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Bioscience DiscoveryVol02, No. 1, Jan. 201L
MORPHOLOGICAL, CUUIURAL AND BIOCHEMICAL CHARACTERISTICS OF
RIIIZOBIUM JAPONICUM SYN AND BMDYRHIZOBIUM TAPONICUM OF'
SOYBEAN
I Dqnd Khansole 6 S1
Post Graduate Department of Botany and Chemistryr
N. F.S. Science College, Nanded-431605 (M.S.)
Received: 10 July 2010; Revised: 22october 2010
Gachande
ABSTRACT
,
Rhizobium joponicum syn. and Brodyrhizobium japonicum was isolated from root
nodules of Soybean (Glycine max L.) on yeast extract manitol agar (YEMA) medium and its
morphological, cultural and biochemical-characteristics were studied. lt was observed that
the colonies were circular, light pink, convex, entire and opac. The bacterium was rod
shaped, aerobic, non-spore forming and motile. lt showed negative chemical reaction for
indole, methyl red, Voges-Proskaur, hydrogen sulphide production, utilization of
carbohydrates and gelatin hydrolysis. While it showed positive reaction for citrate utilization,
catalase dnd ammonia production from peptone and urea.
Key words: Soybean, Rhizobium japonicum syn. Bradyrhizobium ioponicum.
|NTRODUC,I|oN
!
,
(Purohit & Kumar 1998). Therefore the attempt has
Rhizobio are well known for their been made to study the morphological and
'
capacity to establish a symbiosis with legumes. liochemical characters of the bacterium.
Legumes are unique plants which have the ability
to work with certain bacteria i.e. Rhizoibio to MATERIATANDMETHODS
The mature root nodules from Soybean
gather available nitrogen from the soil
atmosphere and convert it to usable ammonia plantwerecollectedfromfieldwashedthoroughly
They under tap water and surface sterilized with 0.1 %
inhabit root lnodules, where they reduce mercuric chloride. Surface sterilized root nodules
atmospheric nitrogen and make it available to were crushed in small quantity of sterile distilted
the plant. Biological nitrogen fixation is a water.Rhizobialsuspensionwasinoculatedonpre
component of sustainable agriculture and sterilized YEMA medium and incubated at 26 t3-eC
Rhizobialinoculantshavebeenappliedfrequently temperature for 10 days. The isolates were
maintained on slopes of YEMA medium as
as bio-fertilizers.
Each major legume group is nodulated described by Graham and Parker (1964) these
by different species of Rhizobium. Soybeans are isolates were used to study the morphological,
nodulated by Rhizobium joponicum (Krichner cultural and biochemical characteristics. Seven
nitrogen and make it available to the plant.
1896; Buchanan 1926). Fred and his associates days old culture was used for Gram's reaction,
(1932) recognized eight cross inoculants group morphology and colony characters. The pattern of
in legumes. The genus Rhizobium was erected by growth was observed in motility agar medium.
Frank (1890) based on its characters to form Growth on Hofer's alkaline medium was examined
nodules on roots of legume plants. This property in YEMA broth with bromothymol blue at PH L0.00
is the only valid test in the identification of the (Hofer 1935).
organism. Apart from it some diagnostic features
Production of indole was noticed in
of Rhizobium could be conveniently not only to inoculated tryptophan broth after 7 days of
determine and identify the organism but also incubationbyaddingcovac'sreagent.Thereduction
delinate different species (Graham and Parker ;il;;i
;"d-rnJ vog"r- proskauer reaction was
1.964; Vincent L97O; Gaur 1975; Mahana 1981)
in glucose phosphate broth by adding
examined
Rhizobium iaponicum syn. Bradyrhizobium- methyl reA a'nA a-napthol solution with KOH
ioponicum is associated with the root nodules of respectively.
- ',,Citrate utilization was observed by
.,,.
Soybean and fixes 100 kg nitrogen/ha/year .i-r.
,","..u,o *.rn ,rorornrro,
L
Gachonde B D ond Khqnsole G S
blue in basal medium. Liquefaction of
gelatin was tested in LT/o gelatin in agar medium
after 14 days of lncubation (Polhman 1931).
Hydrolysis of starch was examined by streaking
the organism on starch with nutrient agar and
iodine solution. The production of ammonia by
the bacterium was eiamined from the inoculated
tube containing peptone water with potassium
nitrate and Nessler's reagent. Production of
hydrogen sulphide gas examine_d by method
described by Zobell and Felt ham 1934. Production
of ammonia:from urea was examined
by
Christensen urea agar with phenol red as an
indicator. Fermentation of carbohydrates was
tested by adding 1.0% Andrade's,indicator in the
basal medium containing peptone water and 2%
sugar. ln case of effect
of PH and temperature,
yeast extract manitol broth tubes were incubated
foi three days and growth was recorded in terms
of turbidity. The Gram's staining technique was
followed as suggested by Graham and Parker
1964. Catalase activity was observed by stirreing
the culture in a drop of hydrogen peroxide (10%
by W/V), while oxidase activity was tested
according to Kovaks (1956).
Table 1: cultural/Morphological and staining characten ol Rhhobium japonicum.
Charapters
Result
Shape
Circu lar
Size
of colony
.i
3.1 mm
Colour/Pigmentation
Whitish pink and glistering
Elevation
Convex
Margin
Regular/entire
Opacity
Opaque
Mdtility
Motile
Bacterium shape
Rod shaped
Oxygen demand
Aerobic
Spore formation
Non spore forming
Gram's nature
Gram - ve
RESULTSAND DISCUSSION
M o rp hol og i ca I Ch a ra cte rs
show any growth on Hofer's alkaline medium at
PH 10.0 with slight growth and evolution of gas
and acid production. According to Ditmer (1930)
The Rhizobium japonicum (Krichner and
those Rhizobia, which produce acid are
Buchanan) Syn. Brodyrhizobium japonicum was
Gram negative, aerobic, non-spore forming and
motile rods. ln general, the colonies we?e circular,
convex, whitish pink and glistering with entire
margin.
considered to be advanced type and those, which
Biochemicol Chorocter
The bacterium showed well-marked growth on
YEMA medium at PH 7.0.However poorgrowth was
observed on Hofer's medium. Mahana et al. (2000),
reported that the Rhizobium isolated from Vigno
mungo L. showed marked variations in growth with
respect to time period on YEMA while they do not
produce alkali, are ancient type.
The bacterium showed positive test for
citrate, production of ammonia and catalase
activity. Nitrate is reduced to nitrite producing
ammonia. Neal and Walker (1935) suggested
rapid nitrate utilization by slow growing root
nodule bacteria. Mahana et ol. 2000 -reported
catalase activity in some isolates from Vigna
mungo.The bacterium is negative for MR-VP and
indole reaction. Similarly, Grahm and Parker
(1964) did not observe MR reduction in all the
isolates of seven rhizobia groups. While Basak
Bioscience DiscoveryVol02, No. L, lan.20LL
and Goyal (1980) also reported that none of
ISSN 2229-3469
the
rhizobial. isolates
indole.
of of seven groups produces
Table2: Biodremical Characters of Rhizobium japonkum
5r.No
Test
Remark
1
Production of indole from tryptophan
Methyl red test
-Ve
2
3
Voges-Proskaur test
-Ve
4
+Ve
6
Ctrate utilization as source of carbon
Hydrolysii of gelatin
Production of ammonia from peptone
7
Production of hydrogen peroxide
-Ve
8
Production of ammonia from urea
rVe
9
Uti
10
PH 7.0
11
Eltect of PH on giowth'of Rhizobium
Effect of temperature on growth of Rhizobium
t2
Catalase test
+Ve
13
Hydrolysis of starch
-Ve
5
-Ve
-Ve
+Ve
lization of carbohydrates
-Ve
Room temp;
Biochemiral teets fcr R*
fuedion otrr**b
&oss
pg*oe
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ldbftyleeadtert
Gochande B D and Khonsole G S
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Fred
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