This document summarizes research on breeding for resistance to bacterial wilt caused by Ralstonia solanacearum in brinjal (eggplant). It discusses: 1. The causal organism R. solanacearum, its characteristics, classification into races and phylotypes. 2. Symptoms of bacterial wilt in brinjal and conditions favoring disease development. 3. Sources of resistance identified in wild relatives like S. torvum and S. sisymbriifolium. 4. Breeding methods used including marker-assisted selection and QTL mapping to identify tightly linked markers and genes controlling resistance.

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2. Ralstonia
solanacearum
Bacterial wilt
Broad host range
& large geographical
distribution
Control measures
Breeding for
resistance
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Broad host range & large geographical distribution

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Control measures

5. University of horticultural sciences , bagalkot
k. R. C. college of horticulture , arabhavi
department of vegetable science
Bacterial wilt resistance breeding in brinjal
Basavaraj S Panjagal
Ph.D in Vegetable Science
Seminar – II
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6. Topic Division
Introduction
Causal organism
Screening for wilt disease Resistance
Sources of Resistance
Mechanism of Resistance
Methods of breeding
Achievements
Conclusion
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7. • Brinjal or eggplant (Solanum melongena L.) belongs to the
family Solanaceae is an important vegetable crop of sub-tropics
and tropics.
• In India, it is one of the most common, popular and principal
vegetable crops grown throughout the country.
(NHB, 2019)
• Ralstonia solanacearum is an important plant pathogen causing
bacterial wilt in brinjal
• Per cent of Yield loss in brinjal is 65- 80 %
Singh et al., 2017
Area
(MHa)
Production
(MT)
Productivity
(t/ha)
India 68.00 13.26 18.68
Karnataka 16.50 0.40 24.67
INTRODUCTION
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8. HISTORY
 Ralstonia solanacearum was reported for the first time in
at the end of 19 century on potato, tobacco, tomato and
ground nut in Asia, South America and Southern USA .
 For first time Bacterium was described as
Bacillus solanacearum by Smith 1896.
Smith, 1896
EPPO, 2014
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9. Causal organism and Phenotypic characteristics
Name : Ralstonia solanacearum (Smith)
Synonyms : Bacterium solanacearum (Smith 1896)
Burkholderia solanacearum (Smith 1914)
Pseudomonas solanacearum (Smith 1896)
Taxonomic position :
Domain : Bacteria
Phylum : Proteobacteria
Class : Betaproteobacteria
Order : Burkholderiales
Family : Burkholderiaceae
Genus : Ralstonia
EPPO, 2014
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10.  Morphology : Single celled, small rod shaped with
rounded ends and single polar flagellum,, gram negative
with 0.5 – 1.5 µm in length.
 Tropical areas : 35°C
 Higher altitudes of tropics and in subtropical areas  27°C
 No growth : >40°C or <4°C
 pH requirements : acid media  favoured growth
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EPPO, 2014
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11. Classification of R. solanacearum
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Five Races
Four Biovars
Four
Phylotypes
EPPO, 2014
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12. Race 1 Race 2 Race 3 Race 4 Race 5
Region Tropical area Tropical area
Of South
America
Tropical,
sub tropical
and
temperate
area
Affected
Crop
tobacco,
tomato, potato,
brinjal and
diploid banana
triploid
banana and
Heliconia
Spp.
tomato and
potato
ginger Morus
species
Cause temperature
optimum
35-37 0C
temperature
optimum
35-37 0C
temperatur
e optimum
27 0C
temperatur
e optimum
35-37 0C
temperature
optimum
35-37 0C
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Phylotypes Geographic origin
Phylotype - I Asia
Phylotype - II America
Phylotype - III Africa
Phylotype - IV Indonesia
EPPO, 2014
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Race 1 :
Different strains were classified into two types:
Type I strains : 90% of strains are pathogenic
 They are irregular, milky and fluidal with a red center
Type II strains : non-/mild pathogenic
 They are smooth and having even colony margins with a red to
orange colored center.
Din , 2016
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14. Disease cycle of Ralstonia solanacearum
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15. Conditions that favour disease development
 Warm temperature and high moisture -
The optimum temperature - 35 ° C and RH- 80%.
• Crop residues
• Injured roots
• Poor and unfertile soil
• Nematodes present in the soil
Rahul , 2015
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16. Symptoms
Leaf drooping
Few days later, sudden and permanent wilt occurs.
Vascular discolouration.
Rotting
A steam of milky white bacterial ooze can noticed when the cut
ends of stem/root is kept in clear container with water.
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17. Wilting of leaves at the end of
plant branch
Wilting of foliage and stunting of plant.
Cross-sections of stems may reveal
brown discoloration of infected tissues
Bacterial streaming in clear water
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18. Bacterial wilt symptoms in egg plant.
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Drench inoculation
Clipping of leaves
Root dip method

20. Ahmed et al., 2019
Hussain et al. (2005),
Disease scoring scale
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21. Sources of resistance
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22. S. torvum S. sisymbriifolium
S. integrifolium
Solanum ferox
Srinivasan 2012
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23. Genetic nature
Monogenic or oligogenic resistance: one or few genes. It
is usually race specific & it is relatively less stable.
Polygenic resistance: It is controlled by many genes & it
is race non-specific. Being quantitative in nature,
expression is affected by genotype, environment & their
interaction.
Cytoplasmic resistance: The genes conferring resistance
are located in the cytoplasm & show maternal resistance.
Sharma, 2009

24. Gene action Population Genotype used Reference
Monogenic
Dominant
F2 DWD1 , DWD2, DWD3 Ajjappalavara et al.
(2008)
F6 RILs MM738 (S) X AG91- 25(R) Lebeau et al., 2013
F2 Malapur x BNDT Kurhade et al. (2017)
Monogenic
Recessive
F2 Solanum melongena var. insanum IC
No. 421463 (R) X Pusa Purple Long
(S)
Uttamrao (2012)
F2 Solanum torvum X Diglipur Local Bainsla et al. (2016)
Polygenic
Recessive
F2 CARI Brinjal 1 X PPL Bainsla et al. (2016)
Complemen
tary gene
action
F2 Manjarigota X BR 14
BR 14 X Pusa Purple Cluster
BB54 Kurhade et al.
(2017)
Digenic
interaction
Double
haploid
MM738(S) X EG 203 (R) Salgon et al. (2018)
Polygenic F2 BR-14 X BND
BR-14 X Kasral
Kurhade et al. (2017)
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25. Steps in breeding for resistance
Development of resistant varieties/hybrids
Selecting for resistance
Inheritance of resistance
Identification of source of resistance
Screening of resistance
Land races
Cultivated variety
Germplasm Line
Mutant
Wild relatives
Jyothi et al. 2012
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26. Breeding strategies for disease resistance
 Marker assisted selection
QTL
Tissue culture methods
a) Somatic hybridization
Effector assisted breeding
Root stock breeding
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27. Marker-assisted selection (MAS)?
• MAS refers to the use of DNA markers that are tightly linked
to target loci as a substitute to assist phenotypic screening
Disease susceptible Disease resistant
Knight, 2014
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28. Objectives.:
1. To study the inheritance of BW resistance in a segregating population derived
from resistant and susceptible eggplant inbred lines.
2. To study the association of SCAR marker linked to BW resistant genes
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29. Material and Methods
E-31 (Highly resistant, Round fruit)
E-32 (Highly susceptible, Round fruit)
 R. solanacearum (race 1) – TZC medium
 Disease index – scoring
 CTAB method –DNA extraction
 SCAR and PCR analysis
P1- 5’-G ACTGCGTACCAATTCAGT T-3’
P2- 5’- G ATGAGTCCTGAGTAACACGATG-3’
 Data analysis
Cao et al., 2009
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30. Table 1: Evaluation of the resistance to bacterial wilt in parents
Table 2: The reaction to bacterial wilt in F1, F2 and BC1 populations
Cao et al., 2009
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31. Fig .1: The PCR amplification of S401 in Parents and F2 segregation individuals
Cao et al., 2009
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32. Fig .1: The PCR amplification of S401 in Parents and F2 segregation individuals
Cao et al., 2009
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33. Fig. 2: The identification of SCAR marker in F2 population. M-Ladder , 1-E-31(R),
2-E-32(s), 3-E-32 x E-31 (R), 6,9,11,16 and 19 susceptible plants of F2,
4,5,7,8,10,12,13,14,15,17,18,20 and 21 resistant plans of F2.
Cao et al., 2009
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Table 3:The identification of the SCAR marker in F1, F2 and BC1 populations

34. Fig. 3: The identification of SCAR marker in resistant plants of F3. M-Ladder , 1-E-31(R),
2-E-32(s), 3-22- resistant plants of F3
Cao et al., 2009
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35. Pandiyaraj et al., 2019
Objectives.:
1. Identification of unique SSR markers tightly associated with the BW resistance
loci
2. To observe the segregation in two diverse F2 populations
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36. Material and methods
Pandiyaraj et al., 2009
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Pandiyaraj et al., 2019
 Phenotyping of parents and F2 – for BW
Disease scoring-0-4 scale, PDI
Screening of SSR markers -390
Data analysis

37. Table 4:Phenotypic Chi-square values of two segregating F2 populations
Table 5:Segregation pattern of 4 SSR markers in 2 segregating F2 population for BW
resistance in eggplant
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38. Table 6: Single marker analysis for four markers with F2 population For BWr
Pandiyaraj et al., 2019
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39. Fig. 4: Validation of SSR-46 marker in parents, F1 and F2 of CARI-1 x Rampur Local
Fig. 5: Validation of emb01D10 marker in parents, F1 and F2 of CARI-1 x Rampur Local
Pandiyaraj et al., 2019
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40. Fig. 7: Validation of emh02E08 marker in parents, F1 and F2 of IIHR-7 x Arka Kushmakar
Fig. 6: Validation of SSR-46 marker in parents, F1 and F2 of IIHR-7 x Arka Kushmakar
Pandiyaraj et al., 2019
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41. Quantitative Trait Loci (QTL)
QTL is defined as “a region of the genome or locus of gene that is
associated with an effect on a quantitative trait”.
QTL mapping is process of locating genes with effects on
quantitative traits using molecular markers
 Requirement for QTL mapping - Mapping Populations
 Types - 1. Mortal Population 2.Immortal populations
• Mortal Population -F2 , F3 BC1F1, BC1F2
• Immortal populations- RILs, NILs, DH and MAGIC
(Multi parent Advanced Generation Inter Cross)
Collard et al.(2005)
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42. Objectives.:
1. To determine the genetic control of resistance to phylotype I strains in a
segregating population of RILs.
2. To map the genes or QTL controlling the resistance
Lebeau et al., 2013
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43. Materials and methods
QTL analysis-R/qtl package of R software and PLABQTL software
Linkage map analysis- MAPMAKER/EXP ver. 3.0b program
DNA extraction and marker analysis (AFLP, SSR, SRAP)
Inoculation and disease assessment (disease scale 1-5 )
Bacterial strains – CMR 134, PSS366, GMI100 and PSS4 (phyl.1)
AG91-25 – R , MM738 -S, F1, F2, BCP1, BCP2, 178 F6 RILS
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Lebeau et al. 2013

44. Table 7. Estimates of mean of parents and progenies for maximum wilting %,
colonization index (CI) and AUDPC for score to study resistance against four
phylotype strains of pathogen
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Lebeau et al. 2013

45. Cont…
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Lebeau et al. 2013

46. Fig. 8. Linkage map 119
markers AFLP, SSR and
SRAP based on 178 F6
families and QTL position
for resistance to
phylotype I strains.
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Lebeau et al. 2013
Lebeau et al. 2013

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The inset corresponds to an enlarged
fragment of the LG2 with ERS1
positioned at the level of the closest
marker
Lebeau et al. 2013

48. Fig. 9: LOD plot for the
SCOAUDPC QTL detection on LG2
for resistance to R. So Strains in
F6 population
*indicate – the degree of distortion
of each marker from mendelian
segregation rations significant at
*p< 0.05, **p< 0.01, ***p< 0.001.
Lebeau et al., 2013
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Somatic Hybridization

50. Objectives.:
1. Morphological and molecular characterization of somatic hybrids derived from
protoplast fusion between S. melongena cv. Dourga and S. aethiopicum.
2. To evaluate the somatic hybrids for fertility and resistance to bacterial wilt in
the field condition
Collonnier et al., 2010
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51. Material and methods
S. melongena cv. Dourga (White half long fruit)
S. aethiopicum group.aculeatum and gilo
 Somatic hybrids : protoplast fusion
 Ploidy level : Flow cytometer
 Hybrid nature: Isozyme analysis and RAPDs pattern
 Tests for bacterial resistance: Screening done in field condition
with artificial inoculation of R.solanacearum
Collonnier et al., 2010
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52. Fig. 10: Flow cytometric analysis of 10 000 DAPI-stained nuclei of A) S. melongena cv. Dourga
B) S. aethiopicum gr. Aculeatum c) and gr. gilo and two tetraploid somatic hybrids D) Dsa-
18a, E) DSa2-2.
Collonnier et al., 2010
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53. Fig. 11: Root –tip metaphasic cell of a tetraploid somatic hybrid (DSa 110) between S.
melongena cv. Dourga and S. aethiopicum gr. aculeatum (2n=4x= 48 chromosomes).
Collonnier et al., 2010
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54. Fig. 12: Electrophoresis
banding pattern of
(A) 6-phosphogluconate
dehydrogenase (6-
Pgd)
(B) Phosphoglucomutase
(Pgm)
(C) Isocitrate
dehydrogenase (Idh)
D – cv. Dourga
Sa- S. aethiopicum (both
aculeatum and gilo)
M- mixture DNA of
cv.Dourga and S.
aethiopicum
1-7 hybrids between
cv.Dourga and
S.aculeatum
8 and 9- hybrids between
cv.Dourga and S. gilo
Collonnier et al., 2010
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55. Fig. 13:
Electrophoresis banding
(A), (B), (C) RAPDs
patterns
D: S. melongena cv
Dourga;
Sa: S. aethiopicum (both
groups aculeatum and gilo
had the same pattern);
M: mixture of DNA from S.
melongena cv
Dourga and S. aethiopicum;
1–7: hybrids DSa 1a, DSa
3a, DSa 4a, DSa 6a, DSa
17, DSa 20a, and DSa 26a;
8 and 9: hybrids DSa2-2
and DSa2-3.
Collonnier et al., 2010
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56. Fig. 14: Flowers and fruits from S. melongena cv Dourga (A), S. aethiopicum gr aculeatum (C), and
their somatic hybrid (B).
Collonnier et al., 2010
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57. Table 10: Plant height, stem diameter, number and length of branches, length and width of leaves
(Means of 30 plants 9S.D.)a
Collonnier et al., 2010
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58. Table 11: Number of flowers per plant, number of fruits per plant, % of flowers setting fruit, fruit
mean weight and fruit yield per plant (Means of 30 plants 9S.D.)a
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59. Table 12: Disease indices (DI) recorded 2, 4 and 8 weeks after root inoculation by race 1 strain
of R. solanacearuma
Collonnier et al., 2010
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60. 6/13/2021 VSC Dept. 60
Effector –Assisted Breeding for Bacterial wilt resistance
Jay et al. (2016)
Plant cell Pathogen Immunity
PRR (Pattern
Recognition receptors)
PAMPS (Pathogen
associated molecular
pattern)
PTI (Pattern/ PAMP
triggered immunity)
R-Gene – R- Protein Pathogen effectors ETI (Effector triggered
immunity)

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MOLECULAR PLANT PATHOLOGY(2018) 19(11), 2459–2472
Morel et al. (2018)
France
Objectives:
To investigate whetherAG91-25 resistance is associated with an effector from GMI1000
AG91-25 Resistant line GMI1000
MM738- Susceptible line
Bacterial strains:
a) GMI1000
b) GMI1000 ripAX2::pCZ367
c) GMI1000 ripAX2::pCZ367::ripAX2 (wild type of ripAX2 in the mutant)
d) PSS4
e) PSS4 :: ripAX2

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Fig 15: The RipAX2 type III effector of Ralstonia pseudosolanacearum is necessary for resistance in
AG91-25 eggplants. The R. pseudosolanacearum GMI1000 wild-type strain and the GMI1000
ripAX2::pCZ367::ripAX2 complemented strain are not able to trigger disease on AG91-25, whereas the
GMI1000 ripAX2::pCZ367 mutant is able to do so.
Fig: 16: The strains GMI1000, GMI1000 ripAX2::pCZ367 and GMI1000
ripAX2::pCZ367::ripAX2 are all able to wilt MM738 susceptible eggplants

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Fig 17: The trans-complementationof the AG91-25 pathogenicstrain PSS4 by RipAX2permits
AG91-25 resistance.
Fig 18: The strains PSS4 and PSS4::ripAX2 are able to wilt MM738 susceptibleeggplants.

64. This technique is promising for combating bacterial wilt without necessitating
the change in genetic backgrounds.
S. torvum is of great interest as a rootstock for eggplant grafting due to its highly
vigorous nature, complete graft compatibility with scions (Moncada et al., 2013),
and resistance to a wide range of soil pathogens, especially R. solanacearum, due
to the presence of mechanical barriers developed in roots.
Along with wild relatives, some suitable bacterial wilt-resistant genotypes have
been identified for rootstock purpose.
Rootstock breeding
Ashok Kumar et al. (2017),
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65. Table 13. Effect of different wild solanum species on percent bacterial wilt
infection of grafted plants
Grafting combinations Bacterial wilt infection
%
Grading
Solanum torvum x Pusa Shyamala 12.22 R
Solanum torvum x Pusa Hybrid - 6 13.47 R
Solanum xanthocarpum x Pusa Shyamala 45.50 S
Solanum xanthocarpum x Pusa Hybrid - 6 48.17 S
Solanum khasianum x Pusa Shyamala 29.60 MR
Solanum khasianum x Pusa Hybrid - 6 31.47 MR
Solanum surathense x Pusa Shyamala 58.52 S
Solanum surathense x Pusa Hybrid - 6 55.30 S
C.D 2.36
SE.m 0.81
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Ashok Kumar et al. 2017
Based on mean performance of the grafted plants with Solanum torvum and Solanum khasianum
were found to be superior and they can be used for resistance against soil borne bacteria.
A new rootstock eggplant cultivar ‘Daizaburou’with high resistance to
bacterial wilt and Fusarium wilt (Yoshida et al. (2004)

66. ACHIEVEMENTS
Sl.
No
Hybrids/Varieties Institute
1 Arka Anand, Arka Nidhi,Arka Keshav, Arka Neelakant, IIHR, Bangalore
2 Pusa Purple Cluster,Pusa Anupama, KT-4 IARI, New Delhi
3 Pant Rituraj, Pant Samrat, Pant Brinjal Hybrid GBPUA&T, Pantnagar
4 Utkala Tarirani , Utkal Madhuri, Utkal Jyoti, Utkal
Anushree
OUA&Technology,
Bhubaneswar
5 Surya KAU, Thrissur, Kerala
6 Hissar Jamuni , Annamalai , JC-1, JC-2 Other SAU’s
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67. Arka Unnathi Arka Harshitha Arka Avinash Arka Anand
https://www.iihr.res.in/varieties
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68. 68
SURYA (SM 6-7)
KAU Varieties
SWETHA (SM 6-6)
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69. 69
HARITHA (SM 141)
Bacterial wilt
resistant Brinjal
variety
KAU Varietis
NEELIMA
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70. Sl.
No
Hybrids/Varieties References
1 Aroman, Sinampire,Taiwan Naga (Japan collection),
ARU- 2C, Improved Muktakeshi, Pusa Purple Round,
Vijay Hybrid, Banaras Giant Green
Swarup (1995)
2 Makra Round, Singhnath Makra, Kata Makra, Pusa
Anupam, Bhagyamati, NDBS-26, BB-40, Sada Lamba,
Melwanki local and Co2
Manna et al. (2003)
3 Utkal Tarini, Utkal Madhuri,Utkal Jyoti, UtkalAnushree
(Odisha University of Agriculture and Technology,
Bhubaneswar)
Sahoo (2015)
4 Kata Begun (Bangladesh collection) Rahman et al. (2011)
5 Arka Nidhi Kumar et al. (2014)
6 VNR 218 (VNR seed company) http://www.iasri.res.
in/aicrpvc
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71. Sl.
No
Breeding lines References
1 EG195 and EG 203 Petran (2013)
2 BEBWRES-05 (from Jharkhand) Kumar et al. (2014)
3 IHR-322, IIHR500-A, BPU-1, IIHR-3, IIHR-5 Gopalakrishnan et al. (2014)
4 2013-070, 2013–080, 2013–064, 2013-090
(Indonesia collection)
Harti et al. (2016)
5 TS-13, V1034885, TS47 (Malaysian collection),
TS-69, TS-87, TS-90 (Indonesia collection)
Taher et al. (2017).
6 CARI-1, IIHR-7 Khapte et al. (2018)
7 IIHR -500-A, IIHR-127, IIHR-575, IIHR-584,
IIHR -667 A, IIHR- 766A, IIHR-817, IIHR- 874,
IIHR-882
Kumar et al. (2019)
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72. LIMITATIONS
 Race specific resistance
 Resistance is temperature dependent
 Lack of prompt and effective screening techniques
 Genetic inheritance of resistance is unclear

73. Broad
host
range
Grafting
Conventional
methods
Patho
profile
Molecular
approaches
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74. “Working Together
Works”
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