Root Response of Mature Live Oaks in Coastal South Carolina to Root Zone Inoculations with Ectomycorrhizal Fungal Inoculants

Site Characterization

The studies were conducted on 7 native live oaks growing at the Penn Center, St. Helena Island, SC. The trees ranged in diameter from 2 to 5 ft. (0.6 to 1.5 m) and had canopy diameters ranging from 60 to 120 ft. (18 to 36 m), respectively. The larger trees were estimated to be between 150 and 250 years old. For presumably more than 100 years, the soil under these trees has been periodically cleared of leaf litter, exposing mineral soil. For the past decade or more, the soil has been compacted by numerous people nearly every weekend having picnics or other gatherings. During these times it is not unusual to find 8 to 12 trucks and cars parked in the shade of these trees. Other than sparse grass there is no other vegetation under these oaks. The trees had symptoms commonly associated with stress, i.e. many dead large branches, low leaf density, short twig growth, and many fruiting bodies of wooddecay fungi on the main trunks, large branches, and root flares. Soil texture is a sandy loam. Chemical analyses showed soil pH of 5.9 and 1.1 percent organic matter in the upper 8 in (20 cm) of soil. Total soil nitrogen was approximately 2000 mg/kg^-1 and phosphorus, potassium, calcium and magnesium were 55, 26, 240, and 29 mg/kg^-1, respectively. Cation exchange capacity was 4.1 cmol/kg^-1 of soil.

Root Response Measurements

Fine roots less than 1/8 in (3 mm) diameter and ectomycorrhizal response to treatment were measured with root-ingrowth cores (RIC) in both studies (6). For these studies, RIC’s were 3-in diameter by 7-in (7.6 by 17.8 cm) long plastic screen-tubes which are open-ended and have 1/ 8 in (3 mm) diameter holes covering 90 percent of the wall surface. A 3-in by 8-in (7.6 by 20.3 cm) diameter soil core was removed from an injection or a vertical mulch location with a special soil extractor tool. The soil was so compacted that many times the soil core extractor had to be driven into the soil with a heavy sledge hammer. The soil from the extractor was screened through a ¼ in (6 mm) mesh screen to remove the roots which were discarded. A treatment-labeled (aluminum tag) RIC was inserted to the bottom of the hole and the root-free, treated soil was used to refill the RIC. Soil was pressed firmly by hand into the RIC. A steel nail was placed on the top of the RIC and then covered with a final inch of treated soil. This soil was then pressed firmly by foot. RICs were buried to minimize vandalism and damage by foot and vehicular traffic. The nail was used so that the RIC could be located later with a metal detector. Normal people activities in the area continued for the duration of the study. Cores were removed after 6 months for the injection study and after 2, 4 and 6 months for the vertical mulch study. A knife was used to cut roots on the outside of the RIC’s. The RIC’s were removed from the soil and their contents dry screened to collect roots which had grown into them. Roots were washed, cleaned of debris, and the few grass roots discarded. Ectomycorrhizal development was visually assessed on the oak roots at 4X magnification then dried at 70°C for 48 hours and weighed. Pt ectomycorrhizae were easily distinguished from the naturally-occurring ectomycorrhizae by their distinctive mustardyellow color, multi-branching habit, and yellow hyphal strands. Visual estimates of ectomycorrhizal development have limitations in that they do not consider the total number of lateral roots supporting ectomycorrhizae, and they only estimate the relative proportion of ectomycorrhizal to nonmycorrhizal roots on the lateral roots. The estimates also do not relate to the size or potential absorbing area of the ectomycorrhizae. This limitation can be overcome by using a derived surrogate for surface-area. The relative root absorbing potential parameter for these studies is derived from the simple formula: fine root wt./ft³ X % ectomycorrhizal development ÷ 10. This derived parameter actually underestimates the physical surface area measurements that have been made on ectomycorrhizae (7, 8) but does serve as a surrogate for surface area or absorbing surface and can serve as an index for relative comparison between treatments (6). All data were processed by Analysis of Variance and significant means were identified with Duncans’ New Multiple Range Test at P=0.05.

Soil Injection Study

Live oaks with 3 to 5 ft. (1.2 to 1.5 m) diameters, each with canopy diameters over 100 ft. (30 m), were used in the injection study. The area under the drip line of each tree was divided into quadrants following the test design used by Smiley et. al. (9). Each quadrant received one of the following four treatments chosen at random:

1. Injectable Pt spores with a yucca surfactant at ¼ lb. (114 gm) (1.1 x 10¹¹spores) per 100 gal. (379 I) of water.

2. Injectable Pt spores at ¼ lb. (114 gm) (1.1 x 10¹¹ spores) plus 5 lb. (2.27 kg) of (12-48-8) water soluble fertilizer with micronutrients per 100 gal. (379 I) of water.

3. Fertilizer alone at the same rate as #2 above.

4. Water only control.

On April 8,1996, injections of 0.5 gal. (1.9 I) of treatment solution to an 8-in (20 cm) soil depth were made on 3 ft. by 3 ft. (0.9 m by 0.9 m) centers in the designated treatment tree quadrant using a motor-driven sprayer with a soil injection nozzle. Injection pressure was 150 psi. Each tree quadrant received 24 injections. Eight injection locations in each quadrant of each tree were selected at random and marked with color-coded flags to designate treatment and to mark the injection spot for placement of RICs. The next day, the 8 RIC’s per treatment per tree were installed directly over the injection spot. A total of 96 RICs were installed in this study. All soil cores were removed in mid-September, 1996 and their contents assessed as described earlier. Root data from the 8 RIC’s from each quadrant treatment were averaged to represent that treatment replicate. Each of the three trees were statistically analyzed as a block. The study was a randomized complete block design.

Vertical Mulch Study

An area under the drip line of four - 2 ft. (0.6m) diameter live oaks, each with canopy diameters of about 60 ft. (18 m) and growing 20 to 25 ft. (6 to 7.6m) apart, was selected. Forty-eight flags spaced 3 to 5 ft. (0.9 to 1.5 m) apart were placed in the area. Twenty-four flag locations were chosen at random for the vertical mulch treatment and the remaining 24 locations were used as controls. The control cores were installed by removing a soil core and inserting a labeled RIC into the hole. The soil in the extractor was screened of roots and, without further treatment, was used to refill the RIC as described in the injection study. The vertical mulch treatment was installed in the remaining locations by removing a soil core, inserting a labeled RIC, and then hand-mixing 3 oz. (85 gm) of a vertical mulch inoculant with the root-free soil from the soil core. The inoculant contains spores of Pt, soil-enriching bacteria, organics, and soil water-managing gels. (It is a product of MycorTree TM Root Saver of Plant Health Care, Inc.) This 10% inoculant to soil mixture was used to refill the RIC as described earlier. A total of 48 RICs were installed in this study. Two, four, and six months after installation, eight cores of each of the two treatments were removed at random and their root contents processed as described. Root data from each RIC was treated as a replicate of that treatment/date. Data were statistically analyzed as a completely randomized design.

Soil Injection Study

Injected water (control) and the fertilizer alone induced similar amounts of fine roots and ectomycorrhizae (Table 1). Naturally-occurring ectomycorrhizae in these treatments was less than 20%. Injected Pt spores alone or combined with the fertilizer stimulated three to four times more fine roots and nearly three times more ectomycorrhizal development than observed in the water control treatment. Fertilizer and Pt spores applied separately induced similar amounts of fine roots but the Pt spore treatment had more than twice the ectomycorrhizal development. About half of the ectomycorrhizae formed in both of the injected spore treatments were formed by Pt. The relative absorbing potential of the fine roots and the ectomycorrhizae produced in response to the injected spore and fertilizer treatments was 7 to 14 times that of the fertilizer alone and the water control treatments, respectively. The combination of spores and fertilizer produced 75% more relative root absorbing potential than Pt spores alone.

Vertical Mulch Study

Probably as a result of the drought, there were few fine roots detected in any of the RIC’s removed two months after installation. Samples assessed after four and six months, however, showed the effects of the vertical mulch treatment on root growth (Table 2). There were no significant differences in either fine roots or ectomycorrhizal development between the two sampling periods for either of the two soil treatments but there were very large differences between soil treatments. Naturally-occurring ectomycorrhizae ranged from 28 to 38% in the control 4 and 6 month samples, respectively. The vertical mulch treatment had about six times more fine roots and twice the ectomycorrhizal development than detected in the control treatment at both sampling periods. About 60 percent of the ectomycorrhizal development in the vertical mulch treatment were formed by Pt. These differences reflected more than a 12 to 8 fold increase in relative root absorbing potential by the vertical mulch treatment over the control for the 4 and 6 months sampling dates, respectively. Fine roots, ectomycorrhizae and extramatrical mycelia of Pt physically penetrated the hydrated water-managing gels in the vertical mulch treatment.