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Plant Pathology

PP-615 Physiological Plant Pathology 2(1-1) Elective

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Practical one 24-09-2021

Mudasir Iqbal

Physiology
the study of plant function and behaviour, encompassing all the dynamic processes of
growth, metabolism, reproduction, defence, and communication that account for plants being
alive

Physiological plant disorders

are caused by non-pathological conditions such as poor light, adverse weather, water-
logging, phytotoxic compounds or a lack of nutrients, and affect the functioning of the plant
system.

Contents of PP-615 Practical course

Fungus cause 70% diseases in plants

Fungal culture, isolation multiplication
In-vitro & In-vivo
In-vitro are done in controlled environment in lab and we use artifical media ( PDA made up
of Agar, Dextrose
Inoculation : Temp for fungus is 20-25 / 27 oC
 Spores / conidia isolation : 106 - 107 spores/ml
Hemocytometer is used for spores / conidia isolation and number estimation
CFU (colony forming unit)
is a unit commonly used to estimate the concentration of microorganisms in a test sample.
The number of visible colonies (CFU) present on an agar plate can be multiplied by the
dilution factor to provide a CFU/ml result.

Bioassay
determination of the relative strength of a substance (as a drug) by comparing its effect on a
test organism with that of a standard preparation.

After extraction of spores it is spored or transfered on plant leaves and left to grow there.
Repeat the spray weekly and observe the symptom development and look for plant growth
parameters
Dry media is prefered over fresh. There can be false +ve results while using fresh media
After this process pathogen is again extracted

1. Fungal spores / conidia (How they penetrate, cause successful infection)

2. Host - Pathogen interactions
3. Succeptiblity and resistance in host against pathogen. Effectors secreted by pathogen
against PR by host. The stronger will win
4. Pthogenicity and virulance : Abality of pathogne to cause disease
Herd immunity :
When body automatically create antibodies after ocurance of a disease
a form of indirect protection from infectious disease that can occur with some
diseases when a sufficient percentage of a population has become immune to an
infection, whether through vaccination or previous infections, thereby reducing the
likelihood of infection for individuals who lack immunity.
5. Management :
Chemical : Chemicals have negative effect on human and environment so they
are highly discouraged
Biocontrol : use of natural processes to control disease.
E.g: Biocontrol/fungi, Trichoderma, Bacilliomicus, namatodes (EPN) are benificial
Some important cultural techniques

Start of course
Important terms :

Symptoms
patterns, expressions

Signs
Physical evidance of pathogen

Major pathogens
1. Fungi : Eukaryotes, heterotrophs, saprotrops, obligate
2. Omycetes: Phytopthora infestans, attack potato
3. Nematodes : Animals having all the system expect digestive and
4. Aging researc is being carried out on nematodes. All are obligate (Plant nematodes)
5. C. elaquas feeds on bacteria and can be grown in lab

Field work :
Farmer reachs out to you
Identification : ( Symptoms, diagnostics etc.)
Colletotrichum has many classes which cause confusion while identification
So you should go for PCR
morphological symptoms are observed under microscope. Molecular. ITS, 18sRRN

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Plant Pathology

PP-615 Physiological Plant Pathology 2(1-1) Elective

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Practical Two 01-10-2021

Mudasir Iqbal

Inoculation

B.Cineria cause Gray Mold disease

We need to isolate it from leaves fruits

1. Sample : Sterilize with NaOCl wash under running tap water 2 - 3 times for few minutes
2. Petri dish with Nutrient media (PDA). Transfer samples in it wrap and incubate at
specific temperature
3. Inoculation vary for different pathogens however dark condition is prefered for all
4. After incubation fungus / mycelia are developed
5. Observe under microscope, there we see reproductive units spores/conidia
6. Now we have to isolate the conidia

Isolation of Conidia
 1. We need particular amount of material obtained from previous work
2. Grow B.Cineria in 9 cm Petri dish
3. Add 5-7ml water on it and dilute then scrap back and forth with the help of needle
4. Take falcon tubes and transfer the material in it use chesse cloth which prevent mycilia
and allow spores/conidia to pass
5. Now we have stock of conidia but we need particular amount 106-107/CFU (colony farming
unit)
6. Hamocytometer is used for colony counting it is called colony counter and have
counting chambers for conidia
7. It is just like slide but have 2 chambers in center which further have small boxes

Counting of conidia

1. Add 10ul on both chambers of slide these are called counting chambers
2. Each chamber have small boxes than again each box have further 20 small boxes
3. Count from 4 to 5 random boxes the no. of conidia than take the average of all to find
the result
4. CFU/ml = (90 (No. of conidia average) / 0.02 (Correction factor)) * 10ul (the material we used for test)
* 1000 (To covert ul to ml)
5. The result is 4.5 * 107 conidia/ml
6. Now as we needed 106. so we have to convert.
7. We have to dilute it 45 times to covert into desired result
8. Take 1ml from stock and add 45ml H2O so that we'll have working results

Host-Pathogen interactions

Conidia lands on leaves but don't cause direct disease this disease development process is
called as Host-pathogen interaction
This is based on resistance and succeptiblity
Resistance : Ability to resist against disease. Host-pathogen interaction is strong
 Succeptibilty : Ability to acquire the disease. Host-pathogen interaction is weak
The host-pathogen interactions if strong there will be battle between host and pathogen
where the stronger will win
E.g: B.Cineria lands on strawberry or any other fruit.
First MAMP / PAMP start
MAMP : Microbial associated molecular patterns
PAMP : Pathogen assocaited molecuar patterns

First layer defence

When pathogen attacks MAMP is activeted as part of defence by plant on surface on plant
The first thing that intiates is PTI (PAMP-Triggered Immunity) and produces R-genes/R-
proteins (Resistant genes) which try to dominate the pathogen
This is First layer Defence
Pathogen secrets genes to battle back called Effectors. to supress the first layer defence
Effectors are protein, the main reason for attack is that pathogen needs food/nutrients from
plant for growth/reproduction

Second layer defence

If pathogen bypass the first layer than plant activates the second layer defence system ETI.
ETI (Effectors triggered Immunity) is activated against the effectors released by
pathogen
ETI Produces the stronger R-proteins if pathogen is still strong enough ETI produces HR
Hyper-senstive response (HR) : In this situation plant kills its own part where the
pathogen has attacked to avoid the further spread of pathogen
Here host becomes dominant due to HR

Floris : studied host-pathogen interactions

He gave concept of gene for gene hypothesis
Hypothesis : Starting guess (Null, alternative are two types) than hypothesis undergoes
testing
1. Observation
2. Hypothesis
3. Experiments (Theoratical then Pratical)
4. Results
 5. Data analysis
6. Theory
7. Conclusion

Pathogen & host evolve at same time. If host have defence evoluation pathogen also
evloves accordingly

Co-existance : Evoluation accordingly / adaptations

Every host have genes against genes of pathogens
E.g: Host have allels RR while pathogen have A & a
Avirulance?
RR - A There will be no disease
Rr - A There will be no disease
RR - a There will be no disease
Rr - a There will be diseae because two alles are small words means pathogen will be
dominant
So in this case there are 25% of chances of disease occurance

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PP-615 Physiological Plant Pathology 2(1-1) Elective

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Practical Three 22-10-2021

Atta Ur Rehman

Pathogen : Always associated with disease

Parasite : May or may not cause disease. Under specific conditions cause disease

Host : Crop or plant invaded by pathogen

Pathogen : Cause damage in palnt and take food nutrients

Normal functions of Plants

Photsynthesis : Pathogen affect it which result in necorsis (death of cells) or chlorosis

(death of chlorophyll)

Three factors Host, Pathogen and environmental conditions are essential for disease
development this is called disease triangle

Each and every pathogen has its range of temperature where it grow and cause disease

Wheat rust : In feburary if there are rains the moisture level will increase and temperature
will decrease which result in rust infection while if any one factor is missing there will be no
disease development . Time is fourth facotr along with Disease triangle

Obligate : Grow only on living host. Rust is obligate parasite it can not be cultured in lab

Facultative : Grow on living as well as non-living media (artificial media)

Effect of temperature on pathogen growth
We need samples collected from filed
Based o symptoms (physical expression) due to disease (due to pathogen) / disorder
(due to environment)
Collect sample with symptoms and label them then take to lab
Make media (substance used to isolate particular pathogen)
Do isolation of pathogen from host for experimental identification
How to make media ?
PDA (potato dextrose agar)
What is concentration of media ?
Media is made in flask
After adding all the materils the total volume should be 1L
Than do sterilization (kill microbes) using Autoclave (sterilization of glassware and
media at 121oC for 15 minutes)
After cooling place in Laminer flow chamber (it is sterlized 99% contaimination free)
Pour media in petri dishes and place the dishes there.
Cut the samples which have symptoms in small pieces
Than disinfect (germ free from other pathogens present on sureface)
Superficial pathogens : Pathogens present on the surface only penetrated pathogens
will cause disease
If not disnfected the superficial pathogens will grow on the media quickly
Use Sodium hypoclhorate NaOCl as disinfectant
Dip samples in first flask which contain distilled water than in disninfectant than again in
third which contains distilled water
You should place 3 to 4 samples on 3 to 4 different Petri dishes air tight and wrap them
to avoid contamination
They are placed in incubator for incubation. Incubator provide optimum conditions for
growthof pathogen
After 7 days you shall see the growth. Then pick slide preparation and adjust at
vdifferent temperature ranges. I.e: 30oC, 35oC and 40oC
We have to see the temperature affect on pathogen growth, temperature is fixed in
incubator
Inspect daily after 12 hours and notes the changes in growth. Color of mycllea / hypha
via visual observation
Than check at various other temperatures ranges
For example pathogen become more vigorious above 30oC. Fin the range i.e: 35-40oC,
40-45oC so on.
 There is a temperature where pathogen cannot cause the disease
Do replication / reproduction petri dishes and sample to avoid errors

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PP-615 Physiological Plant Pathology 2(1-1) Elective

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Practical Four 04-11-2021

Maam Muqadas

Major pathogens of plants : FUngi, bacteria, Nematodes and viruses

Vrisu are obligate. Fungus is obsorpitve

Isolation of bacteria : For this we should be aware of lab saftey rules and instruments

Lab saftey rulse :

1. Washing area : You should know the location of qwashing area

2. All chemicals should be labeled
3. Don not eat or drink anything in lab
4. Use gloves, glasses and lab coat
5. Imporatant : You should know about the working of instrument you are going to use

Instruments :

1. Autoclave : (Pressure cooker type)

Auto -- self, clave -- self locking
121 C and 15 Pa for 15-30 minutes
Work with heat, steam and temperature
For sterilization, disinfection before isolation or anywork. Products are mostly
autoclaved. Plastic plates are not glasswaare are autoclaved
2. Laminer flow : (Biosaftey cabnet) Contamination free working environment. It has
different parts
 Use for media pouring, culturing
It has Cabnet, wrking station, UV lamp, filter
Working station has sections to place materials in center we do our experiment
fFilter (HEPA : High effeciency particulate air). Decontiminate air before it is
escaped.
3. Fridge : For preservation of sample
4. Incubator : grow culture and maintainance of temperature and humidity. For fungi,
bacteria culturing and hatching process of nematodes
5. Incubaotr shaker : pahogen multiplication occur here & for culturing
6. Oven : heating and drying of sample t take dry weight
7. Weighing balance : To weigh weights of samples etc

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Plant Pathology

PP-615 Physiological Plant Pathology 2(1-1) Elective

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Practical Five 29-11-2021

Dr. Atta Ur Rehman

Pathogen : Always associated with disease

Parasite : May or may not cause disease. Under specific conditions cause disease

Host : Crop or plant invaded by pathogen

Pathogen : Cause damage in palnt and take food nutrients

Normal functions of Plants

Photsynthesis : Pathogen affect it which result in necorsis (death of cells) or chlorosis

(death of chlorophyll)

Three factors Host, Pathogen and environmental conditions are essential for disease
development this is called disease triangle

Each and every pathogen has its range of temperature where it grow and cause disease

Wheat rust : In feburary if there are rains the moisture level will increase and temperature
will decrease which result in rust infection while if any one factor is missing there will be no
disease development . Time is fourth facotr along with Disease triangle

Obligate : Grow only on living host. Rust is obligate parasite it can not be cultured in lab

Facultative : Grow on living as well as non-living media (artificial media)

Effect of temperature on pathogen growth
We need samples collected from filed
Based o symptoms (physical expression) due to disease (due to pathogen) / disorder
(due to environment)
Collect sample with symptoms and label them then take to lab
Make media (substance used to isolate particular pathogen)
Do isolation of pathogen from host for experimental identification
How to make media ?
PDA (potato dextrose agar)
What is concentration of media ?
Media is made in flask
After adding all the materils the total volume should be 1L
Than do sterilization (kill microbes) using Autoclave (sterilization of glassware and
media at 121oC for 15 minutes)
After cooling place in Laminer flow chamber (it is sterlized 99% contaimination free)
Pour media in petri dishes and place the dishes there.
Cut the samples which have symptoms in small pieces
Than disinfect (germ free from other pathogens present on sureface)
Superficial pathogens : Pathogens present on the surface only penetrated pathogens
will cause disease
If not disnfected the superficial pathogens will grow on the media quickly
Use Sodium hypoclhorate NaOCl as disinfectant
Dip samples in first flask which contain distilled water than in disninfectant than again in
third which contains distilled water
You should place 3 to 4 samples on 3 to 4 different Petri dishes air tight and wrap them
to avoid contamination
They are placed in incubator for incubation. Incubator provide optimum conditions for
growthof pathogen
After 7 days you shall see the growth. Then pick slide preparation and adjust at
vdifferent temperature ranges. I.e: 30oC, 35oC and 40oC
We have to see the temperature affect on pathogen growth, temperature is fixed in
incubator
Inspect daily after 12 hours and notes the changes in growth. Color of mycllea / hypha
via visual observation
Than check at various other temperatures ranges
For example pathogen become more vigorious above 30oC. Fin the range i.e: 35-40oC,
40-45oC so on.
 There is a temperature where pathogen cannot cause the disease
Do replication / reproduction petri dishes and sample to avoid errors

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