All of rice oligotrophic endophytic Bradyrhizobia in this study were obtained except SUT-R74. 6 Bradyrhizobial strains were obtained from 98 bacterial strains. Bradyrhizobium is found only in rice root, with 10% relative abundance of total Alphaproteobacteria. Endophytic Bradyrhizobia could not be obtained from the monoculture system. Thai rice cultivars, the Thai Bradyrhizobial strains could promote rice growth better than Japanese strains. Three rice cultivars (Pathum Thani 1, Kasalath, and Nipponbare), cultivar Pathum Thani 1 responded only to putative Thai rice endophytic Bradyrhizobia. This phenomenon was not found in Japanese rice cultivars. Non-PB strains are also capable of forming a natural endophytic association with rice. Strains SUT-PR9, WD16, RP5, and RP7 displayed non-PB phenotypes but were genotypically close to PB strains.

1. PREFERENTIAL ASSOCIATION OF ENDOPHYTIC
BRADYRHIZOBIA WITH DIFFERENT RICE CULTIVARS AND
ITS IMPLICATIONS FOR RICE ENDOPHYTE EVOLUTION
NAME: ANAMIKA
ID: 49672

2. Abstract
Plant colonization by Bradyrhizobia is found not only in leguminous plants but also in
non-leguminous species such as rice. To understand the evolution of the endophytic
symbiosis of Bradyrhizobia, the effect of the ecosystems of rice plantations on their
associations was investigated. Samples were collected from various rice(Oryza sativa)
tissues and crop rotational systems. The rice endophytic Bradyrhizobia were isolated in
the basis of oligotropic properties, selective medium, and nodulation on siratro
(Macroptilium atropurpureum). Six Bradyrhizobial strains were obtained exclusively
from rice grown in a crop rotational system. The isolates were separated into the
photosynthetic Bradyrhizobia (PB) and non-photosynthetic Bradyrhizobia (non-PB).Thai
Bradyrhizobial strains promoted rice growth of Thai rice cultivars better than the Japanese
Bradyrhizobial strains. This implies that the rice cultivars posses characteristics that
governs the rice-bacterium associations. To examine whether leguminous plants in a rice
plantation system support the persistence of rice endophytic Bradyrhizobia, isolates were
tested for legume nodulation. All PB strains formed symbiosis's with Aeschynomene
indica and Aeschynomene evenia. On the other hand, non-PB were able to nodulate
Aeschynomene americana, Vigna radiata, and Macroptilium atropurpureum but unable
to nodulate A. indica and A. evenia. Interestingly, the nodABC genes of all of these
Bradyrhizobial strains seem to exhibit low levels of similarity to those of Bradyrhizobium
diazoefficiens USDA110 and Bradyrhizobium species strain ORS285. From these
results, we discuss the evolution of the plant-Bradyrhizobium association, including
nonlegumes, in terms of photosynthetic lifestyle and nod-independent interactions.
Piromyou at el., 2015

4. Oryza sativa
Kingdom : Plantae
• Order : Poales
Family : Poaceae
• Genus : Oryza
Species : sativa

5. Importance of rice in India
 Second largest consuming crop in India.
 Major rice-growing states are West Bengal,
Uttar Pradesh, Andhra Pradesh, Punjab,
Tamil Nadu, Bihar, and Chhattisgarh.
Variety:
 Basmati 217
 Basmati 370
 Dehraduni Basmati(Type 3)

6. Importance of rice in Thailand
 The world's second largest exporter of
rice
 Rain-making ceremonies are common for
rice farmers in Thailand
 Thai Hom Mali - the most delicious
fragrant rice of the world

7. Importance of rice in Japan
 Traditional Japanese meal
Variety of rice
 White Rice (Hakumai)
 Brown Rice (Genmai)
Product of rice
 Rice Vinegar
 Rice Wine (Nihonshu or Sake)

8. NITROGEN FIXATION
Nitrogen fixation is a process in
which nitrogen (N2) in the atmosphere is converted
into ammonia (NH3).

9. Symbiosis
 Symbiosis is close and often long-term
interaction between two different biological
species.
Example
 Frankia that live in alder root

10. ENDOPHYTES
 An endophyte is an endosymbiont, often a bacterium or fungus, that lives
within a plant for at least part of its life cycle without causing apparent
disease.

11. Classification of Bradyrhizobium
Kingdom : Bacteria
Phylum : Proteobacteria
Class : Alphaproteobacteria
Family : Bradyrhizobiacea
Genus : Bradyrhizobium

12. Bradyrhizobium
 Bradyrhizobium species are Gram-negative
bacilli (rod shaped) with a single subpolar
or polar flagellum.
 Slow growing.
 Nitrogen fixing.

14. Rice cultivars, soil sampling, and
endophytic Bradyrhizobial isolation
• Seven different rice
(Oryza sativa L.)
cultivars.
• Rice rhizospheric
soil samples.

• Poly-ethylene
boxes at 4°C.

16. Sterilization method of root
3%
• Sodium Hypochlorite for 5 min.
Soaked
• 70% Ethanol for 5 min.
Rinsed
• Sterilized distilled water at least
five times.

17. Cut
• Root into 3 cm to 5 cm long section.
Control
• To check superficial contamination.
• 200μl rinsed water spread on plate
count agar (PCA) medium.
Isolation
• Root crushed in 1 ml of sterilized water
by sterilized mortar and pestle.

18. To obtain the endophytic Bradyrhizobia from rice
tissues
FIRSTPROTOCOL
• 1 ml of root
homogenate.
• Inoculate.
• Germinated.
• Siratro (M.
atropurpureum)
seeds grown in
plastic growth
pouches.
• Nodule were
crushed.
• Streaked on
arabinose-
gluconate (AG)
agar plates.
SECONDPROTOCOL
• Oligotrophic-based
isolation.
• 1ml homogenate
root added to 50ml
sterilized water for
1 month.
• 0.1 ml of solution
was spread on
BJSM plates.
THIRDPROTOCOL
• 0.1 ml of root
homogenate.
• Spread on BJSM
plates.

19. BOX-A1R PCR were
used to screen for
redundant strains.
Different
bacterial strain
were selected.
Bacterial
identification
by 16S rRNA
gene analysis.

21. Phenotypic characteristics and
nitrogen fixation assay
Bacteria cultivate in the HM agar medium
Cells were grown aerobically at 30°C for 7 days
under a 12-hrs light/12-hrs dark cycle.
Cell pellets were extracted in the dark with cold
acetone-methanol (7:2 [vol/vol]) for 30 min.
Absorbance of the supernatant at wavelengths
from 350 to 850 nm.
Bradyrhizobia were grown at 30°C on YEM
modified agar medium.

22. For 7 days under aerobic conditions on 15-hr light/9-hr dark Cycle.
Lawn of bacteria - analyzed using a Beckman DU40 spectrophotometer
(Cary WinUV scan).
Symbiotic abilities of Bradyrhizobium strains were determined in
Leonard’s jars containing sterilized vermiculite.
1ml bacterial strain, inoculated onto germinated A. indica seed.
The root were used to measure nitrogenase activity by acetylene
reduction assay.

23. 16S rRNA gene, housekeeping gene,
and phylogenetic analyses
Genomic DNA strains grown in HM broth. The
primer pair fD1 and rP2 were used for gene
amplification.
Gene fragments were amplified using a Go Taq
Flexi DNA polymerase kit.
PCR products were purified using a Wizard SV gel
and PCR cleanup system (Promega, Germany).

24. The nucleotide sequences were aligned using the Clustal W
program.
The phylogenetic trees of the 16S rRNA gene and housekeeping gene
sequences were constructed by the maximum- likelihood method
using PhyML.
For comparison, phylogenetic trees were also reconstructed by the
distance neighbor-joining method using the MEGA, version 4.1,
package.
DNA sequences of each gene from related strains in the family
Bradyrhizobiaceae, of other rhizobia, and of outgroups were
obtained from the NCBI database.

25. GUS and DsRed-tagging of endophytic Bradyrhizobia
and monitoring of root colonization
• Bradyrhizobial strains were cultured in HEPES MES (HM) medium at 30°C.
• The Escherichia coli strains were cultured at optimum temperature in Luria-Bertani (LB)
medium.
• Add antibiotic 30 μg/ml gentamicin for E. coli and 200 μg/ml streptomycin for Bradyrhizobium
in the medium.
• Strains were tagged with pCAM120 and pBjGroEL4::DsRed2 by triparental mating on HM agar
plates using pRK2013 as a helper plasmid.
• 100 microlitre of cell suspension spread on plate.
Plates containing 100 μg/ml streptomycin, 100μg/ml spectinomycin, and 50 μg/ml polymyxin B.

26. Surface-sterilized seeds - germinated on a water-agar plate.
After 3 days, rice seedlings were transferred into 80-ml tubes
containing 10 ml of nitrogen- free plant nutrient solution.
3 days after the seedlings were transferred into the growth
medium.
(GUS)-tagged bacteria inoculated into the growth medium.

27. The roots were embedded in 5% agarose gel, and
90-μm-thick sections were prepared using a
vibratome.
Roots of 3-day-old
seedlings were
examined for
bacterial
colonization by GUS
staining.

28. Plant experiments
• A Leonard’s jar assembly was filled with sterilized vermiculite, and
nutrient solutions were applied through a wick to provide nutrients to the
plants.
• One milliliter of 5-day-old inoculum was inoculated onto the seedling at 2
days after transplanting.
• Plants were grown under controlled environmental conditions.
• Dry weight plant were measured after 28 days.

29. Enumeration of endophytic Bradyrhizobia
Incubate for 7days at optimum temperature.
Blue colonies were observed and count the bacterial population density.
Spread on yeast extract-mannitol (YM) plates containing
streptomycin(300μg/ml).
5-bromo-4-chloro-3-indolyl-D-glucuronide (X-Gluc;10μg/ml).
Root was surface-sterilized and crushed.
Diluted in 0.85% (wt/vol) saline solution.

30. Plant nodulation tests
Bradyrhizobial strains
Growth chamber
Sterilized Seed
• grown for 5 days in HM
broth.
• Controlled environmental
conditions.
• Mung bean (Vigna
radiata), soybean (Glycine
max cv. SJ5), and siratro
(M. atropurpureum)
seeds.

31. Sterilized
Nodulation
test
Root
nodules
• Seeds of Aeschynomene
americana (a local Thai
variety), A. indica
(African ecotype), and
Aeschynomene evenia.
• Performed in sterilized
plastic pouches for five
replicates.
• Tested plants were
enumerated at 28 days
post inoculation (p.i.).

33. Bacterial isolation
Endophytic Bradyrhizobial strains (DOA1, DOA9, and SUTN9-2) isolated from A. americana L.
Other isolates Alphaproteobacteria , Firmicutes, Flavobacteria, and Betaproteobacteria.
Total six different strains of putative rice endophytic Bradyrhizobia were obtained.
107 isolates were obtained directly from BJSM culture.
153 bacterial colonies screened to remove the redundant strains using BOX-A1R PCR.
91 colonies were isolated from the crop rotation system.
62 bacterial colonies obtained from the monoculture system.

34. To enhance the opportunity to acquire rice endophytic
Bradyrhizobia
• SUT-R55 was obtained from root
nodules of siratro.
First
• Six Bradyrhizobial strains, were obtain
that is SUT-R3, SUT-PR9, SUT-PR48,
SUT-R55, and SUT-PR64.Second
• Three strains, SUT-R3, SUT-R55, and
SUT-R74.
Third

35. Phenotypic characteristics
 All of the strains formed typical slow-growing
Bradyrhizobium colonies on HM agar plates
 Strains SUT-PR48 and SUT-PR64 could synthesize
the pink/orange pigments
 Not detected - strains DOA1, DOA9, SUTN9-2,
SUT-R3, SUT-PR9, SUT-R55, SUT-R74 and
Japanese bradyrhizobial strains

38. Promotion of growth of rice

40. Endophytic bradyrhizobium population
from different rice cultivars

43. Nodulation test and A. indica
growth promotion
The PB strains, SUT-PR48 and SUT-PR64, produce up to
100 nodules per plant of A. indica and A. evenia.

47. To detect the nodulation gene
 PCR amplification of nodA, nodB,
nodC.
 The primer sets were designed from
B. diazoefficiens USDA110 and
Bradyrhizobium sp. ORS285.
 No specific bands representing nodA,
nodB, and nodC .

49.  All of rice oligotrophic endophytic Bradyrhizobia in this
study were obtained except SUT-R74.
 6 Bradyrhizobial strains were obtained from 98
bacterial strains.
 Bradyrhizobium is found only in rice root, with 10%
relative abundance of total Alphaproteobacteria.
 Endophytic Bradyrhizobia could not be obtained from
the monoculture system.
 Thai rice cultivars, the Thai Bradyrhizobial strains
could promote rice growth better than Japanese
strains.

50.  Three rice cultivars (Pathum Thani 1, Kasalath, and
Nipponbare), cultivar Pathum Thani 1 responded only
to putative Thai rice endophytic Bradyrhizobia.
 This phenomenon was not found in Japanese rice
cultivars.
 Non-PB strains are also capable of forming a natural
endophytic association with rice.
 Strains SUT-PR9, WD16, RP5, and RP7 displayed
non-PB phenotypes but were genotypically close to
PB strains.

54.  Piromyou at el., preferential association of endophytic bradyrhizobia with different rice
cultivars and its implications for rice endophyte evolution, Applied and Environment
Microbiology81:9(2015)3049-3061
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Hattori M, Hattori R, Hattori T. 2013. Soil oligotrophic bacterium Agromonas oligotrophica
(Bradyrhizobium oligotrophicum) is a nitrogen-fixing symbiont of Aeschynomene indica as
suggested by genome analysis. Appl Environ Microbiol 79:2542–2551.
http://dx.doi.org/10.1128/AEM.00009-13.
 Chaintreuil C, Giraud E, Prin Y, Lorquin J, Bâ A, Gillis M, de Lajudie P, Dreyfus B. 2000.
Photosynthetic bradyrhizobia are natural endophytes of the African wild rice Oryza
breviligulata. Appl Environ Microbiol 66:5437–5447. http://dx.doi.org/10.1128/AEM.66.12.5437-
5447.2000.
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