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INTRODUCTION
INTRODUCTION
Plant pathogens are any organisms that cause disease in plants, including bacteria, viruses, nematodes, protists, and fungi. The interest in plant disease pathogens originates from worries about delicate ecosystems and the need to safeguard food supply. This can affect nearly any kind of plant and results in plant diseases that lower the grower's capacity to generate harvests. The majority of bacteria that can harm plants and cause illness are known as plant disease pathogens.
A. Fungal disease indetification
Fungi are eukaryotic, multicellular organisms that belong to a different kingdom than animalia and plantae. It comprises numerous significant species that cause plant diseases including blight, wilt, rot, mildew, canker, smut, or rust that are significant locally or globally. Fungal pathogens have been identified using morphological characteristics including spores and spore-forming structures. The arrangement, size, color, and shape of the spores on the sporophore or in the fruiting body are all familiar to the taxonomy of fungi. Genera monographs or specialized research publications contain descriptions of known species of fungi once a genus has been recognized. The successful isolation of fungi from disease plants depends on several factors:
type of disease tissue (seeds,leaves,stems,roots)
methods of surface sterilization
planting procedure
isolation medium
incubation conditions
B. Bacterial disease identification
Bacteria are minuscule, single-celled organisms without a nucleus. Certain bacteria infect plants or animals and cause sickness. Bacterial pathogen-induced plant diseases significantly reduce crop and forest productivity and result in large annual losses worldwide. Bacterial plant diseases are widespread plant diseases brought on by bacterial microbes that have a major impact on the production of crops, forests, or natural ecosystems. Based on the extent of plant tissue damage and the symptoms they produce which can include vascular wilt, necrosis, soft rot, and tumors bacterial illnesses can be divided into four major groups. Necrosis can be brought on by pathogens that release poisons. Bacterial infections therefore cause significant losses in a variety of crops, impacting productivity and quality globally. Bacterial pathogen-induced plant diseases significantly reduce crop yields and result in large annual losses worldwide. Bacterial plant disease identification can be done by various methods:
testing for bacterial streaming
examination of isolated bacteria
OBJECTIVE
OBJECTIVE
To identify the fungal plant pathogen microscopically.
To identify the bacterial plant pathogen microscopically.
MATERIALS
MATERIALS
Glass slide & cover slip
Plastic dropper dispenser
Inoculating loop
Bunsen burner
Wooden test tube clamps
Lactophenol cotton blue
Crystal violet dye
Safranin dye
Methylene blue dye
Light microscope
Immersion oil
Tissue paper
PROCEDURES
PROCEDURES
A. Fungal pathogen identification
A. Fungal pathogen identification
The work area was wiped with ethanol and heat with bunsen burner.
A sample was take from isolated culture used inoculation needle.
The sample was placed on a clean slide with a drop of Lactophenol cotton blue solution.
A coverslip was placed gently with the help of a needle, to make sure it covers the specimen completely without air bubbles.
The slide was placed on the mechanical stage of the microscope and observed.
B. Bacterial disease identification
B. Bacterial disease identification
The work area was wiped with ethanol and heat with bunsen burner.
A small drop was placed of sterile water on a clean glass slide.
A part of young colony was removed with a cold, sterile loop from the agar medium.
The bacteria was air-dry and heat fix in the slide by passing the slide a few times though a Bunsen flames.
The slide was flooded with crystal- violet and set aside for 60 seconds.
The slide was rinsed under water.
The excess water was drained off.
The lugol's ioidine was flooded and set aside for 60 seconds.
It was washed with 95% ethanol for 30 seconds.
The slide was rinsed under water.
The safranine was counterstain for 10 seconds and rinse with water then dry.
The result was examine at X 100 magnification using oil immersion.
Gram-positive = dark purplish
Gram-negative = red
RESULT
RESULT
- FUNGAL DISEASE IDENTIFICATION
10 X magnification
10 X magnification
40 X magnification
40 X magnification
- BACTERIAL IDENTIFICATION
Gram-negative
Gram-negative
Gram-positive
Gram-positive
DISCUSSION
DISCUSSION
Fungal disease pathogens using Lactophenol blue stain. Lactophenol blue stain is a mounting medium and staining agent used in the preparation of slides for microscopic examination of fungi. It is used to preserve fungal structures and clean tissue while phenol acts as a disinfectant. Apart from that, it is also used to improve the clarity of the fungal cell structure under the microscope. Fungi do not have a fixed cell wall structure like bacteria so they are classified according to colony characteristics and morphology.
Gram staining is necessary for harmful bacterial illnesses. A Gram stain is a lab test used to look for bacteria in certain bodily fluids or at the location of a suspected infection. It can assist direct diagnostic testing and further treatment options and yield rapid findings. There are two types of Gram staining: gram-positive and gram-negative. The physical and chemical characteristics of bacteria's cell walls are used by Gram staining to identify them. The thick coating of peptidoglycan in the cell walls of gram-positive bacteria gives them a blue to purple appearance when stained with Gram stain. Gram-negative bacteria have cell walls that are rich in lipids and have a thin coating of peptidoglycan. They turn crimson to pink when stained with Gram stain as a result.
QUESTIONS
QUESTIONS
Explain why the fungi and bacteria need to be stained in order, to view under microscope?
=The majority of cell types lack natural pigment, making them difficult to observe under a light microscope unless they are dyed. A variety of dyes are applied to increase the visibility of bacterial cells and enhance the contrast and make it much easier to see the bacteria and their internal structures.
What is the usage of oil immersion in viewing bacteria under microscope?
= Immersion oil reduces light refraction and replaces the air gap between the immersion objective lens and cover glass with a high refractive index medium, increasing the microscope's resolving power. This magnification is crucial for studying smaller bacterial structures, such as cell walls, flagella, and pili.
Explain how the bacteria cell wall be stained in gram staining method.
= The Gram staining method uses red or purple staining to distinguish between Gram positive and Gram negative groupings. Gram-positive bacteria stain purple because their cell walls include a thick layer of peptidoglycan, which keeps the crystal violet staining intact.
CONCLUSIONS
CONCLUSIONS
In summary, bacterial and fungal plant pathogens can be identified under a microscope. Effective disease management and the sustainability of agriculture depend on the identification of plant disease pathogens. The ability to differentiate between identical infections, comprehend pathogenicity and host specificity, and monitor new threats all depend on this precise identification. Once the weaknesses of disease-causing pathogens are identified, they can be promptly stopped. The Gram staining method has been employed to identify bacterial plant pathogens. This method's ability to yield data quite quickly and identify the presence of germs is one of its benefits.
REFERENCES
REFERENCES
Bruckner, M. Z. (2007, May 16). Gram staining. Microbial Life Educational Resources. https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
Bacterial Plant Disease | UNDRR. (2023, June 7). Www.undrr.org. https://www.undrr.org/understanding-disaster-risk/terminology/hips/bi0012
LACTOPHENOL BLUE STAIN. (2014). https://www.dalynn.com/dyn/ck_assets/files/tech/SL18.pdf
Mokobi, F. (2022, August 21). Lactophenol Cotton Blue (LPCB) Staining. Microbe Notes. https://microbenotes.com/lactophenol-cotton-blue-staining/
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