Practicall

I~entification of Disease Symptoms.

Objectives: 1. To familiarize with different types of disease symptoms 2. To distinguish diseased plants from healJhy plants 3. To detect disease symptoms early as possible

Specimens: Different types of disease Symptoms 1. Blast disease in rice 2. Anthracnose in chilli'
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3. Powdery mildew in grapes 4. Downy mildew in grapes 5. Canker in citrus 6. Rust in groundnut 7. Leaf spot in cowpea 8. Club root in cabbage 9. Damping off in chilli 10. Vascular wilt in brinjal 11. Die back in citrus 12. Leaf curl in chilli 13. Mossaic disease in cucumber.

Procedure: Observe the different types of diseased symptoms and draw, the figures.

Observation: Identify of the characteristic features of the different disease symptoms.

Practical 2 Preparation 'of Nutrient Media

Materials: Potato, Dextrose, Agar, Distilled Water, Buchner funnel, Autoclave, Conical flask, Cotton wool and Aluminium foil

Composition for 250ml of media Potato Sucrose Agar Distilled water 50g 5g 5g 250ml

Procedure: Peel potato, cut into small pieces and wash in water. Add potato in 125 ml of water and boil until the potato become very soft. Strain the potato using. Mean while place the weighed agar in a 500mlflask Boil this over a'water bath until agar dissolves. To this add the required amount of sucrose and dissolve. Now add the potato extract and stir well. Plug the conical flask containing the media with a cotton wool plug wrapped in aluminium foil. Autoclave the media at 121C, 15 psi for 20 minutes. with 125 ml of water.

Practical 3
Pouring of nutrient media in to petri dishes Objectives: 1. To familiarize with nutrient media pouring techniques 2. To know about precautions during pouring of nutrient media

Procedure: Start lamina flow 30 minutes before'the operation. Take sterilized PDA media away from autoclave or refergerator.

If it is in the solid stage, boil it in the water bath until reach 70C temperature.
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Stop lamina flow and spray 90% alcohol inside the lamina flow. Wash the hands with 70% alcohol. Keep the conical flask containing media inside the lamina flow. Add antibiotic tablet (Penicillin or Streptomycin) to media at 50C. Add 500mg tablet for 1 liter of media. Light the sprit lamp inside the lamina flow. Heat the neck of the conical flask before pouring. Slightly open the petri dish and pour the media at 50 to 40C. After pouring keep the petri dishes lipside down to eliminate moisture condensation. Keep the petri dishes inside the refrigerator until inoc~lation.

Practical 4 Artificial inoculation of Pathogens from diseased plant parts to nutrient media
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Materials: Inoculation needle, forceps, Petri dishes, alcohol (90% & 70%), beakers and
lamina flow

Procedure:
Start the lamina flow 30 minutes before the operation. Red heat the scissors, forceps and inoculation needles
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Cut the affected portion in to small pieces. Transfer it into the 70% alcohol for 30 seconds (surface sterilization). Then transfer it into distilled water. Mob the specimen with filter paper. Stop lamina flow and spray 90% alcohol inside the lamina flow. Wash the hands with 70% alcohol. Transfer the specimen into the petri dish which' containing nutrient media. Seal the petri dish with celotape. Label the petri dish and keep inside the incubator.

Obs.ervation:
1. Observe the mycelial gr~wth 2 to 3 days after inoculation. 2. Stain the !Uycelia and observe the structure of spores and fruiting bodies under !T1icroscope

Practical 5: Isolation and identification of pathogen from disease affected plant parts using

Celotape Impression Method

Objectives: I. To familiarize with isolation and identification of pathogens associated with disease

Materials: Diseased specimens, Celotape, Lacto phenol cotton blue (stain), Scissors, Slides and Microscope .

Impress piece of celotape in diseased symptom and remove from it. Add one drop of stain into that celotape piece. Then re-impressin slide.

Observe under the microscope.

Observation: I. Shapes of the fungal spores 2. Structure of fruiting bodies and mycelia'

Practical 6
Gram staining (Double or Differential Staining)

Equipments: Sprit lamp, alcohol-cleaned Reagents: 1.. Crystal Violet (the Primary Stain)

microscope slide,

2. Iodine Solution (the Mordant) (bIKI Solution) 3. Decolorizer (Ethanol I Ethyl alcohol) 4. Safran in (the Counter Stain) 5. Water (preferably'in a squirt bottle) Specimens: Canker disease in citrus'(Xantho~onas citri) Procedure: Prepare a slide smear Sp\ead the culture with an inoculation loop to an even thin film on a slide. Air-dry the culture (1 to 2 minutes) and fixit or over a gentle flame, while moving the slide in'a circular fashion to avoid localized overheating. The applied heat helps the ceil adhesion on the glass slide to make possible the subsequent rinsing of the smear with water without a significant loss of the culture. Heat can also be applied to facilitate drying the smear. Gram staining I. Place slide on a slide holder or a rack. Flood (cover completely) the entire slide with crystal violet. Let the crystal violet stand for about 60 seconds. When the time has elapsed, gently rinse the excess stain with a stream of water from a tap or a plastic ,water bottle for 5 seconds. Note that the objective of this step is to wash off the stain, not the fixed culture. The specimen should appear blue-violet when observed with the naked eye. 2. Now, flood slide with the iodine solution. Let it stand about 60 seconds as well. When time has expired, rinse the slide with water for 5 seconds and immediately proceed to next step. At this point, the specimen should still be blue-violet. 3. Next step involves addition of the decolorizer, ethanol or ethyl alcohol (95%). This step is somewhat important because using too much decolorizer could result in a false Gram (-) result. Likewise, not using enough decolorizer may yield a false Gram (+) results. To be safe, add the ethanol or ethyl alcohol drop wise until the blue-violet color is no longer emitted from your specimen. As iT!the previous steps, rinse with the water for 5 seconds. 4. The final step involves applying the counter stain, safran in. Flood the slide with the dye. Let this stahd for about 60 seconds to allow the bacteria to 'incorporate the saffranin. Gram positive cells will incorporate little or no counter stain and will remain blue-violet in appearance. Gram negative bacteria, however, take on a pink color and are easily distinguishable from the Gram positives. Again, rinse with wat~r for 5 seconds to remove any excess of dye. ' 5. After blot the slide gently with bibulous paper or allow it to air dry before viewing it under the microscope.

Practical 7 The Rust Disease on Groundnut Objectives: 1. To identify symptoms produced by rust fungi 2. To identify different stages of rust fungi 3. To familiarize with controlJTIeasures

Introduction Leaf rust of ground nut is world wide in distribution and affects groundnut where it is grown. The leaf rust fungus attacks all the above ground parts and causes losses by reducing foliage, root development, yield and quality of groundnut. Symptom The symptoms on ground appear first as orange~yellow powdery spots on the lower side of the leaves. The spots are circular and small. The center of the spots eventually become dry, turn brownish and leaf falls prematurely. Control Crop rotation (cereal-cereal-groundnut). .Application of wettable sulpher (lkg per acre). Application ofchlorothalanil (400g per acre mix with 200 litre of water) Use resistant varieties The Pathogen Class: Basidiomycetes Order: Uredinales Genus: Puccinia Species: Puccinia arachidis The Disease Cycle Because of their reddish brown colour of their spores these fungi are called rusts. The rust form all five stages of spores are called macro cyclic rusts. In rust life cycle is short and completed by only two types of spores (Teliospore ;ind Basidiospore) is called micro cyclic rust. ' . . Different stages of macro cyclic rusts Spermagonia with spermatia . Aecia with aeciospores Uredia with urediospores Telia with teliospores Basidia with basidiospores Observation: 1. . Identification of different stages of rust fungi 2. Identification o~ diseased symptoms

Practical 7 The Rust Disease on Groundnut Objectives: 1. To identify symptoms produced by rust fungi 2. To identify different stages of rust fungi 3. To familiarize with control;neasures

Introduction Leaf rust of ground nut is world wide in distribution and affects groundnut where it is grown. The leaf rust fungus attacks all the above ground parts and causes losses by reducing foliage, root development, yield and quality of groundnut. Symptom The symptoms on ground appear first as orange-:-yellow powdery spots on the lower side of the leaves. The spots are circular and small. The center of the spots eventually become dry, turn brownish and leaf falls prematurely. Control Crop rotation (cereal-cereal-groundnut). .Application of wettable sulpher (1 kg per acre). Application ofchlorothalanil (400g per acre mix with 200 litre of water) Use resistant varieties The Pathogen 'Class: Basidiomycetes Order: Uredinales .Genus: Puccinia Species: Puccinia arachidis The Disease Cycle Because of their reddish brown colour of their spores these fungi are called rusts. The rust form all five stages of spores are called macro cyclic rusts. In rust life cycle is short and completed by only two types of spores (Teliospore ;lnd Basidiospore) is called micro cyclic rust. ' . Different stages of macro cyclic rusts Spermagonia with spermatia Aecia with aeciospores Uredia with urediospores Telia with teliospores Basidia with basidiospores Observation: 1. . Identification of different stages of rust fungi 2. Identification o~ diseased symptoms

Practical 8 Club Root Disease in Crucifers Objectives: 1. To identify symptoms produced by club root fungi
2. To familiarize with control measures -

Specimens: Club root diseased cabbage plants, Permant siide of diseased specimen

Club root fungi affect cruciferous plants.

Symptoms:
Infected plants ~ave pale green to yellowish leaves . . Show wilting in the middle of hot, sunny days but may recover during night. Young plants may be killed by the disease with in a short time after infection, w~ile older. plants remain alive. The characteristic symptom appear on roots and sometime on the under ground part of stem. Symptom consists of small, spindle like, spherical, knobby or clubbed shaped swelling on the roots and rootlets.

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Control:
1. Growing cruciferous crops in fields known to be inf~sted should be avoided; 2. Cabbage should be planted in well-drained soil with the PH 7.2 by adding hydrated lime. 3. Soilfumigatiol1. Chloropicrin, Methyl bromide

Pathogen
Plasmodiophora brassicae

.Observation:
Identification of disease symptoms in Jnfected 'plants both externally,and section (permanent slide) through cross.

Practical 9
Isolation of nematodes from infested soil and plants

Materials: Nematodes infested soil, Sieves 300/l m and 38 /l m mesh, Petri dishes, distilled water, glass.-rod and squash bottle Procedure: Three methods used for isolation of nematodes. 1. Seiving method 2. Baerman funnel method 3. Centrifuge flotation method

Ihe mesh, filter paper,

Take known amount (l00 g, of infested soil in to the beaker. Add water and crush it well "with glass rod. Filter soil suspension through 300/l I? mesh seive. Discard debris. _ Filter the filtrate from 300/l m mesh seive through 38 /l m mesh seive. Nematodes retain in the filter. Wash the nematodes from the seive to beaker. .pla~e filter paper above the fine mesh. Keep the setup in the petri dish. Add nematodes containing solution into fine mesh. Keep for 24 hours.

Diagrams: Annexed Observation: After 24 hours observe the filtrate solution under microscope.

Practical 9
Isolation of nematodes from infested soil and plants

Materials: Nematodes infested soil, Sieves 300ll m and 38 Il m mesh, fihe mesh, filter paper, Petri dishes, distilled water, glass rod and squash bottle Procedure: Three methods used for isolation of nematodes. I. Seiving method 2. Baerman funnel method 3. Centrifuge flotation method

Take known amount (100 g, of infested soil in to the beaker. Add water and crush it well "with glass rod. . Filter soil suspension through 300ll I? mesh seive. Discard debris. Filter the filtrate from 300ll m mesh seive through 38 Ilm mesh seive. Nematodes retain in the filter: Wash the nematodes from the seive to beaker. Plaqe filter paper above the fine mesh. Keep the setup in the petri dish. Add nematodes containing solution into fine mesh .. Keep for 24 hours.

Diagrams: Annexed Observation: After 24 hours observe the filtrate solution under mic.roscope.

Objectives: To familiarize with post harvest diseases To identify control measures. Specimens: 1. Carrot rot - Erwinia carotovora 2. Anthracnose in chill.i - Colletotrichum sp 3. Anthracnose in banana - Colletotrichum sp 4. Bacterial brown spot in beans - Pseudomonas syringae

Observation: Identification of the characteristic features of the different disease symptoms