Isolation and Characterization of Local Isolates of Rhizobia in Nineveh Governorate/Iraq

In this study, thirty-six local isolates of rhizobial bacteria were isolated from root nodes of different leguminous plants collected from different areas in Nineveh Governorate/Iraq. The rhizobial colonies were purified and their agronomic and biochemical characteristics were studied. Results of this study showed that the local isolates had the ability to consume different sugars as a carbon source. Also, the antibiotic sensitivity test showed that only one isolate, Rhizobium leguminosarum bv. phaseoli AS12 was resistant to the antibiotic Azithromycin 15 μg/ml, and the isolate Rhizobium leguminosarum bv. phaseoli AS14 showed resistance to the antibiotic Gentamycin 10 μg/ml, and the rest of the isolates showed different rates of resistance and sensitivity for the rest of the antibiotics. The local isolates showed different NaCl tolerance levels up to 8.0 %, while no tolerance was observed for Rhizobium leguminosarum bv. viciae AS11 isolate at any of the studied concentrations. Results of heavy metal salt tolerance study showed that the isolates of Ensifer meliloti AS34 and Rhizobium leguminosarum bv. viciae AS36 were sensitive to ZnSo4 at a concentration of 25 μg/ml, while the tolerability of the other isolates to the studied heavy salts differed at concentrations higher than 25 μg /ml. All the studied isolates showed a positive result in staining with Congo red and Aniline blue dyes. Maximum exopolysaccharide production was 494 mg/Li by Rhizobium leguminosarum bv. viciae AS35. Antimicrobial activity test showed maximum inhibition zone 17 mm by Rhizobium leguminosarum bv. viciae AS36 against Proteus vulgaris. Keyword: rhizobia, characterization, exopolysaccharide, antimicrobial activity قارعلا/ىونين ةظفاحم يف ايبوزيارلا نم ةيلحم تلازع صيخشتو لزع دمحم يلع دمحا 1 * ، ناطلس يناسح دعر 2 1 مولع مسق ةايحلا قارعلا ،لصوملا ،لصوملا ةعماج ،ةفرصلا مولعلل ةيبرتلا ةيلك ، 2 قارعلا ،لصوملا ،لصوملا ةعماج ،ةفرصلا مولعلل ةيبرتلا ةيلك ،ةايحلا مولع مسق ةصلاخلا : لزع ةسا ردلا هذه يف مت 36 فلتخمل ةيرذجلا دقعلا نم ةيلحم ايبوزيا ر ةلزع ةظفاحمل ةفلتخم قطانم يف ةيمانلا ةيلوقبلا تاتابنلا قا رعلا/ىونين . ايبوزيا رلا تلازع ةليباق ترهظا ةسا ردلا هذه جئاتن .اهل ةيئايميكويابلاو ةيعرزملا تافصلا ةسا ردو ةيبوزيا رلا عرا زملا ةيقنت مت Journal of Education and Science (ISSN 1812-125X), Vol: 30, No: 3, 2021 (83-94) Special Issue for Proceeding of 3 National (1 international conference of biology) (ICBSUM 2021) 5, 6 July College of Education for Pure Science, University of Mosul, Mosul, Iraq. Downloaded from https://edusj.mosuljournals.com/ 84 .نوبراكلل ردصمك ةفلتخم تايركس للاغتسلا ةيلحملا رابتخا يه ةدحاو ةلزع نأب ترهظا ةيويحلا تاداضملل ةيساسحلا Rhizobium leguminosarum bv. phaseoli AS12 تناك نيسيامورثيزأ داضملل ةمواقم 15 ةلزعلا ترهظاو .لم/ما رغوركيام Rhizobium leguminosarum bv. phaseolus AS14 نيسياماتنج يويحلا داضملل ةمواقم 10 لم/ما رغوركيام ، ىرخلاا تلازعلا ترهظاو ظأ .ةيويحلا تاداضملا ةيقب هاجت ةيساسحلاو ةمواقملا نم ةفلتخم تلادعم نم ةفلتخم لمحت تايوتسم ةيلحملا ايبوزيا رلا تلازع تره ىلا لصت مويدوصلا ديرولك حلم 8.0 % ، ةلزعلل لمحت يا ظحلاي مل نيح يف Rhizobium leguminosarum bv. viciae AS11 حلاملاا لمحت ةسا رد ترهظأ .مويدوصلا ديرولك حلم نم زيكرت يلأ نأب ةليقثلا ةلزعلا Ensifer meliloti AS34 ةلزعلاو Rhizobium leguminosarum bv. viciae AS36 زيكرتلا دنع كنزلا تاتيربكـل ةساسح اهنأب 25 لم/ما رغوركيام ، نيح يف نم رثكلاا زيكا رتلل تلازعلا ةيقبل ةنيابتم جئاتنلا تناك 25 وكنوكلا رمحا يتغبصب اهعرا زم تغبطصا تلازعلا عيمج .لم/ما رغوركيام نلاا قرزاو غلب يجراخلا ددعتملا ركسلل ةيجاتنا ىوتسم ىلعأ .نيل 494 ةلزعلا ةطساوب رتل/مغلم Rhizobium leguminosarum bv. viciae AS35 طيبثت ةقطنم رطقب ناك ةيبوركياملا ةداضملا ةيلاعفلل ىوتسم ىلعاو . 17 ةلزعلا ةطساوب ملم Rhizobium leguminosarum bv. viciae AS36 ا ايرتكبلا هاجت ةيضرمل Proteus vulgaris . تاملكلا ةيحاتفملا : .ةيبوركياملا ةداضملا ةيلاعفلا ،يجراخلا ددعتملا ركسلا ،صيخشت ،ايبوزيار


Introduction
Gaseous nitrogen (N2) is abundantly available in the atmosphere. Plants and animals cannot convert it into a biological useful form. Only a few prokaryotes have the ability to utilize the gaseous nitrogen. These organisms reduce the gaseous dinitrogen (N2) to ammonia. This process, known as biological nitrogen fixation, is carried out by the enzyme system nitrogenase. Some nitrogen fixing bacteria like Klebsiella pnumoniae, Azotobacter, and Rhodopseudomonas are able to reduce nitrogen into ammonia as free living organisms. Other organisms fix nitrogen only in symbiotic relationship with a eukaryotic host plant, like rhizobia in legume symbiosis, Frankia in actinorhizal symbiosis and Cyanobacteria in Gunnera symbiosis [1]. Symbiotic nitrogen fixation is of great importance to agriculture since it reduces the need of fertilizer nitrogen.
In agriculture, perhaps rhizobia-legume symbiosis contributes as much as 80 % of the biologically fixed nitrogen [2]. The bacteria of genera Rhizobium, Bradyrhizobium, Azorhizobium, Mesorhizobium and Ensifer, collectively called rhizobia, form symbiotic association with leguminous plants. The bacteria induce the formation of specialized structures, called nodules, on the roots of legume plants. The reduction of atmospheric nitrogen takes place in these nodules. The establishment of symbiotic association takes place in several steps like recognition and infection of root hairs, development and differentiation of root nodules, multiplication of rhizobia and their converts into bacteroids within plant cells and conversion of nitrogen to ammonia. Symbiosis also involves uptake and assimilation of energy rich carbon compounds by bacteroids and utilization of fixed nitrogen by plants [3].
Strains of different rhizobial species possess indigenous plasmids. Megaplasmids have been found in Ensifer meliloti strains. Many of the genes, which are important in symbiosis are plasmid borne. In E. meliloti genes required for nodulation and nitrogenase activity have been mapped on the megaplasmid pSyma. The second megaplasmid, pSymb carries genes involved in exopolysaccharide synthesis and uptake of various nutrients [4]. Researchers revealed that exopolysaccharide (EPS) are key components for the establishment of an effective symbiosis between leguminous plants and rhizobia [5]. Recently, non-toxic rhizobial EPS have been found to have properties suitable for application in various fields, including medicine [6].
Preparation of this medium were done by dissolving 65 gm of this medium and complete the size to 1 litter with distilled water. Adjusting pH at 7.2-7.4 were done.

Gelatin Liquefaction Medium:
This medium was prepared by dissolving 128 gm of this medium in a litter of distilled water, pH was adjusted at 7.2 [12].

Cultural and Microscopic Examination:
Appearance of rhizobial colonies and cultural moisture were examined by necked eye. Gram staining ability and bacterial shape were examined under microscope.

Citrate Utilization Test:
This test was done to determine the capacity of isolates to use various carbohydrate sources e.g. sucrose, glucose and lactose as media for growth. After inoculation and incubation, colour were observed [2].

Gelatin Liquefaction Test:
This test was carried out by inoculation of gelatin medium with fresh rhizobial colonies by stabbing with inoculum needle and incubated at 28±2 °C for 24 hrs. Results recorded after half an hour at 4 °C [13].

Triple Sugar Iron Test:
This test was performed by inculating the medium with a young of rhizobial bacteria and at 28±2 °C for 72 hrs. the positive result was recorded by changing the colour of the medium to dark brown colour [14].

Intrinsic Antibiotic Resistance (IAR) Spectra:
This experiment was carried out to identify sensitivity or resistance of local rhizobial isolates against different antibiotics were used to assay the antibiotic resistance on Millar-Hinton agar plates. Plates containing the discs were incubated at 28±2 °C for 3-7 days [2]. The presence or absence of inhibitory zones around different antibiotic discs was noted. Studied antibiotics as follows; Azithromycin, Gentamycin, Vancomycin, Amikacin, Streptomycin and Erythromycin.

Heavy Metal Sensitivity Test:
To estimate ability of local rhizobial isolates to grow on TY plates containing different concentrations of heavy metal salts, different heavy meatal salts were added to TY plates as follows: Pb (CH3COO), COCl2 and HgCl2 with concentration 500 µg/ml. [15].

Tolerance to Sodium Chloride Salt:
The salinity tolerance of sodium chloride salt was assayed by culturing the rhizobial bacteria on YEMA medium containing different sodium chloride salt concentration as follows: 1.0, 2.0, 3.0 and 4.0%, respectively [2]. cells. Muller agar medium plates were prepared and 0.1 ml of each pathogenic bacterial culture were transferred and spreaded. Wells 6 mm were done and 200 µl of each rhizobial filtrated culture were transferred to the wells. Replicates were done as well as a negative control with 100 µl normal saline [22]. The diameter of the inhibition zone was measured with calipers in mm. The antimicrobial activity was determined by measuring the clear zone around the wells.

Results And Discussion
Roots of different leguminous plants were collected from different areas of Nineveh Governorate/Iraq, and results showed a success of isolating single colonies from smashed nodules after growing on TY plates. The sixty-three different rhizobial isolates and the given names as well as the host plants were recorded in Table 1. A success of isolation of different local rhizobial isolates were also recorded by many researchers [2,13,23].

Reverse Inoculation Test:
Reverse inoculation test study revealed ability of all the thirty-six rhizobial isolates were able to induce root nodules on their specific leguminous plants confirming the success symbiotic relations with their specific leguminous host.

Cultural and Microscopic Examination:
On the basis of morphological characteristics, the isolates were circular in shape with entire margin and milky to watery translucent appearance on TY agar medium. It was found that the colonies of the local rhizobial strains grown showed the convex elevation in Yeast Extract Mannitol. Gram's staining of the isolates was confirmed by microscopic observations and the isolates was found to be Gram negative and rod shaped. Gauri et al., [24] also reported that microscopic examinations of Rhizobium showed that the isolates to be Gram negative. Roychowghury et al. [25] observed his strain under microscopic by Gram staining which showed pinkish colour and red shaped bacteria. isolated rhizobial local strains showed positive results for gelatin liquefaction test with exception with isolates: AS2, AS3, AS4, AS10 and AS11 were unable to liquefaction of gelatin. Kumari et al. [27] also reported different results with variant rhizobial isolates and mentioned that gelatinase enzyme plays an important role during nodule formation. Only one strain AS11 was negative for Triple Sugar Iron test. The variation in enzymatic activities of rhizobial isolates was reported by various workers. It is reported that Rhizobium isolates wich produce these enzymes are considered as best for nodulation and nitrogen fixation [27].

Intrinsic Antibiotic Resistance (IAR) Spectra:
Antibiotic sensitivity study showed that only one isolate i. e., Rhizobium leguminosarum bv. phaseoli AS12 was resistant to Azithromycin 15 µg/ml and the isolate Rhizobium leguminosarum bv. phaseoli AS14 showed resistance to the Gentamycin 10 µg/ml, whereas the other isolates showed different resistance and sensitivity patterns. An IAR spectrum is used for the identification of nodulating strains in reports intended to assess the ecological competitiveness. Kucuk and Kivanc [28] found great variation among chickpea rhizobia with respect to their IAR pattern.

Heavy Metal Salts Tolerance:
Results of tolerance of heavy metal showed that the isolates Ensifer meliloti AS34 and Rhizobium leguminosarum bv. viciae AS36 were sensitive to ZnSO4 at a concentration of 25 µg/ml, whereas the tolerability of the other isolates was different at concentration higher than 25 µg/ml. Datta et al. [29] investigated the effect of metal ions on Rhizobium strains observed that all the strains were sensitive to mercuric chloride.

Tolerance to Sodium Chloride Salt:
All local rhizobial isolates showed good and very good tolerance up to 4.0% of sodium chloride salt with exception with Rhizobium leguminosarum bv. viciae AS11, which was unable to tolerate even 1.0% of sodium chloride salt. Increase salt concertation's up to 4.0% led to moderate rhizobial growth, anyhow no growth was found with isolates AS3, AS10 with sodium chloride concentration 8.0%. Wadhwa et al. [2] found that rhizobial bacteria differ in their capability of sodium chloride salt tolerance. Research showed that some strains may grow at salt concentrations as high as 500 mM NaCl, others may not grow even at low NaCl concentration [28].

Growth on Rhizobial Minimal Medium:
Study of growth of local rhizobial isolates on RMM showed ability of these bacteria to grow on the mentioned medium indicted that these isolates doesn't suffered from any nutritional mutations [16].

Production of Cell Surface Molecules Test:
Results of production of cell surface molecules test revealed ability of local rhizobial isolates to uptake Congo Red and Aniline Blue dyes indicating of production of cellulose fibrils and β-(1-3) glucans. Aniline Blue is known to specifically bind to the curdlan type of polysaccharide, of which beta-(1-3) is a major component [17], [18]. The cell surface molecules are very necessary for rhizobia-legume symbiosis [30]. Clear swarming for each plate spotted with certain local rhizobial isolate gave a good proof for production of β-(1-2) glucans. Absence of beta-(1-2) glucans has been linked to a defective flagellum and resulting absence of chemotactic response which in turn leads to formation of ineffective nodules [19].

Utilization of Different Sugars:
Results of utilization of different studied sugars ability of isolated rhizobia to utilize different sugars viz., glucose, galactose, arabinose, fructose, maltose, xylose, rhamnose and lactose. This results agree with results of Hossain et al. [30], which results revealed that ability of tow rhizobial strains RhBC and NRA1 to utilize different sugars.

Production of Exopolysaccharide:
A cording to examination of rhizobial culture mucoid mixture by necked eye, ten rhizobial isolates were chosen for production of exopolysaccharide production study. These isolates as follows: AS6, AS8, AS10, AS11, AS18, AS21, AS26, AS32, AS35 and AS36. Maximum mean value exopolysaccharide production was by the isolate AS35 which was 494 mg/Li. Minimum production was by the isolate AS21 which mean value was 158 mg/Li. Ghosh et al. [31] study revealed that the maximum EPS production by Rhizobium sp. was 765 mg/Li when they used mannitol as a carbon source. Prabhavati and Malliah [32] research results showed that mannitol gave productivity 4690 mg/Li by Rhizobium sp.

Antimicrobial Activity Test:
After adding 200 µl of fermented filtrate medium, results showed differences in inhibition zone diameter. A maximum average inhibition zone was 17 mm by Rhizobium leguminosarum bv. viciae AS36 against Proteus vulgaris. Average of inhibition zone was 15 mm by Ensifer meliloti against Proteus mirabilis and Proteus vulgaris, respectively. Fig. 1and 2 shows effect of rhizobial fermented filtrate broth on growth of studied pathogenic bacteria. Joseph et al. [33] results study revealed that Rhizobium leguminosarum bv. trifolii produce bacteriocin-like substances and have antimicrobial activity against Salmonella typhi A and B, Pseudomonas aeruginosa, Staphylococcus citrus and Proteus vulgaris. Inhibition zone were 5, 6, 8, 6, 7 and 8 mm, respectively. Deora and Singh [34] research results revealed that rhizobia isolated from Vigna radiate have antimicrobial activity against E. coli and Staphylococcus aureus, which inhibition zone diameter ranged from 13 to 15 mm. Antimicrobial activity of Rhizobium japonicum and Bradyrhizobium japonicum were recorded and the zone of inhibition (in mm) was 8.3 for Aspergillus niger, 7.4 for Fusarium oxysporum and 8 for Alternaria alternata from Rhizobium japonicum and for Bradyrhizobium japonicum the zone of inhibition was 9.2 for Apegillus niger, 7.6 for Fusarium oxysporum and 8.5 for Alternaria alternata [23].

Conclusion
This research work showed ability of isolation of a local Rhzobial isolates that produce exopolysaccharide and some of a local isolates exhibited antimicrobial activity against of studied bacterial pathogens. We recommend that expand the area of isolation may give much more results.