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Accumulation of mineral elements in the rhizosphere and shoots of
Cyclopia and Aspalathus species under different settings of the
Cape fynbos
S.T. Maseko
a
,F.D.Dakora
b,
⁎
a
Department of Crop Sciences, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa
b
Department of Chemistry, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa
abstractarticle info
Available online 19 October 2016
Edited by E Joubert
Several species of the genus Cyclopia are cultivated for the production of Honeybush tea, largely without mineral
fertilization. However, very little is known about the effect of annual harvesting, plant age, and type of planting
material (cuttings vs. seedlings) on the mineral nutrition of Cyclopia. The aim of this study was to evaluate
mineral nutrition in Cyclopia genistoides,Cyclopia subternata,Aspalathus caledonensis,andAspalathus
aspalathoides in relation to (i) plant species, (ii) plant age, (iii) farmer's practice, (iv) planting material, and (v)
toposequence at Koksrivier, Kanetberg, and Kleinberg in the Cape fynbos. A comparison of mineral concentra-
tions in the rhizosphere and non-rhizosphere of 10-year-old C. genistoides at Koksrivier revealed significantly
larger levels of P, Ca, Mg,Cu, Zn, and Mn in the formerrelative to the latter. There were also significantly greater
levels of P, K, Ca,Mg, Na, and Mn in the rhizosphereof 10-year-old C.genistoides compared with 2-year-old plants
at Koksrivier. The levels of P, K, Ca, Mg, Na, Fe, and Mn were significantly greater in the rhizosphere than non-
rhizosphere soil of 5- and 8-year-old C. subternata plants at Kanetberg. Rhizosphere concentration of minerals
were also measured and compared for C.subternata plants raised from cuttings and seedlings at Kanetberg,
and P, Ca, and Cu were greaterin the rhizosphere of plants cultured from cuttings. The concentration of minerals
in the rhizosphere of A. aspalathoides,A. caledonensis,andC.genistoides, which co-occurred within the same tea
plantation at Koksrivier, were significantly different, with P, K, Cu, Zn, and Mn being markedly greater in the
rhizosphere soil of C.genistoides than the two Aspalathus species. Mineral nutrition under farmers' practice of
annual harvesting was compared with unharvested material, and the levels of P, K, Na, Cu, Zn, and Mn were
found to be significantly greater in shoots of the annually harvested plants.
© 2016 Published by Elsevier B.V. on behalf of SAAB.
Keywords:
Rhizosphere
Bulk soil
Cuttings
Toposequence
Koksrivier
Kanetberg
Kleinberg
1. Introduction
The genus Cyclopia consists of 24 species, which occur naturally on a
range of habitats, including mountain peaks, coastal plains, marshy
areas, shale bands, and along perennial streams of the fynbos biome in
the Western and Eastern Cape Provinces of South Africa (Esler et al.,
2014; SAHTA, 2014). Cape fynbos consists of Mountain and Coastal
soils, with the latter underlain by softer sediments of sandstones
(Mitchel et al., 1984; Cowling and Procheş, 2005), and the former
derived from weathering of quartzitic sandstones and fine-grained
shales (Campbell, 1983; Cowling and Procheş,2005). The Cape region
is windy and therefore frequently receives aeolian dust, which is an
important source of nutrients. According to Soderberg and Compton
(2007), dust deposits into the Cape fynbos represents a significant
source of K, Ca, and Zn. Rainwater is also reported to provide nutrients
such as Ca, K, P, Fe, Mn, and Zn from washout of marine and mineral
aerosols (Soderberg and Compton, 2007). Marine aerosols are reported
to supply the fynbos ecosystem with Cl, Na, Mg, SO
4
, and K, while
deposition of local and regional fire ash dust (mineral aerosol) can con-
tributeanadditionalloadofMg,SO
4
and K (Berg and Compton, 2015).
Despite these atmospheric sources of nutrients, soils of the fynbos are
inherently very low in nutrients due largely to their origin from ancient,
weathered, sandstones and shales (Goldblatt and Manning, 2002). Thus,
plants growing on such nutrient-poor soils can be limited by P (Power
et al., 2010), or co-limited by N and P (Maistry et al., 2013). The fynbos
species have therefore developed various mechanisms for enhancing
nutrient uptake, which includes relatively slow growth rates, and
hence low nutrient demand (Abrahamson, 2007; Orians and Milewski,
2007). Such plants also allocate new biomass to the organs that are
involvedin acquiring the most scarce resources such as water and nutri-
ents (e.g., roots) and therefore have a high root/shoot ratio (Marschner,
1995; Poorter and Nagel, 2000). In so doing, they allocate fewer re-
sources to shoot growth because of the proportionally greater allocation
South African Journal of Botany 110 (2017) 103–109
⁎Corresponding author.
E-mail address: DakoraFD@tut.ac.za (F.D. Dakora).
http://dx.doi.org/10.1016/j.sajb.2016.09.007
0254-6299/© 2016 Published by Elsevier B.V. on behalf of SAAB.
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
to functions that improve survival in harsh environments such as the
Cape fynbos (Power et al., 2010; Magadlela et al., 2014).
Of the 24 Cyclopia species, C.genistoides and Cyclopia subternata were
the first to be used for making honeybush tea, an herbal beverage with
many health benefits. Honeybush tea is however now also made from
C. intermedia,C. maculata,C. longifolia, and C. sessiliflora. In fact, about
230 ha are currently under cultivation to tea-producing Cyclopia species
(McKay and Blumberg, 2007; SAHTA, 2014). These plantations are either
established from seeds or cuttings. In 2012, the Honeybush tea industry
was estimated to contribute about twelve million Rands annually to the
South African economy (DAFF, 2013) from exports of 150 to 300 tons be-
tween 2005 and 2009 alone (DAFF, 2013). Extracts from honeybush tea
are used in the production of ready-to-drink beverages, fruit juice mix-
tures, sweets, and in the cosmetic industry (Van Wyk, 2011). The tea is
rich in antioxidants, low in tannins and considered caffeine-free
(Joubert et al., 2011). As a result, it is consumed hugely by health-
conscious individuals. By 2006, honeybush tea consumption was report-
edly increasing at an annual rate of 15–20% (McKay and Blumberg,
2007), a clear indication of a fast-growing market for the product.
So far, Cyclopia plants are cultivated organically, with their growth
and development largely dependent on endogenous soil minerals and
organic matter of the fynbos. Field observations of changes in soil prop-
erties of other perennial tea plants such as Camellia sinensis L. suggest
nutrient depletion after prolonged cultivation (Dang, 2005). A recent
study by Chimphango et al. (2015) reported no decreases in soil P, K,
Mg and Ca, and C in cultivated when compared to uncultivated control
plots of Aspalathus linearis grown organically over a 5-year-period in the
Nieuwoudtville area of the Cape fynbos. However, mineral nutrients
tend to be higher in the rhizosphere relative to bulk soil (Makoi et al.,
2014) due to symbiotic N
2
fixation (Muofhe and Dakora, 1999;
Spriggs and Dakora, 2008; Lötter et al., 2014), cluster root formation
(Lamont, 2003; Maseko and Dakora, 2013a), root exudation of organic
acids (Dakora and Phillips, 2002; Suriyagoda et al., 2010), modification
of rhizosphere pΗ(Muofhe and Dakora, 2000), and plant secretion of
phosphatases (Makoi et al., 2010a; Maseko and Dakora, 2013b). Even
though 48% to 61% of N, P, K, Ca, and Mg were removed in shoots of
Cyclopia genistoides with continuous harvesting without fertilizer sup-
plementation (Joubert et al., 2010b), changes in the mineral concentra-
tions of the rhizosphere and non-rhizosphere soil of Cyclopia have not
yet been explored. Whether annual harvesting and other agronomic
farmer practices deplete soil minerals in Cyclopia plantations, therefore
remains to be directly assessed.
Honeybush tea farmers generate their plants from both seeds and
cuttings. However, Joubert et al. (2010a) found variation in mineral
accumulation in C. subternata established from seeds and cuttings,
with plants from cuttings exhibiting higher K, Mn, and Cu compared
to P, Na, and Zn in seed-raised plants. Maseko and Dakora (2013a)
also found a markedly higher shoot P in cuttings than seedlings of
C. subternata at the same age.
Knowledge of mineral accumulation in the rhizosphere soil and
shoots of all Cyclopia tea species grown with or without fertilization
could lead to better decision-making aimed at mitigating against
possible nutrient mining and soil nutrient depletion, which can nega-
tively affect growth of the tea industry, tea exports, and the economy
and the fynbos ecosystem.
2. Materials and methods
2.1. Description of study sites
The study was carried out at Koksrivier (33° S 18° E, 39 m.a.s.l,
with mean annual rainfall of 661 mm concentrated in the winter
Table 1
Macro- and micronutrient concentrations in rhizosphere and bulk soils of 10-year-old and 2-year-old C. genistoides plants sampled at Koksrivier, Gansbaai, 8-year-old and 5-year-old
C. subternata from Kanetberg farm as well as 2-year-old C. subternata from Kleinberg farm, South Africa. Mean ± SE in same column with dissimilarletters are significant at p≤0.05.
Treatment P K Ca Mg S Na Fe Cu Zn Mn B pH
mg kg
−1
Koksrivier: 10-year-old C.genistoides
Rhizosphere 12 ± 0.8a 33 ± 0.9a 515 ± 23a 100 ± 0.8a 2.62 ± 0.1a 38 ± 3.4a 21 ± 1.1b 0.07 ± 0.0a 0.18 ± 0.0a 0.81 ± 0.1a 0.25 ± 0.0b 4.25
Bulk 4 ± 0.7b 31 ± 2.0a 418 ± 26b 80 ± 3.8b 2.63 ± 0.1a 39 ± 4.3a 46 ± 1.6a 0.04 ± 0.0b 0.11 ± 0.0b 0.14 ± 0.0b 0.45 ± 0.0a 4.48
F-statistics 59.52*** 0.37
ns
7.98** 24.59*** 0.00
ns
0.008
ns
177.1*** 7.81** 23.20*** 95.68*** 20.10***
Koksrivier: 2-year-old C.genistoides
Rhizosphere 7 ± 0.6a 25 ± 3.3a 337 ± 20a 79 ± 5.7a 3.58 ± 0.2a 26 ± 3.3a 37 ± 4.2a 0.06 ± 0.0a 0.20 ± 0.0a 0.43 ± 0.1a 0.26 ± 0.0a 4.32
Bulk 4 ± 0.7b 13 ± 2.1b 275 ± 24a 52 ± 10.1b 2.36 ± 0.2b 12 ± 1.5b 21 ± 1.8b 0.03 ± 0.0b 0.10 ± 0.0b 0.10 ± 0.1b 0.21 ± 0.0a 4.48
F-statistics 8.53* 9.47* 3.76
ns
5.58* 13.53** 14.88*** 12.57** 7.04* 11.07* 5.36* 1.04
ns
Koksrivier: 10-year-vs 2-year-old C.genistoides
Rhizosphere 12 ± 0.8a 33 ± 0.9a 515 ± 23a 100 ± 0.8a 2.62 ± 0.1b 38 ± 3.4a 21 ± 1.1b 0.07 ± 0.0a 0.18 ± 0.0a 0.81 ± 0.1a 0.25 ± 0.0a 4.25
Rhizosphere 7 ± 0.6b 25 ± 3.3b 337 ± 20b 79 ± 5.7b 3.58 ± 0.2a 26 ± 3.3b 37 ± 4.2a 0.06 ± 0.0a 0.20 ± 0.0a 0.43 ± 0.1b 0.26 ± 0.0a 4.32
F-statistics 23.33*** 5.16* 34.09*** 13.27** 14.91** 6.24** 13.45** 0.31
ns
0.28
ns
6.45** 0.06
ns
Kanetberg: 8-year-old C.subternata
Rhizosphere 20 ± 1.8a 77 ± 8.2a 423 ± 52a 136 ± 20a 3.94 ± 0.4a 67 ± 6.1a 142 ± 9.3a 0.09 ± 0.0a 0.27 ± 0.1a 1.47 ± 0.2a 0.31 ± 0.0a 4.13
Bulk 15 ± 1.4b 47 ± 4.6b 262 ± 20b 101 ± 22b 4.27 ± 0.4a 43 ± 2.1b 103 ± 4.1b 0.11 ± 0.0a 0.22 ± 0.0a 0.76 ± 0.2b 0.36 ± 0.0a 4.20
F-statistics 6.14* 10.42* 8.30* 7.09* 0.34
ns
13.75** 14.79*** 0.58
ns
0.54
ns
6.05* 2.19
ns
Kanetberg: 5-year-old C.subternata
Rhizosphere 13 ± 1.1a 135 ± 10a 156 ± 16a 48 ± 3.9a 3.92 ± 0.4b 54 ± 3.2a 85 ± 9.5a 0.08 ± 0.0a 0.29 ± 0.1a 0.44 ± 0.0a 0.23 ± 0.0a 4.17
Bulk 6 ± 0.7b 38 ± 5b 79 ± 1.3b 31 ± 3.6b 5.42 ± 0.6a 19 ± 1.0b 63 ± 2.7b 0.05 ± 0.0b 0.13 ± 0.0a 0.02 ± 0.0b 0.25 ± 0.0a 4.20
F-statistics 29.97*** 71.48*** 22.37*** 10.66* 5.12* 108.69*** 5.20* 22.00*** 2.82
ns
23.95*** 0.33
ns
Kanetberg: 8-year-vs 5-year-old C.subternata
Rhizosphere 20 ± 1.8a 77 ± 8.2b 423 ± 52a 136 ± 20a 3.94 ± 0.4a 67 ± 6.1a 142 ± 9.3a 0.09 ± 0.0a 0.27 ± 0.1a 1.47 ± 0.2a 0.31 ± 0.0a 4.13
Rhizosphere 13 ± 1.1b 135 ± 10a 155 ± 16b 59 ± 6b 3.92 ± 0.4a 54 ± 3.2a 79 ± 7.2b 0.08 ± 0.0a 0.29 ± 0.1a 0.46 ± 0.0b 0.23 ± 0.0b 4.17
F-statistics 11.78** 19.09*** 23.91*** 53.56*** 0.00
ns
3.69
ns
29.13*** 0.59
ns
0.04
ns
27.20*** 13.24**
Kleinberg: 2-year-old C.subternata
Rhizosphere 8 ± 0.4a 52 ± 3.7a 457 ± 3.7a 198 ± 11a 2.80 ± 0.1a 76 ± 4.7b 297 ± 15b 0.14 ± 0.0a 0.95 ± 0.1a 0.77 ± 0.8a 0.24 ± 0.0b 4.42
Bulk 5 ± 0.4b 17 ± 1.6b 538 ± 40a 192 ± 16a 2.35 ± 0.2a 134 ± 14a 437 ± 12a 0.03 ± 0.0b 0.44 ± 0.0b 0.26 ± 0.3b 0.39 ± 0.0a 4.82
F-statistics 27.00*** 73.17*** 4.14
ns
0.07
ns
4.26
ns
14.65* 50.71*** 512.00*** 14.42** 80.93*** 9.26*
104 S.T. Maseko, F.D. Dakora / South African Journal of Botany 110 (2017) 103–109
from months of May to September) and Kanetberg (33° S 21° E, 830
m.a.s.l with mean annual rainfall of 564 mm concentrated during the
winter months of May to September). The tea plants at Koksrivier
were grown in natural settings on sandy soils without ploughing,
fertilization, or irrigation while the two Aspalathus species grew along
the Cyclopia, without having being planted. At Kanetberg, C. subternata
and C. longifolia were established from cuttings taken from superior
mother plants and grown without fertilization on sandy to sandy-
loam soils.
2.2. Soil and plant analyses
A minimum of 5 and maximum of 10 soil samples (0–40 cm depth)
were collected from each farm. The rhizosphere soil closely associated
with the roots (1 to 2 mm) of Cyclopia and Aspalathus was collected to-
gether with non-rhizosphere soil (free from roots) from in-between
rows of Cyclopia and Aspalathus plants. The samples were transferred
into pre-labeled plastic bags and transported to the laboratory, where
the soil was air-dried, sieved (2.0 mm), and analyzed for essential
mineral nutrients.
2.3. Determination of plant-available minerals in bulk and rhizosphere soil
Extractable P, K, Ca, Mg, and Na were determined by the citric acid
method as developed by Dyer (1894) and modified by the Division of
Chemical Services (1956) and Du Plessis and Burger (1964).Following
acid digestion, measurement of P, K, Na, Ca, and Mg were done
directly by aspiration on a calibrated simultaneous inductively coupled
plasma–mass spectrometer (ICP-MS) (IRIS/AP HR DUO Thermo
Electron Corporation, Franklin, Massachusetts, USA).
The determination of S and B in the soil was done by adding 20 g
of soil in 0.01 M Ca(H
2
PO
4
)2·H
2
O extracting solution (FSSA, 1974)
followed by filtering. Sulfur was determined by direct aspiration on a
calibrated simultaneous inductively coupled plasma (ICP) spectropho-
tometer (IRIS/AP HR DUO Thermo Electron Corporation, Franklin,
Massachusetts, USA).
The trace elements Cu, Zn, Mn, and Fe were extracted from soil
using di-ammonium ethylene-diaminetetraacetic acid (EDTA) solution
(Trierweiler and Lindsay, 1969, modified by Beyers and Coetzer,
1971). The extractants were analyzed for Cu, Zn, Mn, and Fe using
ICP-MS spectrometry (IRIS/AP HR DUO Thermo Electron Corporation,
Franklin, MA, USA).
2.4. Plant harvest and processing for shoot and soil nutrient element
analysis
At Koksrivier and Kanetberg farms, young shoots of 10 plants of
different age were harvested, oven-dried (60 °C), weighed, and milled
to a fine powder (0.85 mm sieve) for analysis of major and minor
nutrient elements.
2.5. Measurement of nutrient elements in plant shoots
Measurements of P, K, Ca, Mg, S, Na, Fe, Cu, Zn, Mn, and B
in shoots of Cyclopia and Aspalathus legumes were done using
ICP-mass spectrometry. About 1.0 g of finely ground sample
(0.85 mm) was ashed in a porcelain crucible at 500 °C overnight.
The ash was dissolved in 5 mL of 6 M HCl and placed in an oven
at 50 °C for 30 min, followed by adding of 35 mL deionized
water. The mixture was filtered through Whatman no. 1 filter
paper, and mineral concentration in plant extracts was measured
using inductively coupled plasma–mass spectrometry. The quality
of data collected was checked using standard solutions with certif-
icate of analysis. In place of analyte isotopes to monitor each
element, a known standard was used after every 10 samples.
Sulfur was determined by wet digestion procedure using 65%
nitric acid (high purity grade). In each case, 1 g of milled plant
material was digested overnight with 20 mL of 65% nitric acid in
a 250 mL glass beaker. The beaker containing the extract was
then placed on a sand bath and gently boiled until approximately
1 mL of the extract was left. After that, 10 mL of 4 M nitric acid
(high purity grade) was added and boiled again for 10 min. The
beaker was removed, cooled, and the extract washed completely
into a 100-mL volumetric flask. The extract was filtered through
a Whatman no. 2 filter paper, and the concentration of S in the
sample measured by direct aspiration on a calibrated ICP-MS
(FSSA, 1974).
Statistical analysis was carried out using a STATISTICA software pro-
gram, version 10 (StatSoft Inc., 2010). A 1-way ANOVA was performed
to compare the means of mineral nutrient concentrations in shoots of
each species, between and among years, between slope and type of
planting material. Where significant differences were found, the
Duncan multiple range test (DMRT) was used to separate the treatment
means at p≤0.05.
Table 3
Macro- and micronutrient concentrations in rhizosphere soils of C. subternata,A. aspalathoides and A. caledonensis plants established from Koksrivier, Gansbai, South Africa. Mean ± SE in
same column with dissimilar letters are significant at p≤0.05.
Treatment P K Ca Mg S Na Fe Cu Zn Mn B pH
mg kg
−1
C.genistoides 8 ± 0.24a 46 ± 6a 315 ± 17b 60 ± 6.5a 2.3 ± 0.36a 33 ± 3.2a 22 ± 3a 0.08 ± 0.02a 0.22 ± 0.03a 1.39 ± 0.3a 0.14 ± 0.0a 4.32
A.aspalathoides 5 ± 0.47b 31 ± 4b 316 ± 22b 77 ± 5.0a 3.4 ± 0.62a 26 ± 1.7a 18 ± 2a 0.02 ± 0.00b 0.12 ± 0.01b 0.20 ± 0.1b 0.13 ± 0.0a 4.31
A. caledonensis 2 ± 0.00c 19 ± 2b 434 ± 6a 68 ± 2.5a 2.3 ± 0.13a 31 ± 2.4a 15 ± 1a 0.03 ± 0.00b 0.12 ± 0.01b 0.04 ± 0.0c 0.12 ± 0.0a 4.01
F-statistics 87.60*** 12.64*** 17.80*** 2.76
ns
2.32
ns
1.70
ns
2.07
ns
11.94*** 6.79* 21.29*** 2.35
ns
Table 2
Macro- and micronutrient concentrations in rhizosphere soils of 5-year-old C. subternata plants established from seeds and cuttings at Kanetberg, Barydale, South Africa. Mean ± SE in
same column with dissimilar letters are significant at p≤0.05.
Treatment P K Ca Mg S Na Fe Cu Zn Mn B pH
mg kg
−1
Kanetberg: 5-year-old C.subternata
Seeds 13 ± 1.1b 135 ± 10a 156 ± 16b 59 ± 5a 3.92 ± 0.4a 54 ± 3.2a 79 ± 7.2a 0.08 ± 0.0b 0.29 ± 0.1a 0.46 ± 0.0a 0.23 ± 0.0a 4.12
Cuttings 24 ± 2.7a 98 ± 7b 451 ± 26a 60 ± 4a 3.98 ± 0.4a 60 ± 2.6a 80 ± 5.2a 2.29 ± 0.2a 0.22 ± 0.0a 1.64 ± 0.5a 0.19 ± 0.0a 4.17
F-statistics 13.99*** 8.68* 91.46*** 0.02
ns
0.01
ns
2.52
ns
0.02
ns
124.06*** 0.52
ns
4.94
ns
2.43
ns
105S.T. Maseko, F.D. Dakora / South African Journal of Botany 110 (2017) 103–109
3. Results
3.1. Mineral concentrations in the rhizosphere and non-rhizosphere soils of
Cyclopia species at three locations
A comparison of mineral concentrationsin the rhizosphere and non-
rhizosphere of 10-year-old C. genistoides at Koksrivier revealed signifi-
cantly larger levels of P, Ca, Mg, Cu, Zn, and Mn in the former relative
to the latter (Table 1). By contrast, Fe and B were greater in non-
rhizosphere bulk soil. Except for Ca and B, which showed similar
concentrations in both rhizosphere and bulk soils, the other mineral
elements (P, K, Mg, S, Na, Fe, Cu, Zn, and Mn) were markedly higher in
the rhizosphere of 2-year-old C. genistoides at Koksrivier than bulk soil
(Table 1). Soil analysis showed significantly greater levels of P, K, Ca,
Mg, Na, and Mn in the rhizosphere of 10-year-old C. genistoides
compared with 2-year-old at Koksrivier (Table 1). By contrast, S and
Fe were higher in the rhizosphere of the younger than older plants,
while rhizosphere levels of Cu, Zn, and B were unaltered by plant
age (Table 1).
The concentrations of P, K, Ca, Mg, Na, Fe, and Mn were significantly
greater in the rhizosphere than non-rhizosphere of 5- and 8-year-old
C. subternata plants at Kanetberg (Table 1). While Zn and B showed
similar levels for 5- and 8-year-old plants, S was higher in bulk soil
and Cu was greater in the rhizosphere of the 5-year-old but similar in
the 8-year-old C. subternata (Table 1). Except for K, which showed
increased concentration in the rhizosphere of 5-year-old C.subternata
plants, the levels of P, Ca, Mg, Fe, Mn, and B were all greater in the
rhizosphere of the 8- than 5-year-old plants at Kanetberg (Table 1).
However, rhizosphere levels of S, Na, Cu and Zn were unaltered by
plant age.
At Kleinberg, only P, K, Cu, Zn, and Mn revealed greater concentrations
in the rhizosphere than non-rhizosphere of 2-year-old C.subternata.By
contrast, Na, Fe, and B were much higher in bulk than the rhizosphere
soil of the 2-year-old C.subternata at Kleinberg (Table 1).
3.2. Mineral concentrations in the rhizosphere of C. subternata
cuttings/seedlings
Rhizosphere concentration of minerals were also measured and
compared for C.subternata plants raised from cuttings and seedlings
at Kanetberg (Table 2). Except for K which showed increased levels in
the rhizosphere of plants raised from seed, P, Ca, and Cu were greater
in the rhizosphere of plants cultured from cuttings, while the rhizo-
sphere concentrations of Mg, S, Na, Fe, Zn, Mn, and B were unaltered
by the type of planting material at Kanetberg (Table 2).
3.3. A comparison of mineral concentrations in the rhizosphere of
C. genistoides and two Aspalathus species
The concentration of minerals in the rhizosphere of Aspalathus
aspalathoides,Aspalathus caledonensis,andC.genistoides,whichco-
occurredwithin the same tea plantation at Koksrivier, weresignificantly
different. The levels of P, K, Cu, Zn, and Mn were markedly greater in the
rhizosphere soil of C.genistoides than the two Aspalathus species
(Table 3).However, P and Mn were also muchgreater in A.aspalathoides
than A. caledonensis. While Mg, S, Na, Fe, and B were similar in the
rhizosphere of the three test species, Ca was much higher in the
rhizosphere of A. caledonensis (Table 3).
3.4. Shoot mineral concentrations of C. genistoides and C. subternata in
different settings
Although shoot distributions of P, S, and Zn were unchanged by plant
age, the levels of K, Mg, Cu,and B were greater in 10- than 2-year-old C.
genistoides plants at Koksrivier (Table 4). By contrast, Ca, Na,Fe, and Mn
showed greater concentrations in the 2- than 10-year-old C.genistoides
plants (Table 4).
A similar study done on C.subternata at Kanetberg revealed signifi-
cantly increased concentrations of P, Ca, Mg, Fe, Zn, Mn, and B in shoots
of 7-year-old plants than their 4-year-old counterparts (Table 4). In fact,
shoot levels of P, Ca, Mg, Fe, and Mn were also greater in the 7- than
5- and 6-year-old plants. Only Na and Cu were significantly lower in
7-year-old relative to some or all of the other age groups.
Farmer's practice of annual harvesting was compared with
unharvested, and P, K, Na, Cu, Zn,and Mn were found to besignificantly
greater in shoots of the annually harvested plants (Table 5).Only Ca and
B were increased in shoots of unharvested plants.
When mineral nutrition of C.genistoides,A. caledonensis,and
A.aspalathoides co-occurring in a honeybush tea plantation was
compared at Koksrivier, A.aspalathoides showed increased levels of Ca,
Table 5
Effect of shoot harvesting on macro- and micronutrient concentration of 10-year-old C. genist oides planted at Koksrivier farm. Values (mean ± SE) in same column with dissimilar letters
are significant at p≤0.05.
Practice P K Ca Mg S Na Fe Cu Zn Mn B
mg g
−1
μgg
−1
Harvested 0.08 ± 0.0a 0.9 ± 0.0a 0.3 ± 0.0b 0.1 ± 0.0a 0.1 ± 0.0a 3275 ± 185a 58 ± 1.5a 2.6 ± 0.1a 14.2 ± 0.5a 20.2 ± 1.4a 30 ± 1.1b
Unharvested 0.06 ± 0.0b 0.6 ± 0.0b 0.4 ± 0.0a 0.1 ± 0.0a 0.1 ± 0.0a 1614 ± 76b 60 ± 2.9a 1.3 ± 0.1b 5.1 ± 0.5b 13.6 ± 1.0b 43 ± 1.8a
F-statistics 33.51*** 44.75*** 15.65*** 2.43
ns
4.20
ns
69.07*** 0.35
ns
72.91*** 169.05*** 13.60*** 36.20***
Table 4
Macro- and micronutrient concentration of young shoots of C. genistoides and C. subternata sampled at Koksrivier, Gansbaai, and Kanetberg, Barrydale, South Africa, in 2007. Values
(mean ± SE) in same column with dissimilar letters are significant at p≤0.05.
Plant species/age P K Ca Mg S Na Fe Cu Zn Mn B
mg g
−1
μgg
−1
Koksrivier: C.genistoides
10 0.9 ± 0.0a 9.0 ± 0.2a 3.1 ± 0.1b 1.5 ± 0.0a 0.9 ± 0.0a 3166 ± 92b 51 ± 1.7b 1.8 ± 0.1a 9.2 ± 0.3a 15 ± 0.7b 41 ± 0.7a
20.9 ± 0.0a 8.1 ± 0.3b 3.6 ± 0.1a 0.9 ± 0.0b 1.0 ± 0.0a 3773 ± 135a 58 ± 1.9a 1.5 ± 0.1b 10.4 ± 0.7a 37 ± 2.9a 35 ± 0.8b
F-statistics 0.00
ns
4.60** 12.30*** 132.19*** 1.41
ns
13.75*** 6.26** 4.89** 2.39
ns
54.47*** 27.13***
Kanetberg: C.subternata
7 1.9 ± 0.1a 8.6 ± 0.2b 3.0 ± 0.1a 2.0 ± 0.0a 0.8 ± 0.0c 1730 ± 45bc 90 ± 6.9a 10 ± 0.2bc 14 ± 0.7a 58 ± 7.9a 36 ± 1.1a
6 1.3 ± 0.1b 7.8 ± 0.3c 2.4 ± 0.1b 1.6 ± 0.1c 0.8 ± 0.0 cd 2325 ± 182a 86 ± 7.8bc 5 ± 0.6d 15 ± 0.6a 25 ± 1.9d 29 ± 0.4b
5 1.1 ± 0.0c 8.7 ± 0.2b 2.5 ± 0.1b 1.6 ± 0.0c 0.9 ± 0.0b 1500 ± 0.00c 46 ± 0.0c 13 ± 0.0a 15 ± 0.0a 36 ± 0.0bc 38 ± 0.0a
4 1.4 ± 0.0b 8.6 ± 0.2b 2.5 ± 0.0b 1.8 ± 0.0b 0.8 ± 0.0c 1738 ± 54b 70 ± 4.7c 10 ± 0.3c 8 ± 0.3b 28 ± 1.4 cd 28 ± 0.8b
F-statistics 27.90*** 14.96*** 12.80*** 26.19*** 21.56*** 13.33*** 5.47*** 80.66** 25.44*** 11.08*** 26.18***
106 S.T. Maseko, F.D. Dakora / South African Journal of Botany 110 (2017) 103–109
Mg, Na, Fe, and Mn in its shoots than C.genistoides, similarlevels of K, Fe,
and Mn but greater Ca, Mg, and Na to that of A. caledonensis (Table 6).
Except for K and B, C.genistoides showed lower distribution of all the
other minerals in its shoot.
At Kanetberg, topography was found to affect mineral distribution in
C.subternata (Table 7). Plants from the lower slope showed significantly
increased concentrations of Mg, S, Na, Fe, and Mn in shoots (Table 7).
Only Cu and B, and to some extent P, were higher in plants from the
upper and middle slopes (Table 7). The levels of K, Ca, and Zn in shoots
were unaffected by topography.
C. subternata plants raised from cuttings and seed material differed
in mineral distribution. The cuttings showed increased levels of P, S,
Cu, and Zn than seedlings, while those from seed accumulated more
Ca, Mg, Na, and Fe than cuttings (Table 8). The levels of K, Mn, and B
were unaffected by the type of planting material.
4. Discussions
Various factors including topography, plant species, plant age, type
of planting material, and farmer practices (e.g., annual shoot harvesting
for tea production) can affect the mineral nutrition of plants, and hence
their growth, productivity, and yield. Inthis study, the effect of these pa-
rameters on rhizosphere concentration of mineral elements and their
accumulation in shoots was assessed for the tea legumes, C. genistoides
and C.subternata. In general, the study revealed greater accumulation
of P, K, Ca, Mg, Cu, and Mn in the rhizosphere of C. genistoides and
C.subternata when compared to bulk soil. The test Cyclopia species
probably used various mechanisms including rhizosphere acidification
(Muofhe and Dakora, 2000), which led to increased mobilization and
accumulation of P, K, Mn, Ca, Mg, and Cu in the rhizosphere (Treeby
et al., 1989; Bertin et al., 2003). C. genistoides showed pH 4.25 in the rhi-
zosphere compared to pH 4.48 in bulk soil. Similarly, at Kanetberg,
C. subternata recorded pH 4.13 in rhizosphere compared to pH 4.20 in
non-rhizosphere bulk soil, while at Kleinberg, C. subternata showed
pH 4.42 in its rhizosphere relative to pH 4.82 in non-rhizosphere soil
(Table 1). These decreases in soil pH caused by proton release can influ-
ence the solubilization of P, K, Mn, Ca, and Mg in the rhizosphere
(Muofhe and Dakora, 2000; Rengel and Marschner, 2005; Hinsinger
et al., 2009) and hence their greater accumulation in the rhizosphere
of Cyclopia species. Furthermore, relative to non-rhizosphere bulk soil,
acid, and alkaline phosphatase activity was markedly higher in
rhizosphere soil of 10- and 2-year-old C. genistoides at Koksrivier,
8- and 5-year-old C. subternata at Kanetberg (Maseko and Dakora,
2013b), and 2-year-old C. subternata at Kleinberg. This increased
phosphatase activity could have contributed to enhanced P solubili-
zation and availability in the rhizosphere than bulk soil, as found in
this study.
Older plantations of C. genistoides and C.subternata that were
harvested annually for tea production showed higher accumulation of
P, K, Ca, Mg, Cu, and Mn in the rhizosphere compared to non-
rhizosphere bulk soils at Koksrivier and Kanetberg (Table 1). This was
more likely due to decomposition of plant roots, nodules, and fallen
leaves, as well as root exudates. Deeper rooting system can also permit
better uptake of nutrients from the topsoil and subsoil (Lynch and
Wojciechowski, 2015). The greater P concentration in the rhizosphere
of older C.genistoides and C.subternata plants relative to younger plants
(Table 1) could be due to the higher acid and alkaline phosphatase
activities in the rhizosphere of older Cyclopia plants than younger plants
(Maseko and Dakora, 2013b). However, the greater P in rhizosphere of
older Cyclopia plants could also be due to increased root exudation
and microbial numbers in the rhizosphere of older plants (Garcia
et al., 2001).
We also found that C.subternata plants established from cuttings
showed elevated levels of P in their rhizosphere compared to seedlings
(Table 2),and this matched an increase in acid and alkaline phosphatase
activity in the rhizosphere (Maseko and Dakora, 2013b), thus
suggesting that the greater rhizosphere P was caused by the greater
phosphatase activity. The increase in P-enzyme activity and hence P
availability in the rhizosphere resulted in enhanced P nutrition in plants
from cuttings, as well as in plants harvested annually for tea (Tables 8
and 5). The net result was an increase in symbiotic N nutrition and
better plant growth (Maseko, 2013; Maseko and Dakora, 2015a).
Joubert et al. (2010b) have reported higher accumulation of K, Mn,
and Cu but greater P, Na, and Zn in cuttings than C. subternata plants
raised from seeds. In our study at the Kanetberg mountains,
C. subternata plants raised from cuttings accumulated more P, S, Cu,
and Zn, while those from seed had more Ca, Mg, Na, and Fe. The greater
accumulation of P and Cu in the rhizosphere of C. subternata plants
raised from cuttings supports their higher uptake and accumulation in
plant shoots at Kanetberg (Tables 2 and 8). Similarly, the greater Fe
and Na in the rhizosphere soils of C. subternata plants raised from
seedlings (Tables 2)wasreflected in the higher accumulation of Fe
and Na in shoots of those plants.
At Koksrivier, C. genistoides co-occurred with two Aspalathus species.
Although C. genistoides revealed higher concentrations of P, K, Cu, Zn,
and Mn in the rhizosphere (Table 3), it was the Aspalathus species that
showed greater uptake and accumulation of P, Ca, Mg, Na, Fe, Cu, and
Mn in their shoots (Table 6). As a result, these species also differed
Table 7
Macronutrient concentrations of young C. subternata shoots sampled at Kanetberg, along a soil toposequence of 5-year-old plants. Values (mean ± SE) in same column with dissimilar
letters are significant at p≤0.05.
Topography P K Ca Mg S Na Fe Cu Zn Mn B
mg g
−1
μgg
−1
Upper 1.1 ± 0.0b 8.3 ± 0.1a 2.5 ± 0.1a 1.5 ± 0.0b 0.86 ± 0.0b 1500 ± 0b 46 ± 0.0b 13 ± 0.0a 15 ± 0.0a 36 ± 0.0b 38 ± 0.0a
Middle 1.2 ± 0.0a 8.6 ± 0.2a 2.3 ± 0.1a 1.5 ± 0.0b 0.90 ± 0.0ab 1500 ± 0b 46 ± 0.0b 13 ± 0.0a 15 ± 0.0a 36 ± 0.0b 38 ± 0.0a
Lower 1.1 ± 0.0b 8.7 ± 0.2a 2.6 ± 0.1a 1.7 ± 0.0a 0.94 ± 0.0a 1669 ± 60a 87 ± 6.9a 8 ± 0.4b 15 ± 0.7a 57 ± 4.6a 34 ± 0.9b
F-statistics 5.05** 1.89
ns
3.12
ns
14.52*** 4.11** 8.04** 33.83*** 138.41*** 0.13
ns
20.95*** 20.34***
Table 6
Macro- and micronutrient concentration in young shoots of C. genistoides,A. caledonensis and A. aspalathoides sampled from Koksrivier farm established in 2005. Values (mean ± SE) in
same column with dissimilar letters are significant at p≤0.05.
Species P K Ca Mg S Na Fe Cu Zn Mn B
mg g
−1
μgg
−1
C.genistoides 0.09 ± 0.0b 0.8 ± 0.0a 0.4 ± 0.0b 0.09 ± 0.0c 0.10 ± 0.0a 3773 ± 135b 60 ± 1.9b 1.5 ± 0.1b 10 ± 0.7a 37 ± 2.9b 35 ± 0.8a
A.caledonensis 0.12 ± 0.0a 0.6 ± 0.0b 0.4 ± 0.0b 0.16 ± 0.0b 0.08 ± 0.0b 2003 ± 109c 68 ± 1.7a 2.0 ± 0.1a 12 ± 0.8a 78 ± 4.1a 18 ± 0.5c
A.aspalathoides 0.07 ± 0.0c 0.6 ± 0.0b 0.6 ± 0.0a 0.19 ± 0.0a 0.10 ± 0.0a 6114 ± 161a 68 ± 3.5a 1.5 ± 0.1b 10 ± 0.6a 67 ± 5.6a 27 ± 0.4b
F-statistics 67.64*** 30.77*** 57.58*** 81.31*** 23.07*** 227.49*** 5.29** 12.99*** 2.79
ns
22.88*** 211.73***
107S.T. Maseko, F.D. Dakora / South African Journal of Botany 110 (2017) 103–109
significantly in their symbiotic functioning as A.aspalathoides obtained
about 91.06% ± 3.0% of its N nutrition from symbiosis, compared to
86.24% ± 2.7% by A. caledonensis and 73.71% ± 2.0% by C.genistoides
(Maseko andDakora, 2015b). Increased shoot concentration of minerals
such as P, Ca, Mg, Na, Fe, Cu, and Mn has been reported to enhance N
2
fixation in legumes (Makoi et al., 2010b), and this probably explains
the significantly higher percent N derived from fixation by the
Aspalathus species when compared to Cyclopia at the Koksrivier. In
general, plant species differ in the mechanisms employed for mineral
acquisition. For example, Maseko and Dakora (2013b) found that of
the three co-existing legume species at Koksrivier, there was a large
mass of cluster roots on A.aspalathoides, but with few (or none) on
C.genistoides and A. caledonensis. This could explain the greater shoot
concentration of P, Ca, Mg, Na, Fe, Cu, and Mn in the Aspalathus species.
The presence of cluster roots expands the surface-volume ratio in soils,
thus promoting the solubilization and uptake of Fe, Ca, P, Mn, and Zn
(Lamont, 2003).
The distribution of minerals in shoots of C. subternata at Kanetberg
was probably affected by management practices such as regular tea har-
vest and topography. Compared to plants not harvested, there were
greater levels of P, K,Na, Cu, Zn, and Mn in shoots of Cyclopia harvested
annually for tea production (Table 7), a finding consistent with the data
of Hawkins et al. (1999). While the abundance of these minerals in
shoots could improve tea quality and enhance its health benefits for a
higher market price, it has potentialfor nutrient mining. Thus, the annu-
al harvesting of Cyclopia for tea production has the potential to remove
more nutrient elements from the already fragile, nutrient-poor fynbos
soil. The net result would be wealth creation at the expense of the
environment, if no fertilizers are applied to Cyclopia tea plantations.
Variations in soil nutrient levels and shoot mineral distribution as a
result of topography are caused largely by runoff and underground
water movement, which create differences in nitrification, mineraliza-
tion, and moisture in soils of different toposequence (Stewart et al.,
2014; Zhang et al., 2014). In this study, there was greater concentration
of P in the middle slope, and higher levels of Mg, S, Na, Fe, and Mn at the
lower toposequence (Table 7). Clearly, plant growth and development
is strongly influenced by environmental factors such as toposequence
and farmer practice of frequent plant harvest for tea. While in general
nutrient uptake and assimilation by plants is a function ofmineral avail-
ability in the rhizosphere, the data from this study show that nutrient
supply in the rhizosphere does not always reflect uptake by roots. For
example, the concentrations of P, K, Ca, Na, Cu, and Mn in the rhizo-
sphere of C.subternata developed from cuttings were significantly
greater than those from seedlings (Table 2). However, greater levels of
only P and Cu (but not K, Ca, Na, or Mn) were found in shoots (Table 8).
Acknowledgments
The South African Research Chair in Agrochemurgy and Plant
Symbioses, the National Research Foundation, and the Tshwane
University of Technology are acknowledged for supporting the research
of FDD, and for providing a bursary to STM. We are grateful to the
farmers (Mr. Fritz Joubert and Mrs. Rica Joubert at Koksrivier, and
Mr. Matie Taljaard and Mrs. Erica Taljaard at Kanetberg) for allowing
us to collect plant and soil samples from their commercial Honeybush
tea plantations.
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