Mycorrhizae of Landscape Trees Produced in Raised Beds and Containers

Sample collection

A total of 23 tree species were sampled between May 3 and June 8,1996, from an established tree farm at Walt Disney World in Lake Buena Vista, Florida (Table 1). The majority of the trees were of exotic origin, with only Magnolia grandiflora, Persea borbonia, and the 3 Quercus species being native to central Florida. Nine species were sampled from plants grown both in containers and raised beds, 6 species were sampled from containers only, and the remaining 8 species were sampled from embedded plants only. Five trees were sampled from each tree species in each growth environment where it occurred.

Containers (60 cm high and 70 cm in diameter, or 24×28 in.) were constructed of perforated and corrugated aluminum sheets formed into cylinders and secured with 2 sheet metal screws. Containers were placed on pieces of woven plastic cloth (to provide drainage and restrict root growth into soil) and filled with a blend of approximately 35% sandy topsoil, 35% green pine bark, and 30% biosolids compost. Five-gallon-size trees were purchased from commercial nurseries and transplanted into the containers a minimum of 2 years prior to sampling. Containers were irrigated daily with a spray stake emitter.

Trees grown in the ground were on raised beds (30 cm [12 in.] high) in a sandy soil that was topdressed annually with approximately 5 cm (2 in.) of biosolids compost. Trees were planted as either 5- or 10-gallon-size plants and had been grown in the raised beds for a minimum of 2 years prior to sampling. Embedded trees were irrigated daily by overhead irrigation.

The same fertilizer regime was used for trees grown in containers and in raised beds. Fertility was maintained by the application of 10-5-10 granular fertilizer (2.5% ammoniacal N, 3.51% soluble organic N, 3.99% water-insoluble N, 1.51% Mg, and 1.55% Fe) at the rate of 0.97 kg N 100 m^–2 (2 Ib 1,000 ft^–2) for each of 3 applications made each year during February, June, and September. The fertilizer was broadcast on the top of the containers or placed in a 1.8-m-wide (6-ft) band adjacent to embedded trees.

Samples were collected by driving a sharpened steel pipe (4.5 cm in diameter, or 1.8 in.) to a depth of 25 cm (9.8 in.). Two samples were collected within each container or, for trees grown in raised beds, around the dripline of each tree. Samples were placed in plastic bags and kept cool until they were returned to the laboratory, where they were stored at 4°C (39°F). In the laboratory, the 2 samples from each tree were combined and mixed thoroughly before a 100-g (3.5-oz) subsample was removed for further processing. Roots and spores of AM fungi were extracted from the subsample by decanting and wet sieving (sieve size ranged from 45 to 1,000 μm, or 0.002 to 0.039 in.), followed by sucrose-density-gradient centrifugation for separation of spores (Sylvia 1994).

Clearing and staining protocols

Roots collected on the 1,000-μm (0.039-in.) sieve were cleared in 1.8 M KOH for 1 hour at 80°C (176°F) and washed in 3 changes of water. Roots of woody plants are often difficult to clear because they contain high levels of phenolic materials; therefore, 2 additional protocols subsequent to KOH treatment were evaluated on one sample from each plant species: 1) 30% H₂O₂ for 10 minutes at 50°C (122°F) and 2) 3% NaOCI, acidified with several drops of 5 M HCI, for 50 seconds at ambient temperature. Following clearing, roots were washed in 5 changes of water, acidified with HCI, and then stained with trypan blue for 30 minutes at 80°C (176°F) (Sylvia 1994). Clearing with NaOCI proved most satisfactory and was used for the preparation of the remainder of the samples.

Mycorrhizal root colonization, root diameter, and AM spore number

Cleared roots were spread evenly on a scribed, plastic Petri dish and total and colonized root lengths were estimated with a dissecting microscope using the gridline-intersect method (Giovannetti and Mosse 1980). Intersections were scored positive for AM only if arbuscules or vesicles were present, and positive for EM only if evidence of a fungal mantle or Hartig net was observed. Any questionable fungal structures within the roots were evaluated at 400× magnification with a compound microscope. To quantify fine-root diameters, 10 randomly collected root pieces of each species were mounted in water on a microscope slide and measured at 400× magnification with an eyepiece micrometer. The supernatant of the final centrifuge wash was placed in a scribed Petri dish, and AM spores were counted under a dissecting microscope. Again, any questionable structures were further evaluated at 400× magnification.

Mycorrhizal root colonization

The proportion of root length colonized by mycorrhizal fungi ranged from 0% for Cinnamomum camphora (in containers), Ulmus parvifolia, and 2 Quercus species, to 83% for Bambusa ventricosa growing in raised beds (Figure 1A, Table 2, and Table 3). The growth environment did not have a consistent effect on root colonization (Figure 1A).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Mycorrhizal root colonization (A), AM spore number (B), and root diameter (C) of landscape trees samples both in containers and raised beds at Walt Disney World on May 3 or June 8,1996. Bars represent the mean of 5 replicates ± S.E.M.

The majority of trees sampled formed mycorrhizae of the arbuscular type. We did, however, sample 5 species that are in plant families dominated by ectomycorrhizae. Quercus laurifolia and Q. virginiana were not colonized (Table 3). Quercus myrisinifolia (Table 2) and Cedrus deodara (Table 3) had low levels of colonization, and the colonized regions had distinct mantles of an ectomycorrhiza. In contrast, Taxodium mucronatum was colonized by AM fungi in both containers and in raised beds.

AM spore number

Spores of AM fungi were present in all samples, but mean numbers ranged from < 20 to nearly 500 spores 100 g^–1 (3.5 oz^–1) (Figure 1B, Table 2, and Table 3). More than 75% of the spores observed were Glomus spp., with the remainder belonging to the family Gigasporineae. Samples from containers tended to have more spores (146 ±75 spores 100 g^–1,or 3.5 oz^–1) than samples from the raised beds (76 ± 24 spores 100 g^–1, or 3.5 oz^–1).

Fine root diameter

Mean root diameters ranged from < 500 μm (0.019 in.) for Ulmus parvifolia, Bambusa spp., and Gordonia lasianthus to > 1,000 μm (0.039 in.) for Quercus virginiana, Robinia idahoensis, and Taxodium mucronatum (Figure 1C, Table 2, and Table 3). When comparing plants growing in both environments, samples from the raised beds had greater root diameters (914 ± 97 μm, or 0.036 ± 0.004 in.) than samples from containers (684 ± 65 μm, or 0.027 ± 0.003 in.). No significant relationship was found between root coarseness and root colonization or AM sporulation.