7. 7
Jain et al. (2012)
Virus
Record of emergence
Acronym Year Crop Place Key symptoms
Capsicum chlorosis
virus
CaCV 2006 Chilli Karnataka Apical necrosis
Groudnut bud
necrosis virus
GBNV 1968 Groundnut Andhra
Pradesh
Bud necrosis
Irish yellow spot
virus
IYSV 2002-03 Onion Maharashtra Chlorotic lesion
Peanut yellow virus PYSV 1991 Groundnut Andhra
Pradesh
Yellow spot
Watermelon bud
necrosis virus
WBNV 1991-92 watermelon Karnataka Bud necrosis
Table: Tospoviruses recorded in India
8. 8
Group: Group V ((-)ssRNA)
Order: Unassigned
Family: Bunyaviridae
Genus: Hantavirus
Genus: Nairovirus
Genus:Orthobunyavirus
Genus Phlebovirus
Genus Tospovirus
Bunyaviridae
Wikipedia
9. 9
Size: 80-120nm
Shape: Icosahedral
Genetic material
• Nuclic acid(-ssRNA)
• Tripartite genome(L,M and S)
Large(L)(~8.9kb) -Replacase associated protiens
Medium(M)(~4.8kb) -Movement and glycoprotiens(Gn/Gc)
Small(S)(~2.9 kb) -Nonstructural and Nuclocapsid Protiens
Holkar et al.(2015)
10. 10
Tospovirus as a group, have a wide host ranges including Economically important or
Minor crop plants as well as numerous weed species and some native plants.
TSWV : >900 sp
INSV : >300 sp
Transmission:
• Mechanically transmissible with difficulty
• No seed transmission
• Exclusively transmitted by Thrips
Biological Diversity of Tospo
• Symptom variant
• Virulence variant
• Differences in thrips specificity and
transmissibility
• Ability to break host resistance
Pappu et al.(2009)
11. 11
• virus vectors introduced in to new areas
because of the increased worldwide
movement of germplasm through seed
and other propagative material in global
trade and agriculture.
•To ensure the safe movement of
germplasm across the borders diagnosis
plays an important role in preventing
virus introduction.
Viruses and insects
do not carry passports
12. 12
Thrips transmission
-Circulative persistent manner of transmission
-I and II instar larvae are effective in transmission
-No transovarial transmission
-Adults acquiring the virus is Ded end of
epidemiology
Whitfield and Rotenberg (2015)
Family: Thripidae
Sub family: Thripinae
13. 13
General features of thrips
• Small size
- difficult to detect
• Polyphagous
- feed on a broad range of plant species
- feed on different parts of the plant
(pollen, flower structures, leaves, stems)
• Show habitat infidelity
- extraordinary ability to adapt
- can expand geographic range
- can spread to new crops
• Have superior reproductive output
- produce many off springs
• Have propensity to ‘overwinter’ on a
broad range of plant species
- survive through out the year
• Vectors of viruses
- Spread virus diseases
14. 14
• Provides greater flexibility in identification
• Increased sensitivity and specificity for rapid diagnosis of virus
• Epidemiological studies and spread of disease
• In plant quarantine and seed certification and
• Breeding programs
15. 15
Detection based on biological
properties
Detection methods based on viral coat protein
Detection methods based on virus nucleic acid
• Symptomatology
• Transmission tests
• Physical properties
• Microscopy
• Precipitation and agglutination tests
• Immunosorbent electron microscopy
• Enzyme-linked immunosorbent assay
• Nucleic acid hybridization assays
• Polymerase chain reaction
16. 16
Symptomatology
Symptoms on plants commonly are used to characterize a disease having viral
aetiology and for roguing of diseased plants in an attempt to control the disease
Factors influencing sytmptoms
• Virus strain
• Host plant cultivar/variety
• Time of infection and
• The environment
Often confusion with virus symptoms
• Unfavorable weather conditions
• Soil mineral/nutrient imbalances
• Infection by nonviral pathogens
• Damage caused by
insect/mite/nematode pests
• Air pollution
• Pesticides (particularly herbicides).
17. 17
Necrotic rings on leaf
stem necrosis
concentric rings and patchy color
on fruit of tomato.
18. 18
necrosis on developing fruit.
chlorosis and dieback of shoot bud and shoot necrosis chlorotic ring on fruit
19. 19
necrosis on tomato leaves
uneven ripening of tomato fruits
Chlorosis on chilli leaves
Chlorosis on fruits of chili.
Capsicum chlorosis virus on tomato and chili
21. 21
Transmission tests
• It is still used in many laboratories as an important
assay in virus diagnosis and maintaining virus
cultures.
• Indicator hosts and host range study can also be
performed by this transmission study.
• For tospviruses thrips acts as vector
vector transmission
Mechanical transmission
Graft transmission
22. 22
Table2 : Experimental host range studies of tomato Tospovirus isolates
Umamaheshwaran et al. (2003), Indian Phytopathology
23. 23
Test plants Local symptoms Systemic symptoms
WBNV GBNV WBNV GBNV
Arachis hypogea nosy cll, nll nosy ly, bn
Citrullus lanatus cv. Sugar Baby ly. m nosy bn nosy
Solanum lycopersicum nosy ly nosy ly, cs, bn
Nicotiana benthamiana cs, m cs, m m, w m, w, sn
Vigna unguiculata
cv. Pusa Komal
cs cll, nll nosy vn, sn
Vigna unguiculata
cv. C-152
yp cs, ns nosy vn, sn
Holkar et al.(2016), J. Plant Biochem. Biotech.
Note: bn-bud necrosis, cll-chlorotic local lessons, cs-chlorotic spots, ly-leaf yellowing, m-mottling, ns-necrotic spots,
nll-necrotic local lesions, nosy-no symptoms, sn-stem necrosis, vn- veinal necrosis, w-wilting, yp-yellow patch
Symptoms on cowpea
25. 25
Physical Properties of WBNV in Sap
DEP: Cow pea induce numerous local lesions (>100lesions/leaf) at 1:1 dilution
of sap, at 1:100 dilution no lesion was observed on the inoculated leaves.
LIV: N. benthamiana leaves stored in 4-6°C were infective up to 24 days of
storage in contrary, the infected leaves stored at -80°C remain infective upto 120
days.
TIP: The WBNV lost its infectivity, when sap inoculum is exposed to ≥40°C
Holkar et al.(2016), J. Plant Biochem. Biotech.
26. 26
Electron microscopy (EM) provides very useful information on
the morphology of the virus particles
It is commonly used for virus detection
The efficiency of virus visualization can be improved in
combination with serology
ISEM combines the specificity of serological assays with the
visualization capabilities of the EM
Virus particles are selectively “trapped” on antibody-coated
grids with little contaminating host-plant material. Hence, the
technique is more sensitive for detecting viruses than the leaf
dip method.
29. 29
Enzyme-linked immunosorbent assay
It was introduced to plant virology by Clark and Adams (1977).
Due to its adaptability, sensitivity, and economy in use of reagents,
ELISA is used in a wide range of situations
Many variations of ELISA have been developed and fall into two broad
categories: “direct” and “indirect” ELISA procedures.
30. 30
Indirect ELISA using monoclonal antibody MAb-WNSs to react with the NSs proteins of different
tospoviruses.
Positive reactions : WSMoV, CaCV, CCSV, MYSV, IYSV and TYRV.
No signals were recorded in the samples of N. benthamiana inoculated with TSWV and PCFV.
A healthy plant of N. benthamiana (H) was used as a negative control.
Chen et al.(2011), Acta Hort. 901
31. 31
• Immunoblotting techniques use antibodies (or other specific ligands in
related techniques) to identify target proteins among a number of
unrelated protein species. They involve identification of protein target via
antigen-antibody (or protein-ligand) specific reactions.
• Proteins are typically separated by electrophoresis and transferred onto
membranes (usually nitrocellulose).
• The membrane is overlaid with a primary antibody for a specific target and
then with a secondary antibody labeled, for example, with enzymes or with
radioisotopes.
• When the ligand is not an antibody, the reaction can be visualized using a
ligand that is directly labeled.
Dot blot is a simplified procedure in which protein samples
are not separated by electrophoresis but are spotted directly
onto membrane.
32. 32
(i) The target nucleic acid is spotted and immobilized onto
nitrocellulose
(ii) The free binding sites on the membrane are blocked with a
nonhomologous DNA (usually salmon sperm or calf-
thymus DNA) or protein (usually bovine serum albumin or
nonfat dried milk),
(iii) Hybridization is allowed to take place between the bound
viral nucleic acid and the probe (which is free in the
hybridization solution)
(iv) The nonhybridized probe is removed from the membrane
by a series of washing steps at defined stringency, and
(v) The target sequences are assayed by detecting the
reporter molecule in the hybridized probe.
Hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA.
33. 33
Technique used in molecular biology to amplify a
single copy or a few copies of a piece of DNA
across several orders of magnitude
It is an easy and cheap tool to amplify a focused
segment of DNA, useful for such purposes as
the diagnosis and monitoring of genetic diseases
RT- PCR generally used for Tospoviruses Thermocyclar
35. 35
In 2009, TSWV, the type species of Tospovirus, was found on sweet pepper in central Taiwan (Zheng
et al., 2010).
A new disorder showing symptoms of mottle and deformation on leaves and fruits was observed on
net house-cultured sweet peppers
In this study, the causal agent was identified and characterized.
36. TP-PCSV
C- CaCV
W-WSMoV and
H-healthy control.
Electron micrographs of spherical enveloped viral
particles
Serological relationships of Pepper chlorotic spot virus
37. 37
Reverse transcription-polymerase chain reaction (RT-PCR)
(A) RT-PCR using degenerate primers of potyviruses,
tobamoviruses and tospoviruses, and specific
primers of Cucumber mosaic virus (CMV).
(B) Sequencing strategy for PCSV S RNA
(C) Evaluation of primer specificity and
(D) effectiveness in field detection .
Label of lanes: TP-PCSV-TwPep3;
P- Potato virus Y;
PM- Pepper mild mottle virus;
C- CaCV;
CM- Cucumber mosaic virus;
W- WSMoV;
H- healthy control;
The specific primers ( FJJ2011-198,
FJJ2011-199, FJJ2011-201 and FJJ2011-202)
designed from the identified sequences were used
for amplification of the intergenic region (IGR) and
the 5 and 3 terminal regions.
39. 39
The virus disease triangle
Tospovirus
Thrips Host
• Thrips control strategies may be a better
option to disrupt the disease triangle
• Thrips not a vector but a mobile host
42. 42
• The number of viruliferous thrips in a thrips population is one of the most important factors in
the spread of tospovirus disease(Yudin et al., 1990).
• To identify viruliferous thrips, both serological and molecular methods have been commonly
used.
• reliable protocol for the identification of tospovirus in thrips and thrips species is required for
epidemiological study and disease management
46. 46
Sensitivity of the dot blot and RT-PCR methods for detection of tospoviruses.
Extracts of Tomato necrotic ringspot virus (TNRV)-infected Thrips palmi (Karny)were serially
diluted in non-viluriferous thrips extract and detected by dot blot analysis (a1–a3) and RT-PCR
(b1–b3)
47. 47
Detection of viruliferous and viruliferous/transmitter thrips by dot blot analysis, RT-PCR and
leaf-disk assay.
48. 48
Identification of tospoviruses and thrips species in individual thrips collected from pepper,
tomato, watermelon and cucumber production fields in thailand.
49. 49
Case study conclusion
• This protocol provides immunological and molecular based assey for detection and accuracy in
identification of tospovirus and thrips species
• The non viruliferour thrips due to many factors like stages of aquired thrips, virus replication and
translocation, amount of virus that reaches the salivary glands.
• Viral genetic factors like that determines the success of transmission: specific mutation in the
Gn/Gc protein, mutation in NSs coding region,
• Thrips palmi was the most abundant thrips sp. found in pepper and cucurbit field. C. claratis in
tomato fields.
• This study help in accurate identification of all four tospoviruses(CaCV, MYSV, TNRV and WSoMV)
and four thrips species(C. Claratus, F. intonsa, S. dorsalis, T. Palmi)
• Viruliferous, non viruliferous thrips and thrips sp. could help in understanding the population
dynamics of thrips and tospovirus sp. Which are important factors in epidemiology and spread of
tospovirus.
50. 50
Charoenvilaisiri et al.(2013), Journal of Virological Methods
Comparison of tospovirus sp. identification by conventional RT-PCR and Multiplex RT-PCR
51. 51
Strategies aimed at preventing virus infection by:
(i) Eradicating the source of infection to prevent the virus from reaching the crop,
(ii) Minimizing the spread of the disease by controlling its vector,
(iii) Utilizing virus-free planting material and
(iv) Incorporating host-plant resistance to the virus.
52. 52
Disease management approaches
1. Conventional approaches
2. Biotechnological approaches
• Indexing and certification programme
• Disease free planting material
• Oil spray
• Cross protectioon
• Barrier crops
• Reflective mulches
• Natural resistance and Breeding
• Alteration of date of sowing
• Use of Antiviral Principals
• Vector management
• Use of reflective mulches
Protien based:
Pathogen Derived Rsistance(
• CP mediated
• Replicase mediated
• Movement protien mediated
Nuclic Acid base:
• Antisense RNA technology
• Gene silencing technology
Matthews Plant Virology by Roger Hull
58. 58
• Host plant resistancs is the most sustainable solution to reduce the virus diseases
• Few promising genes impact resistance are SW-5 and SW-7 were used in commercial Cv.
62. 62
(a) The pRAP vector-supplied scFv cDNAs from a phage display library with the coding sequences
of the murine signal sequence (k) (N-terminal) and a C-terminal cMyc tag (for detection) and
KDEL (for cytoplasmic stability).
(b) Using the NcoI and NotI sites for cDNA cloning, cMyc and KDEL were retained.
(c) The k signal sequence, however, was removed. The pRAP vector also supplied the scFv with
35S promoter sequences and a nos terminator.
(d) Final cloning between left (L) and right (R) borders of pBINPLUS was performed using AscI and
PacI sites and yielded the plant transformation vector pBINRAP-N that was subsequently used
for Agrobacterium-mediated plant transformation.
Cloning scheme of the previously isolated scFv fragment genes targeting the TSWV N gene
into the pBINRAP vector system
63. 63
Resistance against the TSWV in transgenic N. benthamiana plant lines expressing the anti TSWV scFv
64. Efforts must continue to refine existing management approaches and develop
more cost effective, durable and environmentally friendly control tactics.
To know natural host ranges of some tospovirus
Excessive and continuous use of low cost pyrethroid, carbamates, OP resulted in
build up of insecticide resistance vectors. Major challenge is to reduce
insecticide and increase cost effective.
Efforts should be need to identify new sources of tospovirus resistance and use of
HPR and PDR- not accepted by people.
Application od IDM (fragmented land, lack of co-ordinated efforts to address
risk factors involved )IDM practice become ineffective when virus outbreaks.
Use of sanitary and cultural measures may not be cost effective.
Challenges
65. 65
Tospovirus causes major losses in Economically important crops
No direct Management available
Detection method include Biological, Physical and Biochemical, serology and molecular
approach
Recent methods include ISEM, ELISA and PCR based methods fro detection.
Conventional methods include development of transgenic that involves use of Healthy
planting material, Barrier crops, mulches, resistant varieties
Recent methods include transgenic derived from pathogen
