INTRODUCTION

Plant disease diagnosis is a vital process that involves identifying diseases based on visible
symptoms and signs of pathogens on plants. It’s essential to distinguish between biotic factors,
like fungi and bacteria, and abiotic factors, such as nutrient deficiencies, extreme weather, or
environmental contaminants, as both can produce similar symptoms. Correctly identifying the
cause is crucial for effective disease management and crop protection, as pathogens often
mimic environmental stress. Certain diseases, such as crown gall caused by Agrobacterium
tumefaciens and powdery mildew on cucurbits, are easily detectable through visual
examination. However, some instances may require more thorough analysis, including
microscopy or reference to disease encyclopedias, especially when visible symptoms are
insufficient (Agrios, 2005; Cooke, Jones, & Kaye, 2006). The rise of digital technologies has
made disease diagnosis more accessible, with apps like Plantix enabling users to quickly
identify potential diseases by photographing affected plants. This provides farmers, researchers,
and extension agents with the ability to make faster decisions (Nelson & Bushe, 2006). This lab
examines both traditional and modern plant disease diagnosis methods, combining visual
assessment, microscopy, and mobile app diagnostics to offer a deeper understanding of plant
health and pathogen impacts.

OBJECTIVES
1. To identify the fungal plant pathogen microscopically

2. To identify the bacterial plant pathogen microscopically

MATERIALS
1. Glass Slide
2. Cover Slip
3. Glass Dropper Dispenser
4. Inoculation Loop
5. Inoculation
6. Bunsen Burner
7. Lactophenol Cotton Blue Solution
8. Crystal Violet Dye Solution
9. Safranin Dye Solution
10. Methylene Blue Dye Solution
11. Light Microscope
12. Immersion Oil
13. Tissue Paper
PROCEDURE
A. Identifying Fungal Pathogens

Preparing fungal specimens for microscopic observation

a) Preparing a fungal specimen from fresh plant tissue with a cottony appearance or
signs of pathogen structures
1. A sample was taken by gently scraping the diseased tissue with an inoculation
needle.
2. The sample was carefully placed on a slide in a drop of lactophenol cotton blue dye.
3. A coverslip was then gently placed over the specimen using a needle, ensuring
complete coverage without air bubbles.
4. The slide was placed on the microscope's mechanical stage for observation.
5. Sketches were made of the observations in the spaces provided.

b) Preparation of fungal specimens from isolated culture

1. A sample was taken from the isolated culture using an inoculation needle.
2. The sample was placed on a clean slide in a drop of Lactophenol cotton blue solution.
3. A coverslip was gently placed over the sample using a needle, ensuring complete
coverage without air bubbles.
4. The slide was placed on the microscope's mechanical stage and observed.
5. Sketches were made of the observations in the spaces provided.
6. A few areas of the slide were sketched using the 10X or 40X objective lens.

B. Bacterial pathogen identification

1. A glass slide was gripped with wooden test tube clamps, and its surface was degreased
with alcohol over a Bunsen burner. It was then placed on a metal rack or staining stand with the
degreased surface facing upwards and allowed to cool.
2. A small drop of water was placed onto the cooled slide (a properly degreased slide
would be wet, and a small loopful of bacterial culture was mixed into it, creating a thin
suspension. A film layer (smear) was made with the needle of the inoculating loop and left to air
dry.
3. The prepared slide was heat-fixed over a Bunsen burner. Methylene blue was applied to
the fixed smear until it was fully covered, and the smear was left to stain for 1-2 minutes.
4. The stained smear was washed with tap water to remove any excess methylene blue
and then allowed to dry.
5. During microscopy, the 40x objective lens was used initially, followed by the 100x
objective lens with immersion oil. A drawing was made of the observed microscopic field.
6. After the microscopic observation was complete, all used objective lenses were cleaned
with benzene (alcohol was avoided for this purpose as it can dissolve the lens adhesives).
RESULT
Table

1. Fungal Disease Identification

Sample From Fresh Plant Tissue

Figure: 40x of powdery mildew on mango leaf Figure: 100x of powdery mildew on mango
leaf
Sample From Isolated Culture

Figure: 40x of hyphae on mango red rust Figure: 100x of hyphae on mango red rust
 2. Bacterial Identification

Figure: 40x of bacteria on rubber leaf Figure: 100x of bacteria on rubber leaf
QUESTIONS
1. Explain why the fungi and bacteria need to be stained in order to view under
microscope?
Fungi and bacteria need to be stained before being viewed under a microscope because
their natural structures often lack sufficient contrast to be clearly visible under a light
microscope. Staining enhances the visibility of cellular components such as cell walls,
hyphae, spores, and bacterial cells, making it easier to distinguish between different
pathogens and observe their characteristics. For fungi, stains like lactophenol cotton
blue highlight key structures such as hyphae and spores, while for bacteria, stains like
methylene blue help visualize individual bacterial cells by coloring them, thus improving
contrast against the background. Without staining, the cells of both fungi and bacteria
are typically transparent and difficult to differentiate, making identification and diagnosis
challenging. Staining not only improves visibility but also aids in the identification of
pathogens by enhancing their structural details (Harris, 2008; Preece & Horgan, 2010).

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

Oil immersion is used in microscopy to improve resolution and clarity when viewing
bacteria. It involves placing a special immersion oil with a refractive index like that of
glass between the objective lens and the slide. This technique helps to reduce light
refraction as light passes through the specimen, which would otherwise distort the
image. By minimizing light loss, oil immersion allows for higher magnification and clearer
views of the fine details of bacterial cells. This is particularly important for viewing
smaller organisms like bacteria, where high resolution is needed to distinguish between
various cell shapes, sizes, and arrangements (Luna, 2004). The use of oil immersion
increases the numerical aperture of the objective lens, resulting in a sharper and more
detailed image, essential for accurate bacterial identification and analysis (Bancroft &
Gamble, 2008).
 3. Explain how the bacterial cell wall be stained in gram staining method.
In the Gram staining method, bacterial cell walls are stained through a series of steps
that differentiate bacteria into two groups: Gram-positive and Gram-negative, based on
their cell wall structure. The procedure begins with applying crystal violet dye to the
bacterial smear, which stains all bacterial cells. Next, iodine is added, which forms a
complex with the crystal violet, making it more difficult to wash out. After this, the smear
is treated with alcohol or acetone, which acts as a decolorizer. This step is crucial
because it removes the crystal violet-iodine complex from Gram-negative bacteria, but
Gram-positive bacteria, which have thicker peptidoglycan layers in their cell walls, retain
the dye. The final step involves applying safranin, a counterstain, which stains the Gram-
negative bacteria a red or pink color, while the Gram-positive bacteria remain purple.
The Gram staining method is essential for bacterial identification, as the structural
differences in the bacterial cell wall are critical for determining the appropriate treatment
(Willey, Sherwood, & Woolverton, 2017).

CONCLUSION
The objective of this experiment, which aimed to identify fungal and bacterial plant pathogens
through microscopy, was successfully achieved. By carefully applying aseptic techniques,
staining methods, and performing microscopic observations, we were able to identify and
differentiate between fungal and bacterial pathogens. For fungal pathogens, the use of
lactophenol cotton blue dye improved the visibility of structures such as hyphae and spores,
allowing for detailed observation and documentation of their unique characteristics. For bacterial
pathogens, methylene blue staining and oil immersion microscopy provided clear views of
individual bacterial cells, aiding in their identification. This experiment emphasized the
importance of proper sample preparation and staining techniques to enhance contrast and
reveal structural details that are typically invisible under a light microscope. Through this
process, we gained valuable hands-on experience in diagnosing plant diseases, which is an
important skill in plant health management.
REFERENCES
Agrios, G. N. (2005). Plant pathology (5th ed.). Elsevier Academic Press.

Cooke, B. M., Jones, D. G., & Kaye, B. (2006). The epidemiology of plant diseases. Springer.

Nelson, D., & Bushe, R. (2006). Using mobile technology in plant disease diagnosis.
International Journal of Agricultural and Biological Engineering, 2(4), 23-30.

Harris, R. L. (2008). Plant pathology: A practical approach. Springer.

Preece, T. F., & Horgan, E. (2010). Fundamentals of microbiology. Wiley-Blackwell.

Bancroft, J. D., & Gamble, M. (2008). Theory and practice of histological techniques (6th ed.).
Churchill Livingstone.

Luna, L. G. (2004). Manual of histologic staining methods of the Armed Forces Institute of
Pathology (3rd ed.). McGraw-Hill.

Willey, J. M., Sherwood, L. M., & Woolverton, C. J. (2017). Prescott's microbiology (10th ed.).
McGraw-Hill.