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Isolation of pathogens fungi from plant diseases leaves 3.. Isolation of pathogens fungi from plant diseases leaves 3.. Isolation of pathogens fungi from plant diseases leaves 2.1.. Incu

1 Fungi 1 Preparation of media for fungi (WA, PDA)

2 Isolation of pathogens fungi from plant diseases (leaves)

3 Observation of some plant fungal diseases

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2 Bacteria 1 Preparation of medium for bacterium culture King’B and SPA

2 Observation of some bacteria diseases.

3 Oozing test – Identify bacterial and fungus diseases.

4 KOH Test – Identify positive gram and negative gram bacteria.

5 Isolation of bacteria from diseases.

6 HR test – Hypersensitive reaction.

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3 Viruses 1 Observation of some plant viral diseases.

2 PTA- ELISA methods to detect PRSV on Cucubit plant.

3 Mechanical transmission test (inoculation) of PRSV on healthy plant

4 Check results of isolation both fungi and bacteria culture.

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4 Nematodes - Part 1:

1 Preparation of 10 plates of PDA (for sub culture of fungi).

2 Preparation of KOH 3%.

3 Observation the isolation of fungi and bacteria.

4 HR test from cultured bacteria.

5 Check ELISA results.

- Part 2:

1 Observation of Root knot nematode (cabbage, carrot, tomato).

2 Staining of roots for observation of nematodes inside the roots.

3 Extraction of nematodes from soil and plant samples.

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PRACTICAL LAB LESSON 1

I Date and place

- Date: 14/11/2018

- Place: Laboratory of Plant Pathology Department

II Main activities

1 Preparation of media for fungi (WA, PDA)

2 Isolation of pathogens fungi from plant diseases (leaves)

3 Observation of some plant fungal diseases

III Methodology and results

1 Preparation of media for fungi ( WA, PDA)

a WA( Water Agar) media:

- WA (2%) consists of 20 g agar in 1L of water and is recommended as the substrate forthe germination of conidia used to initiate single spore cultures

- For this experiment, we need 200 ml water and 5 g of agar to create the media for fugalculture

- Hyphal growth is sparse on this medium so it is suitable for cultures from which singlehyphal tips are to be taken for the initiation of new colonies

b PDA (Potato Dextrose Agar) media:

- PDA is a carbohydrate rich medium which contains 20 g dextrose, 20 g agar and thebroth from 250 g white potatoes made up to 1 L with tap water

- For this experiment, we use 200ml water, 4 g dextrose (glucose), 4 g agar, 50 g potato

- Procedure for making PDA media culture:

+ Step 1- Preparation of PDA media in bottle: weigh the component of PDA media followthe recipe, boil the potato and filter through cheesecloth, leaving some sediment in thebroth Mix the component with each other in a big bottle

+ Step 2- Sterilization of media in autoclave (121oC, 15-30 minutes)

+ Step 3- Pouring the melting media in laminar: before pouring, we add some drop ofantibiotics solution in media bottle to inhibit the non-target bacteria will grow in medialaminar

2 Isolation of pathogens fungi from plant diseases (leaves)

2.1 Isolation procedures:

Step 1: Wipe the work area with 70% ethyl alcohol

Step 2: Dip equipment (forceps and knife or scalpel) in 70% ethyl alcohol and bunsenburner to sterilization

Step 3: Surface sterilise leaf tissue by dipped in 70% ethyl alcohol for 5 seconds, rinsing

in sterile water and damp-drying on sterile paper tissue

Step 4: Aseptically cut small pieces from the margin of the healthy and diseased tissue,and transfer them to a WA media follow the lab’s experiment

Step 5: Incubate the plates at room condition

Step 6: Check plates each day for fungal colonies development

Step 7: Make a final identification using pure cultures grown from a single germinatedspore or a hyphal tip

2.2 Incubation of plant fungal diseases in PDA media

- Diseases for incubation:

+ Southern blight in corn- Bipolaris maydis- WA media

+ Corynespora spot in tomato leaves- Corynespora cassiicola- WA media

Southern blight in corn after 2 days Corynespora spot in tomato after 2 days

Southern blight in corn after 6 days Corynespora spot in tomato leaves

after 6 days

2.2 Daily check for incubation:

Table 1: Result of observing fungal disease development in 6 days

Diseases Fragments After 1 day After 2 days After 6 days

Southern blight

in corn

1 Full sides Full sides Full sides

3 3 branches Full sides Full sides

 Conclusion: From the data of table, it can be seen that with the same environment

(WA), the fungi causing southern blight in corn grows and develops well and faster thanthat causing corynespora spot in tomato After 6 days observing, all fragments of corn leafhave fungi develop full sides surrounding the fragments and nearly full, whereas only 1st

fragment extracted from tomato has fungi grow full sides and the other fragments existnothing

3 Observation of some plant fungal diseases

3.1 Observation symptoms and signs of some plant fungal disease

a Anthracnose in chilli - Colletotrichum capsici.

c Corynespora spot in tomato fruit - Corynespora cassiicola

Symptom:

Big, dark, sunken spots occur occasionally

on the surface of fruit The myceliumappears surrounding the spot of fruit

d Rust in maize – Puccinia maydis Ber.

The disease mostly occurs in leaf blade Early stage, disease spot has yellow color and irregularly

distribution in leaf Later on, the spot

is bigger and containing the brown powder The spot floats on the leaf surface

red-e Rust in ground nut - Puccinia arachidis Speg

Symptom:

The disease spots have round shape andsmall size The lower epidermis of leaf iscracked with the appearance of sporangium

in orange, red color Upper epidermis, thedisease stains with yellow color andburning the leaf

f Anthracnose in dragon fruit - Colletotrichum gloeosporioides

Symptom:

Anthracnose disease on dragon fruit wascharacterized with reddish brown lesionsand chlorotic haloes symptoms on stem aswell as fruit These lesions had browncenters and then coalesced to rot

g Canker in ornamental peach - Cytospora leucostoma

The bark is killed, and when removed, theunderlying tissue is orange to brown incolour, and often has a strong, sour smell

h Corynespora spot in tomato leaves - Corynespora cassiicola

Symptoms:

Small, light brown, circular spots develop

on the leaves The spots have brown centres,surrounded by a prominent yellow halo.That causes extensive areas of leaf death

i Freckle in banana - Phyllosticta cavendishii.

Symptoms:

Dark brown to black spots develop onleaves Spots are usually on the uppersurface of the leaf and occasionally formdense aggregations The spots are rough tothe touch That causes leaf yellowing Theblack speckled strips extend from the midribtowards the leaf margin

3.2 Observation the isolation of plant fungal diseases on microscope

Due to lack of time and low technique, we observed only 3 samples of plant fungaldisease on microscope.

a Anthracnose in chilli - Colletotrichum capsici.

I Date and place

- Date: 15/11/2018

- Place: Laboratory of Plant Pathology

II Main activities

1 Preparation of medium for bacterium culture ( King’B, SPA)- Most of bacteria live in SPA media

2 Observation of some bacteria diseases

3 Oozing test – Identify bacterial and fungus diseases

4 KOH Test – Identify positive gram and negative gram bacteria

5 Isolation of bacteria from diseases

6 HR test – Hypersensitive reaction

III Methodology and results

1 Preparation of medium for bacterium culture (King’B, SPA)

1.1 Sucrose peptone agar (SPA) media preparation:

- The composition of SPA media:

Chemical agents Theoretically recipe Experimental recipe

1.2 King’ B medium preparation:

- The compositions of King’B media:

Chemical agents Theoretically recipe

2 Observation of some bacteria diseases

2.1 Huanglongbing/citrus greening - Candidatus Liberibacter asiaticus

* Symptoms: The common symptoms of

yellowing of the veins and adjacent tissues;

followed by splotchy mottling of the entire leaf,

premature defoliation, dieback of twigs, decay of

feeder rootlets and lateral roots, and decline in

vigor, ultimately followed by the death of the

entire plant

2.2 Citrus canker - Xanthomonas citri

* Symptoms: The lesions appear on the lower

surface of leaves as small, pinpoint, water-soakedand slightly raised spots first The lesions expandand thicken over time and protrude from both leafsurfaces As the lesion develops, the tissuebecomes spongy or corky, and the color changesfrom tan or brown, to grey or white, usuallysurrounded by a greasy, water-soaked margin and

a yellow halo

2.3 Gall of Rose and Tea stem - Argobacterium tumefaciens

* Symptoms: Rough galls are different size on

twigs, branches Galls appear singly or as

groups and form around wounds on the main

trunk Galls develop as small swellings across

and grow into smooth, spherical, and dark

knots

2.4 Bacterial wilt in tomato and cucumber - Ralstonia solanacearum

Figure 5: Wilting in tomato Figure 6: Wilting in cucumber

* Symptoms:

Bacterial wilt causes a rapid wilt and death of trees The trees may have wilted foliage The foliage appears dull green and hangs almost vertically Leaves usually become slow chlorotic and remain attached

on tree Darkening of the systemically infected leaf veins occurs

2.5 Bacterial leaf blight (BLB) - Xanthomonas oryzae

* Symptoms: The leaves turn grayish green and roll up.

As the disease progresses, the leaves turn yellow to

straw-colored and wilt, leading whole tree to dry up and

die

2.6 Bacterial leaf streak (BLS) - Xanthomonas oryzae

* Symptoms: Linear lesions occur

between leaf veins These streaks are lightbrown and yellowish gray The lesions aretranslucent when held against the light.Entire leaves may become brown and die

2.7 Black rot - Xanthomonas campestris pv.Campestris

* Symptoms:

- Initial symptoms are irregular, dull, yellow blotches

that appear on the edges of leaves

- As the disease progresses, these blotches expand into

V-shaped areas with the V-shaped areas are initially

yellow, but eventually become brown and necrotic (i.e.,

dead) in the center with a yellow border or halo

- Later, affected plants tend to show symptoms of

wilting

3 Oozing test

3.1 Procedures for Oozing test in lab:

Step 1: Obtain one pot of diseased tomato and cucumber plant (bacterial wilt disease).Step 2: Remove soils under water tap Cut the aboveground portion of the stem near thesoil line from diseased plants

Step 3: Sterilize the equipments with alcohol

Step 4: Cut through the rinsed main stem using a scalpel into small fragments of ~ 3-5 cm.Step 5: Insert the cut end of the stems into a sterile-water blank Forces will help to standthe stems on the water blank

Step 6: Observe both stems for about 1-2 minutes

3.2 Results of the experiment:

Describe the results of experiment: Observed

the white bacterial steam flows out the root of

tomato crop

4 KOH Test – Indentify bacterial and fungus disease:

- Finished in lesson 4

5 Isolation of bacteria from diseases:

5.1 Procedures for bacteria isolation:

a For leafy diseased isolation:

Step 1: Prepare and sterilize working area and your hands

Step 2: Flame-sterilize scalpel & forceps

Step 3: Excise a piece of infected leaf containing both healthy and diseased tissues

Step 4: Surface sterilize the leaf piece in for 90o in 5 sec (in a petri dish), rinse in sterilewater (in another petri dish) and dry on paper tissue

Step 5: Place the leaf piece in a drop of sterile water in a sterilized slide and macerate(using a flame-sterilized forces or scalpel) the tissue until forming a green suspension.Step 6: Using a flame-sterilized transfer loop, transfer 2-3 loopfuls of this suspension toanother drop of sterile water and mix thoroughly

Step 7: Sterilization the flame transfer loop, take a loopful of the diluted suspension andstreak onto 1 PDA plate using a three way dilution streak technique

Step 8: Daily record the growth of bacteria in cultural media

b For stem diseased isolation:

Step 1,2,3 are similar

Step 4: Surface sterilize the provided stem fragment in 95% ethanol

Step 5: Remove two end of the fragment and insert the cut fragment into a sterile watervial Leave the vial for 10 minutes

Step 6: Using a newly flame-sterilized transfer loop, take a loopful of the stem fluid andstreak onto PDA plate using a three way dilution streak technique

Step 7: Daily record the growth of bacteria in cultural media

c Diseases for bacteria isolation in experiment

- Rice leaf blight - Xanthomonas oryzae

- Citrus canker - Xanthomonas citri

- Bacterial wilt in cucumber - Ralstonia Solanacearum

Figure 1: Rice leaf blight Figure 2: Citrus canker Figure 3: Bacterial wilt

 Conclusion: These pictures are taken after 5-day observation While the fluid of citrus

canker and rice leaf blight are streaked into PDA media, the fluid of bacterial wilt isstreaked into WA media due to lack of PDA media After 5-day observation, it can be seenthat bacteria of citrus canker in PDA media develops well and faster than that of Thestreak of citrus canker in PDA can be seen quite clearly The bacterial wilt in WA mediahas no phenomenon

5.2 Observation of bacterial streaming for leaf spot/canker/vascular diseases on

micro-scope

5.2.1 Preparation for observation:

Step 1: Excise a small rectangular section from a typical and new lesion (~1/2 x1/2 cm) The section should include healthy and diseased tissues

Step 2: Place the section on a clean glass slide

Step 3: Cut the section into 2 equal halves using a sharp scalpel Move them apart from each other (~ 1 mm)

Step 4: Cover the tissues with a coverslip and flood them with sterilized water from the edge of the coverslip

Step 5: View the edges of the tissues using 10X to 40X objectives If the cause of an

active lesion is bacterial, a cloud of bacteria will usually stream out from the cut edge of tissue

5.2.2 Results of experiment:

- Describe the experiment’s results:

The stream of bacteria flows out of the leaf blade

as the cloud.

6 HR Test – Hypersensitive reaction

- Finished in lesson 4

PRACTICAL LAB LESSON 3

I Date and place

- Date: 16/11/2018

- Place: Laboratory of Plant Pathology Department

II Main activities

1 Observation of some plant viral diseases

2 PTA- ELISA methods to detect PRSV on Cucubit plant

3 Mechanical transmission test (inoculation) of PRSV on healthy plant

4 Check results of isolation both fungi and bacteria culture

III Methodology and results

1 Observation of some plant viral diseases.

1.1 Mosaic disease of Zucchini caused by Zuchini yellow mosaic virus (ZYMV)

+ ZYMV are RNA virus which belong to viral family Potyviridae

+ The vector for this virus is aphid (Aphid-borne Potyvirus)

+ CYSDV is RNA virus which belongs to viral family Closteroviridae

+ The major vector for CYSDV is the whitefly - Bemicia tabac)

+ CVYV is RNA virus which belongs to viral family Potyviridae

+ The major vector for CVYV is also the whitefly - Bemicia tabaci

Symptoms:

The leaves occur yellowing from the

margin to the vein after that the margin

turns to brown and wilt When the leaf

blade is yellow, the vein of leaf still

remains green There is the distortion of

the leaf in some seriously diseased leaves

1.3 Mosaic disease of cucumber:

+ CMV is RNA virus which belongs to viral family Bromoviridae

+ The major vector for CMV is also aphid.