Arbuscular Mycorrhiza Status of Gold and Uranium Tailings and Surrounding Soils of South Africa's Deep Level Gold Mines: I

South African Journal of Botany 73 (2007) 218–225 www.elsevier.com/locate/sajb

Arbuscular mycorrhiza status of gold and uranium tailings and surrounding soils of South Africa's deep level gold mines: I. Root colonization and spore levels ⁎ C.J. Straker a, , I.M. Weiersbye b, E.T.F. Witkowski b

a Restoration and Conservation Biology Research Group, School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa b Restoration and Conservation Biology Research Group, School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa Received 11 December 2006; accepted 13 December 2006

Abstract

Surveys of arbuscular mycorrhiza fungi (AMF) root colonization and spore status of slimes dams of South Africa's deep level gold mines in the North–West province were undertaken in the late summer of 1999. Five indicator host plant species [Asclepias fruticosa L., Cynodon dactylon (L.) Pers., Atriplex semibaccata R. Br., Phytolacca octandra L. and Asparagus laricinus Burch.] were sampled from the surrounding natural soils (veld) and dams of three age categories (recently-revegetated-RV, old-revegetated-OV and never-revegetated-NV) within the different zones of each dam (top, lower slopes, retaining wall, toepaddocks). Host species and broad substratum type (slimes, slimes-polluted veld and veld) showed significant effects on AM root parameters. Overall, A. fruticosa and A. laricinus were the most mycotrophic hosts on RV, OV and NV slimes and C. dactylon and A. semibaccata in the veld. On RV sites there were negligible differences between host species and zones in mycorrhizal parameters, whereas on OV and NV sites most parameters were significantly different, with significant interactive effects between host species and zones. No differences in spore densities between host species or age categories were found but total spore densities between zones were significantly different. NV slopes had the lowest pH, aerial cover, organic matter and total P but highest potential acidity and weak relationships were found between zone pH and mycorrhizal colonization parameters. These results are discussed in relation to the factors affecting distribution of AMF on these sites and the options relating to AMF inoculum introduction in rehabilitation programmes. © 2006 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Management of arbuscular mycorrhizas; Mine waste rehabilitation; Pollution; Restoration ecology

1. Introduction by using ‘high-input’ methods that involve intensive leaching, liming, fertilization and irrigation prior to planting with a suite of Tailings or slimes dams, consisting of pulverized high pyrite pasture grass species. At great cost to the gold industry, these rock waste from the mining of gold and uranium, are a significant methods have proven ecologically and economically unsustain- environmental hazard in South Africa (Van As et al., 1992). The able (Witkowski and Weiersbye, 1998b; Weiersbye et al., 2006). main environmental hazards from gold-tailings dams are, (i) water The introduced and naturally-colonizing vegetation of slimes pollution from acid mine drainage (AMD) and, (ii) wind-borne dams and the adjacent slimes-polluted soils (toepaddocks; Fig. 1) dust clouds (Blight, 1991; Van As et al., 1992; Mizelle et al., in two old mining regions (North–West and Free State provinces) 1995). Until recently, grassing of slimes dams has been considered was surveyed over a six-year period (Weiersbye et al., 2006). Less the most effective means of reducing wind-caused erosion and than 5% of the almost 400 plant species identified were found to protecting the surrounding environment. Historically, the South be species actively introduced during grassing. The longevity of African gold mining industry has facilitated slimes dam grassing cover on grassed slopes was also found to be largely limited to the lifetime of the introduced individuals as pasture grass regener- ⁎ Corresponding author. Tel.: +27 11 717 6322; fax: +27 11 717 6351. ation by seed was negligible (Witkowski and Weiersbye, 1998b). E-mail address: [email protected] (C.J. Straker). Less than five introduced species remained on slopes within four

Fig. 1. Diagram of slimes dams and the surrounding environment divided into the sampling substrata and zones. years after the withdrawal of supplemental amelioration (Weiers- further exacerbated by the high fertilization regimes, precluding bye et al., 2006). Despite the actual contribution to cover on the development of suitable AM symbioses (Eason et al., 1999). slopes being extremely low, there was a high species diversity of The aims of this survey were, (i) to assess mycotrophy of natural colonizers with most individuals detected on heavily both the introduced and naturally-colonizing vegetation of gold polluted toepaddocks, retaining walls and on the flatter surfaces of slimes dams, slimes-polluted soils (toepaddocks) and adjacent the dams (berms and tops; Fig. 1). natural soils (veld) and, (ii) to determine levels of natural Successful rehabilitation of degraded and polluted substrata immigration and introduction of AMF via vegetating efforts, is dependent on an understanding of plant establishment and and (iii) the reproduction of the AMF. All the aims are of value succession, factors which are strongly influenced by dynamic in establishing suitable management regimes for sustainable soil components such as organic matter and microbial vegetation on slimes dams and slimes-polluted soils. communities. Arbuscular mycorrhizal fungi (AMF) are an essential component of the soil/plant community and an aim of 2. Materials and methods many rehabilitation programs is to accelerate the predominance of mycotrophic plant populations in order to create more stable 2.1. Slimes dams sites and vegetation history communities (Smith et al., 1998). Mycorrhizal inoculation of disturbed/degraded sites is important in promoting the domi- The study was carried out during late summer at gold mines nance of mycotrophic species, which would lead to a more rapid in the North–West province of South Africa. All the mines rate of succession (Smith et al., 1998). Rehabilitation of receive summer rainfall and winter frosts. The North–West disturbed sites tends to attract ruderal non-mycotrophic or province slimes dams are situated on doleritic and sandy soils facultatively mycotrophic plants, which preclude the survival of overlying dolomites at an altitude of ∼1300 m a.s.l. The main mycotrophic seedlings and the introduction of mycorrhizal grassland (veld) type in the mine lease areas is dry transitional propagules (Reeves et al., 1979). However, slimes dams present Cymbopogon-Themeda (with some development of a mixed a unique situation in that they are highly disturbed by erosive grassy false Karoo veld), surrounded by xeric grasslands forces and are biologically depauperate polluted environments (klipveld) and Acacia karroo savanna (Kooij et al., 1990). in which nitrogen, phosphorus and surface water-availability is Five plant species were assessed from ten high (N0.5%) low, whereas the availability of transition metals, metalloids and pyrite dams derived from three gold plants within a region of radionuclides is high (Witkowski and Weiersbye, 1998b; 400 km2. The dam slopes were classed according to their Weiersbye et al., 1999). The dominant natural vegetation of rehabilitation history; (i) recently-vegetated (RV): amelioration such sites is not ruderal, but consists of slow-growing woody, (lime, fertilizers, seeds and irrigation) had ceased between 1.5 semi-woody and perennial species (Weiersbye et al., 2006) and 3 years previously; (ii) old-vegetated (OV): amelioration which in natural environments would be expected to be highly had ceased N4b8 years previously, and (iii) never-vegetated mycotrophic (Smith and Read, 1997). However, the current (NV): dams on record as never having been ameliorated. All the high-input grassing regime actively introduces ruderal species dams surveyed, with the exception of one RV dam (established and exotic pasture grass species with shallow, fibrous root- in 1985), were first established between 1956 and 1982, and systems. The low mycotrophy of these plant forms would be comprised similar parent material. All the dams except NV had 220 C.J. Straker et al. / South African Journal of Botany 73 (2007) 218–225 received similar liming, fertilization and irrigation regimes for 2.5. Soil analyses establishment of similar suites of pasture grasses and herbs (Witkowski and Weiersbye, 1998a). In addition, only slopes of The dams were visually assessed with respect to percentage similar aspect opposite relatively undisturbed grasslands were aerial cover of vegetation, percentage litter and percentage bare- included in the survey. Eighteen slopes of south-westerly and ground. Three replicate cores (0–20 cm depth) were taken from south-easterly aspects were selected (OV: n=7; RV: n=5 and each zone of the surveyed slopes (including the adjacent veld). NV: n=6). The slopes were divided into 6 physical zones: top After drying at 40 °C, sub-samples of slimes and soil were (for the OV treatment only), upper slope, mid to lower slope, analyzed for (a) pH in water and in 0.1 M KCl, (b) total potential retaining wall, toepaddock (a 20 m to 50 m wide area of slime- acidity, (c) conductivity and (d) total organic matter content contaminated soil around the base of the dam) and veld using standard methods (Anderson and Ingram, 1993). Total P (rangelands) N100 m away from the edge of the toepaddock and Ca was determined using X-ray Fluorescence Spectrometry. (Fig. 1). Upper slopes were not assessed as these are of more recent origin, wetter, tending to alkaline pH and are seldom 2.6. Statistical analyses actively vegetated (Witkowski and Weiersbye 1998b). Data were analysed by two- and three-way ANOVA and 2.2. Host species sampled and sampling strategy Student Newman Keuls multiple range tests (SNK), using SAS (SAS Institute, 1985). Because comparisons were unbalanced Lists of species originally planted on the slimes dams, and due to the inconsistent occurrence of species on the selected sites, planting regimes are described by Weiersbye et al. (2006).Fiveof type III sums of squares were used in the analyses. All percentage the common perennial species were chosen to be indicators for the data were arcsin-transformed prior to statistical analyses. Data are AM status of the substrata. Entire root-systems were excavated presented as untransformed means and standard errors. together with the encompassing substratum from every zone of each slope. Three replicates of each species per zone were 3. Results excavated as far as possible (occasionally 3 replicates could not be found on a particular zone, and the ‘top’ zone was only available 3.1. Plant cover for the OV class). The indicator hosts consisted of a perennial grass, two perennial herbs and two perennial forbs. The grass was Overall estimates of percentage aerial live cover was highest Cynodon dactylon (L.) Pers., a facultatively-evergreen perennial on all zones of RV sites, whereas on the lower slopes of the OV pasture species (a naturalized alien) with a spreading growth habit, dams surveyed N15% live cover remained (Table 1). With the fibrous root system and adventitious rooting at stem nodes. The exception of the toepaddocks, the zones of NV sites had the perennial herbs were Atriplex semibaccata R. Br., an evergreen lowest aerial cover. Overall estimates of the percentage litter semi-woody perennial (alien) with a central tap-root and a trailing was highest on RV sites; on slopes consisting mostly of standing habit and Phytolacca octandra L., a short-lived deciduous peren- biomass of dead pasture grasses, with large amounts of litter nial herb (alien) with a stout starchy tap-root. The perennial forbs having washed off onto the retaining walls and toepaddocks. were Asclepias fruticosa L., a short-lived deciduous perennial forb (indigenous) with a central tap-root and Asparagus laricinus 3.2. Soil physical and chemical conditions Burch., a deciduous perennial monocotyledon (indigenous) with a shrubby habit and a tuberous root system. C. dactylon, The high pyrite content of all the dams surveyed is reflected in P. o cta nd ra and A. semibaccata were introduced during slimes the low pH and high potential acidity of the substrata (Table 1). dam vegetation operations, and are also colonizers of slimes. Both Potential acidity was highest on NV lower slopes, followed by A. fruticosa and A. laricinus are common natural colonizers of OV lower slopes and both NV and OV toepaddocks. Lowest acidic slimes and slimes-polluted soils (Weiersbye et al., 2006). potential acidity levels were found on RV sites, undoubtedly due to the effects of liming, which was also reflected in the neutral pH 2.3. Assessment of root AM status values and high Ca levels of the RV sites. The topsoil of the natural soils in the region are also acidic (pH ∼4.8–5.3 in KCl). Plant roots were severed from shoots, packed with the Soil organic matter was low in all substrata including the natural encompassing substratum into plastic bags and stored at 4 °C in veld soils, but lowest on NV slimes. The high levels of fertilizer the dark. Roots were immediately cleared and stained by the application that occurs during grassing was reflected by P levels method of Koske and Gemma (1989) and intraradical status of on RV slopes almost twice those of the natural soils. On NV arbuscles, vesicles, hyphae and total colonization determined by dams total P levels are extremely low but still higher than those of the modified intersect method of McGonigle et al. (1990). some natural nutrient-poor ecosystems in South Africa (e.g. Witkowski and Mitchell, 1987). The conductivity of NV slimes 2.4. Assessment of AM spore density is ∼30× that of the natural veld and only slightly lower in OV slimes. Total Ca levels on RV dams are extremely high due to the AM extraradical spore density was assessed by the centrifu- addition of lime, but decline in time so that Ca levels in OV gation method of Tommerup (1994) from 50 g substratum sam- slimes approach those of NV slimes (still however 5× those of the ples separated from around the host roots. natural soils). C.J. Straker et al. / South African Journal of Botany 73 (2007) 218–225 221

Table 1 Broad characteristics of the slimes dam zones (means±S.E.) of different age categories, and unpolluted veld at the study sites Recently-vegetated slimes dams Old-vegetated slimes dams Never-vegetated slimes dams Veld Lower slope Retaining Toepaddock Lower slope Retaining Toepaddock Lower slope Retaining Toepaddock wall wall wall Vegetation % Bare 59.5±8.6 41.3±7.2 48.3±7.0 91.7±2.4 80.7±10.1 61.5±7.8 99.7±0.3 98.3±0.7 67.5±3.0 – ground % Litter 6.3±2.2 20.0±5.1 20.0±7.4 1.9±0.6 1.3±0.7 14.0±6.6 0.0±0.0 1.0±0.4 5.6±0.8 – % Aerial 52.0±9.5 75.0±5.0 61.7±4.8 12.7±2.9 25.6±11.3 45.1±8.0 0.9±0.4 2.3±0.61 49.6±4.2 – cover

Slime & soil Organic 2.1±0.5 – 1.5±0.1 3.9±1.7 – 1.1±0.2 0.8±0.5 – 0.9±0.3 3.2±0.2 matter (%) pH (water) 6.8±0.1 – 6.8±0.2 3.4±0.5 – 3.5±0.1 2.5±0.2 – 3.7±0.1 5.2±0.3 pH (KCl) 6.7±0.1 – 6.6±0.1 3.4±0.5 – 3.4±0.1 2.5±0.2 – 3.6±0.1 4.8±0.2 Acidity 49.0±11.0 – 245.0±102.0 5051.0±1552.0 – 1054.0±118.0 26423.0±6145.0 – 1862.0±49.0 302.0±24.0

(μgH2SO4/g)

Conductivity 1000.0±32.0 – 2325.0±250.0 2142.0±149.0 – 978.0±378.0 2950.0±703.0 – 500.0±50.0 119.0±33.0 (μmhos/cm)

Total P (μg/g) 850.0±220.0 – 280.0±90.0 170.0±50.0 – 80.0±20.0 90.0±30.0 – 90.0±20.0 470.0±140.0 Total Ca 1.6±0.5 – 1.0±0.1 0.7±0.3 – 0.2±0.1 0.5±0.2 – 0.5±0.1 0.1±0.0 (mg/g)

3.3. Root AM assessment percentage colonization on slimes-polluted veld. The highest percentage of colonization overall occurred in A. semibaccata Two-way ANOVA analysis (host species and broad and C. dactylon in veld, followed by A. laricinus and substratum type) showed a significant effect of host species A. fruticosa in slimes-polluted soil (toepaddocks) and veld. on all AM attributes except proportion of hyphae (p=0.0674), The percentage arbuscules was low in all hosts (data not and of broad substratum type on percentage total colonization shown), but the proportion of arbuscules to other intraradical (Table 2). There were also significant interactions between host structures was uniformly highest in A. laricinus, regardless of species and substratum type for percentage total colonization. substratum. A. fruticosa and A. semibaccata had higher A. laricinus had the highest percentage colonization on slimes proportions of arbuscules on slimes than on the other substrata, (although not significantly different to the other species), wherea C. dactylon had lower proportions of arbuscules on whereas both A. laricinus and A. fruticosa had similarly high slimes. All species except A. semibaccata on veld and

Table 2 Mycorrhizal colonization of 5 host species (mean±S.E.) and the relative proportions of intra-radical mycorrhizal structures across broad substratum types Plant species Asclepias Atriplex Cynodon Phytolacca Asparagus All species Probability values-two-way ANOVA: fruticosa semibaccata dactlyon octandra laricinus Host species (H) Substratum (S) H⁎S Slimes Total colonization (%) 30.1±5.4 a 25.5±7.0 a 33.8±7.3a 19.1±5.5a 44.1±9.8a 30.5±4.2 0.0255 0.0019 0.0203 Proportion arbuscules (%) 1.4±0.8b 1.8±1.8b 1.0±0.6b 0.0±0.0b 5.8±2.1a 2.0±1.0 0.0143 0.9743 0.7412 Proportion vesicles (%) 26.4±6.6a 29.1±7.1a 29.2±5.3a 24.0±8.9a 16.9±4.1 a 25.1±2.3 0.0255 0.2977 0.5021 Proportion hyphae (%) 72.3±6.8a 69.1±7.5a 69.8±5.4a 76.0±8.9a 77.3±4.6 a 72.9±1.6 0.0679 0.4754 0.2335

Slimes-polluted veld Total colonization (%) 45.1±3.3 a 11.4±6.2 b 21.9±7.1b 9.2±1.2b 58.8±6.2a 29.3±21.8 Proportion arbuscules (%) 0.3±0.3a 0.0±0.0a 5.3±4.2a 0.0±0.0a 5.1±3.6a 2.1±1.3 Proportion vesicles (%) 24.8±7.4 a 29.7±18.0 a 46.2±8.1 a 31.4±14.4 a 27.9±6.9 a 32.0±8.3 Proportion hyphae (%) 74.9±7.6 a 70.3±18.0 a 48.6±7.5 a 68.7±14.4 a 67.0±6.1 a 65.9±10.1

Veld Total colonization (%) 44.0±9.1a 72.7±1.9a 70.5±11.1a NP 51.8±5.6a 59.8±7.0 Proportion arbuscules (%) 0.0±0.0a 1.0±1.0a 3.4±1.9a . 7.3±4.2a 2.9±1.6 Proportion vesicles (%) 9.0±4.2b 62.8±6.2a 43.8±9.1a . 15.9±7.5b 32.9±12.5 Proportion hyphae (%) 91.0±4.2a 36.2±6.1c 52.9±8.4bc . 76.8±9.7ab 64.2±12.2 Different superscripts per row are significantly different (SNK, pb0.05) and significant p values (two-way ANOVA) are in bold. NP=not present. 222 C.J. Straker et al. / South African Journal of Botany 73 (2007) 218–225

C. dactylon on slimes-polluted soil tended to have a lower Table 4 proportion of vesicles to hyphae on all substrata. Results of two-way ANOVA (probability values) for the effects of host species and slimes dam zone (lower-mid slopes, retaining walls, toepaddocks and Two-way ANOVA analysis (host species and slimes dam age adjacent veld) on mycorrhizal colonization and relative proportions of intra- category) showed significant differences between host species radical mycorrhizal structures and history class for percentage colonization (Table 3), but no Host species Zone Host⁎Zone interactive effects. On RV and OV dams, A. laricinus had the highest percentage colonization, whereas on NV dams both Recently-vegetated Total colonization (%) 0.3110 0.1141 0.2212 A. fruticosa and A. laricinus had the highest percentage colo- Proportion arbuscules (%) 0.6525 0.7014 0.6430 nization. Whereas there were no significant differences in per- Proportion vesicles (%) 0.0887 0.5723 0.6184 centage colonization between vegetation history classes for Proportion hyphae (%) 0.2576 0.9380 0.4333 A. fruticosa and A. semibaccata, there was an overall trend to highest levels on RV sites. There was a significant decline in Old-vegetated Total colonization (%) 0.0400 0.0001 0.0177 percentage colonization in C. dactylon, P. octandra and Proportion arbuscules (%) 0.1012 0.8630 0.8754 A. laricinus in the order RVNOVNNV sites. With the exception Proportion vesicles (%) 0.5414 0.0184 0.0887 of A. semibaccata and P. octandra, the proportion of arbuscules Proportion hyphae (%) 0.7762 0.0837 0.0206 was higher in the vegetation of OV sites, although not significantly so. In contrast, the proportion of vesicles tended Never-vegetated Total colonization (%) 0.6909 0.0001 0.0049 to be higher (n.s) on RV sites for most species except Proportion arbuscules (%) 0.3635 0.4937 0.6716 C. dactylon which showed significantly higher levels on NV Proportion vesicles (%) 0.0872 0.5141 0.5003 sites. Overall, the apparently most mycotrophic species were Proportion hyphae (%) 0.0779 0.2741 0.5942 A. laricinus and A. fruticosa (in RV, OV and NV slimes and Significant p values are in bold. slimes-polluted substrata) and in the veld, C. dactylon and A. semibaccata (Tables 2 and 3). interactive effects on RV sites. In contrast, two-way ANOVA At a finer scale, data were also analyzed for RV, OV and NV demonstrated significant differences between host species and sites individually (Table 4). Two-way ANOVA indicated no zone for % colonization, and between zones for the proportion significant differences between host species and zones and no of vesicles, on OV sites. On NV sites, two-way ANOVA

Table 3 Mycorrhizal colonization (means±S.E.) of five host species and the relative proportions of intra-radical mycorrhizal structures on slimes of different age categories Recently-vegetated (RV) Old-vegetated (OV) Never-vegetated (NV) Probability values–two way ANOVA Host (H) Age Class (A) H⁎A Asclepias fruticosa: Total colonization (%) 49.6±3.3a 32.6±5.1a 31.6±9.2a 0.0001 0.0001 0.4160 Proportion arbsucules (%) 0.0±0.0a 1.7±0.85a 0.0±0.0a 0.4095 0.3864 0.6932 Proportion vesicles (%) 26.7±6.0a 27.4±7.4a 20.8±11.5a 0.2315 0.1219 0.2336 Proportion hyphae (%) 73.2±6.0a 70.9±7.7a 79.2±11.5a 0.1298 0.1867 0.2607

Atriplex semibaccata: Total colonization (%) 31.9±10.3 a 9.3±3.3a 15.9±7.9a Proportion arbuscules (%) 2.3±2.3a 0.0±0.0a 0.0±0.0a Proportion vesicles (%) 47.8±12.0a 7.8±5.3a 22.0±16.8a Proportion hyphae (%) 49.9±11.9 ab 92.2±5.3 a 78.0±16.8ab

Cynodon dactlyon: Total colonization (%) 49.0±13.2a 24.2±5.6 b 16.0±9.1 b Proportion arbuscules (%) 0.63±0.63 a 3.3±2.5 a 3.3±3.3 a Proportion vesicles (%) 34.9±8.6 b 29.8±5.7 b 64.0±6.5 a Proportion hyphae (%) 64.5±8.3 a 67.0±5.8 a 32.7±9.5 a

Phytolacca octandra: Total colonization (%) 37.7±15.0 a 15.7±6.1 b 8.9±2.3 b Proportion arbuscules (%) 0.0±0.0 a 0.0±0.0 a 0.0±0.0 a Proportion vesicles (%) 34.8±18.2 a 24.7±9.3 a 23.9±14.5 a Proportion hyphae (%) 65.2±18.2 a 75.3±9.3 a 76.1±14.5 a

Asparagus laricinus: Total colonization (%) 77.8±6.3 a 54.2±7.2 a 22.7±12.2 b Proportion arbuscules (%) 2.9±2.0 a 8.2±3.0 a 0.0±0.0 a Proportion vesicles (%) 28.5±7.7 a 19.7±3.8 a 21.2±19.0 a Proportion hyphae (%) 68.6±7.3 a 72.1±3.5 a 78.8±19.0 a Different superscripts per row are significantly different (SNK, pb0.05) and significant p values are in bold (results of two-way ANOVA). C.J. Straker et al. / South African Journal of Botany 73 (2007) 218–225 223

in infection parameters between host species, zones and pH were found with significant interactions (Tables 2 and 3). A critical biological determinant of infection by AMF is the degree of host mycotrophy. For example, in tall grass prairie, it is the perennial warm-season C4 grasses and forbs which benefit significantly from mycorrhizal symbiosis, whereas biomass production of the (less-highly colonized) cool-season C3 grasses is unaffected (Wilson and Hartnett, 1998). Furthermore, annuals in this system are generally less responsive to mycorrhizal colonization than the perennials and also less colonized. In the present survey, all the plant species sampled were perennials, and could be grouped on the basis of rooting system morphology — an accepted regulator of mycotrophy (Smith and Fig. 2. AMF spore density (means±S.E.) in different zones of (■) recently- vegetated slimes, (▲) old-vegetated slimes and (●) never-vegetated slimes. Inset Read, 1997), with coarse-rooted species of wide diameter represents average values from all vegetation age/class slimes. appearing to be more mycotrophic (Fitter and Merryweather, 1992). Based on root morphology, and on the observations that showed significant differences between zones for % coloniza- shorter-lived perennial and pasture species are less mycotrophic tion, and significant interactions between host species and NV (van der Heijden, 2002), A. laricinus and A. fruticosa would be zone for % colonization. expected to be the most mycotrophic hosts in the less favourable growing environment of the slimes and slimes-polluted 3.4. AMF spore density substrata. The data support this supposition in that A. laricinus has the highest colonization levels in both slimes and slimes- Overall, the density of spores was highest on toepaddocks, polluted veld followed by A. fruticosa on slimes-polluted veld decreasing with distance up the slope until flat surfaces of slimes (Table 2). However, in ecosystem studies there is generally a were reached, where spore density again increased, approaching great range in mycorrhizal infection which appears at all scales that of the toepaddocks (Fig. 2). However, this overall pattern was of observation. Each plant species has its own infection strongly biased by the extremely high spore densities on NV potential and even closely related plant species support different toepaddocks (Fig. 2). In contrast, upon RV and OV dams the degrees of mycorrhizal infection when growing together (Allen density of spores was similarly very low across all zones, only and Allen, 1992b; Fitter and Merryweather, 1992; Vanden- increasing slightly on the flat tops of OV dams (the tops of RVand koornhuyse et al., 2003). Allen and Allen (1992b) assessed a NV dams were not assessed as either no vegetation or extensive single plant species to be colonized by up to 15 AMF species in reedbeds were present). However, two-way ANOVA demonstrat- a single transect whereas Vandenkoornhuyse et al. (2003), using ed no significant differences in spore density between host species molecular techniques, estimated an average of six AMF species and broad substratum, nor any interactions between the two. in three grassland plants with, however, distinctly different There were no significant differences in spore density between AMF communities in individual host plants. Distribution of sloped and flat slimes, or sites of different vegetation history class infection may thus be fractal, with a pattern that is complex and or any interactions between these (three-way ANOVA). apparently stochastic, but which may be governed by strong deterministic processes (Fitter and Merryweather, 1992). 4. Discussion Vandenkoornhuyse et al. (2003) found that additions of nitrogen and lime had a marked effect on the composition of the plant In general, a pattern of highest levels of total colonization community which changed the overall fungal community al- and proportion of arbuscules was observed in veld vegetation though without change in the fungi colonizing each plant. Thus, (Table 2). Some host-AMF combinations exhibited apparent environmental gradients constitute deterministic variables and survivorship distributions across slimes dams of different vege- influence root growth and the fungi themselves. Chemically, tation history classes, and these distributions may be associated there is a strong gradient which exists on slimes dams with clear with the type of host (Tables 2 and 3). For example, the more differences between zones in the levels of organic matter, short-lived herbaceous hosts P. octandra and C. dactylon were, acidity, conductivity, P and Ca (Table 1) and micro- and similarly to all other species, more mycotrophic on recently potentially toxic elements (Witkowski and Weiersbye, 1998b). ameliorated (RV) slimes, but in contrast to the perennial woody All of these factors would impinge on the distribution of AMF hosts, exhibited significant declines in AM infection levels on and act as selective forces favouring only those strains from the less ameliorated OVand NV sites (Tables 2 and 3). On RV sites, veld populations which are able to survive and propagate on the where rehabilitation treatments had created more uniform slimes. Veld populations of AMF would therefore be more chemical characteristics across zones, no differences in infection diverse than those of slimes-polluted soils and slimes dams. A parameters between host species or zones were found. However, higher diversity of AMF would facilitate the simultaneous on NV sites where no such alteration of chemical characteristics infection of veld plants by a wider range of strains and this had been imposed, and on OV sites, which would have reverted situation could be manifested as higher total percentage to chemical gradients similar to NV sites, significant differences colonization. Physical conditions (steepness of slope angle 224 C.J. Straker et al. / South African Journal of Botany 73 (2007) 218–225 and erosion.) on slimes slopes would also militate against the homogeneous slopes would reduce AMF refugia and increase retention of both plant hosts and AM inoculum, also reducing AMF mortality, thus inhibiting the establishment of an effective the pool of AMF able to colonize the remaining plants. Another infection network on these dams and contributing to the observed biological determinant of AM distribution and levels may relate low levels of root infection. to the differences in host plant size and disjunction in pheno- The slimes-polluted soils around the base of the dams phase that occurred between conspecific hosts over small (toepaddocks) contained the highest density of AMF spores distances on the sites surveyed. Although we harvested plants of (Fig. 2). Spore counts are not always a reliable measure of similar size and phenophase, the generally unfavorable condi- mycorrhizal status because of their often “patchy” distribution, tions for growth on slimes leads to plants being smaller than leading to high variances, and because an AMF may be an their conspecifics on the adjacent veld, whereas in toepaddocks effective root colonizer and symbiont but a poor sporulator it is higher levels of water and nutrients often results in plants being then under-represented in a spore count. Nevertheless, spore larger (Weiersbye et al., 2006). Since reproduction in plants is counts are a useful marker of the activity of AMF in a system size-dependent, plants on slimes can be expected to exhibit a especially in successional studies. For example, on surface coal delayed onset of first reproduction (i.e. longer juvenile period). mines there was a succession of spore populations after reclama- Since the costs of reproduction may divert carbon from the tion, with species richness low soon after reclamation but roots, AM infection levels in reproductive plants could be lower increasing erratically over 5 years and stabilizing at about 10 than those in pre- and post-reproductive plants. species (Gould and Hendrix, 1998). A pattern of veldNtoepaddocksNtopNretaining wallsNslopes This survey is a comparative indicator of the mycorrhizal was seen throughout the study, with the measured indicators of status of slimes dams and slimes-polluted soil versus that of AM infection and reproduction on dam tops approaching those of unpolluted soil late in the growing season of the vegetation and toepaddocks and veld. This strongly indicates that the flat tops it does not attempt to describe the biology of the symbiosis. and berms (flat areas, 4–8 m wide around the dam that reduces Current studies involve the isolation and assessment of the the length of slopes; Fig. 1) of dams are a relatively favourable potential of the AM inoculum present in these polluted sites to zone for AMF, probably due to the semi-permanent nature of infect plants and produce a positive effect on host plant growth organic matter and hosts, and possibly also the dominance of the in slimes. It is also not valid to directly compare total spore vegetation by perennial herbaceous, semi-woody and woody production in the rhizosphere of a host species on and off-slimes species rather than grasses (Weiersbye et al., 2006). The over- as the same host may be in distinctly different phenological riding factor affecting AMF communities on slimes therefore phases. However, levels of spore viability may be a more useful appears to be a combination of the physical and chemical indicator of the regeneration potential of the AMF community at constraints inherent in colonizing and persisting on steep any one time and these are being assessed in a follow-up study. erodable slopes on which safe-sites (organic matter, host plants) Given the low AM status of the slimes dams indicated by this are temporary and on RV at least, the hosts consist of facultative study, and the fact that on vegetated slimes dam slopes, the and non-mycotrophic pasture species. The high levels of P on RV AMF infection levels decline once amelioration ceases and the sites may also have contributed to the relatively lower proportion community of pasture species collapses, the introduction of of arbuscules and higher proportion of vesicles seen in most appropriate (ie slimes-tolerant) AMF inoculum during vegeta- roots. Despite the many constraints to natural colonization of NV tion establishment appears to be vital to hasten the succession to slopes by hosts and microbes, there was evidence of AM infec- dominant perennial mycotrophs and so promote more stable tion and these can be considered to be extremely tolerant host- plant communities on these sites (Johnson, 1998; van der AM combinations. AMF can rapidly invade disturbed sites, Heijden et al., 1998). In addition to enhancing nutrient acqui- primarily by wind but also by animals (Allen and Allen, 1992a) sition, AMF may facilitate host growth in polluted soil substrata but NV slopes would be unattractive to biotic dispersal agents. It by contributing to pollutant immobilization, as the vesicles and is therefore not surprising that the unameliorated sites with arbuscules of AMF in C. dactlyon from South African gold and extreme chemical and physical constraints to both plant and uranium tailings have a higher affinity for radionuclides and microbial immigration and survival should show the lowest heavy transition metals than the surrounding root tissues levels of AM. Spore density and species diversity of AMF can be (Weiersbye et al., 1999). The continual inundation by slimes logarithmically related to size of individual plants, with either onto soils around the base of slimes dams would have subjected larger shrubs (or clumps of shrubs) having greater trapping the component biota to intensive selection pressures for abilities or a greater variety of habitats within a root system to decades, and this study has demonstrated that these substrata enhance local spore production (Allen and Allen, 1992a). In are sites of active AMF reproduction from which suitable AMF colonization of disturbed habitats, there is an initial establishment can be isolated for use in inoculum production for slimes dams phase (initial spore trapping, then development of a hyphal rehabilitation in South Africa. network) and subsequent sporulation. The development of a runner hyphal network from invading spores in initiating new Acknowledgements infections has been shown to be extremely important (Allen and Allen, 1992a). The non-vegetated dams had the most bare- This study was funded by Anglo American Corporation ground and least aerial cover and litter (Table 1). The presence of (1997–1998) and then AngloGold Ltd as part of our ongoing only small, sparse and widely dispersed vegetation patches on programme: Containment of Pollution from Gold and Uranium C.J. Straker et al. / South African Journal of Botany 73 (2007) 218–225 225

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