10. agri sci - ijasr - characterization - gauri

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).


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


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).


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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standardized single disk method. Am J Clin Pathol; 45: 493-6.

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fixationof soyabeans. Jour of weed sci. 33, 427-430.

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production using various starchy substances as carbon substrates. Braz. J. Microbiol. 38: 208-

216.

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symbiotic efficiency of nitrogen-fixing rhizobia and host plants. Process Nat. Acad. Sci. USA,

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6. Goring CAI and Laskowski DA (1982). The effects of pesticides on nitrogen transformations in

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Selenite-Reducing -Proteobacteria Isolated From a Bioreactor. Curr. Microbiol. 55: 455-460.

12. Kucuk, C. and Kivanc, M. (2008). Preliminary Characterization of Rhizobium Strains.Isolated

from Chickpea Nodules. Afr. J. Biotechnol., 7(6): 772-775.

13. L’taief B, Sifi B, Gtari M, Zaman-Allah M and Lachaal M (2007). Phenotypic and molecular

characterization of chickpea rhizobia isolated from different areas of Tunisia. Can. J. Microbiol.

53: 427-434.

14. Lawson, I.Y.D. Muramatsu, K. and Nioh, I. (1995). Effect of organic matter on the growth,

nodulation and nitrogen fixation of soybean grown under acid and saline conditions. Soil Sci.

Plant Nutr.41:721-728.

15. Lupwayi, N. and Haque, I. 1994. Legume-Rhizobium Technology Manual. Environmental

Sciences Division International Livestock Centre for Africa. Addis Ababa, Ethiopia. 1-93

16. Maâtallah J, Berraho EB, Sanjuan J and Lluch C (2002). Phenotypic characterization of rhizobia

isolated from chickpea (Cicer arietinum) growing in Moroccan soils. Agronomie, 22: 321-329.

17. Martyniuk S, Wozniakowska A, Martyniuk M and Oron J (1999b). Interaction between chemical

dressings and Rhizobium inoculant on pea seeds. Prog. Plant Prot. 39(1): 120-125.

18. Martyniuk S, Oron J, Mertyniuk M and Woznıakowska A (2002). Effects of interactions between

chemical seed dressings and Bradyrhizobium japonicum on soybean seeds. Arch. Acker-Pfl.

Boden. 48: 305-310.

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characteristics of Mesorhizobium species isolated from chickpea (Cicer arietinum L.) growing

areas of Ethiopia Afric J BiotechnolVol. 11 (29), pp. 7483-7493

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Tolerance of Vicia faba Plants under Salinity Stress. Afr. J. Biotechnol., 4(3): 210-222.

21. Ramadoss S and Sivaprakasam K (1991). Effect of fungicides and insecticides on Rhizobium and

Rhizobium-cowpea symbiosis. Soil Fertil. 54(2): p. 282.

22. Sadowsky MJ, Keyser HH and Bohlool BB (1983). Biochemical Characterization of fast- and

slow-growing rhizobia that nodulate Soybeans. Int. J. Syst. Bacteriol. 33: 716-722.


23. Saraf, M. and Dhandhukia, P. (2005). Response of Sinorhizobium meliloti toHigh Salt

Concentration and Effect of Added Osmotica. J. Microb. World, 7: 250-257.

24. Stowers MD (1985). Carbon metabolism in Rhizobium species. Annu. Rev. Micrbiol. 39:89-108.

25. Vincent J.M., (1970), A manual for the practical study of the root nodule bacteria.45.

26. Wei G.H., Tan, E.T., Zhu, M.E., Wang. E.T., Han, S.Z. and Chen, W.X. 2003. Characterization

of rhizobia isolated from legume species within the genera Astragalus and Lespedeza grown in

the Loess Plateau of China and description of

RhizobiumloessensespInt.J.Evol.Microbiol.53:1575-

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


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.


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.


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.


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.


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.

10. agri sci - ijasr - characterization - gauri

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