MANAGING CUCUMBER MOSAIC VIRUS IN CUCUMBER.ppt

This presentation provides a comprehensive overview of Cucumber Mosaic Virus (CMV) and its management strategies in cucumber. It covers the genome organization, symptomatology, and subgroups of CMV, as well as its transmission by aphids and other vectors. The presentation discusses cultural, chemical, and biotechnological approaches for managing CMV, along with breeding strategies for resistance and the inheritance pattern of CMV resistance genes. This resource is useful for students, researchers, and professionals in plant pathology, horticulture, and breeding who are interested in disease management and crop improvement.

1. COLLEGE OF HORTICULTURE, BENGALURU
Name of Student : Deepika
ID.NO: UHS23PGD474
Degree programme: II Ph.D. (Hort.)
Department: Vegetable Science
Seminar-I
APPROACHES FOR MANAGING CUCUMBER MOSAIC
VIRUS IN CUCUMBER
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Major Advisor : Dr. Meenakshi Sood
Associate Professor
Department: Vegetable Science
College of Horticulture, Bengaluru

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3. 15/09/25 Dept of VSC 3
Cucumber Production
Fusarium wilt
Downey mildew
Powdery mildew
Anthracnose
Whitefly
Leaf minor
Mites
Aphids
Cucumber more than 20 viruses (Caciagli, 2008)
and
60-100 % loss (Akbar et al., 2015)
(Akbar et al., 2015)

4. Rank Virus
1 Tobacco mosaic virus
 (TMV)
2 Tomato spotted wilt virus
 (TSWV)
3 Tomato yellow leaf curl virus
 (TYLCV)
4 Cucumber mosaic virus
 (CMV)
5 Potato virus Y
 (PVY)
6 Cauliflower mosaic virus
 (CaMV)
7 African cassava mosaic virus
 (ACMV)
8 Plum pox virus
 (PPV)
9 Brome mosaic virus
 (BMV)
10 Potato virus X
 (PVX)
Cucumber mosaic virus
 
Beth et al., 2011
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5. Bottle gourd
PumpkinsZucchini and squash
Snake gourd

Cucumber
Widespread nature
High infectivity
Primary
transmission
by aphids
Disease
management
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6. FLOW
OF
SEMINAR

7. •Botanical Name :
   
Cucumis sativus L.
• Family :
   
Cucurbitaceae
• Chromosome No :
   
2n = 14
• Origin : India (De Candolle,1886)
• Progenitor : Cucumis sativus var. hardwickii

Introduction
Fig. 1. History of the spread of cucumbers around the world from its center of diversity in India
Weng, 2021
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8. CUCUMBER NUTRITIONAL COMPOSITION HEALTH BENEFITS
1.Hydration and weight
management
2.Promotes digestion
3.Improve skin health
4.Improve bone health
5.Improve heart health
6.Helps in diabetes management
7.Helps in eyes’ well-being
8.Anti-inflammatory properties
9.Oral health and alleviates bad
breath
 Bhuyan et al., 2024
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9.  Food and Agriculture Statistics, 2022
Area and Production
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China with 7,27,79,781 tonnes
2 largest exporter, Utter
Pradesh and Karnataka (5 )

10. Cucumber
Cucumber mosaic virus
Papaya ring spot virus
Tomato leaf curl New Delhi virus
Melon necrotic spot virus
Tobacco streak virus
Zucchini yellow mosaic virus
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Akbar et al., 2015

11. Virus Family Genus Vector Research Findings
Cucumber mosaic virus BromoviridaeCucumovirus Aphid
Yin et al., 2024
Zucchini yellow mosaic virusPotyviridaePotyvirus Aphid
Melon necrotic spot virusTombusviridaeGamma carmovirusChytrid fungus
Cucumber leaf spot virus TombusviridaeAureusvirus Fungus Olpidium radicale
Cucumber vein yellowing virusPotyviridaeIpomovirus Whitefly
Tomato leaf curl New Delhi
virus
GeminiviridaeBegomovirus Whitefly
Cucumber Green Mottled
Mosaic Virus
VirgaviridaeTobamovirus Infected seeds Rajapaksha et al.,
2019
Tobacco Ring Spot Virus SecoviridaeNepovirus Nematodes (Xiphinema
americanum)
Rajapaksha et al.,
2019
Papaya Ring Spot Virus PotyviridaePotyvirus Aphid Rajapaksha et al.,
2019
Table :1 Major viruses infecting cucumber and their vector
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12. Virus Family Genus Vector Research Findings
Zucchini tigre mosaic virus Potyviridae PotyvirusAphids Kumari et al., 2021
Groundnut bud necrosis BunyaviridaeTospovirusThrips Venkataravanappa et al.,
2021
Cucurbit aphid-borne yellows virus LuteoviridaePolerovirusAphids Ivanova et al., 2022
Cucurbit chlorotic yellows virusClosteroviridaeCrinivirusWhite fly Orfanidou et al., 2014
Lettuce infectious yellows virusClosteroviridaeCrinivirusWhite fly Abrahamian and Jawdah
2014
Cucurbit yellow stunting disorder virusPotyviridae IpomovirusWhitefly Abrahamian and Jawdah
2014
Cucumber leaf spot virus TombusviridaeAureusvirusZoospores of the
fungus, Olpidium
bornovanus
Segundo et al., 2001,
Pospieszny et al., 2004
Tobacco streak virus BromoviridaeIlarvirusThrips tabaciKrishnareddy et al., 2001
Cntd…..
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13. Cucumovirus
CMV virions are icosahedral particles 29nm in diameter, composed of 180
subunits of a single capsid protein and 18 per cent RNA (Bujarski et al., 2019)
CMV's genome comprises
three positive-sense single-
stranded RNAs (Sinha et al.,
2021)
Family : Bromoviridae
Genus :Cucumovirus
Transmitted by Aphids
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Sinha et al., 2021

14. Fig. 2. Genome organization of Cucumber mosaic virus
Codes for single protein, known as 1a helps in
the replication of the viral genome
Encodes 2a protein that has characteristic RNA-
dependent RNA polymerase motifs that helps in
viral replication
2b protein counters the host’s post-transcriptional
gene silencing mechanism and plays a role in long-
distance movement of the virus
Two proteins movement protein (MP) and essentially
required for cell to cell movement of the virus
Coat protein (CP) helps the encapsidation of the
genomic RNAs into virus particles, important in
aphid transmission and affecting expression of
symptoms
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Sinha et al., 2021

15. CMV subgroups
Based on nucleotide sequence data
Subgroups II Subgroups I
Based on the 5′ non-coding
sequence of RNA 3
Subgroup IA Subgroup IB
RNA5 is a mixture of the terminal
307 and 304 nucleotides regions of
RNAs 2 and 3.
Satellite RNA
CMV replication
Inducing necrosis
Chlorosis
Bright yellow mosaic.
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Sinha et al., 2021

16. CMV’s host range
1. Originally detected in 1916 in cucurbits
Tennant et al., 2018
3. CMV has been found infecting more than 1200 plant species belonging to at least 100 different
families
2. It was first reported in cucumber in 1934 (Price, 1934)
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4. Medicinal plants like African basil, betel vine, geranium , Korean mint, lemon grass, holy basil,

Indian long pepper, and vanilla (Sinha et al., 2021)5. Ornamental plants like gomphrena, petunia, lilium and narcissus ( Navlinskiene and
Samuitiene, 2021)
 

Vinodhini et al., 2020

17. Weeds
Vector for disease transmission
Non persistent manner of virus transformation
Vectors
Seeds
Aphids
Reported in common bean, Vigna unguiculata and V. radiata
(Babovic et al., 1997 and Abdullahi et al., 2001) pea and faba
bean (Lantham et al., 2001); chickpea, tomato (Park et al.,
2001); alfalfa (Jones et al., 2004)
Dodder
Li et al., 2020
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18. Symptoms of cucumber mosaic
virus in cucumber
Qiu et al., 2017
Fig. 3. Model for Fd I functions during viral infections
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A number of other symptoms are reported such
1.Mottling of leaves or flowers
2.Stunting or yellowing of entire plants
3.Flecking
4. Dwarfing and fern leaf (Pratap et al., 2008).
Some of the intermediate symptoms include
Blight
Shoestring
Ringspot
Fruit woodiness and necrosis of fruits.

19. Symptom Observation
Indicator Plants
2. Serological Methods
Enzyme-Linked Immunosorbent Assay (ELISA)
DAS-ELISA (Double Antibody Sandwich ELISA)
TAS-ELISA (Triple Antibody Sandwich ELISA)
Lateral Flow Assay (LFA)
3. Molecular Methods
Polymerase Chain Reaction (PCR)
Reverse Transcription PCR (RT-PCR)
Real-Time PCR (qRT-PCR)
Loop-Mediated Isothermal Amplification (LAMP)
4. Nucleic Acid Hybridization
Northern Blotting
Dot-Blot Hybridization
1. Biological Method
Detection Methods of Cucumber Mosaic Virus (CMV)
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20. Sinha et al., 2021
Fig. 4. Worldwide distribution of cucumber mosaic virus
1.Isolates of subgroup II are
frequently reported in temperate
regions
2. Whereas those of subgroup I
are found in East Africa, the
Mediterranean, California,
Brazil and Australia.
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21. Table 2. Disease incidence of cucumber mosaic virus in South India
Sl.NoVirus Area Disease Incidence(%) Findings
1.
Cucumber Mosaic Virus
Bagalkot (Karnataka) 13.33-66.66 Tejashwin et al., 2019
Bagalkot (Karnataka) 21.31
Revadi and Pati , 2016
2.
 
 
 
Belagavi (Karnataka) 22.79
Haveri (Karnataka) 23.34
Dharwad (Karnataka) 23.94
3. Cucumber Mosaic Virus in gherkinKolar (Karnataka) 52.49
Venkatesh et al., 2016
  Bengaluru rural (Karnataka) 21.64
4. Cucumber Mosaic Virus in gherkin
Bangalore urban Bengalore north 23.34
Kavyashri et al., 2016
Bangalore rural Doddaballapur 17.15
Chikkaballapura (Karnataka) 31.63
Kolar rural (Karnataka) 51.27
Tumkur Sira (Karnataka) 35.62
Chittoor (Andhra Pradesh) 40.27
5. Cucumber mosaic virus
Anantha Nambi Kuruchchi
(Tamilnadu)
67.50
Santhoshinii et al.,
2021
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Virus disease management strategies
Risk-
reducing
measures
Genetic
resistance
RNA-
based
resistance
Cross
protection
Exclusion
Quarantine
Virus-free seed
Avoidance
Eliminate alternate hosts
Eliminate green-bridge
Sowing date
Eradicate
Identify infected plants
Rouging infected plants
Vector management
Application of pesticide
Biological control of vectors
Barrier to minimize vector movement
siRNA-based resistance
Spray induced gene silencing
CRISPR-based virus resistance
Tatineni and Hein, 2022

23. Fig.5. Integrated Aphids Management
Chemicals used for controlling
the aphids
1.Imidacloprid 0.08%,
2.Thiamethoxam 0.01%, and
3.Acetamiprid 0.01%.
4. Acephate 75 SP at 350g a.i./ha
5.Imidacloprid 17.8 SL @ 0.2 g/L
(Landge et al., 2024)
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24. Fig.6. Cases of insecticide resistance in aphids (top 10 species) documented in the Arthropod Pesticide Resistance
Database
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24
(Bass and Nauen , 2023)
Myzus persicae
Aphis gossypii
Fig. 7. Expressed resistance mechanisms for pesticides in Myzus persicae
Esterase –
Resistance for
organophosphates ,
pyrethroids
Mutation GABA gated
chlorine channel–
Resistance for
pyrethroids
Modification in
cuticle - Reduce
the penetration of
chemical

25. Wide range of host
Non - persistence
transformation
Chemical control
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26. Inheritance and QTL mapping of cucumber mosaic virus resistance in cucumber
(Cucumis sativus L.)
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science, China
Lixue Shi, Yuhong Yang, Qing Xie, Han Miao, Kailiang Bo, Zichao Song, Ye
Wang,Bingyan Xie, Shengping Zhang and Xingfang Gu
Objective: To know the inheritance and identify QTLs
linked to CMV resistance
Case study : 1
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27. 1. Plant materials and population development
Materials and methods
‘02245’ (resistant)
‘65G’ (susceptible)
F1
SSD
Recombinant inbred line
2. Inoculations and virus detection by DAS-ELISA
1g Infected tobacco
plants
Grinding
10 mL of 0.03 M phosphate
buffer (pH 7.0)
Inoculation
Carborundum
powder
true leaf of cucumber
seedlings
Repeated after

2–3 day
0 = no symptoms
1 = apical leaves with dispersed vein clearing
or slight mottling
3 = mosaic patches and/or necrotic spots on

leaves
5 = moderate mosaic patches and moderate
distortion of the three youngest leaves
7 = apical meristem exhibiting mosaic
9 = extensive leaf mosaic patches and severe
distortion of all leaves even plant death
3. 0–9 infection ranking system
4. Disease indices (DI) for the RIL population
Using by the formula:
DI = ∑[(s×n)/(S×N)]×100%
(s: disease grade, n: number of plants in the disease grade, S:
highest disease rating scale, N: total numbers of plants)
5. Linkage map construction
1,228 SSR primers
JoinMap1 4.0
6. Inheritance analysis and QTL mapping
Statistical calculations by Microsoft1 Excel 2007.
QTL analysis was performed by the interval mapping (IM) and
multiple-QTL model (MQM) methods with MapQTL1 4.0
Shi et al., 2018
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28. EnvironmentParental Lines (Mean+S.E)F1
(Mean+SE)
RIL Population
P1 ('65G')P2 ('02245') Mean+SE.SD SkewnessKurtosis
June 2016 50.43+0.6915.75+0.6335.80+0.3537.33+1.1013.090.11 -0.19
June 2017 48.14+0.2511.11+0.0034.72+0.1230.76+0.819.67 -0.51 -0.18
Table 3. The Disease indices of the parental lines, the F1 and RIL populations.
Fig. 8. Symptoms of the susceptible
parental line ‘65G’, the resistant line
‘02245’ and their F1 hybrid progeny
after they were inoculated with
CMV
Result and Discussion
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Shi et al., 2018

29. Fig. 10. DAS-ELISA results for CMV in the leaves of P1
(‘65G’), P2 (‘02245’) and F1 plants and RIL population
Fig. 9. Frequency distribution of the DI among the
‘65G’ב02245’ RIL population. The frequency distribution in
June 2016 and 2017 each presented a normal distribution
ranging from resistant to susceptible phenotypes
Quantitative inheritance
15/09/25 Dept of VSC 29Shi et al., 2018

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Shi et al., 2018
EnvironmentPopulationQTL ChromosomeMarker IntervalLOD R
2
(%)
June 2016 RIL cmv6.1 Chr.6 SSR9-56-SSR11-17711.58 31.7
June 2017 RIL cmv6.1 Chr.6 SSR9-56-SSR11-17710.09 28.2
Table 4. QTL controlling CMV resistance and its effect on cucumber seedlings
Fig 11. QTL controlling CMV resistance and its effect on cucumber seedlings during two years. One QTL was detected
at
the same location in June 2016 and 2017; this QTL was identified on chromosome 6

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Inferences
1.CMV resistance in cucumber control by many gene
2.cmv6.1, is identified as a major QTL
3.The markers SSS95-6 and SSS11-177 are tightly linker with the QTL

32. 1.Among cucurbits, resistance to CMV is believed to be mostly recessive
2.Wasuwat and Walker (1961) reported that resistance in the cultivar SMR 12 is controlled by a
single dominant gene
3.Kooistra (1969) suggested that resistance was due to three dominant genes
4.Wang et al. (2006), Huang (2007) and Munshi et al. (2008) reported that resistance is
controlled by a single recessive gene
Inheritance of CMV disease resistance in cucumber
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QTL of CMV disease resistance in cucumber
Wang et al. (2006) identified seventeen QTLs that control resistance in ‘F-3’ lines; these QTLs were
named cmv1 through 17. cmv17 was determined to be a primary locus that controls resistance to
CMV, and this locus explained 67.3% of the variance.

33. Genetics of resistance to Cucumber mosaic virus in Cucumis sativus var. hardwickii R.
Alef ( Munshi et al., 2008)
Screened 31 accessions
1.IC-202049 : 19.25 %
2.IC-277000 : 24.27 %
3.IC-277048 : 6.33 %
4.IC-331444 : 14.90 %
5.IC-331619 : 16.27 %
6.IC-331628 : 7 %
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33

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35. A new strategy to control Cucumber mosaic virus using fabricated NiO-nanostructures
Aly Soliman Hamed Derbalah and Mohsen Mohamed Sharkawy
Objective: To study the direct antiviral activity of NiO-nanostructures against CMV under greenhouse

conditions
To study the effect of applied NiO-nanostructures to certain growth parameters of cucumber
Case study : 2
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Kafrelsheikh University, Kafr Elsheikh, Egypt

36. Derbalah and Elsharkawy, 2019
Materials and methods
1. Fabrication of NiO nanostructures
One-pot hydrothermal synthetic approach
(Ni(NO
3
)
2
.6H
2
O) (1 mM)
50 mL of Milli-Q water
Dropwise addition
1 mM (NH
4 )
2 HPO
4
Autoclave 150 °C for 10 h
Green precipitate
water and ethanol
wash
90 °C and
annealed at 450
°C for 5 h.
Foliar and drenching: 150 μg/L before
24 h of CMV inoculation on Beit Alpha
2. Cucumber mosaic virus inoculation
Scale:
0 = no symptoms
2 = mild malformation and mosaic of two leaves
4 = leaf malformation and mosaic of four leaves
6 = leaf malformation and mosaic in all leaves
8 = all leaves showing mosaic and dwarfing;
10 = severe dwarfing and malformation
3. RT-PCR analysis for gene expression study
Gene Forward primer Reverse primer
pod TCAGGATGGGAAATCTCGAC CGTGGCCAACTCATACACAC
pal1 ATGGAGGCAACTTCCAAGGA CCATGGCAATCTCAGCACCT
pr1 TGCTCAACAA ATGCGAACC TCATCCACCCACAACTGAAC
Table 5. Forward and reverse primers sequence for pod, pal1 and pr1 genes
4. Evaluation of plant growth characters
Data analysis
Analysis of variance (ANOVA) using XLSTAT statistical
analysis software (Addinsoft)
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37. Results and Discussion
Fig. 12. The FE-SEM of constructed NONS B) FE-SEM captures the distribution of NM with the homogeneous

formation of head and trunk Derbalah and Elsharkawy, 2019
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A
Control Foliar spray Soil drench
Control Foliar spray Soil drench
Fig. 13. Disease severity (A) and Cucumber mosaic virus accumulation (B) in cucumber plants treated with NONS
(150 μg/L ) relative to non-treated control plants at 1 and 2 weeks post inoculation.
15/09/25 Dept of VSC 38Derbalah and Elsharkawy, 2019

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PR1
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Control Foliar spray Soil drench
Control Foliar spray Soil drench Control Foliar spray Soil drench
Fig. 14. Expression of defence related genes in leaves cucumber plants treated with NONS after two and four
days of inoculation with CMV. Statistical analysis was done among each time point (2 and 4 days).
Derbalah and Elsharkawy, 2019
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PA
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40. The effect of mixing NONS with the infectious sap of CMV in cucumber plants
Equal volume of CMV sap and NONS (150 μg/L) kept it for 10 min
4 weeks old cucumber plants
Disease severity and virus titer compared with untreated plants
Treatments Disease severity CMV concentration
NONS 1.80 + 0.02b 0.29 + 0.01 b
Control 9.0 + 0.32 1.4 + 0.11
Table .7 Direct antiviral activity of NONS (150 μg/L)against CMV on cucumber plants.
*Different letters indicate significant differences by Fisher’s LSD at P ≤ 0.05.
Derbalah and Elsharkawy, 2019
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41. Table 8. Effect of NONS on some growth characters of cucumber plant
Treatment Fresh weight (g) Dry weight (g) Number leaves
Foliar spray 23.30 ± 0.88a 1.80 ± 0.14 a 7.80+ 0.32 a
Soil drench 22.70 ± 0.46a 1.70 ± 0.18 a 7.50 ± 0.46a
Control 18.30 ± 1.11b 0.80 ± 0.01 b 5.80 ± 0.68 b
Different letters indicate significant differences by Fisher’s LSD at P ≤ 0.05
Derbalah and Elsharkawy, 2019
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42. 15/09/25 Dept of VSC 42
1.The application of NONS increased the expression of pod,
pr1 and pal1 genes and enhanced the growth characters of
cucumber compared to untreated plants.
2.Finally, NONS application could be used as an alternative
control strategy of CMV infection in cucumber plants.
Inferences

43. Glycine betaine (GB)
Chitosan (CHT)
Environmentally sustainableCost-effective
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44. Improving Regulation of Enzymatic and Non-Enzymatic Antioxidants and Stress-
Related Gene Stimulation in Cucumber mosaic cucumovirus-Infected Cucumber
Plants Treated with Glycine Betaine, Chitosan and Combination
Ahmed R. Sofy , Rehab A. Dawoud, Mahmoud R. Sofy, Heba I. Mohamed,
Ahmed A. Hmed and Noha K. El-Dougdoug
Objective : To study the efficacy of GB and CHT either combination or in alone in the
protection of cucumber plants against CMV infection
Case study : 3
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Al-Azhar University, Nasr City, Cairo, Egypt

45. CHT (1 g) Water (40 ml) + 1 M acetic acid (9 ml)Volume make up 100 ml with pH 5.2
Materials and Methods
1. Plant Materials
Sofy et al., 2020
2. Preparation of glycine betaine and chitosan solutions
Seeds of cucumber plants (Cucumis sativus L. cv. Beit–alpha)
The glycine betaine (GB) solutions were prepared using distilled water and GB granular powder
3. CMV inoculation at cotyledon stage
∑(Disease grade × Number of plants in each grade)

(Total number of plants × Highest disease grade) × 100
DS (%) =
Scale:
0 = No symptoms
2 = Vein clearing
4 = Mild mosaic
6 = Severe mosaic
8 = Severe mosaic and blisters
10 = Severe mosaic, blisters and malformation
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46. 4. Experimental Design
Eight groups
Three replicates in which the replicate consisted of 10 plants per group
1 group: Sprayed with water considered as a control without any treatment (absolute control)
2 group: Spraying the leaves (about 10 mL/ plant) with GB (50 mM),
3 group: Spraying the leaves (about 10 mL/ plant) with CHT (1%)
4 group: Spraying the leaves (about 10 mL /plant) with GB+CHT (1:1 v/v),
5 group: CMV and group 1
6 group: CMV and group 2
7 group: CMV and group 3
8 group: CMV and group 4
Sofy et al., 2020
Cntd…..
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47. 6. Measurements
Growth Indices
Biochemical Measurements
Enzymes Activity
7. Statistical Analysis
SPSS (Social Science version 26.00)
Shoot length, fresh and dry weight of shoot and leaf area
Photosynthetic Pigments: Chlorophyll a, b, Carotenoids and Melanaldehyde

Phytohormones: Indole acetic acid, Gibberellic acid and Salicylic acid

SOD, POX, PPO, CAT, LOX, APX and chitinase
Sofy et al., 2020
Cntd…..
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48. Results and discussion
Fig. 15. Effect of foliar spray by glycine
betaine (GB), chitosan (CHT) and
combination (Co) on plant growth of
cucumber plant under absolute control
(AC) and CMV infection (challenge
control, ChC) after 21 days of
inoculation. Means with the different
letters (a, b, c, d, e, f, g) are significantly
different at the 0.05 level among
treatments according to Tukey’s test
Sofy et al., 2020
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48
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Healthy Infected Healthy Infected
Healthy Infected Healthy Infected

49. Healthy Infected Healthy Infected
Healthy Infected Healthy Infected
Fig. 16. Effect of foliar spray by glycine
betaine (GB), chitosan (CHT) and
combination (Co) on photosynthetic
pigments (A–C) and malondialdehyde
(MDA) (D) content in leaves of
cucumber plant under absolute control
(AC) and CMV infection (challenge
control, ChC) after 21 days of
inoculation.
Means with the different letters (a, b, c,
d, e, f, g) are significantly different at
the 0.05 level among treatments
according to Tukey’s test.
Sofy et al., 2020
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d
e
h
y
d
e

50. S
a
l
i
c
y
l
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c

a
c
i
d




I
n
d
o
l
e

a
c
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c

a
c
i
d


G
i
b
b
e
r
e
l
l
i
c

a
c
i
d
Healthy Infected Healthy Infected
Healthy Infected
Fig. 17. Effect of foliar spray by glycine
betaine (GB), chitosan (CHT) and
combination (Co) on phytohormones
content in leaves of cucumber plant
under absolute control (AC) and CMV
infection (challenge control, ChC) after
21 days of inoculation. Means with the
different letters (a, b, c, d, e, f, g) are
significantly different at the 0.05 level
among treatments according to Tukey’s
test
Ahmed et al., 2020
15/09/25 Dept of VSC 50

51. Fig. 18. Effect of foliar spray by glycine betaine (GB), chitosan (CHT) and combination (Co) on enzymatic antioxidants
content in leaves of cucumber plant under absolute control (AC) and CMV infection (challenge control, ChC) after 21
days of inoculation. Means with the different letters (a, b, c,d, e, f, g) are significantly different at the 0.05 level among
treatments according to Tukey’s test.
Sofy et al., 2020
15/09/25 Dept of VSC 51




S
u
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e

d
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m
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o
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e


P
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x
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a
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e
Healthy Infected
Healthy Infected Healthy Infected

52. C
a
t
a
l
a
s
e
L
i
p
o
x
y
g
e
n
e
a
s
e

A
s
c
o
r
b
a
t
e

p
e
r
o
x
i
d
a
s
e
Fig. 19. Effect of foliar spray by glycine betaine (GB), chitosan (CHT) and combination (Co) on enzymatic antioxidants
content in leaves of cucumber plant under absolute control (AC) and CMV infection (challenge control, ChC) after 21
days of inoculation. Means with the different letters (a, b, c,d, e, f, g) are significantly different at the 0.05 level among
treatments according to Tukey’s test.
Sofy et al., 202015/09/25 Dept of VSC 52
Healthy Infected
Healthy Infected Healthy Infected

53. I
N
F
E
R
E
N
C
E
Sofy et al., 202015/09/25 Dept of VSC 53
Avoid the harmful effect of CMV
Reduction in the morphological
characters
Reduction in photosynthetic
pigments
Increased endogenous proline
Increased ROS contents
1.Photosynthesis
2.Expression of
stress related gene
such as PR1 and
PAL1
3.Antioxidant
machinery
By modulatingIncreases the content
of SA and JA
GB, CHT and combination
Cucumber mosaic virus
Caused

54. Mechanism of RNAi
1.RDRs are characterized by the presence of a
catalytic DLDGD domain and can
synthesize dsRNA from single- stranded (ss)
RNA templates by primer independent and
primer dependent mechanisms.
2. Plays important roles in antiviral immunity
by producing viral dsRNA
3. Involved in host resistance mechanism
15/09/25 Dept of VSC 54
Targeted silencing of mRNA

55. Cucumber RDR1s and cucumber mosaic virus suppressor protein 2b association directs
host defence in cucumber plants
Reenu Kumari, Surender Kumar, Diana Leibman, Bekele Abebie, Yulia Shnaider,
Shou-Wei Ding and Amit Gal-On
Objective: To study the role of RDR1s in defence activities against CMV infection in cucumber
Case study : 4
15/09/25 Dept of VSC 55

56. Experimental procedures
1. Plant growth conditions and virus inoculations
3. Western blotting
2. Generation of CMV-2b mutants
4. Protoplast isolation and transfection
5. RT-qPCR
Cucumber cultivars Beit Alfa and Shimshon were
maintained under growth conditions 25 with a 14-
℃
hr photoperiod and plants at the cotyledon stage with
an emerging first true leaf were inoculated
mechanically with CMV
First ATG of the coding frame, that is, ATG was
changed to ACG (methionine to threonine)
Kumari et al., 2021
15/09/25 Dept of VSC 56

57. Kumari et al., 2021 15/09/25 Dept of VSC 57
200 μl of protoplasts containing 2 × 10
6
cells was mixed gently with plasmids (30
μl) and 500 μl of PEG (35%) was added to this mixture. The mixture was then
incubated at room temperature for 5 min. Transfected protoplasts were incubated
in the dark at 26 ± 2 for 68 hr.
℃
2b and CP genes were cloned at NcoI/BamHI
sites of pSAT6-EGFP-N1 vector
CsRDR1a/1b/1c1 genes were cloned in the SalI/BamHI,
XhoI/BamHI, or EcoRI/BamHI sites of pSAT6-ECFP-C1
vector respectively
Contrasts for Subcellular localization study in protoplasts

58. Fig. 20. Expression levels of
CsRDR1s (CsRDR1a, CsRDR1b
and CsRDR1c) in cv. Beit Alfa
(BA) at 7 and 14 days post
inoculation (dpi) with CMV or
the CMVΔ2b mutant
R
E
S
U
L
T
S
Kumari et al., 2021
15/09/25 Dept of VSC 58
C
s
R
D
R
1
a
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s
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1
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1
b

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1
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e
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n

59. Fig. 21. Immunodetection of CMV coat protein (CP) in CMV- and CMVΔ2b-inoculated cv. Beit Alfa
and cv. Shimshon (SH) plants at 7 and 14 days post inoculation (dpi) using CP- specific antiserum
Kumari et al., 2021
15/09/25 Dept of VSC 59

60. Fig. 22. Effects of CsRDR1s on the accumulation of CMV and CMV Δ2b in cucumber protoplasts. (a–c) CMV and
CMVΔ2b accumulation in cucumber protoplasts transfected with pSAT6-eCFP constructs of CsRDR1a eCFP, CsRDR1b
eCFP and CsRDR1c eCFP or empty pSAT6-eCFP vector (EV) along with in vitro transcripts of CMV or CMVΔ2b
mutant. CMV and CMVΔ2b levels were determined by reverse transcription (RT) PCR of cucumber protoplasts at 68 hr
after transfection. Virus accumulation was calculated with reference to CMV transfected control samples
Kumari et al., 2021
15/09/25 Dept of VSC 60
R
e
l
a
t
i
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e

a
c
c
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n

o
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61. R
e
l
a
t
i
v
e
e
x
p
r
e
s
s
i
o
n

o
f

C
s
R
D
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1
b

R
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l
a
t
i
v
e

e
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p
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s
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o
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1
a

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e

e
x
p
r
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s
s
i
o
n

o
f

C
s
R
D
R
1
c
Fig. 23. Effects of CsRDR1s on the accumulation of CMV and CMV Δ2b in cucumber protoplasts. Accumulation of
CsRDR1a, CsRDR1b, and CsRDR1c was verified by quantitative RT-PCR
Kumari et al., 2021
15/09/25 Dept of VSC 61

62. Fig. 24. A model describing the
regulation of the cucumber
defence system as an equilibrium
between CMV-2b virulence and
the RNA-silencing activity of
cucumber RDR1s.
Kumari et al., 2021
15/09/25 Dept of VSC 62

63. 15/09/25 Dept of VSC 63
Bio control agent

64. 15/09/25 Dept of VSC 64
Bio control agent

65. 15/09/25 Dept of VSC 65
Nano particle application

66. 15/09/25 Dept of VSC 66
Spray inducing gene silencing RNAi
The vaccinated plants showed significant reduction in
symptoms (51.15%) at 14 days post inoculation (dpi)
and (29.9%) at 21 dpi (Urvashi et al., 2024): IIHR-
Dept of Biotechnology.

67. 15/09/25 Dept of VSC 67
1.Virus vector carrying a gene
2.Intercistronic region between the 3a
and 3b genes of the CMV-HL RNA3
genome
3.Induce RNAi
Virus-induced gene silencing (VIGS)

68. Spray induced gene silencing (SIGS) Virus induced gene silencing (VIGS)
15/09/25 Dept of VSC 68
RNAi
Activation
of RNAi

69. 15/09/25 Dept of VSC 69
9

70. 15/09/25 Dept of VSC 70
Transgenic tomatoes were obtained by Agrobacterium tumefaciens-mediated transformation with
expression vectors with a 5 proteins of cmv.
Fig. 25. Diagrammatic representation of a plant expression vector
Miao et al., 2018

71. 15/09/25 Dept of VSC 71

72. 15/09/25 Dept of VSC 72
Gene editing
1.eIF4E gene family, (eukaryotic translation initiation factor 4E)
2.Susceptible gene
3.9 base editing CRISPR/ cas9
4.Increase the resistance

73. 15/09/25 Dept of VSC 73
C
onclusion
Cultural
practices
Resistant
varieties RNAi
Genome
editing
Vector
control
Integrate Disease Management
An efficient ecosystem approach to crop production and protection

74. 15/09/25 Dept of VSC 74