molecular characterization of fusarium fujikuroi associated with bakanae disease of rice in india...
TRANSCRIPT
Molecular characterization of Fusarium fujikuroi associated with bakanae disease of rice in India
Dr. Bishnu Maya BashyalScientist
Division of Plant PathologyICAR-Indian Agricultural Research Institute,
New Delhi-110012
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Bakanae: an emerging disease of rice in India
Rice (Oryza sativa L.) is one of the most important food crops of India. Total area under
rice production is 43.64 million hectares with the production of 97 million tones. India is
the largest producer and exporter of basmati rice in the world.
About 70% of basmati rice is produced in India, where, it shares 7.76 million hectares with
production of 71.10 lakhs tonnes and contributes 15449.6 crores in rice export.
Bakanae disease of rice is emerging as a potential threat in India (Anonymous, 2007). High
incidence of bakanae disease has been observed in Pusa-1121, Pusa-1509, Pusa-2511, CSR-
30, Dehradoon Basmati and Pakistani Basmati (Anonymous, 2013).
In India, bakanae disease had been considered as of minor importance earlier and work
has been carried out only on resistance evaluation and fungicidal management (Sharma et
al., 1998; Pannu et al., 2009).
The disease causes upto 40% losses in the yield. Most of the aromatic rice varieties are
susceptible to the disease. Therefore, it can decrease the production of basmati rice to
28.44 lakhs tones worth for 6179.84 crores loss in export per year, which signifies its
importance in near future.
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
How does the disease look like?The typical symptoms of bakanae are slender, chlorotic and abnormally elongated primary leaves.
However, crown rot is also seen, resulting in stunted rice plants.
In crops reaching maturity infected plants show tall lanky tillers bearing pale green flag leaves
Infected plants usually have small numbers of tillers and plants die in few weeks.
Occasionally infected plants survive until maturity but bear empty panicles.
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Different symptoms produced by F. fujikuroi in rice seedlings ; A: elongation; B: rotting; C:elongated and rotted plants
Research gaps and objectives
An understanding of the pathogen’s population structure, its aggressiveness, and its genetic diversity is required for the development and implementation of effective management strategies. However, no studies have characterized the Indian population of F. fujikuroi.
The objectives of this study were to determine the morphological, pathological, and molecular diversity, as well as the mating types of the F. fujikuroi population isolated from symptomatic bakanae-diseased rice plants in India.
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Geographical distribution of Fusarium fujikuroi isolates of this study
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Bakanae disease in rice variety Pusa Basmati -1121 I
CA
R -
Ind
ian
Agr
icu
ltu
ral
Res
earc
h I
nsti
tute
States Districts % Disease incidence
Variety grown
Haryana Karnal 2 CSR 30
Punjab Fatehgarh 10 1401
Uttar Pradesh
Aligarh 15 1401
Buladshaher 7 2511
Gautam Budha Nagar
3 1509
Uttarakhand Haridwar 02 Dehradoon Basmati
Udham Singh Nagar
02 Pakistani Basmati
Incidence of bakanae disease in different rice varieties
Bakanae disease in different rice varieties
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Survey for Bakanae disease of rice I
CA
R -
Ind
ian
Agr
icu
ltu
ral
Res
earc
h I
nsti
tute
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Methodology
• Collection of diseased samples
Diseased samples were collected from farmer’s fields from different states of India. No specific permissions were required for the collection, farmers happily permitted as they were also feeling concern about the disease.
• Fungal isolates and maintenance
A total of 63 different Fusarium infected rice samples were collected from the farmers field of different Basmati growing states of India (Punjab, Haryana, Uttarakhand, Uttar Pradesh and Bihar).
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Cultural and morphological variability All the 63 isolates of F. fujikuroi were grown on potato dextrose agar (PDA) and carnation leaf agar (CLA) medium.
Pathogenicity test
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Seed inoculation in variety Pusa Basmati 1509
Molecular characterizations
• Genomic DNA extraction• Universal Rice Primers and Polymerase chain reaction• Mating type evaluation
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Morphological characterization of Fusarium fujikuroi isolates
Isolates classified based on radial growth at 5 days after inoculation>6 cm: fast growing4-5.9 cm: Moderate growing3-3.9 cm: slow growing
Isolates classified based on pigment color
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Isolates of Fusarium fujikuroi showing different colours: a: Yellowish white; b: pink and c: purple
Characterization of F. fujikuroi isolates for their virulence
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Virulence
Categories
Isolates name Percentage
(%)
Moderately virulent
(2-25 %)
F204, F228, F341, F273, F301a, F302 , F237a, F284, F308,
F309, F249, F252, F252a, F253, F282, F299, F347
26.98
Highly virulent
(26-50 %)
F206a, F216a, F218a, F220, F229, F267, F278a, F301,
F210a, F232, F303, F320, F328, F343, F255a, F350, F352
26.98
Most virulent
(51-100 %)
F203, F206, F219, F224, F231, F234, F259, F268, F269,
F276, F338, F304, F306, F314, F338a, F343a, F210, F284a,
F310, F250, F256, F319, F322, F327, F337, F337a, F255,
F348, F351
46.04
Details of Universal Rice Primers (URP) used to amplify DNA of Fusarium fujikuroi isolates
Primers Primer sequence Total no. of bands
Polymorphic bands
Monomorphic bands
Polymorphism (%)
URP 1F ATCCAAGGTCCGAGACAACC 8 6 2 75%
URP 9F ATGTGTGCGATCAGTTGCTG 12 11 1 91.67%
URP 32F TACACGTCTCGATCTACAGG 14 14 0 100%
URP 6R GGCAAGCTGGTGGGAGGTAC 15 15 0 100%
URP 13R TACATCGCAAGTGACACAGG 9 9 0 100%
URP 17R AATGTGGGCAAGCTGGTGGT 16 16 0 100%
Total 74 71 3 94.44%
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Genomic DNA amplification of all 63 isolates of F. fujikuroi produced 74 bands, of which 71 showed polymorphism
Diversity analysis of Fusarium fujikuroi isolates
DNA finger print profile of Fusarium fujikuroi isolates obtained with primer URP-17R
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 M
Fig. from left to right- M:molecular marker 1kb; Fusarium isolate code 1 to 63 203,204,206,206a,218a,219,220,224,228,229,231,234,259,267,268,269, 341, 273, 276,278a,338, 301,301a, 302,303,304,306, 314, 237a,338a,343a, 210,210a,232,284,284a,309,310,308,249,250,252,252a,253,256,282, 319,320,322,327, 328, 337,337a,343,255,255a,299,347,348,350,351,352
DNA finger print profile of Fusarium fujikuroi isolates obtained with primer URP-32F
The maximum number of bands (16) were obtained using the URP 17R primer
Diversity analysis of Fusarium fujikuroi isolates
Coefficient0.65 0.72 0.80 0.87 0.95
F203 F206a F216a F234 F268 F229 F220 F231 F259 F218a F224 F228 F338a F309 F273 F301 F302 F304 F306 F343a F210 F210a F232 F284 F278a F338 F301a F314 F237a F219 F267 F269 F341 F276 F284a F249 F310 F308 F322 F343 F327 F328 F252 F252a F253 F256 F319 F299 F320 F337 F337a F255 F255a F250 F282 F331 F204 F347 F348 F350 F351 F352 F303
• III• IV
• I
• a
• b
• c
• II
Dendrogram obtained after combined analysis of Fusarium fujikuroi isolates collected from different states of India
Plot of genetic distances and geographical distances derived using the Mantel test in F. fujikuroi isolates from six states of India (geographical distances means distances based on longitudinal-latitudinal coordinates)
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Subcluster a comprised 27 isolates from Punjab Isolates collected within the geographical distances of 300–600km were more variable compared to isolates collected from larger distances
Amplification of MAT-1 and MAT-2 locus of the Fusarium fujikuroi isolates collected from different states of India (Isolates 1-17, 19-24, 26-32, 55-56 collected from Punjab; 25, 33-39: Uttar Pradesh; 18, 57, 58: Uttarakhand; 40-54: Haryana; 59-62: Bihar and 63: Jammu and Kashmir)
Mating type distribution in Indian population of Fusarium fujikuroi inciting bakanae disease of rice
Perithecia of Fusarium fujikuroi
GMAT 1a- 5’ GTTCATCAAAGGGCAAGCG 3’GMAT 2b- 5’ TAAGCGCCCTCTTAACGCCTTC 3’GMAT 2C- 5’ AGCGTCATTATTCGATCAAG 3’GMAT 2d- 5’ CTACGTTGAGAGCTGTACAG 3’
•Among the 63 isolates evaluated 18 (28.57%) were identified as MAT-1 and 45 (71.42%) as MAT-2. •Distribution of MAT-2 population was highest in Haryana (86.67%), followed by Punjab (71.0%) .•Effective population no. Ne(mt) for mating type was 89% of the total population.•Study on female sterility needs to be conducted.
800 bp MAT-2300bp MAT-1
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Conclusions
The present study generated significant information on morphological, pathogenic and molecular variability in Indian population of F. fujikuroi, which could be utilized for the development of resistant varieties of rice.
Further, study also confirms the presence of both the mating types of F. fujikuroi in India, therefore, existence of sexual reproduction in this species could not be avoided.
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
Acknowledgements
Director, IARI
Joint Director Research, IARI
Dean & Joint Director Education, IARI
Co-ordinator, School of Crop Protection
Head of the Division
Mr. Dheeraj Kumar
Sapna, Kirti
IC
AR
-In
dia
n A
gric
ult
ura
l R
esea
rch
Ins
titu
te
