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Appendix Lab lessons Fungi Main content Preparation of media for fungi (WA, PDA) Isolation of pathogens fungi from plant diseases (leaves) Observation of some plant fungal diseases Bacteria Preparation of medium for bacterium culture King’B and SPA Observation of some bacteria diseases Oozing test – Identify bacterial and fungus diseases KOH Test – Identify positive gram and negative gram bacteria Isolation of bacteria from diseases HR test – Hypersensitive reaction 11 11 11 13 Viruses Observation of some plant viral diseases PTA- ELISA methods to detect PRSV on Cucubit plant Mechanical transmission test (inoculation) of PRSV on healthy plant Check results of isolation both fungi and bacteria culture 14 16 17 - Part 1: Preparation of 10 plates of PDA (for sub culture of fungi) Preparation of KOH 3% Observation the isolation of fungi and bacteria HR test from cultured bacteria Check ELISA results - Part 2: Observation of Root knot nematode (cabbage, carrot, tomato) Staining of roots for observation of nematodes inside the roots Extraction of nematodes from soil and plant samples 18 Nematodes Page 2 17 19 PRACTICAL LAB LESSON I Date and place - Date: 14/11/2018 - Place: Laboratory of Plant Pathology Department II Main activities Preparation of media for fungi (WA, PDA) Isolation of pathogens fungi from plant diseases (leaves) Observation of some plant fungal diseases III Methodology and results 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 for the germination of conidia used to initiate single spore cultures - For this experiment, we need 200 ml water and g of agar to create the media for fugal culture - Hyphal growth is sparse on this medium so it is suitable for cultures from which single hyphal 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 the broth from 250 g white potatoes made up to L with tap water - For this experiment, we use 200ml water, g dextrose (glucose), 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 follow the recipe, boil the potato and filter through cheesecloth, leaving some sediment in the broth 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 of antibiotics solution in media bottle to inhibit the non-target bacteria will grow in media laminar 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 bunsen burner to sterilization Step 3: Surface sterilise leaf tissue by dipped in 70% ethyl alcohol for 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 germinated spore 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 days Corynespora spot in tomato after days Southern blight in corn after days Corynespora spot in tomato leaves after days 2.2 Daily check for incubation: Table 1: Result of observing fungal disease development in days Diseases Southern blight in corn Corynespora spot in tomato Fragments After day After days After days 3 Full sides sides branches No No No Full sides Full sides Full sides branch No No Full sides Full sides Full sides Full sides No No  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 than that causing corynespora spot in tomato After days observing, all fragments of corn leaf have fungi develop full sides surrounding the fragments and nearly full, whereas only st fragment extracted from tomato has fungi grow full sides and the other fragments exist nothing 3 Observation of some plant fungal diseases 3.1 Observation symptoms and signs of some plant fungal disease a Anthracnose in chilli - Colletotrichum capsici Symptoms: Plant diseases characterized by very dark, sunken lesions, containing spores b Powdery mildew - Erysiphe cichoracearum De Candolle Symptom: The white powder layer covers the all the leaf blade The diseased leaf turns from green to yellow color Plant grow slowly, reducing the quality of products c Corynespora spot in tomato fruit - Corynespora cassiicola Symptom: Big, dark, sunken spots occur occasionally on the surface of fruit The mycelium appears surrounding the spot of fruit d Rust in maize – Puccinia maydis Ber Symptom: 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 redbrown powder The spot floats on the leaf surface e Rust in ground nut - Puccinia arachidis Speg Symptom: The disease spots have round shape and small size The lower epidermis of leaf is cracked with the appearance of sporangium in orange, red color Upper epidermis, the disease stains with yellow color and burning the leaf f Anthracnose in dragon fruit - Colletotrichum gloeosporioides Symptom: Anthracnose disease on dragon fruit was characterized with reddish brown lesions and chlorotic haloes symptoms on stem as well as fruit These lesions had brown centers and then coalesced to rot g Canker in ornamental peach - Cytospora leucostoma Symptoms: The bark is killed, and when removed, the underlying tissue is orange to brown in colour, 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 on leaves Spots are usually on the upper surface of the leaf and occasionally form dense aggregations The spots are rough to the touch That causes leaf yellowing The black speckled strips extend from the midrib towards 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 samples of plant fungal disease on microscope a Anthracnose in chilli - Colletotrichum capsici Description: b Powdery mildew - Erysiphe cichoracearum De Candolle Description: c Corynespora spot in tomato fruit - Corynespora cassiicola Description: PRACTICAL LAB LESSON I Date and place - Date: 15/11/2018 - Place: Laboratory of Plant Pathology II Main activities Preparation of medium for bacterium culture ( King’B, SPA)- Most of bacteria live in SPA media Observation of some bacteria diseases Oozing test – Identify bacterial and fungus diseases KOH Test – Identify positive gram and negative gram bacteria Isolation of bacteria from diseases HR test – Hypersensitive reaction III Methodology and results 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 Sucrose 20(g) 4(g) Peptone 5(g) 1(g) K2HPO4 0.5(g) 0.01(g) MgSO4.H2O 0.25(g) 0.05(g) Agar 20(g) 4(g) Distilled water (L) 0.2 (L) 1.2 King’ B medium preparation: - The compositions of King’B media: Chemical agents Agar Proteose peptone No Glycerol, C.P K2HPO4 MgSO4 Distilled water Theoretically recipe 15 g 20 g 10 ml 1.5 g 1.5 g 1L 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-soaked and slightly raised spots first The lesions expand and thicken over time and protrude from both leaf surfaces As the lesion develops, the tissue becomes spongy or corky, and the color changes from tan or brown, to grey or white, usually surrounded 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 10 * Symptoms: Linear lesions occur between leaf veins These streaks are light brown and yellowish gray The lesions are translucent 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 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 the soil 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 stand the stems on the water blank Step 6: Observe both stems for about 1-2 minutes 3.2 Results of the experiment: 11 Describe the results of experiment: Observed the white bacterial steam flows out the root of tomato crop KOH Test – Indentify bacterial and fungus disease: - Finished in lesson 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 90 o in sec (in a petri dish), rinse in sterile water (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 to another drop of sterile water and mix thoroughly Step 7: Sterilization the flame transfer loop, take a loopful of the diluted suspension and streak onto 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 water vial Leave the vial for 10 minutes Step 6: Using a newly flame-sterilized transfer loop, take a loopful of the stem fluid and streak 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 12 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 is streaked into WA media due to lack of PDA media After 5-day observation, it can be seen that bacteria of citrus canker in PDA media develops well and faster than that of The streak of citrus canker in PDA can be seen quite clearly The bacterial wilt in WA media has 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 equal halves using a sharp scalpel Move them apart from each other (~ 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 13 HR Test – Hypersensitive reaction - Finished in lesson PRACTICAL LAB LESSON I Date and place - Date: 16/11/2018 - Place: Laboratory of Plant Pathology Department II Main activities Observation of some plant viral diseases PTA- ELISA methods to detect PRSV on Cucubit plant Mechanical transmission test (inoculation) of PRSV on healthy plant Check results of isolation both fungi and bacteria culture III Methodology and results 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) 14 Symptoms: The virus incites yellow mosaic, severe malformation, blisters, extreme reduction in the size of leaf lamina, necrosis, and severe plant stunting 1.2 Yellowing disease of zucchini caused by mix of Cucurbit yellow stunting disorder virus (CYSDV) and Cucumber vein yellowing virus (CVYV) + 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 15 Symptoms: CMV stunts plant growth and causes leaves to be distorted, reduced in size, curled or rolled, and show a yellow mosaic or mottle pattern with chlorotic patches 1.4 Mosaic disease of Amaryllidaceae family Symptoms: The mosaic symptom occurs on the leaf, some mosaic has yellow color and surrounding surface of the leaf 1.5 Yellow leaf curve disease of tomato caused by Tomato yellow leaf curve virus (TYLCV) + TYLCV is DNA virus which belongs to Germiniviridae viral family + The vector for this virus is white flies ( Bemissia tabaci) 16 Symptoms: Plants are stunted with shoots becoming erect Leaflets of compound leaves are reduced in size and pucker Leaflets curl upwards, become distorted, and have prominent yellowing along margins and/or interveinal regions Flowers wither Diseased plants set very few and small fruit than healthy plants 1.6 Necrotic ringspot disease of tomato (new disease) caused by tomato necrotic ringspot virus (TNRV) Symptoms: Infected leavesare cupped and malformed withcircular spots to mm in diameter Young leavesmay be straplike and mottled Symptoms are variable within the same plant Infected plants may be defoliated by midharvest and eventually die, often after a severe winter The disease spreads slowly in the field 1.7 Bunchy top disease of banana caused by Banana bunchy top virus ( BBTV) + BBTV is DNA virus which belongs to viral family Nanoviridae + The vector for this virus is banana aphid (Pentalonia nigronervosa) 17 Symptoms: The leaves become progressively dwarfed, upright and bunched at the top of the plant, with wavy and chlorotic margins that tend to turn necrotic PTA-ELISA to detect PRSV on Cucubit ( first step) * ELISA – Enzyme Linked Immuno Sorbent Assay - For this experiment, we focus on the first step of PTA- ELISA - Procedures (following a PTA-ELISA- Plate Trapped Antigen Indirect ELISA): + Grind ~ 100 mg leaf tissue in ml carbonate buffer using mortar and pestle + Add 100 µL of extract per well using a micropipette + Incubate the plates ON at 4-6OC in a moistened plastic box + Next day Empty the plates and wash 3x (3 minutes each) with washing buffer Plant proteins and virions (if present) will non-specifically bind onto wells Mechanical transmission test (Inoculation) on PRSV on plant 3.1 Procedures for mechanical inoculation - Step 1: Wash hands with water and soap - Step 2: Prepare the inoculums: + Remove from the diseased papaya plant one or more pieces (~ 2-5 g) of younger leaves showing clear symptoms + Grind with volumes of ice-cold buffer using mortar and pestle + Dilute the slurry in ice-cold buffer to give a final tissue concentration of 1/5 -1/10 + Filter by squeezing the plan suspension through several layers of cheesecloth + Add carborundum, add % (w/v) to the final leaf suspension - Step 3: Dip a cotton swab into the suspension and apply the liquid on to the leaves by firmly and gently one-way strokes from petiole to tip while supporting the leaf from beneath Inoculate the entire leaf surface, dipping the swab frequently in the liquid; firmly but gently rub only once or maximum twice on the same area Excessive pressure will result in lethal damage to the epidermal cells from the carborandum; however, too little pressure will not wound the cells for viral entry - Step 4: Immediately after plant inoculation, i.e after maximum 2-5 mins, rinse the leaves with tap water from the squeeze bottle (The abrasive - inoculum mix is hard to remove, if dried up, and it can obscure symptom observation.) 18 - Step 5: Place the inoculated plants in an insect-free greenhouse or growth room Avoid excessively high temperature, at least during the next 12 h Keeping the inoculated plants in a humid environment for some hours reduces the chance for wilting - Step 6: Check inoculated plants after 1, 2, 4, 7, 14 days Local symptoms may occur after only days and symptoms of systemic infection after 1-2 weeks Record the symptoms 3.2 Results of experiment: + Results for young papaya plant (After 11 days of inoculation) Conclusion: After 11-day observation, we realize that seedling and have disease symptoms caused by PRSV while the tissue of papaya leaf of seedling is damaged The leaves of papaya seedling have size reduction, and the leaf of papaya seedling start having yellow mosaic symptoms + Results for Chenopodium amaranticolor (Indicator plant) (After days of inoculation) Conclusion: After days of inoculation of indicator plant, the plan express the symptom as yellow lesion on the leaf, we observed that there are 3/5 inoculated leaves express this symptom Then, the indicator plant start to die * Some picture through observation process - Inoculated papaya seedling: Papaya seedling Papaya seedling Papaya seedling 19 - Indicator plant After day After days Check results of isolation both fungi and bacteria culture - Finished in class PRACTICAL LAB LESSON I Date and place - Date: 20/11/2018 - Place: Laboratory of Plant Pathology Department II Main activities - Part 1: Preparation of 10 plates of PDA ( for sub culture of fungi) Preparation of KOH 3% Observation the isolation of fungi and bacteria HR test from cultured bacteria Check ELISA results - Part 2: Observation of Root knot nematode (cabbage, carrot, tomato samples) Staining of roots for observation of nematodes inside the roots Extraction of nematodes from soil and plant samples 20 III Methodology and results * Part 1: Preparation of 10 plates of PDA (for sub culture of fungi) - Finished in Biosafety cabinet Preparation of KOH 3% - Finished on lab with 200mL of KOH 3% - Result for KOH test to identify positive gram and negative gram bacteria Rice leaf blight Bacterial wilt Citrus canker Positive or negative Negative gram Negative gram No result gram bacteria Observation the isolation of fungi and bacteria - Finished on the lab HR test from cultured bacteria - Finished on the lab Check ELISA results - Finished on the lab, the results obtained is not good * Part 2: Observation of Root knot nematode (cabbage, carrot, tomato samples) Root of carrot Root of tomato Root of cabbage Staining of roots for observation of nematodes inside the roots Step 1: Clearing Root Tissue - Wash roots with water and place them in a 150 ml beaker Large root systems may be cut into sections for staining - Add 50 ml of tap water and an appropriate amount of Javen solution to clear the root tissue Soak the roots for minutes in the Javen solution and agitate occasionally Step 2: Staining Nematodes in Root - Rinse the roots with running tap water for about 45 seconds and then immerse them in water for 15 minutes to remove any residual Javen which may affect staining with acid fuschin - Drain the water and transfer the roots to a glass beaker with 30-50 ml of tap water - Add ml of stock acid-fuschin stain solution to the water and boil for about 30 seconds on a hotplate or in a microwave oven To prepare stock acid-fuschin solution, dissolve 3.5 g acid fuschin in 250 ml acetic acid and 750 ml distilled water - Cool the solution to room temperature, drain the stain solution, and rinse the roots in running tap water 21 Extraction of nematodes from soil and plant samples 3.1 Using a Baermann funnel - Description for Baermann funnel: A funnel is fitted with a wire screen which supports a sample of soil, wrapped in paper tissue or cheesecloth The sample is immersed in water, allowing migration of nematodes downward into the funnel The nematodes collect in a length of tubing attached to the funnel and secured with a clamp 3.2 Observe the nematodes under the compound microscope: 3.2.1 Describes the morphological structure of Nematode 22 Nematode male individual Nematode female individual 3.2.2 Results for five observed Nematode individuals 23