EJBR2017v7i1art9 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2017; 7 (1): 9-21 Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt Mohamed H. Abd-Alla, Abdelwahab E. Elenany, Taha R. Mohamed, Manal El Zohri, Ibrahim M. Nafady* Department of Botany and Microbiology, Faculty of Science, Assiut University, Assiut 71516, Egypt *Corresponding author: Ibrahim M. Nafady; Tel.: 20 1001376883; Fax: 20 88 2342708; E-mail: mhabdalla@aun.edu.eg, imanafady@yahoo.com ABSTRACT This study was devoted to exploring the natural nodulation and nitrogen fixation of wild legumes grown in different Egyptian habitats. These habitats are representative to four phytogeographical regions. Sites that inhabited by Melilotus indicus, Medicago polymorpha, Trifolium resupinatum, Trigonella hamosa and Vicia sativa in each region were selected for study. High nodulation, nitrogen fixation and plant biomass were recorded in plants grown at Nile region and Oases compared with those at Mediterranean region and Sinai. The inhibition in nodulation and potential of nitrogen fixation in legumes at MR and S were attributed to drought and low soil fertility. Differences in species, regions or their interaction have significant effect on nodulation, legheamoglobin, nitroginase activity and biomass of nodules, shoots and roots; the magnitude of effect due to different species was the greatest. Five rhizobial isolates (Sinorhizobium fredii, Rhizobium mesosinicum, Rhizobium daejeonense, Rhizobium huautlense, Rhizobium alamii) recovered from root nodules of the five species were identified by 16S rRNA gene sequence. The indigenous rhizobia of legumes grown at MR and S expected to be exhibit higher tolerance to the existing harsh environmental conditions. These rhizobia can be used as inoculants for crop legumes under unfavo- rable environmental conditions of agroecosystems or recently reclaimed desert. Keywords: Nodulation; Nitrogen fixation; Wild legumes; Legheamoglobin; Rhizobia. 1. INTRODUCTION The leguminous plants constitute one of the largest families of the flowering plants, consisting of ca. 730 genera and ca. 19,400 species [1]. It is an extremely diverse family with worldwide distribution, encompassing a wide range of life forms, from arctic alpine herbs and temperate or tropical perennial shrubs to annual xerophytes and equatorial giant trees [2]. Legumes play a vital role in agro-ecosystems based on their ability to form a symbiosis with soil rhizobia that fix atmospheric nitrogen [3, 4]. Biologically fixed N2, either asymbiotic, associative, or symbiotic, is considered a renewable resource that should constitute an integral part of sustainable agro-ecosystems globally [5, 6]. Rhizobium spp. are Gram-negative soil bacteria that have a profound scientific and agronomic significance due to their Received: 10 October 2016; Revised submission: 09 December 2016; Accepted: 29 December 2016 Copyright: © The Author(s) 2017. European Journal of Biological Research © T.M.Karpiński 2017. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.224013 10 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 ability to establish nitrogen-fixing symbiosis with leguminous plants, which is of major importance for the maintenance of soil fertility [7, 8]. Nitrogen fixation by legumes, play an important role in sustaining crop productivity and soil reclamation of the semi-arid areas [9, 10]. It is well documented that wild leguminous plants inhabiting any region show adaptability to the environment and fix the atmospheric nitrogen more efficient than the cultivated legumes in that region [11-13]. Little information has been reported on natural nodulation of wild legumes [14-16]. One fascinating application of rhizobia of wild legumes is using it as inoculums for crop legumes. Many reports proved that some rhizobia of wild legume are more efficient in nitrogen fixation activity with other hosts than their compatible hosts [17, 18]. It has been reported that cross inoculation of crop legumes with rhizobia isolated from wild non- crop legumes enhanced nodulation and nitrogen fixation [19]. Nodule formation and nitrogen fixation of legumes are strongly affected by sub-optimal soil conditions, such as temperature extremes, salt stress, high or low soil pH, low water content, pesticide application and nutrient deficiency [20]. Environ- mental differences also strongly affect demographic processes like germination and seedling recruitment, which in turn affect genetic differentiation among plant populations [21], often due to random processes such as founder effects or genetic drift [22]. Hence, the hereditary structure of any plant species reflects its interaction with the environment [23]. Genetic diversity is strongly influenced by reproductive mode and mating system [24, 25]. In addition, genetic diversity is assumed to increase with abiotic and biotic heterogeneity and in stressful environments [26, 27]. Thus, ecological components such as temperature and precipitation also affect genetic diversity [28, 29]. The ability of species to respond to changes in the environment will ultimately determine survival in a particular habitat [30, 31]. Egypt extends across large areas of land comprising several geographical regions differ topographically, climatically and environmentally in general. Egypt is the meeting point of floristic elements belonging to at least four different regions: the African Sudano-Zambezian, the Asiatic Irano- Turanian, the Afro-Asiatic Saharo-Arabian, and the Euro-Afro-Asiatic Mediterranean [32]. Egyptian farmers facing a problem in providing crops with required nutrients, especially nitrogen, due to inadequate supply of mineral nitrogen fertilizers or the costs of these fertilizers. However, there is an urgent need to find alternatives on the base that soil fertility strongly depends on metabolic activities of microbes. Efficient symbiotic nitrogen fixation reduces the level of the requirement for external input of mineral nitrogen fertilizers. As nodule activity is known to vary diurnally and seasonally, also nodulation and nitrogen fixation of wild legumes could be varied depending on their habitat. Hence, improving our knowledge around the ecological distribution of wild legumes is a topic of utmost importance to better understand how to preserve it, increase their import and select the most efficient nitrogen-fixing wild legumes. Therefore, the present research aimed to study the biodiversity and biogeography of rhizobia associated with some wild legumes and their ability to fix atmospheric nitrogen. 2. MATERIALS AND METHODS 2. 1. Soil sampling and analysis Soil samples from each site at four phytogeographical regions were collected for the physical and chemical analyses. Three soil samples were collected from profiles of 0-50 cm depth, pooled together to form a single composite sample, and carried to the laboratory in plastic bags. The samples were then spread over sheets of paper and left to dry in the air. Dried soils were passed through a 2 mm sieve and packed into paper bags for analysis. Soil texture was determined according to Allen et al. [33], organic matter according to Walkley and Black [34], soil sodium and potassium according to Williams and Twine [35], calcium and magnesium according to Johnson and Ulrich [36], chlorides according to Hazen [37], sulphates according to Black et al. [38], phosphates according to Woods and Mellon [39], bicarbonates accor- ding to Piper [40], nitrate according to Markus, McKinnon and Buccafuri [41], electric conductivity according to Jackson [42]. The pH value and soil water content also were determined. 11 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 2.2. Vegetation sampling and preparation The current study was carried out along two successive years 2010-2011. The studied stands were chosen at locations inhabited by five wild nitrogen fixing legumes namely: Melilotus indicus (L.) All., Medicago polymorpha L., Trifolium resupinatum L., Trigonella hamosa L. and Vicia sativa L. One site at each of four Egyptian phytogeographical regions was chosen for this study: Assiut site in NR (27º 08’ N, 31º 20’ E) Al- Kharga site representing Oases (25º 32’ N, 30º 37’ E), Burg Al-Arab site in MR (30º 57’ N, 29º 37’ E) and Saint Katherine site in South Sinai (28º 33’ N, 33º 56’ E). At each site three individuals (as replicates) from the same population of every plant species were collected, and separated into roots and shoots. The shoots and roots were washed several times with distilled water, blotted gently with filter paper and were quickly weighted for fresh biomass (FW) determination and oven-dried at 70 oC for 48 hour to determine the dry biomass (DW). 2.3. Assessment of nodulation Nodulation was assessed by up-rooting the plant, washing away adhering soil particles, and counting the number of nodules present. Nodules fresh and dry biomasses were determined. Some nodules from another individuals were also detached to check for the presence of red pigment (leghaemoglobin). 2.4. Determination of leghaemoglobin in nodule cytosol One gram of fresh nodules was rinsed thoroughly with distilled water and immediately hand ground in an ice chilled mortar with 5 ml of distilled water. Nodule homogenates were filtered through four layers of cheesecloth and the filtrate was centrifuged at 500 x g for 2 min to remove nodule debris. The resulting supernatant was centrifuged at 12,000 x g for 15 min to sediment the bacteroids. Leghaemoglobin levels in the super- natant, the 'nodule cytosol', were determined colorimetrically as described by LaRue and Child [43], using Unico UV-2100 spectrophotometer. The colorimetric assay was standardized using freshly preapaerd Hemetrol reagent (solution of cyanmet- hemoglobin titrated exactly according to recom- mendations of BioMerieux, Marcyletoile, 69260 Carbon nieres les Bains, France) 2.5. Determination of nitrogenase activity Nitrogenase activity was determined in detached roots, using gas chromatograph as descri- bed by Abd-Alla [44], (Thermo Scientific TRACE GC Ultraequipped with FID detector and Capillary column CP-PoraBOND Ufused silica plot 25 m × 0.32 mm, df = 7 m). The excised nodulated roots were placed in 500 ml bottles sealed with a rubber septum. 50 ml of air were taken and the same volume of acetylene gas introduced into the bottle, incubated at 37 ˚C then samples from root atmosphere in bottles were with-drawn and injected to the gas chromatograph. Afterwards nodules of each individual root were counted and nodules fresh and dry mass were estimated. A calibration curve was constructed using pure ethylene. 2.6. Determination of proline content Free proline was determined in fresh tissues according to method of Bates, Waldren and Teare [45], shoots samples (30 mg) were homogenized in 6 ml 3% sulfosalicylic acid, then filtered through filter paper. After addition of acid ninhydrin and glacial acetic acid, the resulting mixture was heated for 1 h in water bath at 100 ºC. The reaction was stopped by using ice bath. The mixture was extracted with toluene and mixed vigorously. The chromophore containing toluene was aspired from the aqueous phase and the absorbance measured at 520 nm. Proline concentration was determined using calibration curve. 2.7. Isolation of rhizobial strains Twenty representative sites inhabeted by the studied species (one site for each species at each of the four phytogeographical regions) were chosen for rhizobial isolation. The strains of rhizobia were isolated from root nodules of M. indicus, M. poly- morpha, T. resupinatum, T. hamosa and V. sativa. The isolates were grown on yeast extract mannitol 12 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 agar (YEMA) medium and incubated at 28 ˚C [46] on an orbital shaker at 120 rev per min for three days. 2.8. Molecular identification of rhizobia From bacterial cultures using SDS/CTAB lysis and phenol/chloroform extraction method Ausubel et al. [47], DNA extracted sent to South Korea, Solgent Co., Ltd Bio industry Development for PCR-amplified at using primer pairs 16S (1492R 5' TACGGYTACCTTGTTACGACTT 3' and 27F 5' AGAGTTTGATCMTGGCTCAG 3'). The sequence reads were edited and assembled using BioEdit version 7.0.4 (www.mbio.ncsu.edu/ BioEdit/bioedit.html) and clustal W version 1.83 (http://clustalw.ddbj.nig.ac.jp/top-e.html). BLAST searches were done using the NCBI server at www.ncbi.nlm.nih.gov/blast/Blast.cgi. The rhizobial isolates identified and recoded in GenBank. 2.9. Nucleotide sequence accession numbers The nucleotide sequences of the rhizobial isolates namely, Sinorhizobium fredii, Rhizobium huautlense, Rhizobium daejeonense, Rhizobium alamii, Rhizobium mesosinicum were deposited in the GenBank nucleotide sequence database under accession number [GenBank: KF879914.1, KF87 9916.1, KF879917.1, KF879920.1, KF879921.1, respectively]. 2.10. Statistical analysis Data were subjected to statistical analysis using SPSS package (version 19). One-way ANOVA, followed by Duncan multiple range test were employed and the differences between means deemed to be significant at p < 0.05. Correlation analyses were carried between soil variables and some parameters estimated in plants. Factorial ANOVA was carried to achieve the effect of species, regions and their interaction on different parameters estimated in plants and η2 was calculated as: η2 = SSbetween / SStotal. 3. RESULTS 3.1. Soil The data recorded in Table 1 revealed that there are significant differences between the physical and chemical properties of soils of the four phytogeographical regions. Soil of NR was characterized by the highest values of water content, K+ and organic matter (OM), while soil of MR was characterized by higher content of HCO3 - . Compare to other regions, soil of Saint Katherine (S) have high concentrations of Na+, Mg+2 and SO4 -2 , and hence the TSS in the soil was high. The soil of the NR and O were rich in NO3 -, while the soil of the NR and MR were rich in PO4 -3. The pH value of MR and S soil was high as compared with the other regions. 3.2. Nodulation The present study proved that the nodulation differ significantly among the studied plant species and as affected by the habitats at the four studied phytogeographical regions. Nodules number in all legumes inhabiting NR, O and MR increased significantly compared with those grown at Sinai (Fig. 1). Amongst the studied species, T. resu- pinatum showed the highest nodules number averaging about 223 nodule/individual plant compared with less than 70 in the four other legumes. The highest fresh and dry weight of root nodules was recorded in most species grown at the MR. Across all species, the fresh weight of nodules for plants sampled from MR was more than 1.5-fold of those sampled from other regions. The maximum fresh and dry weight of nodules was recorded in M. indicus (non-significant increase) (Fig. 2). Although there was a significant positive correlation between nodules fresh and dry biomass (r-value= 0.891**), both did not depend on the nodules number. Variations between species exerted the greatest magnitude of effect on the number of nodules where η2 = 0.904 compared with η2 = 0. 064 for the effect of regions. Nodules fresh and dry biomass affected by differences between habitats more than their number (Table 3). 13 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 3.3. Leghaemoglobin content and nitrogenase activity Leghaemoglobin content of root nodules and nitrogenase activity (acetylene reduction) differed significantly among the studied plant species as affected by the phytogeographical regions. The significant increases in leghaemoglobin content were recorded in legumes grown at S and NR (Fig. 3). The highest leghaemoglobin content and nitrogen-fixing activity were estimated in T. resupinatum (Figs. 3, 4). Despite there are non-significant differences in the content of leghaemoglobin between T. resupinatum, T. hamosa and V. sativa, interestingly the nitrogenase activity in T. resupinatum was about 10-fold of the activity in the both other species. Legumes grown at NR were characterized by a significant increase in nitrogenase activity compared with those at other regions. In all studied species, the content of leghaemoglobin was strongly correlated with the concentration of PO4 3- and water content (WC%) of soil, while it negatively correlated with SO4 2-, NO3 -, TSS, pH, Na+, Ca2+ and Mg2+ (Table 2). Nitroginase activity was positively correlated with the concentration of Cl-, PO4 3- and WC% of soil, while the activity negatively correlated with SO4 2- (weak –ve r-values), NO3 -, TSS, HCO3 -, pH, K+, Ca2+ and Mg2+. As shown in Table 3, variation in species have the greatest magnitude of effect on legheamoglobin content (η2 = 0.878), while its effect on nitrogenase activity reduced (η2 = 0.487) in favor of differences in habitats or regions (η2 = 0.299). Table 1. Some physical and chemical properties of soils at different studied habitats. NR, Nile region (Assiut); O, Al-Kharga Oases; MR, Mediterranean region (Burg Al-Arab); S, South Sinai (Saint Katherine); TSS, Total soluble salts; OM, Organic matter. Values are means± SD, n=5. Regions Parameter NR O MR S Soil texture Clay Clay loam Loam Loam Water content % 30.82±2.29 d 20.80±0.76 c 13.73±0.88 b 8.47±0.70 a pH (in 1:5 extract) 7.68±0.13 a 7.78±0.09 a 7.98±0.02 b 8.09±0.02 b E.C. (mS/cm) 0.365±0.06 ab 0.45±0.07 b 0.33±0.06 a 0.57±0.09 c TSS% 0.117±0.02 ab 0.144±0.02 b 0.107±0.02 a 0.182±0.03 c OM % 1.61±0.08 c 1.23±0.11 bc 0.82±0.16b 0.47±0.05 a Na+ m g/ g so il 0.13±0.01 a 0.15±0.03 a 0.13±0.01 a 0.19±0.02 b K+ 0.10±0.02 c 0.01±0.00 a 0.04±0.00 b 0.04±0.01 b Ca+2 0.34±0.04 a 0.45±0.03 b 0.48±0.01 b 0.48±0.03 b Mg+2 0.09±0.04 a 0.09±0.04 a 0.12±0.02 a 0.29±0.04 b Cl- 0.83±0.20 a 0.89±0.18 a 1.14±0.18 b 1.12±0.14 b HCO3 - 2.44±0.30 a 2.44±0.31 a 3.25±0.32 c 2.75±0.26 ab NO3 - 0.37±0.02 c 0.35±0.06c 0.22±0.02 b 0.08±0.01a SO4 -2 0.43±0.05 a 0.38±0.04 a 0.34±0.01 a 0.85±0.04 b PO4 -3 0.06±0.001b 0.043±0.001a 0.063±0.004 b 0.039±0.002 a Means with different letters are significantly different according to Duncan comparisons (P < 0.05). 14 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 Figure 1. Nodule number (nodule/plant) of the legumes M. indicus, M. polymorpha, T. resupinatum, T. hamosa and V. Sativa inhabiting different phytogeographical regions of Egypt (NR= Nile region; O = Oases; MR = Mediterranean region; S = Sinai). The values are mean ± SD, n = 3, means of each species with different letters are significantly different at P< 0.05. Figure 2. Nodule fresh and dry biomass (g/plant) of five legumes inhabiting different phytogeographical regions of Egypt. Statistics as in Fig. 1. Figure 3. Leghaemoglobin content (mg g-1 nodule FW) in five legumes inhabiting different phytogeographical regions of Egypt. Statistics as in Fig. 1. Figure 4. Nitrogenase activity (µ mole C2H4 h -1) of five legumes inhabiting different phytogeographical regions of Egypt. Statistics as in Fig. 1. 3.4. Proline content The data recorded in Figure 5 shows that differences in habitat conditions have a significant effect on the proline content in the five wild leguminous plants. High proline content was recorded in plants grown at MR and S compared to those grown at the NR and O. It is clear that M. indicus and T. hamosa contain high proline content compared with M. polymorpha, T. resu- pinatum and V. sativa. On the bases of pooled data, proline content in M. indicus, T. hamosa and V. sativa was significantly higher than that in M. polymorpha and T. resupinatum. Also, the proline content in plants at MR and S was significantly higher than that in plants at NR and O. Correlation analyses of proline content in shoots of leguminous plants with soil variables showed undefined trend. Proline content negatively correlated with soil K+ (r-values between -0.796 and -0.870), while it positively and weakly correlated with Na+. A significant positive correlation resulted between proline content in M. indica and concentration of NO3 - in the soil, but a significant r-value resulted in case of M. polymorpha. Against what was expected, a very weak correlation has been found between proline content in all studied plants and TSS in the soil (Table 2). Also, differences between species have the major magnitude of effect on proline content of shoots (η2 = 0.77) rather than changing habitat conditions (Table 3). 15 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 Table 2. r-values of linear correlation analyses between some parameters estimated in different leguminous plants and soil variables (WC = soil water content; TSS = total soluble salts). Para- meter Plant species Soil variables Cl- SO4 -- PO4 -- HCO3 -- NO3 - WC TSS pH Na+ K+ Ca++ Mg++ N od ul e fr es h bi om as s M. indicus -0.060 -0.511 0.253 0.544 0.313 0.249 -0.310 -0.900* -0.268 -0.624 -0.166 -0.573 M. polymorpha 0.984** -0.596 0.964* -0.579 -0.999** 0.975* -0.115 -0.767 -0.430 -0.680 -0.365 -0.255 T. resupinatum 0.792 -0.289 0.975* 0.155 -0.847 0.720 -0.555 -0.262 -0.047 -0.645 0.160 -0.578 T. hamosa 0.853 -0.175 0.968* -0.454 -0.622 0.774 -0.547 -0.148 0.008 -0.588 -0.668 0.005 V. sativa 0.997** -0.465 0.993** 0.553 -0.798 0.840 -0.397 -0.690 -0.105 -0.613 -0.252 -0.172 N od ul e dr y bi om as s M. indicus -0.312 -0.303 -0.062 0.677 0.544 -0.056 -0.332 0.670 -0.463 -0.032 0.335 -0.247 M. polymorpha -0.896 0.957* -0.618 0.950* 0.781 -0.915* -0.847 0.091 -0.358 0.038 0.545 -0.503 T. resupinatum -0.374 0.482 -0.598 0.667 0.851 -0.986** -0.641 0.198 -0.479 -0.109 0.360 -0.368 T. hamosa 0.187 0.020 0.447 0.643 -0.350 0.108 -0.223 0.956* -0.624 -0.029 -0.274 -0.003 V. sativa -0.347 -0.210 -0.180 0.501 -0.084 -0.266 -0.728 0.292 -0.551 0.016 0.677 -0.588 L eg he m og lo bi n M. indicus 0.964* 0.122 0.808 -0.172 -0.348 0.361 -0.953* -0.369 -0.937* 0.074 -0.631 -1.000** M. polymorpha 0.996** -0.785 0.862 -0.772 -0.955* 0.999** -0.178 -0.940* -0.984** 0.041 -0.588 -0.928* T. resupinatum -0.049 -0.925* 0.644 -0.333 -0.884 0.817 -0.692 -0.769 -0.840 -0.008 -0.357 -0.985** T. hamosa -0.372 -0.982** 0.393 -0.089 -0.869 0.690 -0.998** 0.121 -0.798 0.123 -0.190 -0.699 V. sativa 0.572 -0.986** 0.660 0.272 -0.954* 0.855 -0.786 -0.861 -0.865 0.118 -0.399 -0.885 N it ro gi na se ac ti vi ty M. indicus 0.840 -0.495 0.990** -0.174 -0.495 0.798 -0.099 -0.950* 0.025 -0.174 -0.495 -0.272 M. polymorpha 0.874 -0.303 0.998** -0.283 -0.957* 0.851 0.588 -0.594 -0.129 -0.258 -0.721 0.063 T. resupinatum 0.985** 0.249 0.672 -0.039 -0.525 0.574 0.132 -0.456 0.141 -0.141 -0.327 -0.173 T. hamosa 0.977* 0.143 0.767 -0.626 -0.267 0.554 -0.282 -0.783 0.287 -0.151 -0.067 -0.183 V. sativa 0.780 -0.259 0.669 -0.133 -0.468 0.736 0.232 -0.706 0.172 -0.196 -0.736 0.172 P ro li ne M. indicus -0.193 0.706 -0.409 0.911* 0.995** -0.863 0.267 -0.849 0.306 -0.825 0.236 -0.025 M. polymorpha 0.981** -0.848 0.802 -0.836 -0.917* 0.989** -0.036 -0.300 0.139 -0.870 -0.036 0.315 T. resupinatum 0.296 -0.347 0.704 0.811 -0.409 0.045 -0.222 0.210 0.513 -0.796 0.452 -0.043 T. hamosa 0.600 0.933* -0.044 0.195 0.612 -0.482 0.007 -0.250 0.548 -0.815 -0.702 0.489 V. sativa 0.834 0.100 0.771 0.507 -0.332 0.446 0.037 -0.255 0.457 -0.841 -0.025 0.386 **:Correlation is significant at P< 0.01. *:Correlation is significant at P< 0.05. Figure 5. Proline content (mg/g FW) in shoots of the five studied legumes. Statistics as in Fig. 1. Figure 6. Shoot and root fresh biomass (g/plant) of the five studied legumes. Statistics as in Fig. 1. 16 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 Table 3. F-values of factorial ANOVA for the effect of species, regions and their interaction; and eta-square (η2) calculated for each factor. Species Region Species*Region Parameter F-value η2 F-value η2 F-value η2 Nodule number/ plant 3848.442 0.904 456.853 0.064 52.825 0.030 Nodule fresh biomass 385.634 0.835 63.956 0.083 12.414 0.065 Nodule dry biomass 421.846 0.776 106.467 0.117 20.856 0.092 Leghemoglobin 700.039 0.878 126.794 0.095 5.310 0.016 Nitroginase activity 178.100 0.487 182.088 0.299 29.166 0.192 Proline 649.648 0.770 218.976 0.156 22.627 0.064 Shoot fresh biomass 212.259 0.634 137.211 0.246 13.410 0.096 Shoot dry biomass 231.130 0.666 135.545 0.235 10.976 0.076 Root fresh biomass 196.687 0.805 28.338 0.070 9.493 0.093 Root dry biomass 136.973 0.770 29.391 0.099 6.414 0.086 All F-values are significant at p < 0.001. Figure 7. Shoot and root dry biomass (g/plant) of the five studied legumes. Statistics as in Fig. 1. 3.5. Plant biomass The fresh and dry biomass of collected legumes was affected by phytogeographical regions. The highest fresh and dry biomass of the shoot and root system was recorded in the wild plants collected from NR followed by O, MR and S. Amongst species, T. resupinatum grown at NR attained the highest fresh biomass while M. polymorpha attained the highest dry biomass (Fig. 6, 7). Calculating the ratio of rootdry biomass: shootdry biomass indicated that M. indicus (0.202), M. polymorpha (0.214) and T. resupinatum (0.384) inhabiting MR and T. hamosa (0.323) and V. sativa (0.446) inhabiting O have the highest ratios. The lowest root/shoot ratios were recorded for all studied species inhabiting NR (0.042 – 0.113). Averaging across all habitats, T. hamosa has the highest ratio (0.211), while M. indicus (0.129) and M. polymorpha (0.130) have the lowest ratios. As indicated by η2 in Table 3, the plant, especially root, biomass is greatly related to the variations between species rather than regions or species*regions. 3.6. Rhizobial isolates The rhizobial isolates recovered from different wild legumes collected from different habitats were identified by 16S rRNA gene sequence. The root-nodule bacteria that was isolated from M. indicus, M. polymorpha, T. resupinatum, T. hamosa, V. sativa and had been classified into five species namely: Sinorhizobium fredii, Rhizobium mesosinicum, Rhizobium daejeonense, Rhizobium huautlense, Rhizobium alamii, respec- tively. 17 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 4. DISCUSION The present study clearly indicates that there is a critical role of the habitat conditions on nodulation and nitrogen fixation of wild legumes as indicated by the significant differences in number and biomass of the nodules between individuals of each studied species at different regions. Generally, soils of all habitats at the four phytogeographical regions where the wild legumes studied were not saline and the TSS were less than 0.2% (concentration of Na+ was less than 0.02%). Soil at Assiut (NR) and for some extent at Kharga (O), with high water content, organic matter, and nutrients such as K+, Ca+2 and PO4 -3 represents the best habitat for nodulation, nitrogen fixation and growth of plants. This could be attributes to the clay or clay loamy soil at NR and Oases, respectively. The desert soils of Egypt are inherently low in organic matter due to the arid or hyper-arid climate and historically low vegetation cover, and at all regions the OM were ranging from 0.5-1.6%. The fine textured soils at NR and O were characterized by relatively high level of organic matter, and hence increasing water retention capacity, K+, Ca+2 and Mg2+. Such conditions are essential for survival of rhizobia in the soil and support the process of root hair infection and nodule development. This study supports what have been found by Rao and Venkateswarlu [48] that not the high Indian desert soil temperature but the low organic matter and poor soil moisture were the major factors that reduced the numbers of different micro-organisms. The poor nodulation and nitrogen fixation of wild legumes grown in sandy soils of MR and S could be attributed to the coarse soil, decreasing content of organic matter and water shortage. However, the nodule fresh biomass positively correlated, while its dry biomass negatively correlated with the soil water content; but both of the fresh and dry biomass negatively correlated with TSS. Another an important factor affecting nitrogen fixation is the temperature. The studied five legumes are annual herbs and their height ranging from 10 to 60 cm [49]; so they complete their life cycle nearly through the winter (the samples collected on April). The average maximum temperature of five months prior to sampling (from December to April) at Assiut, Kharga, Burg Al-Arab and Saint Katherine was 23 ± 2, 23 ± 2, 19 ± 1 and 15 ± 3 °C; while the average minimum temperature was 8 ± 2, 10 ± 2, 10 ± 1 and 4 ± 2 °C, respectively. As Abdel Gadir and Alexander [50] reported in Egyptian sandy soils, the temperature near the soil surface was 59°C when the air temperature was 39°C. However, the soil temperature decreased rapidly with depth, being moderate 35 °C, at 15 cm. Every bacterium has its own optimum conditions, under which it grows at its best. For most rhizobia, the optimum temperature range for growth is 28-31 °C, and many are unable to grow at 37°C [12]. Also, temperature plays an essential role on the exchange of molecular signals between rhizobia and their partners, thus reducing nodulation [51]. However, low tempe- rature may be critical factor reducing nodulation and nitrogen fixation activity in all species at Saint Katherine. Water, and its availability, is one of the most critical environmental factors that affect the growth and survival of micro-organisms. Drought is one of the most common stresses soil microorganisms have to face. The responses of bacterial cells to drought can be: shrinkage of the bacterial cytoplasm and capsular layers, increase in intracellular salt levels, crowding of macromolecules, damage to external layers (pili, membranes), changes in ribosome structure, and decrease in growth [52]. A shortage of water supply can slow the growth of the nodule and accelerate its senescence. So these results compatible with Ralston and Imsande [53] who reported that nodules in dry soils lose water faster than the vascular system can supply it and hence suffer water stress. Shortage in water supply to the nodules may result in collapse of cells near the surface creating impaired diffusion which reduce the adverse effects of drought. This also reflects why the water content of nodules is too low in plants inhabiting the MR sandy formations and South Sinai. Previous study indicated that harmful effects of water deficit can be alleviated by increasing K2+ supplementation [54]. Water stress is quickly reflected as changes in hormonal content [55]. The nodules are an active site of synthesis of auxins and cytokinins. Therefore, it is likely that nodules, besides the supply of organic N, are a source of cytokinins that makes the plant more tolerant to water stress [56]. The results indicated that there is a significant variation in rooting development 18 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 between the studied species as reflected on the root: shoot ratios. Plants inhabiting the NR, with the highest soil water content, have the lowest root: shoot ratios and vice versa for those inhabiting Sinai where the soil is relatively dry. However, in dry soil the plants tend to increase the extinction of lateral and sinker roots for mineral uptake and water absorption; and translocation of photosynthats for this vital purpose will affect negatively on that transported to nodules. Nitrogenase activity is decreased signifi- cantly, accompanied by the decrease in respiratory activity of the root nodules [57, 58]. A limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of nitrogenase activity in alfalfa nodules [59]. In addition, the transport of fixed nitrogen out of the nodule is decreased possibly due to an insufficient supply of photo- synthates from stems and leaves under stress [60]. Leghaemoglobin content of nodule cytosol was also severely inhibited by drought stress so the leghaemoglobin content in this study was high in NR followed by O and low in MR and S. This decline was attributed to the induction of protease activity [54]. Legumes tend to maintain a level of O2 within their nodules that can support respiration but is sufficiently low to avoid inactivation of nitroginase [61]. Despite leghaemoglobin act as a buffer for nodule O2, Denison and Harter [62] adduced that it stores only enough O2 to support nodule respiration for a few seconds. Gas permeability in nodules decreases under drought or upon exposure to nitrate, and as the permeability decreases may be there is no need to further leghaemoglobin. This may explain why the content of leghaemoglobin increased significantly in nodules of all studied species inhabiting NR, while it negatively correlated (significant –ve r-values in M. polymorpha and V. sativa) with the concentrations of NO3 - in the soil. Proline considered as an indicator for response to environmental stresses and it accumulates in relatively large quantities under stress conditions [63, 64]. Proline content was relatively high in shoots of wild legumes grown at MR and S, and this may be attributed to the low soil water content found at these habitats. Also, this proved that the plants at MR and S, at period of sampling, were more exposed to environmental stresses. The strong –ve correlation between contents of proline in shoots of all studied legumes and K+ concentrations in the soil, have lead to a suggestion that availability of K+ may suppress proline synthesis. Rhizobial isolates recovered from wild legume plants grown in S and MR such as Medicago polymorpha (Rhizobium mesosinicum ASU8) and Trigonella hamosa (Rhizobium huautlense ASU3), are of good traits, such as tolerant to high salt, drought and temperature level. Isolation of root-nodules bacteria of wild legumes growing in arid region is very attractive and promising. These indigenous rhizobia are characterized by wide host ranges that offer these legumes ecological benefit. Therefore, successful isolation of rhizobia from such environment will definitely result in obtaining good rhizobia candidates for establishing successful symbioses in extreme environments useful for production of crop legumes. It is well documented that native rhizobia can form nodules with other wild or cultivated crop legumes, and can be utilized for genetic manipulation to improve and perform of symbiotic characters of other root nodule bacteria with crop legumes [19, 65] 5. CONCLUSION The present investigation revealed that the natural nodulation and nitrogen fixation of wild legumes are drastically affected by habitat. In contrast to Mediterranean region and Sinai, wild legumes inhabiting Nile region and Oases were characterized by high nodulation and nitrogen fixation. Rhizobium mesosinicum ASU8 and Rhizobium huautlense ASU3 isolated from root nodules of Medicago polymorpha and Trigonella hamosa grown at Sinai and Mediterranean region are expected to be more tolerant to harsh environmental conditions than rhizobia from cultivated legumes. These rhizobia could be valuable in agricultural practice, specifically in the inoculation of crop legumes grown under unfavorable conditions or in the new reclaimed soil. 19 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 ACKNOWLEDGEMENTS This research was supported by Assiut University Fund. AUTHORS’ CONTRIBUTION MHA-A: Conception, design of the work and Critical revision of the article; AEE: Data collection and revised the manuscript; TRM: Data analysis and interpretation the experimental; ME: Drafting the article; IMN: carried out the practical experiments. The final manuscript has been read and approved by all authors. TRANSPARENCY DECLARATION The authors declare no conflicts of interest. REFERENCES 1. Lewis GP, Schrire B, Mackinder B, Lock M. Legumes of the world. Royal Botanic Gardens Kew, 2005. 2. Van Rhijn R, Vanderleyden J. The rhizobium-plant symbiosis. Microbiol Rev. 1995; 59: 124-142. 3. Mortier V, Holsters M, Goormachtig S. Never too many? How legumes control nodule numbers. Plant Cell Environ. 2012; 35: 245-225. 4. Abd-Alla MH, El-Enany AW, Nafady NA, Khalaf DM, Morsy FM. Synergistic interaction of Rhizobium leguminosarum bv. viciae and arbuscular mycorrhizal fungi as a plant growth promoting biofertilizers for faba bean (Vicia faba L.) in alkaline soil. Microbiol Res. 2013; 169: 49-58. 5. Abd-Alla MH. Nodulation and nitrogen fixation in faba bean (Vicia faba L.) plants under salt stress. Symbiosis. 1992; 12: 311-319. 6. Ferreira de Araujo AS, Figueiredo MVB, Monteiro RTR. Potential of biological nitrogen fixation as indicator of soil pollution. In: Couto GN, ed. Nitrogen fixation research progress. Nova Science Publishers, New York. 2008: 1-13. 7. Somasegaran P, Hoben HJ. Handbook for rhizobia: methods in legume rhizobium technology. Springer, Berlin Heidelberg New York, 1994. 8. Yates RJ, Howieson, JG, Reeve WG, Brau L, Speijers J, Nandasena K. Host-strain mediated selection for an effective nitrogen-fixing symbiosis between Trifolium spp. and Rhizobium leguminosarum biovar trifolii. Soil Biol Biochem. 2008; 40: 822-833. 9. Abd-Alla MH, Voung TD, Harper JE. Genotypic differences in dinitrogen fixation response to NaCl stress in intact and grafted soybean. Crop Sci. 1998; 38: 72-77. 10. Serraj R, Adu-Gyamfi J, Rupela OP, Drevon JJ. Improvement of legume productivity and role of symbiotic nitrogen fixation in cropping systems: overcoming the physiological and agronomic limitations. In: Serraj R, ed. Symbiotic nitrogen fixation: prospects for enhanced application in tropical agriculture. 2004; 67-97. 11. Walsh KB. Physiology of the legume nodule and its response to stress. Soil Biol Biochem. 1995; 27: 637-655. 12. Zahran HH. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev. 1999; 63: 968-989. 13. Kumari BS, Ram MR, Mallaiah KV. Studies on nodulation, biochemical analysis and protein profiles of Rhizobium isolated from Indigofera species. Malay J Microbiol. 2010; 6: 133-139. 14. Rejili M, Mahdhi M, Ferchichi A, Mars M.. Natural nodulation of five wild legumes in the south of Tunisia. Plant Biosystems. 2009; 143: 34-39. 15. Bécquer CJ, Prévost D. Nodule formation potential in forage and grain legumes from rhizobia indigenous to Sancti Spíritus, Cuba. Cuban J Agricult Sci. 2014; 48: 301-307. 16. Kucuk C, Cevheri C. Nodulation study of natural forage legume in semiarid region, Turkey. Pak J Biol Sci. 2014; 17: 535-539. 17. Lalani WTI, Van Holm LHJ, Kulasooriya SA. Rhizobiology and nitrogen fixation of some tree legumes native to Sri Lanka. Biol Fertil Soils. 2000; 30: 535-543. 18. Yates RJ, Howeieson JG, Nandasena KG, O’Hara GW. Root-nodule bacteria from indigenous legumes in the north Western Australia and their interaction with exotic legumes. Soil Biol Biochem. 2004; 36: 1319-1329. 19. Zahran HH. Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology. J Biotechnol. 2001; 91: 143-153. 20. Abd-Alla MH, Nafady NA, Khalaf DM. Assessment of silver nanoparticles contamination on faba bean- Rhizobium leguminosarum bv. viciae-Glomus aggregatum symbiosis: implications for induction of 20 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 autophagy process in root nodule agriculture. Ecosyst Environ. 2016; 218: 163-177. 21. Montesinos A, Tonsor SJ, Alonso-Blanco C, Xavier Pico F. Demographic and genetic patterns of variation among populations of Arabidopsis thaliana from contrasting native environments. PLoS ONE. 2009; 4: e7213. 22. Lawton-Rauh A. Demographic processes shaping genetic variation. Curr Opin Plant Biol. 2008; 11: 103-109. 23. Andrew RL, Wallis IR, Harwood CE, Foley WJ. Genetic and environmental contributions to variation and population divergence in a broadspectrum foliar defence of Eucalyptus tricarpa. Ann Bot. 2010; 105: 707-717. 24. Loveless MD, Hamrick JL. Ecological determinants of genetic structure in plant populations. Annu Rev Ecol Syst. 1984; 15: 65- 95. 25. Hamrick JL, Godt MJW. Allozyme diversity in plant species. In: Brown AHD, Clegg MT, Kahler AL, Weir BS, eds. Plant population genetics, breeding and genetic resources, Sinauer, Sunderland, MA, USA, 1989; 43-63. 26. Nevo E. Evolution of genome-phenome diversity under environmental stress. Proc Nat Acad Sci USA. 2001; 98: 6233-6240. 27. Kis-Papo T, Kirzhner V, Wasser SP, Nevo E. Evolution of genomic diversity and sex at extreme environments: fungal life under hypersaline Dead Sea stress. Proc Nat Acad Sci USA. 2003; 100: 14970-14975. 28. Zhao N-X, Gao, Y-B, Wang J-L, Ren A-Z, Xu H. RAPD diversity of Stipa grandis populations and its association with some ecological factors. Acta Ecol Sinica. 2006; 26: 1312-1319. 29. Liu Q, Yao X, Pi L, Wang H, Cui X, Huang H. The ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis. Plant J. 2009; 58: 27-40. 30. Helmuth B, Kingsolver JG, Carrington E. Biophysics, physiological ecology, and climate change: does mechanism matter? Annu Rev Physiol. 2005; 67: 177-201. 31. Harley CDG, Hughes AR, Hultgren KM, Miner BG, Sorte CJB, Thornber CS, et al. The impacts of climate change in coastal marine systems. Ecol Lett. 2006; 9: 228-241. 32. El Hadidi MN. A historical flora of Egypt: a preliminary survey In: Davis WV, Walker R, eds. Biological anthropology and the study of Ancient Egypt. London: British Museum Press. 1993; 144- 155. 33. Allen SE, Grimshaw HM, Parkinson, JA, Quarmby C. 2372020 Chemical analysis of ecological materials. Oxford: Blackwell Scientific Publication, 1974. 34. Walkley A, Black, IA. An examination of the Degtjareff method for determining organic carbon in soils: effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci. 1934; 63: 251-263. 35. Williams CH, Twine ME. Flame photometric method for sodium, potassium and calcium. In: Paech K, Tracey MV, eds. Modern methods of plants analysis. Berlin: Springer-Verlag, 1960. 36. Johnson CM, Ulrich A. Analytical methods for use in plant analysis. US Dept Agric Calif Univ Agric Inform Bull, 1959. 37. Hazen A. On the determination of chloride in water. Am J Chem. 1989; 2: 409-425. 38. Black CA, Evans DD, White JL, Ensminger LE, Clark FE. Methods of soil analysis. Part 2. Agronomy. Series No. 9. Madison, Wisconsin, USA: Am Soc Agron, Inc. 1965: 1102-1116. 39. Woods JT, Mellon MG. Chlorostannous-reduced molybdophosphoric blue colour method, in sulfuric acid system. In: Jackson M L. Soil chemical analysis. Prentice-Hall International Inc. London. 1941: 141-144. 40. Piper CS. Soil and plant analysis. New York, NY, USA: Interscience, 1947. 41. Markus DK, McKinnon JP, Buccafuri AF. Automated analysis of nitrite, nitrate and ammonium nitrogen soils. New Jersey Agr Exp Stanford Publ. N.P. 1982: 15117-15184. 42. Jackson ML. Soil chemical analysis. Prentice hall of India Private Ltd, New Delhi, 1967. 43. LaRue TA, Child JJ. Sensitive fluorometric assay for leghaemoglobin. Anal Biochem. 1979; 92: 11- 15. 44. Abd-Alla MH. Nodulation and nitrogen fixation in interspecies grafts of soybean and common bean is controlled by isoflavonoid signal molecules translocated from shoot. Plant Soil Environ. 2011; 57: 453-458. 45. Bates L, Waldren RP, Teare, ID. Rapid determination of free proline for water-stress studies. Plant Soil. 1973; 39: 205-207. 21 | Abd-Alla et al. Nodulation and nitrogen fixation of some wild legumes from differing habitats in Egypt European Journal of Biological Research 2017; 7 (1): 9-21 46. Vincent JM. A manual for the practical study of root-nodule bacteria. IBP Handbook No. 15, Blackwell Scientific Publications, Oxford, 1970. 47. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JD, Struhl K. Current protocols in molecular biology. New York: John Wiley and Sons, 1987. 48. Rao AV, Venkateswarlu B. Microbial ecology of the soils of Indian desert. Agric Ecosyst Environ. 1983; 10: 361-369. 49. Boulos L. Flora of Egypt. Vol. 1 (Azollaceae – Oxalidaceae). Al Hadara Publishing, Cairo, Egypt, 1999. 50. Abdel Gadir AH, Alexander M. Procedures to enhance heat resistance of Rhizobium. Plant Soil. 1997; 188: 93-100. 51. Zhang F, Smith DL. Application of genistein to inocula and soil to overcome low spring soil temperature inhibition of soybean nodulation and nitrogen fixation. Plant Soil. 1997; 192: 141-151. 52. Potts M. Desiccation tolerance of prokaryotes. Microbiol Rev. 1994; 58: 755-805. 53. Ralston EJ, Imsande J. Entry of oxygen and nitrogen into intact soybean nodules. J Exp Bot. 1982; 33: 208-214. 54. Abd-Alla MH, Abdel Wahab AM. Response of nitrogen fixation, nodule activities, and growth to potassium supply in water-stressed broad bean. J Plant Nutr. 1995; 18: 1391-1402. 55. Hsiao CT. Plant responses to water stress. Ann Rev Plant Physiol. 1973; 24: 519-570. 56. Phillips DA, Torrey JG. Research in grain legume improvement. Span. 1973; 16: 9-11. 57. Weisz PR, Denison RF, Sinclair TR. Response to drought stress of nitrogen fixation (acetylene reduction) rates by field-grown soybean. Plant Physiol. 1985; 78: 525-530. 58. Gerosa-Ramos ML, Parsons R, Sprent JI, James EK. Effect of water stress on nitrogen fixation and nodule structure of common bean. Pesquisa Agropecuária Brasil. 2003; 38: 339-347. 59. Naya L, Ladrera R, Ramos J, Gonzalez E, Arrese- Igor C, Minchin FR, Becana M. The response of carbon metabolism and antioxidant defenses of alfalfa nodules to drought stress and to the subsequent recovery of plants. Plant Physiol. 2007; 144: 1104-1114. 60. Huang CY, Boyer JS, Vanderhoef LN. Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials. Plant Physiol. 1975; 56: 228-232. 61. Kuzma MM, Hunt S, Layzell DB. Role of oxygen in the limitation and inhibition of nitrogenase activity and respiration rate in individual soybean nodules. Plant Physiol. 1993; 101: 161-169. 62. Denison RF, Harter BL. Nitrate effects on nodule oxygen permeability and leghemoglobin. Plant Physiol. 1995; 107: 1355-1364. 63. Hsu SY, Hsu YT, Kao CH, 2003. The effect of polyethylene glycol on proline accumulation in rice leaves. Biol Plant. 1995; 46: 73-78. 64. Kavi Kishore PB, Sangam S, Amrutha RN, Laxmi PS, Naidu KR, Rao KRSS, Rao S, Reddy KJ, Theriappan P, Sreenivasulu N. Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance. Curr Sci. 2005; 88: 424-438. 65. Bhargava Y, Murthy JSR, Rajesh Kumar TV, Narayana Rao M. Phenotypic, stress tolerance and plant growth promoting characteristics of rhizobial isolates from selected wild legumes of semiarid region, Tirupati, India. Adv Microbiol. 2016; 6: 1- 12.