Physiologia Plantarum 153: 149–160.

2015 © 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317

Effects of simulated acid rain on germination, seedling

growth and oxidative metabolism of recalcitrant-seeded
Trichilia dregeana grown in its natural seed bank
Chandika Ramlall, Boby Varghese, Syd Ramdhani, Norman W. Pammenter, Arvind Bhatt,
Patricia Berjak and Sershen*

School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Durban 4001, South Africa

Correspondence Increased air pollution in a number of developing African countries, together

*Corresponding author, with the reports of vegetation damage typically associated with acid precip-
e-mail: naidoose@ukzn.ac.za
itation in commercial forests in South Africa, has raised concerns over the
Received 18 March 2014; potential impacts of acid rain on natural vegetation in these countries. Recal-
revised 10 April 2014 citrant (i.e. desiccation sensitive) seeds of many indigenous African species,
e.g. must germinate shortly after shedding and hence, may not be able to avoid
doi:10.1111/ppl.12230 exposure to acid rain in polluted areas. This study investigated the effects of
simulated acid rain (rainwater with pH adjusted to pH 3.0 and 4.5 with 70:30,
H2 SO4 :HNO3 ) on germination, seedling growth and oxidative metabolism in
a recalcitrant-seeded African tree species Trichilia dregeana Sond., growing in
its natural seed bank. The results suggest that acid rain did not compromise T.
dregeana seed germination and seedling establishment significantly, relative
to the control (non-acidified rainwater). However, pH 3.0 treated seedlings
exhibited signs of stress typically associated with acid rain: leaf tip necro-
sis, abnormal bilobed leaf tips, leaf necrotic spots and chlorosis, reduced
leaf chlorophyll concentration, increased stomatal density and indications of
oxidative stress. This may explain why total and root biomass of pH 3.0 treated
seedlings were significantly lower than the control. Acid rain also induced
changes in the species composition and relative abundance of the different
life forms emerging from T. dregeana’s natural seed bank and in this way could
indirectly impact on T. dregeana seedling establishment success.

Introduction dramatic increase in the quantities of sulfur dioxide and

Acid rain has been described as one of the most seri-
ous environmental threats associated with industrializa-
tion and, more recently, climate change (Glass et al.
1979, Fan and Wang 2000, Wyrwicka and Skłodowska
2006, Zhang et al. 2007, Liu and Liu 2011). Natural
rain is slightly acidic due to dissolved atmospheric car-
bon dioxide, however, the progressive increase in indus-
trial activity and combustion of fossil fuels has led to a
nitrogen oxide released into the atmosphere (Glass et al.
1979, Fan and Wang 2000). These compounds undergo
oxidation reactions in combination with ozone to form
strong acids (e.g. sulphuric and nitric acid), which when
dissolved into rainwater decrease its pH (Glass et al.
1979, Wyrwicka and Skłodowska 2006, Zhang et al.
2007). Acid rain is defined as rainwater with a pH <5.6
(Ferenbaugh 1976, Wyrwicka and Skłodowska 2006).

Abbreviations – chl, chlorophyll; DAS, days after sowing; FW, fresh weight; LAR, leaf area ratio; LN, liquid nitrogen; PVP,
polyvinylpyrrolidone; RCD, root collar diameter; SEM, scanning electron microscopy; SLA, specific leaf area; TAA, total aqueous
antioxidant.

Physiol. Plant. 153, 2015 149

 The general effects of acid rain on plants include foliar
injury (Hogan 1998, Fan and Wang 2000), interference
with metabolic functioning (Gabara et al. 2003), direct
impacts on seed germination (Fan and Wang 2000),
seedling emergence and recruitment (Fan and Wang
2000, Benitez-Malvido et al. 2001). Indirect impacts
include accelerated leaching of nutrients from the soil
and mobilization of heavy metals (Zhang et al. 2007),
and changes in the symbiotic microbial community
(Pennanen et al. 1998).
Seeds exposed to acid rain or shed onto soil with
a history of exposure to acid rain are likely to experi-
ence one or more of the following symptoms/effects:
the physiologically disruptive effects of acid toxicity, a
reduction in the availability of nutrients and/or a subse-
quent decline in water uptake (Eamus et al. 1989). This
could have serious ecological ramifications, as seeds
are the reproductive propagules of plants and require
sufficient amounts of water to facilitate radicle emer-
gence and subsequent seedling establishment (Bewley
and Black 1994). Seed responses to acid rain are well
documented in desiccation tolerant orthodox-seeded
species (e.g. Eamus et al. 1989, Fan and Wang 2000)
but at the time of the present study there were no
published reports on the effects of acid rain on ger-
mination or seedling establishment in desiccation
sensitive recalcitrant-seeded species. The influence of
post-harvest seed physiology on plant responses to acid
rain is an important ecological consideration. Orthodox
(i.e. desiccation tolerant) seeds (Roberts 1973) acquire a
variety of mechanisms involved in desiccation tolerance
(Pammenter and Berjak 1999) that allow them to persist
in the seed bank (for years) until favorable conditions
permit germination (Bewley 1997, Ooi 2012), and
hence could potentially avoid direct exposure to acid
rain. Conversely, recalcitrant (i.e. desiccation sensi-
tive) seeds (Roberts 1973) remain metabolically active
throughout their development and constitute part of the
transient (short-lived) seed bank (Farnsworth 2000), as
they lose viability quickly if germinative development is
interrupted/impeded due to the unavailability of water
(Pammenter and Berjak 1999). As a consequence of this,
recalcitrant seeds may therefore not be able to avoid
the exposure to acid rain. Even if recalcitrant seeds
germinate successfully after shedding, their recruit-
ment into the seedling bank may be compromised by
exposure to acid rain. Seedling recruitment is a major
requirement for the persistence of any population of
plant species (Benitez-Malvido et al. 2001, Chesser and
Brewer 2011).
Increased air pollution in a number of developing
African countries, together with the reports of vegeta-
tion damage typically associated with acid precipitation
150
in commercial forests in South Africa (van Tienhoven
and Scholes 2003), has raised concerns over the poten-
tial impacts of acid rain on wild vegetation in these
countries (Emberson et al. 2001, 2003). As explained
above, recalcitrant seeds of many indigenous African
species may be particularly susceptible to the effects
of acid rain. Hence, this study which investigated the
effects of simulated acid rain (70:30 equivalent basis
H2 SO4 :HNO3 at pH 3.0 and 4.5) on germination and
seedling establishment in a recalcitrant-seeded African
tree species Trichilia dregeana Sond., while growing
in its natural seed bank. Trichilia dregeana is an ever-
green tree with widespread distribution, stretching from
Pondoland, KwaZulu-Natal and Mpumalanga in South
Africa, through Swaziland, into Zimbabwe and north-
wards into tropical Africa (Pooley 1997). In countries like
South Africa, T. dregeana occurs in forests that are within
close proximity of industrial and urban centers (e.g. in
Mpumalanga) with exceptionally high levels of air pol-
lution (Siversten et al. 1995) and evidence of vegetation
damage by acid rain (van Tienhoven and Scholes 2003).
This study investigated the effects of simulated acid
rain on T. dregeana germination and seedling vigor
and viability. A major objective of the study was to
relate T. dregeana seedling vigor and viability responses
to oxidative metabolism, as biochemical measures
of oxidative stress allow for the investigation of the
effects of acid rain, often before any physiological
consequences can be observed (Koricheva et al. 1997).
Free radicals are a common consequence of most
stresses in plants and their seeds (Koricheva et al. 1997,
Wyrwicka and Skłodowska 2006, El-Maarouf-Bouteau
and Bailly 2008), and are important in interpreting the
effects of abiotic stresses as free radicals can lead to
damage of a variety of biomolecules (Wyrwicka and
Skłodowska 2006). Plants and their propagules possess
enzymatic and non-enzymatic antioxidant defense sys-
tems (Koricheva et al. 1997, Gabara et al. 2003, Liu
and Liu 2011) but abiotic stresses can impair these,
leading to uncontrolled ROS production and eventual
cell and organismal death (El-Maarouf-Bouteau and
Bailly 2008).
Additionally, T. dregeana seeds and recovered
seedlings were exposed to simulated acid rain while
growing with species in its natural seed bank. The
objective here was to investigate whether acid rain
altered competition dynamics between T. dregeana
and its natural neighbor species. Competition among
plant species frequently intensifies under conditions
of abiotic stress, often favoring alien species due to
their relatively higher stress tolerance (Benitez-Malvido
et al. 2001, Ooi 2012). Invasive species can alter the
Physiol. Plant. 153, 2015
colonization ability of indigenous seedlings and dis-
rupt the vegetation structure and seed bank dynamics
(Benitez-Malvido et al. 2001, Ooi 2012). Even though
competition is a major determinant of reproductive
success (Classen et al. 2010), its effects are often ignored
in studies investigating the impacts of climate change on
seeds/seedlings.
Materials and methods
Soil and plant material
The top ±100 mm of soil was collected from beneath
the canopy of seeding T. dregeana trees growing on the
grounds of the University of KwaZulu-Natal, in Durban,
South Africa (June 2012). After removal of all T. dregeana
seeds, the soil was used to fill twelve 25 l (top diameter
of 0.32 m) pots to capacity. The pots were lined with
coarse building stone up to 100 mm from the base to
facilitate drainage, before they were filled to capacity
with collected soil (300 mm). Mature T. dregeana seeds
were then harvested directly from the parent trees and
40 of these were sown (at a depth of 15 mm) within each
pot. The number of T. dregeana seeds sown was based
on observations of seed densities (per m2 ) beneath T.
dregeana parent trees and they were sown with the aril
intact to simulate natural conditions.
Treatment with simulated acid rain
Rainwater was collected at a roof drainage point close
to the parent plants, throughout the period of the study.
Solutions of 1 N H2 SO4 and 1 N HNO3 , mixed in a ratio
of 70:30 (after Kelly and Strickland 1987), were used to
adjust the pH of this rainwater (pH ∼6.3) to 3.0 and 4.5.
The T. dregeana seedlings that emerged from the sown
seeds were grown in combination with species emerging
from the seed bank for 168 days, during which time they
were watered with non-acidified (pH ∼6.3) or acidified
(to pH 4.5 and 3.0) rainwater.
Before the T. dregeana seeds were sown, the pots were
watered to field capacity with their respective rainwa-
ter treatments daily for a week and placed under a rain
shelter. After T. dregeana seeds were sown, each pot
was watered with 350 ml of acidified/non-acidified rain-
water every 2–3 days (in the afternoon), until signs of
T. dregeana first leaf emergence. The pots were there-
after watered with 600 ml of acidified/non-acidified rain-
water for the remainder of the experiment. Pots were
always watered from a height of approximately 0.5 m
using a watering can fitted with a rose to prevent exces-
sive soil disturbance and ensure foliar exposure. A soil
fertilization regime was introduced after 1 month to pre-
vent nutrient deficiency effects typically associated with
Physiol. Plant. 153, 2015
pot studies. Six grams of Fisher’s Multifeed (Multifeed™;
Plaaskem, Boksburg, South Africa; 190 g nitrogen, 82 g
phosphorous and 158 g potassium kg –1 ) was dissolved in
2.4 l of rain water, and 600 ml of this mixture was applied
directly to the soil in each pot for a week. Thereafter, the
normal watering regime continued until the experiment
was terminated.
Other factors that could have influenced plant
growth such as light intensity, soil moisture content and
soil pH (measured on the day of watering events as
well 2 days thereafter) were monitored throughout the
experiment.
Growth measurements for T. dregeana seedlings
Growth measurements were carried out for individual
T. dregeana seedlings after complete opening of the
first leaf across all pots, up until and including, the
day of the final harvest [168 days after sowing (DAS)].
During the course of the experimental period T. dregeana
germinants were monitored for emergence of the first
leaf. These data were then used to calculate cumulative
first leaf emergence: the percentage of seedlings that
produced a first leaf on any particular sampling day,
added to the percentage associated with the previous
sampling day, and velocity of first leaf emergence: time
(days) taken to reach 50% of the final cumulative first leaf
emergence. Stem height (cm), root collar diameter (RCD)
(mm) and leaf number were also measured weekly,
throughout the seedling growth period.
At the final harvest (168 DAS), the roots, stems and
leaves of five seedlings (at least one seedling from each of
the four pots) from all treatments were weighed individ-
ually and dried in an oven for 72 h in foil bags at 70∘ C
to determine the dry mass. The root and shoot lengths
(mm) were measured before dry weight estimations. At
the final harvest, leaf area was measured using a CID leaf
area meter (CI-202; CID Bio-Science, Inc., Camas, WA).
Five leaves from each of five seedlings were measured
to calculate specific leaf area (SLA) and leaf area ratio
(LAR). The SLA was based on the ratio of the leaf area to
the leaf dry weight, whereas the LAR was calculated as
the ratio of leaf area to the total seedling dry weight.
Biochemical studies
Various biochemical assays were carried out on
T. dregeana seedlings from acidified treatments and
the control 168 days after planting (DAP). Seedlings
were randomly selected from different pots across each
treatment, freed of soil by rinsing with distilled water
and immediately used for assays.
151
Superoxide assay
Root and leaf samples from each of six seedlings (at least
one seedling from each of the four pots) across all treat-
ments were pre-weighed, ground using a pre-chilled pes-
tle and mortar with 100 mg of insoluble polyvinylpyrroli-
done (PVP) (Sigma-Aldrich, Steinheim, Germany) and
3 ml of 50 mM chilled phosphate buffer (pH 7.8). The
homogenate was transferred into a pre-chilled Eppen-
dorf and centrifuged at 18 620 g at 4∘ C for 15 min. The
resulting supernatant was assayed for intracellular super-
oxide activity, measured in terms of nitrite formation
from hydroxylamine in the presence of superoxide (after
Elstner and Heupel 1976). The amount of superoxide
produced [expressed as μmol g –1 fresh weight (FW)]
was calculated using a standard curve constructed using
0.1–50 μM sodium nitrite (Sigma-Aldrich), dissolved in
phosphate buffer.
Hydrogen peroxide assay
Root and leaf samples from each of six seedlings (at
least one seedling from each of the four pots) across
all treatments were pre-weighed and ground in a
pre-chilled pestle and mortar with 100 mg of insoluble
PVP (Sigma-Aldrich), 2 ml of 50 mM chilled phosphate
buffer (pH 6.5) and 1 ml of 1 mM hydroxylamine. The
homogenate was transferred into a pre-chilled Eppen-
dorf, centrifuged at 3420 g at 4∘ C for 25 min and then
assayed for intracellular hydrogen peroxide (H2 O2 )
production according to Jana and Choudhuri (1982).
The amount of H2 O2 produced was calculated using the
extinction coefficient, 0.28 μmol –1 cm –1 , and expressed
as μmol g –1 FW.
Total aqueous antioxidant assay
Total aqueous antioxidant (TAA) activity was estimated
for root and leaf samples from four seedlings (each
from a different pot) across all treatments. Leaf/root sam-
ples were pre-weighed before being transferred to a
pre-chilled pestle and mortar and ground with 50 mg of
insoluble PVP (Sigma-Aldrich). Total antioxidants were
extracted in 1 ml of chilled extraction buffer (50 mM
potassium phosphate buffer containing 1 mM CaCl2 ,
1 mM KCl and 1 mM EDTA, pH 7.0). The homogenate
was transferred into a pre-chilled Eppendorf and cen-
trifuged at 21 380 g at 4∘ C for 30 min. The resulting
supernatant was extracted and kept on ice before being
assayed for TAA, measured in terms of the depletion
of the 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic
acid (ABTS) radical in the presence of the antioxidant
extract (after Re et al. 1999). The decolorization of the
working solution was measured before and 120 s after
152
addition of 20/40 μl of the antioxidant extract. A stan-
dard curve constructed using 0.05–1.0 mM Trolox™
(Sigma-Aldrich), dissolved in the extraction buffer, was
used to express sample TAA as Trolox equivalents,
expressed as g –1 FW.
Leaf chlorophyll content
Leaf chlorophyll (chl) content was measured across all
treatments using one 1.77 cm2 leaf disc from each of
four seedlings (each from a different pot; only alive
leaf material was sampled). Immediately after excision,
using a cork borer, discs were ground using a pestle and
mortar with liquid nitrogen (LN) and chl was extracted
immediately in 5 ml of 80% acetone, in the dark (after
Arnon 1949). After 6 h, the leachate was filtered and its
absorbance was read spectrophotometrically at 663 nm
for chla and 645 nm for chlb (Arnon 1949). Chlorophyll
a, b and total chl contents were thereafter expressed
on an area basis. Colorimetric pigment assays, such
as the one used here (Arnon 1949), are sufficient pro-
vided that the stability of the pigment extract is checked
because extraction with acetone solutions can lead to
chl degradation to pheophytin by co-extracted acids
(Rodriguez-Amaya and Kimura 2004). This was unlikely
to have occurred here, since the absorbance of the ace-
tone chl filtrates from T. dregeana leaves remained stable
(to the third decimal) for as long as 6 h after filtration
(when kept in the dark).
Scanning electron microscopy and stomatal density
At the final harvest, four leaves, of comparable age, were
taken from different seedlings across all treatments and
processed for visualization of the adaxial leaf surface
via scanning electron microscopy (SEM). Leaves were
cut into 1 cm2 squares and snap-frozen in LN before
being freeze-dried, coated with gold for 10 min using
a Polaron SC500 Sputter Coater (Leica Microsystems,
Wetzlar, Germany) and viewed using a Zeiss Ultra Plus
Scanning Electron Microscope (Zeiss, Jena, Germany).
Images of the adaxial leaf surface were captured at a
constant magnification and field of view. The number of
stomata (opened and closed) was then counted, keeping
the sample area constant. Counts were carried out on
four leaf discs across all treatments and these were
averaged to yield a mean stomatal density.
Characterization of species emerging from
the native seed bank
At the final harvest, the species arising from the seed
bank were identified to genus and species rank (in most
Physiol. Plant. 153, 2015
cases). These species were classified into the following Table 1. Summary of viability assessments for Trichilia dregeana seeds
life forms: graminoids, herbs and shrubs. All taxa were treated with natural (control) and acidified (pH 4.5 and pH 3.0) rain-
water for 168 days. Values represent the mean (±SD) of four replicate
also categorized as either indigenous or alien. These pots, each sown with 40 seeds. a Trichilia dregeana germinants that
data were used to compare seedling abundances of eventually established as seedlings. b Trichilia dregeana germinants that
the different taxa and life forms across the acid rain formed seedlings that subsequently died during the 168-day experi-
treatments and the control. mental period. c Average number of days from shoot emergence until
√
death. P > 0.05, when arcsine transformed percentages were com-
pared across treatments, within categories (ANOVA).
Statistical analyses
Control pH 4.5 pH 3.0
The data were analyzed using PASW 18 statistics version
18.0.3 (SPSS Inc., Chicago,√ IL). Viability and germi- Germination (%) 40.6 ± 12.5 35.6 ± 6.6 41.8 ± 10.1
nation percentiles were arcsine transformed and Seedling establishment 17.5 ± 6.1 17.5 ± 8.4 11.3 ± 6.3
then subjected to ANOVA. ANOVA was used to test for (%)a
Seedling mortality (%)b 7.5 ± 3.5 3.8 ± 4.3 10.0 ± 3.5
inter-treatment differences in biomass, intracellular
Seedling residence time 48.0 ± 4.1 56.0 ± 9.9 41.8 ± 8.3
superoxide and hydrogen peroxide production, chloro- (days)c
phyll content, TAA and stomatal density. Means for all Exhibiting delayed shoot 15.6 ± 3.8 14.4 ± 8.5 20.6 ± 8.8
analyses of variance were separated using a LSD post hoc emergence (%)
test. An independent-samples t-test was performed to
test differences between the root and shoot lengths of
30

Cumulative shoot emergence (%)

seedlings. Where data did not meet ANOVA assumptions,
control pH 4.5 pH 3
even after transformation, a Kruskal–Wallis test was 25 a
applied. All differences were considered significant at a
20
the 0.05 level.
15

Results and discussion 10

The effects of acid rain on recalcitrant-seeded species are 5

virtually unknown. In this study, recalcitrant seeds of T.
dregeana were sown with species in their native seed
bank and treated with natural (pH ∼6.3) and acidified
(pH 4.5 and 3.0) rainwater over a period of 168 days.
Trichilia dregeana seed and seedling responses
to acid rain
Germination in a number of tree species was significantly
reduced when seeds were treated with simulated acid
rain of pH 2.0 (e.g. Fan and Wang 2000). The germina-
tion totality and percentage of T. dregeana germinants
that eventually established as seedlings (i.e. survived the
168 day experimental period) were comparable across
the control and acidified treatments (Table 1). Interest-
ingly, seedling mortality (% seedling death) was observed
in control and acidified pots and seedling residence
time (i.e. the average number of days from shoot emer-
gence until death) was comparable among the control
and acid rain treatments (Table 1). These data suggest
that seedling mortality and the low seedling establish-
ment rates observed [<26% in the control and acidified
treatments (Table 1)] may be reflective of a natural phe-
nomenon in T. dregeana which does not appear to be
exacerbated under conditions of acid rain.
Cumulative first leaf emergence as well as first leaf
emergence velocity (both of which are defined in the
0
72 78 84 90 96 102 108 114 120 126 132 138 144 150 156 162 168
Days after sowing
Fig. 1. Cumulative first leaf emergence in Trichilia dregeana seeds sown
within their native seed bank. Seeds and subsequent seedlings were
treated with non-acidified (control) and acidified (pH 3.0 and pH 4.5)
rainwater for 168 days. Values represent the mean (±SD) of four replicate
pots, each sown with 40 seeds. P = 0.78 when final values were tested
for inter-treatment differences (ANOVA).
section Materials and methods) did not differ signifi-
cantly across the control and treatments (Fig. 1). Given
the fact that T. dregeana seeds are recalcitrant, the
relatively low velocity of leaf emergence (>100 days
across the control and treatments) was surprising (Fig. 1).
Trichilia dregeana seeds possess chlorophyllous cotyle-
dons which usually emerge from the soil shortly after
radicle protrusion and our observations suggest that
these cotyledons can remain viable for at least up to
4 months after planting (Fig. 2). These cotyledons in
providing embryonic axes with resources for survival
during root establishment possibly negate the urgency
for leaf emergence, which is usually an urgent priority
in species in which the cotyledons (or endosperm)
perish shortly after radicle protrusion (Roqueiro et al.
2013). Young leaves are particularly sensitive to acid

Physiol. Plant. 153, 2015 153


Fig. 2. A pH 3.0 treated Trichilia dregeana seedling showing a
below-ground shoot (left) and a control seedling showing an emerged
shoot (right) after 168 days. Bar = 3 cm.
rain and by implication render seedlings sensitive to
acid rain-induced mortality (Wyrwicka and Skłodowska
2006, Liu and Liu 2011). This phenomenon of delayed
shoot emergence, which was exhibited by both acid
rain-treated and control germinants in this study, has
not yet been reported for recalcitrant seeds and in
T. dregeana may represent a strategy for opportunistic
seedling establishment: retain the chlorophyllous cotyle-
dons until the root is well established and only produce
true leaves months after producing a root (Fig. 2). This
growth strategy may have reduced the impacts of acid
rain on T. dregeana seedlings in this study (Table 1).
However, only seedlings occurring in the acid
rain-treated pots, particularly pH 3.0, exhibited signs
A B
Fig. 3. Evidence of leaf damage displayed by Trichilia dregeana seedlings treated with acidified rainwater for 168 days: (A) signs of leaf tip necrosis
which occurred in pH 3.0 and 4.5 treatments; (B) necrotic spots and (C) leaf chlorosis in pH 3.0 treated seedlings.
154
of stress typically associated with acid precipitation:
leaf tip necrosis and abnormal bilobed leaves (Fig. 3A),
necrotic spots (Fig. 3B) and chlorosis (Fig. 3C). Acid
rain at pH < 3.5 can induce visible foliar injury (e.g.
necrotic spots on the adaxial leaf surface) (Hogan 1998),
with younger leaves being more prone to anatomical
damage than older ones (Wyrwicka and Skłodowska
2006, Liu and Liu 2011). Sant’Anna-Santos et al. (2006)
found Genipa americana seedlings treated for as little
as 10 days with pH 3.0 acid rain to exhibit damage to
leaf morphology and micromorphology. Further analy-
ses of T. dregeana leaf surfaces via SEM revealed that
the control leaves possessed fewer and more widely
distributed stomata on the adaxial surface than the
acid-treated leaves (pH 4.5 and 3.0; Fig. 4). Kumaravelu
and Ramanujam (1998) also found that an increase
in rain acidity correlated with an increase in adaxial
stomatal frequency in Vigna radiata seedlings but this
enhancement in stomatal frequency with acid rain
exposure is yet to be explained.
Additionally, leaf total chlorophyll (chl) and chla con-
tent in pH 3.0 treated seedlings were significantly lower
than in control seedlings (Fig. 5). Leaf chlorophyll con-
tent is often strongly influenced by the pH of rain (Kumar-
avelu and Ramanujam 1998, Sant’Anna-Santos et al.
2006, Wyrwicka and Skłodowska 2006). Chlorophyll for-
mation may be decreased by acid rain due to foliar leach-
ing of nutrient elements, especially magnesium which
contributes to the structure of chlorophyll (Fan and Wang
2000). Qiu and Liu (2002) found that acid rain induced
a decrease in chlorophyll content of Dimorcarpus lon-
gana leaves which was attributed to a reduction in chla
content, while chlb was less sensitive to acid rain. This is
in agreement with the results obtained for pH 3.0 treated
seedlings in this study (Fig. 5).
This reduced light harvesting capacity and abnormal
leaf anatomy (described earlier) in pH 3.0 treated leaves
may explain why total biomass in pH 3.0 treated seedling
was significantly lower than the control and pH 4.5
C
Physiol. Plant. 153, 2015
 10
A Chl a Chl b Total chlorophyll

Chlorophyll content cm–2

8 a
ab
b
6 a ab
b
4
a a a
2

0
Control pH 4.5 pH 3.0

Treatments

B
Fig. 5. Leaf chlorophyll content (chla , chlb and total) for leaves of
Trichilia dregeana seedlings treated with non-acidified (control) and
acidified (pH 4.5 and pH 3.0) rainwater for 168 days. Values represent the
mean (±SD) of four replicate leaves. Columns labeled with different letters
are significantly different when compared across treatments, within
categories (P = 0.02 for chla and P = 0.02 for total; ANOVA).

Root Stem Leaves

0.4

C 0.3
a AB
Biomass (g)
B
0.2
a a
a
a
0.1 a

a b b
0
Control pH 4.5 pH 3.0
Treatments

Fig. 6. Total biomass and biomass partitioning for Trichilia dregeana

Fig. 4. Adaxial leaf surface of Trichilia dregeana seedlings treated
with (A) non-acidified (control) rainwater and rainwater acidified to
(B) pH 4.5 and (C) pH 3.0 for 168 days. Bar = 20 μm. Arrows point to
the stomata. Average stomatal densities differed significantly (P = 0.04;
ANOVA; n = 4) across treatments (5 ± 3 stomata mm
–2 for control; 11 ± 4
seedlings treated with non-acidified (control) and acidified (pH 4.5 and
pH 3.0) rainwater for 168 days. Values represent the mean (±SD) of five
replicate seedlings. Columns labeled with different letters (uppercase for
total and lowercase for partitioning) are significantly different (P > 0.05
for stems and leaves; P = 0.003 for roots and P = 0.01 for total; ANOVA).

stomata mm –2 for pH 4.5 and 10 ± 2 stomata mm –2 for pH 3.0).
(Fig. 6). Reduced growth in response to acid rain is
a common response in plants (Fan and Wang 2000).
Though there were no significant differences in RCD,
root and shoot length, number of leaves, leaf area, SLA
and LAR across acidified rainwater treatments and the
control (data not shown), it was interesting to note that
root biomass in pH 3.0 treated seedlings was significantly
lower than in the control (Fig. 6).
As interception of acid rain is primarily at the leaf
level, percolation through the soil can affect roots
(Salisbury and Ross 1978). Those authors suggest that
acidic soil, like that found after acid rain has fallen,
may limit plant growth simply because H+ , the acidic
part of a molecule, is toxic to roots. Certain soil types
exhibit a natural buffering capacity (James and Riha
1986) and while differences in soil pH were not strik-
ing (P = 0.24) when measured 2 days after a watering
event (5.83–6.34 in pH 3.0, 6.55–7.02 in pH 4.5 and
6.66–7.06 in the control), soil pH in acid rain-treated
pots was significantly (P = 0.03) lower than the control
and pH 4.5 pots on watering days (3.83–4.18 in pH 3.0,
5.55–5.62 in pH 4.5 and 6.71–6.96 in the control). This
reduction in soil pH immediately after a watering event,
when combined with direct exposure to percolating low
pH acid rain, may explain the significant reduction in
root biomass in the pH 3.0 treated seedlings relative
to the control and pH 4.5 (Fig. 6). This suggestion is
supported by the fact that root intracellular superoxide
levels in pH 3.0 treated seedlings were significantly
higher than the control and pH 4.5 (Fig. 7). In addition,
intracellular leaf and root H2 O2 levels in pH 3.0 treated
seedlings were higher than the control and pH 4.5
(Fig. 8), which may explain why the significant reduc-
tion in root and total biomass observed in pH 3.0 treated
seedlings did not manifest in those exposed to pH 4.5
(Fig. 6). This heightened production of ROS in the roots
and leaves of pH 3.0 treated seedlings was accompanied

Physiol. Plant. 153, 2015 155

Intracellular superoxide 1 Roots Leaves 12 Roots Leaves
a
a 10 a

Trolox equivalent
0.8 a

(µmol g–1 FW)

(µmol g–1 FW)

a 8
0.6 b a
6
0.4 4
a ab b
a
a
0.2 2

0
0 Control pH 4.5 pH 3.0
Control pH 4.5 pH 3.0

Treatments Treatments

Fig. 7. Intracellular superoxide production for roots and leaves of
Trichilia dregeana seedlings treated with non-acidified (control) and
acidified (pH 4.5 and pH 3.0) rainwater for 168 days. Values represent
the mean (±SD) of four replicates. Columns labeled with different letters
are significantly different, across treatments, within organs (P < 0.001 for
roots; ANOVA).
Intracellular hydrogen peroxide
Fig. 9. Total antioxidant activity, in terms of Trolox equivalents, of roots
and leaves from Trichilia dregeana seedlings treated with non-acidified
(control) and acidified (pH 4.5 and pH 3.0) rainwater for 168 days.
Values represent the mean (±SD) of four replicates. Columns labeled
with different letters are significantly different across treatments, within
organs (P < 0.03 for roots; ANOVA).

25 Table 2. Above-ground biomass production of different life forms in

Roots Leaves b non-acidified (control) and acidified (pH 4.5 and pH 3.0) treatments after
20 168 days. All values represent means for four replicate pots per treat-
(nmol g–1 DW)

ment, and exclude Trichilia dregeana. Values followed by different letters

15
are significantly different when compared within life form categories,
10 a across pH treatments (P < 0.05; ANOVA). P < 0.05 in all cases where values
a
were compared across life forms, within rain water categories.
5 b
a a
Mean above-ground biomass (g)
0
Control pH 4.5 pH 3.0 Control pH 4.5 pH 3
Treatments
Graminoids 20.38 ± 3.60a 9.34 ± 5.61b 19.36 ± 8.12ab
Herbs 12.63 ± 9.52a 22.23 ± 5.42a 10.67 ± 6.88a
Fig. 8. Intracellular hydrogen peroxide production by roots and leaves Shrubs 9.99 ± 4.93a 8.01 ± 3.67a 10.19 ± 5.51a
of Trichilia dregeana seedlings treated with non-acidified (control) and Total biomass 43.00 ± 7.12a 38.56 ± 4.42a 40.22 ± 3.08a
acidified (pH 4.5 and pH 3.0) rainwater for 168 days. Values represent the
mean (±SD) of four replicates. Columns labeled with different letters are
significantly different across treatments, within organs (P < 0.04; ANOVA).
in its interaction with established vegetation, given
that competition can reduce both seedling vigor and
by a rise in root and leaf total antioxidant activity relative
survival (Fayolle et al. 2009). Also, since competition
to the control (Fig. 9), but ROS production under abiotic
among plant species frequently intensifies under con-
stress conditions can exceed antioxidant capacity, lead-
ditions of abiotic stress, often favoring alien species
ing to uncontrolled ROS production and eventual cell
(Benitez-Malvido et al. 2001, Ooi 2012), we investigated
death (El-Maarouf-Bouteau and Bailly 2008). Increased
whether acid rain is likely to alter T. dregeana’s neighbor
H2 O2 production, in particular, has been implicated
species composition, abundance and biomass produc-
in initiating responses that signal programmed cell
tion. This was accomplished by sowing T. dregeana seeds
death and cellular damage (El-Maarouf-Bouteau and
in soil containing the soil seed bank typical of the area in
Bailly 2008).
which the T. dregeana parent trees (from which the seeds
The data discussed above suggests that that the detri-
were harvested) were growing.
mental effects of relatively low pH (pH 3.0 as opposed to
The neighbor species emerging from the seed bank
pH 4.5) acid rain on T. dregeana seedling root and total
included graminoids, herbs and shrubs and their com-
biomass accumulation may be related to the oxidative
bined above-ground biomass did not differ significantly
stress, reduction in light harvesting capacity and leaf
across the control and acid rain-treated pots (Table 2).
anatomical abnormalities exhibited by pH 3.0 treated
Across life forms, graminoids exhibited the highest com-
seedlings.
bined above-ground biomass in control and pH 3.0
treated pots, whereas herbs exhibited the highest com-
Seed bank responses to acid rain
bined above-ground biomass in pH 4.5 pots (Table 2).
Seedling establishment is one of the most important Furthermore, herbs were the most abundant life form
events in a plant’s life cycle and is particularly sensitive across acid rain and control pots but their abundance

156 Physiol. Plant. 153, 2015

Table 3. Species that emerged from Trichilia dregeana’s native seed bank when it was treated with natural (control) and acidified (pH 4.5 and pH
3.0) rainwater for 168 days. Values represent the sum of absolute values for four replicate pots. P values labeled with an asterisk indicate significant
differences when abundance and number of taxa (for individual pots) were compared across the control and treatments, within life forms (P < 0.05;
Kruskal–Wallis test).

Abundance

Species Family Indigenous/alien Control pH 4.5 pH 3.0 Significance

Graminoids
Digitaria ternata (A.Rich.) Stapf Poaceae Indigenous 0 0 2
Panicum maximum Jacq. Poaceae Indigenous 22 8 19
Combined abundance 22 8 21 0.04*
Number of taxa 2 1 2 0.51
Herbs
Phaulopsis imbricata (Forssk.) Sweet Acanthaceae Indigenous 8 9 6
Bidens pilosa L. Asteraceae Indigenous 0 4 0
Mikania natalensis DC. Asteraceae Indigenous 4 3 0
Tridax procumbens L. Asteraceae Alien 0 1 1
Commelina africana L. Commelinaceae Indigenous 3 2 0
Dalechampia capensis A.Spreng. Euphorbiaceae Indigenous 1 1 1
Hibiscus calyphyllus Cav. Malvaceae Indigenous 0 3 1
Sida cordifolia L. Malvaceae Indigenous 43 41 29
Centrella asiatica (L.) Urban Mackinlayaceae Alien 13 13 15
Oxalis corniculata L. Oxalidaceae Indigenous 24 20 17
Phyllanthus myrtaceus Sond. Phyllanthaceae Indigenous 5 2 2
Lantana rugosa Thunb. Verbenaceae Indigenous 1 1 2
Combined abundance 102 100 74 0.03*
Number of taxa 9 12 9 0.43
Shrubs
Justicia sp. Acanthaceae Indigenous 0 6 3
Berkheya umbellata DC. Asteraceae Indigenous 6 3 7
Chromolaena odorata (L.) R.M.King & H.Rob. Asteraceae Alien 3 0 2
Erigeron floribundus (Kunth) Schultz-Bip. Asteraceae Alien 3 2 2
Argemone mexicana L. Papaveraceae Alien 0 2 2
Solanum mauritianum Scop. Solanaceae Alien 1 0 0
Solanum nodiflorum Jacq. Solanaceae Alien 1 3 1
Combined abundance 14 16 17 0.67
Number of taxa 5 5 6 0.34
Total abundance for all life forms 138 124 112 0.02*
Total number of taxa for all life forms 16 18 17 0.74

declined significantly at pH 3.0, whereas graminoid
abundance, particularly Panicum maximum, declined at
pH 4.5 (Table 3). However, this decline in P. maximum
[often found in abundance within soil seed banks in
South Africa, e.g. Sand Forest seed banks (Kellerman
and van Rooyen 2007)] abundance at pH 4.5 was not
accompanied by an increase in herb abundance. These
data suggest that under conditions of moderately acidic
rain herbaceous species like Sida cordifolia and Bidens
pilosa (an aggressive alien invasive) may thrive in terms of
biomass productivity, displacing graminoids. However,
the opposite may occur under conditions of severe acidic
rain, as S. cordifolia declined in abundance at pH 3.0
whereas graminoid abundance remained comparable to
the control.
The total number of taxa remained relatively
unchanged across the control and treatments, irrespec-
tive of life form but the total plant abundance (for all life
forms combined) was significantly lower in the acid rain
treatments, being lowest at pH 3.0 (Table 3). Leading on
from above, T. dregeana seedlings may encounter lower
levels of competition from certain graminoid species
(e.g. P. maximum) under conditions of moderately acidic
rain and from certain herb species (e.g. S. cordifolia)
under conditions of severe acidic rain, purely as a
consequence of these neighbors being present in lower
abundances. It must be noted though that these changes
in competition dynamics will be based largely on the
species composition of T. dregeana’s seed bank: while
some species such as Solanum mauritianum Scop. may

Physiol. Plant. 153, 2015 157

not emerge under acid rain conditions, others such as
Justicia sp., Digitaria ternate (A.Rich) Stapf. and Arge-
mone mexicana may exhibit enhanced emergence when
exposed to acid rain (Table 3).
Interestingly, of the seven alien taxa encountered, five
emerged from the seed banks in control pots whereas six
emerged in the acid rain-treated pots (Table 3). This sug-
gests that the alien species usually found in T. dregeana’s
natural seed bank appear to be just as, if not more, toler-
ant of acid rain. This is disconcerting, as alien species
like the broad leaf forb Centrella asiatica (L.) Urban,
which occurred at relatively high densities in the pH
3.0 pots are good competitors in a number of environ-
ments as a consequence of their superior stress tolerance,
growth and photosynthetic rates and resource captur-
ing abilities (Fayolle et al. 2009). Abiotic stresses, espe-
cially when applied to mixed species assemblages, can
be synergistic (e.g. Walck et al. 1999) and future climate
change-related plant studies should consider this.
The acid rain-induced changes in species composi-
tion and life form abundance ratios described above
can impact on competitive interactions which naturally
occur at high frequencies (Goldberg and Barton 1992)
and do influence seedling recruitment levels, plant dis-
tribution patterns, relative abundances, species diversity
and community structure (Goldberg and Barton 1992,
Fayolle et al. 2009). However, given the limited research
on seed banks of South African subtropical coastal forest,
it is only when seed bank behavior is investigated in situ
that the present results can be better interpreted.
Concluding remarks
Guy et al. (2013) showed that while acidic deposition
is not likely to be a short-term threat to vegetation, cur-
rent and future deposition does represent a significant
long-term threat. The results of this study suggest that
acid rain did not compromise T. dregeana seed ger-
mination and seedling establishment significantly, rela-
tive to the control (non-acidified rainwater). However,
acid rain-treated seedlings, particularly pH 3.0, exhib-
ited anatomical and biochemical signs of stress typi-
cally associated with acid precipitation. Additionally,
seedlings treated with pH 3.0 acid rain appear to have
experienced oxidative stress at the leaf and root level
and this may explain why total and root biomass in
these seedlings were compromised relative to the con-
trol. Interestingly, acid rain also induced changes in the
species composition and relative abundance of the dif-
ferent life forms emerging from T. dregeana’s natural
seed bank and in this way could indirectly impact on
T. dregeana seedling establishment success. Future stud-
ies on the effects of various climate change scenarios on
158
recalcitrant seed germination and seedling establishment
should also accommodate for the potential interactive
effects of competition.
The persistence of any population of plant species is
ultimately dependent on seedling establishment (Fayolle
et al. 2009, Chesser and Brewer 2011). Trichilia dregeana
is a long-lived tree species and appears to naturally dis-
play low seedling establishment levels. However, under
conditions of low pH acid rain (i.e. pH 3.0) these levels
may be further reduced by leaf anatomical abnormali-
ties, reduced light harvesting capacity, enhanced oxida-
tive stress, reduced biomass accumulation and altered
competitive interactions.
Acknowledgements – The assistance of Anushka
Moothoo-Padayachie for the biochemical analyses and
Sariksha Ramlall throughout this project is gratefully
acknowledged. Teddy Govender’s assistance with identifi-
cation of germinants is also acknowledged. This research
was supported by the National Research Foundation (South
Africa).
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