Food Chain and Food Web (Ecosystem) EVS, B. Pharmacy 1st Year, Sem-II
MOLECULAR CHARACTERIZATION OF ELITE LINES FOR BLB RESISTANCE IN RICE
1. ACHARYA N.G RANGAAGRICULTURAL UNIVERSITY,
AGRICULTURAL COLLEGE, BAPATLA.
Credit Seminar : Molecular Characterization of Elite Lines for
BLB Resistance in Rice.
Course no. : GP 591 ( MASTER’S SEMINAR)
SUBMITTED TO : SUBMITTED BY:
Dr. T. Srinivas N. Ruth Hepsi Sindhura,
Professor and Head BAM-20-24,
Department of GPBR M.Sc. 1st year
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Agricultural College, Bapatla
Department of Genetics and Plant Breeding
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Agricultural College, Bapatla
Department of Genetics and Plant Breeding 1
Contents
1. Introduction of RICE
2. BLB and it’s Symptoms
3. Impact of BLB
4. Screening Methods
5. Resistance genes against BLB
6. Commercially released Varieties
7. Case studies
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• Scientific name – Oryzae sativa L.
• Chromosomal number – 2n=2x=24
• Rice is a Self pollinated, Monocot with Hypogeal
type of germination.
• It is a Semi aquatic, Annual grass.
• It is a short day plant (ie., it requires 10 to 12 hrs of
light period at the flowering stage).
• Inflorescence of paddy is called Panicle and stem is
called Culm.
• Fruit type of Rice is Caryopsis.
RICE ( Oryzae sativa L.)
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• Rice ( Oryza sativa L.) is the world's second most important crop after wheat.
• It is the central to the lives of billions of people around the world, possibly the oldest
domesticated grain (~10,000 years).
• Rice is the staple food for half of the world’s population, growing rice is the largest
single use of land for producing food, covering 9% of the Earth's arable land.
• Rice provides 21% of global human per capita energy and 15% of per capita protein.
• Calories from rice are particularly important in Asia, especially among the poor,where
it accounts for 50-80% of daily caloric intake.
• Unfortunately rice production is impeded by several diseases of fungal, bacterial and
viral origin, of which most drastic is bacterial leaf blight caused by Xanthomonas.
Introduction :
M. H. M. Mubassir et al.,
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Bacterial leaf blight
• Domain : Bacteria
• Phylum : Proteobacteria
• Class : Gamma Proteobacteria
• Order : Xanthomonadales
• Family : Xanthomonadaceae
• Genus : Xanthomonas
• Species : oryzae
Trinomial name : Xanthomonas oryzae pv. oryzae (Xoo.)
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• BLB first observed by a farmer of Japan in year 1884
(Tagami and Mizukami 1962).
• BLB is a gram negative bacteria and causes disease as
rods, 1.2 × 0.3-0.5 μm.
• The bacterium is of straight rods, with a single polar
flagellum (Swings et al. 1990).
• They are single, occasionally in pairs but not in chains.
• An Extracellular Polysaccharide (EPS) is present which
helps in protection of bacteria from desiccantion and
also facilities rain borne dispersal.
• Their colonies in nutrient agar are pale yellow as they
produce an yellow coloured soluble pigment called
XANTHOMONADIN.
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Disease symptoms
Kresek / wilt :
• This is the most destructive stage and also called
seedling blight or wilt phase syndrome.
• Young plants are more susceptible which shows
sudden wilting.
• The bacteria multiplies in vascular bundles ,
exudation of bacterial ooze at the cut end portion
of leaf.
• In general, temperatures at 25 – 30°C and relative humidity >70% favours the
disease development.
The symptoms of the disease may vary depending upon growth stage of the crop.
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Leaf blight:
• This symptoms will appear 4-6 weeks after
transplanting.
• Appearance of small circular water soaked
spot on the margins of the leaf & this spots
also extends to leaf sheath, also later this
spots turn yellow & becomes necrotic &
results in drying of leaf.
• Under humid conditions creamy white
color bacterial oozes comes out from the
young lessions in the morning hours.
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• Disease also attacks leaves, leaf sheath and grains of rice plants as a result yields are
reduced.
• Infection particularly at the booting, heading and milk stages reduces the yield.
• Due to its high damage to the rice fields, a lot of studies and observations has
already been done for controlling this disease but effective control measures are yet
to be found.
• So besides cultural practices, chemical control, biological control, disease forecasting,
use of resistant cultivars is the best cost effective approach for controlling this
disease.
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Impact of BLB
• Causes significant yield loss of 20 to 30%, but based on weather conditions this can
reach as high as 80 % (Noh et al., Ou, 1985).
• In Punjab, Haryana and Western Uttar Pradesh states of India, major epidemics
occurred in 1979 and 1980.
• Epidemic covering 10,000 acres of BPT 5204 broke out in Kurnool district during 2010
and since then the diease is appearing regularly in that region and other adjoining
areas (Yugander et al. 2014).
• A sever disease outbreak occurred in Thanjavur district of Tamilnadu during early
2014 and varieties like BPT 5204, ADT 43, CR 1009 all were severely affected (Laha et
al. 2014).
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• The Morphological and Molecular Screening both are widely used approach in the
recent scenario for identifying BLB Resistant cultivars.
• Morphological screening is done to measure the disease severity by considering
the percentage of diseased leaf area.
• Molecular screening is by the use of markers and to identify the disease Resistant
genotypes and for their confirmation.
SCREENING
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Morphological screening
Preparation of Bacterial Inocula :
• The strain responsible for BLB disease is
collected.
• This isolate is maintained in slants containing
peptone sucrose agar (PSA) medium.
• After growing Xoo. on PSA media for 72 hrs at
30°C, the innoculum is prepared by mixing the
cultured bacteria with 10 ml sterile distilled
water in a slant.
• The concentration of bacterial suspension was
adjusted to 108 CFU/ml prior to inoculation
using sterile distilled water.
M H. M. Mubassir et al.,
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Methods of inoculation
NEDDLE-PRICKING METHOD:
• About 100 needles are mounted on a rubber stopper or any kind of supporting
materials.
• To inoculate needles are dipped in bacterial suspension and gently pricked into
the leaf vein.
• Not practical for large-scale field inoculation.
• Root and basal part of the seedlings are dipped in a bacterial suspension before
transplanting.
• This method is used to test for seedling wilt or Kresek phase of bacterial blight.
DIPPING METHOD:
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CLIPPING METHOD :
• Takes advantages of the fact that BLB is a vascular disease, by Clipping off inoculum is
directly deposited in the infection court.
• Approximately 1-2 cm of the leaf tip is cut when it reaches 5 inches of length.
• The instrument used to inoculate the rice plant with the bacterial Inocula is scissors and
before using the scissor it should be sterilized using 70% ethanol.
• The scissors is dipped into the bacterial suspension and used to cut the leaf of the plant.
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SPRAYING METHOD :
• Disease development was usually slow.
• Spraying limits the invasion of the bacterial pathogen to natural openings unless
wounds on the leaf blade are present.
• Bacterial inoculum at a concentration of 10⁸ - 10⁹ bacterial cells/ml is sprayed onto
the plants.
• This method of inoculation is not practical during dry season when humidity is very
low for bacterial cells to survive.
• To cut several leaves in a hill, a small plastic bottle
containing the inoculum and attached to a garden
clipper is used for inoculation (Kauffman et al., 1973).
• About 2000 plants can be inoculated with inoculation
clippers per man- hour per day.
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Scoring system ( FIELD TEST ) :
SCORE CHARACTER CHROMOSOME
1 1-5 % Resistant (R)
3 6-12 % Moderately resistant
(MR)
5 13-25 % Moderately susceptible
( MS)
7 26-50 % Susceptible (S)
9 51-100 % Highly Susceptible (HS)
IRRI, SES 2014
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• The total of 42 genes conferring resistance to BLB has been identified in rice
(Vikas and Bhatia et al,. 2017).
• Among them, 9 genes namely Xa1, Xa3/Xa26, xa5, Xa10, xa13, Xa21, Xa23, xa25
and Xa27 have been cloned (Nino-Liu et al,. 2006; Liu et al,. 2011; Tian et al,.
2014; Wang et al., 2015)
• And 5 genes namely Xa4, xa5, Xa7, xa13 and Xa21 have been reported as major
resistance genes (Singh et al., 2015).
• Xa 21 gene of Rice confers a broad spectrum resistance to almost all the races of
Xoo.
RESISTANCE GENES AGAINST BLB :
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Commercially released MAS Rice varieties in India:
• Minghui 63 (Xa21) is the first BLB resistant rice cultivar developed by MAS in China.
(Chen et al., 2000).
• RP BIO 226 (Improved Samba Mahsuri) was derived from introgression of xa5, xa13
and Xa21 into a premium quality rice variety (Samba Mahsuri) in India (Sundaram et
al., 2008).
• Other varieties or improved genotypes include,
PR 106 Lalat
Samba Mahsuri Tapaswini
Type 3 Basmati Swarna
PAU 201 IR 8
Pusa Basmati 1
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Identification of BLB resistant genes in some rice varieties
for development of High yielding Bacterial Leaf Blight
tolerant types
K. Majumder et al., 2018
Case study 1
Journal of Environmental Biology. NASS RATING: 6.78
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MATERIALS AND METHODS :
• Performed Randomised block design with 3 replications at Agricultural
Experimental farm, University of Calcutta.
• A total of 61 Genotypes (58+3) including resistant and susceptible checks
were screened in field condition by artificial inoculation using IX020 strain of
Xoo for 2 years (Kharif 2016 and 2017).
IRBB 7 (Xa7)
IRBB 60 ( Xa4+xa5+xa13+Xa21)
IR 24 (No resistant gene)
Checks
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These varieties were also genotyped for 7 SSR markers tagged with major
BLB resistant genes, i.e., Xa4, xa5, xa13 and Xa7.
GENE CHARACTER CHROMOSOME MARKERS
Xa4 Dominant 11 RM 224 + MP1
xa5 Recessive 5 RM 13 + RM 153
xa13 Recessive 8 RM 264 + RM 230
Xa7 Dominant 6 RM 251
MOLECULAR CHARACTERIZATION :
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Varieties Resistant genes
IR 64 Xa4, xa5, xa13, Xa7
Ratna Xa4, xa5, xa13, Xa7
Surjamukhi Xa4, xa5, xa13, Xa7
Kalinga-2 Xa4, xa5, xa13, Xa7
Zheshan-2 Xa4, xa5, xa13, Xa7
IR-68144-2b-2-2-3-1-127 Xa4, xa5, xa13
Azucena Xa4, xa5, xa13
Conclusions:
All expressed similarly, thus presence of gene Xa7 was not found critical for
resistant reaction.
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Case study 2
Surya Kanth Mishra et al., 2016
Morphological and Molecular marker based Identification of
rice (Oryzae sativa L.) genotypes for BLB Resistance.
Ecology Environment and Conservation NASS RATING: 5.41
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Materials and methods :
Total of 30 Genotypes were studied.
Phenotyping:
• Inoculums of BLB were used to create artificial epiphytotic conditions at all 3 stages
(viz. Seedling, tillering, adult plant stage) .
• An ave. of 5 leaves per plant was inoculated using Clipping method.
Resistance - < 3.0 cm Xa4 - RM224
Moderate resistance - (3.1 -6.0) cm. xa13 - RG136
Susceptible - > 6.0 cm Xa21 - pTA248
Genotyping: Screened for identification of BLB resistant genes (Xa21, xa13 and Xa4).
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Results :
From morphological data
Lesion length Genotypes Response BLB genes
Less than 3 cm 13 lines Resistant
4 with all 3 genes
9 with atleast 2
3.01 to 6.00 12lines
Moderately
resistant
12 with 1 gene
atleast
More than 6 cm 5 lines Susceptible No resistant genes
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Amplification product of Xa21 gene
7 Genotypes out of 30 showed the appropriate amplification of 1000 bp fragment
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Amplification product of xa13 gene
21 Genotypes out of 30 showed the appropriate amplification of 1000 bp fragment
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Amplification product of Xa4 gene
15 Genotypes out of 30 showed the appropriate amplification of 150 bp fragment
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Conclusions :
• The genotypes having two or more BLB resistance genes can be effectively used
as a donor parent in breeding programmes to develop durable BLB resistance in
rice germplasm.
• Because it is estimated that rice genotypes with only one BLB resistance genes
were found susceptible to BLB disease.
• In our study, we have identified thirteen Indian rice genotypes having two or
three BLB resistance genes.
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Morpho – Molecular Screening for Bacterial Leaf Blight
Resistance in Some Rice Lines and Varieties
M. H. M. MUBASSIR et al., 2016
Case study 3
Journal of Plant Sciences NASS RATING: 3.61
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• 10 IRRI Advanced lines and 17 varieties of rice from BRRI, BINA and Gazipur including
Resistant (IRBB21) and susceptible (BR-11) checks.
MATERIALS AND METHODS :
• Inoculation of Active strain Xoo. (BXO-09) done on 5 inches length paddy by Clipping
method.
• After 21 days, data was taken for measurement of BLB (seedling stage).
1. Morphological Screening (PHENOTYPING) :
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2. Molecular Screening (GENOTYPING) :
• PCR amplification done by using three RFLP primers (RG136, RG556 and pTA248).
• Marker analysis done.
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Conclusions
• 7 could be utilised to develop BLB Resistant rice varieties using MAS
and backcrossing.
• 3 were resulted moderately resistant could be tested further.
• To improve grain quality and bacterial leaf blight resistance, this
molecular characterization information could be helpful for further
planning.
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Marker assisted selection of Rice (Oryzae sativa L.) Genotypes
for Bacterial Leaf Blight disease resistance
K. Sowmya and P. Sindhumole 2016
Case study 4
Electronic Journal of Plant Breeding NASS RATING: 5.14
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MATERIALS AND METHODS :
• Xa5 is an important recessive bacterial blight resistant gene.
• Main objective of the present study was MAS of rice germplasm accessions for
xa5 gene, using RM122, a closely linked microsatellite marker.
• Forty traditional rice genotypes from the germplasm of Division of Plant
Breeding and Genetics, Regional Agricultural Research Station, Pattambi, were
initially screened in field for BLB resistance.
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Results and Discussion
Field screening:
• 10 seeds each of the selected genotypes were germinated by top of the filter paper
method.
• After 4th day, the seedlings were transplanted to disposable paper cups filled with soil.
• Three seedlings were planted per cup and they were irrigated daily.
• Natural occurrence of BLB disease was observed in these seedlings one week after
planting onwards.
• Presence of BLB pathogen in seedlings of various genotypes with symptoms, was
confirmed by ooze test.
• Out of the total forty varieties, only twenty varieties which had either no or less
symptoms of BLB disease, were used for marker assisted selection.
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Molecular Screening
• DNA isolation of tender leaves of various genotypes is done.
• PCR amplification and marker analysis is done.
• Molecular analysis done by using Plant Direct Gen Amp PCR kit method.
Specific marker RM 122, indicating the presence of xa5 gene.
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Conclusions :
Varieties Treatment
number
Karuthakuruka T1
Kuruva T8
Kokkankoli T19
Kochuvithu T12
Punjaparathu T14
• These 5 Genotypes Showed band for xa5 gene during gel electrophoresis and showed
no symptoms of BLB.
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Varieties Treatment number
Kadamakudi Pokkali T2
Cheriya Oorpandy T4
Karavala Kochuvithu T5
Chuvanna IR8 T6
Arupatham Kuruva T11
Gandhasala I T13
Pandi Chempan T16
Jeerakasala T18
• These 8 Genotypes Showed band for xa5 gene resistance and exhibited some symptoms
of BLB also in subsequent generations
• Indicating breakdown of resistance to BLB in these Genotypes.
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