Cogon grass (Imperata cylindrica) and Madre de Cacao (Gliricidia sepium)

Leaves Ethanolic Crude Extract as a Potential Anti-Bacterial

Agent Against Xanthomonas oryzae pv. Oryzae,
Causing Bacterial Blight Disease on Rice

A Science Investigatory Project

Submitted to the Science and Technology Department
In Fulfillment of the Requirements
In Research II

Submitted by:
Ariana Marie Alinsunurin
Abigail Articona
Florence Bernette Doctora
Shem Rose Gaudite

Submitted to:
Mrs. Cynthia Doral Laurente
Research IV Teacher
Ramon Torres National High School
Bago City
Problem Being Addressed

Rice Plantation and farming, the most common and important industries in the country,
especially in Negros. According to the information given by Agrimag, The Department of
Agriculture (DA) awarded Bago City, Negros Occidental as the top rice-producing city of
Region VI at the Rice Achievers Awards (RAA) at the Philippine International Convention
Center, Pasay City last May 30.

Despite these achievements, diseases and manifestation of pests are one of the major
concerns in the rice industry. On a report done by Guadalquiver 2019, she stated that, “Rice
farmers in Negros Occidental suffered losses amounting to PHP11.544 million brought by
various crop pests in the past months. A report released by the Office of the Provincial
Agriculturist (OPA) showed the infestations were caused by rice black bugs, rats, and bacterial
leaf blight between the period November 2018 and February 2019.”

One of the disease mentioned is the Rice bacterial blight, also called bacterial blight of rice,
deadly bacterial disease that is among the most destructive afflictions of cultivated rice. In
severe epidemics, crop loss may be as high as 75 percent, and millions of hectares of rice are
infected annually (Yen). According to a Related Literature, students from Doña Hortencia, La
Carlota City, stated that Negros is facing a lot of problems regarding the Blight Disease. In
addition to this, according to an article written by Yen on the website Britannica, methods of
controlling rice bacterial blight are limited ineffectiveness. Chemical control has been largely
ineffective in minimizing bacterial blight because of safety concerns, practicality, and bacterial
resistance. That is why the researchers wants to test the potential of Cogon grass (Imperata
cylindrica) and Madre de Cacao (Gliricidia sepium) Leaves Ethanolic Crude Extract in
controlling the growth of Xanthomonas oryzae pv. Oryzae,that causes Bacterial Blight Disease
on Rice.
B. Goals and Expected Outcomes/ Hypothesis

The main objective of this study is to determine the effect of Cogon grass and Madre de
Cacao Leaves Ethanolic Crude Extract on controlling the growth of Xanthomonas oryzae pv.
Oryzae, that causes Bacterial Blight Disease on Rice. In specific, this study aims:

 To produce an alternative Anti-bacterial agent using Cogon grass and Madre de Cacao
Leaves Ethanolic Crude Extract
 To measure the bacteria`s radial growth upon the application of the extract
 To measure the bacteria`s percent of inhibition upon the application of the extract
 To compare its effect on the commercial ones
 To determine if there is a significant difference on the bacteria`s radial growth and
percent of inhibition on, TREATMENT A- 10 ml Cogon grass extract ,TREATMENT
B- 10 ml Madre de Cacao Leaves Extract ,TREATMENT C- 5 ml Cogon grass extract
and 5 ml Madre de Cacao Leaves Extract ,TREATMENT D- 2.5 ml Cogon grass extract
and 7.5 ml Madre de Cacao Leaves Extract ,TREATMENT E- 2.5 ml Madre de
Cacao Leaves Extract and 7.5 Cogon grass extract ,TREATMENT F- 10 ml
commercial product, and TREATMENT G- 10 ml sterilized Distilled water .

Significance Of The Study

This study bears the following importance:

The result of this investigation will provide agriculturists and farmers information that Cogon
grass extract and Madre de Cacao Leaves Extract can be used to control the growth of the
bacteria. Also as a way of promoting organic farming using a natural control system rather
than using commercial ones that can cause environmental and health problems. The result
of this data would provide for researchers to conduct other similar studies using other variables
found in this study.
C. METHODOLOGY

A. Research Design

Control group design will be used in this study with five experimental groups
(varying proportion of Cogon grass and Madre de Cacao leaves extract) and two control
groups (commercial one and distilled water). The experiment will be laid in Completely
Randomized Design (CRD) with seven treatments replicated three times for a total of 21
experimental units. The zone of inhibition and the radial growth of the bacteria applied with the
varying leaf extract proportions, commercial one (positive control) and distilled water
(negative control) will be measured and compared after the implementation of
intervention. Between group differences will be used to determine treatment effects.

B. Materials and Equipments

The leaves of Madre de Cacao and Cogon grass will be collected at Sta. Catalina
Phase 4, Brgy. Lag-Asan , Bago City. The Commercial antibacterial agent (still unknown)
will be bought at DC CRUZ TRADING. Other equipments and materials such as 4
graduated cylinders , 21 test tubes, 2 beakers, stirring rod, ethanol, and analytical balance
will be provided by the institution in which the researchers will do the extraction method, 21
petri dishes, 7 spray bottles, vials, hand gloves, face mask, and laboratory gown will be
bought at local drug stores. The bacteria sample will be gathered at Philrice Negros.

C. Procedure

C1. Drying of Cogon grass and Madre de Cacao leaves

The collected Cogon grass and Madre de Cacao leaves will first be sun dried. After
the 3 days of drying, the dried leaves will be brought at the institution in which the
researchers will continue the experimentation, the leaves will then be cut into smaller pieces,
and will be dried again for 2 days, but this time they will use a special machine

C2. Adding of Ethanol

 The dried leaves will be transferred into separate beakers. The beaker containing
Cogon grass will be combined with ethanol and the beaker containing Madre de Cacao will
also be combined with ethanol . After that, the beakers will be covered and will be
sealed properly and it will also be labeled according to the name of the plant. The dried
leaves will be soaked with ethanol for 2 days.

C3. Extraction of the leaves

The leaves will be extracted using a Rotary Evaporator.

C4. Sterilization of test tubes, and petri dishes

The petri dishes and test tubes will be placed on boiled water and this will be
done to kill all the bacteria present in it.

C5. Preparation of Treatments

The culture medium will be provided by the laboratory in which the researchers will
conduct the experiment. The researchers will measure the Cogon Grass and Madre de Cacao
extract using a graduated cylinder. Specific ratio of Cogon Grass extract and Madre de
Cacao extract, distilled water and commercial agent will be added to each of the 15 ml
culture medium and will allow to cool off for 20 minutes at 45 degree celsius before
dispensing it into Petri Dishes. Seven set-ups will be prepared with three replicates.

 TREATMENT A- 10 ml Cogon grass extract + 15 ml culture medium

 TREATMENT B- 10 ml Madre de Cacao Leaves Extract + 15 ml culture medium
 TREATMENT C- 5 ml Cogon grass extract and 5 ml Madre de Cacao Leaves Extract
+ 15 ml culture medium
 TREATMENT D- 2.5 ml Cogon grass extract and 7.5 ml Madre de Cacao Leaves
Extract + 15 ml culture medium
 TREATMENT E- 2.5 ml Madre de Cacao Leaves Extract and 7.5 Cogon grass extract
+ 15 ml culture medium
 TREATMENT F- 10 ml commercial product + 15 ml culture medium
 TREATMENT G- 10 ml sterilized Distilled water + 15 ml culture medium
C6. Transferring of Xanthomonas oryzae pv. Oryzae to petri dishes

The researchers will be guided by the authorized personels of the laboratory in

which they will conduct the study.

C7. Data Collection

The researchers will measure the initial and final growth of the bacteria by taking
its width and length using the Vernier caliper. After the two days incubation period, the
radial growth and percent of inhibition of the bacteria will be calculated.

C8. Statistical tool

 The treatment MEAN AND STANDARD DEVIATION will be used to describe the
data as computed based on the three replications.
 One-way Analysis of Variance (ANOVA) will be used in the study to determine
whether there is a significant difference among the radial growth and percent of
inhibition of the bacteria using the different treatments.
 Duncan Multiple Range Test will be used to determine which of the means are
equal and which are significantly different.

C9. Proper Disposal

After the experiment, materials and equipment used in the experiment will be
washed thoroughly. The materials and equipment will be placed in their proper places. The
area that was used during the experiment will be properly sanitized.

D. REFERENCES:

(BAGO CITY “THE RICE GRANARY OF NEGROS OCCIDENTAL” AWARDED AS

TOP RICE-PRODUCING CITY IN WESTERN VISAYAS)
 Guadalquiver, N. (2019). Pests bring P11.5-M losses to NegOcc rice farmers. Philippine
News Agency.

Yen, H. (n.d.). bacterial blight of rice. encyclopedia Britannica.

Kritz, Ben. Pressreader. December 30, 2016.

https://www.pressreader.com/philippines/manila-times/20161230/281861528170493 (accessed
August 18, 2018)

"Sugar Cane Success Story, Philippines." NETAFIM, n.d.

SUBHANI, MUHAMMAD NASIR. "Efficacy of Various Fungicides against Sugarcane

Red Rot (Colletotrichum falcatum)." RESEARCH GATE, 2008.