1. Genomic properties of Potato virus M occurring in northern plain of India
AKSHAY KATIYAR
Advanced Centre for Plant Virology
Division of Plant Pathology
Indian Agricultural Research Institute

2. POTATO (Solanum tuberosum)
Third most important food crop of the world
India, second largest producer share about 12.2% in world potato production after China (FAOSTAT data, 2014).
Viruses are the major problem in Potato crop causing yield loss upto 90%.
HEALTHY
VIRUS INFECTED
Major production from India (85% )
Uttar Pradesh
West Bengal
Bihar
Karnataka
Madhya Pradesh
Gujarat
Jharkhand,
Punjab

3. Viral and viroid diseases of potato
Alfalfa mosaic virus
Andean potato latent virus
Andean potato mottle virus
Arracacha virus B - Oca strain
Beet curly top virus
Cucumber mosaic virus
Eggplant mottle dwarf virus
Potato aucuba mosaic virus
Potato black ringspot virus
Potato deforming mosaic virus
Potato latent virus
Potato leafroll virus
Potato mop-top virus
Potato rugose mosaic
Potato stem mottle virus
Potato spindle tuber viroid
Potato yellow dwarf virus
Potato yellow vein virus
Potato yellowing virus
Potato virus A
Potato virus M
Potato virus S
Potato virus T
Potato virus U
Potato virus V
Potato virus X
Potato virus Y
Sowbane mosaic virus
Tobacco mosaic virus
Tobacco necrosis virus
Tomato yellow mosaic virus
Wild potato mosaic virus
Tobacco rattle virus
Tobacco streak virus
Tomato black ring virus
Tomato mosaic virus
Tomato spotted wilt virus
Potato Viruses in INDIA
Potato leafroll virus
Potato virus A
Potato virus M
Potato virus S
Potato virus X
Potato virus Y
Tomato leaf curl virus
Groundnut bud necrosis virus
Viruses are the major threat in potato production and a total of 36 viruses and viroids are reported worldwide in potato (Tiwari, 2012; Raigond, 2013) that significantly reduces the yield of potato.

4. Detection of Potato virus in the field samples collected from North region of India by ELISA
PVX
PVY 27/57
PVM 18/78
PVS 9/21
PLRV
PVA 14/21
PVX 28/146
PVY 34/179
PVM 33/179
PVS 24/179
PLRV 1/146
PVA 1/57
PVX 2/8
PVY 10/36
PVM 10/44
PVS 0/8
PLRV 0/8
PVA 5/44
PVX 118/410
PVY 141/463
PVM 74/490
PVS /490
PLRV 31/133
PVA 24/330
TOTAL
PVX 148/564 (26%)
PVY /796(29%)
PVM 169/852(20%)
PVS 54/698 (8%)
PLRV 52/348 (14%)
PVA /513 (8%)
PVX
PVY 22/61
PVM 34/61
PVS
PLRV 20/61
PVA 0/61
Uttar Pradesh
Madhya Pradesh
West bangal
Bihar
Delhi

5. Specific Detection of six RNA viruses infecting Potato in India
PVM
PVS
PVX
PVY
PVA
PLRV
INOCULATION OF DIFFERENT POTATO VIRUSES IN GLASS HOUSE
300 bp
500 bp
1000 bp
M PVX PVY PVA PVS PVM PLRV
1008 bp
800 bp
616 bp
504 bp
445 bp
345 bp
Specific Detection of six RNA viruses infecting Potato in India
(Mandal et al., 2012)
(Shilpi et al., 2013)
(Jeevalatha et al.,2013)
Sap Inoculation

6. Question : Why Potato virus M ?
PVM has been characterized in India only on the basis of their biological properties, transmission, host range and yield loss.
Causing yield loss Between 15% and 45% (CPRI Annual Report )
Infection results in significant decrease in size and number of tubers.
PVM incidence increased continuously during the period of study
Availability of only six complete genome sequence all over the world.
No sequence data was available of PVM from India.
PVM stains have remarkable diversity as per existing reports.
First reported in Solanum tuberosum; from the U.S.A.; by Schultz and Folsom (1923); Bagnall et al. (1956)..
Symptoms: Causes mottling, mosaic, crinkling and abaxial rolling of leaves, and stunting of shoots..
Healthy
Infected

7. Genus : Carlavirus Family : Flexiviridae Particle Genome Transmission
Potato virus M
Family : Flexiviridae
Particle : Filamentous,Flexuous rods,
650x12 nm, not enveloped,
usually straight (to slightly curved)
Genome : Single stranded RNA, positive-sense,
5’ cap, 6 ORFs, 3’ poly A tail
Size approx. 8.5 kb
Transmission : Vector an insect; Myzus persicae
Non-persistent manner.
Mechanical inoculation, grafting Not transmitted by seed and pollen
Host : Natural: mainly solanaceae (potato)
Experimental: chenopodiaceae, leguminosae and solanaceae
Distribution : World wide
Genome
REPLICASE
TGB1
TGB2
TGB3 CP
NAB
(A)n3'UTR
5'UTR
Transmission

8. STRATEGY AND PRIMER USED FOR COMPLETE GENOME
Schematic representation of the strategy used for the amplification and List of primers used for amplification of complete genome of Indian isolate of Potato virus M (PVM-Del-144) isolated from Solanum tuberosum
Question : Is our isolate is different from other submitted PVM genome?

9. PVM-Del-144
Percent sequence identity of Potato virus M-Del-144 isolate with the other isolates reported worldwide
Isolates
Country
Accession no
Genome
length (bp)
Complete
genome
5' UTR
3' UTR
ORF1
nt/aa
ORF2
ORF3
ORF4
ORF5
ORF6
Gansu
China
JN835299
8,520
92.8 92
96.2
92.1/93.5
93.0/96.9
94.8/97.2
94.7/95.3
93.2/98.0
97.6/96.5
VIRUBRA 4/007
Czech Republic
HM854296
8,474
92.2
61.3
39.4
92.1/93.3
92.8/94.7
93.9/97.2
95.8/95.3
93.9/96.7
92.7/91.3
M57
Poland
AY311395
8,522
93.6
93.3
98.1
93.1/94.2
94.7/99.1
93.0/95.4
94.7/98.6
96.8/96.5
Uran
AY311394
93.4
93.0/93.9
93.1/96.9
95.1/97.2
93.2/93.7
94.0/98.0
97.3/96.5
DSMZ PV0273
Germany
EU604672
8,523
93.9
71.8
93.2/93.9
93.6/96.5
94.8/96.3
96.2/99.0
93.3/92.1
Russian wild
Russia
D14449
8,533
93.1
74.6
92.3/92.4
94.3/97.8
94.5/97.2
92.7/89.0
95.6/97.7
91.0/87.8
Phylogenetic tree of Potato virus M isolates based on the
maximum parsimony analysis of complete genome.
Our isolate shown in highlighted portion

10. Coat protein gene specific primers
Host reactions difference between Indian isolate of Potato virus M (PVM-Del-144) and other reported isolates.
Phaseolus vulgaris
Solanum lycopersicum
RT-PCR confirmation
Host Range study
BM455F 5' ATCTGAAATAGTGAGTATGGG 3'
BM587R 5' GC CAC CTT GGT TAC GTG CTT 3'
Coat protein gene specific primers
Datura metel
Solanum tuberosum
M +ve H
Plant species
Symptoms
References
PVM-Del-144
Other isolates
Solanum tuberosum
Abaxial leaf rolling, necrosis
and stunting of plant
Mottle, mosaic, crinkling and rolling of leaves and stunting of shoots
Huimin Xu et al., 2010
Phaseolus vulgaris
Faint grayish local lesion that turned to dark brown in advanced stage
Local necrotic lesion on primary leaves
Bangall et al.,1956, 1959;Kowalska and was,1976; Slack,1983; Edwardson and Christie,1997
Datura metel
Local lesion on leaf non systemic
Local chlorotic spotting and systemic chlorotic mottling and rugosity
Bangall et al.,1956, 1959;Kowalska and was,1976; Slack,1983;
Edwardson and Christie,1997
Nicotiana benthamiana
Twisting of leaves
Symptomless
S.Flatken et al., 2008
Solanum lycopersicum
Yellowing of leaves
Gomphrena globosa
Non host
Local cholrotic rings or necrotic spots
C.Hiruki
Chenopodium quinoa
Chlorotic local lesions
N. Glutinosa
No infection
N. Tabacum
Bangall et al.,1956, 1959;Kowalska and was, 1976; Slack,1983; Edwardson and Christie,1997

11. Disease diversity at field
PVM-7
PVM-6
PVM-4
Disease diversity at field
Question : Is there is any genetic diversity in PVM genome?
Indication of the existence
of molecular diversity
Collect symptomatic plant leaves from IARI experimental field
Confirmed presence of potato virus M by RT PCR with CP specific primer
Out of seven sample three samples were found positive (4,6,7)
But sample No 4 is not giving amplification if we tried any other primer of PVM genome M
Partial genome amplification of PVM
3200
5100
4700
M H +ve
915
Coat protein gene amplification of PVM
AMPLIFICATION VARIATION WITH DIFFERENT SETS OF PRIMER

12. Detection of Potato virus M in the field samples collected from different region of India by direct antigen-coated enzyme-linked immunosorbent assay using commercial polyclonal antisera
Place
Year of collection
No. positive/
No. tested (%)
OD range for
positive samplesa
Jalpaiguri
Vaishali
Patna
IARI field-1
2012
7/10 (70)
9/21(42.8)
9/22(40.9)
10/27(37.0)
IARI field-2
5/85 (5.9)
IARI field-3
IARI field-4
2014
19/53 (35.8)
IARI field-5
Meerut
2013
17/160 (10.6)
13/20(65.0)
Balia
4/22 (18.2)
Gajipur
2/13 (15.4)
Kolkata
3/8 (37.5)
Agra
4/25 (16)
Mathura
3/6 (50)
Jaunpur
4/33 (12.1)
Mirjapur
4/15 (16.7)
Hatrus
1/21 (4.8)
0.321
Kannauj
Indore
Ujjain
1/11 (9.1)
18/31(58.0)
16/30(53.33)
0.158
Total
154/698 (22.0)
Map of India showing the major potato growing area in North India from
where infected potato samples were collected and percent of potato virus M
(PVM) incidence in those areas evaluated based on serological diagnosis
aThe OD value was taken at A405 nm after 1 h of addition of substrate.
Samples with absorbance value more than two times of healthy control
( ) were considered as ELISA positive.

13. Cloning and sequencing
s.no
Isolate
Collected area
Genome location
Size(bp)
Accession no 1
PVM-Del-144
IARI , Delhi
Complete genome
8523
KJ194171 2
M34
coat protein
915
KF471070 3
Jau-33
Jaunpur, UP
coat protein+3’UTR
1300
KJ473993 4
Gaj-13
Gazipur,UP
KJ473992 5
Del-147
KJ462137 6
Bal-21
Baliya, UP
KJ462133 7
Mir-12
Mirjapur, UP
KJ462134 8
Del-123
KJ462135 9
Del-134
KJ472136 10
Mat-12
Mathura, UP
KJ569697 11
PVM-Del-133
KJ569696 12
Agf-5
Agra, UP
KJ919964 13
Kan-16
Kannauj, UP
KJ919965 14
Hat-12
Hathrus, UP
KJ919966
RNA ISOLATION
PCR/RT-PCR (cp primer)
TA vector
Cloning
Sequencing

14. Potato Virus M : Genetic diversity
KJ (Del-144) India
100%
SCALE
KF (M34) India
99%
KJ (Del-134) India
95%
KJ (Del-123) India
90%
KJ (Del-147) India
85%
KJ (AGF-5) India
98%
80%
KJ (Kan-16)India
75%
KJ (Del-133) India
76%
77%
70%
KJ (Hat-12)India
97%
KJ (Jau-33) India
96%
KJ (Bal-21) India
94%
KJ (Mir-12) India
KJ (Mat-12) India
KJ (Gaj-13) India
93%
92%
Maximum parsimony tree of Indian isolates of Potato virus M
PVM-o
PVM-d
Sequence Identity Matrix of coat protein gene sequence of Indian isolates
ICTV
Species
Demarcation
criteria

15. Validation of specificity of primers
Designing of specific primers for the specific detection of PVM-d isolate
PRIMER
PRIMER SEQUENCE
Tm (C)
Amplicon length
BM761F    
5` AGCAAGAGAGCGAGCACATC 3` 60
737 Bp
BM764R  
5`CTCGGAGTGGGCCTCCTT3` 59
Validation of specificity of primers
by using the cloned DNA M
M: Marker
Lane 1 : Gaj-13
Lane 2 : Del-144
Lane 3 : Del-133
Lane 4 : Bal-21
Lane 5 : Del-147
Lane 6 : Hat-12
Lane 7 : Del-123
1500
500

16. Conclusions
first time the complete genome sequence of an Indian isolate of Potato virus M.
our isolate (PVM-Del-144) is different from all other previously reported PVM isolates based on their biology as well as on their molecular properties.
PVM incidence in the potato fields in India was found 22% which is almost two times higher than reported from Iran (Fatemeh et al. 2014) and it is continuously increasing from last three years.
Existence of two different genotypes of Potato virus M in India and we have developed the primers for the specific detection of both the genotypes.

17. Research ahead:
Need to carry out further study on Genetic diversity and their distribution
Current study shows the existence of biological variants: PVM strains in India need to be well characterised
Need to develop infectious clone of PVM for study of viral gene function and host pathogen interaction

18. Acknowledgements Contribution
Dr. Alok Kumar (Post Doctoral Scientist, ILRI, Ethiopia, Africa)
Guidance
Dr. Bikash Mandal (Principal Scientist, IARI, N. Delhi)
Dr. Anirban Roy ( Sen. Scientist Advanced Center for Plant Virology, N. Delhi)
Dr. G.P. Rao (Principal Scientist, IARI, N. Delhi)
Dr. V. K. Baranwal (Incharge, Advanced Center for Plant Virology,N. Delhi)
Financial support
Department of Biotechnology & ICAR
Travel & accommodation
Indian Virological Society

19. Thank you