Root processing and assessment of root colonization

Root processing involved removing 40 to 50 fine (?2mm width), fibrous, healthy appearing roots from each soil sample. Roots were washed with water and a commercial softener (Calgon�, Benckiser, CT) to remove soil particles, wrapped in mesh paper and placed into plastic tissue capsules. Roots were then fixed using a solution of 50% ethanol prior to staining procedures.

AM fungal colonization was assessed by staining roots in trypan blue using a modification of the method of Koske and Gemma (1989) with the potassium hydroxide (KOH) concentration adjusted for woody and succulent plant roots. Because succulent plant roots were thinner than woody plant roots, the concentration of KOH used was 1.5 % for the succulent plant roots and 4% for the woody plant roots. To assess the possible presence of DSE, double staining was performed with Sudan IV staining (Barrow and Aaltonen 2001) after staining with trypan blue; then roots were stored in acidic glycerol until preparation of slides. Approximately 30 root segments of 1cm were mounted on slides by placing them horizontally across the slide with several drops of acidic glycerol solution, and covered with a cover slip. Slides were examined using a light microscope at 40X magnification. Percent root colonization was quantified following the magnified intersections method of McGonigle et al. (1990) by using a compound microscope fitted with an ocular crosshair. At each intersection of a root and the vertical axis of the crosshair, the root was analyzed for the process of AM fungal structures such as hyphae, vesicles, arbuscules, coils, colonization by DSE, colonization by other fungi or no colonization and the result recorded until at least 100 intersections were assessed.

Assessment of AM fungal diversity

A subsample of 250 cm 3 of each soil sample was mixed with 250 cm 3 of #12 and #20 silica sand (1:1) to set up trap cultures that were grown in the greenhouse. This technique has shown to promote AM fungal spore production in desert soils (Stutz and Morton 1996). Trap cultures were established following the protocol reported by Stutz and Morton (1996). Surface-sterilized 656 ml Deepots� (Stuewe and Sons, OR) were placed in benches with a mixture of planting media 1:1:2 of sterilized silica sand #20 and #12 and sample soil per each Deepot. Around 40 to 50 surface-sterilized Sudan grass (Sorghum sudanese) seeds were added into the Deepots and covered by wet silica sand #12. Trap cultures were grown in the greenhouse at ASU Polytechnic campus and watered as needed over the course of 3 months until flowering and subsequent dry down. After drying down, length of stems was measured and stems were harvested. New surface-sterilized Sudan grass seeds were placed into the depots and covered by wet silica sand #12. The second generation of trap cultures was grown and watered as needed during 3 months until flowering. ParEX� (Vigoro Industries, Il) slow release Nitrogen fertilizer (20-0-20 in IBDU) was top dressed once and biological control (Gnatrol�, Valent Products, CA) applications for fungus gnats were applied as a drench as needed.

After 2 generations of trap cultures, watering was stopped and cultures dried down. Plant tops were clipped and cultures removed and placed in self-closing plastic bags and stored at 4� C until analysis. Spore analysis was done with a 100 cm3 subsample of the trap cultures soil by using wet sieving and a sucrose density gradient centrifugation (Daniels and Skipper 1982). Spores were washed into a petri dish for examination with the stereomicroscope. Healthy spores were separated into different morphotypes based on shape, color, size, surface conditions, spore contents and no evidence of parasitism. Spores were mounted on slides in polyvinyl alcohol lactic acid-glycerol (PLVG) (Koske and Tessier 1983) and PVLG mixed 1:1 (v/v) with Melzer�s reagent to be compared with voucher specimens and descriptions from the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM) (Morton et al. 1993). Species richness was measured as the numbers of AM fungal species present at each site.