The field site was in the Rothrock State Forest (PA, USA), in the Valley and Ridge physiographic province on a north to north-east facing lower slope with an elevation of approximately 420 m (77°52′ W Long, 40°42′ N Lat). Annual precipitation in 1998 and 1999 was 97.7 cm and 98.5 cm, respectively. There was a moderate to severe drought during most of the growing season in 1999. The site had been dominated by a mixture of Quercus spp., including Q. montana Wildenow, Q. alba, Q. coccinea Münchhausen, and Q. rubra (in decreasing order of basal area, Rothrock State Forest timber sale records, sale #5–74 BC3). This stand was partially shelterwood harvested in 1974. In 1990, the site burned in a fire that killed most vegetation but did not burn the lower layers of duff and roots. All remaining trees greater than 5 cm in diameter were removed in a salvage logging operation in 1990. Vegetation on the site is dominated by scattered A. rubrum and Quercus spp. stump sprouts emerging from a dense shrub layer consisting of (in decreasing order of percentage cover) Dennstaedtia punctilobula (Michx.) T. Moore, Vaccinium spp., Comptonia perigrina (L.) J. M. Coult., Kalmia latifolia L., Galussacia bacatta (Wang) K. Koch, grasses, forbs, sedges, Gaultheria procumbens L., Lycopodium sp., and other ferns.
In the yr 1 planting, two treatments were established: Near-Quercus and Near-Acer. For the Near-Quercus treatment, we selected stump sprouts of Q. montana and cleared a 2 × 2 m plot on the south side of the stump at canopy drip line. Stump sprouts were defined as a stump of at least 20 cm diameter with at least one living sprout of at least 2 m in height. Near-Quercus plots were an average of 5.4 m from the nearest A. rubrum stump sprout. Near-Acer plots were similarly located adjacent to sprouted stumps of A. rubrum, but had the additional criterion of being at least 9 m from the nearest ectomycorrhizal tree, stump sprout, or seedling. Plots were sprayed with glyphosate herbicide 2 wk before planting and hand-cleared approximately 2 d before planting. Eleven replicates of each treatment were established, of which 10 from each treatment had surviving seedlings by the end of the first growing season.
Acorns were collected from a single Q. rubra in State College, PA, USA in the autumn of 1997 and stored at −1°C over winter. Acorns were surface sterilized in 10% household bleach for 5 min, planted in Pro-Mix BX potting soil on 27 May 1998 (Premier Brands Inc., Riviere-du-Loup, Quebec, Canada), and kept continually moist until radicle emergence. On 2 and 3 June 1998, germinated acorns with < 2 cm of emerged radicle were planted, four acorns per plot, in a square arrangement at approximately 30 cm spacing and 2 cm depth. Immediately after planting, a 60 × 60 × 30 cm tall, 1.25 cm mesh hardware cloth cage was placed over seedlings to reduce herbivore damage. Following the full expansion of the first pair of leaves on each seedling, the cotyledons were removed to maximize seedling growth responsiveness to environmental conditions and to reduce the risk of seedling mortality during post-germination seed predation. Seedlings were watered on three occasions during early establishment with tap water.
One seedling from each plot was destructively harvested 3 October 1998 to measure mycorrhizal infection. Seedlings were removed from the soil with care to minimize damage to root systems; the extremely stony soil made extraction of intact soil cores impossible. Because of damage to root systems during extraction from soil, all data on mycorrhizal infection are on a percentage basis, rather than absolute values, and root mass is not presented. Seedlings were harvested on a cool, overcast day and placed in moist toweling. After returning to the lab, seedlings were soaked in water for 24 h to loosen remaining soil, then gently washed under running water. Intact root systems were stored under refrigeration for up to 5 d before measuring mycorrhizal infection. For a separate experiment (unpublished) an additional seedling was harvested from four plots from each treatment, but at least one seedling was left in every plot.
Seedlings remaining from the yr 1 plantings were left in place for a second year. Plots were weeded in the spring of 1999, and cages replaced with 30 cm diameter, 120 cm tall, open-topped tubes of 2.5 cm mesh chicken-wire to reduce deer browse on larger seedlings. Seedlings were harvested on 4 October 1999.
The yr 2 planting was similar to yr 1, with one added treatment. Acorns were also planted in proximity to Quercus spp. stumps that had not resprouted, hereafter referred to as the ‘Near-Dead-Quercus’treatment. Dead Quercus stumps were selected with similar criteria used for the Near-Acer treatment: a minimum of 20 cm diameter and at least 9 m from the nearest ectomycorrhizal tree, stump sprout or seedling. All stumps were either Q. montana or Q. alba. Both Near-Quercus and Near-Dead-Quercus seedlings in the yr 2 planting averaged 4.7 m from the nearest A. rubrum stump sprout. Acorns were collected from a single tree near the study site in 1998, stored as in yr 1, and placed in peat moss to germinate on 21 May 1999; planting took place on 27 and 28 May 1999. Cages were somewhat smaller in footprint; but taller than in yr 1 (30 × 30 × 60 cm tall) with three acorns planted in an equilateral triangle at about 15 cm distance. Seedlings were watered with tap water on two occasions during early establishment. All surviving seedlings were harvested 3–4 October 1999. Harvesting procedures were as in yr 1.
A subsample of fine roots from each seedling was examined for vesicular–arbuscular mycorrhizal (VAM) infection. Roots were cleared for 30 min in 10% KOH at 121°C, rinsed in water, acidified for 5 min in 5% HCl, and stained for at least 24 h in 45% H2O, 50% glycerol, 5% acetic acid, 0.01% trypan blue. Stained roots were examined as in Koide & Mooney (1987), with a root recorded as VAM if both vesicles and hyphae were present within a section of root. Ectomycorrhizal infection was also quantified in stained samples, with all samples where a mantle was present recorded as ectomycorrhizal.
Stem and leaf tissues were digested and analysed for N and P. Dried (70°C) and ground tissue (c. 0.1 g samples) was digested in 5.0 ml H2SO4 and 5.0 ml 30% H2O2 for 60 min at 400°C. Samples that retained colour after 30 min of digestion received an additional 3.0 ml H2O2 and a total of 90 min at 400°C. After digestion, N was quantified with the Nessler method (Jensen, 1962), and P was quantified with the molybdo-phosphate method (Wantanabe & Olsen, 1965).
The glasshouse experiment was a 2 × 2 factorial design, with VAM inoculum (±V) and sorghum (Sorghum bicolor, ±S). Each treatment combination was replicated in 10 pots, each containing one tree seedling. Pots (3.8 l ‘Tall Ones’, Steuwe and Sons, Corvallis, OR, USA) were filled with a 1 : 3 mix of soil and sand. Soil was collected from an agricultural field, dried, autoclaved for 60 min at 121°C, and permitted to rest for several weeks before planting to reduce possible phytotoxic effects of soil heating (Rovira & Bowen, 1966). The VAM pots (+V) received 200 ml of Glomus intraradices pot culture mixed into soil in the upper 1/2 of each pot. Pot culture contained soil, sand, root fragments, and VAM spores (c. 225 spores ml−1) in which mycorrhizal sorghum had been previously grown. NonVAM pots (−V) received 100 ml of water that had been mixed with pot culture soil and passed through a 63-µm soil sieve (Koide & Li, 1989). All pots were established on 8 January 2000.
Nine sorghum seeds were planted in each +S pot on 8 January 2000. Pots in which fewer than four sorghum germinated 14 d after initial planting were planted with an additional 4–6 sorghum seeds. Pots with more than four sorghum were thinned to four plants per pot on 3 February 2000, so that all pots had four sorghum plants. Nonsorghum treatment pots (−S) were established simultaneously and treated identically to +S pots, except for the planting of sorghum. Pots were watered once per week with 200 ml of 300 p.p.m. N as NH4NO3 and twice per week with water.
Acorns were obtained from the same lot as those used in yr 2 of the field experiment. Acorns were surface sterilized as above and planted into a coarse vermiculite seedbed on 3 February 2000. Germinated acorns with radicles of 1–15 mm in length were transplanted into pots on 11 February 2000.
No Q. rubra mortality occurred during the study. Seedlings were harvested on 14 April 2000. All seedlings had completed two flushes of growth and had set bud at time of harvest. Roots and soil were removed from pots, soil was gently shaken free from roots, and roots were washed by immersion in water. Post-harvest measurements of growth and nutrient status were made as in the field experiment. VAM infection was quantified as in the field experiments but with an additional 30 min clearing period in hot 10% KOH following autoclaving to improve clearing.
For a bioassay of mycorrhizal inoculation potential, smaller pots (600 mL) of soil from the +V−S treatment were established concurrent with the main experiment. These pots were treated exactly as the main experiment, except that only 100 mlof nutrient solution was given weekly in order to compensate for smaller pot size. Four maize seeds (Zea mays cv. Bodacious) were planted in each pot on 11 February 2000. Maize emerged by 16 February 2000 and was harvested on 7 March 2000 for quantification of VAM infection. Maize roots were stained as above, except with 15 min clearing in 10% KOH. Sorghum roots removed from pots during thinning were also stained to detect VAM infection.
Sorghum shoots were clipped just above soil in order to reduce competitive effects of sorghum on seedling growth on 25 February 2000. Shoots were then re-clipped every 7–14 days. After 13 March 2000 sorghum failed to regrow after clipping in some pots.
The field experiment was analysed using either t-tests (yr 1 planting) or analyses of variance (ANOVAs) with protected least significant differences (PLSD) for means separation (yr 2 planting), provided that the assumptions of the tests were met. Data were checked for normality and homoscedasticity using Shapiro-Wilks and Levenne’s tests, respectively. Where needed, Box-Cox transformations were used. Data that could not be successfully transformed were analysed using nonparametric statistics (Mann–Whitney U for two sample tests and Kruskal–Wallis with Nemenyi’s test for means separation of three sample tests; Zar, 1999). Data and standard errors are presented untransformed in graphs and tables for ease of interpretation.
Results from the glasshouse study were analysed as a 2 × 2 factorial fixed model analysis of variance (ANOVA). All data were examined for normality and homogeneity of variances and transformed with Box-Cox transformations as needed. In the glasshouse study, the proportion of root length infected had highly heterogeneous variances, as all −V+S seedlings and nine of 10 −V−S seedlings had 0% infection. The frequency of infection (number of seedlings with infection) was therefore analysed with a χ2 statistic. Proportion of root length infected was compared between infected seedlings in the +V−S and +V+S treatments using a t-test for unequal variances.
Statistics were performed in SAS version 7, except for Box-Cox transformations which were determined with Minitab version 12.