FACULTY OF PLANTATION AND AGROTECHNOLOGY

UNIVERSITI TEKNOLOGI MARA (MELAKA), JASIN CAMPUS

BACHELOR OF SCIENCE IN PLANTATION TECHNOLOGY

AND MANAGEMENT

AT220

AGR514

CROP DISEASE MANAGEMENT

LAB REPORT 6

IDENTIFICATION OF PLANT DISEASE PATHOGENS

LECTURER’S NAME:

MADAM NURUL WAHIDA RAMLI

SUBMISSION DATE:

24 JANUARY 2023

PREPARED BY:

NAME NO MATRICS CLASS

MUHAMMAD NUR ALIFF BIN RAMLAN 2022771787
MOHAMAD ZAKI BIN GADIN 2022771721
NURUL ASYIQIN BINTI ABD GAFAR 2022917661 M3AT2203E
NURUL SYAMIRA SYAMIM SELAMAT 2022745973
RAJA NURUL SYIFA AINA BINTI RAJA DAHLAN 2022900717
INTRODUCTION

Significant financial losses in the global agriculture sector are brought on by plant diseases. It
is essential to keep an eye on plant health and spot infections early in order to prevent disease
spread and develop effective management techniques. Before effective control techniques
can be provided, the condition must be quickly and accurately diagnosed. It is the initial phase
of any illness investigation. The distinctive characteristics of the diseased plant are used to
make the diagnosis. Before making a diagnosis, the pathogen must be located. The natural
look of an afflicted plant species, its local air and soil environment, the cultural circumstances
in which it is growing, the pathogens listed for the region, and the pathogen's capacity to cause
illness are all things that a skilled diagnostician must be knowledgeable with. In most cases,
it is important to isolate and artificially cultivate the pathogen in order to accurately identify and
analyse infections. It is crucial to examine the pathogen under a microscope. Microscopic
inspection is typically needed to identify the pathogen and diagnose the condition. The
pathogen slides were created utilizing isolated cultures or sick tissue.

A. Fungal diseases identification

To appropriately identify the cause of plant diseases and get accurate findings, appropriate
isolation techniques must be used. Several elements must be present for fungus to be
successfully isolated from sick plants:
1. Type of diseased tissue (seeds, leaves, stems, roots)
2. Method of surface sterilization
3. Plating procedure
4. Isolation medium
5. Incubation conditions

Fungal infections are initially recognized by their physical traits, such as spores and spore-
forming structures. For instance, most fungal infections that cause leaf diseases generate
spore-forming structures like perithecia, pycnidia, acervuli, sporangiophores, or conidiophores
that may be examined under a microscope together with the fungus body (mycelium) to a
certain extent. For those who are familiar with fungi taxonomy, the shape, size, colour, and
arrangement of the spores on sporophores or in fruiting bodies, as well as the colour of the
sporophores or fruiting bodies, are sufficient characteristics to suggest the class, order, family,
and genus to which the specific fungus belongs. Once the genus of the fungus has been
determined, descriptions of the known species can be obtained in monographs of the genus
or research publications. Such host indices can be used as shortcuts for rapidly determining
the names of fungal species that might be pertinent to the fungus in question, as there are

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often lists of infections that attack a certain host plant. Conversely, host indexes only serve as
a beginning point for identifying hosts; in the end, identities must be discovered via the use of
monographs and other more specialized literature. Books, keys, and manuals should be kept
on hand in diagnostic labs as resources, and online access to a wealth of scientific journal
articles on taxonomy and identification is available.

B. Bacterial disease identification

Bacteria are little creatures with just one cell. There are over 200 distinct bacteria that
cause plant diseases. They must be viewed under a high-magnification microscope due
to their tiny size. When there are a lot of cells present, plants may occasionally be observed
"oozing" bacteria and other organic waste products. Bacteria have the ability to multiply
quickly, mostly through a process known as binary fission. One cell divides into two during
this step, and two cells divide into four during the next stages. Under ideal circumstances,
one bacterial cell may quickly grow into millions, and populations can double in as little as
20 minutes. Pure cultures of phytopathogenic bacteria are seldom obtained on isolation
medium (even on semi-selective media). Pathogen colonies will typically prevail if the
isolates were produced from new infections and plant material. Materials with advanced
symptoms and old samples are frequently invaded by a succession of saprophytic fungi
and fast-growing bacteria. There are frequently non-target saprophytes that can be
mistaken for infections. Even when using selective and diagnostic medium, colony
morphology alone should never be utilized for diagnosis as several common saprophytes'
features can be readily mistaken with those of phytopathogenic bacteria. Selective or
semi-selective medium make it easier to identify suspicious colonies early in Phyto
bacteriology. King's Medium B is the diagnostic medium that is most frequently utilized.
The luminous pseudomonas, both pathogenic and non-pathogenic, may be distinguished
from one another when colonies are inspected under short-wavelength UV light. The target
bacteria display diagnostic traits on a variety of additional diagnostic media, including
complicated carbon sources that are used by a small group of microorganisms.
Morphological characteristics have minimal taxonomic significance in bacteria because
they are too basic to offer meaningful taxonomic information. The majority of bacterial
classifications are based on their biochemical and physiological characteristics.

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There are several ways to identify bacterial plant diseases:

1. Testing for bacterial streaming

Most phytopathogenic bacteria have flagella, which are visible in fresh material and aid in
their ability to move. Even said, the lack of bacterial oozing does not guarantee that a
bacterium is not to blame for the lesion. Because they are frequently hidden by or mistaken
for other particulate matter like latex, plastids, and starch granules, bacteria can also be
challenging to spot in some plant species.

2. Examination of isolated bacteria

a) Colony appearance
Based on colony shape during isolation, phytopathogenic and saprophytic bacteria
are to be distinguished in the early phases of pathogen identification. Specific
phytopathogenic bacteria have typical colony shape, growth rate, color, and
appearance on various isolation media.
b) Gram Staining
The two kinds of bacteria are Gram-positive and Gram-negative. Gram stain
techniques may be used to differentiate groups since the division is based on
variations in the makeup of the cell wall. After purification, the first step in identifying
a bacteria is gram staining. The majority of phytopathogenic bacteria are gram-
negative rods, either facultatively anaerobic gram-negative rods or gram-negative
aerobic rods. Actinomycetes and related species are comprised of gram-positive
phytopathogenic bacteria.

OBJECTIVES

1. Microscopically detect the fungal plant pathogen

2. Microscopically detect the bacterial plant pathogen

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MATERIALS

• Glass Slide
• Cover Slip
• Glass Dropper Dispenser
• Inoculating Loop
• Inoculation
• Bunsen Burner
• Wooden Test Tube Clamps
• Lactophenol Cotton Blue Solution
• Crystal Violet Dye Solution
• Safranin Dye Solution
• Methylene Blue Dye Solution
• Light Microscope
• Immersion Oil
• Tissue Paper

PROCEDURES

You will look at several pre-made slides in this project, as well as create a few of your own.

A. Fungal pathogens identification

preparing fungal specimens for microscopy observation

a) Preparing a fungal specimen from freshly harvested plant tissue that appears cottony or
has pathogen structures,
1. Use an inoculation needle to carefully scrape a sample from the affected tissue (Figure
7a).
2. Carefully put the sample and a drop of the lactophenol cotton blue dye on the slide
(Figure 7b).
3. Next, carefully apply a coverslip over the specimen with the aid of a needle, ensuring
sure that it is free of air bubbles (Figure 7c).

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 Figure 7
4. Set your slide on the microscope's mechanical stage and start watching.
5. In the areas provided, draw what you are seeing.

b) Preparing fungal specimens from isolated cultures

1. Use an inoculation needle to collect a sample from the isolated culture.
2. Add a drop of Lactophenol cotton blue solution to the sample on a clean slide.
3. Carefully place a coverslip over the specimen with the aid of a needle, checking sure
there are no air bubbles.
4. Set your slide on the microscope's mechanical stage and start watching.
5. In the areas provided, draw what you are seeing.
6. Using the 10X or 40X objective, sketch a few portions of this slide.

B. Bacterial Pathogen Identification

Simple staining of bacteria

1. Hold a glass slide firmly in place with wooden test tube clamps, degrease the surface
with alcohol over a Bunsen burner, and then place the slide on a metal rack or staining
stand with the degreased surface facing upward.
2. Place a little drop of water on the slide (a slide that has been thoroughly degreased
will be moistened), and then stir in a small amount of bacterial culture. In this technique,
a thin suspension will be created. With the inoculating loop's needle, create a film layer
(smear), then let it dry.
3. Use the Bunsen burner to heat up your preparation.
4. After the fixed smear is completely coated, add methylene blue and let it sit for a few
of minutes to stain.

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 5. To get rid of extra methylene blue, wash the smear with tap water.
6. Let the slide dry.
7. When doing a microscope, start with 40x and then 100x objective lenses. Utilize
immersion oil in the latter scenario. Create a representation of the tiny field that was
seen.
8. Once microscopic examination is complete, thoroughly clean all utilized objective
lenses with benzene (do not use alcohol for this as it may destroy the adhesives in the
lenses).

Gram-staining Procedures

1. Put one sterile drop of water on each clean microscope slide.

2. With a cool, sterile loop, remove a portion of a nascent colony from the agar media.
3. Cover the slide with the bacteria. Just enough to make the smudge visible.
4. Pass the slide through a Bunsen flame four times to air-dry and heat-fix the germs on
it but be careful not to overheat it.
5. Pour crystal violet all over the slide and leave it alone for 60 seconds.
6. Rinse with water that is flowing.
7. Empty the surplus water out.
8. Pour Lugol's iodine over the mixture and let it sit for 60 seconds.
9. Rinse for 30 seconds in 95% ethanol.
10. Use water to rinse.
11. Dry with a towel.
12. Safranine counterstain for 10 seconds.
13. Water-based rinsing and drying.
14. Use oil immersion to do an 100x magnification examination. Gram-positive means dark
purple. Red is for gram-negative.

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RESULTS

As you examine the slides, make drawings in the areas provided at the bottom of this activity.

1. Fungal disease identification

Sample from fresh plant tissue

10x magnification 40x magnification

Sample from isolated culture

10x magnification 40x magnification

2. Bacterial identification

40x magnification 100x magnification (oil immersion)

Gram staining

40x magnification 100x magnification (oil immersion)

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QUESTIONS

1. Explain why the fungi and bacteria need to be stained in order, to view under microscope?

To improve the visibility of the cell or of certain components of the cell when seen via a
microscope. Staining the cells in a sample is another option for highlighting metabolic
activities and distinguishing between living and dead cells in the sample.

2. What is the usage of oil immersion in viewing bacteria under microscope?

In light microscopy, immersion oil is used to increase the quality of the images. When
compared to a similar design that does not employ immersion oil, a microscope lens
system that incorporates the usage of microscope immersion oil will provide an image that
is clearer and more distinct.

3. Explain how the bacterial cell wall be stained in gram staining method.

Flood the slide (bacterial cell) with crystal violet for 60 seconds, then rinse under running
water and drain excess water. After that, flood with Lugol's iodine for 60 seconds before
washing with 95% ethanol for 30 seconds, rinsing with water, and blow drying. Then, for
10 seconds, counterstain with safranine, rinse with water, and dry. Finally, using oil
immersion, examine at 100x magnification.

REFERENCES

ZAITON SAPA, N. M. (n.d.). Practical Laboratory Manual For Crop Disease Management .