Physiological characterization of rhizobial strains

Latrach & al./ Appl. J. Envir. Eng. Sci. 3 N°4(2017) 353-364

Physiological characterization of rhizobial strains nodulating alfalfa (Medicago sativa) isolated from soils of Southeastern Morocco L. Latrach(a,b), M. Mouradi(a,b), M. Farissi(c), A. Bouizgaren(b) and C. Ghoulam(a) (a)

Unit of Biotechnology and Symbiosis Agro-physiology, Faculty of Sciences and Techniques, Cadi Ayyad University, PO. Box 549, Gueliz 40000 Marrakech, Morocco. (b) Unit of Plant Breeding, National Institute for Agronomic Research (INRA), PO. Box 533, Gueliz 40000, Marrakech, Morocco. (c) Laboratory of Biotechnology & Sustainable Development of Natural Resources, Polydisciplinary Faculty, Sultan Moulay Slimane University, PO Box: 592, Beni-Mellal, 23000 Morocco.

*Corresponding author: E-mail: [email protected]; [email protected] Received 4 Oct 2017, Revised 3Nov 2017, Accepted 21Nov 2017

Abstract The legume-rhizobia symbiosis provides the necessary nitrogen for plants growth and contributes to the improvement of soil nitrogen level. However, environmental constraints prevailing in many Moroccan regions constitute the limiting factors for this symbiotic interaction. For this reason, the present work aims to select some rhizobial strains nodulating alfalfa (Medicago sativa L.) for their performance under environmental stresses. Rhizobial strains nodulating alfalfa were isolated from soils of Tafilalet region (Arfoud 1, Arfoud 4, Aoufous and Rich) and purified in yeast extract mannitol (YMA) medium. After nodulation test, 20 strains were able to induce root nodulation in alfalfa and they were tested for their tolerance to high salinity, water deficit, acidity and temperature. These strains were also assessed for their abilities to solubilize inorganic phosphorus using Pikovskaya (PVK) medium. The results indicated that the studied strains developed different behaviors in response to all considered abiotic stresses. The positive correlations between strains behaviors and origin were noted. In general, strains from Arfoud1 and Arfoud 4 were qualified as the most tolerant to salt stress and can grow under extreme pH levels (4 to 10). Whereas, strains from Rich were the most tolerant to water deficit. Results recorded on PVK medium mentioned that some strains (Arf1RL1, RYRL9 and Arf4RL16) showed a high potential of inorganic phosphorus solubilization. Some strains such as Arf1RL1 and Arf4RL13 both high tolerance level to considered environmental constraints and high potential of inorganic phosphorus solubilization. Keywords: Medicago sativa; rhizobia, nodule; osmotic stress; salt stress.

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1. Introduction Alfalfa (Medicago sativa L.) is the most cultivated forage legume in the world due to its high nutritional quality, high protein content and positive effects on soil fertility [1]. Indeed, alfalfa is a crop that has a very favorable influence on soil fertility by contributing to the incorporation of nitrogen in pastoral ecosystems with beneficial economic impacts, helping to reduce or limit the use of chemical fertilizers with nitrogen-fixing symbiosis involving rhizobial strains [2]. In Morocco, this crop occupies over than 400,000 hectares, over 25% of the total area devoted to forage crops [3]. About 50% of the total feed units provided by alfalfa. This plant is characterized by its high production potential, its high quality and could also provide a greater degree of flexibility usage than most of the other forage plants (green chop, hay, grazing, dehydrated meal, ensilage) [4]. Moroccan populations of this species are widely used in the local traditional agro-ecosystems, oasis and mountains. They are characterized by a great diversity, biotic stresses tolerance and their adaptation to different pedoclimatic conditions [4,5]. Nevertheless, climate change induced prolonged drought combined with water deficit and rain irregularity that have adverse consequences on the restrictions and stability of agricultural production [6]. Similarly, soil salinity mapping in arid and semi-arid ecosystem, is a limiting factor for plant production, particularly in thirsty crops, such as alfalfa [7]. All these situations require a selection of host genotypes of alfalfa tolerant to drought and salinity conditions, in order to improve its production and persistence. This crop constitutes the main crop in these arid and semi-arid regions of Morocco and contributes to the socioeconomic development of these regions and provides plant proteins intended for use in animal feed. The inoculation of alfalfa with rhizobial strains tolerant to salinity and water deficit conditions has been reported to improve the total plant biomass, especially, under salinity and drought [5,8]. Previous studies have widely addressed the effects of different environmental factors on the symbiotic nitrogen fixation (SNF). The major influencing factors on SNF include water deficit, phosphorus deficiency, salinity, soil pH and temperature [9]. Thus, the physiological characterization of different strains of alfalfa-nodulating rhizobia, will be helpful to determine the extent of physiological variations between strains and also to exploit these variations for selecting strains with high performance of nitrogen fixation under different conditions. Therefore, the aim of the present study is to evaluate the tolerance of rhizobial strains nodulating Moroccan alfalfa populations, to different abiotic factors: salinity, drought, pH, temperature and phosphorus deficiency.

2. Materials and methods 2.1. Geographical data of studied area The bacterial strains used in this study were isolated by trapping method from root nodules of alfalfa (Medicago sativa L.) cultivated in different soils from the Southeastern oasis of 354

Latrach & al./ Appl. J. Envir. Eng. Sci. 3 N°4(2017) 353-364 Morocco “Tafilalet” This region is characterized by severe environmental conditions. Local populations of alfalfa are widely used in this area. It is characterized by extreme pedo-climatic conditions, high temperatures, lack of irrigation water, soil and water salinity. Soil samples were collected from the Ziz Valley. Three different sites were selected as sampling sites: Rich (High Atlas Ziz basin), Aoufous oasis (intermediate zone) and Erfoud (plain zone) (Figure 1). At each site, soil samples were collected from plot of alfalfa. 2.2. Rhizobia isolation from root nodules Alfalfa seeds were germinated in pots filled with soil sample collected fromthe Ziz Valley (one soil sample per pot). One month after seeding, under complete asepsisconditions , detached alfalfa root nodules were rinsed with sterile deionized water, sterilized with sodium hypochlorite (0.3%) and rinsed several times with sterile deionized water. The disinfected nodules were separately crushed in the presence of 0.5 mL of sterile deionized water using a sterile glass rod. The obtained homogenate was cultivated on Petri dishes containing Yeast Extract Mannitol (YEM) Agar supplemented with Congo red. The Petri dishes were incubated at 28 °C for three to seven days.

Figure 1. Collection areas of the soil samples used for the trapping of the rhizobia. 2.3. Purification of rhizobia Isolates Before transplanting the rhizobial colonies,the loop was sterilized by holding it into a flame until the wire glows red from loop to holder. Once the loop has had few moments to cool, the colonies were picked out and uniformly streaked on Petri dishes containing the YEM medium, then they were incubated at 28°C for 48 h. This process was repeated multiple times until single pure colonies were visible. 355


2.4. Nodulation test The nodulation test allows to confirm that the isolates are rhizobia. It is an approach for evaluating the ability of the strains to produce efficient nodules. Seeds of alfalfa (Medicago sativa L.) were surface sterilized with 6% sodium hypochlorite solution for 5 min, rinsed several times with sterile deionized water and germinated in pots filled with a mixture of sterile sand and peat (sterilized at 200°C for 2 hours) with proportionsof 3:1, respectively. Twenty days after germination, various bacterial strains were used to inoculate different plant roots. Three replicates of each isolate, were arranged in greenhouse and regularly irrigated with deionised water and N-free nutrient solution was added once a week. A total of 16 rhizobiastrains were selected by the nodulation test and were used for the physiological characterization. 2.5. Physiological characterization of the isolates 2.5.1. Evaluation of NaCl tolerance To evaluate the NaCl tolerance of the isolates, we prepared the YEM medium containing NaCl at: 0%,0.5%, 1%, 1.5%, 2%, 2.5%, 3 %, 3.5%, 4%, 4.5%, 5%, 5.5% and 6%. Each rhizobial strain was grown in YEM liquid medium for 48 h. Then, we divided the Petri plates into 8 compartments. Each compartment was inoculated with 10 µLof a cultivated bacterial solution on a YEM medium with the same concentration of NaCl (8 rhizobia strains per Petri plate). Three replicates per strain were considered. The result was recorded 7 days after incubation at 28°C. 2.5.2. Evaluation of water deficit tolerance The tolerance of isolates to the water deficit induced by PEG 6000, was performed by assessing the development of the strains on YEM medium containing PEG 6000 at: 0%, 6%, 6.5%,7%, 7.5% and 8%. After cultivation of each rhizobial strains in YEM liquid medium for 48 h. Using the same methode described in the previous test. Each compartment was inoculated with 10 µLof a cultivated bacterial solution on a YEM medium for each concentration of PEG 6000. Three replicates per strain were also excuted. The result was recorded 7 days after incubation at 28 °C. 2.5.3. Evaluation of pH tolerance The strains were tested for their tolerance to extreme pH on solid YEM medium withpH valuesadjusted to: 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5; 9; 9.5 and 10, and the controls with pH of 6.8. Three replicates per strains were also excuted. The result was recorded 7 days after incubation at 28 °C. 2.5.4. Assessment for temperature tolerance

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To determine the optimum and maximum growth temperatures, strains were grown on the solid YEM medium and incubated at different temperatures: 4 °C, 10 °C, 20 °C, 28 °C, 37 °C and 50 °C. The result was recorded 7 days after incubation. 2.5.5. Evaluation of phosphate solubilization Phosphate solubilization test was conducted on Pikovskaya medium [10] with tricalcium phosphate as the sole phosphorus source in the medium. The Petri dishes were inoculated with 10 µLof a cultivated bacterial solution from fresh precultures with 3 replicates for each strain. The Petri dishes were incubated at 28 °C. The result was recorded 7days after incubation, by measuring the diameter of solubilization (halozone + colony) and the diameter of the bacterial colony. The solubilization index was determined using the fellowing formula [11]:

3. Results and discussion 3.1. Morphological characteristics In total,20 rhizobial strains were isolated and purified from the soil of the Tafilalet region. These strains showed morphological characteristics that are consistent with the described aspects of Ensifer meliloti [12]. The colonies were circular shaped, translucid, whitish, convex, viscous, having a smooth surface or shiny and they develop after 1 to 7 days of incubation at 28 °C (Figure 2).

Figure 2. Appearance of rhizobia colonies after several subcultures.

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3.2. Nodulation test The results of nodulation test (Figure 3) showed that only 16 strains were able to infect the roots and induced nodules development after 30 days of inoculation. Therefore, they have a high capacity or infectivity to induce nodulation of alfalfa. This confirms that the isolated strains from alfalfa nodules were rhizobia, other strains could be Agrobacterium or other bacteria. Indeed, host specificity is one of the major characteristics of the rhizobia-legume symbiosis. Indeed, the ability of the strains to form nodules with host plant (alfalfa) is the basic test to confirm the purification and the belonging of our strains to rhizobia group. Many studies have demonstrated that the variation in nodulation could be due to the density and incompatibility of rhizobia and soil factors that hinder the efficiency of rhizobia [13,14].

Figure 3. (a) Setting up nodulation test. (b) The nodulation of alfalfa by the strain Arf1RL1. 3.3. Tolerance of tested rhizobia strains to the salinity The majority of the tested rhizobia strains presented a range of tolerance levels to salinity. This tolerance is variable depending on the strains and their geographical origin (Table 1). The strains isolated from Arfoud 1 and Arfoud 4regions presented the highest tolerance levels to salinity. Regarding the strains in the regions of Aoufous and Rich (Ait Beni Yehya), they have almost the same tolerance limit that were 2.5% NaCl, except for RhRL7which appear to be more sensitive to salt in comparison with the other studied strains. In fact, Arf1RL1 and Arf4RL13 from Arfoud region were qualified as the most salt tolerant strains. They were able to grow at 5.5% and 6% NaCl respectively. Similar results were noted by Jebara [15], Abbas [16] and Jida and Assefa [17]. They mentioned that the fast-growing rhizobia can tolerate higher concentrations of 2% and the tolerance limits may considerably vary from one species to another and even between strains of the same species. Tafilalet region was classified as an area threatened by increasing soil salinization. Salt tolerance which characterizes the studied strains could be related to the isolation site salinity and could be an indication of an adaptation to osmotic stress which is due to the increase of the concentration of ions and the aridity of the soil during dry periods. This remarkable osmotolerance noted for these two strains could also 358


be related to their ability to accumulate osmoprotectants compatible solutes. Similar results were reported by Maatallah et al., [18] for strains Mesorhizobium nodulating chickpeas in Morocco and Mohammed et al., [19] for Acacia nodulating strains in Libya. In parallel to our results, isolated rhizobia of woody plants (Acacia, Prosopis, etc.) can tolerate NaCl concentrations of 3 to 5% [20]. Furthermore, Thami Alam et al., [21] found that strains of Ensifermeliloti isolated from alfalfa in southern Morocco were able to grow in the presence of 10% NaCl.Rhizobia halotolerant cells can survive in the presence of high salinity in pure culture [22] or in the soil [20]. This tolerance capability is due to the accumulation of organic osmolytes protectors such as amino acids like proline, betaine and glutamate or carbohydrates like trehalose, sucrose and others to maintain turgor of the cell and to limit the damage caused by salt [23]. The tolerance limite of strains to salt wasfrom 1.5% for AufRL7 strain until 6% for Arf4RL13. In fact almost all of the tested strains tolerate NaCl concentration of 2% and 88% of strains can grow on a medium containing 2.5% salt. Above this concentration the number of tolerant strains decreases to 6% at a salt concentration of 6% (Figure 4). Table1. Margin of tolerance of rhizobia, nodulating alfalfa at NaCl, PEG, pH and temperature. NaCl

PEG

pH

(%)

(%)

(unit)

Arf1RL1

5

8

4 - 10

4 - 28

Arf1RL2

2.5

7

5-9

10 - 37

Arf1RL3

2.5

7.5

5-9

10 - 37

Arf1RL4

2.5

7

5-9

10 - 37

AufRL5

2.5

8

5-9

10 - 37

AufRL6

2.5

8

5-9

10 - 37

AufRL7

1.5

7

5-9

10 - 37

AufRL8

2

7,5

5-9

10 - 37

Rich-

RyRhL9

2.5

8

5-9

10 - 37

Ait Beni

RyRhL10

2.5

8

5-9

10 - 37

Yehya

RyRhL11

2.5

8

5-9

10 - 28

(RY)

RyRhL12

2.5

8

5-9

10 - 37

6

8

4 - 10

4 - 37

2.5

7

5-9

10 - 28

2.5

7.5

5 - 9.5

10 - 28

3

7

5-9

10 - 28

Area

Arfoud 1 (Arf1)

Aoufou s (Aouf)

Strains

Arf4RhL1 3 Arfoud 4 (Arf4)

Arf4RhL1 4 Arf4RhL1 5 Arf4RhL1 6

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T (°C)

Tolerent strains (%)


100% 80%

60% 40% 20% 0% 0

0,5

1

1,5 2 2,5 3 3,5 NaCl concentration (%)

4

5

6

Figure 4. Tolerance range of isolated rhizobia nodulating alfalfa at different NaCl concentrations.

3.4. Tolerance of rhizobia strains to PEG 6000 induced water deficit The study of the reaction of rhizobia strains to osmotic stress induced by the presence of polyethylene glycol (Table 1), showed that all of the tested strains presented normal growth at 7% PEG 6000. Above this concentration the number of tolerant strains decreased by half at 8% PEG (Figure 5). The strains isolated from Rich region showed a more homogeneous reaction with higher tolerance level to the PEG. The tolerance of rhizobia to osmotic stress is directly related to their ability to accumulate solutes and modify their cell morphology [21]. Other studies showed that Gram-negative bacteria such as Rhizobium synthesize

Tolerant strains (%)

lipopolysaccharide at their wall that provide a protective role against desiccation [24].

100% 80% 60% 40% 20%

0% 0

6

6,5 7 PEG concentration (%)

7,5

8

Figure 5. Tolerance range of rhizobia strains nodulating alfalfa to the studied PEG concentrations. 360


3.5. Tolerance of the isolated rhizobia strains to extreme pH levels The results of the pH tolerance of all strains of the collection are shown in Table 1. For the pH between 5 and 9, all of the tested strains presented an optimum development of 100% (Figure 6). Macroscopic observation of these bacteria culture on YEM medium with different pH levels showed that they identically behave such that when they are grown on control medium (pH 6.8). However, Arf1RL1 and Arf4RL13 strains were able to grow at pH of 4 to 10. Our results are in agreement with those of Ali et al., [25] and Harun et al., [26] who reported a tolerance range of pH 4 to pH 10 for some rhizobia strains. Similarly, Indrasumunar et al., [27] reported a tolerance of Bradyrhizobium japonicum strains on media with a pH level of 3.8.


100%

80% 60% 40% 20% 0% 4 4,5 5 5,5 6 6,5 6,8 7 7,5 8 8,5 9 9,5 10 pH

Figure 6. Tolerance range of the strains nodulating alfalfa at different pH values of YEM.

It was suggested that the tolerance of the strains to acidity or alkalinity may be due to the pH level of the origin site soil. In our case, the pH at the KCl (Table 2) measured for different studied sites soils were slightly alkaline. In addition, several studies showed that the Rhizobium is characterized by a large difference in the pH tolerance. A tolerance of alkalin pH was observed for some Rhizobium strains nodulating Lens culinaris that can tolerate a pH of 10 [28]. The physiological and biochemical mechanisms of adaptation of rhizobia under acidic conditions are numerous. These mechanisms include, among others, exclusion and expulsion of protons H+ [29], accumulation of polyamines, the high potassium content and glutamate cytoplasm of stressed cells and the change of the lipopolysaccharide composition [23].

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Table 2. pH KCl of different sites soils used for isolation of rhizobia. Area

Arfoud 1

Aoufous

pHKCl

7.60

7.23

Riche

Arfoud 4

Yahya 7.46

7.41

3.6. Tolerance of tested rhizobia strains to temperature Results indicating the range of temperature tolerance of the studied rhizobia strains (Table 1) showed that most strains can grow at temperatures from 4 to 37 °C. Outside this temperature range, no growth was noticed (Figure 7). Some rhizobia cannot tolerate temperatures higher than 28 °C and it was the case for the strains: Arf1RL1, RyRL11, Arf4RL14, Arf4RL15 and Arf4RL16, which did not grow at temperatures above 28 °C. Also, only Arf1RL1 and Arf4RL13 showed an ability to grow at 4 °C.


100% 80% 60% 40% 20% 0% 4°C

10°C

20°C 28°C 37°C Temperature (°C)

50°C

Figure 7. Strains nodulating alfalfa tolerance to different temperatures studied. In accordance with our findings, Rashid et al., [28] reported that rhizobia are mesophilic bacteria which can grow at temperatures between 4 °C and 37 °C and most strains of the optimum temperature for growth is 28 °C. However, several strains of Ensifer meliloti could tolerate temperatures of 40 °C [30]. As well, mutant strains of rhizobia showed a high ability to survive extreme temperatures of 60 °C [31]. However, several authors have reported that there is no correlation between the temperature of isolation the site and tolerance of strains to heat stress [19]. In this study, we found that strains isolated from Tafilalet region, characterized by hot and arid conditions, tolerate a temperature of 4 °C and do not support against the high temperatures above 37 °C. High temperatures affect the differentiation of Bacteroides of rhizobia as well as the nodules functioning and therefore the N2-fixing ability [32]. This effect is manifested by dehydration and degradation of enzymes of the metabolic pathway of bacteria. In contrary, low temperatures lead to the gelling of the water in the cells, sometimes irreversible inactivation of the enzymes inhibits the expression of Nod genes and thereby infection and nodulation [33].

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3.7. Assessment of phosphate solubilization capacity in tested rhizobia strains Phosphate solubilization test showed that 87.5% of rhizobia nodulating alfalfa tested were able to solubilize the insoluble inorganic phosphate (Table 3). This solubilization was revealed by the presence of a halo around the bacterial colony. The variation in the diameter of the halo showed that there is a variability in the phosphate solubilizing capacity among the tested strains. Among the tested rhizobia, 3 strains (Arf1RL1, RyRL9 and Arf4RL16) were more efficient, compared to the others, with solubilization index(SI) varying from 2.83 to 3.5. Contrary, the two strains (RyRL10 and Arf4RL13) showed relatively low P solubilizing activity. The obtained solubilization index with alfalfa rhizobia was somewhat in agreement with results reported by Marra et al., [34] who reported that bacteria nodulating legumes are often more efficient in solubilizing the inorganic phosphate through their ability to produce gluconic acid involved in this phenomenon. Recently, an Iranian team considered that native rhizobia were able to solubilize phosphate with solubilization indexes between 3.06 for Rhizobium leguminosarun bv. Viciae and 1.41 for Mesorhizobium cicero, M. mediterraneum and E. meliloti [35]. Moreover, Sridevi et al., [36] isolated a bacterial strain of the genus Rhizobium from root nodules of Crotalaria with high tricalcium phosphate solubilizing capacity ranging from 2.40 to 2.70. Table 3. Solubilization of phosphate by rhizobia nodulating alfalfa. Diameter

Diameter Area

Arfoud 1

Aoufous

Riche Yahya

Arfoud 4

strains

Halo Diameter / Colony Diameter

Colony (cm)

Halo + Colony

Arf1RL1

0.40

1.40

3.50

Arf1RL2

1.00

1.90

1.90

Arf1RL3

0.60

1.40

2.33

Arf1RL4

0.80

1.30

1.63

AufRL5

1.00

1.90

1.90

AufRL6

1.10

2.10

1.91

AufRL7

0.70

1.40

2.00

AufRL8

0.60

1.30

2.17

RyRL9

0.60

1.70

2.83

RyRL10

1.60

1.60

1.00

RyRL11

0.70

1.60

2.29

RyRL12

0.60

1.50

2.50

Arf4RL13

2.00

2.00

1.00

Arf4RL14

0.70

1.40

2.00

Arf4RL15

0.70

1.50

2.14

Arf4RL16

0.60

1.80

3.00

4. Conclusion Our results allowed us to conclude that tolerance of the studied rhizobia isolates significantly varied according to their habitat of origin. Rhizobia strains isolated from Tafilalet region 363


presented high osmotolerance level in media with up to 6% NaCl and 8% PEG. The strains were also able to grow over a relatively wide pH and temperature ranges between 4 and 10 and from 10 °C to 28 °C respectively. Also, 87.5% of the tested strains of our collection were able to solubilize insoluble phosphate form. .Arf1RL1 and Arf4RL13 strains from Arfoud region were qualified as the most tolerant to salinity, water deficit and temperature in comparison to other strains.

References 1. C. Huyghe, Fourrages. 174 (2003) 145-162. 2. F. Zakhia, P. De Lajudie, Agronomie., 21 (2001) 569-576. 3. FAO (Food, Agriculture Organization of the United Nations)., FAO, Rome (2004). 4. A. Bouizgaren, Edition INRA 2007. 5. M. Mouradi, M. Farissi, A. Bouizgaren, B. Makoudi, A. Kabbadj, A.A. Very, H. Sentenac, A. Qaddoury, C. Ghoulam, Arid Land Res. Manag., 00 (2016) 1-16. 6. H.B. Shao, L.Y. Chu, C.A. Jaleel, P. Manivannan, R. Panneerselvam, M.A. Shao, Crit. Rev. Biotechnol., 29 (2009) 131-151. 7. M. Farissi, A. Bouizgaren, M. Faghire, A. Bargaz, C. Ghoulam, Seed Sci.Tech., 39 (2011) 389-401. 8. L. Latrach, M. Farissi, M. Mouradi, B. Makoudi, A. Bouizgaren, C. Ghoulam, Turk. J. Bot., 38 (2014) 320-326. 9. H. Mhadhbi, V. Fotopoulos, N. Djebali, A. N. Polidoros, M.E. Aouani, J. Agron. Crop Sci., 195 (2009) 225-231. 10. R.I. Pikovskaya, Microbiologiya., 17 (1948) 362-370. 11. M. Edi-Premono, A.M. Moawad, P.L.G., Vlek, Indonesian J. Crop Sci., 11 (1996) 13-23. 12. D.J. Brenner, T.S. James, Manuel of systematic bacteriology second edition. (2005) 261-266. 13. J. Slattery, D. Pearce, In: D. Herridge, (Ed,) (2002). 14. K. Habtegebrail, B.R. Singh, Nutr. Cycl. Agroecosystem., 75 (2006) 247-255. 15. M. Jebara, J.J. Drevon, M.E. Aouani, Agronomie., 21 (2001) 601-605. 16. M. Abbas, M. Monib, A. Rammah, M. Fayez, N. Hegazi, Agronomie., 21 (2001) 517-525. 17. M. Jida, F. Assefa, Afr. J. Biotechnol., 11 (2012) 7483-7493. 18. J. Maâtallah, E. Berraho, J. Sanjuan, C. Lluch, J.Agro., 22 (2002) 321-329. 19. S.H. Mohammed, A. Smouni, M. Neyra, D. Kharchef, A. Filali-Maltouf, Plant Soil., 224 (2000) 171183. 20. K.V.B.R. Tilak, N. Ranganayaki, K.K. Pal de R, A.K. Saxena, C.S. Nautiyal, S. Mittal, A.K. Tripathi, B.N. Johri, Curr. Sci., 89 (2005) 136-150. 21. I. Thami-Alami, N. Elboutahiri, S.M. Udupa, Options Méditerranéennes. 92 (2010) 265-269. 22. O.N. Reva, V.V. Smirnov, B., Petterson, F.G. Priest, Int. J. Syst. Evol. Microbiol., 52 (2002) 101-107. 23. J.A.C. Vriezen, J. Frans, de Bruijn, K. Nusslein, App. Environ. Micro., 73 (2007) 3451-3459. 24. J.C. Den Herder, R. Vanhee, V. De Rycke, M. Corich, M. Holsters, S. Goormachtig, Mol. Plant Microbe Interact., 20 (2007) 129-137. 25. S.F. Ali, L.S. Rawat, M.K. Meghvansi, S.K. Mahna, J. Agric. Bio. Sci., 4 (2009) 13-18. 26. M. Harun, M.A. Sattar, M.I. Uddin, J.P.W. Young, Electronic J. Environ. Agric. Food Chem., 8 (2009) 602-612. 27. A. Indrasumunar, W. Menzies Neal, P.J. Dart, Soil Biol. Biochem., 48 (2012) 135-141. 28. M.H. Rashid, H. Schäfera, J. Gonzaleza, M. Winka, Systematic Appl. Micro., 35 (2012) 98-109. 29. O.N. Kurchak, N.A. Provorov, B.V. Simarov, Russ. J. Genet., 37 (2001) 1025-1031. 30. N. El boutahiri, I. Thami-Alami, S.M.U. dupa, BMC Microbiol., 10 (2010) 1471-2180. 31. B.E. Boboye, B.A. Ogundeji, H. Evbohoin, 2011. Adv. Biosci. Biotechnol., 2 (2011) 255-262. 32. R.F. Denison, E.T. Kiers, FEMS Microbiol. Lett., 237 (2004) 187-93. 33. S. Phadtare, K. Yamanaka, M. Inouye, ASM Press, Washington, D.C. (2000) 33-45. 34. L.M. Marra, S.M. de Oliveira, C.R.F.S. Soares, F.M.S. Moreira, Sci. Agric., 68 (2011) 603-609. 35. H.A. Alikhani, N. Saleh-Rastin, H. Antou, Plant Soil., 287 (2006) 35-41. 36. M. Sridevi, K.V. Mallaiah, N.C.S. Yadav, J. Plant Sci., 2 (2007) 635-639.

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