arbuscular mycorrhizal fungi enhance seedling growth in two ... - FIU

Int. J. Plant Sci. 163(4):559–566. 2002. 䉷 2002 by The University of Chicago. All rights reserved. 1058-5893/2002/16304-0007$15.00

ARBUSCULAR MYCORRHIZAL FUNGI ENHANCE SEEDLING GROWTH IN TWO ENDANGERED PLANT SPECIES FROM SOUTH FLORIDA Jack B. Fisher1,*,† and K. Jayachandran‡ *Fairchild Tropical Garden, Coral Gables, Miami, Florida 33156, U.S.A.; †Department of Biological Sciences, Florida International University, Miami, Florida 33199, U.S.A.; and ‡Department of Environmental Studies and Southeast Environmental Research Center, Florida International University, Miami, Florida 33199, U.S.A.

Arbuscular mycorrhizal fungi (AMF) are reported and described in the fine roots of two federally listed endangered plant species: Amorpha crenulata Rydb. (Fabaceae) (crenulate lead plant or lusterspike indigobush) and Jacquemontia reclinata House ex Small (Convolvulaceae) (beach jacquemontia or beach clustervine). Wildgrown plants have typical Arum-type arbuscular mycorrhizae. Seedlings of these species were grown in pots with various native soil treatments under greenhouse conditions. Native mixed AMF from soil and roots growing in the natural communities pine rockland and beach back dune, respectively, were multiplied on Sudan grass and pigeon pea nurse cultures. Native sandy soil is low in available phosphorus (P), ranging from 8–18 ppm at the surface 0–5 cm to 2 ppm at 70 cm. AMF significantly increased the dry weight and total P content of seedlings growing on native soil. Additions of phosphate but without AMF also promoted seedling growth. Soil microbe filtrate had no effect on J. reclinata but did increase growth of A. crenulata, possibly by increased potential for Rhizobium inoculum for nitrogen-fixing nodules in this legume. Keywords: arbuscular mycorrhizal fungi, endangered species, Amorpha crenulata, Jacquemontia reclinata, phosphorus uptake.

Introduction

of AMF in their roots growing in the natural habitat and to clarify the degree of benefit that these species derive from AMF colonization. Other studies of endangered plants have found that AMF vary in their significance to plant growth and survival. AMF were required for survival of seedlings of Astragalis applegatei under nursery conditions (Barroctavena et al. 1998), whereas Penstemon haydenii appeared to be little associated with AMF under natural conditions (Flessner and Stubbendieck 1992). In a survey of endangered Hawaiian plants, Koske and Gemma (1995) found a wide range of responsiveness in seedlings inoculated with AMF and growing in artificial potting media. More recently, a survey of tropical and subtropical native plants in Brazil found variation in responsiveness to both AMF and added phosphorus fertilizer when these plants were grown on infertile native soil (Siqueira and Saggin-Ju´nior 2001). They also found quite different responses among species of the same genus. These results indicate that generalizations regarding AMF dependency of endangered species are inappropriate. Each species must be investigated individually before decisions on its mycorrhizal requirement can be made and implemented in restoration projects. In the greater Everglades ecosystem, which includes the coastal and pine rockland habitats of the two species under study, phosphorus (P) is a significant pollutant (U.S. Fish and Wildlife Service 1999). These habitats are all environments low in available P. Consequently, restoration efforts must be mindful of keeping additions of P to a minimum. Colonization of roots by AMF increases the uptake of P and allows plants to thrive in soils with low levels of available P (Smith and Read 1997). In the Everglades context, we designed our research to verify that plant growth in native soils that are low in available

Two rare plant species growing in the subtropical region of southeast Florida are listed as endangered, and restoration actions have been recommended (U.S. Fish and Wildlife Service 1999). Jacquemontia reclinata House ex Small (Convolvulaceae) (beach jacquemontia or beach clustervine) grows in seven small isolated populations along the Atlantic Ocean coast behind sand dunes and in the transitional zone to coastal pineland and hammock. Fewer than 733 plants remain, with most in two large populations, making this one of the most endangered plants in Florida (Lane 2001). Anthropogenic activities such as coastal development have eliminated most of this coastal habitat. Amorpha crenulata Rydb. (Fabaceae) (A. herbaceae Walter var. crenulata [Rydb.] Isely) (crenulate lead plant or lusterspike indigobush) grows in six sites (including one not protected) in the highly endangered pine rockland community that has largely been destroyed by farming and urbanization of metropolitan Miami. More than 1000 plants exist mainly in two populations (Fisher 2000). As part of the effort to better understand the biology of these endangered species and to improve success in restoration efforts, we examined the role of arbuscular mycorrhizal fungi (AMF) in the species’ growth, since it is widely known that AMF develop symbiotic relationships with most vascular plants (Smith and Read 1997). We sought to verify the presence 1 Author for correspondence; telephone 305-665-2844; fax 305665-8032; e-mail [email protected].

Manuscript received July 2001; revised manuscript received November 2001.

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P is enhanced by AMF. We also use the addition of P (in the form of phosphate) to the native soil as a standard to evaluate the effect of AMF on plant growth. Since additions of fertilizer will not be advisable, the P equivalence of AMF treatment will be a valuable index for restoration managers to consider. Because of the extreme rarity of these endangered plants and their habitats, there is an urgency to apply botanical and horticultural research to the pressing needs of conservation (St. John 1993; Affolter 1997). Our findings will aid in planning future restoration strategies and improving methods of propagating plants used for restoration. For convenience, only the generic name is used when referring to these two species when there is no ambiguity.

Material and Methods Habitat The research site for Jacquemontia is a nature preserve at Crandon Park on the northern end of Key Biscayne, a barrier island in Miami–Dade County, Florida. The plant community is a mix of back dune species (e.g., Cocoloba uvifera [L.] L., Uniola paniculata L., and Caesalpinia bonduc [L.] Roxb.) and coastal pine rockland species (e.g., Myrica cerifera L., Pithecellobium keyense Britton ex Britton & Rose, and Sabal palmetto [Walter] Lodd. ex Schult. & Schult. f). The research site for Amorpha is a remnant of undisturbed natural pine rockland vegetation in Tropical Park, Miami–Dade County, Florida. The overstory is the Dade County slash pine, Pinus elliottii Engelm. var. densa Little & Dorman. The following plants grow within 10 m of the collection site: Pithecellobium keyense Britton ex Britton & Rose, Quercus virginiana Mill., Rhus copallinum L., Sabal palmetto (Walter) Lodd. ex Schult. & Schult. f., Serenoa repens (Bart.) Small, and Smilax auriculata Walter.

Soil Soil samples for physicochemical analysis were collected within 50 cm of the plant. Soil analysis was carried out by a reputable local commercial testing service. For both species, soil used to fill pots for greenhouse experiments was dug from 5–20-cm depths in nearby identical habitats but in areas free of the endangered species. Soil was sieved through 6-mm mesh to remove stones and large root fragments.

Root Samples Soil and larger surface roots were carefully excavated at the base of wild plants. The finest feeder roots with clear organic connection to the mother plant were fixed in 70% ethanol. Root samples of experimental plants were weighed before being fixed in ethanol. Each root sample was cleared in KOH, bleached with ammoniated H2O2, and stained with trypan blue in acidic glycerol (Brundrett et al. 1996) to determine presence of AMF.

AMF Inoculum Nurse cultures of native AMF from each of the two habitats were maintained separately in 4-L pots containing fresh, unpasteurized soil from the native habitat of the species of interest. Host plants were pigeon pea and Sudan grass. Nurse cultures were at least 12 wk old before use. The inoculum samples showed heavily colonized root fragments and many AMF spores. Soil and root fragments were mixed well and used as a mixed AMF inoculum. A part of the same inoculum was steam pasteurized for 2 h one time and used as an inoculum control for all treatments except treatment 2 (table 1).

Greenhouse Study Seeds were collected from cultivated plants in the ex situ conservation collection at Fairchild Tropical Garden. Seeds were surfaced sterilized with 1.0% sodium hypochlorite solution, scattered on the surface of new inorganic Perlite in plastic pots, and placed under periodic mist watering. Seedlings at the cotyledon or first true leaf stage were transplanted into 6.4 # 25-cm plastic pots (D40 Deepots, Stuewe and Sons), each filled with 600 g of native sandy soil (for Jacquemontia) that was steam pasteurized twice (brought to 90⬚C on the first and third days). For Amorpha, pots were filled with 300 g of native sandy soil mixed with 300 g of sieved natural limestone gravel that was similarly steam pasteurized. Inclusion of gravel gave a better-drained mix that was more like the natural site. A layer of paper towel prevented loss of soil from bottom drainage holes. The treatment numbers and compositions are noted in table 1. Each pot received 20 g of either fresh or steam pasteurized inoculum. Soil filtrate consisted of 50 mL per pot of a soil solution derived from 300 g of fresh soil shaken in 2 L of distilled water and filtered through Whatman no. 1 filter paper. Phosphate treatments consisted of five treatments on alternating weeks beginning 3–4 wk after transplanting. This brought the total available P additions (in the form of KH2PO4) to the

Table 1 Treatments Used in Pot Experiments Treatment 1 2 3 4 5 6

Description Control AMF + microbes 5 ppm available P 10 ppm available P 20 ppm available P Filtrate (microbes only)

Note.

Contents of pot Pasteurized Pasteurized Pasteurized Pasteurized Pasteurized Pasteurized

soil soil soil soil soil soil

+ + + + + +

pasteurized inoculum live inoculum pasteurized inoculum pasteurized inoculum pasteurized inoculum pasteurized inoculum

+ + + +

PO4 additions PO4 additions PO4 additions soil filtrate

Soil and AMF from the native habitat of each species. See details in “Material and Methods.”

FISHER & JAYACHANDRAN—ENDANGERED PLANT ARBUSCULAR MYCORRHIZAE pot to 5, 10, and 20 ppm (based on 600 g of total soil). Treatments were as follows: control (soil + steamed inoculum), AMF (soil + fresh inoculum), filtrate (control + soil filtrate), and phosphorus (control + added KH2PO4). Since we wanted to relate effects of AMF inoculation (which included soil microbes) to additions of PO4, we did not include soil filtrate (treatment 6) to the PO4 treatments (treatments 3–5). In one Amorpha experiment (fig. 3B), 50 mL of Hoagland’s nutrient solution (made without PO4) was added three times to all pots because the seedlings became chlorotic from apparent nitrogen deficiency. All other plants were purposely not fertilized with Hoagland’s in order to stress the plants nutritionally as would be the case in natural habitats.

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Each treatment was replicated in eight to 10 pots. The pots each had one seedling and were randomly arranged in frames and grown on benches in a glasshouse under ca. 50% shade. Experiments were repeated twice, starting during the months of May and June. Cross contamination was prevented by separation of pots and care in watering. Plant height from cotyledonary node to shoot apex was measured at irregular intervals, but variations due to branching in Amorpha and the twining habit of Jacquemontia limited the usefulness of these measurements. At the end of the experiment, fresh and dry weights of roots and shoots were determined and used to evaluate treatment response. A small fresh root sample was also fixed in ethanol after its fresh weight

Fig. 1 Experimental plants and roots cleared and stained with trypan blue. A–C, Amorpha plants after 21 wk. A, Representative plants (from left to right: control, AMF, P, and filtrate treatments). B, AMF in cortex of Amorpha. C, Arbuscules in cortical parenchyma of Amorpha. D–F, Jacquemontia plants after 16 wk. D, Representative plants (from left to right: control, filtrate, AMF, and P at 5, 10, and 20 ppm). E, AMF in root, vesicles dark. F, Arbuscules in cortical parenchyma. a, arbuscule; h, hypha; v, vesicle. Bars p 50 mm in B and E; 100 mm in C and F.


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was a yellow-orange sand; available P p 4–8 ppm and pH p 7.3–7.4. Organic matter was 4%–8%.

AMF Colonization Wild-collected roots of both species had many septate and presumably saprophytic fungi on their surfaces. Similar surface fungi also occurred irregularly in most of the experimentally treated plants, although at lower density. Nonseptate (rare and irregular septa) hyphae were observed in the root cortex of wild-collected and experimental plants. Defining arbuscules and vesicles were observed attached to these nonseptate hyphae. In both species, most AMF hyphae were found in the longitudinal intercellular spaces of the cortex, mainly from the midcortex to the endodermis (fig. 1B, 1E). Single arbuscules filled individual cells (fig. 1C, 1F) and were concentrated in younger regions of fine roots. Vesicles tended to occur in older regions of fine roots (fig. 1E). Roots of Jacquemontia were difficult to clear and observe because of scattered pigmented epidermal cells (gray cells in fig. 1E) and laticifers in the cortex of even the thinnest roots. Root hairs occur irregularly (in surface patches) in both species. Fig. 2 Dry weight of Jacquemontia plants after 16 wk of growth; see table 2 for significant differences among treatments. A, Experiment started in late June; no Hoagland’s addition. B, Another experiment started in early May; no Hoagland’s addition.

was measured. The final dry weight of the root was calculated based on the addition of the sample’s proportion of the whole root system. Phosphorus content of tissues was determined from pulverized dried tissues of all replicates. Digestion and analysis of total P followed the dry combustion and colorimetric method (Solorzano and Sharp 1980). Statistical analysis was carried out using SPSS Base 10.0 statistical software (SPSS, Chicago). Differences between all treatment means of dry weight and P concentrations were tested with a one-way ANOVA. The data were natural log transformed if homogeneity of variances could not be assumed (Levene statistic). A post hoc comparison of the differences between means was made using either a conservative Bonferroni test if the Levene statistic indicated homogeneity of variances or a Games-Howell test if variances were not homogeneous even after transformation.

Results Soils The native beach back dunes site of Jacquemontia had deep (150 cm) sandy soil. In the surface 0–5 cm (three samples, n p 3), P (weak Bray test) p 8–18 ppm and pH p 7.3–7.5. At 5–10 cm depth (n p 3), P p 4–10 ppm and pH p 7.6–7.7. At 70 cm depth (n p 1), P p 2 ppm and pH p 8.1. Organic matter declined from 7% near the surface to 1% at 70 cm depth. The native pine rockland site of Amorpha had shallow sandy soil over a bed of oolitic limestone. The surface 0–5 cm was a white to gray sand (n p 3); P (weak Bray test) p 8 ppm and pH p 6.8–7.3. The soil at 5–20 cm depth (in scattered pockets)

Growth Effects of AMF Jacquemontia. The two experiments had similar results (figs. 1A, 2) after 16 wk of growth. The later experiment produced somewhat smaller plants, possibly because it was started almost 2 mo later (in late June) and was exposed to less sunlight because of increased cloudiness in the later rainy season (fig. 2). Table 2 shows the treatment effects on growth that have potential ecological significance: promotion of shoot and root dry weight (biomass) by AMF versus control (treatment 1 vs. treatment 2), promotion by AMF versus soil filtrate (treatment 2 vs. treatment 6), and equivalence of AMF effect to level of added phosphate (treatment 2 vs. treatments 3–5). Except Table 2 Comparison of Dry Weights in Treatments with Potential Ecological Significance

Jacquemontia: Figure 2A: Root Shoot Figure 2B: Root Shoot Amorpha: Figure 3A: Root Shoot Figure 3B: Root Shoot

AMF 1 control (2 vs. 1)

AMF 1 filtrate (2 vs. 6)

AMF p ppm P (2 vs. 3, 4, 5)

ns +

ns +

ns (5, 10, 20) 5, 10, 20

+ +

+ +

20 10, 20

+ +

+ +

20 10, 20

+ +

ns ns

10 only 10 only

Note. Two replicate experiments for each species; ns p not significantly different (5% level); + p significant difference; 10 only p only one concentration used in this experiment.

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Table 3 Ratio of Root : Shoot Dry Weights Treatment

Jacquemontia: Figure 2A Figure 2B Amorpha: Figure 3A Figure 3B

1

2

3

4

5

6

ANOVA

0.88A 1.06ABCE

0.56A 1.49ABE

0.50A 0.78ACDE

0.52A 0.52CDE

0.53A 0.59ACDE

0.98A 0.99ABCDE

ns s

2.28A 2.99A

1.55A 1.57AB

4.10A …

2.63A 0.63B

2.41A …

2.80A 2.31AB

ns s

Note. Two replicate experiments for each species; ratios with same letters are not significantly different; s p significant difference at 5% level; ns p not significantly different; ellipses indicate treatment not done.

for the roots in figure 2A, AMF promoted growth more than control and more than filtrate (non-AMF microbes). This AMF promotion was equivalent to the addition of phosphate at various levels (available P p 5–20 ppm). The base level of available P in the pasteurized soil was 10 ppm. The ratio of shoot to root dry weight was calculated for all plants. Table 3 shows that the root : shoot ratio was either equivalent for all treatments (fig. 2A in table 3) or various for all treatments (fig. 2B in table 3) depending on the experiment. However, the root weight data may be overestimated. While washing soil from the roots, we found that it was impossible to remove all soil without the loss of fine roots. Small particles of sand and organic matter were bound to the surface of all roots by fungal hyphae, as seen with magnification. Treatments 2 and 6 were especially problematic, although saprophytic fungal hyphae occurred in all treatments. Much of this unwashed soil detached after drying and was separated from the roots with a brush. Nevertheless, treatment 2 roots were never completely free of dark particles, presumably overestimating root weight. Amorpha. The unpotted plants are shown in figure 1D. Plants inoculated with AMF (treatment 2) were larger than control (treatment 1) and soil filtrate (treatment 6) in one experiment (fig. 3A in table 2) and only larger than the control in the other (fig. 3B in table 2). The effect on dry weight of AMF is equal to that of added available P at 10 and 20 ppm (treatments 4 and 5). We had problems with soil attachment similar to but not as extreme as Jacquemontia. For the root : shoot ratios, there were no significant differences among treatments in one experiment (fig. 3A in table 3). AMF was smaller than in the control in the other experiment (fig. 3B in table 3). The number of root nodules was counted for each plant in one experiment (fig. 3A), and there were no significant differences among treatments (numbers are not shown). However, there was a moderate positive linear relationship between nodule number and shoot dry weight (linear regression, adjusted r 2 p 0.379), with the greatest number of nodules in treatment 2 (mean p 3.1, n p 10). The mean nodule number was 2.3 in treatment 6 and 1.2 in treatment 1.

Jacquemontia. Because of low dry weights per shoot, pairs of plants were pooled for P analysis, but care was taken to record the replicate numbers and their pooled weights for later calculation of total shoot P. Thus, most P calculations are based on n p 4 or 5. Both shoot total P and tissue concentration were increased by AMF and additions of PO4 (fig. 4). In both experiments, AMF was equivalent to added available P at 20 ppm. Statistical analysis revealed that AMF inoculation significantly increased both tissue P concentrations and uptake per shoot compared with control and microbial filtrate treatments (table 4). Amorpha. In treatment 1 in figure 1A, only two plants were large enough for a P assay using our techniques because of the small size of plants. In the remaining treatments, eight to 10 plants were assayed by pooling pairs of replicates; thus, only four to five values were obtained per treatment. There was no statistical difference between AMF and control and filtrate treatments (table 4). In figure 5, the PO4 addition had

Phosphorus Uptake Effects of AMF We chose to analyze the concentration of P in only shoot tissues because of the unreliable determination of root weights (especially in treatment 2). Concentration of P in the tissue (mg/0.1g) and total shoot P (mg/shoot) were calculated.

Fig. 3 Dry weight of Amorpha plants after 21 wk of growth; see table 2 for significant differences among treatments. A, Experiment started in late May; no Hoagland’s addition. B, Another experiment started in early May; Hoagland’s solution added three times.

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Fig. 4 Effect of treatments on the phosphorus concentration and total content in shoots of Jacquemontia; pairs of replicate plants were pooled for P analysis so that each bar is the mean of four samples; measurements made after 16 wk of growth. A, Experiment started in late June. B, Another experiment started in early May.

a significant effect to increase both shoot total and tissue concentrations of P compared with AMF and filtrate. The tissue concentration of P in AMF was significantly less than control but equal to filtrate in figure 5 and table 4. Plants grown with higher available P (10 and 20 ppm) had more P in both tissues and per shoot than those with AMF and filtrate.

Discussion We report for the first time that both of these endangered species have a symbiotic association with AMF in their wild habitats. The fungi form typical Arum-type arbuscules within cortical parenchyma cells of roots (Smith and Smith 1997). This is expected since many of the associated plants growing nearby have been shown to be mycorrhizal, e.g., Serenoa (Fisher and Jayachandran 1999), Zamia (Fisher and Vovides 2002), Uniola (Sylvia et al. 1993), and Myrica (Semones and Young 1995). Other species in the same genus or family are also colonized by AMF, e.g., Convolvulaceae: Jacquemontia (Koske and Gemma 1990; Koske et al. 1992) and Ipomoea (O’Keefe and Sylvia 1993) and Fabaceae-Papilonoideae:

Amorpha (Wilson and Hartnett 1998), Cajanus (Olsen and Habte 1995), and Pithecellobium (Herrera and Ferrer 1980). The soil in both habitats is relatively low in available P (8–18 ppm at the surface 5 cm and 2–8 ppm at greater depths). Both species have feeder roots near the surface (0–10 cm) during the rainy season (June–November), which is when roots were collected to verify presence of AMF. Tap roots grow deeper but were not exposed or studied in detail at protected sites. The Hawaiian endemic Jacquemontia sandwicensis, two introduced species of Ipomoea, and several species of Fabaceae were all growing in the same coastal strand community in Hawaii and were all colonized by AMF (Koske and Gemma 1990, Koske et al. 1992). In J. sandwicensis, AMF hyphae were present on the surface of rhizomes collected from the wild (Koske and Gemma 1990). In Jacquemontia reclinata, AMF clearly promote seedling growth and P uptake by shoots when grown on these native soils with low to moderate levels of available P. The growth of Ipomoea batatas was also promoted by AMF (O’Keefe and Sylvia 1993). We found that root measurements were unreliable because of difficulty in cleaning soil particles off roots. This surface rhizosphere sheath may have been associated with the AMF hyphae (St. John et al. 1984) or other soil microorganisms. The inconsistent and nonsignificant variations in root : shoot ratio were likely due to this difficulty. Our observation that more soil particles adhere to AMF roots compared with control roots suggests that AMF extend their hyphal network beyond the P depletion zone, which increases nutrient uptake from these poor soils. In Amorpha, which is a nitrogen-fixing legume, AMF significantly promoted growth as did high available P. In one experiment (fig. 3B), soil filtrate had an effect equivalent to AMF, possibly by promoting Rhizobium inoculation, although this was only weakly supported by differences in nodule numbers. However, we found no support for a positive AMF effect on P accumulation in the shoot. The filtrate was equivalent to AMF, while the control had greater P accumulation (fig. 5A). It is possible that P accumulated at higher concentrations in roots than in shoots, but we did not collect data on this. The interaction between AMF and nitrogen-fixing nodules (which require high levels of P) is complex and appears to be complimentary in noncrop woody legumes such as Cajanus (Olsen and Habte 1995) and Acacia (Martin-Laurent et al. 1999). The growth of Amorpha canescens, a prairie species, was significantly promoted 6 wk after inoculation with native AMF (Wilson and Hartnett 1998). Both habitats (coastal and pine rocklands) are fire prone. Seedlings of both endangered species form a taproot system, and mature plants survive and resprout from the rootstock after wild fires or artificial mowing (U.S. Fish and Wildlife Service 1999). However, the relationship between mycorrhizal colonization and postfire responses is unknown for these species. Restoration of these two U.S. federally listed species was proposed and includes artificial outplanting to augment the few remaining populations and to introduce new populations into historically appropriate habitats that are now protected (U.S. Fish and Wildlife Service 1999). Our demonstration that AMF occur in and promote growth of these species will aid in developing best methods to produce the propagules used in

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Table 4 Comparison of Shoot Phosphorus Levels in Biologically Significant Treatments

Jacquemontia: Figure 4A: P total P concentration Figure 4B: P total P concentration Amorpha: Figure 5A: P total P concentration Figure 5B: P total P concentration

AMF 1 control (2 vs. 1)

AMF 1 filtrate (2 vs. 6)

AMF p ppm P (2 vs. 3, 4, 5)

+ +

+ +

20 20

+ +

+ +

20 20

ns ns

ns ns

5, 10 5

? ?

ns ns

Only 10 Only 10

Note. Two replicate experiments for each species; ns p not significantly different (5% level); + p significant difference; ? p sample size too small.

restoration efforts. Plants of the endangered legume Astragalis applegatei survived on artificial, sterilized potting medium (peat + perlite) only if inoculated with native soil containing AMF (Barroetavena et al. 1998). Inoculated plants had both mycorrhizae and nodules, both of which were absent in control, uninoculated plants. In experimental seedlings and cuttings grown on artificial media, Hawaiian plants inoculated with a species of Glomus formed mycorrhizae and were “usually larger” than control plants that were not inoculated (Koske and Gemma 1995). Thus, inoculation of nursery stock with AMF can promote growth and substitute for additions of phosphate on native soils. However, acceptance of phosphate fertilization is very unlikely for plants grown for Everglades ecosystem restoration since P is such a significant pollutant in these habitats (U.S. Fish and Wildlife Service 1999). The use of native AMF is an ecologically sound method for conservation horticulture and will be a valuable tool in future restoration plans. We presume that nursery-grown seedlings may have improved survivorship when they are later outplanted if they are first colonized by AMF. This was demonstrated with Uniola in dune restorations of a similar habitat in south Florida (Sylvia 1989; Sylvia et al. 1993). However, promotion of growth of potted plants by AMF does not necessarily indicate improved survivorship. Of two Hawaiian species with increased survival due to AMF inoculation, one had significantly larger seedlings, but the other was not significantly different in size from the control plants (Koske and Gemma 1995). Therefore, the hypothesis of AMF-enhanced survival in Jacquemontia and Amorpha seedlings must still be tested in situ under natural field conditions. Fig. 5 Effect of treatments on the phosphorus concentration and total content in shoots of Amorpha; measurements made after 21 wk of growth. A, Experiment started in late May; pairs of replicate plants were pooled for P analysis so that each bar is the mean of five samples. B, Another experiment started in early June; in treatment 1, only two plants were measured; in treatments 2–4, eight to 10 plants were measured.

Acknowledgments We thank Elena Pinto-Torres, Paul Fenster, and Marianne Vanevic for technical assistance and Carl Lewis for helpful

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comments on the manuscript. The Montgomery Botanical Center permitted use of its pine rockland site. We appreciate the cooperation of the Natural Areas Management of Miami– Dade County Park and Recreation Department, which allowed collections under permit 12. Research was supported in part

by U.S. Fish and Wildlife Service grant 1448-40181-99-G-173 and by Florida Department of Agriculture and Consumer Services contract 5619. This article is Southeast Environmental Research Center contribution 168 and Florida International University Tropical Biology Program contribution 46.

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