Antibacterial Effect of Cattle Leech (Hirudinaria graulosa) Midgut Fluid Against

Xanthomonas oryzae pv. Oryzae

Proponents: *Macatangay, J. T., Villanueva, N. C., Escarnuela, A. E., Foronda, G. J. A.,

Gacutan, L. L. M., Go, A. B. H.

Research Adviser: Mr. Earl Francis Merilles

Life Science: Individual Category

______________________________________

Research Plan

A. Rationale

The world loses large percentage of crops caused by biotic stresses like pest, bacterial

disease, etc. One of which is the most important problems in rice, the Xanthomonas oryzae pv.

Oryzae, generator of the bacterial blight disease in rice crops (ex Ishimaya, 1922). As early as

1950’s, the Philippines has been declared of bacterial blight disease manifestation in rice (Oryza

sativa L.) produced by Xanthomonas oryzae pv. Oryzae (Mew T. et al., 1992) . In the Philippines,

the bacterial leaf blight causes the rice crop loss in wet season about generally 22% and roughly

7% in dry season (Exconde, 1973). The Xanthomonas oryzae pv. Oryzae penetrates over

hydathodes, stomata or wounds of the rice plant. Through mechanical contact, in irrigation water,

and in rain the bacteria are spread (Devadath & Dath, 1985). Once the leaf is infected by the

bacteria, it becomes greyish or whitish then eventually dies (Reddy, 1984). And because the

Philippines is close to the Pacific Ocean where the number of typhoons pass each year, it is

difficult for farmers to control the bacterial blight disease because the rain spreads the bacteria

Xanthomonas oryzae pv. Oryzae. in the yield.

 The chemical control of Xanthomonas oryzae pv. Oryzae developing bacterial leaf blight

disease was started with precautionary use of Bordeaux mixture (hydrated lime and copper

sulfate) and verification of several antibiotics, mercury and copper compounds (Mizukami and

Wakimoto, 1969). Laboratory examinations revealed that streptomycin derivates and mercuric

compounds are effective but damage rice grains when sprayed in the field at the heading stage

(Mizukami and Wakimoto, 1969). Nevertheless, in Asia’s tropical monsoon, chemical control is

impractical and no authentically bactericide is commercially available for disease control of

Xanthomonas oryzae pv. Oryzae (Lee et al., 2003; Ou, 1973). To make a contribute for the

chemical control of bacterial blight disease, the researchers found an antibacterial agent from a

parasite that may inhibit the growth of Xanthomonas oryzae pv. Oryzae.

Hirudinaria graulosa,

In the Philippines, leeches are considered pests and are usually shied upon. These creatures are not

edible and sometimes destroy crops. But, on the other hand, leeches have therapeutic and medicinal

uses. Leeches is extremely abundant worldwide, both in species and the number of its individuals. It

is a parasite that sucks the blood of its victim. The 25% of its species are the predator of small

vertebrates. Commonly, most species of leech are found in fresh water or terrestrial habitat and some

are found in the marine water (Aloto D et al., 2018).

Leeches is also used in medicine for bloodletting. Even today they are still used to reduce fluid

pressure in tissues caused by, for example, a snake bite. It is also used to eliminate pools of

coagulated blood (Aloto D et al., 2018). This leads the researchers to use the cattle leech for

eradicating one of the most destructive bacteria causing diseases in rice crop industry.
In the recent study, the midgut fluid of cattle leech has been known as an antibacterial agent to

both gram positive and gram negative bacteria (Galula, 2003). This resulted the researchers to

use the cattle leech’s midgut fluid against destructive bacteria Xanthomonas oryzae that causes

bacterial leaf blight disease in rice crops.

This study aims to determine if cattle leech midgut fluid has an effect and has the potential to

eliminate Xanthomonas oryzae pv; to find an alternative way to give solution to the rice

industry's problems, helping improve economic production and to support human needs

properly.

B. Engineering Goals/Expected Outcomes/Hypotheses

B.1. Engineering Goals

Generally, the current study will determine the antibacterial property, effectiveness and

the amount of Cattle Leech Mdgut fluid to eliminate Xanthomonas oryzae.

Specifically, the study will find solutions to the following questions:

• How will the antibacterial compound found in the cattle leech midgut fluid affect the

Xanthomonas oryzae?

• How long will the antibacterial compound be effective against the Xanthomonas

oryzae?

• How long will it take for the antibacterial compound to affect the Xanthomonas oryzae?

• How will the application of the antibacterial compound affect the Xanthomonas oryzae

in terms of time of growth?

 • What does the antibacterial compound contain that could possibly eliminate

Xanthomonas oryzae that causes bacterial blight?

• What are the harmful side effects upon the application of the antibacterial compound?

The current study will investigate the effectivity of the antibacterial compound found in

Cattle Leech Midgut fluid in eliminating Xanthomonas oryzae. This may be used to

improve the production of rice by treating the bacterial blight caused by Xanthomonas

oryzae.

B.2. Expected Outcomes

The outcome of this study will be also useful to the following:

Benefits the farmers. The study will help the farmers to increase the yields of crops and

reduce the number of infected crops caused by Xanthomonas oryzae.

Suppliers and Consumers. The application of the study will help the suppliers in

producing more rice. The consumers will also benefit for there will no be rice shortage.

B.3. Hypotheses

The current study will be guided by the following hypotheses:

• Null Hypothesis

There is no antibacterial effect in the Cattle Leech Midgut fluid that can eliminate

Xanthomonas oryzae causing bacterial blight.

 • Alternative Hypothesis

There is an antibacterial effect in the Cattle Leech Midgut fluid that can eliminate

Xanthomonas oryzae causing bacterial blight.

• Cause and Effect

If the Cattle Leech Midgut fluid is applied to the Xanthomonas oryzae, then its

antibacterial compound has the potential to inhibit the growth of Xanthomonas oryzae.

C. Procedures

C.1 Collection of Materials

Large mature cattle leeches will be cultured and collected at Philippine Bureau of Plant

Industry, Manila. Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf

blight will be collected from the diseased plants of rice. The specimens will be prepared

for isolation. While polymyxin B will be bought for commercial antibacterial agent.

C.2 Isolation of Cattle leech Midgut Fluid

Large mature cattle leeches will be soaked in 15% ethanol and then will be dissected

under the binocular dissecting microscope. The fluids in the leech midgut will be

extracted using heparinized tubes, suctioned and will be secured in the sterilized bottles.

C.3 Isolation of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae will be isolated from the diseased plants of rice.

Infected leaf pieces of rice will be excised with sterile scalpel. The leaf surface will be

sterilized with 1% Clorox (Sodium hypochlorite) for three minutes and then will be

washed with sterile distilled water (SDW). Leaf pieces of rice leaves after drying on
 sterile blotting paper will be transferred to nutrient agar (NA) medium and will be

incubated at room temperature (25-27°C) for 72hrs (Jabeen et al., 2012).

C.4 Testing the effectiveness of Cattle Leech Midgut Fluid

The cattle leech midgut fluid with different solutions will be applied in each Xanthomonas

oryzae pv. oryzae and will be observed for 7 days. The results will be collected and recorded.

C.5 Collection of Data

Data such as the growth of the Xanthomonas oryzae pv. oryzae, the presence of midgut

fluid and the inhibition of the bacteria will be collected and interpreted.

Flowchart of procedures

Collection of Materials

Gathering mature cattle Collecting bacterial leaf blight-

leeches diseased rice plants

Preparation for Experiment

Isolating cattle leech Preparing midgut fluid Isolating Xanthomonas

midgut fluid with polymyxin B oryzae pv. oryzae
 Experimentation

Applying cattle leech midgut fluid

with different solutions in each
Xanthomonas oryzae pv. oryzae

Data Collection and Analysis

D. Research Design

Treatments Replicates

T1. 25% Cattle Leech Midgut Fluid

75% Polymixin B R5

T2. 50% Cattle Leech Midgut Fluid

50% Polymixin B R3

T3. 75% Cattle Leech Midgut Fluid

25% Polymixin B R4
 T4. 100% Polymixin B R1

T5. 100% Cattle Leech Midgut Fluid R2

E. Risk and Safety

The cattle leech midgut fluid may harm the researchers upon direct contact with skin and

eyes. Cattle leeches may harm the researchers during the experimentation.

Xanthomonas oryzae pv. oryzae is a bacteria that might harm the researchers when

mishandled. Supervisor’s guide and help will be needed in the whole experiment.

F. References

Yang, Ying. "Characterization of Broad-spectrum Antibiotic Resistance Genes in

Wastewater Treatment Reactors through Metagenomic Approaches."

doi:10.5353/th_b5312323.

Ghasemie, Elham, Mostafa Kazempour, and Ferydon Padasht. "Isolation and

Identification of Xathomonas Oryzae PV. Oryzae the Causal Agent of Bacterial Blight of

Rice in Iran." Journal of Plant Protection Research 48, no. 1 (2008). doi:10.2478/v10045-

008-0006-9.
 Aloto, Desta, and Eyob Eticha. "Leeches: A Review on Their Pathogenic and Beneficial

Effects." Journal of Veterinary Science & Technology 09, no. 01 (January 24, 2018).

doi:10.4172/2157-7579.1000511.

In the Philippines, leeches are considered pests and are usually shied upon. These creatures are not

edible and sometimes destroy crops. But, on the other hand, leeches have therapeutic and medicinal

uses. Leeches is extremely abundant worldwide, both in species and the number of its individuals. It

is a parasite that sucks the blood of its victim. The 25% of its species are the predator of small

vertebrates. Commonly, most species of leech are found in fresh water or terrestrial habitat and some

are found in the marine water (Aloto D et al., 2018).