In vitro and greenhouse inoculation of Pseudomonas fluorescens
against Rice blast fungus Magnaporthe grisea:
Potential Biological Control
CHAPTER I
THE PROBLEM AND A REVIEW OF THE RELATED LITERATURE
Introduction
Considering the fact that the Philippines is an agricultural country, one of its main
agricultural product is rice (Oryza sativa). Another fact is that a large part of diet of the
Filipino diet dwells on rice. Filipinos consume large amounts of rice that they still need to
import from nearby Asian countries.
With the scarcity of rice in some Philippine provinces, the presence of rice
pathogens attacking rice seedlings is a big burden not only for farmers, but for all
Filipinos as it threatens their way of living. The most destructive enemy of rice is
Magnaporthe grisea, a fungus that causes rice blast disease. It is estimated that rice blast,
is the leading cause of rice loss and is responsible each year fro killing enough rice to
feed the Filipino people.
As of now, M. grisea outbreaks in Philippine rice fields are controlled through the
application of expensive and health-hazardous fungicides.
With this, the researchers seeks to find out the Biological agent potentials of
Pseudomonas fluorescens against rice blast fungus Magnaporthe grisea.
Conceptual Framework and Schematic Diagram of the Study
The study, “In vitro and greenhouse inoculation of Pseudomonas fluorescens
against Rice blast fungus Magnaporthe grisea: Potential Biological Control”, was
based on the concept that fungi that causes diseases are antagonized by some bacteria
used to manage some soil and waterborne pathogens.
The study is focused on the concept that P. fluorescens has antagonistic growth
potentials for rice blast fungus M. grisea. The study will undergo in vitro and greenhouse
applications.
Schematically shown on Figure 1 is the thrust of this study.
Potential
Biological Control
In vitro application
Pseudomonas
fluorescens
In vitro inhibition
assays
Rice blast fungus
Measuring the zones
of inhibition
Magnaporthe
grisea
Greenhouse
application
Test Subject
Bioassay for bio
control of plant
pathogen
Rice Seedlings
(Oryza sativa)
Inoculation of M.
grisea in greenhouse
Figure 1. Schematic Diagram of the Study
Biological Pest
Control
Statement of the Problem
This study aims to achieve one main objective this is to determine if
Pseudomonas fluorescens is a potential biological pest control against rice blast fungus
Magnaporthe grisea under in vitro and greenhouse experimentation.
Specifically, this study seeks to answer the following questions
 Is
Pseudomonas fluorescens effective biological control against rice blast
fungus Magnaporthe grisea?
 Is
there a significant difference between the rate of growth inhibition of
Magnaporthe grisea if commercial fungicides or biological control agent
Pseudomonas fluorescens are applied?
Significance of the Study
The study tends to answer the current problem, to determine if Pseudomonas
fluorescens is a potential biological pest control against rice blast fungus Magnaporthe
grisea under in vitro and greenhouse experimentation.
The realization of this study will help most Filipino farmers who are suffering
from M. grisea outbreaks within their rice fields. This study will also answer the rice
scarcity in some Philippine provinces. If taken into consideration, this study will stop M.
grisea outbreaks and replace hazardous and expensive fungicides in the market.
Scope and Limitation
This study is only limited on the in vitro and greenhouse application of
Pseudomonas fluorescens against rice blast fungus Magnaporthe grisea. This study will
be conducted at the Philippine Rice Research Institute – CARAGA situated in Agusan
del Norte and at the Department of Environment and Natural Resources – Surigao City
Chapter.
CHAPTER II
METHOD
EXERIMENTAL DESIGN AND TREATMENTS
The experiment was laid out in a Randomized Complete Block Design(RCBD)
wherein the treatments were replicated three times. The treatments were as follows:
In vitro assessment
1. Control – M. grisea with no applied treatments
2. M. grisea treated with commercial fungicides
3. M. grisea treated with potential Biocontrol agent P. fluorescens.
Greenhouse application
1. Control – Rice
seedlings, inoculated with M. grisea, with no
applied treatments
2. Rice seedlings, inoculated with M. grisea, treated with commercial
fungicides.
3. Rice seedlings, inoculated with M. grisea, treated with potential
Biocontrol agent P. fluorescens.
PARTICIPANTS
Magnaporthe grisea
Magnaporthe grisea
M. grisea is an ascomycete fungus. It is
an extremely effective plant pathogen as it can
A conidium and conidiogenous cell of M.
grisea
Scientific classification
Kingdom: Fungi
reproduce both sexually and asexually to
produce
specialized
infectious
structures
known as appressoria that infect aerial tissues
and hyphae that can infect root tissues. Also
Phylum:
Ascomycota
Class:
Sordariomycetes
commonly known as rice blast fungus, rice
Order:
Sordariomycetes incertae
sedis
rotten neck, rice seedling blight, blast of rice,
Family:
Magnaporthaceae
Genus:
Magnaporthe
Species:
M. grisea
oval leaf spot of graminea, pitting disease,
ryegrass blast, and johnson spot, is a plantpathogenic fungus that causes an important
disease affecting rice.
Binomial name
Magnaporthe grisea
(T.T. Hebert) M.E. Barr
Pseudomonas fluorescens
P. fluorescens has multiple flagella. It has an
extremely versatile metabolism, and can be found in the
soil and in water. It is an obligate aerobe but certain
strains are capable of using nitrate instead of oxygen as a
final electron acceptor during cellular respiration.
Pseudomonas fluorescens
Scientific classification
Kingdom: Bacteria
Phylum:
Proteobacteria
Class:
Gamma
Proteobacteria
Order:
Pseudomonadales
Some P. fluorescens strains (CHA0 or Pf-5 for
Family:
Pseudomonadaceae
example) present biocontrol properties, protecting the
Genus:
Pseudomonas
roots of some plant species against parasitic fungi such as
Species:
P. fluorescens
Fusarium or Pythium, as well as some phytophagous
nematodes
Binomial name
Pseudomonas fluorescens
(Flügge 1886)
Migula, 1895
DATA ANALYSIS
Using the single factor ANOVA, the results of the experiment were analyzed tabulated
and plotted in a graph. The results were used in the interpretation of the zones of inhibition in
order to determine if the chosen variables are effective as Biocontrol agent against rice blast
fungus M. grisea.
PROCEDURE
Sample Collection
Rice seedlings was secured from the Department of Environment and Natural Resources
and was brought to the DENR greenhouse. Rice blast pathogen Magnaporthe grisea and
potential biocontrol agent Pseudomonas fluorescens were secured from the Philippine National
Collection of Microorganisms(PNCM) and the International Rice Research Institute (IRRI)
respectively from the University of the Philippines – Los Baños, Laguna. The test participants
were then brought to the Caraga Regional Science High School Bio. Chem Laboratory for
experimentation and analysis and was kept at room temperature.
External Morphological Observation
External Morpholological observation of the rice seedlings (Oryza sativa) were done and
taken into consideration. It was made sure that no signs or strains of the rice blast fungus M.
grisea or any plant pathogens are present in the rice seedlings.
Sterilization
All the instruments and laboratory apparatuses used in the conduct of the experimentation
were thoroughly sterilized. Glassware were wrapped in white bond paper and was placed in the
hot oven for about 120 ° C for 2 hours.
A
Bioassay for Biological Control of Plant Pathogen
Rice seedlings were continually grown in the greenhouse and inoculated with
Magnaporthe grisea under conditions optimal for the development of disease
Measuring the growth of M. grisea
Using a Vernier caliper, the length of infection of M. grisea in each treatment
were measured after 3 weeks of growth. The size of the rice blast fungal infection in the
rice seedlings were thoroughly observed among the different treatments. Observations
were then recorded and tabulated in a graph.
B
In Vitro Inhibition Assay
Different bacterial isolated treatments were tested for their ability to inhibit the
growth of M. grisea, a soil and waterborne plant pathogen that can cause rice blast and
blight. Zones of growth inhibition can be detected around strains placed at two of the four
positions on the plate.
Petri Dish Preparation
Petri dishes were prepared; 3 for the control set-up, 3 for the application of
commercial fungicides and 3 for the application of the potential biocontrol P. fluorescens.
Each Petri dish were divided into five (5) or six (6) quadrants and the test participants and
control were tested.
Incubation of the Petri Dishes with treatments
The Petri dishes with applied treatments were incubated at 30 ° C within 24 hours.
Reading the Zones of Inhibition
After 24 hours of incubation, zones of inhibition around the cup cylinders were
measured and recorded. Only the zones showing clear areas were measured for the
diameter using a Digital Vernier Caliper. The results were then tabulated and compared.
Decontamination and Disposal of Microbes
All the materials like laboratory glass wares, pipettes, used Petri dishes which
came in contact with the microorganisms tested in this study were washed thoroughly
with detergent and water. After such, all these materials were subjected to autoclaving at
maintained temperature at least 121 – 132 degrees Celcius for 30 – 40 minutes. Then the
contaminated materials were allowed to cool. The working area was cleaned and
disinfected with alcohol.
Data Gathering Technique
All factors that influenced the zones of inhibition of the selected microorganisms
were considered. Digital Vernier Caliper was used for gathering of data. The variable
indicator such as the zone of inhibition (length of the diameter measured in millimeter) in
each plant sample was carefully measured, recorded and tabulated.