LAB REPORT 5

ISOLATION OF PLANT PATHOGEN

INTRODUCTION

It is frequently necessary to isolate and cultivate pathogens from diseased specimens in order to identify them. Isolation is the process of separating a plant pathogen from its source or host and cultivating it in pure culture on a synthetic medium. Pathogen cultures could also be required for other reasons. The primary components of plant pathogens are bacteria, fungus, viruses, viroids, and phytoplasms.

FUNGAL

Fungal infections can cause observable symptoms by infecting several plant components, including roots, stems, leaves, flowers, and fruits. It is often possible to obtain a pure culture by direct transfer to a growth medium. Simply contacting the spores with a sterile, fine-gauge inoculating needle—which can be dry or moistened by first stabbing it into the sterile medium—and then streaking onto a plate or slope will do this. A number of variables affect the ability to successfully isolate fungus from diseased plants, including the type of diseased tissue, the plating process, the isolation medium, the incubation conditions, and the surface sterilization technique.The presence of one or more types of fungus or spores is ubiquitous in many habitats, allowing fungi to spread widely.

BACTERIA

An important first step in identifying the source of plant disease is the isolation of bacteria from affected plant material. Bacteria are tiny organisms that often only have one cell. Plant diseases are known to be caused by about 200 different species of bacteria. Bacteria use a technique called binary fission to multiply quickly. One bacterial cell can grow to thousands in a matter of hours, and under the right circumstances, the population can double in just 20 minutes.

OBJECTIVE

To isolate the plant pathogenic fungi and bacteria from symptomatic plant samples.

MATERIALS

Nutrient agar and inoculating loop

Plant disease sample

Potato Dextrose Agar (PDA)

Scalpel

Sterile tissue paper

Sterile distill water

70% ethyl alcohol

10% sodium hypochlorite

PROCEDURES

A) Fungal isolation from stem

The work area was wiped with 70% ethyl alcohol
Forceps and scalpel were dipped in 70% ethyl alcohol and flame dried
Surface of stem tissue was sterilized by wiping with soft paper that had been dipped in 70% ethyl alcohol for 5 seconds.
Next, sterile water had been used to rinsed and damp-dry on sterile tissue paper.
Small pieces was aseptically cut from the margin of the healthy and diseased tissue.
The sample was transefered to PDA by placed the piece near the side of the plant and the plate was incubated for 24 hours.
A final identification was made using pure cultures grown from a single germinated spore or a hypal tip .
Fungal pathogen was identified based on morphological features and classified.

B) Bacteria isolation

The plant tissue was places in a test tube contained 3 ml of a liquid and allow diffusion at room temperature for 5 minutes.
The appropriate agar media was streaked by leachate using a wire loop for obtained single colonies.
A series of 1:10 dilution of the leachate was made in sterille water and plated by spreaded 0.1 ml on the surface of dried agar plates using a sterile L- shaped glass rod.
The agar plate was incubated for 24 hours.
Bacteria pathogen was identified based on morphological features and classified.

RESULT

Macroscopic features

Fungal

Creamy-white and dark green colony
Circular and large shape
The elevation is raised
Margin is even
AFPA +, CREA -

Bacteria

Clear colony
Small and punctiform shape
The elevation is flat
Surface is smooth and glistening

DISCUSSION

The fungal colonies' outcomes from this experiment are dark green and creamy white in hue. The chili sample utilized to differentiate between healthy and sick samples is nearly completely covered by its big shape. This is a result of the decision being made after the allotted time. As a result, fungal growth is rapid and saturates the nutrient order. In terms of bacteria, they create tiny, punctiform, transparent colonies. However, because the bacteria are destroyed by the heated inoculation loop, the results are not observed on the full nutrient. It can also result from actions made after the deadline, which prevents the colony's growth from reproducing.

QUESTIONS

Why we need to keep the healthy tissue along with symptomatic tissue during isolation?

= This seeks to verify the existence of infections and guarantee a correct diagnosis. In order to differentiate harmful organisms from potentially present saprophytic or ambient microbes, healthy tissue acts as a control. It aids in separating the main disease from opportunistic microbes or secondary invaders, which might emerge only after the main infection has debilitated the plant.

If you get more than one type of bacteria or fungus species on the media, what next you need to do?

= Each species must be isolated in order to produce a pure culture. Morphological identification is one approach that can be applied. Under a microscope, every growth characteristic is inspected, including colony color, shape, size, and texture. It offers early hints for recognizing various creatures. Extensive documentation of the isolation procedure is maintained, encompassing colony appearance, biochemical and molecular outcomes, and pathogenicity assays. Accurate identification and further studies on illness management will be aided by these data.

What are the different between fungi and bacteria colony on media?

= The difference between fungal and bacterial colonies on media is that bacteria are unicellular organisms while fungi can consist of unicellular or multicellular organisms. Second, bacteria have a smooth or rough appearance and fungi have a fuzzy appearance. Third, bacteria also have defined margins while fungi have filamentous margins. For bacteria it can grow in pH 5 to 9 while for fungi it can grow in pH 5 to 6.

What can be concluded from the activities above?

= Things that can be deduced from the activity on fungi and bacteria colonies give different characteristics and colors.

CONCLUSIONS

In conclusion, we were able to isolate plant pathogenic fungi and bacteria from symptomatic plant samples. The characteristics of fungi and bacteria have also been identified from the results obtained. fungi and bacteria have different characteristics and their own uniqueness. Isolation of plant pathogens is an important step in diagnosing and managing plant diseases. It can help in detecting the presence of new pathogens and monitoring their growth.

REFERENCES

Alsohaili S A, Bani-Hasan B M. Morphological and molecular identification of fungi isolated from different environmental sources in the Northern Eastern desert of Jordan. Jordan Journal of Biological Sciences, 2018, 11(3).
CD Genomics. (n.d.). How to Distinguish Bacteria and Fungi: From Morphology to Sequencing - CD Genomics. Www.cd-Genomics.com. https://www.cd-genomics.com/microbioseq/how-to-distinguish-bacteria-and-fungi-from-morphology-to-sequencing.html
Dianna Gellar. (2023, October 19). How to Distinguish Bacteria and Fungi: From Morphology to Sequencing. PharmiWeb.com. https://www.pharmiweb.com/article/how-to-distinguish-bacteria-and-fungi-from-morphology-to-sequencing
Reynolds, J. (2016). Bacterial colony morphology. Biology LibreTexts. https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/08%3A_Bacterial_Colony_Morphology

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