SHRIKANT RAWAT BUAT PLANT PATHOLOGY SYNOPSIS

i am shrikant rawat student of buat PLANT PATHOLOGY

1. Synopsis seminar on Shri Kant Rawat ID.NO:2037 Department of Plant Pathology College of Agriculture, BUAT, BANDA 210001 (UP) Characterization of Yellow Mosaic Viruses infecting kharif crops

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. INTRODECTION Yellow Mosaic Virus(YMV) and Leaf C url Disease(LCD) in infected kharif pulses ,vegetables and weeds Mungbean [Vigna radiata (L.) Wilczek] is native to India or Indo-Burma region and is third most important pulse crop of India after chickpea and pigeonpea (Anonymous, 2017; Sundaram, 2010). It belongs to family Febaceae and genus Vigna. It is self-pollinated diploid crop (2n=22) with genome size 574Mbp (Gupta and Pratap 2016 ).

4. Cont.… It is also known as green gram, green bean, mash bean, golden gram and greensoy, (Markam et al., 2018). In India, MYMV was first reported from the mungbean fields of Indian Agricultural Research Institute (IARI), New Delhi during 1950s ( Nariani , 1960). The disease is caused by yellow mosaic virus (YMV) belonging to begomovirus genus and Geminiviridae family. They are single-stranded DNA viruses with bipartite genomes and are transmitted by means of whitefly (Bemesia tabaci).

5. Cont … The visible symptoms appear as scattered yellow-color spots on the young leaves which later turns into a yellow mosaic pattern and ultimately results in complete yellowing, drying and withering of leaves The pods on the infected mungbean plant become smaller in size, yellowing of the leaves decreases the photosynthetic efficiency which ultimately manifested as severe yield penalty ( Malathi and John, 2009).

6. OBJECTIVES Survey for the incidence of mungbean yellow mosaic and leaf curl disease in Banda locality. Molecular characterization of viruses exhibiting symptoms of yellow mosaic and leaf curl in kharif pulses , vegetables and weeds.

7. TECHNICAL PROGRAMME Survey for the incidence of mungbean yellow mosaic disease in Banda location Survey will be carried out to assess the mungbean yellow mosaic virus disease incidence in major mungbean growing areas 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 .

8. Cont.… The per cent disease incidence will be calculated randomly in different locations in an area of 5×5 m in the field by counting the number of plants infected out of total number of plants examined in each sector using the formula given be Per cent disease incidence = The infected samples collected during the survey will be brought to the laboratory and tested for the presence of Begomovirus by PCR technique using MYMV specific primers .  

9. Cont.… Table- S1: list of crops and weeds used in the study   S. No. Name of crops Diseases Diseases Source 1 Mungbean Yellow Mosaic Virus (YVM) Leaf Curl (LC) BUAT Banda 2 Urd/Black gram Yellow Mosaic Virus (YVM) Leaf Curl (LC) BUAT Banda 3 Spong gourd Yellow Mosaic Virus (YVM) Leaf Curl (LC) Kanwara, Village 4 Wild kakdi Yellow Mosaic Virus (YVM)   Pipri Village 5 Pumpkin Yellow Mosaic Virus (YVM) Leaf Curl (LC) Kanwara, Village 6 Bhringaraj Yellow Mosaic Virus (YVM)   BUAT Banda 7 Croton Yellow Mosaic Virus (YVM)   BUAT Banda 8 Bitter gourd Yellow Mosaic Virus (YVM)   Mawai Buzurg 9 Cucmber Yellow Mosaic Virus (YVM) Leaf Curl (LC) Pipri Village

10. Cont.… 10 Rajama Yellow Mosaic Virus (YVM) Leaf Curl (LC) BUAT Banda 11 Okra Yellow Mosaic Virus (YVM) Leaf Curl (LC) BUAT Banda 12 Cowpea Yellow Mosaic Virus (YVM) Leaf Curl (LC) BUAT Banda 13 Bean Yellow Mosaic Virus (YVM) Leaf Curl (LC) BUAT Banda 14 Pigeonpea Yellow Mosaic Virus (YVM)   BUAT Banda 15 Chilli   Leaf Curl (LC) BUAT Banda 16 Tomato   Leaf Curl (LC) BUAT Banda 17 Pointed gourd   Leaf Curl (LC) BUAT Banda 18 Ivy gourd   Leaf Curl (LC) BUAT Banda

11. Cont … Molecular characterization of Mungbean yellow mosaic virus DNA extraction by CTAB method Isolation of total DNA from virus infected mungbean will be done by CTAB method with slight modification as given below Kollar et al . (1990). 200 Mg Leaves Sample add 250 µl CTAB Buffer Grind in Pastel and Motel Transfer in 2 ml Centrifuge tube maintain CTAB Buffer and add 2µl Beta Marceptoethenol

12. Cont.. 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 Transfer 200 µl Clean Supernatant and add 300 µl Isopropanol (Chilled ) Centrifuge 9000 rpm for 15 minutes at 4 C  

13. Cont … Discard Supernatant and 200 µl 70 percent Ethanol and Centrifuge 10000 rpm at 4 C 5 minutes after Ethanol Discard Keep tube open for 24 hours to evaporate ethanol Add 100 µl TE Buffer Keep at -20 for storage and after quantify  

14. Cont … B. Qualitative and quantitative analysis of DNA The quality and quantity of DNA will be analysed by running 2 µl of sample mixed with 2 µl of 10X loading dye in 1 per cent agarose gel . The DNA from the isolate produced clear sharp bands in one per cent agarose gel indicating the good quality of DNA. The DNA will be quantified by comparing with the 100 bp size marker.

15. Primer   Table 1. Details of the primers designed to get the complete coat protein gene and movement protein gene of four bogemoviruses known to infect different leguminous host in India and expected size of amplicons . Primer ID Primer sequence 5’……3’ AT (0C) ~size of DNA Fragment to be amplified Name of the virus to be detected/DNA component HYMV-CP-F ATG CTT GCA ATT AAG TAC TTG CA 56 1050 bp HgYMV/DNA A MYMV-CP-F ATG GG (T/G) TCC GTT GTA TGC TTG 54 1000bp MYMV/DNA A HYMV-MPF ATG GAG CAT TAT TCC GGT GCA 64 900bp HgYMV/ DNA B MYMV-MPF ATG GAG AAT TAT TCA GGC GCA 58 900bp MYMV/ DNA B MYMIVMPF ATG GAA AAT TAT TCA GGT GCA 53 900bp MYMIV/ DNA B

16. Cont … C. PCR reaction mixture Corbett research gradient PCR supplied by JH BIO innovation Pvt. Ltd., Bengaluru was used for cyclic amplification of DNA of Mungbean yellow mosaic virus. The master mix required will be prepared from components obtained from Bangalore Genei , Bengaluru and Eppendorf , Germany and was distributed into 0.2 ml PCR tubes and volume was made up to 20 µl with sterile distilled water as mentioned below.

17. Cont … Components Volume (µl) Taq buffer (10 X 2.00 dNTP (1mM) 1.00 Forward Primer (5 pM ) 1.00 Reverse Primer (5 pM ) 1.00 DNA template (40 ng ) 1.00 Taq polymerase (5 u/µl) 0.30 Sterile distilled water 13.70 Total 20.00

18. Cont … Procedure/Steps of PCR Denaturation The DNA template is 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 is 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 is 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.

19. Cont … Agarose gel electrophoresis Separation of PCR amplified products by agarose gel electrophoresis. Requirements Electrophoretic unit, gel casting, gel comb, power pack and UV- transilluminator Agarose 1 % Bromophenol blue Ethidium bromide (0.5 µg/ml) 50X TAE (stock) : Tris -free base - 60.5 g Glacial acetic acid - 14.25 ml

20. Cont … 0.5 M EDTA - 25 ml . Made up the volume to 250 ml, pH - 8.0. Working solution (1X TAE): 20 ml of 50X TAE will be made up to 1000 ml by using distilled water . Procedure One gram of agarose will be weighed and added to a 250ml conical flask containing 100 ml of 1X TAE buffer. The agarose will be melted by heating the solution in an oven and the solution will be stirred to ensure even mixing and complete dissolution of agarose . The solution was cooled to about 50 C.

21. Cont … Two to three drops of ethidium bromide (0.5 µg/ml) were added. The solution will be mixed and poured into the gel casting platform after inserting the comb in the trough. While pouring sufficient care will be taken for not allowing the air bubbles to trap in the gel. The gel will be allowed to solidify and the comb will be removed after placing the solidified gel into the electrophoretic apparatus, containing sufficient buffer (1X TAE) so as to cover the wells completely.

22. Cont … The amplified products (8-10 µl) to be analysed will be carefully loaded into the sample wells, after adding bromophenol blue with the help of micropipette. Electrophoresis will be carried out at 80 volts for 45 minutes. Ethidium bromide stained DNA bands will be viewed under UV- transilluminator and documented using a gel documentation system.

23. EXPECTED OUTCOMES