EVALUATION OF GLIRICIDIA SEPIUM (KAKAWATE) LEAF EXTRACT AS A

POTENTIAL BIOCONTROL AGENT AGAINST FUSARIUM OXYSPORUM F. SP. CUBENSE

TROPICAL RACE 4 IN CAVENDISH BANANA
G. O. GINER, M. M. MABAYLAN, N. J. L. SISON
CATEGORY: LIFE SCIENCE
SUBCATEGORY: AGRICULTURAL SCIENCES
REGION, CITY/MUNICIPALITY: REGION XI, PANABO CITY
CORRESPONDING AUTHOR’S EMAIL: g.giner1897@gmail.com

ABSTRACT

Commercial fungicides are environmentally harmful and biologically invasive. Thus organic fungicides are
essential. The study was conducted to evaluate the potential of Gliricidia sepium (Kakawate) leaf extract in
controlling the radial growth of Fusarium oxysporum f. sp. cubense tropical race 4 causing Fusarium wilt of banana
and compare it with commercial treatment – 33% Formalin.
A Complete Randomized Design was used, having six treatments: T 0 – PDA with SDW, T1 – 25% G.
sepium leaf extract, T2 – 50% G. sepium leaf extract,T3 – 75% G. sepium leaf extract, T4 – 100% G. sepium leaf
extract and T5 – 33% Formalin, replicated thrice per treatment. The experiment employed poisoned food technique
and was observed for seven days. Data for radial growth and percent of inhibition were analyzed using Analysis of
Variance (ANOVA) and Tukey’s test.
Results showed that G. sepium leaf extract is an effective organic-based fungicide against the radial growth
of Foc TR4. 25% and 50% concentrations of G. sepium leaf extract has significantly decreased the radial growth
and percent of inhibition of Foc TR4 while the 75% and 100% concentrations having the most potent results, on par
with the T5 – 33% Formalin.
G. sepium yielded far promising results to combat Foc TR4 and is a viable substitute to commercial
fungicides because it has antifungal properties that outpaces the growth of Foc TR4.

of bananas, such as the Fusarium wilt, is a concern to

INTRODUCTION all banana growers and producers.

Fusarium wilt is one of the major
problems faced by banana growers in the Philippines
and all over the world (Onyago, 2005). This disease
is caused by Fusarium oxysporum f. sp. cubense, a
typical vascular disease, infecting banana crops
resulting to huge economic losses (Daly and
Walduck, 2006). The fungus infects through roots,
colonizes the rhizomes and eventually blocks the
vascular system of the pseudostems, resulting in plant
death (Visser et al, 2004). FOC is consisted of four
races previously identified (Vicente, 2004) but
tropical race 4 is the most virulent of all (Sutherland,
2013). This affects 80% of the total production of
bananas all over the world, mostly the Cavendish
variety (Van Brunschot, 2006)
With revenues of more than 260 million US
dollars annually, banana cultivar Cavendish is the
second top fruit export (Macabaso, 2011), a
consistent dollar earner (BOI, 2009), and is the
country’s second major agricultural export industry
(BAS, 2013), thus diseases that affect the production
Exporters are pushing for intensified actions
against diseases such as Fusarium wilt affecting
banana production in Mindanao (Sunstar, September
2013). Due to environmental hazards caused by the
chemical fungicides, farmers are encouraged to use a
safer and environmental friendly bio-control agent,
utilizing plant raw materials.
Gliricidia sepium known as Kakawate
(common), Madre de Cacao (local) is a medium-size,
semi-deciduous tree that typically grows to 10 m (33
ft) (occasionally reaching 15 m (50 ft)) in height,
with a broad canopy (Nazli et al., 2011).
According to the Agroforestry Database in
2009 and to De Boer et al (2005), G. sepium
(Kakawate) has been shown to have high antifungal
activity. G. sepium contains various phytochemicals
like flavonoids, sterols, alkaloids, glycosides, tannins,
saponins,(Sahin et al, 2004) medicarpin, coumarin,
and coumaric acid have been isolated and
characterized from the plant that showed antifungal
and antibacterial properties (Jose and Reddy, 2010). C. Preparation of the Laboratory Equipment

Phytcohemicals such as flavonoids,
effectively controls fungal pathogen such as
Fusarium oxysporum f. sp. cubense (Terreros and
Adriana., 2009). Medicarpin increases the release of
phytoalexins (Soby et al., 2006) which is released by
plants to combat pathogenic infection (Hasegawa et
al., 2010). Coumarin and coumaric acids both
prevent the entrance of nutrients into a fungal cell
(Geweelly, 2009) thus resulting in the fungus’s cell
lysis or death (Alvim et al., 2005). Saponins causes
loss of membrane integrity (Zhang and Xu, 2006) and
is lethal to fungal pathogens (De Lucca et al., 2006)
The safety cabinet and other materials were
cleaned and disinfected using 70% ethyl alcohol. The
laboratory glass wares were washed with running tap
water and detergent solution and autoclaved for 30
minutes under 1210C to make sure there were no
other microorganisms that might contaminate the
result..
D. Collection of Samples

For this reason, the study was conducted to

evaluate G. sepium leaf extract in controlling the
radial growth and percent of inhibition of Foc TR4
and to compare its efficacy with the commercial
treatment-33% Formalin.

This study develops possible substitutes to

commercial fungicides that can effectively outpace
Fusarium wilt in bananas. This organic fungicide
offers a safer substitute for synthetic fungicides that
poses biohazards. The outcome of the study is
beneficial and provides a new source of information
on the potent antifungal properties of G. sepium leaf
extract which introduces effective, cheaper, and an
organic way to outpace Foc TR4.
METHODOLOGY
A. Location and Duration of the Study
Figure 1. G. sepium leaves
Fresh leaves of G. sepium were collected
from a secluded backyard in Brgy Cagangohan,
Panabo City on September 14, 2013. Representative
plant was submitted to the Department of Agriculture
- Agricultural Training Institute for proper plant
identification.
Formalin, a commercial fungicide was
provided by the Plant Pathology Section of Stanfilco
Research Technical Services (STARTECHS), DOLE
Biotech compound

The study was conducted at Plant Pathology E. Extraction of C. maxima rind

Section of Stanfilco Research Technical Services
(STARTECHS) at the Dole Biotech Compound,
Brgy. Alejal, Carmen, Davao del Norte. It was
conducted on August 8, 2013 – September 28, 2013.

B. Experimental Design and Data Analysis

The study was laid out in a Complete

Randomized Design (CRD) with six treatments
replicated three times. The following treatments
were:
T0- Potato Dextrose Agar (PDA) with Sterilized
Distilled Water (SDW)
T1- 25% G. sepium (Kakawate) leaf extract solution
T2- 50% G. sepium (Kakawate) leaf extract solution
T3- 75% G. sepium (Kakawate) leaf extract solution
T4- 100% G. sepium (Kakawate) leaf extract
T5 - 33% Formalin (Commercial treatment)
Figure 2. C. maxima rind extract
G. sepium was prepared and weighed
using a triple-beam balance. Two kilograms of G.
sepium leaves were then soaked in one liter of 95%
ethanol for 48 hours in a sealed and airtight
container. After 48 hours, the soaked leaves were
then submitted to DOST Laboratory at Davao City
for a Rotary Evaporation extraction and
phytochemical analyses at University of Immaculate
Conception. Two kilograms of G. sepium leaf can
produce 1.1 L of extract.

F. Phytochemical Analysis

G. sepium leaves were analyzed at UIC-SRC and

at the Department of Science and Technology,
Industrial Technology Development Institute –
Standards and Testing Division. Both analyses were
performed by the personnel of the UIC – SRC and Figure 3. Cultured F. oxysporum f. sp. cubense TR4
DOST ITDI-STD separately. Experimentation

G. Preparation of Different Treatments

The different treatments were prepared using the

required volume of G. sepium leaf extract to the
required volume of Potato Dextrose AGAR (PDA).
T0- Potato Dextrose Agar (PDA) with Sterilized
Distilled Water (SDW)
T1- 10 mL of 25% G. sepium (Kakawate) leaf extract
solution + 15 mL PDA
T2- 10 mL of 50% G. sepium (Kakawate) leaf extract
solution+ 15 mL PDA
T3- 10 mL of 75% G. sepium (Kakawate) leaf extract
solution + 15 mL PDA
T4- 10 mL of 100%G. sepium (Kakawate) leaf extract
+ 15 mL PDA
T5 - 33% Formalin (Commercial treatment)
H. Preparation of Culture Media mixed with
Specific Treatments
The culture media was prepared by dissolving
125 grams of PDA for 500 mL of distilled water in a
container provided with magnetic stirrer. 100 mL of
the culture media was then subjected to sterilization
by autoclaving at 1210 Celsius at 15 psi. Specified
amounts of leaf extracts and commercial fungicides
were added and allowed to cool off for 20 minutes at
450C before dispensing it into petri plates.
I. Preparation of the Cultured F. oxysporum f.
sp. cubense Tropical Race 4
One week-old pure culture of F. oxysporum
f. sp. cubense tr. 4 (fig. 8) was provided by the Plant
Pathology Section of Stanfilco Research Technical
Services (STARTECHS) at the Dole Biotech
Compound, Brgy. Alejal, Carmen, Davao del Norte
Figure 4. Mixing of G. sepium extract with PDA
The study employed poisoned food technique,
where the fungi will be intoxicated with the nutrient
medium (Singh et al, 2005). Using a sterilized cork
borer, an agar disc was cut into the week-old sample
of FOC and placed in a separate petri dish. 15 mL
PDA was combined with 10 mL G. sepium extract
and gently mixed by swirling the test tube. 3.75 ml
lactic acid was then added to the test tube to lessen
contamination after which the solution was placed in
the petri dish. The culture disc was aseptically placed
at the center of the petri plate. Then, the petri plates
were properly sealed and stored in an isolation room
for seven days of observation.
J. Observation and Data Gathering
Radial Growth
The researchers observed the onset of radial growth
and measured its growth in mm after the seven-day
inoculation period. This refers to the growth of the
fungus on the culture medium in its cross-sectional
diameter in millimeter (N). After seven days of
incubation period, the radial growth was observed
and calculated using the following formula (Johnson
and Sekhar, 2012):
 [(N1+N2)/2]2(0.8) – 8 = Radial Growth (mm2)
Percent of Inhibition of the growth of the fungal
pathogen by the treatment
The cultures were observed for seven days.
Percent inhibition was determined using the formula
of Johnson & Sekhar (2012);

Where C represents the radial growth in the control

and T represents the radial growth on the treated
samples.
K. Disposal of the experimental FocTR4
treated with different concentrations of G.
sepium
Figure 5. . Radial growth of F. oxysporum f. sp.
cubense TR4 on different concentrations of G.
sepium (Kakawate) extract for duration of
observation within 7 days.

The pure cultured and the experimental Foc

TR4 was autoclaved under 121 psi for 15 minutes.
Then, it was disposed by the authorized Plant
Pathology personnel in the company’s disposing
center.

H. Disposal of the Extract

Leftover of G. sepium leaf extract was

sprayed over a large open area with plants, away
from waterways and creeks. Since the extract is
organic, it doesn’t pose harm to the surrounding
plants and animals.
Figure 6. Percent of Inhibition of G. sepium
(Kakawate) against the growth of Foc TR 4
RESULTS AND DISCUSSION
The study yielded three important results
summarized in the Table 1.

Treatments Radial Percent of No. of

Growth1 Inhibition1 Days
(mm2) (Radial
Growth)
T0 1217.20d 0c 3b
T1 412.87c 66.08b 4c
T2 146.20b 87.99b 5d
T3 0a 100a 0a
T4 0a 100a 0a Figure 7. Number of days before Radial Growth
T5 0 a
100 a
0 a onset.

** ** **
**- Means in column having the same letter
superscripts are not significantly different at 5% level
by HSD.
1
Taken after seven days of incubation.

Figure 8. T0-PDA with SDW
Figure 9. T1-25% G. sepium concentration
Figure 10. T2-50% G. sepium concentration
Figure 11. T3-75% G. sepium concentration
Figure 12. T4-100% G. sepium concentration
Figure 13. T5-33% Formalin
Radial Growth
As shown on Table 1, graphically presented
in Figure 5, radial growth was significantly decreased
even at 25% leaf extract (T1) with radial growth of
412.87 mm2 as compared to the To (PDA with SDW)
at radial growth of 1217.20 mm2
Increasing concentrations of the leaf extract
resulted in a great decrease of radial growth (T2, T3,
and T4), the most notable being T3 and T4. The
concentrations T3 and T4 exhibited no radial growth
of Fusarium oxysporum f. sp. cubense Tropical Race
4. Interestingly, T3 (75% leaf extract) and T4 (100%
leaf extract) have the same effectiveness with the T5
(33% Formalin) which is the control. This means that
G. sepium (Kakawate) leaf extract is very effective in
controlling the radial growth of F. oxysporum f. sp.
cubense tropical race 4.
There is significant difference as revealed by
Tukey’s test. The test showed that among the
following treatments: T0, T1 and T2 have significant
difference while T3,T4 and T5 are not significant. This
means that T3 and T4 (75% and 100% of G. sepium
leaf extract respectively) have comparable effect in
T5 (commercial fungicide) in controlling the radial
growth of Fusarium oxysporum f. sp cubense tropical
race 4.
Percent of inhibition
The results indicated that with increasing
concentration of Kakawate leaf extracts, the higher
the percent of inhibition.
It also shows that percent of inhibition of G.
sepium (Kakawate) leaf extract against the fungus –
Fusarium oxysporum f. sp cubense Tropical Race 4
on T3 and T4 is the same with T5, which is the control.
It shows that T3, and T4 has 100% inhibition of the
fungal pathogen.
Analysis of variance revealed that there are
highly significant differences with the treatment
means. Using Tukey’s test, it revealed that T0, T1 and
T2 were significantly different from one another at
5% level of significance while T3 and T4 are
statistically the same at 5% level of significance.
No. of Days
The number of days before radial growth
commenced increased significantly as the
concentrations of G. sepium leaf extract was
increased, excluding the T3 and T4. Results also
showed that even at 25% concentration of G. sepium
can delay the radial growth for a day, 50%
concentration can delay two days, while the 75% and
100% concentrations can completely prevent the
growth of the fungal pathogen. This means that G.
sepium leaf extract is effective in controlling the
radial growth of Fusarium oxysporum f. sp cubense
tropical race 4. Its efficacy is inversely proportional,
the higher the concentration the lesser the survival of
FOC as the days getting longer. This is because of the
presence of phytochemicals such as flavonoids,
saponins medicarpin, coumarins, and coumaric acid
that acted as a pathogen resistance (Adetuyi F. C. et
al, 2012).
 Phytochemical analyses revealed that G.
sepium contains flavonoids, sterols, alkaloids,
glycosides, tannins and saponins,.These compounds
hold the most possible mechanisms in suppressing
the radial growth and the percentage of inhibition
while delaying the onset of the occurrence of
Fusarium oxysporum f. sp. cubense Tropical Race 4.
Flavonoid acts as a powerful antioxidant (Hussin et
al., 2009) and is cytotoxic to fungal cells (Plochmann
et al., 2007). Sterol and alkaloids induces necrosis or
death of the tissues (O’Brien, 2000) and inhibits cell
proliferation in fungal pathogens (Corcoran and
Scott, 2006). Glycosides are readily converted to
biologically active compounds in response to
pathogen attack (Lattanzio, 2006) while tannins
inhibit extracellular hydrolases from invading
pathogens, thus preventing their rapid development in
the cell (Onyilagha, 2004). Saponins will intoxicate
the cell (Figen Mert-TŸrk, 2005), arresting the cell in
mitosis (Matson, 2011) thus the cell cannot reproduce
(Blagonsky, 2007).
Several studies also revealed that G. sepium
has medicarpin which exhibited potent anti-fungal
activity towards Fusarium spores, by inhibiting their
germination and hyphal growth, (Cherif et al., 2007)
and has cytotoxic abilities (Ngamrojanavanich et al.,
2007). Coumarins, specifically esculetin, inhibit cell
growth and cell cycle progression by inducing arrest
of the G1 phase in cells. (Lacy, A. and O’Kennedy R.,
2004). Coumaric acid, on the other hand, shows
strong antioxidant properties (Urbaniak et al.,2012).
CONCLUSION
Based on the results of the study, G. sepium
is effective organic based fungicide in outpacing the
radial growth, increases the percent of inhibition and
delays the onset of fungal growth of Fusarium
oxysporum f. sp. cubense TR4.
Recommendation
Based on the formulated
conclusions, the following recommendations were
deduced:
A. It is recommended to use G. sepium
(Kakawate) leaf extract as an organic based
fungicide against the growth of F.
oxysporum f. sp. Cubense tropical race 4
causing Fusarium wilt of banana
B. It is recommended to use Gliricidia sepium
(Kakawate) leaf extract to mix up with other
potential plant parts such as bark, roots and
flowers.
C. It is recommended to conduct further study
to Gliricidia sepium (Kakawate) leaf extract
as an organic based biocontrol agent in
preventing and controlling the growth of
other fungal pathogens in mangoes and
pineapples.
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