ASSESSING LOCAL DIVERSITY OF ARBUSCULAR MYCORRHIZAL FUNGAL SPECIES IN AN EXPERIMENTAL URBAN LANDSCAPED SITE

1. Seven 9.2 × 9.2 m experimental plots were established in April 1999 at the site after vegetation was removed by discing.

2. The plants were propagated by stem cuttings and grown in containers in a mixture of native soil, bark, sand, TurfliteTM (Sierra Cascade, Chemult, OR) and slow release fertilizer.

3. Water is applied by drip irrigation at the base of each plant at an average rate of 2401 L per plant per year.

4. All woody plants were fertilized during April of each year with 200 g of controlled release 20N-4.4P-4K plus micronutrients fertilizer .

5. Winter and summer broadcast applications of pre-emergent herbicide (XL 2G, Dow Elanco Specialty Products, Indianapolis, IN) were made to each plot to inhibit weed seed germination. Plots were regularly weeded by hand thereafter.

6. Two plots at the site were sampled,the soil was removed with a sanitized hand shovel to a depth of 10 cm and stored in sealed plastic bags at 4ºC.

7. Trap cultures were established by mixing a 250-cm3 portion of each soil sample with 250 cm3 of a 1:1 mixture of autoclaved #12 and #20 silica sand and placing the mixture in a D40 Deepot™ that had been pre-treated with a 10% household bleach solution .

8. The pots were planted with 50-60 seeds of Sorghum bicolor (L.) Moench, and the plants were grown in a greenhouse for four months, then the grass was clipped and the pots were re-seeded

9. After another four months of growth, the soil was air dried, removed from the pots and stored in sealed plastic bags at 4ºC until further processing. .

10. AMF spores were isolated from each trap culture using the sucrose density gradient centrifugation method .

11. A thoroughly mixed 100-cm3 soil subsample was removed from each trap culture. The soil was homogenized in a blender with water for 1 min, and the liquid was decanted into a 500-µm sieve stacked on a 45-µm sieve.

12. The top sieve was rinsed thoroughly and the organic matter from the bottom sieve was decanted into a beaker.

13. This material was then placed in a centrifuge tube loaded with a 20%/60% sucrose gradient. The tubes were spun in a swinging arm centrifuge at 1500 g for 5 min.

14. The supernatant was poured off into a fine sieve, rinsed with water and washed into a petri dish.

15. The supernatant was poured off into a fine sieve, rinsed with water and washed into a petri dish. The washing was examined using a stereo microscope

EFFECTS OF PRUNING ON ROOTS, MYCORRHIZAE, AND SOIL RESPIRATION OF TWO LANDSCAPE SHRUBS

1. Seven experimental plots (9.2 m × 9.2 m) were established at the site in 1999 and each planted with 3.8 L container-grown clones of two common landscape shrubs

2. Water was applied at an average rate of 2401 L per shrub per year via drip emitters at the base of each shrub.

3. Three pruning treatments were performed in a completely randomized design; Shrubs in two of the plots were sheared; shrubs in two plots were headed back; shrubs in two plots were renewal pruned

4. Roots from the two shrub species were sampled in February and May to determine the effects of these treatments on root length density, root biomass and AMF colonization

5. The plots were sampled in February, just before the first renewal pruning and the second heading back treatment, and three weeks after a shearing treatment. They were sampled again in May, 15 wk after renewal pruning and heading back, and immediately after a shearing treatment.

6. The soil cores from each depth were stored separately in plastic bags on ice and transported to the laboratory, where they were kept at 4ºC until processing.

7. Wet weight of all live fine roots (less than 2 mm in diameter) was determined and a portion (ca. 0.1 g) of roots from each sample was removed and fixed in 70% ethanol for later quantification of root length and arbuscular mycorrhizal colonization.

8. The remaining roots were dried in an oven for at least 48 h and weighed. Total root biomass for the sample was determined by calculating the percentage of fresh roots removed for AMF colonization analysis and adding this percentage to the final dry weight of the remaining sample.

9. The AMF colonization subsample was cleared and stained according to a modification of the method of Koske and Gemma .

10. The fixed roots were rinsed in water and cleared in 5% potassium hydroxide at 90º C for 15 min. The roots were then rinsed thoroughly and bleached in alkaline hydrogen peroxide for 1 h to overnight, then acidified in 1% hydrochloric acid and stained in 0.05% trypan blue in acidic glycerol in an autoclave at 121º C for 3 min.

11. The stained roots were mounted on glass slides and scanned to determine percent colonization using the magnified intersections method of McGonigle et al. .

12. Each slide was scanned at 400 and a total of 100 root intersections were scored for percent colonization by AMF hyphae, arbuscules, vesicles, and hyphal coils. This percentage was multiplied by the RLD to determine the length of root colonized per cm3 of soil.

13. Soil CO2 efflux was measured at each sampling location to quantify the impact of the treatments on soil respiration rates. PVC collars (10 cm diameter × 10 cm length) were placed in the soil immediately adjacent to the root sampling locations at least one day prior to sampling.

14. CO2 efflux rates were measured with an LI-6200 portable photosynthesis system (LI-COR, Lincoln, NE) just prior to soil coring. Measurements were taken once every 30 s for 2 min and averaged for a 2 min mean.