Growth and Iron Sequestering of Pin Oak (Quercus palustris) Seedlings Inoculated with Soil Containing Ectomycorrhizal Fungi

Methods and Materials

Stratified pin oak seeds were obtained in bulk from a commercial seed company (F.W. Schumacher Co., Inc., Sandwich, Massachusetts). Only undamaged seeds that sunk during a float test in water and that were within 20% of the mean weight of the bulk acorns were used. Seeds were surface sterilized on May 31,1993, in a 30% (v/v) H₂O₂ solution for 15 minutes, washed afterwards 3 times in deionized H₂O (dH₂O), planted singly in each of 280 10-cm³ pots containing pasteurized coarse vermiculite, and placed in a greenhouse with iron-free glass at the Plant Sciences Laboratory at the University of Illinois at Urbana/ Champaign. Seedlings were grown for 2 months at 25 ± 4°C daytime and 18 + 2°C nighttime temperatures, under ambient light conditions, watered daily with reverse osmosis-treated H₂O, and fertilized bimonthly with a 20:20:20 Peters brand fertilizer (nitrogen at 473 ppm) containing a full-strength, modified Hoaglands solution without iron (16). After 2 months, 120 seedlings of uniform size were selected for the study.

The experimental design was a 2:3:2 factorial: 1) acidic and alkaline media, 2) soil inoculum from 3 sites, and 3) inoculation with either natural or autoclaved soil inoculum. Seedlings were randomly distributed among the 12 treatments (10 seedlings per treatment combination) and transplanted into 1-gal plastic containers filled with a pasteurized 1:1:1 (v/v/v) peat moss:coarse vermiculite:calcined clay soil medium maintained under either acidic (pH = 5.5) or alkaline (pH = 7.5) regimes. The soil medium initially had a pH of approximately 5.5; thus, no adjustment was required for the acidic treatment. The alkaline treatment soil was adjusted with 3 kg CaCO₃ per m³ to produce a pH of 7.5. The soil medium was measured every 2 to 3 weeks throughout the study for pH changes with a Fisher Accumet 610 pH meter (Fisher Scientific, Pittsburgh, Pennsylvania) using a 1:1 soil:dH₂O slurry. The experimental soil media were adjusted to initial pH treatment levels every 3 to 5 weeks. No soil medium ever deviated more than 0.7 pH units from initial treatment pH levels. The pH of the acidic treatment increased with time and the soil medium pH was lowered in each container through the application of 250 mL of a 0.0014 MH₂SO₄ solution. The soil medium in the alkaline treatment decreased in pH with time, and the pH was increased by applying 300 mL of lime water that contained 1.18 g of CaCO₃ per L of reverse-osmosis deionized H₂O to each container.

The soil used for inoculation was collected from 3 different locations: 1) a pin oak stand at the Iroquois County (Illinois) Wildlife Area; 2) under 4 chlorotic pin oak trees on the campus of the University of Illinois at Urbana/Champaign; and 3) under 4 non-chlorotic pin oak trees on the campus of the University of Illinois at Urbana/Champaign. Soil at each site was collected by taking approximately 150 soil cores, 2 cm in diameter, to a 15 cm depth; the cores were subsequently bulked by site. Natural soil was collected 1 to 2 days prior to inoculating the artificial soil media. The soil pH values were 4.9 at the native pin oak stand, 7.0 at the chlorotic urban site, and 5.8 at the non-chlorotic urban site. Soil inoculum was incorporated at a 1.13 (v/v) inoculum to medium ratio. Soil that was autoclaved at 137°C for 2 hours was incorporated into the control containers at the same 1:13 (v/v) inoculum to medium ratio. Experimental seedlings had no sign of ectomycorrhizae prior to inoculation.

On August 3,1993, inoculated seedlings were placed in a greenhouse room designed for plant and symbiotic microbe studies. The air supply was filtered through Air Guard type dp 2-40 filters (Air Guard Industries Inc., Louisville, Kentucky), ambient light was supplemented with Sylvania L41000 Metal Halide lamps (GTE Sylvania Inc., Manchester, New Hampshire) to maintain a 16-hour photoperiod with a minimum of 400 μmol/m²/sec of photosynthetic photon flux, and seedlings were watered daily or as needed with water deionized by reverse osmosis. The greenhouse floor was washed with a commercial bleach solution containing sodium hypochlorite weekly to reduce the risk of contamination of the experimental seedlings by mycorrhizal fungi from soil on the floor. Temperatures were maintained at 25 ± 4°C daytime and 18 ± 2°C nighttime. Nutrients were supplied weekly with a Hoaglands solution (16) modified so that iron was applied as FeCI₃ at 100 ppm.

After the seedlings were 7 months old (5 months after inoculation), half of the seedlings from each treatment combination (n = 5) were randomly selected for harvesting to estimate biomass and ectomycorrhizal abundance, and half were overwintered for 3 months in a dormant state in a cold room set at 3-4°C. The soil moisture content of the overwintered seedlings was monitored and the seedlings were watered as needed to maintain a moist substrate. Ectomycorrhizal infection was assayed on 3 seedlings per treatment using the methods of Daughtridge et al. (9). Lateral roots were evenly divided into 4 groups, 1 of which was randomly selected for examination under a dissecting scope. Each lateral root was examined and considered infected if at least 1 ectomycorrhizal root tip was observed. The percentage of infection was determined by dividing the number of infected laterals by the total number of laterals and multiplying by 100. Biomass was determined from shoot and root tissue of 3 seedlings per treatment. Samples were dried for 48 to 72 hours at 75°C until a consistent dry mass was observed for each sample.

Winter-dormant seedlings were placed in a greenhouse on March 3, 1994, and grown for 2 additional months. Afterwards, 4 seedlings per treatment were harvested for biomass determinations and 5 seedlings per treatment for estimation of the concentration of iron in leaf tissue. Biomass was estimated as for previously harvested seedlings. Approximately 0.45 g (± 0.05 g) of leaf tissue was used to estimate plant iron concentrations. Tissue analyzed for nutrient concentration was ground in a Wiley mill to pass through a 0.5 mm mesh screen and placed in 1.5 cc polyethylene vials that were rinsed 3 times in dH₂O, acid washed, and rinsed again 3 times in dH₂O. Neutron activation analysis (NAA) was performed using the University of Illinois TRIGA reactor. Samples and standard reference materials, from the National Institute of Standards and Technology (NIST), were placed into clean 1.5 cc polyethylene vials. The vials were irradiated at a neutron flux of 1.1 x 10¹² n/cm²/s for a period of 3 hours. After a 1 -month decay, the samples were counted for 3 hours each using a hyperpure germanium detector. Elemental calibration was done using MST-certified solutions. The usual comparator method was used to calculate final concentrations. NAA values for Fe agreed very well with the NIST standard reference materials. Typical detection limits for Fe were 40 ppm.

Analysis of variance was used to test for significance of mean differences among the treatments at the 0.05 probability level. A Fisher’s protected least significant difference was used to separate mean differences among treatments significant at the 0.05 probability level. Means significantly different at the 0.10 probability level were also discussed.

Results

Discussion

Foliar iron concentrations were higher in seedlings grown in the alkaline substrate and infected with ectomycorrhizae by inoculation with soil than in the control seedlings grown without ectomycorrhizae in the alkaline treatment. The inoculated seedlings in both pH treatments had a similar tissue iron concentration. This suggests that ectomycorrhizal fungi may increase the iron concentration of pin oak trees under alkaline conditions. Ectomycorrhizal fungi probably increase iron concentration in other plants under iron-limiting conditions (15,18,19,21,22,34). For example, Clement et al. (1977, as cited in 13) and Le Tacon (20) discovered that Austrian pine seedlings grown in calcareous media were sometimes chlorotic when ectomycorrhizae were absent. In comparison, no ectomycorrhizal Austrian pine trees were chlorotic and a genetic tolerance was ruled out. Mycorrhizal root mats have also been correlated with higher soil iron concentrations in comparison with soil locations not containing these mats (11).

Inoculation of the media with soil containing ectomycorrhizae clearly enhanced growth of pin oak seedlings in comparison to control seedlings. This indicates that there are benefits to pin oak seedlings from ectomycorrhizal symbioses. Thus, ectomycorrhizae may have contributed to enhanced growth of the seedlings grown in alkaline substrate. However, seedlings in an alkaline medium and inoculated with soil from the urban chlorotic pin oak site and control seedlings inoculated with soil from this site had similar mean biomass values, suggesting that ectomycorrhizal populations at this site may have been less effective in influencing plant growth than ectomycorrhizal fungi from the other sites tested. The ectomycorrhizal symbiont Pisolithus tinctorius has been demonstrated to increase the growth of pin oak in controlled experiments (1,24). Pisolithus tinctorius has also been found associated with pin oak trees in urban areas (25). Vesicular arbuscular mycorrhizae (VAM) also form on pin oak (42), though we did not determine the VAM status of the experimental pin oak seedlings.

Lapeyrie (18) recently reviewed the tolerance of woody plants growing in calcareous soil and cited 4 studies in which seedlings grown in sterile, calcareous media were chlorotic. A reduction in chlorosis and mortality occurred in some cases after inoculation with ectomycorrhizal fungi. The health of inoculated seedlings on calcareous media was similar to seedlings on sterile acidic media. It has been demonstrated that ectomycorrhizal fungi produce hydroxamate siderophores (39). Siderophores have been associated with an increased availability of iron for plants (2,5,12,33).

Acidic soil medium favored a higher iron concentration in pin oak leaf tissue than did the alkaline treatment. Iron is more mobile in acidic soil systems than in alkaline systems (32). Ferric iron is more commonly associated with alkaline soil systems and readily precipitates into unavailable forms with low solubility (38). Biomass was also consistently greater in pin oak seedlings grown under acidic conditions during the first and second years of growth. These findings are consistent with observations that the native soil habitat of pin oak is acidic (10).

We found no differences among soils from the 3 sites in their capacity to form ectomycorrhizal roots on pin oak seedlings. The taxa of ectomycorrhizae were not determined so we do not know which specific fungi colonized the root systems in the various treatments. Mycorrhizal fungi are known to have wide to narrow ranges of pH tolerance (13). The observations in this study that seedlings inoculated with urban soils had both light and dark colored ectomycorrhizae, while the pin oak forest soil inoculum produced dark ectomycorrhizae, and that alkaline soil from the chlorotic urban pin oak site did not increase biomass of inoculated seedlings compared with autoclaved soil inoculum from the same site suggests that ectomycorrhizal taxa differ in occurrence among the sites.

Conclusions

Results of this study clearly demonstrate that pin oak-infective ectomycorrhizal propagules occurred in both an acidic and an alkaline urban soil from under pin oaks. Ectomycorrhizae were associated with increased iron uptake and growth of seedlings after 2 growing seasons in an alkaline soil, but not an acidic soil, suggesting the importance of ectomycorrhizae in improving the iron nutritional status of pin oak trees in alkaline soils. It is possible that pin oak chlorosis is mitigated in many alkaline urban soils by ectomycorrhizae. Alternatively, one could speculate that chlorosis of pin oak trees is more pronounced on alkaline soils lacking ectomycorrhizal fungi. The determination of such a pattern is possible with sampling of pin oak roots for ectomycorrhizal assessment and analysis of pH and other properties of associated soils across a wide range of urban sites.