International Journal of Scientific Research in Knowledge, 3(11), pp. 0270-276, 2015

Available online at http://www.ijsrpub.com/ijsrk

ISSN: 2322-4541; ©2015; Author(s) retain the copyright of this article

http://dx.doi.org/10.12983/ijsrk-2015-p0270-0276

270

Full Length Research Paper

Isolation and Biochemical Characterization of Bacteria Isolated from Groundnut

(Arachis hypogaea L.) in Sudan

Abdelmalik O. A. Idris*1,2; Awad M. Abdel Rahim2; Abdel Moneim E. Suleiman3

1Department of Biology, Faculty of Education, University of Gadarif, Sudan 2Faculty of Sciences, University of Gezira, Sudan

3Faculty of Sciences, University of Hail, Kingdom of Saudi Arabia

*Corresponding Author: E-Mail: malikidris1977@hotmail.com; Mobiles: 00249912953363 - 00249123232431

Received 28 August 2015; Accepted 14 November 2015

Abstract. Using of commercial bacteria showed unacceptable results in inoculation trials. This necessitates looking for locally

isolated nitrogen fixing bacteria to use as inoculants. This study was done to isolate and select the most efficient bacteria from

groundnut (Arachis hypogaea) root nodules. In this study three bacterial strains were isolated from El-Gadarif, El-Gezira and

El-Obied soils and pot experiments were done. The study showed that, the field nodules were found distributed on both main

and lateral roots of groundnut. The colour of nodules obtained from El-Gadarif soil was pink, while the nodules obtained from

El-Gezira and El-Obied soils were of a white colour. All strains were Gram negative, white, circular, viscous, did not absorb

Red Congo indicator, produced yellow colour and acidified Yeast Extract Mannitol Agar with bromothymol blue, fast

growing, grew in Peptone Glucose Agar medium. All bacterial strains induced nodule formation to groundnut and unable to

form nodules in Phaseolus valgaris and Vigna senesis. Inoculation of groundnut by the isolates enhanced groundnut growth.

The strain isolated from groundnut nodules in El-Gadarif soil was found the best for groundnut growth promotion. The isolates

in the present study showed high symbiotic effectiveness. The study concluded that the strains isolated were fast growing

Rhizobia like. Isolation and application of local nitrogen fixing bacteria may resolve the competitiveness and environmental

conditions variation and fluctuation problems in the field.

Keywards: Biofertilization, nitrogen, inoculants

1. INTRODUCTION

Restoring, maintaining and increasing soil fertility are

major agricultural priorities in many parts of the

developing world where soils are inherently poor in

plant nutrients, and the demand for grain food and raw

materials is increasing rapidly. Sustainable production

of crops cannot be maintained by using chemical

fertilizers alone. Nutrients need to be added from

other sources such as organic manure and biofertilizer

for providing greater stability in production and

improving soil fertility (Mosharof et al., 2012).

In recent years there is increasing in fertilizers

cost, this increases crop cost. In addition chemical

fertilizers have harmful effect on the environment,

therefore a process known as inoculation or

biofertilization instead developed. Rhizobial

inoculants are known to be an alternative to the use of

industrial nitrogen fertilizers and a mean to maintain

or improve soil fertility (Peoples et al., 1995; Alves et

al., 2003; Chalk et al., 2006). There is considerable

interest in improving nitrogen fixation in legumes to

increase soil fertility, particularly in the developing

countries (Castro et al., 1999). The isolation and

selection of elite strains are very important because

the effective rhizobial strains can be used as

inoculants for effective nodulation (Dhery and

Dreyfus, 1991; Lanier et al., 2005; Bogino et al.,

2006). The competitive ability of an inoculant strain is

an important factor determining the success of

rhizobial inoculation (Triplett, 1990). Knowledge

about the indigenous population is necessary for the

selection and application of inoculant strains (Saleena

et al., 2001). More over long and inefficient

commercial channels, particularly where the product

is imported, may mean that farmers are applying

largely dead inocula that were of dubious efficacy

when they were alive. Previous studies in Sudan

recommended that further researches are required to

study the response of groundnut (Arachis hypogaea)

in Gazira scheme and rainfed areas, to Rhizobium

inoculation (Gadalla et al., 2010). In a study

conducted in the rainfed areas of the Gadarif State to

evaluate some rhizobium strains for nitrogen fixation

Idris et al.

Isolation and Biochemical Characterization of Bacteria Isolated from Groundnut (Arachis hypogaea L.) in Sudan

271

in the groundnuts it was found that the effect of the

new introduced strains did not reflected on the yield of

the crop but it is clear that strain TAL1371 has its

effect only on the pods weights and the shelling out

tern percent (Saeed et al., 2007). Cultivation of

groundnut in Western Sudan is still lacking nitrogen

fertilizers, hence three imported strains were

compared to local strains. The results indicate that, the

imported Rhizobium strain has no benefits for

groundnut production in Western Sudan. It was

recommended that nitrogen fixation by groundnut in

this area should be directed to selection and

identification of the most effective rhizobia strains

from the adapted local population (Ali, 2003). Also

Loynachan and Hadad (1984) stated that if facilities

permit, the field work in Sudan will be extended to

important production areas other than Wad Medani.

They showed that a logical choice for future work is

El-Obeid region which characterized with the low

number of native rhizobia and the potential for

groundnut expansion.

Therefore this work was conducted to isolate and

study the efficiency of the locally isolated bacteria

compared to the reference commercial strain.

2. MATERIALS AND METHODS

2.1. Isolation, purification and culture of bacteria

Nodules from groundnut (Arachis hypogae L.) were

collected in August 2011 from soils in El-Obied –

North Kordofan (Western Sudan), El-Gezira (Central

Sudan) and El-Gadarif (Eastern Sudan) (Fig 1). To

collect the groundnut nodules the whole plants were

pulled up carefully without detaching the nodules.

Collected nodules were washed with sterile water and

then surface sterilization was done using 70% ethanol

and 0.1% HgCl₂ and repeatedly washed with sterile

water. After surface sterilization, nodules were

crushed and then the resulting suspension was

streaked onto yeast extract mannitol agar (YEMA) at

pH 6.8, the medium contains (g / l): mannitol, 10;

K₂HPO₄, 0.5; MgSO₄.7H₂O, 0.2; NaCl, 0.4; yeast

extract, 1; agar, 20 with Congo Red or Bromothymole

Blue. Purity was assured by routine plating on YEMA

supplemented with Congo red and the uniform

colonies were selected. The basal growth medium is

that described by Vincent (1970). After repeated sub-

culturing, pure culture was obtained from a single cell

and preserved in 40% glycerol at -20 °C. For

experimental purposes growth was incubated at 28 °C

for ten days. Growth was observed daily

(Somasegaran and Hoben, 1994).

Colony shape was determined on YEMA. Acid or

alkali production was visually determined on the same

medium supplemented with bromothymol blue as a

pH indicator. All strains were maintained in yeast

extract mannitol medium (Vincent, 1970) and

transferred to fresh slant every month. The characters

of the isolated strains were compared with reference

strain (TAL169) which was obtained kindly from

National Center for Research, Sudan. Primary cultures

in 10 ml medium were taken from agar slants and

incubated at 28 °C with shaking until the early

logarithmic phase was reached. These cultures

provided inocula for the experimental cultures that

were also grown with shaking at 28 °C in 100 ml

flasks, with 1% inoculums. The isolated strains were

cultured in yeast extract mannitol broth for seven

days. The inoculum was applied on the pots

containing germinated groundnut, haricot beans

(Phaseolus valgaris) and cowpea (Vigna senesis) at an

initial population level of 1.8 x 106 CFU (Cell forming

unit)seed-1 (Castro et al., 1999). The experiments were

carried in Food Technology, Microbiology Laboratory

in Faculty of Natural Resources, University of

Kordofan, Sudan.

North Kordofan El-Gezira El-Gadarif

Fig. 1: Study area

International Journal of Scientific Research in Knowledge, 3(11), pp. 0270-276, 2015

272

2.2. Pots experiments

The nitrogen fixation ability of the different isolated

strains was evaluated in 15 cm diameter ×15 cm deep

pots containing 1200 gm of soil taken from El-Obied ,

El-Gadarif and El-Gazira. Pots were covered with

aluminum foil and sterilized in a forced air oven at

180 °C for 4 hours. The seeds were incubated to

germinate in sterile Petri dishes containing 0.9% (w/v)

water agar for 2 to 3 days at 28°C. After germination,

four seedlings were sown in each pot. Two weeks

after germination, the number of plants was thinned to

two per pot (Ngo Nkot et al., 2011). Treatments were

compared with inoculated plants and uninoculated

controls. Each treatment was replicated twice in a

completely randomized design. Pots were inoculated

with the isolates using the four strains; the three

isolated and the reference strain. All pots were placed

outside under natural conditions of light, temperature

and humidity. The pots were watered as required with

non-sterile tap water. Plants were watered and

harvested 8 weeks after sowing. Plant growth was

determined by comparing branch number, plant

height, shoot fresh weight and shoot dry weight of the

shoot system. The dry weight was made when the

shoot system was left to dry at 70°C for more than 72

hour in an air dry oven and then weighed as was

described by Athar and Johnson (1996). The percent

of relative symbiotic effectiveness (RSE) of the

isolates was calculated according to Lalande et al.

(1990) as:

X100 The RSE% =

The RSE (%) values were rated as: i - highly effective = >80%; іі - effective = 50 – 80%; iіі- less effective = 35 –

<50%; iv- ineffective = <35%.

Table 1: Identification of the different bacterial isolates and the reference strain

Gram

stain

R.C absorption Growth in

PGA

B.T.B+

YEMA

PH

B.T.B+

YEMA

reaction

B.T.B+

YEMA

color

Colony

color

Colony

shape

Strains

-ve No + 5.55 acid yellow white circular El-Gadarif

-ve No + 5.60 acid yellow white circular El-Gezira

-ve No + 6.36 acid yellow white circular El-Obied

-ve No + 5.24 acid yellow white circular Reference

B.T.B = Bromothymol blue indicator R.C= Red Congo indicator YEMA = Yeast extract mannitol agar PGA = Peptone glucose agar

Table 2: Effect of the different isolates and the reference strain on groundnut (Arachis hypogaea) growth in El-Obied soil

*Each values mean of two replicates.

*Values in column share same superscript letters show no significant differences as separated by Duncan Multiple Range Test.

2.3. Data analysis

The results were submitted to analysis of variance

(ANOVA) by using sigma stat software program

version 2.0. When there were significant, means were

compared with Multiple Range Test at P <0.05.

3. RESULTS AND DISCCUSIONS

3.1 Isolation and purification of the isolates

Isolates were obtained from groundnut nodules from

El-Gadarif, El-Gezira and El-Obeid. The nodules

obtained from the field differ in their abundance,

distribution and colour. The Gezira nodules were

found the highest in number and distributed in the

lateral roots. The nodules obtained from El-Obied

groundnuts were also distributed in the lateral roots

more than the main root. Both El-Gezira and El-Obeid

nodules were white in their colour the fact that

suggests that these bacteria strains are inefficient.

After series of streaking pure cultures of the isolated

strains were obtained from different soil nodules (El-

Obied, El-Gadarif and El-Gezira). The colonies of the

isolates were white, Gram negative, circular, viscous

and easily stuck to the inoculating loop and growth

Idris et al.

Isolation and Biochemical Characterization of Bacteria Isolated from Groundnut (Arachis hypogaea L.) in Sudan

273

was observed after two days. These characters were in

agreement with Bergy and Holt (1994) who described

these bacteria as rods 0.5 - 0.9 x1.2 – 3.0 µm., Gram

negative, mobility occurs by one polar. Colonies are

circular, white and convex. They produce an alkaline

reaction in mineral salt medium containing mannitol

or many other carbohydrates. Bacteroids in root

nodules are slightly swollen rods with rare branching

and coccus form in Arachis. It was also stated by

Akuma (2010) that all of their tested isolates were

gram negative and rod shaped bacteria with little or no

absorption of Congo red grown on YEMA-CR

medium. All isolates in this study including the

reference strain were grown well on peptone glucose

Agar after incubation for three days. However

previous studies documented that Rhizobia do not

grow in this media but, recently it was found that

some strains of Rhizobia grow in peptone glucose

agar medium as reported by Sharma et al. (2010).

Jordan (1984) also reported that slow growing

rhizobia were produce alkaline while fast growing

rhizobia produce acid. Moreover, Wolde-meskel et al.

(2004) have also reported the presence of fast growing

alkaline producing and slow growing acid producing

strains that were isolated from native woody legumes

in southern Ethiopia. The Gezira isolate grew better

than the others and appear rigid somewhat. The

isolates gave yellow colour and acidified the YEMA

medium containing bromothymol blue indicator

(B.T.B). These last characters distinguish

Bradyrhizobium from Rhizobium which gave blue

colour and alkaline the medium containing

bromothymol blue. On the other hand the isolates did

not absorb Red Congo indicator which distinguish

Rhizobia and other bacteria (Barbara and Thomas,

1983). The formation of acid and yellow colour in

YEMA medium are characters of fast growing

Rhizobium, so our isolates can be classified as fast

growing. In Sudan Habish and Kheiri (1968) found

that groundnut Rhizobium isolates were fast growing

and acid producing which agreed with the results in

this study. All these characters were compared with

the reference strain (Table 1).

3.2. Pot experiments

In all pots no nodule was formed when Phaseolus

valgaris and Vigna senesis were used. This agrees

with Habish and Kheiri (1968) who reported that their

isolates did not nodulate cowpea, which is not

characteristic of the cowpea group. However Hadad

and Loynachan (1985) reported that individual

isolates of Rhizobium from six legumes: groundnut

(Arachis hypogaea), mung bean (Vigna radiata), lubia

(Dolichos lablab), cowpea (Vigna unguiculata),

pigeonpea (Cajanus cajan) and bambara groundnut

(Voandzeia subterranea) were obtained from four

locations in Sudan. All isolates were able to nodulate

each of the six legumes when grown in sterile

vermiculite. On the other hand when groundnut in the

different soils inoculated with the different strains,

nodules were appeared and there were significant

differences (P ) between the different strains

except between El-Gadarif and El-Obied strains there

were no significant differences (Table 2). However all

isolated strains gave nodule number more than the

reference strain. The plant heights for groundnut was

also found significantly vary among the isolated and

the reference strains. All strains showed highly

symbiotic effectiveness, the best symbiotic

effectiveness in this study was obtained when

groundnut inoculated with El-Gadarif strain which

was correlated to the pink colour of the nodules

mentioned before.

In study done in Ethiopia nine locally isolated

Bradyrhizobium spp. (Arachis) strains out of 19

strains displayed the highest score of 80 – 116%

effectiveness of symbiotic nitrogen fixation (Akuma,

2010). So our isolated strains were found highly

effective than the Ethiopian strains. These results

confirm Deshwal et al. (2003) results that showed

that, Peanut seeds coated with Bradyrhizobium strains

were significantly enhanced seed germination,

seedling biomass, nodule number, nodule fresh

weight, average nodule weight compared to

uninoculated and uninfected controls. Also

Nodulation and top dry weights of groundnuts have

been shown to be positively correlated to Rhizobium

efficiency in nitrogen fixation (Wynne et al., 1980).

They also reported that nodulation, plant weight,

nitrogenase activity and nitrogen content of

greenhouse-grown groundnut were significantly

correlated with each other. The isolated rhizobia in

this study varied significantly in the number of

nodules and top dry weights produced. The positive

correlation observed between nodulation and shoot

dry weight of groundnut suggests that the two

parameters evolve together (Ngo Nkot et al., 2011).

Alemayehu (2010) reported a positive correlation

between nodulation and shoot height of V. faba.

Inoculation with different strains of Bradyrhizobium

significantly improved shoot fresh and dry weight,

number of pods and nodules, seed quality and yield.

4. CONCLUSION

The bacteria isolated in this study are like fast

growing Rhizobia nodulate groundnut only. They

promote groundnut growth better than the commercial

one. El-Gadarif isolate was the best in groundnut

growth enhancement and it can be tested in the field to

International Journal of Scientific Research in Knowledge, 3(11), pp. 0270-276, 2015

274

insure its effectiveness and competitiveness and used

as inoculant.

ACKNOWLEDGEMENT

The main author would like to thank DAAD for

financial support of this work and laboratory of

Biochemistry and food technology, Faculty of Natural

Resources, University of Kordofan, Sudan for

providing space in the lab to achieve this work.

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Dr. Abdelmalik O. A. Idris Assistant professor, University of Gadarif, Faculty of Education,

Department of Biology. PhD in Biosciences and Biotechnology (Microbiology). He is DAAD Alumni

and DAAD scholarship holder (2012). His research interest in Microbiology, Molecular Biology and

Biotechnology.

Prof. Dr. Awad M. Abdel Rahim is Professor, Faculty of Sciences, University of Gezira, Sudan. His

interest is Microbiology.

Prof. Dr. Abdel Moneim Sulieman has been awarded his PhD in 2001 and pursued a postdoctoral

fellowship at University of Kobe, Japan. He is expert in “Food Science and Technology, his main

concern is food microbiology. Professor at Faculty of Sciences, University of Hail, Saudi Arabia.