pb experiments protocols

8/8/2019 PB Experiments PROTOCOLS

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Detailed experiments of Phytophthora blight disease of pigeonpea

Experiment No. PB- 001-10

Title: Collection, purification and maintenance of pure couture of Pdc.

Completed

Repetition: Yes

S.

No

Isolate

name

Location State Year of

collection

Year of

isolation

Single

spore Pathogen

1 Pdc-1 ICRISAT, BP 15B Andhra pradesh 2009 2009 No Yes

2 Pdc-2 ICRISAT, BP 2 Andhra pradesh 2009 2009 No Yes

3 Pdc-3 ICRISAT, BP 7A Andhra pradesh 2009 2009 No Yes

4 Pdc-4 ICRISAT, BP 7C Andhra pradesh 2009 2009 No Yes

5 Pdc-5 ICRISAT, BW 8 Andhra pradesh 2009 2009 No Yes

6 Pdc-6 ICRISAT, RCE 12B Andhra pradesh 2009 2009 No Yes

7 Pdc-7 ICRISAT, RCE 24 Andhra pradesh 2009 2009 No Yes

8 Pdc-8 ICRISAT, RCW 1 Andhra pradesh 2009 2009 No Yes

9 Pdc-9 ICRISAT, RCW 19A Andhra pradesh 2009 2009 No Yes

10 Pdc-10 ICRISAT, BR 1A Andhra pradesh 2009 2009 No Yes

11 Pdc-11 ICRISAT, BR 4D Andhra pradesh 2009 2009 No Yes

12 Pdc-12 ICRISAT, BR 2J Andhra pradesh 2009 2009 No Yes

13 Pdc-13 ICRISAT, RP 8A Andhra pradesh 2009 2009 No Yes

14 Pdc-14 ICRISAT, RP 8C Andhra pradesh 2009 2009 No Yes

15 Pdc-15 ICRISAT, RM 1 Andhra pradesh 2009 2009 No Yes

16 Pdc-16 ICRISAT, BM 1 Andhra pradesh 2009 2009 No Yes

17 Pdc-17 ICRISAT, BM 25 Andhra pradesh 2009 2009 No Yes

18 Pdc-18 ICRISAT, BIL 7A Andhra pradesh 2009 2009 No Yes

19 Pdc-19 ICRISAT, BUS 14B Andhra pradesh 2009 2009 No Yes

20 Pdc-20 ICRISAT, RL 33 Andhra pradesh 2009 2009 No Yes

21 Pdc-21 ICRISAT, RL 26 Andhra pradesh 2009 2009 No Yes

22

Pdc-22

Naganpally, Bodhan (11),

Nizamabad

Andhra pradesh

2009 2009 No No

23 Pdc-23 Vargoti, Ambajogai (32), Beed Maharastra 2009 2009 No No

24 Pdc-24 Silomba, Ambajogai (34), Beed Maharastra 2009 2009 No No

25 Pdc-25 Murad, Latur Maharastra 2009 2009 No No

26 Pdc-26 Alni, Osmanabad Maharastra 2009 2009 No No

27 Pdc-27 ARS, Gulbarga Karnataka 2009 2010 No No



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Title: Pathogenicity of the Pdc isolates

Objective: To prove the pathogenicity of the Pdc isolates

Methods and materials:

Inoculation technique

raych

Inoculum form

3. Host- Growth stage: 15 days old

Variety- HY3C

Environment: Controlled environment (Growth chamber- Temp- 15 & 25)Design of experiment

Treatments: 6+1Replication: 3

No. of plants/ potObservation

Temp: 15 & 25oC

RH: Nil

Light: 12 hrsProgress of disease development

0 day- inoculation

1 day- incubationTypical symptoms development

Termination of the experiment

80% plant showing the typical PB symptoms

Precautions:Flooding with sterilized water

Plating of water contaminants

Exposing plats in experiment environments to catch contaminants.

4. Data analysis: Collection and compilation.

Conclusion: Whether it needs repetition- Yes


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Title: Purification of Pdc isolates by Single spore isolation

Objective: To purify the isolates of all Pdc by single sporangial/ single tip isolation


1. Media used: V8 juice agar

2. Inoculum form: Sporangial/ hyphal form

3. Age of culture used: 7-10 days old on media

4. Environment: Laminar flow (under microscopic field)

p: 26+2Light: 12 hrs

5. Materials used: Cork borer, inoculation needle, ocular cutter, media

Time line:

Spreading of suspension of Pdc on 2% agar: 9.08.10 No. of isolates: 5

Pickup spores: 10.08.10

Spreading of suspension of Pdc on 2% agar: 12.08.10

no.of isolates: 5



no.of isolates: 5



no.of isolates: 5Pickup spores: 20.07.10


No. of isolates: 5Pickup spores: 24.07.10


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Title: Evaluation of inoculation techniques and PB development

Objective: To identify the most effective inoculation technique for PB development.

4.1: On 10 day old seedlings


1. Inoculation technique1.1. Scattered inoculum on soil

1.2. Soil mixing

1.3. Soil drenching

1.4. Stem bit staging/ attaching

1.5. Stem bit insertion into the soil near root zone

1.6. Stem scratching/scrapping and applying inoculum

1.6.1. Stem bit attachment

1.6.2. Cotton swabbing

1.6.3. Inoculum brushing on scrapped portion

1.7. Leaf scar

1.8. Toothpick

2. Inoculum form: Mass multiplied on grains

Stem bits (Plant debris)3.Host- Growth stage: 10 days old plants grown on sterile red soil

Varity- HY3C

4.Environment: Controlled environment Growth chamber- Temp- 25Greenhouse- temp-28

5. Design of experiment

Treatments: 10+1

Replication: 3No. of plants/ pot:5

6. Observation

6.1. Temp: Max & minRH

Light

6.1.2. Progress of disease development0 day- inoculation


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1 day- incubation

Typical symptoms development

6.1.3. Termination of the experiment 80% plant showing the typical PB symptoms

6.1.4. Precautions: Flooding with sterilized water

Plating of water contaminantsExposing plats in experiment environments to catch contaminants.

7. Data analysis: Collection compilation and statistical analysis with graphs,dendograms ete.

8. Conclusion: Whether it needs repetition- Yes with Pdc14 single sporangial culture

Repeated twice

Once with six methods: (Following methods)

2

nd

time with 6 methodsTime line:

6 methods repetition

Date of sowing and inoculum multiplication on grains: 5th july

Date of inoculation: 19.07.10

Scattered inoculum on soil

Soil mixing

Soil drenching

Stem bit insertion into the soil near root zone

Stem bit staging/ attaching without injury

Stem bit staging/ attaching with injury

Date of observations: 20.07.10 onwards


4.2: On one month old seedlings

Only one method used: soil mixing with inoculum on grain

Repeated with 4 techniques


1. Inoculation technique1.1. Scattered inoculum on soil


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1.2. Soil mixing

1.3. Soil drenching

1.4. Inoculum on grain insertion near the root zone


3.Host- Growth stage: 30 days old plants grown on sterile red soilVarity- HY3C

4.Environment: Controlled environment Growth chamber- Temp- 25

Greenhouse- temp-28


Treatments: 4+1

Replication: 3

No. of plants/ pot:5

6. Observation6.1. Temp: Max & min

RHLight

6.1.2. Progress of disease development

0 day- inoculation


6.1.3. Termination of the experiment

80% plant showing the typical PB symptoms6.1.4. Precautions: Flooding with sterilized water



7. Data analysis: Collection compilation and statistical analysis with graphs,

dendograms ete.

8. Conclusion: Whether it needs repetition- Yes with Pdc14 single sporangial culture

the most effective inoculation technique for PB development.

Time line:

Date of sowing: 11.06.10

Date of inoculum multiplication: 9.7.10


Date of observations started: 14.7.10


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Title: Effect of growth stage on PB development

Objective:

1. To identifies the most vulnerable stage of growth of pigeonpea against PBdevelopment.

2. To identify the resistance stage of plant growth against PB

3. To identify the correlation between growth stage and PB symptom development.


1.Inoculation technique: Best method form inoculation techniques (Experiment: 003-

10)

2.Inoculum form: Mass multiplied grains or plant derby

3.Host- Growth stage: 7, 15, 30, 45, 60, 75, 90Varity- HY3C

4. Environment: Controlled environment Growth chamber- Temp- 25

Greenhouse- temp-28

5. Design of experimentTreatments: 7+1

Replication: 3

No. of plants/ pot:56. Observation

6.1.1. Temp: 24-28oC

RH: No needLight: 12 hrs


0 day- inoculation

1 day- incubation period (IP)Latent period (LP)

Typical symptoms on different plant parts

Type of symptoms on foliar and root6.1.3. Termination of the experiment




7.Data analysis: Collection compilation and statistical analysis with graphs etc.

8. Conclusion: Whether it needs repetition- Yes/ no: Yes


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Time line:

Host- Growth stages:7, 15, 30, 45, 60, 75, 90 (5th july- 28 sept.)Growth stages: 15, 30, 45 days

Environment: GH, GC

Date of sowing: 5th july

No of pots: 9 big pots 8 1/2 inch

Age of plants to 28th sept: 90 days

Date of sowing: 19 july


No of pots: 9 big pots 8 1/2 inch

Date of sowing: 2nd Aug

Age of plants to 28

th

sept: 60 days No of pots: 9 big pots 8 1/2 inch

Growth stages: 15, 30, 45 days

Date of sowing: 16th Aug

Age of plants to 3th October: 45 days

No of pots: 6+6 (12) big pots 6

Date of sowing: 30th Aug



Date of sowing: 14th sept



Date of inoculum multiplication: 14th sept




No. of plants: 5



Date of inoculation on to pots: 28th sept


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Title: Standardization of the inoculum conc./quantity, method and form

of inoculum during inoculation methods

Objective:

1. To determine the quantity of inoculum needed for maximum infection

2. To determine the suitable form of inoculum for disease development.


1.Inoculation technique: Best method form inoculation techniques (Experiment: 003-10)

2.Inoculum form: Mass multiplied grains & plant derby (5, 10, 20, 30, 40 g/ 1 kg soil)

3.Host- Growth stage: 10 days

Variety- HY3C

4.Environment: Controlled environment- Growth chamber- Temp- 25

Greenhouse- temp-285.Design of experiment:

Treatments:

Replication: 3

No. of plants/ pot: 5

6. Observation

6.1. Temp: Max & min

RH

Light6.2. Progress of disease development

0 day- inoculation

1 day- incubation period (IP)Latent period (LP)

Typical symptoms on different plant parts

Type of symptoms on foliar and root

6.3. Termination of the experiment 80% plant showing the typical PB symptoms

6.4. Precautions: Flooding with sterilized water




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8. Conclusion: Whether it needs repetition- Yes

Time line:

Gains sterilization:

6.07.10

Debris (stem) sterilization: 6.07.10Quantity/conc.: 5, 10, 20, 30, 40g/ half kg soil

Each conc. replications: 3

Each flask 100ml: 10g/half kg soil (5 pot)Total flasks: 15 flasks

Environment: 2

Total flasks: 15+15=30

Forms: pp grain, stem bitsDate of inoculation on to media: 8.7.10

Pots sowing: 15+15+6=36 for grains

Total pots: 36+36= 72 pots

Seeds: 6-7seeds/potDate of sowing: 06.7.10

Date of inoculation: 19.7.10Date of observations started: 20.7.10


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Title: Variability studies of Pdc isolates collected from different geographicallocations

Objective:

1. To study the diversity of Pdc isolates at morphological, pathological and

molecular characterization.

2. To established the taxonomic group of the Pdc in India

7.1. Morphological variability

7.1.1. Cultural and morphological character of the different isolate of Pdc

Objective:

To determine the cultural and morphological characters and variation in Pdc isolates


1. Media used: V8 juice agar

2. Inoculum form: 8 mm disc of 7-10 days old culture

3. Age of culture used: 7-10 days old of each isolate on media

4. Environment: Laminar flow (under microscopic field)

4.1. Temp: 26+2

Light: 12 hrs

5. Materials used: Cork borer, inoculation needle, ocular cutter, media

6. Design of experiment: CRD

Replication: 3


7. Observation


Light

7.2. The radial growth rate of the isolate at every 24hrs.Observations on color, size and shape of the fungal colony as appeared

Mycelial color and type (septate/ coenocytic).

Hyphal width, structure and texture.


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7.3. Termination of the experiment

7.4. Precautions: prevention from contamination



7.1.2. Study the different methods for rapid sporangial production and zoospore

release

Objective:

To found the rapid sporangial production methods


1. Media used: V8 juice agar and broth, pigeonpea grains


3. Methods: 3-4 methods

3.1 Culture bits in sterile distilled water

3.2 Inoculum suspension in sterile distilled water

3.3 Inoculum on grains in sterile distilled water

3.4

4.Environment: Incubators

4.1 Temp: 26+2

4.2 Light: 12 hrs

5. Materials used: Cork borer, inoculation needle Petriplates


Replications: 3

7. Observation


Light

7.2. Sporangial formation time, color, size, shape and structure.Zoospore size, color, releasing time and conditions.




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7.1.3. Study the conditions favorable for oospores formation by different methods

Objective:

To found the favorable environment for oospore production


1. Media used: V8 juice agar and broth, pigeonpea grains2. Age of culture used: 7-10 days old of each isolate on media

3. Methods: 3-4 methods


4.1 Temp: 26+2

4.2 Light: 12 hrs



Replications: 3

7. Observation


Light

7.2. Oospore formation time, color, size, shape and structure.






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7.1.4. Study the effect of different media and temperature on Pdc isolate based on

cultural and morphological characters.

Objective:

To found the suitable media and temperature for Pdc growth


1. Media used: V8 juice agar, oat meal agar, PDA, pigeonpea ground meal agar


3. Method: 5-8mm disc of 7 day old culture on fresh media


4.1 Temp: 15, 20, 25, 30 & 35oC

4.2 Light: 12 hrs


5.1 Isolates: Representative locations: 4


Treatments: 7 media+ 5 temp= 12 + 4 isolates

Replications: 3

7. Observation


Light

7.2. The radial growth rate of the fungus at every 24hrs on media and at temp.Observations on color, size and shape of the fungal colony as appeared

Mycelial color and type (septate/ coenocytic).

Hyphal width, structure and texture.





7.2. Pathogenic variability

Objective: To Study the variation in virulence of the Pdc isolates


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1. Inoculation method: Best method from previous experiments


2.1: No. of isolates: 273.Host- Growth stage: 15 days old plants grown on sterile red soil

Varity- HY3C

4.Environment: Controlled environment Growth chamber- Temp- 25Greenhouse- temp-28


Treatments: 1

Replication: 3No. of plants/ pot:5

6. Observation


RHLight


1 day- incubation

Typical symptoms development

6.1.3. Termination of the experiment 80% plant showing the typical PB symptoms




8. Conclusion: Whether it needs repetition-Yes with all Pdc single sporangial cultures

7.3. Molecular characterization

Objective: Characterization of Pdc pure culture at molecular level


Isolate: ICRISAT isolate of Pdc-14

Design of experiment: 1. Isolation of DNA from Pdc isolate by gene specific primers2. Cloning of amplified product in suitable vector system.


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3. Sequencing of cloned fragment either in house or by commercial

serviceData analysis: Sequence analysis for making dendogram and sequence identity

Conclusion: Whether it needs repetition- Yes/no


Title: Effect of inoculum depth on pigeonpea

Objective: To study the relationship between inoculum depth and PB disease


1. Inoculum form: Mass multiplied grains- Pdc 14

2.Inoculation methods:1. Infected grains on the bottom of the pot

2. Infected grains on the middle of the pot

3. Infected grains on the 2-5 cm below the surface.4. Infected grains on the top surface.

5. Infected grains along with pigeonpea seed at the time of sowing.

6. Infected grains mixed in soil and after sowing (soil mixing before sowing).

3. Host- Growth stage: Sowing on inoculated soil

Varity- HY3C4. Environment- Controlled environment Growth chamber- Temp- 25

Greenhouse- temp-28

5. Design of experiment:



6. Observation

Temp: Max & minRH

Light6.1 Progress of disease development

After germination

1 day after germination- incubation period (IP)

Latent period (LP)Typical symptoms on different plant parts


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Type of symptoms on foliar and root6.2 Termination of the experiment

80% plant showing the typical PB symptoms6.3. Precautions: Flooding with sterilized water


Exposing plats in experiment environments to catch contaminants.7. Data analysis: Collection compilation and statistical analysis with graphs, dendograms

ete.

8. Conclusion: Whether it needs repetition- Yes/no

Tima line: Date of inoculation:

Date of observation:


Title: Effect of Pdc and Fu both on pigeonpea to know the

aggressiveness of Pdc and Fu in greenhouse

Objective: To found which pathogen (Pdc and Fh) is more dominating on each other


1. Inoculum form: Pure culture of Pdc and Fu

2. Host- Growth stage: 10 days old seedlingVarity- HY3C

3. Environment- Controlled environment Growth chamber- Temp- 25

Greenhouse- temp-284. Design of experiment

Treatments: 1. Pdc alone 2. Fu alone 3. Pdc and Fu both

Replication: 3No. of plants/ pot: 5

5. Observation

Temp: Max & minRH

Light

5.1 Progress of disease development

0 day after inoculation


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1 day after inoculation- incubation period (IP)

Latent period (LP)

Typical symptoms on different plant partsType of symptoms on foliar and root

Which is more aggressive Pdc or Fu

6 Termination of the experiment 80% plant showing the typical PB symptoms

Precautions: Flooding with sterilized water


7. Data analysis: Collection compilation and statistical analysis with graphs, dendograms

ete.



Host range


19/28


Title: Effect of environment factors for pathogenicity of Pdc on development of PB

Objective: To Study the factors responsible for development of PB incidence atgreenhouse conditions


1. Inoculation method: Best method from previous experiments

2. Inoculum form: Mass multiplied on grains- Pdc 14

3.Host- Growth stage: 15 days old plants grown on sterile red soil

Varity- HY3C

4.Environment: Controlled environmentFactors: Temp- 20, 25, 30 & 35oC

RH: 45, 60, 75, 90 & 100%Light: 2000, 5000, 10000 & 15000 lux

Moisture: water logged condition, medium water & water stress


Treatments: 4+5+4

Replication: 3

No. of plants/ pot: 56. Observation


RHLight


0 day- inoculation


6.1.3. Termination of the experiment

80% plant showing the typical PB symptoms6.1.4. Precautions: Flooding with sterilized water



7. Data analysis: Collection compilation and statistical analysis with graphs,

dendograms ete.

8. Conclusion: Whether it needs repetition-Yes with all Pdc single sporangial cultures


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Title: Screening of pigeonpea for host plant resistant

Objective: To find out resistant germplasm/ variety resistant or tolerant against PB


1. Inoculation technique: Soil mixing

2. Inoculum form: Mass multiplied on grains or debris

3.Host- Growth stage: 10 days old plants grown on sterile red soilVarity/germplasm: As much as available form the previous

study as well as new


Greenhouse- temp-285. Design of experiment


No. of plants/ pot:5

6. Observation


Light


1 day- incubation

Typical symptoms development6.1.3. Termination of the experiment


6.1.4. Precautions: Flooding with sterilized waterPlating of water contaminants



8. Conclusion: Whether it needs repetition- Yes/ No


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12.2: Mini-core accessions of pigeonpea water logging resistant and water logging

sensitive lines


1. Inoculation technique: Inoculum on grain insertion near the root zone


3.Host- Growth stage: 30 days old plants grown on sterile red soil

Varity- 10




No. of plants/ pot:

6. Observation

6.1. Temp: 25+3oCRH: Nil

Light: 12 hrs


1 day- incubation

Typical symptoms development6.1.3. Termination of the experiment


6.1.4. Precautions: Flooding with sterilized waterPlating of water contaminants



8. Conclusion: Whether it needs repetition- Yes with Pdc14 single sporangial culturethe most effective inoculation technique for PB development.

Time line:

Date of sowing: 17.06.10

Date of inoculum multiplication: 9.7.10



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Date of observations started: 16.7.10


Title: Role of biochemical and histo-pathological component for HPR of

pigeonpea

Objective: To study the exact biochemical mechanism for HPR in pigeonpea

12.1. Role of biochemical components for HPR


1.Inoculation technique: Best method form inoculation techniques (Experiment:003-10)


3.Host- Growth stage: 10 days

Variety- HY3C (Control) and one resistant variety


5.Design of experiment:

Treatments:

Replication: 3

No. of plants/ pot: 52 hrs after inoculation, sample taken for biochemical assays form inoculated and

control. Then after every 2 hrs gap the assay continued.

6. Observation


RHLight

6.2. Change of biochemical components

0 day- inoculation2 hrs, 4, 6, 8..

Typical biochemical changes

6.3. Termination of the experimentWhen data not varying


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6.4. Precautions: Same environment should be maintained through out the experiment.

7.Data analysis: Collection compilation and statistical analysis with graphs ete.


12.2. Changes of biochemical components after inoculation of Pdc in pigeonpea




3.Host- Growth stage: 10 daysVariety- HY3C



Treatments:Replication: 3


2 hrs after inoculation, sample taken for biochemical assays form inoculated andcontrol. Then after every 2 hrs gap the assay continued.

6. Observation


RHLight

6.2. Change of biochemical components

0 day- inoculation

2 hrs, 4, 6, 8..Typical biochemical changes


When data not varying6.4. Precautions: Same environment should be maintained through out the experiment.

7.Data analysis: Collection compilation and statistical analysis with graphs ete.



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Title: Effect of Pdc on pigeonpea at field

Objective: 1. To implement standardized greenhouse inoculation technique (s) in

field

2. To study the incidence and severity in field level

13.1. Field inoculation: Implementation of greenhouse techniques in field




3.Host- Growth stage: 30 daysVariety- HY3C

4.Environment: Field


Treatments: Soil mixingPlot: 9x9

ObservationTemp: Max & minRH

Light

Progress of disease development

After inoculation

1 day after inoculation- incubation period (IP)

Latent period (LP)Typical symptoms on different plant parts

Type of symptoms on foliar and root


80% plant showing the typical PB symptomsPrecautions: Flooding with sterilized water


Exposing plats in experiment environments to catch contaminants.Data analysis: Collection compilation and statistical analysis with graphs, dendograms

ete.

Conclusion: Whether it needs repetition- Yes/no


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13.2. Succession of fungi (off session/ crop session)

13.2.1 From plant parts

Objective:To found the sequence of occurrence of fungi on pigeonpea

infected plants during crop season


1. Time of collection: Every 15 DAS infected plant samples

2. Media used: Selective media for Fusarium & Phytophthora

3.Host- Growth stages: 15DAS- till full maturity of the crop


Field no: RL- 17Area: 0.3 ha


Treatments:

Replication:

6. Observation

Temp: Max & min

RHLight

Identification of fungus

After inoculation2 day after inoculation- growth of the fungus

Identification based on colony color, type and spore characters

Sub culture of the fungi from infected parts on selective media


7-10 Days after inoculation

7. Data analysis: Collection compilation and statistical analysis with graphs.



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13.2.1 From soil samples

Objective:To found the occurrence of fungi on pigeonpea field before

sowing and after harvesting


1. Time of collection: Before sowing & after harvesting

2. Media used: Selective media for Fusarium & Phytophthora

3. Place of collection and depths: Four corners and one centre; 5, 10, 15, 20 cm


Field no: RL- 17

Area: 0.3 ha

5.Design of experiment:Treatments: 4

Replication: 3

6. Observation

Temp: Max & minRH

Light


After inoculation

2 day after inoculation- growth of the fungus

Identification based on colony color, type and spore charactersSub culture of the fungi from infected parts on selective media






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13.3. Survey of pigeonpea field

Objective: Survey for the incidence of disease on different pigeonpea

growing areas


1. Time of Survey: During the early crop season

2. Collection of samples: The infected plant parts at the time in those areas

Media used: Selective media for Fusarium & Phytophthora

3.Place of collection: Any place in the field



6. Observation

Latitude & Altitude of that placeWeather parameters on those areas

Type of soil

Irrigation facilitiesVariety


After inoculation2 day after inoculation- growth of the fungus

Identification based on colony color, type and spore characters

Sub culture of the fungi from infected parts on selective mediaTermination of the experiment





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Title: Mode of inheritance and allelic relationship of genes for resistance to

PB

pb experiments protocols

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