilting and dead. isease severity (%) was
expressed as formula developed by
Bdliya and Dahiru (2006):
Disease severity (%) = [∑ (ni × vi) ÷ (V × N)] ×100
Where: ni= number of plants with the
respective disease rating, vi= the disease
rating, V= the highest disease rating (5),
and N= the total number of observed
plants.
Measuring the fresh and dry weights:
For estimating fresh weight and dry
weight of all treatments, ten plants of
each treatment were randomly taken,
washed, left to dry and then their fresh
weight was recorded. Then, these plants
were dehydrated at 60°C for three days
and the dry weight was determined.
Statistical analysis:
All recorded data
were analyzed statistically, Fisher's least
significance difference test at
p
=0.05 was
used to compare the average values of
treatments (Gomez & Gomez, 1984).
Results and Discussion
In vitro
study:
Tested concentrations of
the ethanol
extracts of
C. sinensis
,
C.
reticulate
,
P. granatum
and
C.
camphorm
were caused varied pathogen
growth inhibition zones
in vitro
(Table
2). In compared with other plants,
inhibition zones were the highest in case
of use of orange extracts at 20%.
C.
reticulate
at 20% caused the least
inhibition zones (1.37 mm). Results also,
showed that the use of orange produced
the highest decreasing of the bacterial
pathogen growth (2.2 mm) followed by
P. granatum
and the lowest one is
C.
reticulate
extract. These results agree
with those reported by others study Abo-
Elyousr and Asran (2009), Ranjit
et al
.
(2012) and El-Ariqi
et al.
(2005).
Numerous investigators indicated that the
plant contains a number of chemicals
groups such as tannins and flavonoids
(Ranjit
et al
., 2012; Kapoor, 2001). The
bacterial growth inhibition caused by
plant extracts possibly explicated on the
basis of the act of antimicrobial
secondary metabolites present in the
plant.
Influence of certain plant extracts on
seed germination percentage:
Tested
81
plant extracts enhanced the tomato seed
germination percentage compared to
infected control, the treatment with
C.
sinensis
extract
was the best one which
increased the seed germination
percentage to 84.0%, followed by
P.
granatum
and
C. camphorm
extracts
especially at 20% concentrations (Figure
1). Among all tested treatments,
C.
reticulate
achieved the lowest increasing
percentage of the seed germination
(48%).
Table 2: In vitro impact of some concentrations of certain plant extract on Ralstonia solanacearum.
Treatments
Concentration
Zone of inhibition
(mm, dia.)
C. sinensis, Orange
15
1.8 de
20
2.6 a
C. reticulate, Tangerine
15
1.37 f
20
1.87 d
P. granatum, Pomegranate
15
1.77 de
20
2.23 b
C. camphor, Camphor
15
2.03 c
20
1.7 e
Control
0.0 g
Values in the same column have a similar superscript letter are not significantly
different according to LSD test at P=0.05.
Figure 1: Influence of tested concentrations of certain plant extracts on seed germination percentage of
tomato cv. Super Marmande in vitro. Bars indicate the standard error.
Effect of certain plant extracts on
diseases severity under greenhouse
conditions:
Data in Table (3) revealed
that all treatments either applied after or
before inoculation reduced the disease
severity compared to control,
C. sinensis
achieved the highest disease reduction
percentage (89.6%) followed by
P.
granatum
concentration (85.6%) while
C.
reticulate
and
C. camphorm
after
inoculation recorded the lowest disease
reduction percentage (27.5%). Gaind and
Budhiraja (1967) mentioned that the
extracts of certain plants were found to
be effective in inhibiting the activity of a
broad spectrum of bacteria. Also, Abo-
Elyousr and Asran (2009) found that the
application of extracts of datura, garlic
and nerium to the soil at two days before
inoculation or the time of inoculation,
and two days after inoculation by the
pathogen, have significantly reduced the
0
20
40
60
80
100
Citrus
sinensis
Citrus
reticulate
Punica
granatum Cinnamomum
camphorm
Healthy
Control
Infected
Control
Seed germination (%)
Treatments
20% 15%
82
disease severity of bacterial wilt of
tomato plants under greenhouse
conditions. As well as the use of these
plant extracts could induce resistance in
the plants against the pathogen or
enhanced the peroxidases, phenylalanine
ammonia lyase, pathogenicity-related
proteins and phenolic substances against
the bacterial wilt of tomato (Hassan
et
al
., 2009).
Table 3: Efficacy of plant extracts 20% concentrations to control tomato bacterial wilt disease under the
greenhouse conditions.
Treatments
Time of application
from inoculation
Disease incidence
(%)
Control efficacy
(%)
C. sinensis, Orange
Before
6.8 c
89.6
After
9.5 c
85.5
C. reticulate, Tangerine
Before
25 b
61.8
After
47.5 d
27.5
P. granatum, Pomegranate
Before
16.25 c
75.2
After
8.8 b
85.6
C. camphor, Camphor
Before
25.75 b
60.17
After
47.5 d
27.5
Infected Control
82.25 a
-
Values in the same column have a similar superscript letter are not significantly different according to LSD
test at P=0.05.
Table 4: Efficacy of plant extracts concentrations on biomass of tomato plants after inoculation with Ralstonia
solanacearum under the greenhouse conditions.
Treatments
Time of application from inoculation
Fresh weight
Dry weight
C. sinensis, Orange
Before
43.67 a
16.2 ab
After
23.08 c
14.77 abc
C. reticulate, Tangerine
Before
23.10 c
11.7 cde
After
19.73 d
9.495 e
P. granatum, Pomegranate
Before
39.33 b
12.85 bcde
After
24.93 c
13.3 bcd
C. camphor, Camphor
Before
24.23 c
13.48 bcd
After
24.67 c
10.99 de
Infected Control
12.05 e
7.95 f
Healthy Control
44.73 a
16.13 ab
Values in the same column have a similar superscript letter are not significantly different according to LSD test at
P=0.05.
Effect of specific plant extracts on
tomato plants biomass under
greenhouse conditions:
All tested plant
extracts in the two different times of
application were caused increasing of
shoot fresh and dry weights compared
with control (Table 4). The highest fresh
weight was recorded in tomato plant
treated with
P. granatum
followed by
orange before inoculation. Also, the
highest dry weight was recorded in
infected control and
P. granatum
. Our
results are agreed with a previous study
of Abo-Elyousr and Asran (2009). In
conclusion, we can recommend that use
of plant extracts of
C. sinensis
,
C.
reticulate
,
P. granatum
and
C.
camphorm
to control of
R. solanacearum
under greenhouse conditions.
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