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. Isolate Location State Year of Year of Single

No name collection isolation spore Pathogenicity
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 Naganpally, Bodhan (11), Andhra pradesh
Pdc-22 Nizamabad 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
 Experiment No. PB- 002-10

Title: Pathogenicity of the Pdc isolates

Objective: To prove the pathogenicity of the Pdc isolates

Methods and materials:

1. Inoculation technique
ar spray
drench

1. Inoculum form

      3.   Host- Growth stage: 15 days old

                                              Variety- HY3C

4. Environment: Controlled environment (Growth chamber- Temp- 15 & 25)

4. Design of experiment
Treatments: 6+1
Replication: 3
No. of plants/ pot
5. Observation
6.1. Temp: 15 & 25oC
                   RH: Nil
                   Light: 12 hrs
6.2. Progress of disease development
                   0 day- inoculation
                   1 day- incubation
                   Typical symptoms development
6.3. Termination of the experiment
                   ≥ 80% plant showing the typical PB symptoms
6.4. Precautions:
       Flooding with sterilized water
                   Plating of water contaminants
                  Exposing plats in experiment environments to catch contaminants.

5. Data analysis: Collection and compilation.

6. Conclusion: Whether it needs repetition- Yes

     
 Experiment No. PB- 003-10

Title: Purification of Pdc isolates by Single spore isolation

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

Methods and materials:

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)

Temp: 26+2
              Light: 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
Pickup spores:                                                                                13.08.10

Spreading of suspension of Pdc on 2% agar:                                 16.07.10

no.of isolates:                                                                                  5
Pickup spores:                                                                                17.07.10

Spreading of suspension of Pdc on 2% agar:                                 19.07.10

no.of isolates:                                                                                  5
Pickup spores:                                                                                20.07.10

Spreading of suspension of Pdc on 2% agar:                                23.07.10

No. of isolates:                                                                               5
Pickup spores:                                                                                24.07.10
Experiment No. PB- 004-10

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

Methods and materials:

1. Inoculation technique
           1.1. 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- 25
                        Greenhouse- temp-28
      5. Design of experiment
              Treatments: 10+1
  Replication: 3
     No. of plants/ pot:5
    6.  Observation
         6.1. Temp: Max & min
                RH
                Light
         6.1.2. Progress of disease development
                   0 day- inoculation
                   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 contaminants
          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 with Pdc14 single sporangial culture
 
Repeated twice
Once with six methods: (Following methods)

2nd time with 6 methods

Time 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

Experiment No. PB- 004-10

4.2: On one month old seedlings

Only one method used: soil mixing with inoculum on grain

Repeated with 4 techniques

Methods and materials:

1. Inoculation technique
           1.1. Scattered inoculum on soil
      1.2. Soil mixing
     1.3. Soil drenching
     1.4.  Inoculum on grain insertion near the root zone 

2. Inoculum form: Mass multiplied on grains

                                   
3. Host- Growth stage: 30 days old plants grown on sterile red soil
                                            Varity- HY3C
4. Environment: Controlled environment Growth chamber- Temp- 25
                        Greenhouse- temp-28
      5. Design of experiment
              Treatments: 4+1
  Replication: 3
     No. of plants/ pot:5
    6.  Observation
         6.1. Temp: Max & min
                RH
                Light
         6.1.2. Progress of disease development
                   0 day- inoculation
                   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 contaminants
          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 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 inoculation: 13.7.10
Date of observations started: 14.7.10
 Experiment No. PB- 005-10

Title: Effect of growth stage on PB development

Objective:
1. To identifies the most vulnerable stage of growth of pigeonpea against PB
development.
2. To identify the resistance stage of plant growth against PB
3. To identify the correlation between growth stage and PB symptom development.

Methods and materials:

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, 90
                               Varity- HY3C

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

                                 Greenhouse- temp-28
5. Design of experiment
          Treatments: 7+1
          Replication: 3
         No. of plants/ pot:5
6. Observation
          6.1.1. Temp: 24-28oC
                    RH: No need
                    Light: 12 hrs
          6.1.2. 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
          6.1.3. Termination of the experiment
                    ≥ 80% plant showing the typical PB symptoms
          6.1.4. Precautions: Flooding with sterilized water
                    Plating of water contaminants
                    Exposing plats in experiment environments to catch contaminants.

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

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

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
Age of plants to 28th sept:                                                         75 days
No of pots:                                                                               9 big pots 8 1/2 inch
Date of sowing:                                                             2nd Aug
Age of plants to 28th 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
Age of plants to 28th sept:                                                         30 days
No of pots:                                                                               6+6 (12) big pots 6”
Date of sowing:                                                             14th sept
Age of plants to 28th sept:                                                         15 days
No of pots:                                                                               6+6 (12) big pots 6”
Date of inoculum multiplication:                                     14th sept
Date of sowing:                                                             21th sept
Age of plants to 28th sept:                                                         7 days
No of pots:                                                                               6+6 (12) big pots 6”
No. of plants:                                                                            5
Date of sowing:                                                             14th sept
Age of plants to 28th sept:                                                         15 days
Date of inoculation on to pots:                                              28th sept
Experiment No. PB- 006-10

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.  

Methods and materials:

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-28
5. Design of experiment:
    Treatments:
    Replication: 3
    No. of plants/ pot: 5

6. Observation

    6.1. Temp: Max & min

           RH
           Light
    6.2. 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
          Plating of water contaminants
          Exposing plats in experiment environments to catch contaminants.

7. Data analysis: Collection compilation and statistical analysis with graphs etc.
8. Conclusion: Whether it needs repetition- Yes

Time line:
Gains sterilization:                                                                    
6.07.10                                   
Debris (stem) sterilization:                                                         6.07.10
Quantity/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 bits
Date 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/pot
Date of sowing:                                                             06.7.10                       
Date of inoculation:                                                                   19.7.10
Date of observations started:                                                     20.7.10
Experiment No. PB- 007-10

Title: Variability studies of Pdc isolates collected from different geographical

locations
                                                                      
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

Methods and materials:

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
    No. of plants/ pot: 5

7. Observation

   7.1. Temp: Max & min

           RH
           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.
    7.3. Termination of the experiment
    7.4. Precautions: prevention from contamination
8. Data analysis: Collection compilation and statistical analysis with graphs etc.

9. Conclusion: Whether it needs repetition- Yes

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

Objective:

To found the rapid sporangial production methods

Methods and materials:

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

2. Age of culture used: 7-10 days old of each isolate on media
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
6. Design of experiment: CRD
Replications: 3

7. Observation

    7.1. Temp: Max & min

           RH
           Light
    7.2. Sporangial formation time, color, size, shape and structure.
Zoospore size, color, releasing time and conditions.
   7.3. Termination of the experiment
         
   7.4. Precautions: prevention from contamination
8. Data analysis: Collection compilation and statistical analysis with graphs etc.

9. Conclusion: Whether it needs repetition- Yes

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

Objective:

To found the favorable environment for oospore production

Methods and materials:

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

2. Age of culture used: 7-10 days old of each isolate on media
3. Methods: 3-4 methods
4. Environment: Incubators
4.1 Temp: 26+2
4.2 Light: 12 hrs
5. Materials used: Cork borer, inoculation needle Petriplates
6. Design of experiment: CRD
Replications: 3

7. Observation

    7.1. Temp: Max & min

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

   7.3. Termination of the experiment

         
   7.4. Precautions: prevention from contamination

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

9. Conclusion: Whether it needs repetition- Yes

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

Methods and materials:

1. Media used: V8 juice agar, oat meal agar, PDA, pigeonpea ground meal agar
2. Age of culture used: 7-10 days old of each isolate on media
3. Method: 5-8mm disc of 7 day old culture on fresh media
4. Environment: Incubators
4.1 Temp: 15, 20, 25, 30 & 35oC
4.2 Light: 12 hrs
5. Materials used: Cork borer, inoculation needle Petriplates
5.1 Isolates: Representative locations: 4
6. Design of experiment: CRD
Treatments: 7 media+ 5 temp= 12 + 4 isolates
Replications: 3

7. Observation
    7.1. Temp: Max & min
           RH
           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.
   7.3. Termination of the experiment
         
   7.4. Precautions: prevention from contamination

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

9. Conclusion: Whether it needs repetition- Yes

7.2. Pathogenic variability

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

Methods and materials:
1. Inoculation method: Best method from previous experiments
    
2. Inoculum form: Mass multiplied on grains
2.1: No. of isolates: 27                                   
3. Host- Growth stage: 15 days old plants grown on sterile red soil
                                            Varity- HY3C
4. Environment: Controlled environment Growth chamber- Temp- 25
                        Greenhouse- temp-28
      5. Design of experiment
              Treatments: 1
  Replication: 3
     No. of plants/ pot:5
    6.  Observation
         6.1. Temp: Max & min
                RH
                Light
         6.1.2. Progress of disease development
                   0 day- inoculation
                   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 contaminants
          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 with all Pdc single sporangial cultures

7.3. Molecular characterization

Objective: Characterization of Pdc pure culture at molecular level

Methods and materials:

Isolate: ICRISAT isolate of Pdc-14

Design of experiment: 1. Isolation of DNA from Pdc isolate by gene specific primers
                        2. Cloning of amplified product in suitable vector system.
 3. Sequencing of cloned fragment either in house or by commercial
service
Data analysis: Sequence analysis for making dendogram and sequence identity

Conclusion: Whether it needs repetition- Yes/no

Experiment No. PB- 008-10

Title:  Effect of inoculum depth on pigeonpea

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

Methods and materials:

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- HY3C
4. Environment- Controlled environment Growth chamber- Temp- 25
                        Greenhouse- temp-28
5. Design of experiment:
Treatments: 6+1
Replication: 3
No. of plants/ pot: 5
6. Observation
Temp: Max & min
RH
Light
6.1 Progress of disease development
After germination
1 day after germination- incubation period (IP)
Latent period (LP)
Typical symptoms on different plant parts
Type of symptoms on foliar and root
6.2 Termination of the experiment
≥ 80% plant showing the typical PB symptoms
6.3. Precautions: Flooding with sterilized water
Plating of water contaminants
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:    

      
Experiment No. PB- 009-10

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

Methods and materials:

1. Inoculum form: Pure culture of Pdc and Fu

                         
2. Host- Growth stage: 10 days old seedling
  Varity- HY3C

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

                        Greenhouse- temp-28
4. Design of experiment
Treatments: 1. Pdc alone 2. Fu alone 3. Pdc and Fu both
Replication: 3
No. of plants/ pot: 5
5. Observation
Temp: Max & min
RH
Light
5.1 Progress of disease development
0 day 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
Which is more aggressive Pdc or Fu
6 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.
7. Data analysis: Collection compilation and statistical analysis with graphs, dendograms
ete.
8. Conclusion: Whether it needs repetition- Yes/no

Experiment No. PB- 010-10

Host range
Experiment No. PB- 011-10

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

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

greenhouse conditions

Methods and materials:

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 environment
Factors: 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
      5. Design of experiment
              Treatments: 4+5+4
  Replication: 3
     No. of plants/ pot: 5
    6.  Observation
         6.1. Temp: Max & min
                RH
                Light
         6.1.2. Progress of disease development
                   0 day- inoculation
                   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 contaminants
          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 with all Pdc single sporangial cultures
Experiment No. PB- 012-10

Title: Screening of pigeonpea for host plant resistant

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

Methods and materials:

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 soil
                                            Varity/germplasm: As much as available form the previous
study as well as new
4. Environment: Controlled environment Growth chamber- Temp- 25
                                                                        Greenhouse- temp-28
      5. Design of experiment
              Treatments: 1+1
  Replication: 3
     No. of plants/ pot:5
    6.  Observation
         6.1. Temp: Max & min
                RH
                Light
         6.1.2. Progress of disease development
                   0 day- inoculation
                   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 contaminants
          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

Experiment No. PB- 012-10

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

Methods and materials:

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

2. Inoculum form: Mass multiplied on grains

                                   
3. Host- Growth stage: 30 days old plants grown on sterile red soil
                                            Varity- 10
4. Environment: Controlled environment Growth chamber- Temp- 25
      
5. Design of experiment
              Treatments: 10+1
  Replication: 2
     No. of plants/ pot:
    6.  Observation
         6.1. Temp: 25+3oC
                RH: Nil
                Light: 12 hrs
         6.1.2. Progress of disease development
                   0 day- inoculation
                   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 contaminants
          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 with Pdc14 single sporangial culture
the most effective inoculation technique for PB development.

Time line:
Date of sowing: 17.06.10
Date of inoculum multiplication: 9.7.10
Date of inoculation: 15.7.10
Date of observations started: 16.7.10

Experiment No. PB- 013-10

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 

Methods and materials:

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

2. Inoculum form: Mass multiplied grains or plant derby

                         
3. Host- Growth stage: 10 days
                                   Variety- HY3C (Control) and one resistant variety

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

                                                                   
5. Design of experiment:
    Treatments:
    Replication: 3
    No. of plants/ pot: 5
    2 hrs after inoculation, sample taken for biochemical assays form inoculated and
control. Then after every 2 hrs gap the assay continued.
  
6. Observation

    6.1. Temp: Max & min

           RH
           Light
    6.2. Change of biochemical components
           0 day- inoculation
           2 hrs, 4, 6, 8…..
          Typical biochemical changes
   6.3. Termination of the experiment
          When data not varying
   6.4. Precautions: Same environment should be maintained through out the experiment.
         
7. Data analysis: Collection compilation and statistical analysis with graphs ete.

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

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

Methods and materials:

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

2. Inoculum form: Mass multiplied grains or plant derby

                         
3. Host- Growth stage: 10 days
                                   Variety- HY3C

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

                                                                   
5. Design of experiment:
    Treatments:
    Replication: 3
    No. of plants/ pot: 5
    2 hrs after inoculation, sample taken for biochemical assays form inoculated and
control. Then after every 2 hrs gap the assay continued.
  
6. Observation

    6.1. Temp: Max & min

           RH
           Light
    6.2. Change of biochemical components
           0 day- inoculation
           2 hrs, 4, 6, 8…..
          Typical biochemical changes
   6.3. Termination of the experiment
          When data not varying
   6.4. Precautions: Same environment should be maintained through out the experiment.
         
7. Data analysis: Collection compilation and statistical analysis with graphs ete.

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

Experiment No. PB- 014-10

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

Methods and materials:

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

2. Inoculum form: Mass multiplied grains or plant derby

                         
3. Host- Growth stage: 30 days
                                   Variety- HY3C

4. Environment: Field
                                                                   
5. Design of experiment:
    Treatments: Soil mixing
    Plot: 9x9
  
Observation
Temp: Max & min
RH
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
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.
Data analysis: Collection compilation and statistical analysis with graphs, dendograms
ete.
Conclusion: Whether it needs repetition- Yes/no

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

Methods and materials:

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                           
4. Environment: Field  
Field no: RL- 17
Area: 0.3 ha                                                             
5. Design of experiment:
    Treatments:
    Replication:
 6. Observation
Temp: Max & min
RH
Light
Identification of fungus
After inoculation
2 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
Termination of the experiment
7-10 Days after inoculation

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

8. Conclusion: Whether it needs repetition- Yes/no
13.2.1 From soil samples

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

sowing and after harvesting

Methods and materials:

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
4. Environment: Field  
Field no: RL- 17
Area: 0.3 ha                                                             
5. Design of experiment:
    Treatments: 4
    Replication: 3
 6. Observation
Temp: Max & min
RH
Light
Identification of fungus
After inoculation
2 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
Termination of the experiment
7-10 Days after inoculation

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

8. Conclusion: Whether it needs repetition- Yes/no
13.3. Survey of pigeonpea field
Objective: Survey for the incidence of disease on different pigeonpea
growing areas

Methods and materials:

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
4. Environment: Field  
5. Design of experiment:
6. Observation
Latitude & Altitude of that place
Weather parameters on those areas
Type of soil
Irrigation facilities
Variety

Identification of fungus
After inoculation
2 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
Termination of the experiment
7-10 Days after inoculation

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

8. Conclusion: Whether it needs repetition- Yes/no
Experiment No. PB- 015-10

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

PB