RawatFinal Synopsis PPT.pptx PATHOLOGY BUAT AGRICULTURE

HERE IS RAJU KUMAR VERMA UPLOADING OUR PPT SYNOPSIS

1. Synopsis seminar on Shri Kant Rawat ID. No: 2037 Department of Plant Pathology College of Agriculture, BUAT, BANDA- 210001 (UP) Characterization of Begomoviruses causing Yellow Mosaic and Leaf Curl Diseases in Different Host-Plants

2. ADVORY COMMITTEE ADVISORY COMMITTEE S. No. Name & Designation Department Member 1. Dr. H. S. Negi Assistant Professor Plant Pathology , College of Agriculture, BUAT, Banda Major Advisor 2. Dr. Dharmendr a Kumar Professor Plant Pathology, College of Agriculture, BUAT, Banda Co-Advisor 3. Dr. Mukesh Kumar Mishra Assistant Professor Entomology, College of Agriculture, BUAT, Banda Co-Advisor 4. Dr. C. M. Singh Assistant Professor Genetics and Plant Breeding, College of Agriculture, BUAT, Banda Co-Advisor

3. INTRODUCTION Yellow Mosaic Disease (YMD) and Leaf Curl Disease (LCD) are infecting leguminous and cucurbitaceous crops of the Kharif season. YMD and LCD has been reported to be caused by Mungbean Y ellow Mosaic Virus (MYMV), Mungbean Y ellow Mosaic India Virus (MYMIV) and Horse gram Y ellow Mosaic Virus ( HgYMV ) belonging to Begomovirus genus and Geminiviridae family (Ilyas et al., 2010). They are single-stranded DNA viruses with bipartite genomes and are transmitted by means of whitefly ( Bemesia tabaci ). In India, MYMV was first reported from the mungbean fields of Indian Agricultural Research Institute (IARI), New Delhi during 1950s ( Nariani , 1960).

4. Cont.… In India, the leaf curl disease of mungbean and urd bean was first time reported in 1968 from Pantnagar, Uttrakhand (Singh, 2013). Losses from yellow mosaic disease upto an extent of 85 % has been reported from different mungbean regions of Asia (Karthikeyan et al ., 2014; Naimuddin et al ., 2016). MYMV incidence ranging from 5.88 – 28.54% has been reported in French bean from Karnataka ( Archith et al ., 2017). Mungbean leaf curl caused considerable loss in mungbean up to 40 per cent in 33 districts of Uttar Pradesh as per the survey conducted by Nene and Singh (1972).

5. OBJECTIVES Survey for the occurrence of yellow mosaic and leaf curl diseases in Banda locality. Symptomatology of the yellow mosaic and leaf curl diseases of different host-plants. Molecular detection of Begomoviruses from samples showing symptoms of yellow mosaic and leaf curl.

6. TECHNICAL PROGRAMME Survey for the occurrence of yellow mosaic and leaf curl diseases in Banda location Survey will be carried out to record the incidence of yellow mosaic and leaf curl diseases from the pulses and vegetable growing villages of Banda locality namely Pipri , Mawai Buzurg , Chahitara , Kanwara and BUAT Banda during kharif 2022. The diagnosis of the disease in the field will be based on symptoms on the plants.

7. The per cent disease incidence will be calculated randomly in different locations in an area of 5×5 m in the field using the formula Percent disease incidence = 100 The infected samples collected during the survey will be brought to the laboratory to test for the presence of Begomovirus by PCR technique using specific primers .  

8. Table- 1: Crops to be studied for symptoms & collection of samples of YMD and LCD   S. No. Name of crops Family Diseases 1 Mung bean Fabaceae Yellow Mosaic (YM) & Leaf Curl (LC) 2 Urd bean Fabaceae Yellow Mosaic (YM) & Leaf Curl (LC) 3 French bean Fabaceae Yellow Mosaic (YM) & Leaf Curl (LC) 4 Cowpea Fabaceae Yellow Mosaic (YM) & Leaf Curl (LC) 5 Dolichos bean Fabaceae Yellow Mosaic (YM) & Leaf Curl (LC) 6 Pigeon pea Fabaceae Yellow Mosaic (YM) 7 Sponge gourd Cucurbitaceae Yellow Mosaic (YM) & Leaf Curl (LC) 8 Wild kakdi Cucurbitaceae Yellow Mosaic (YM) 9 Pumpkin Cucurbitaceae Yellow Mosaic (YM) & Leaf Curl (LC)

9. Cont.… S.No . Name of crops Family diseases 10 Bitter gourd Cucurbitaceae Yellow Mosaic (YM) 11 Cucumber Cucurbitaceae Yellow Mosaic (YM) & Leaf Curl (LC) 12 Pointed gourd Cucurbitaceae Leaf Curl (LC) 13 Ivy gourd Cucurbitaceae Leaf Curl (LC) 14 Chilli Solanaceae Leaf Curl (LC) 15 Tomato Solanaceae Leaf Curl (LC) 16 Bhringraj Asteraceae Yellow Mosaic (YM) 17 Croton Euphorbiaceae Yellow Mosaic (YM)

10. Molecular characterization of YMD and LCD causing viruses DNA isolation In 200 mg leaves sample add 250 µl CTAB Buffer Grind in Mortar and pestle. Transfer in 2 ml Centrifuge tube maintain CTAB Buffer and add 2µl Beta Merceptoethanol . Put at 65 C in water bath in 45 minute and put at 10 Minute at room temperature Centrifuge 12000 rpm for 10 minutes at 28 C Transfer 500µl Supernatant in 1.5 ml Centrifuge tube and add 300 µl PCI mix at Room Temperature for 15 minutes, Centrifuge 10 minutes 11000 rpm Transfer 400 µl Clean Supernatant in 1.5 ml Centrifuge tube and Equal Volume of CI mix, Centrifuge 5 minute at 11000 rpm Next step in repeat same as upper step

11. Transfer 200 µl Clean Supernatant and add 300 µl Isopropanol (Chilled) Put at the -21 C for 1 hours or for 10 to 20 minutes Centrifuge 9000 rpm for 15 minutes at 4 C Discard Supernatant 4 C and 200 µl 70 % Ethanol and Centrifuge 10000 rpm at 4 C 5 minutes after Discard Ethanol Keep tube open for over night to evaporate ethanol Add 100 µl TE Buffer Keep at -20 C for storage DNA qualitative and quantitative analysis using nanodrop

12. Procedure of PCR Denaturation The DNA template will be heated to 94°C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA. Annealing The mixture will be cooled to anywhere from 50-70°C. This allows the primers to bind (annealing) to their complementary sequence in the template DNA. Extension The reaction will be then heated to 72°C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.

13. Table 2. Details of the primers to get the complete coat protein gene and movement protein gene of three Begomoviruses and expected size of amplicons . Primer ID Primer sequence 5’……3’ AT ( C ) ~size of DNA Fragment to be amplified Name of the virus to be detected/ DNA component MYMV-MPF ATG GAG AAT TAT TCA GGC GCA 58 900bp MYMV/ DNA B MYMV-CP-F ATG GG (T/G) TCC GTT GTA TGC TTG 54 1000bp MYMV/DNA A MYMIVMPF ATG GAA AAT TAT TCA GGT GCA 53 900bp MYMIV/ DNA B AC3PF AC3PR TTA TGA TTC GAT ATT GAA TTA ATA CTG AAG TGTGGG TGT AGC TAT 48 450bp MYMIV/DNA A HYMV-MPF ATG GAG CAT TAT TCC GGT GCA 64 900bp HgYMV / DNA B HYMV-CP-F ATG CTT GCA ATT AAG TAC TTG CA 56 1050 bp HgYMV /DNA A

14. Agarose gel electrophoresis PCR amplified products will be separated by agarose gel electrophoresis. Requirements Electrophoretic unit, gel casting, gel comb, power pack and UV-transilluminator Agarose 1 to 3 % Ethidium bromide (0.5 µg/ml) 50X TAE (stock) Working solution (1X TAE): 20 ml of 50X TAE buffer will be made up to 1000 ml by using double distilled water.

15. DNA bands will be viewed under UV-transilluminator and documented using a gel documentation system.

16. EXPECTED OUTCOMES Virus causing the mosaic and leaf curl symptoms will be characterized and will be helpful in devising further strategies to work on these diseases in different pulses and vegetable crops of kharif season to be studies in present investigation.