Bacterial Diversity in Soils under Artemisia sieberi and Noaea mucronata plants in the Negev Desert
Water and nutrient availability are the major limiting factors of biological activity in arid and semi-arid ecosystems, therefore, perennial plants have developed different ecophysiological adaptations to cope with the severe harsh conditions. Artemisia sieberi has a special mechanism of chemical-compound excretion beneath the plant; Noaea mucronata has no allelopathic effect on its surroundings. We examined the influence of these two different shrubs on soil microbial diversity. Soil samples were collected monthly, from December 2006 to November 2007, near canopies of both shrubs (0-10?cm depth). Samples were grouped by seasons, winter (December 2006, and January and February 2007), spring (March, April, and May 2007), summer (June, July, and August 2007), and autumn (September, October, and November 2007). DNA extraction: DNA was extracted from the soil samples using a BioLAB kit according to the manufacturer's purification protocol. DNA was then purified by electrophoresis on 1% agarose gel and isolated with a NucloTrap gel extraction kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol. PCR: 16S rRNA genes were amplified from extracted DNA samples. The components for each 50 ?l total reaction were: 0.6-1 DNA ?l template, 1.5 U Taq DNA polymerase (Red Taq; Sigma, St. Louis, MO), 5 ?l Taq buffer )Sigma), 6.25 ?g bovine serum albumin (BSA) (Roche Diagnostics, Mannheim, Germany), 2 ?M PCR nucleotide mixture (dNTP) (Larova, Telpow, Germany), 2 mM MgCl2 (Amresco, USA), and 50 ?M bacterial primer pair 341F (GCC TAC GGG AGG CAG CAG) and 907R (GTT TGA TCM TGG CTC AG). The program included initial denaturation at 95?C for 5 min, followed by 33 cycles of denaturation at 94?C for 30 s, annealing at 60?C for 30 s, and elongation at 72?C for 30 s. Cycling was completed with a final elongation step at 72?C for 5 min. The PCR reaction was performed in the T-gradient Thermocycler (Whatman Biometra, Gottingen, Germany). The PCR products were visualized on 1.5% agarose gel and purified with a Nucleospin plasmid purification kit (Macherey-Nagel) according to the manufacturer's purification protocol. Cloning: Purified DNA products were then ligated into plasmids using the Topo PCR cloning kit, which includes the pCRII-TOPO TA vector (Invitrogen Life Technologies, Carlsbad, CA). Sequencing: Sequencing was carried aout on an ABI 3730XL BigDye terminator v3.1 cycle sequencer at the Genome Center at Washington University. |  |  | |