CHARACTERIZATION OF MESORHIZOBIUM SP. ISOLATED FROM
ROOT NODULES OF CICER ARIETINUM
GAURI, ASHOK KUMAR SINGH AND MEENAKSHI BAMANIA
Department of Microbiology, Dolphin (PG) Institute of Biomedical & Natural Sciences, Dehradun, India
ABSTRACT
This study was carried out to be investigate the effect of salt, pH, antibiotics, carbohydrate and
fungicides on growth of Mesorhizobium isolates obtained from root nodules of chick pea plants from
different sites of Dehradun. After morphological and biochemical test, 40 isolates were selected. Isolates
were subjected to different (2%, 3%..........up to 8%) NaCl concentrations and found that all isolates grew
up to 4% NaCl concentration, variation starts from 5% NaCl and salt tolerance ability was reduced with
increasing salt concentration. All isolates grew at pH 4.0 to pH 8.5 and none of the isolates at pH 9.0.
Ten carbohydrates were used for describing carbohydrate utilization pattern. Most of the isolates
catabolize nine carbohydrates while only 47.5% showed resistance to dextrose. All of the isolates were
showed sensitivity to Meropenem, Netillin, Ceftriaxone and Amikacin antibiotics. All isolates were
tested for their tolerance to four fungicides. The effect of the fungicides on the isolates was variable,
depending on the fungicide and isolate. Further studies are needed to study the genes involved in salt,
pH, carbohydrates, fungicides, antibiotic resistance and the relationship between these genes and the
symbiotic genes.
KEYWORDS : Antibiotics, Carbohydrate, Chick pea, Fungicides and Mesorhizobium.
INTRODUCTION
The earth’s population grows annually by 1.4% and is expected to double in the next fifty
years. This increase in population necessitates a simultaneous enhance in food production to maintain the
dietary intake of the growing human population in an environmentally sustainable manner. This demand
for higher crop production also implies a higher demand for fixed nitrogen (Graham & Vance,2000).
Chemically produced nitrogen fertilizers can provide this nitrogen, but they are expensive to produce in
addition to being harmful to the environment. This damage to the environment includes changes in the
global nitrogen cycle, loss of nitrous oxides to the atmosphere, acid rain, nitrate pollution of ground
water & induced leaching of soil nutrients. An inexpensive & environmentally friendly alternative to
nitrogen fertilizer is biological nitrogen fixation (BNF), which is a process where by nitrogen gas in the
atmosphere is converted into biologically useful & utilizable source of nitrogen for plants. The majority
of the world’s land based biological nitrogen fixation can be accounted for by the symbiotic nitrogen
fixation relationship between leguminous plants & rhizobia. The advantages of this type of BNF have led
to numerous studies investigating the diversity & identity of the associated bacterial symbionts.
International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol.2, Issue 3 Sep 2012 142-154 © TJPRC Pvt. Ltd.,
143 Characterization of Mesorhizobium Sp. Isolated from Root Nodules of Cicer Arietinum
Although symbiotic nitrogen fixation by legumes is generally the dominant source of nitrogen
input in soil for imparting fertility but soil stresses, pose a severe yield constraint in obtaining plant
growth and development (Lawson et al, 1995). Environmental stresses adversely affect symbiotic
nitrogen fixation in both tropical and temperate soils (Graham, 1981). Limiting rhizobial survival and
persistence in soils and reduce nodulation.Typical environmental stress faced by the legume nodules &
the symbiotic partners may include water stress, salinity, soil pH, temperature, heavy metals, fungicides
etc (Kucuk & Kinvanc,2008).
This study determines the diversity of the Mesorhizobium that nodulates Chick pea through
their growth characteristics, salt and ph tolerance, intrinsic antibiotic resistance, carbon source utilization
and fungicide resistance pattern. This could lead to an improvement in the selection of isolates adapted
to the climatic conditions.
MATERIALS AND METHODS
Collection of Samples
Root nodulating bacterial strains were isolated from the root nodule of Cicer arietinum (chick
pea) according to Vincent (1970), collected from different sites of Dehradun (U.K).
Isolation and Characterization of Root Nodulating Bacteria
The collected nodules were first surface-sterilized with 95% ethanol, then with 0.1% mercuric
chloride and finally washed thoroughly with distilled water. strains were obtained by streaking the
crushed root nodules on YEMA plates and incubated at 28±2°C .After 2 days of incubation, Rhizobium
colonies were obtained. Further streaking, spreading and visual characterization of colony morphology
helped in isolation of pure cultures..Biochemical characterization of recovered isolates were done
according to Bergey’s Manual of Determinative Bacteriology (Holt et. al., 1994) All the test were carried
out with 03 replicates
Salt and pH tolerance
The ability of the isolated Rhizobial strain to grow in different concentration of salt was tested
by streaking them on YEMA medium containing 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0% and 8.0%
(wt/vol) NaCl. Differences in pH tolerance were tested in YEM agar. The pH was adjusted to 4.0, 5.0,
5.5, 6.0, 6.5, 7.0, 8.0, 8.5 and 9.0 with HCl or NaOH. All the plates were incubated at 28°C during 72
hours and plates containing basal YEMA medium were used as controls.
Strains were considered salt tolerant and resistant to acidity when growth was similar to the growth in the
control plates.
Antibiotic Resistance Pattern of Isolates
All the isolates were tested for antibiotic sensitivity by Kirby-bauer disc diffusion method
(Bauer et al; 1966) on YEMA. Cultures were inoculated by swabbing with standard inoculums
(corresponding to 0.5 Mc Farland tube over the entire agar surface. The agar surface was allowed to dry
Gauri, Ashok Kumar Singh & Meenakshi Bamania 144
for 3-5 minutes before applying the antibiotic disc using sterile forcep. The plates were incubated at 300C
for 48 hours, sensitive to an antibiotic was detected by zone of inhibition around the disc. The following
antibiotic discs were used, Cefalexin(CN30), Meropenem(MRP10), Polymixin-B(PB50), Netillin(NET30),
Amikacin(AK30), Ceftriaxone(CTR30), Clindamycine(CD2), Streptomycin(S25), Amoxycilline(AMX25),
Ampicilline(AMP25).
Determination of Carbohydrate Utilization
To determine the capability of isolates to use carbohydrates (Mannitol, Dextrose, Maltose,
Galactose, Mannose, Sucrose, Raffinose, Arabinose, Sorbitol, Lactose.) fresh culture was spread on
YEMA plates. The agar surface was allowed to dry for 3-5 minutes before applying the carbohydrate
disc using sterile forcep. The plates were incubated at 300C for 48 hours. Carbohydrate resistance was
detected by zone of inhibition around the disc.
Determination of Fungicide Tolerance
The effect of fungicides on the growth of strains on YEMA medium were tested as described
by Bollich et al, (1985) using measurement of diameters of the growth inhibition zones. The fungicides
used were:- Thiram (75%), Mancozeb (75%), Zineb (75%), Carbendazim (50%). The concentrations
used were 25, 50, 75 and 100 g/l. The cultures were spread over the entire agar surface four disc of filter
paper from different concentrations were applied using sterile forcep. The plates were incubated at
300C.The diameters of the inhibited growth zones formed were measured after 24 hours. A plate with
filter paper disc without any fungicide was used as control.
RESULTS
Isolation and Characterization of Mesorhizobium Sp.
Bacterial isolates were recovered from chick pea root nodules collected from different sites of
Dehradun. All isolates showed colonies similar in appearance with large mucoid (LM) and large watery
(LW) with diameter of 2-4.5 mm, round, white colored till 3-4 days of growth and turning yellowish in
color after 4 days. All isolates were gram negative rods (Table 1) Selected isolates were catalase positive
as confirmed by the liberation of oxygen around the bacterial colonies. Positive results for oxidase and
urease were also observed by different isolates. None of the strain showed growth on medium containing
methylene blue (0.1%), gentian violet (0.1%), glucose peptone agar and citrate agar. All isolates showed
growth on lactose peptone agar. Positive results were obtained from starch hydrolysis assay, when the
inoculated plates were subjected to iodine test, which was indicated by a clear zone around the colonies.
All the isolates were gelatinase negative as there was no clearing zone formed around the colonies . On
the basis of these tests forty isolates were designated as BCR1-BCR14, ECR1-ECR4, TCR1-TCR5,
SCR1-SCR8, DCR1-DCR5, MCR1-MCR2 and HCR1-HCR2.(Table 2.0).
145 Characterization of Mesorhizobium Sp. Isolated from Root Nodules of Cicer Arietinum
Effects of Salt and Ph on Different Mesorhizobium Isolates
Tolerance of rhizobial isolates to NaCl concentration showed variation among strains. All
isolates showed growth at YEMA containing 2%, 3% and 4%,of NaCl. isolate. At higher concentrations,
number of tolerant strains decreased rapidly and only eight isolates i.e. BCR2, ECR2, SCR3, SCR5,
SCR6, DCR3, DCR4, & DCR5 tolerated NaCl concentrations of 5%, 6%, 7%, & 8% (Figure 1)
Differences in pH tolerance are shown in Figure2. All isolates except SCR5 grew at pH 4.0 to 8.5. All
were found to resist at pH 9.0.
Antibiotic Resistance Pattern of Recovered Isolates
All isolates were sensitive to Meropenem, Netillin, Amikacine, and Ceftriaxone. Resistance to
Ampicillin, Streptomycin, Amoxycilline, Clindamycin, Polymyxin-B, and Cefalexin was recorded for
only 45%, 5%, 57.5%, 25%, 10% and 12.5% of the isolates, respectively. Isolates BCR13, BCR14,
SCR4, SCR5, SCR6, SCR7, DCR3, DCR4, MCR1, and HCR1 were found to be tolerant to almost all
antibiotics. Maximum resistance were observed with Amoxycilline and Ampicillin, the most potent
antibiotics that allow the growth of only few isolates (Figure3).
Carbohydrate Utilization Pattern of Mesorhizobium Isolates
Almost all of the isolates were able to catabolize a large variety of carbon substrates (Tables
4.7). All tested strains grew on Mannitol, Lactose, Sucrose, Sorbitol, Arabinose, Galactose, Mannose,
Maltose, and Raffinose except Dextrose. Only 47.5% isolates utilized Dextrose. All isolates utilized
Sorbitol except TCR2 & DCR7 and only DCR7 was also unable to utilized Mannose. 97.5% of the
isolates utilized Raffinose and Mannitol (Figure4).
Effects of Different Concentration of Fungicides on Mesorhizobium Isolates
The results presented in Figures 5-8 shown the effect of four fungicides namely Mancozeb,
Zineb, Thiram & Carbendazim .All parameters depends on the tested fungicide and applied
concentrations. Almost 98% isolates were found to be sensitive against Mancozeb, Thiram and Zineb.
SCR4, SCR2, and MCR1 were found to be resistant at 25, 50, 75, concentrations, respectively against
Mancozeb. DCR5 and MCR1 were resistant to all concentrations of Zineb. At lower concentration
(25mg/l) BCR11, TCR5, SCR1, SCR6, SCR8 were showed no zones against Thiram.Most of the isolates
showed resistance behavior while using Carbendazim.. Twenty three isolates ie. BCR1, BCR2, BCR7,
BCR9, BCR12, BCR13, BCR14, ECR1, TCR1, TCR2, TCR3, TCR5, SCR1, SCR2, SCR3, SCR4,
SCR6, SCR7, SCR8, DCR3, DCR4, HCR1 and HCR2 showed resistance to all tested concentrations
used in present study.
DISCUSSIONS
The root nodule bacteria were isolated from Cicer arietinum (chick pea) plants growing in
different regions of Dehradun. Microscopic examination showed that the isolates were gram negative
rods. Biochemical characterization revealed that the selected isolates were oxidase, catalase and urease
Gauri, Ashok Kumar Singh & Meenakshi Bamania 146
positive. Isolates were able to utilize citrate, which complements with the findings of Lupwayi and
Hague (1994). None of the isolate showed growth on medium containing dyes i.e methylene blue and
gentian violet at 1% concentration, which correlates with the earlier studies by Wei et.al. (2003), who
indicated that Rhizobial cells were unable to grow in the presence of these two dyes. All the isolates were
able to grow on lactose peptone agar and growth was absent on glucose peptone agar. The isolates were
not producing gelatinase enzyme and it is shown by Hunter (2007) that negative gelatinase activity is a
feature of Rhizobium. Positive results were obtained when the isolates were subjected to medium
containing starch. De Oliveria et. al (2007) also observed that Rhizobium strains obtained from different
sources can utilize starch.
Growth of the rhizobial strains were not much affected as the concentration of the salt increases
from 2%- 4%. High concentration of NaCl solution can give high competitive value in the rhizosphere to
survive and nodulate the host plants in harsh environmental conditions particularly at high concentrations
of salt in the soil. This finding is in line with the report of Saraf and Dhandhukia (2005), who found that
Sinorhizobium meliloti growth was not completely inhibited by 4% of NaCl concentration. Rabie and
Alamadini (2005) also stated that the growth of rhizobium was not affected by low and moderate levels
of salinity. Rhizobium has been reported to grow the best at neutral pH i.e. 7.0. All the isolates grown
very well on pH 4.0-8.5. No growth was observed in pH 9.0. Our results were in agreement with
previous studies (Kucuk et al, 2006; Baoling et al, 2007).
The results on the resistance patterns of the isolates to ten antibiotics reported high level of
resistance against Ampicillin, Amoxycilline, Clindamycine and Streptomycin and least with
Meropenem, Netillin, Amikacine, and Ceftriaxone .Several studies (Maâtallah et al., 2002;Küçük and
Kıvanç, 2008) also observed great variation among chickpea rhizobia with respect to their intrinsic
antibiotics resistance pattern (IAR). Sensitivity of isolates to antibiotics may be due to the fact that these
bacteria have not been exposed to these antibiotics in natural environments. Depending on the
differences in antibiotic resistance pattern, this technique could be successfully employed in ecological
studies particularly in the recovery and enumeration of rhizobia introduced into soil.
It has been a well-established fact that Rhizobium can utilize a wide variety of carbon sources
for growth, an effective tool to characterize the isolates. Results of the present study ion utilization of
different C sources showed that the strains isolated from Chickpea nodules were able to utilize Mannitol,
Lactose, Sucrose, Sorbitol, Arabinose, Galactose, Mannose, Maltose, and Raffinose as carbon sources.
Similar results were also recorded by Sadowsky et al., 1983 and Stowers, 1985 in case of the Rhizobium
strains. Fast-growing rhizobia were able to grow on a large variety of carbon substrates whereas slow-
growing rhizobia were more limited in their ability to use diverse carbon sources. However, our result
shows that the majority of tested slow-growing chickpea rhizobia were able to use a broad range of
carbohydrates. This is in line with the result of other studies (Matalah et al., 2002; L’taief et al., 2007
Mulissa Jida and Fassil Assefa., 2012). It is very interesting to notice that the types of carbohydrates
utilized also varied among chickpea rhizobia. Such characteristics are usually used as diagnostic features
for root nodule bacteria (Küçük and Kıvanç, 2008; Hungria et al., 2001).
147 Characterization of Mesorhizobium Sp. Isolated from Root Nodules of Cicer Arietinum
Nitrogen fixation by root nodulating isolate of Rhizobium contributes a significant input into the
many types of farming systems. For instance, even 80% of total N in pasture legume plants can be
supplied by root nodule bacteria (Goring and Laskowski, 1982). Fungicides applied as seed dressings
protect seeds against fungal pathogens and pests (Martyniuk et al., 1999b). In the case of leguminous
plants, treatment of seeds with Rhizobium inoculants is also very important (Martyniuk et al., 2002).
When Rhizobium bacteria are inoculated on chemically treated seeds of crop, their survival and capacity
to induce symbiosis can be markedly reduced due to possible toxic effects of fungicides on these bacteria
(Rennie and Dubetz, 1984).
Therefore, the effect of fungicides on legume nitrogen fixation is of great importance In present
study, all the isolates were tested for their tolerance to Mancozeb, Carbendazim, Thiram and Zienb
fungicides. Most of the isolates showed sensitivity to Thiram and resistance to Carbendazim. Many
studies have indicated differential effect of fungicides on Rhizobium, nodulation and nitrogen fixation
(Fox et al., 2007; Ramadoss and Sivaprakasam 1991).
The results obtained are a part of our successful efforts to contribute to screen bacteria from root
nodules which can be future candidates for increasing productivity of agriculture crops which is at
decline presently. The results on screening of Mesorhizobium strains resistant to various antibiotics and
also to determine optimum level of tolerance for fungicides and other stress conditions may also help to
grow crops in highly polluted soils.
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149 Characterization of Mesorhizobium Sp. Isolated from Root Nodules of Cicer Arietinum
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APPENDICES
Table 1: Morphological and biochemical characteristics of all recovered isolates from Chickpea
CHARACTERS RESULTS Bacterium shape Rod shaped Size of colony 3.1mm
Color Whitish pink and glistering
Elevation Convex Margin Regular/entire opacity Opaque
Oxygen demand Aerobic Spore formation Non spore forming Gram staining Negative
1% Methylene blue treatment
Negative
1%Gentian violet treatment
Negative
Growth on Glucose peptone agar
Positive
Growth on Lactose peptone agar
Positive
Gelatin hydrolysis Negative Fluorescence assay Negative Starch hydrolysis Positive
Nitrate reduction test Positive Triple sugar iron test A/K, A/A, K/A
Catalase test Positive Oxidase test positive Unease test positive
Gauri, Ashok Kumar Singh & Meenakshi Bamania 150
Table 2 Mesorhizobium isolates obtained from different sites of Dehradun. PLACE→
BHAUWALA
ENGENRING
TILWARI
SURVEY
DHARAPUR
MANDU-
HATHI- No. Of
CROP↓ ENCLAVE ESTATE
WALA BARKALA
ISOLATES
BCR 1- 14
14
ECR 1- 4 4
CHICK TCR 1-5
5
PEA SCR 1-8
8
PLANTS DCR 1-5
5
MCR 1-2
2
HCR 1-2
2
NO. OF 14 4 5 8 5 2 2 40
ISOLATES
0
20
40
60
80
100
02
34
56
78
PE
RC
EN
TA
GE
OF
TO
LE
RE
NT
&
SE
NS
ITIV
E
CONCENTRATION OF SALT
Tolerent
sensitive
Figure1 Effects of salt concentrations on growth of Mesorhizobium sp.
151 Characterization of Mesorhizobium Sp. Isolated from Root Nodules of Cicer Arietinum
0
20
40
60
80
100
o 5 55.5 6
6.5 7 88.5 9
PE
RC
EN
TA
GE
O
F T
OL
ER
EN
T &
SE
NS
ITIV
E
DIFFERENT PH
Tolerent
Sensitive
Figure 2 Effect of pH on growth on Mesorhizobium sp.
0
20
40
60
80
100
PE
RC
EN
TA
GE
O
F T
OL
ER
AN
T &
SE
NS
ITIV
E
CONCENTRATION OF ANTIBIOTICS .
Tolerant
Sensitive
Figure3 Effects of antibiotics on growth of Mesorhizobium sp.
Gauri, Ashok Kumar Singh & Meenakshi Bamania 152
0
20
40
60
80
100P
ER
CE
NT
AG
E O
F T
OL
ER
EN
T &
SE
NS
ITIV
E
CONCENTRATION OF CARBOHYDRATES
Tolerent
Sensitive
Figure 4 Effects of carbohydrates on growth of Mesorhizobium sp.
0
20
40
60
80
100
025
5075
100
PE
RC
EN
TA
GE
O
F T
OL
ER
AN
T &
SE
NS
ITIV
E
CONCENTRATIONS OF MANCOZEB
Tolerant
sensitive
Figure5 Effects of mancozeb on growth of Mesorhizobium sp.
153 Characterization of Mesorhizobium Sp. Isolated from Root Nodules of Cicer Arietinum
0
20
40
60
80
100
025
5075
100
PE
RC
EN
TA
GE
OF
TO
LE
RA
NT
&
SE
NS
ITIV
E
CONCENTRATIONS OF ZINEB
Tolerant
Sensitive
Figure6 Effects of zineb on growth of Mesorhizobium sp.
0
20
40
60
80
100
025
5075
100
PE
RC
EN
TA
GE
O
F T
OL
ER
AN
T &
SE
NS
ITIV
E
CONCENTRATION OF THIRAM
Tolerant
Sensitive
Figure7 Effects of thiram on growth of Mesorhizobium sp.
Gauri, Ashok Kumar Singh & Meenakshi Bamania 154
0
20
40
60
80
100
025
5075
100
PE
RC
EN
TA
GE
O
F T
OL
ER
AN
T &
SE
NS
ITIV
E
CONCENTRATIONS OF CARBENDAZIM.
Tolerant
Sensitive
Figure 8 Effects of carbendazim on growth of Mesorhizobium sp.
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