25, -0.5, -1 and -1.5 MPa; pH tolerance [47] at pH 3.0, 3.5, 4.5, 5.5, 7.0, 9.0 and 9.5 (Homopipes buffer 25 mM used for pH range of 3-5, and for pH range 9-9.5 [pKa 7.5 at 25°C] and the MES buffer used for pH range 5-7 [pKa 6.1 at 25°C]); and PD0332991 chemical structure intrinsic antibiotic [47] and heavy metal tolerance [47] were determined on solid YEM medium containing the following filter-sterilized antibiotics or heavy metals (all μg/ml): chloramphenicol (25 and 100), spectinomycin (15 and 50), streptomycin (10 and 25) and tetracycline (10 and 25); CdCl2.2H2O (5 and 20), MnCl2 (300), HgCl2 (20) and ZnCl2 (200). After 7 days of incubation at 28°C, the bacterial growth

was compared to controls. Isolate genotyping Bacterial DNA was extracted by a simple boiling method. Bacteria were grown in TY agar [48] petri dishes at 28°C for 2 days. Cells were suspended in 25 μl of sterile distilled water and followed by 25 μl of freshly prepared lysis-buffer containing 0.1 N NaOH and 0.5% SDS. The mixture was boiled in a water bath for 15 min. Then, 200 μl of TE (10 mM Tris-HCl

and 0.1 mM EDTA) was added to the mixture, which was then centrifuged for 15 min at 12,000 g. The supernatant formed by the aqueous phase that contained clear and suspended DNA was transferred to new sterile tubes. For the rhizobia species assignment, the 16S rDNA gene of the isolates was amplified using primers fD1 and rD1 with an annealing temperature of 58°C and restricted with RsaI. Based on RsaI restriction

LY2109761 cost pattern, the isolates were assigned to either S. meliloti or S. medicate [2, 49, 50], by comparing their pattern with the restriction pattern of the reference strains S. meliloti (USDA, learn more NRRL-45) and S. medicae (ABT5). PCR targeting repetitive DNA sequences (rep-PCR) such as repetitive extragenic palindromic sequences (REP) [51] and enterobacterial repetitive intergenic consensus sequences (ERIC) [52] were performed according to de Bruijn [15] with minor modifications. Since BOX primer did not reveal any polymorphism in S. meliloti [53], it was not used in this study. The amplification was carried out in tubes containing 25 μl of final reaction volume. The reaction mixture contained Amoxicillin 2.5 μl of DMSO (100%), 14.65 μl of sterile distilled water, 2.5 μl of PCR buffer (10×), 1.25 μl of dNTPs (2 mM), 0.55 μl of REP primers [51] (Rep1 5′-IIIICGICGICATCIGGC-3′ and Rep2 5′-ICGICTTATCIGGCCTAC-3′; 0.3 μg each) or 0.44 μl of ERIC primers [51] (Eric1 5′ATGTAAGCTCCTGGGGATTCAC-3′ and Eric2 5′AAGTAAGTGACTGGGGTGAGCG-3′; 0.3 μg each) and 0.4 μl (2U) of Taq polymerase. After the addition of 2 μl (50 ng) of DNA, the reaction mix was placed on a thermocycler (Mastercycler, Eppendorf, Germany) and subjected to PCR cycles: 95°C for 7 min, followed by 35 cycles of 94°C for 1 min, 53°C for 1 min and 65°C for 8 min, and followed by final elongation at 65°C for 8 min.