AGR 554 - PRACTICAL REPORT

FACULTY OF PLANTATION AND AGROTECHNOLOGY
UNIVERSITI TEKNOLOGI MARA CAWANGAN MELAKA KAMPUS JASIN
BACHELOR OF SCIENCE AGROTECHNOLOGY (HONS) AGRONOMY
______________________________________________________________________
PROGRAM CODE : AT222
GROUP : M3AT222 5B
COURSE CODE : AGR 554
COURSE NAME : PLANT PATHOLOGY
SEMESTER : MARCH 2022 – JULY 2022
LECTURER’S NAME : MADAM NURUL WAHIDA BINTI RAMLI
SUBMISSION DATE : 22ND JULY 2022
GROUP MEMBER :
NAME STUDENT’S ID
ATIQAH BINTI ROSLAN 2020995321
NUR AINI BINTI MOHAMAD 2020991389
NURUL LYANA BINTI MOHAMAD KAMAL 2020982347
SITI NUR WAHIDAH BINTI BUSTAMAM 2020957813

TABLE OF CONTENT
1.0 PRACTICAL 1 – RECOGNITION OF DISEASE SYMPTOMS AND PREPARATIONS OF
CULTURE MEDIA ..................................................................................................................... 1
1.1 INTRODUCTION ......................................................................................................... 1
1.2 OBJECTIVES .............................................................................................................. 2
1.3 APPARATUS & MATERIALS....................................................................................... 2
1.4 METHODS................................................................................................................... 3
1.5 RESULTS/ OBSERVATIONS ...................................................................................... 4
1.6 DISCUSSION .............................................................................................................. 8
1.7 QUESTION.................................................................................................................. 9
1.8 CONCLUSION............................................................................................................10
1.9 REFERENCES ...........................................................................................................10
2.0 PRACTICAL 2 – COLONIES OF COMMON FUNGI AND BACTERIA ..........................12
2.1 INTRODUCTION ........................................................................................................12
2.2 OBJECTIVES .............................................................................................................13
2.3 APPARATUS & MATERIALS......................................................................................13
2.4 METHODS..................................................................................................................14
2.5 RESULTS/ OBSERVATIONS .....................................................................................11
2.6 DISCUSSIONS...........................................................................................................14
2.7 QUESTIONS...............................................................................................................15
2.8 CONCLUSIONS .........................................................................................................17
2.9 REFERENCES ...........................................................................................................18
3.0 PRACTICAL 3 – ISOLATIONS AND OBSERVATIONS OF PATHOGENIC BACTERIA
…………………………………………………………………………………………………….19
3.1 INTRODUCTION ........................................................................................................19
3.2 OBJECTIVES .............................................................................................................20
3.4 METHODS..................................................................................................................21
3.6 DISCUSSIONS...........................................................................................................28

3.7 CONCLUSIONS .........................................................................................................31
3.8 RECOMMENDATIONS...............................................................................................31
3.9 REFERENCES ...........................................................................................................32
4.0 PRACTICAL 4: KOCH POSTULATES (FUNGUL ISOLATION AND OBSERVATION).33
4.1 INTRODUCTION ........................................................................................................33
4.2 OBJECTIVES .............................................................................................................34
4.4 METHODS..................................................................................................................35
4.6 DISCUSSIONS...........................................................................................................37
4.7 QUESTIONS...............................................................................................................40
4.8 CONCLUSIONS .........................................................................................................42
4.9 RECOMMENDATIONS...............................................................................................42
4.10 REFERENCES ...........................................................................................................43
5.0 PRACTICAL 5: KOCH POSTULATES (INOCULATION OF PATHOGEN INTO
HEALTHY PLANT)...................................................................................................................44
5.1 INTRODUCTION ........................................................................................................44
5.2 OBJECTIVES .............................................................................................................45
5.4 METHODS..................................................................................................................46
5.6 DISCUSSIONS...........................................................................................................49
5.7 QUESTIONS...............................................................................................................50
5.8 CONCLUSIONS .........................................................................................................50
5.9 REFERENCES ...........................................................................................................51
6.0 PRACTICAL 6: PATHOGENICITY TEST (DISEASE SCORE, DISEASE SEVERITY,
AND VIRULENCE LEVEL).......................................................................................................52
6.1 INTRODUCTION ........................................................................................................52
6.2 OBJECTIVES .............................................................................................................53

6.4 METHODS..................................................................................................................54
6.6 DISCUSSIONS...........................................................................................................58
6.7 CONCLUSIONS .........................................................................................................59
6.8 REFERENCES ...........................................................................................................59
7.0 PRACTICAL 7: HOW PATHOGEN ATTACKS PLANT (FORMATION OF
APPRESSORIUM)....................................................................................................................60
7.1 INTRODUCTION ........................................................................................................60
7.2 OBJECTIVES .............................................................................................................61
7.4 METHODS..................................................................................................................62
7.6 DISCUSSIONS...........................................................................................................64
7.7 QUESTIONS...............................................................................................................65
7.8 CONCLUSIONS .........................................................................................................66
7.9 REFERENCES ...........................................................................................................66
8.0 PRACTICAL 8: TRICHODERMA (FUNGUS) AS BIOLOGICAL CONTROL AGENT)...67
8.1 INTRODUCTION ........................................................................................................67
8.2 OBJECTIVES .............................................................................................................68
8.4 METHODS..................................................................................................................69
8.6 DISCUSSIONS...........................................................................................................72
8.7 QUESTIONS...............................................................................................................73
8.8 CONCLUSIONS .........................................................................................................74
8.9 REFERENCES ...........................................................................................................74

1
1.0 PRACTICAL 1 – RECOGNITION OF DISEASE SYMPTOMS AND
PREPARATIONS OF CULTURE MEDIA
1.1 INTRODUCTION
A plant disease is referred to any change in the function or structure of a plant
that results in observable symptoms. Several plant diseases are easily identifiable
based on the host plant's unique symptoms, whereas others may not be. It is
required an organized diagnostic approach to identify. The identification of the
causative agent based on the production of symptoms is critical for preliminary
identification since different pathogens create diverse symptoms. We may observe
overlapping symptoms between fungal, bacterial, and viral illnesses based on
symptom observation. Culture media or known as growth media is an essential
component in plant disease detection since it is used to develop microbial pathogens
and achieve clean cultures. It provides nutrition, growth factors, and energy to the
microbial pathogen. Culture media are used in non-sterile raw material and finished
product quality control testing, as well as microbial contamination (sterility) testing in
applications such as hygiene monitoring, sterilization process verification, and
determining the effectiveness of preservatives and antimicrobial agents.

2
1.2 OBJECTIVES
➢ To identify the common signs of plant disease.
➢ To produce the fundamental medium for isolating fungi and bacteria
1.3 APPARATUS & MATERIALS
➢ Samples plant infected
a) Blight - Extensive area of diseased fruit or leaves.
b) Chlorosis - Yellow-green colour of foliage due to destruction or lack of
production of chlorophyll
c) Dieback - Generalized shoot death.
d) Gall - Swollen area of non-differentiated tissues (tumor) caused by an
infection.
e) Mosaic - Chlorotic pattern, ringspots, and mottles in leaves, petals,
f) Necrosis- Dead tissue.
g) Rot- Portion of plant destroyed by disease. Root rot is rotted roots.
h) Wilt- Loss of turgor in a plant or plant part.
➢ Agar medium with potato dextrose (1 liter)
➢ Agar medium in air (1 liter)
➢ Petri dish (120 pieces)
➢ Parafilm
➢ Tape

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1.4 METHODS
1. With the help from internet, every infected disease as stated in the materials &
apparatus were being search.
2. The main symptoms of the infected plant species were recorded.
3. For the second part, the media that has been prepared by lab assistant was
poured into the petri dish.
4. The media then were left to cooled by itself at room temperature.
5. After that, the petri dish was closed with it lids.
6. The closed petri dishes were then arranged in the plastic and being sealed with
tape.

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1.5 RESULTS/ OBSERVATIONS
1. General Symptoms of Plant Diseases
Disease
name
Picture Symptoms Possible
causal agent
Blight
Figure 1.5.1 Rice
bacterial leaf blight
➢ It promotes seedling wilting
as well as yellowing and
drying of leaves.
➢ The leaves become greyish
green and roll up when
infected.
Bacterium
xanthomonas
oryzae pv.
oryzae
Chlorosis
Figure 1.5.2 Chlorotic leaf
distortion
➢ The major sign of chlorosis is
yellowing leaves.
➢ Chlorotic plants may only
display symptoms on one or
two branches, or they may be
damaged throughout the
plant.
➢ The leaf tissue appears light
green, while the leaf veins
remain green.
➢ The leaf tissue is light white to
pale yellow, and the leaf size
is stunted.
➢ The leaf margins might be
burnt or develop dark,
angular patches between the
veins, and the leaves may
prematurely wither and drop.
Fungus,
Fusarium
denticulatum

5
Dieback
Figure 1.5.3 Mango
Dieback Disease
➢ The continuous and
downward drying out or dying
down of the twig/branch
➢ Usually, the entire branch
dies.
➢ Twig/branch/stem cankers
are also a common symptom.
Fungus
Lasiodiplodia
theobromae
Gall
Figure 1.5.4 Crown gall
➢ Roundish, rough-surfaced
galls (woody tumour like
growths),
➢ The galls are cream-colored
or greenish at first, then
become brown or black.
Bacterium
agrobacterium
tumefaciens
Mosaic
Figure 1.5.5 Bean
common mosaic virus
➢ On normally green leaves,
veins have an amorphous
mosaic pattern of bright
yellow, green, and dark
green stripes.
➢ Additionally, the foliage may
twist and wrinkling.
Potyvirus

6
Necrosis
Figure 1.5.6 Soybean
vein necrosis disease
➢ Demonstrate venous
cleansing (venous color
lightening).
➢ A chlorosis (jaundice).
➢ On the impacted leaves,
there are mosaic patterns
(i.e., spots of light and dark
color).
➢ Around the veins of the
leaves, symptoms first
appear before spreading
elsewhere.
➢ Brown veins and leaves
signal necrosis, which
signifies death.
Pathogens
Tospovirus
Rot
Figure 1.5.7 Fusarium
root rot
➢ The disease first shows as
red to reddish-brown lesions
on stems and main roots.
➢ Affected areas might merge
and eventually turn brown.
➢ Stunted or poor growth
Fungus
fusarium
solani f. sp.
phaseoli
Wilt
Figure 1.5.8 Vascular wilt
disease of tomato
➢ Wilting-The plants will begin
to wilt. It usually begins with a
single leaf or branch at the
top of the plant.
➢ Yellowing- Lower leaves will
become yellow, frequently
starting on only one side.
Fusarium
oxysporum f.
sp. lycopersici

7
Yellowing, like wilting, will
continue up the plant.
➢ Wilted Leaves-Wilted leaves
will dry and fall.
➢ Discolored Stem- dark brown
stripes on elongated stems.
2. Media preparation
Figure 1.5.9 Pour agar media into petri dish

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1.6 DISCUSSION
Pathogens or causative agents are hosts for the appearance of symptoms
that express disease, pests, or the environment. Primary symptoms (direct and
immediate modifications in tissues impacted by a pathogen as well as other
causative factor or secondary (indirect and later physiological effects on host tissue
caused by activity at a location other than the site of original infection). It is important
to understand that because symptoms are "host responses" to an irritant, a variety
of substances or even abiotic variables might create a certain symptom. Yellowing,
wilting, dieback, galls, and blight are examples of symptoms. Symptoms alone
should not be used to make a diagnosis because a symptom, such as wilting, might
be caused by several factors (drought, borer, root rot, etc.) Furthermore, symptoms
on one section of the plant may occur because of damage on another area of the
plant. Clearwing borer eating on the stem, for example, can produce leaf wilt.

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1.7 QUESTION
a) Explain the importance of symptoms observation for plant disease identification.
➢ If diseases and disease-causing substances are not adequately
recognized, disease control efforts can waste resources. This may
result higher losses in plant. As a result, accurate disease diagnosis is
essential. Plant pathologists frequently utilize symptoms to diagnose
disease concerns. A pathogen's identification is important for
developing a management strategy.
b) Determine three (3) types of agar media for fungal and bacterial identification.
Microorganism Agar Media
Fungal ➢ Potato Dextrose Agar General media
➢ Sabouraud Dextrose Agar General media
➢ Yeast Malt Agar General media
Bacteria ➢ Nutrient Agar General media
➢ Tryptic Soy Agar General media
➢ Brain Heart Infusion Agar General media

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1.8 CONCLUSION
As conclusion, diagnosis is a type of hypothesis testing in which the
hypothesis is simply the identified of the disease, and a great diagnostician goes
through several rounds of the scientific process. Plant infections and disorders
cause plants to struggle, kill them, or impair their capacity to live and reproduce.
Plant disease refers to any abnormal condition that affects the aspect or function of
a plant. Pathogens are responsible for plant diseases. As a result, a pathogen is
always related to a disease. Disease, in other words, is a symptom induced by the
invasion of a pathogen capable of survival, reproduction, and dissemination. Plant
disease diagnosis involves consideration of many biotic and abiotic elements that
may be implicated in disease pathogenesis, as well as a thorough understanding of
host plant symptoms and indications. Many factors can affect any condition,
including the host's status, cultural history, meteorological conditions, soil, and
general site features.
1.9 REFERENCES
1. AGR554 LABORATORY MANUAL: Lab manual 1: Recognition of disease
symptoms and preparation of culture media
2. Amar, H., & Road, S. (n.d.). Culture Media Preparation. Retrieved from How to
Make Growth Media Formulations:
https://www.mt.com/in/en/home/applications/Laboratory_weighing/Culture-
Media-Preparation.html#overviewaf
3. Chlorosis in trees & shrubs: symptoms, causes & treatment. (2020). Retrieved
from Independent tree: https://www.independenttree.com/chlorosis/
4. Chlorotic leaf distortion. (n.d.). Retrieved from Causal organism: Fusarium
denticulatum Nirenberg and O’Donnell:
https://keys.lucidcentral.org/keys/sweetpotato/key/Sweetpotato%20Diagnotes/
Media/Html/TheProblems/DiseasesFungal/ChloroticLeafDistortion/CLD.htm
5. Clark, C., Buckley, & Lori. (n.d.). Chlorotic Leaf Distortion. Retrieved from LSU
College of Agriculture:

11
https://www.lsuagcenter.com/topics/crops/sweet_potatoes/diseases/chlorotic-
leaf-distortion
6. Crown gall. (2022). Retrieved from University of minnesota extension:
https://extension.umn.edu/plant-diseases/crown-gall
7. Fusarium Root Rot. (n.d.). Retrieved from Utah Vegetable Production & Pest
Management Guide: https://extension.usu.edu/vegetableguide/tomato-pepper-
eggplant/fusarium-root-rot
8. Hauser, J. (2011). Techniques for Studying Bacteria and Fungi. Carolina
Biological Supply Company, 3-31.
9. International Rice Research Institute (IRRI). (n.d.). Retrieved from Bacterial
blight: http://www.knowledgebank.irri.org/decision-tools/rice-doctor/rice-doctor-
fact-sheets/item/bacterial-blight
10.Mango common dieback. (2021). Retrieved from Northern territory government:
https://industry.nt.gov.au/publications/primary-
industrypublications/newsletters/regional-newsletters/rural-review/nt-rural-
review-november-2021/mango-common-dieback
11.Schwartz, H., David H., G., Franc , G., & Harveson, R. (2016). High planis IPM.
Retrieved from Bugwoodwiki:
https://wiki.bugwood.org/HPIPM:Fusarium_Root_Rot
12.Sileshi, F., Galano, T., & Biri, B. (2016). Plant Disease Diagnosis Practical
Laboratory Manual. ResearchGate, 1-35.
13.Stokes, C., & Meadows, I. (2021). Fusarium Wilt of Tomato. Retrieved from NC
State Extension Publications: https://content.ces.ncsu.edu/fusarium-wilt-of-
tomato

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2.0 PRACTICAL 2 – COLONIES OF COMMON FUNGI AND BACTERIA
2.1 INTRODUCTION
When describing bacteria and fungi, morphological characteristics are crucial.
A useful technique frequently employed by scientists to recognize and describe them
is colonies’ morphology. The only way to observe bacteria is with a microscope since
they are tiny, microscopic organisms. They are prokaryotic creatures with only one
cell. They are invisible to our unaided vision. On agar media in Petri plates, it can be
observed when it forms colonies. A visible collection of bacterial cells growing on a
bed of solid agar is known as a bacterial colony. It is presumable that bacterial
colonies develop from a single bacterial cell and multiply into numerous bacteria by
binary cleavage. Millions of bacterial cells with a similar genetic makeup make up a
colony. Hence, when counting bacteria, bacterial colonies were used as a unit.
Yeast, filamentous fungi, and other microorganisms are included in the
category of eukaryotic organisms known as fungi. The fungus thrives in warm, humid
environments. Their morphological and molecular properties can be used to classify
them. Grow fungus on solid medium, such as potato dextrose agar, makes it simple
to detect morphological characteristics (PDA). Fungus that are frequently found in
laboratories are grown in PDAs as media. A fungus forms a colony when it develops
on solid media. Diverse varieties of fungi have different fungal colonies with different
morphologies. Fungal colonies can be used to study traits like pigmentation, texture,
and other attributes. Therefore, in this laboratory report, the various bacterial and
fungal colonies will be observed on agar media and the structure of fungus also will
be observed under a microscope.

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2.2 OBJECTIVES
➢ To examine various bacterial and fungal colonies on agar media.
➢ To examine the structure of fungus under a microscope.
➢ Fungi cultures (PDA) which is Fusarium species
➢ Bacteria cultures (Nutrient Agar)
➢ Light microscope
➢ Slides and cover slip
➢ Lactophenol
➢ Distilled water
➢ Inoculating needle

14
2.4 METHODS
1. With naked eyes, the fungi cultures (PDA) which is Fusarium species were
observed and recorded for its color, pigmentation and the size.
2. From the actively growing area, the small part of the Fusarium species was
taken by using inoculating needle and placed on the slide.
Figure 2.4.1 Inoculating loop
Figure 2.4.2 Fungus place on the slide
3. A drop of lactophenol and then a drop of distilled water was added on the slide.
4. A slide was covered with its cover slip.
Figure 2.4.3 Cover the slide with cover slip.
5. The structure under microscope was observed.
6. Under microscope, the shapes of the fungus spores which is Fusarium that has
been observed were recorded.
7. The color, pigmentation and the opacity of the species were recorde

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Sample Colony Spore Description
Figure 2.5.1 Fusarium
oxysporum.
Fungi colony ➢ Septate
Asexual spores
➢ Macroconidia:
Foot-shaped basal cells and
tapering, curved, thin-walled,
straight to slightly curved,
typically with three or four
septa.
➢ Microconidia:
Ellipsoidal and lacking a
septum. By basipetal division,
they are created from phialides
in the false head. In cases of
subsequent infections, they are
crucial.
➢ Chlamydospores:
The microbiota of the rhizosphere is
well-represented by Fusarium
oxysporum. All strains of
saprophytes exist, however some
are well known for causing root rot or
wilt in plants, while others are
regarded as non-pathogenic.
Different techniques have been
devised to categorize Fusarium
oxysporum strains based on
phenotypic and genetic features.
➢ Form: Circular
➢ Size: Punctiform
➢ Margin / border: Filiform
➢ Surface: Wrinkled
➢ Opacity: Translucent
➢ Colour (pigmentation): Purple

12
Circular with substantial walls.
It is created from hyphae or,
alternatively, through hyphae
cell alteration. In soils where it
functions as an inocula in
primary infection, it is
significant as a resistance
organ.
Figure 2.5.2 Colletotrichum
gloeosporioides.
Fungi colony Needle-shaped
➢ The asexual stage of the
fungus, which produces spores
(conidia) in huge quantities on
damaged plant tissues,
including dead and rotting leaf
litter that collects at the base of
the plant, produces spores
(conidia).
➢ By splashing water, spores
embedded in a slimy and
compact matrix spread to
nearby plants.
One of the most prevalent diseases
of the fungus Colletotrichum is
Colletotrichum gloeosporioides
(Anthracnose). Around the world, it
produces bitter rot in a range of
crops, particularly perennials in the
tropics. Although it can also survive
as a saprophyte, it serves as a
secondary invader of damaged
tissue.
➢ Form: Circular
➢ Margin / border: Lobate

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➢ Surface: Dull
➢ Colour (pigmentation): Buff
Figure 2.5.3 Colletotrichum
truncatum.
Fungi colony Allantoid
➢ In the presence of unrestricted
moisture and under ideal
conditions, fungal spores will
begin to grow.
➢ The ideal temperature for
Colletotrichum species to
develop and infect differs,
however high conditions are
typically preferred.
➢ Spores typically create an
infectious structure
(appressorium) on the surface
plant tissue once they have
germinated.
With the majority of the more than
200 species that are known to
spread disease in plant crops all
over the world, Colletotrichum is one
of the most significant genera of
plant pathogenic fungus. Particularly
significant post-harvest diseases of
fruit and vegetable crops grown in
tropical and subtropical climates are
Colletotrichum species, which cause
anthracnose.
➢ Form: Circular
➢ Margin / border: Entire
➢ Surface: Glistening
➢ Colour (pigmentation): White

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2.6 DISCUSSIONS
Morphological characteristics are significant when defining bacteria and fungi.
Colonies’ morphology is a helpful method frequently employed by scientists to
categorize and describe them. Bacteria are tiny, microscopic organisms that can
only be seen under a microscope. They are single-celled prokaryotic organisms.
Without our assistance, we cannot see them with our eyes. It can be seen when it
develops colonies on agar material in Petri plates. Fungi are a group of eukaryotic
creatures that include filamentous fungi, yeast, and other microbes. The fungus
prefers a warm, moist environment to grow. It is possible to categorize it using its
morphological and molecular characteristics. Developing fungus on a solid medium,
like potato dextrose agar, makes it easier to identify morphological characteristics
(PDAs). So, the various bacterial and fungal colonies will be observed on agar media
and the fungus’s structure also will be observed under a microscope. In this lab
practical, the fungi cultures (PDA) which is Fusarium species were observed and
recorded for its color, pigmentation, and the size with naked eyes and it was
observed under microscope. Based on the result, there are different types of fungal
colonies which are Fusarium oxysporum, Colletotrichum gloeosporioides, and
Colletotrichum truncatum according to the form, size, margin, surface, opacity, and
colour (pigmentation). All these fungal colonies were reproduced by different shape
of spores. For example, Fusarium oxysporum (septate), Colletotrichum
gloeosporioides (needle-shaped), and Colletotrichum truncatum (allantoid).

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2.7 QUESTIONS
Briefly explain the different characteristic of fungal and bacteria colony that can be
used for genus and species identification
Characteristics Fungal colony Bacteria colony
1) Definition A fungus colony is represented
by the mass of hyphae that
resembles a thread.
The discernible cell mass of a
single bacterial colony is what
is known as a bacterial colony.
2) Number of
cells
Fungal colonies may make up
unicellular or multicellular
organisms.
Bacterial colonies are made up
of single cells.
3) Composition Fungal hyphae made up of a
single spore make up fungus
colonies.
A mass of bacteria cells that
developed from a single
bacterial fraction make up
bacterial colonies.
4) Colony size The majority of hyphal-producing
fungal colonies are huge.
Small bacterial colonies.
5) Appearance Colonies of fungi have a hairy
look.
The surface of bacterial
colonies can be smooth or
bumpy.
6) Margin Fungal colonies have
filamentous edges.
The boundaries of bacterial
colonies are set.
7) Texture Colonies of fungi resemble
powder.
Bacterial colonies have a
glossy, wet appearance.

16
8) Shape Colonies of fungi are rhizoid or
filamentous.
Bacterial colonies can be round
or asymmetrical.
9) pH
compatibility
pH 5 to 9 is the growth range for
bacterial colonies (optimum 7).
Colonies of fungi flourish at pH
5 –6.

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2.8 CONCLUSIONS
As conclusion, we have observed and recorded on the fungal colony which is
Fusarium oxysporum. So, this fungus was reproduced by spore which is septate.
The spores were divided into three types. First, macroconidia that straight to slightly
curved, typically with three or four septa. Second, microconidia that has ellipsoidal
and lacking a septum. Third, chlamydospores. It is circular with substantial walls and
were created from hyphae or, alternatively, through hyphae cell alteration. The
fungus Fusarium oxysporum is a good representative of the rhizosphere's
microbiota. All saprophyte strains exist, however some are well known for wilting or
producing root rot in plants, while others are thought to be non-pathogenic. Fusarium
oxysporum strains have been categorized using a variety of methods based on their
phenotypic and genetic characteristics. As a result, we succeeded in observing and
recording the fungus colony which Fusarium oxysporum based on the
characteristics such as form (circular), size (punctiform), Margin (filiform), surface
(wrinkled), opacity (translucent), and colour pigmentation (purple).

18
2.9 REFERENCES
1. Laboratory manual 2: Colonies of Common Fungi and Bacteria.
2. Website:
3. CD Genomics MicrobioSeq. (2022). Retrieved from How to Distinguish Bacteria
and Fungi: From Morphology to Sequencing: https://www.cd-
genomics.com/microbioseq/how-to-distinguish-bacteria-and-fungi-from-
morphology-to-
sequencing.html#:~:text=Bacterial%20colonies%20consist%20of%20unicellular
,made%20up%20of%20fungal%20colonies.&text=Bacterial%20colonies%20co
nsist%20of%20a,up%
4. Hudson Robotics. (2021, December 7). Retrieved from Why Are Colonies
Important in the Study Of Microbiology: https://hudsonrobotics.com/why-are-
colonies-important-in-the-study-of-microbiology/
5. Nursery Production Plant Health & Biosecurity Project . (n.d.). Retrieved from
The biology and management of Colletotrichum diseases in production
nurseries: https://www.horticulture.com.au/globalassets/hort-
innovation/resource-assets/ny11001-colletotrichum-diseases.pdf
6. Samanthi, D. (2017, July 11). Different Between.com. Retrieved from Difference
Between Bacterial and Fungal Colonies:
https://www.differencebetween.com/difference-between-bacterial-and-vs-
fungal-colonies/
7. Wikipedia The Free Encyclopedia. (2022, June 10). Retrieved from Fusarium
oxysporum f.sp. ciceris:
https://en.wikipedia.org/wiki/Fusarium_oxysporum_f.sp._ciceris#:~:text=Fusariu
m%20oxysporum%20is%20a%20common,tapered%20and%20curved%20apic
al%20cell.

19
3.0 PRACTICAL 3 – ISOLATIONS AND OBSERVATIONS OF
PATHOGENIC BACTERIA
3.1 INTRODUCTION
Microbes living everywhere including on surface and inside of the plants.
Bacteria is one of those species. Bacteria is the simplest organism that only have a
single-cell with size range 1-2um and cannot be seen with naked eyes (Vidaver &
Lambrecht, 2004; Al-Mohanna, 2016). Bacteria that host inside or outside surfaces
may be beneficial and harmful to the plant. For this practical, the bacterial
pathogenicity was focus as it cause many diseases to plant that eventually lead to
higher loss of yield if do not detect at early stage. These pathogens can enter the
plants through many paths such as natural opening or wounds, soil, contaminated
water, farm equipment, etc. and could survive many years in association with
alternate hosts (Karim, et al., 2018).
Bacterial disease diagnosis depends on characteristic of the symptom
occurrences, isolation and presume infectious agents and physiological or molecular
test. One of the simplest tests to recognize the disease causes by bacteria is
streaming test as it is inexpensive, environmentally friendly and can be carry out
either in field or laboratory. Next, the serial dilution was one of the techniques was
used to recognize and know the present microorganism population. The dilution was
able to harvest pure strains of the single species that separate it from the mixed
population. From serial dilution, the test then being continued by streaking the
dilution of organism to a solid medium (Water Agar). Both of serial dilution and
streaking test used Water Agar that contain appropriate nutrients to develop the
organism growth (Blaize, et al., 2022). Hence, it can be said that streaming test was
to detect the present of bacteria, while serial dilution and streaking test were dividing
the mixed colonies of bacteria to from single colonies.

20
3.2 OBJECTIVES
➢ Testing for bacterial streaming.
➢ Serial dilution and streaking of bacterial suspension on agar medium.
➢ Observation of bacterial structure under microscope.
➢ Chili with infected symptoms
➢ Distilled water
➢ Sterile water
➢ Small beakers
➢ Tubes with racks
➢ Inoculation loop/ wire loop
➢ Bunsen burner with lighter
➢ Glass rod
➢ Scalpel
➢ Forceps
➢ 70% ethanol
➢ Petri dishes with Water Agar
➢ Glass slides
➢ Cover slips
➢ Methylene blue for staining
➢ Tissue paper

21
3.4 METHODS
A. Testing for Bacterial Streaming
1. The chili that has visible diseases symptoms were collected.
2. 10ml of distilled water were put into a beaker.
3. A square of tissue that has half disease tissues and half healthy tissue were
being cut from the chili act as a sample.
4. The sample then being put into the beaker containing distilled water for 30
seconds.
5. The sample in the beaker then being observed.
Figure 3.4.1: Tissue of chili immersed in the distilled water for bacteria streaming
test.

22
B. Serial Dilution and Streaking of Bacterial Suspension on Agar Medium.
i. Preparation of stock culture
1. 10ml of distilled water were put into a beaker.
2. A square of the infected part of chili tissue was cut and then being soaked
into the beaker contained distilled water.
3. The tissue of the infected part then being crushed into smaller pieces by
using sterile scalpel.
4. The crushed chili tissues were left to settle on itself for 1 minutes (original
stock of bacteria suspension).
Figure 3.4.2: Pieces of chili tissue immersed in the distilled water for stock
culture.
ii. Serial dilution procedure
1. 9ml of sterile water of 10X serial dilution were being prepared and poured
into 3 tubes.
2. The tube then being labelled as tube 1, 2 and 3.
3. 1ml of bacterial suspension from the beaker then being transferred into the
tube 1 by using the sterile pipette.
4. After that, the tube was being shake for several minutes so that the solution
able to mix well.

23
5. Next, 1ml of mixture from Tube 1 was transferred into the test Tube 2 by
using another sterile pipette.
6. The mixture in Tube 2 also being shake so that it will mix well.
7. 1ml of mixture from Tube 2 was transferred into the Tube 3 by using new
sterile pipette and it also were shake well for several minutes.
8. Then, 100ul of the dilution sample from Tube 1, 2 and 3 were transferred
into the petri dishes that contain Water Agar.
9. The Water Agar in the petri dish then being closed by its lids and sealed
with parafilm.
10. The culture then being incubate in the incubator for 24 hours and lastly
bacterial growth were being observed.
iii. Streaking of bacterial suspension
1. The inoculation loop was heated by using the Bunsen burner until the loop
was red in color and let it cool by itself.
2. One loopful of bacteria were collected from the third suspension dilution.
3. The loop then being streaked on the Water Agar media by drawing few
straight lines and after that the plate was turned in clockwise rotation.
4. The inoculation once again being heated with Bunsen burner for the
sterilization process.
5. Next, one of the straight lines from first streaked was chosen for the new
streaked.
6. Steps 3 and 4 were repeated until 4 streaked was drawn.
7. The Water Agar in the petri dish then being closed by its lids and sealed
with parafilm.
8. The culture then being incubate in the incubator for 24 hours and lastly
bacterial growth were being observed.

24
Figure 3.4.2: The summary of the procedure.
C. Observation of Bacterial Structure under Microscope.
1. The Bunsen burner was lighted to sterilize the inoculation loop and the
inoculation loop was heated until it turned red.
2. The inoculation loop then being cooled by its own in room temperature.
3. A drop of distilled water was added onto the slide.
4. A loopful of bacterium from the Water Agar was added and spread carefully on
the slide.
5. The slide was hold using forceps and being dried and heat fixed by using
Bunsen burner.
6. After that, the methylene blue was added on the slide for 30 seconds.
7. Next, the methylene blue was washed away by using sterile water.
8. The slide was being left to dry by its own.
9. Lastly, the bacterium on the slide being observed under the microscope.

25
Practical Result/ Observation
Streaming of
Bacteria
Figure 3.5.1 The streaming of bacteria.
(Source: Google Image)
Serial Dilution
of Bacteria
Figure 3.5.2 Serial dilution 1 after 2 days.

26

27
Streaking of
Bacterial
Suspension
Figure 3.5.5 Streaking of bacterial suspension after 2 days.
For the streaming of bacterial test, the image was from Google Image as the
camera cannot capture the real image. For the serial dilution test and streaking of
bacteria, as can be seen in Figure 3.5.2 until Figure 3.5.5, there is no changes in
detected on the Water Agar and it will further discuss in the discussion section.

28
3.6 DISCUSSIONS
Bacteria is one of the microorganisms either can be useful or harmful towards
other organism including plants. To figure out and known either the disease caused
by bacteria or not, many test can be carry out such as, bacterial streaming, serial
dilution, streaking of bacterial suspension. These methods were most popular
method because they are inexpensive, easy, save time, eco-friendly, and can be
carry out in the field or laboratory (Ivery & Lunos, 2015).
For the first experiment, which is the streaming of bacterial the tissue from
the chili fruit that has half infected and half healthy tissue was selected and
immersed into the distilled water for 30 second. The cloudy secretion of bacterial
streaming from chili tissue can be seen but our camera cannot capture the real
photo. The cloudy streaming proved the present of bacteria on the infected chili
tissue. Hence, the photo was being replaced by the photo from Google Image as
being stated in Figure 3.5.1.
For the second and third experiment, which the serial dilution and streaking
test, another tissue from the same chili fruit were selected and the tissue was cut
into small pieces when they were immersed in the distilled water. The purpose was
to obtain single colony from the mixed colonies of bacteria. The summary of each
procedure was as being shown in Figure 3.4.2. From the observation after 3 days,
there is no present of bacterial in all serial dilution of 1, 2 and 3 and streaking test
as being shown by the Figure 3.5.2, Figure 3.5.3, Figure 3.5.4, and Figure 3.5.5,
respectively. The result of serial dilution should be as Figure 3.6.1 and streaking test
should be as Figure 3.6.2. below.

29
Figure 3.6.1 The expected result for serial dilution test and shows the
number of colonies decreasing from first dilution to the last dilution.
Figure 3.6.2 The expected result for streaking test in which the colonies
decreasing or thinning as the streaking moves clockwise.

30
But unfortunately, the result present in this experiment were not as expected from
both figures above. This result may be because there is an error occur during the
experiment was conducted. The first factor may cause by the glass rod and
inoculation rod that were used for each experiment was still hot when spreading the
culture on the Water Agar media in which resulted the death of bacteria or other
microbes. This is because both high and low temperatures affect the growth of
microorganism (Wei, 2020). The second factor may be cause by there were no
present of bacteria on the selected tissue used even though from the same fruits.

31
3.7 CONCLUSIONS
In a nutshell, all the experiment which are streaming, serial dilution
preparation and streaking test of bacteria that being carry out in this practical are
simple as it is not consumed much time and procedure were very easy. The
experiment for the first objective was successful as the bacteria present was
detected because of cloudy streaming were detected. Next, the serial dilution
preparation with test and streaking test by spreading it on Water Agar media were
not successful as there were no colonies detected and present on the media after 3
days of experiment. Hence, the observation of the bacteria under the microscopes
cannot be carry out and the third objective also fail to be fulfilled.
3.8 RECOMMENDATIONS
➢ First and foremost, students need to ensure that the inoculation loop and glass
rod that were used for streaking and spreading of the microorganism dilution on
media was cool so that the microorganism were not death and can grow healthily
on the media as the media already contain enough and sufficient nutrient for their
growth.
➢ Next, student need to make and choose proper tissue on the sample of fruits or
plant before carrying out the experiment as different infected tissue may cause
by different organism even though the symptoms were same when being
observed by naked eyes.
➢ Finally, student need to prepare and study first before conducting the experiment
to make sure that the steps that being stated in the procedure can be carry out
successfully.

32
3.9 REFERENCES
1. Al-Mohanna, M. T., 2016. Chapter 34: Bacterial Introduction. In: s.l.:s.n., pp.
679 - 692.
2. Blaize, J. F., Suter, E. & Corbo, C. P., 2022. Serial Dilutions and Plating:
Microbial Enumeration. Journal of Visualized Experiments (JoVE).
3. Ivery, M. L. L. & Lunos, A., 2015. Bacterial Streaming Test for Rapid Diagnosis
of Vegetable Bacterial Diseases, Louisiana: LSU AgCenter Department of
Plant Pathology & Crop Physiology.
4. Karim, Z., Hossain, M. S. & Begum, M. M., 2018. Ralstonia solanacearum: A
threat to potato production in Bangladesh. Fundamental and Applied
Agriculture, Volume 3, pp. 407-421.
5. Vidaver, A. K. & Lambrecht, P. A., 2004. Bacteria as plant pathogens, Lincoln,
Nebraska, USA: University of Nebraska.
6. Wei, A. A. Q., 2020. The Effect of Temperature on Microorganisms Growth
Rate.

33
4.0 PRACTICAL 4: KOCH POSTULATES (FUNGUL ISOLATION AND
OBSERVATION)
4.1 INTRODUCTION
Koch postulates are four criteria designed to determine or verify whether
microorganism are causes of a specific disease. It was being develop by the Robert
Koch in 19th century (Segre, 2013) has been widely used in many industries such
as medicine, agriculture, and pharmaceutical. This method is the first reliable
method established to recognized microorganism as the cause of disease (Bhunjun,
et al., 2021). The 4 rules are;-
i. The symptoms produced by the microorganism must be associated with the
diseases.
ii. The microorganism/ pathogen must be isolated from the infected plant and
grown in pure culture.
iii. The isolated pathogen must be inoculated on healthy host and produced similar
symptoms with the infected host
iv. The pathogen must be re-isolated from the inoculated host and grown in the
pure culture.
For this practical, the focus was to isolate the fungal pathogen from the
infected host which is chili fruits. There are two mechanisms used to isolate the
pathogen from the sample, which are direct isolation and surface sterilization. Direct
isolation is simply transferred a mould from the natural habitat to the pure culture in
the laboratory (Al-Mohanna, 2016). Surface sterilization was done to make the
surface of the sample free from certain microorganism (Felek, et al., 2015) and thus
only selected fungi were able to present on the sample.

34
4.2 OBJECTIVES
➢ To learn how to carry out the direct isolation and surface sterilize techniques of
fungal isolation.
➢ To observe the fungal growth of the direct isolation and surface sterilize
techniques on the PDA media.
➢ Infected chili anthracnose
➢ 70% ethanol
➢ Bunsen burner
➢ Forceps
➢ Scalpel
➢ Distilled water
➢ Sterile distilled water
➢ Small beaker
➢ PDA media
➢ Parafilm
➢ Sterilized tissue paper
➢ Glass slide
➢ Cover slips
➢ Methylene blue for staining

35
4.4 METHODS
Fungal Isolation
A. Direct Isolation Technique
1. A square of 5x5 mm2 that containing half infected part and half healthy
part was being cut from the chili.
2. The sample then being transferred on the PDA media.
3. After that, the PDA media in the petri dishes was closed with it lid and the
parafilm was used to seal the sample.
4. The culture then was incubated in the incubator at room temperature.
5. The observation was being done after mycelia growth were visible on the
PDA media.
B. Surface Sterilization Technique
1. 10ml of ethanol was poured into the beaker.
2. A square of 5x5 mm2 that containing half infected part and half healthy
part was being cut from the chili.
3. The sample then was dipped into the ethanol for 3 minutes.
4. After that, the sample being rinsed three times with sterile distilled water.
5. Next, the rinsed sample was being dried by using sterile tissue paper to
remove excess water.
6. After that, the PDA media in the petri dishes was closed with it lid and the
parafilm was used to seal the sample.
7. The culture then was incubated in the incubator at room temperature.
8. The observation was being done after mycelia growth were visible on the
PDA media.
Fungal Observation

36
1. Macroscopic – The color of colony, pigmentation, and growth diameter of the
fungus was observed.
2. Microscopic – The shaped of spores produced by the fungus was observed
under light microscope.

37
a) Macroscopic characteristic
Direct Isolation Surface Sterilization
Figure 4.5.1 The result of direct isolation
of chili tissue after 3 days.
Figure 4.5.2 The result of surface
sterilization of chili tissue after 3 days.
The area that has activity in which there like white staining on the surface
of PDA medium in Figure 4.5.1 were larger than the Figure 4.5.2. Besides that,
on the surface of PDA media of Figure 4.5.1 have some brown spot while there
was no brown area on the PDA media of the Figure 4.5.2. This show that the
activity of microorganism on the direct isolation are more active than the activity
of microorganism at the surface sterilization.
b) Microscopic characteristic
There was no result for the microscopic characteristic observation under the light
microscope as there was no shaped of spores, etc. that were able to be
recognized as fungus.
4.6 DISCUSSIONS

38
Isolation is one of the techniques to prepare pure culture free from any
contamination and ready for the identification (Al-Mohanna, 2016). Isolation can be
categorized into two methods which are direct isolation and surface sterilization. For
fungi, Sabouraud's dextrose agar and potato-dextrose agar (PDA) are commonly
used media as it able to suppress bacterial contamination and supplied appropriate
nutrients to the fungi species. From this experiment, the objectives were to learn
how to carry out direct isolation and surface sterilization techniques of fungal
isolation and observe the fungal growth by macroscopic and microscopic vison on
the PDA media.
For the first experiment, both of direct isolation and surface sterilization
observation can be done because there were changes on the PDA media surfaces.
As Figure 4.5.1 shows, there were like some activity on the media as they were more
surface look like white staining and there some brown area around the sample of
chili tissue for the direct isolation. Meanwhile, for the surface sterilization, only the
area around sample of chili tissue has white staining, while other areas do not show
any changes on the PDA media surfaces as being show in the Figure 4.5.2. This
proved that the activity of microorganism on the direct isolation techniques are more
active than surface sterilization techniques as some microorganism or fungus have
already been killed during the sterilization process by using 70% alcohol.
For the microscopic observation, the fungus or microorganism structure or
shape of spores under the light microscope for both direct isolation and surface
sterilization were do not detected even though with the help of lecturer. This
condition may happen because the selected sample of the PDA media do not
contain microorganism or fungus when collecting the sample on the PDA media.
Next, it’s also caused by the student do not know how to use the light microscope
efficiently. Besides that, the sample also may be contaminated with other organism,
and thus make the wanted microorganism or fungus that need to be investigate was
suppress by them. Lastly, the time to observe the sample may be too early as some
microorganism required more time to growth even though it has suitable

39
environment and sufficient nutrients. Therefore, Figure 4.6.1 and Figure 4.6.2 below
was expected result for this experiment, which is to observe the Colletotrichum sp.
as were the major cause of anthracnose disease on the fruits.
Figure 4.6.1 Colletotrichum sp. (40x) (Ridzuan, et al., 2018)
Figure 4.6.2 Colletotrichum sp.

40
4.7 QUESTIONS
a) Briefly explain why there are two techniques to isolate fungal pathogen from
infected plant.
Techniques Explanation
Direct
Isolation
➢ It means by simply transfer the tissue that contain mold
from the sample to a pure culture in the laboratory.
➢ This technique was done so that researcher able to
observe the growth diameter, color of colony, and
pigmentation easily, but difficult to identify the specific
causative agent for the diseases.
➢ Usually used to identify the fungus characteristic by
macroscopic or naked eyes only.
Surface
sterilization
➢ It means the surface of tissue sample was being washed
by the standard alcohol so that the surface of the sample
free from certain microorganism.
➢ For example, there was dead circular spot that on sample
and assumed certain fungus, but when the sample directly
put onto the media, the result that will come out may be
not the causative agents but other fungus.
➢ This means that, this technique was used to narrow down
a certain fungus and do not cause other fungus effects the
expected result.
➢ Besides, it will be able to kill the fungus on the surface but
not the fungus inside the sample.
➢ This technique also used to avoid the contamination of
bacteria on the tissue sample.

41
b) Give the name of fungal pathogen that commonly associated with fruit
anthracnose diseases.
There were many fungal species that cause anthracnose disease of fruit
such as Diplocarpin sp., Elsinoe sp., and most popular are Colletotrichum
sp. The table below show several fungi that cause anthracnose disease to
fruits.
Fruit Fungus
Mango ➢ Colletotrichum gloeosporioides also known as
Glomerella cingulata
Papaya ➢ Colletotrichum gloeosporioides
Olive ➢ Colletotrichum gloeosporioides and Colletotrichum
acutatum.
Banana ➢ Colletotrichum musae
Chili ➢ Fruit – Colletotrichum acutatum and Colletotrichum
gloeosporioides
➢ Leaf and stem – Colletotrichum coccodes and
Colletotrichum dematium
Strawberry ➢ Colletotrichum acutatum
(Than, et al., 2008; NC State Extension Publications, 2014; Sarkar, 2016)

42
4.8 CONCLUSIONS
In a nutshell, Koch’s Postulate was very important in the agriculture industry
as it was able to help the researchers determine the disease of the plant cause by
specific causative agents. Therefore, the isolation process of the sample from the
fruits or plant parts needs to be do carefully so that researchers were able to get the
expected results in the end of the process. The two famous mechanism of isolation
which are direct isolation and surface sterilization were used in this practical. For
microscopic observation, both of direct isolation and surface sterilization shows
some activity of microorganism on the PDA surface, but sadly for microscopic
observation under light microscope the observation does not have the expected
result. Thus, the expected result in this practical which was to see shape of spores
of Colletotrichum sp. under microscope are unsuccessful. Finally, the first objective
of this practical was carried out successfully, meanwhile the second objective was
failed as the microscopic observation do not have any result.
4.9 RECOMMENDATIONS
➢ The students need to carefully select the sample on the PDA media so that they
will be able to get the expected results.
➢ Before carrying out the experiment, student need to study the procedure
provided in the manual so that they can carry out the experiment smoothly.
➢ Besides that, student also need to always sanitize their hands, environment, and
equipment that will be used in the experiments so that the culture would not be
contaminated.
➢ Student also needs to know how to use light microscope efficiently so that they
were able to carry out the experiment smoothly in the future as this is some of
basic skill when dealing and working in the laboratory of plant pathogens.

43
4.10 REFERENCES
1. Al-Mohanna, M., 2016. Methods for Fungal Enumeration, Isolation and
Identification. Iraq: University of Al-Qadisiyah.
2. Bhunjun, C. S. et al., 2021. Importance of Molecular Data to Identify Fungal Plant
Pathogens and Guidelines for Pathogenicity Testing Based on Koch’s
Postulates. Pathogen, Volume 10.
3. Felek, W., Mekibib, F. & Admassu, B., 2015. Optimization of explants surface
sterilization condition for field grown peach (Prunus persica L. Batsch. Cv.
Garnem) intended for in vitro culture. African Journal of Biotechnology, 14(8), pp.
657-660.
4. “HOW MOULDS CAN BE ISOLATED.” Mycology Web Pages, website.nbm-
mnb.ca/mycologywebpages/Moulds/Isolation.html. Accessed 19 July 2022.
5. NC State Extension Publications, 2014. Anthracnose Fruit Rot of Strawberry,
North Carolina: NC State University and NC A&T State University.
6. Ridzuan, R. et al., 2018. Breeding for Anthracnose Disease Resistance in Chili:
Progress and Prospects. International Journal of Molecular Sciences, 19(10).
7. Sarkar, A. K., 2016. Anthracnose diseases of some common medicinally
important fruit plants. Journal of Medicinal Plants Studies, 4(3), pp. 233-236.
8. Segre, J. A., 2013. What does it take to satisfy Koch’s postulates two centuries
later? Microbial genomics and Propionibacteria acnes. Journal Invest Dermatol,
pp. 2141-2142.
9. Than, P. P. et al., 2008. Chilli anthracnose disease caused by Colletotrichum
species. Journal of Zhejiang University Sciences, Volume 9, pp. 764-778.

44
5.0 PRACTICAL 5: KOCH POSTULATES (INOCULATION OF
PATHOGEN INTO HEALTHY PLANT)
5.1 INTRODUCTION
The term "pathogenicity" refers to a pathogen's capacity to inflict disease, and
testing calls for the fulfilment of a series of criteria known as Koch's postulate
requirements. Artificial inoculation is a common laboratory technique for studying
pathogenic microorganisms because it makes it simpler to manage the
environmental factors that can cause infection. It is also one of the most important
criteria for determining putative pathogens as the etiological agents of plant
diseases. This entails the growth of lesions after an appropriate host-made infection
in a greenhouse environment. On rare occasions, pathogenic testing on plant parts
can be done in controlled lab settings. The need for using new and useful methods
of identifying harmful bacteria is indicated by the difficulty of choosing the kind of
susceptible host and the proper settings. Pathogenicity is the attribute or state of
having the potential to cause disease, whereas virulence is the capacity of an
organism to cause disease, or the level of pathogenicity within a group or species.
Therefore, in this laboratory report, the pathogenicity test will be conducted on the
plant samples and the inoculated fruits will be observed for control, wounded, and
unwounded treatment.

45
5.2 OBJECTIVES
➢ To conduct the pathogenicity test on the plant samples.
➢ To observe the inoculated fruits for control, wounded, and unwounded treatment.
➢ 3 samples of healthy fruits of chilli
➢ Pure culture of plant pathogen (Colletotrichum spp)
➢ Sterile water
➢ Distilled water
➢ Scalpel
➢ Forceps
➢ Clear plastic container to store the fruit
➢ Bunsen burner
➢ 70% ethanol
➢ Tissue paper
➢ Beakers

46
5.4 METHODS
1. By using mycelial plug technique, the pathogenicity test was conducted with
treatments which are wounded, unwounded and control.
2. The plant samples which are chillies were surface sterilized by using 70% ethanol
for 3 minutes and the chillies also were rinsed three times with sterile distilled
water.
3. With sterilized tissue paper, the plant samples were dried.
4. An artificial wound was made on the chillies by pin prick the chilli to a 2 mm depth
by using scalpel.
Figure 5.4.1 Make an artificial wound on the chilies.
5. The chilies were inoculated with uncultured Potato Dextrose Agar (PDA) for
control.
6. At the centre, the inoculated chillies were put in a tray with 100 mL of water and
partially the fruits were sealed with a clear plastic bag to maintain relatively high
humidity.
7. At 27±1°C, the inoculated chillies were incubated for 9 days.
8. The three replicates were prepared for each treatment which is wounded,
unwounded and control on the same chilli fruit.
➢ Wounded treatment: Agar with pathogen were put on the lesions.
➢ Unwounded treatment: Agar with pathogen were put on the chilli without
lesions.
➢ The chilli fruits were inoculated with uncultured PDA (no pathogen).

47
Figure 5.5.1 The condition of
inoculated chilies after 9
days.
Figure 5.5.2 The condition at
the chili fruits were inoculated
with uncultured PDA (no
pathogen).

48
chili at unwounded treatment
chili at wounded treatment
Based on the observation, the three replications of chilies were affected by
Colletotrichum sp. at the wounded treatment which is agar with pathogen were put
on the lesions and the chili fruits were inoculated with uncultured PDA (no pathogen).
There is not any affected on the second treatment which is unwounded treatment
(agar with pathogen were put on the chili without lesions).

49
5.6 DISCUSSIONS
The ability of a pathogen to cause disease is referred to as "pathogenicity,"
and testing necessitates meeting a set of requirements known as the Koch postulate
requirements. Because artificial inoculation makes it simpler to control
environmental conditions that can cause infection, it is a typical laboratory approach
for researching harmful bacteria. So, the pathogenicity test was conducted on the
plant samples and the inoculated fruits were observed for control, wounded, and
unwounded treatment. In this lab practical, the three replications of chilies were
prepared for each treatment which is wounded (agar with pathogen were put on the
lesions), unwounded (agar with pathogen were put on the chili without lesions), and
control (the chili fruits were inoculated with uncultured PDA which is no pathogen.
Based on the result, agar containing pathogen was applied to the wounds on the
three replications of chilies that were damaged by Colletotrichum spp., and the chili
fruits were then infected with uncultured PDA (no pathogen). The second treatment,
known as the unwounded treatment, is unaffected (agar with pathogen were put on
the chili without lesions). This is because finding bacteria that are thought to be the
etiological agents of plant diseases is based on the results of pathogenic testing. So,
the meaning is the test give affect more in the bacterial pathogen which on the third
treatment by using uncultured pathogen. The Potato Dextrose Agar (PDA) is used
in counting mold and isolating yeast from diverse samples, and it is the most popular
growing media for bacteria and fungus. A simple, all-purpose medium with plenty of
nutrients, potato dextrose agar encourages the growth of mold spores and the
generation of color.

50
5.7 QUESTIONS
Give two main functions of pathogenicity test.
➢ To observe the pathogens that might be the cause of plant illnesses.
➢ To prevent the spread of infection by manipulating the environment
through laboratory fake inoculation techniques.
5.8 CONCLUSIONS
As a conclusion, we have observed and recorded on the three replications of
the chilli fruits according to the three treatments which is wounded treatment,
unwounded treatment, and inoculated with uncultured PDA (no pathogen) which is
called pathogenicity test. The process by which infection results in disease is known
as pathogenesis. Pathogenicity is the attribute or state of having the potential to
cause disease, whereas virulence is the capacity of an organism to cause disease,
or the level of pathogenicity within a group or species. While virulence is a phrase
that evaluates harmful qualities, pathogenicity is a qualitative term that refers to an
"all-or-nothing" idea. On three separate occasions, the wound of chiles wounded by
Colletotrichum spp. was rubbed with agar harbouring pathogens, and the chillies
were subsequently infected with uncultured PDAs (no pathogens). Wound-free
treatment, the second course of action, was unaffected (according to the pathogen
placed on the wound -free chili). As a result, we succeeded in observing and
recording the the inoculated chillies in the plastic container after 9 days that have
been affected by Colletotrichum spp.

51
5.9 REFERENCES
1. Laboratory manual 5: Inoculation of Pathogen into Healthy Plant
2. Alspaugh, J. A. (2017). The Pathogenic Potential of a Microbe. PMC PubMed
Central, 15-17. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322344/
3. Biology LibreTexts. (2021, April 6). Retrieved from Characteristics and Steps
of Infectious Diseases:
https://bio.libretexts.org/Courses/Manchester_Community_College_(MCC)/Re
mix_of_Openstax%3AMicrobiology_by_Parker_Schneegurt_et_al/12%3
4. A_Microbial_Interactions_Flora_Pathogenicity_and_Epidemiology/12.02%3A
_How_Pathogens_Cause_Disease
5. Dalynn Biologicals. (n.d.). Retrieved from Potato Dextrose Agar:
https://www.dalynn.com/dyn/ck_assets/files/tech/PP85.pdf
6. MBInfo Defining Mechanobiology. (2018). Retrieved from Bacterial
Pathogenesis: https://www.mechanobio.info/pathogenesis/
7. Varadi, L., Luo, J. L., Hibbs, D. E., Perry, J. D., Anderson, R. J., Orenga, S., &
Groundwater, P. W. (2017). Methods for the detection and identification of
pathogenic bacteria: past, present, and future. Royal Society of Chemistry,
4818-4832.

52
6.0 PRACTICAL 6: PATHOGENICITY TEST (DISEASE SCORE,
DISEASE SEVERITY, AND VIRULENCE LEVEL)
6.1 INTRODUCTION
Pathogenic elements include pathogen presence, pathogenicity (virulence
and aggressiveness), adaptability, dissemination efficiency, survival efficiency, and
reproductive ability. Typically, infectious diseases are evaluated by assessing
disease severity. Disease severity is referring as the percentage of critical host
tissues or organs that are covered by symptoms or lesions or affected by the
disease. Severity is determined by the number and size of lesions. The capacity of
a disease or microorganism to infect or harm a host is referred to as virulence. The
ability of a disease to infect a resistant host in the context of gene for gene systems,
most typically in plants, is referred to as virulence. In this laboratory practical, we
able to examine the pathogenicity of the fungus that causes anthracnose on chilli.
By using a given disease score and disease severity (DS) formula from (Cooke,
2006) as guidelines. Each isolate's virulence level was evaluated according to the
approach of Charoenporn et al. (2010), with minimal modifications.

53
6.2 OBJECTIVES
➢ To examine pathogenicity of the fungus that causes infected on chilies
➢ To rate and evaluate disease severity in an infected chilli
➢ Infected chili fruits
➢ Tissues paper
➢ Plastic container
➢ Forceps
➢ Pens and paper
➢ Calculator

54
6.4 METHODS
1. Samples of infected chilies prepared in Practical 5.
➢ Disease score table and formula
2. One of the chili samples from was take out from plastic container and put on
the tissue paper by using forceps.
3. The disease severity score of the sample was discussed among the group
members.
4. Steps 2 and 3 were repeated to decide the disease severity score.
5. The disease score of the chilies sample was calculated and determined by
using Table 6.4.1 below.
Table 6.4.1 Disease Score for Chilli Anthracnose
Disease severity
score
Symptom’s description
0 No infection
1 1 – 2% of the fruit area produced necrotic
3 3 – 5% of the fruit area produced necrotic lesion
9 >25% of the fruit area produced necrotic lesions
6. The following formula (Cooke,2006) was used to calculate disease severity
(DS):
𝐷𝑆 =
Σ(𝑎 × 𝑏)
𝑁. 𝑍
× 100%
Where Σ (a×b) = Sum of contaminated fruits and their related scoring scale
N = The total number of fruits sampled
Z = The highest possible score scale

55
7. The level of virulence of the sample being determined by the following Table
6.4.2.
Table 6.4.2 Level of Virulence Based on Ds Value
DISEASE SEVERITY VALUE VIRULENCE LEVEL
DS = 0 Avirulence
DS ≤ 30.00 Low virulence
DS > 30.00-60.0 Moderate virulence
DS > 60.00-80.00 High virulence
DS > 80 Very high virulence

56
Figure 6.5.1 (a, b, c) The result of infected chilies from Practical 5.
A
B
C
A
B
C
C
B
A
a b
c

57
Calculation:
Disease severity score:
A= 5 + 1 + 9
B= 1 + 1 + 9
C= 3 + 1 + 7
𝑫𝑺 =
𝟓 + 𝟏 + 𝟗 + 𝟏 + 𝟏 + 𝟗 + 𝟑 + 𝟏 + 𝟕
𝟗 × 𝟗
× 𝟏𝟎𝟎%
𝐷𝑆 =
37
81
× 100% = 𝟒𝟓. 𝟔𝟖%
➢ Disease severity (DS) = 45.68%
➢ Virulence Level = Moderate virulence

58
6.6 DISCUSSIONS
In this laboratory, we measure the disease score, disease severity and
virulence level of the fungus (Colletotrichum species) which causing anthracnose of
chili. By using a given disease score, disease severity (DS) formula from (Cooke,
2006), and virulence level method by Charoenporn et al. (2010) as guidelines. 3
fruits sampled was used and it showed 9 highest possible disease score scale.
Meanwhile, disease severity (DS) was indicated 45.68%. By following the DS value,
the pathogenicity experiment revealed that one category of virulence level which is
moderate virulence where 45.68 > 30.00-60.0. Experiments on pathogenicity Chili
fungus found that, while all species were pathogenic on chili fruits after injuring the
fruit cover, the majority generated a low rate of non-wounded fruit infection.

59
6.7 CONCLUSIONS
Pathogenicity testing was performed to determine the disease score parasitic
nature of the fungus Colletotrichum spp. that causing anthracnose of chili. The
pathogenicity experiment was carried out using chilli fruits that had been intentionally
infected with a pure culture of the fungus. The degree of pathogenicity was assessed
by comparing the disease symptoms developed in each treatment to the control.
The result was showed moderate virulence with 45.68% of disease severity. Lastly,
Colletotrichum spp. may commence the anthracnose disease as the primary
organism, but additional fungi may relate to the infected site as secondary
microorganisms, making the disease more complex and causing significant product
loss.
6.8 REFERENCES
1. AGR554 LABORATORY MANUAL: Lab manual 6 (pathogenicity test (disease
score, disease severity and virulence level)
2. Groenewald, J., Crous, P., Ades, P., Nasruddin, A., Mongkolporn, O., Taylor,
P., & De Silva, D. (2019). Identification, prevalence, and pathogenicity of
Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA
Fungus.
3. Kumar, P., Nayak, B., Pratap, D., & Chandra, K. (2021). Pathogenicity of
Colletotrichum capsci on chilli by seed inoculation technique. Journal of
emerging technologies and innovative research (JETIR), 30-33.
4. Moe Oo, M., Lim, G., A Jang, H., & Oh, S.-K. (2017). Characterization and
Pathogenicity of New Record of Anthracnose on Various Chili Varieties Caused
by Colletotrichum scovillei in Korea. Mycobiology, 184-191.
5. Syifaudin, S., Firmansyah, M., & Budi, S. (2022). Pathogenicity test of fungal
leaf blight on sengon seedlings at permanent nursery dramaga bogor. IOP
Conference Series: Earth and Environmental Science, 1-9.

60
7.0 PRACTICAL 7: HOW PATHOGEN ATTACKS PLANT (FORMATION
OF APPRESSORIUM)
7.1 INTRODUCTION
To spread illness and ensure their own existence, pathogens must infect their
hosts. Since the protoplast of plant cells contains a variety of materials, pathogens
must first break through the exterior barriers created by the cuticle or cell walls to
access the protoplast (Forces et al., n.d.). The pathogen must be able to overcome
these barriers if it is to survive and continue feeding on the plant. In response to the
pathogen's presence and activities, the plant develops structures and chemical
compounds that hinder the pathogen's growth or survival (Forces et al., n.d.).
Finding and entering through a natural opening, an injury, or a direct mechanical
attack are all possible ways to penetrate the host tissues by the pathogen.
Appressorium is one of the mechanisms used by the fungus to enter the host
directly. Fungal infections use structures known as appressoria to press against and
adhere to the surface of plants to infect them (Thilini Chethana et al., 2021).

61
7.2 OBJECTIVES
➢ To induce the formation of appressorium using slide which is by culture
technique.
➢ Culture of fungal pathogen (Colletotrichum spp.)
➢ Potato Dextrose Agar (very thin slice)
➢ Glass slide
➢ Petri dish
➢ Cover slip
➢ Microscope
➢ Parafilm
➢ Inoculation loop

62
7.4 METHODS
1. 10 mm x 10mm of thin PDA was cut and placed on the glass slide carefully.
2. The glass slide was placed inside an empty petri dish.
3. The Colletotrichum spp conidia or spores were taken from the sporulating
cultures by using inoculation loop and were inoculated at each edge of the agar.
4. The inoculated agar was covered with cover slip to allow formation of the
appressoria.
Figure 7.4: The glass slide were placed in petri dish.
5. After 3-4 days, the formation of the appressoria were observed under
microscope.

63
Figure 7.5.1: The formation of fungus which seen by naked eyes.
Figure 7.5.2: The formation of appressorium under microscope.

64
7.6 DISCUSSIONS
Based on the observation under microscope, the result of this experiment was
recorded as appressoria under microscope. Appressoria is the product from fungi
that help them to penetrate the epidermal tissue of plant host. Appressorium is the
specialized cell commonly of many fungal plant pathogens that is used to infect plant
host. From the observation above, the shape and the structure of appressoria had
been observe. Appressoria were flattened and had hyphal pressing organs. From
these organs, a minute infection peg grows and penetrates the body by employing
turgor pressure powerful enough to punch through Mylar. Appressoria also had a
thread like structure that help them to easily penetrate to the plant cell. When the
thread like structure at the end of the edge penetrate the plant cell, it can easily
spread and give damaged to the plant cell.

65
7.7 QUESTIONS
Explain how different pathogen attacks plant using “mechanical forces”.
➢ To infect a plant, a pathogen must be able to enter and pass through the
plant, take nutrients from the plant, and deactivate the plant's defense
mechanisms (Forces et al., n.d.). Different pathogens having different
mode of action to survive. Mechanical forces refer to the mechanical
pressure that applied to the plant’s surface to penetrate (Forces et al., n.d.).
➢ For fungus, the hypha or radical in contact with the host grows as the
fungus touches it, establishing contact, and the flattened, bulb-like
structure known as the appressorium is formed. By doing so, the pathogen
is firmly fastened to the plant and the area of adhesion between the two
organisms is increased. A small sprouting point known as the penetration
peg emerges from the appressorium and advances into and through the
cuticle and cell wall. (Note, n.d.).
➢ Meanwhile, the stylet, which is used by nematodes to pierce plant surfaces,
is propelled back and forth while applying mechanical pressure to the cell
wall. Nematode uses suction to first cling to the plant's surface, which it
produces by contacting the plant. The nematode moves its body to a
position vertical to the cell wall after adhesion has been achieved. The
nematode then pushes its stylet forward as the back portion of its body
sways or slowly spins around in a circle, keeping its head still and attached
to the cell wall. The stylet punctures the cell wall after many successive
thrusts, and either the stylet or the complete nematode enters the cell
(Abbas, 2016).

66
7.8 CONCLUSIONS
As the conclusion, the objective which to induce the formation of the fungus
by using culture technique is successful since the appressorium of the
Colletotrichum sp. is capable to be seen under microscope. The appressorium is
important for the fungus to penetrate the host so that it can spread its spores for the
survival purpose. The flattened, thickened tip of a hyphal branch is the
appressorium, via which some parasitic fungus connects to and enter their host.
When the appressorium attached to the host, the penetration peg can be emerging.
7.9 REFERENCES
1. Abbas, A. (2016). How Pathogen attacks Plants. January 2014.
2. Forces, M., By, E., On, P., Tissues, H., Weapons, C., Pathogens, O. F., In, E.,
Disease, P., Degradation, E., Cell, O. F., Substances, W., Degradation, E.,
Substances, O. F., In, C., Cells, P., Toxins, M., Plant, I. N., That, T., Wide, A.
A., … Plants, H. (n.d.). Interação Planta Patógeno (1).
3. Köhl, J., Kolnaar, R., & Ravensberg, W. J. (2019). Mode of action of microbial
biological control agents against plant diseases: Relevance beyond efficacy.
Frontiers in Plant Science, 10.
4. Note, L. (n.d.). The mode of attack of plant-by-plant pathogens. Cellulose.
5. Thilini Chethana, K. W., Jayawardena, R. S., Chen, Y. J., Konta, S.,
Tibpromma, S., Abeywickrama, P. D., Gomdola, D., Balasuriya, A., Xu, J.,
Lumyong, S., & Hyde, K. D. (2021). Diversity and function of appressoria.
Pathogens, 10(6), 3–6.
6. Zin, N. A., & Badaluddin, N. A. (2020). Biological functions of Trichoderma spp.
for agriculture applications. Annals of Agricultural Sciences, 65(2), 168–178.

67
8.0 PRACTICAL 8: TRICHODERMA (FUNGUS) AS BIOLOGICAL
CONTROL AGENT)
8.1 INTRODUCTION
Through the application of natural enemies, biological management is a safe
and efficient way to lessen or prevent the impacts of pests and illnesses. By applying
a biocontrol agent (BCA), often a fungus, bacterium, or virus, or a combination of
these, to the plant or soil, disease can be controlled biologically. The biocontrol agent
works to stop the pathogen from infecting the plant or from becoming established in
the plant. Due to its well-known biological control mechanism, Trichoderma spp.
have been extensively employed in agricultural applications (Zin & Badaluddin,
2020). Due to its great potential to inhibit the growth of other fungal diseases,
Trichoderma species are being used more and more as biocontrol agents.
Trichoderma species control the speed of plant development and strongly inhibit the
growth of plant pathogenic bacteria (Zin & Badaluddin, 2020). Using the dual culture
technique on PDA plates, Trichoderma isolates will be tested for their capacity to
prevent the growth of other pathogens. On a petri plate with solidified PDA media,
the Trichoderma and the pathogen will be inoculated side by side.

68
8.2 OBJECTIVES
➢ To determine the potential of Trichoderma as potential biological control agent of
fungal pathogen.
➢ Trichoderma culture (biocontrol agent)
➢ Colletotrichum species (pathogen)
➢ 2 PDA media
➢ Scalpel
➢ Bunsen Burner
➢ 70% ethanol
➢ Tissue paper
➢ Small beaker

69
8.4 METHODS
1. The scalpel was dipped onto the ethanol liquid and dried using tissue paper. Then
the scalpel was heated using the Bunsen burner.
2. After the scalpel had cooled, 5 mm x 5 mm of the Trichoderma (biocontrol agent)
culture was cut and placed 1 cm away from the margin of the PDA media.
3. Step 1and 2 was repeated to inoculate the Colletotrichum (pathogen) into the same
media as the biocontrol agent.
4. For the control, only Colletotrichum was placed on the PDA plate.
5. Both plates were incubated in the incubator for seven days.
6. The suppression effects of the Trichoderma species isolated was determined and
calculated using Percentage Inhibition in Radial Growth (PIRG) formula.
𝑷𝑰𝑹𝑮 =
𝑹𝟏 − 𝑹𝟐
𝑹𝟏
× 𝟏𝟎𝟎%
Whereas;-
R1 = Radial growth of pathogen in the absence of the antagonist (control)
R2 = Radial growth of pathogen in the presence of the antagonist/ Trichoderma
(treatment)

70
Figure 8.5.1 The control culture.

71
Figure 8.5.2 The dual culture.
Calculation:
𝑷𝑰𝑹𝑮 =
(𝟐. 𝟓𝒄𝒎 − 𝟑. 𝟎𝒄𝒎)
𝟐. 𝟓𝒄𝒎
× 𝟏𝟎𝟎%
𝑷𝑰𝑹𝑮 = 𝟐𝟎%

72
8.6 DISCUSSIONS
Based on the Dual culture method that has been done, it shows a clear sign
of inhibition zone. The calculated PIRG shows that the percentage is -20%. Which
can clearly justify that the Trichoderma able to inhibit the growth of the
Colletotrichum. This also shows that the antagonist activity of the Trichoderma. In
the Figure 8.5.1 which is the control plate, the Colletotrichum shows the darker color
for the base and whitish spores on the surface. The measurement of the
Colletotrichum radius is 3 cm. Meanwhile in the Figure 8.5.2, there were less dark
color of the Colletotrichum which convey the meaning that Trichoderma inhibited the
growth of Colletotrichum. The radius measurement for Figure 8.5.2 is 3 cm for the
Trichoderma while it was 1 cm away from the Colletotrichum. Thus, the PIRG is -
20%.

73
8.7 QUESTIONS
Briefly explain the different modes of action of the antagonist (biological control agent) to
inhibit or control the growth of fungal pathogen.
Modes of Action Explanation
Mycoparasitism A particular biocontrol agent (BCA) specifically attacks
the pathogen and kills it or its propagules.
Antibiosis In small doses, antibiotics can harm or kill other pathogen
since they are microbial poisons. It has been
demonstrated that some microorganism-produced
antibiotics are very effective against plant infections and
the diseases they cause.
Metabolite
production
Other metabolites produced by numerous biocontrol-
active microorganisms can inhibit the growth and activity
of pathogens. Among these metabolites are lytic
enzymes, which may degrade polymeric substances like
DNA, chitin, proteins, cellulose, and hemicellulose.
Competition Many times, microorganisms are deficient in rhizosphere
and soil nutrients. For colonizing the photosphere and
rhizosphere, microbes must compete for resources.
Plants receive nutrients from exudates, leachates, and
senesced tissue.
(Köhl et al., 2019)

74
8.8 CONCLUSIONS
As the conclusion, the objective of this experiment which is to determine the
potential of Trichoderma as potential biological control agent of fungal pathogen is
acceptable. It is because, as the observation which shows the clear sign of inhibition
by Trichoderma. Trichoderma is capable to inhibit the growth of the Colletotrichum
where the Percentage Inhibition Radial Growth is -20%. About 20% of
Colletotrichum’s radius wad inhibited by the Trichoderma. Thus, it is proven that
Trichoderma isolates is capable to prevent the growth of other pathogen and may
act as biological agent for Colletotrichum species.
8.9 REFERENCES
1. Köhl, J., Kolnaar, R., & Ravensberg, W. J. (2019). Mode of action of microbial
biological control agents against plant diseases: Relevance beyond efficacy.
Frontiers in Plant Science, 10.
2. Zin, N. A., & Badaluddin, N. A. (2020). Biological functions of Trichoderma spp.
for agriculture applications. Annals of Agricultural Sciences, 65(2), 168–178.

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