MATERIALS AND METHODS
Habitat and ecology of the studied species – Gossia
acmenoides and G. bidwillii are small to medium trees up to
~18 m tall, which occur in drier scrub patches and
rainforests. Gossia acmenoides has a brown/green bark, shedding
in patches; terminal buds silky. Leaves are oval to elliptical quickly
tapering to a pointed or rounded tip, simple, opposite, blade moderately
glossy above, translucent, venation distinct, old dots visible with the
naked eye (Snow et al., 2003). Unlike G. bidwillii , the leaves ofG. acmenoides are not sticky when crushed. It is a hardy plant
with dense and glossy foliage. On the other hand, G .bidwillii have almost round, very smooth, shiny and a cinnamon
smell when crushed (Fig. 1).
Plant dosing trial of G. bidwillii – Gossia
bidwillii plants, approximately 20 cm in height, were obtained from
Coastal Dry Tropics Landcare (Pallarenda Road, Townsville, Queensland)
and cultivated in a temperature and humidity-controlled glasshouse.
Plants were kept at 20 ºC and 80% Relative Humidity (RH), with and
13:00 hours of PAR light (1600 μM photons s-1) at the
Central Glasshouse Services at The University of Queensland, Brisbane,
Australia. After three weeks, the plants were transferred into 15 cm
pots containing a ratio of 9:1 mixture of Composted Pine Bark 5–10 mm
and Coco Peat (Bassett Barks Pty Ltd, Queensland, Australia). The media
was mixed with low-level fertilizers and other augments consisting (per
m3) of 1.2 kg Yates Flowtrace, 1 kg iron sulphate
heptahydrate
(FeO4SO4·7H2O), 0.1 kg
superphosphate
(Ca(H2PO4)2), 1.5 kg
gypsum (CaSO4) and 1.5 kg dolomite
(CaMg(CO3)2). The composition of the
Flowtrace was 24 wt% iron (Fe) as FeSO4, 14 wt% sulfur
(S) as SO4, 0.75 wt% copper (Cu) as
CuSO4, 0.5 wt% manganese (Mn) as MnSO4,
0.2 wt% zinc (Zn) as ZnSO4, 0.04 wt% molybdenum (Mo)
as Na₂MoO₄, 0.033 wt% boron (B) as
Na2B4O7 and also
contains zeolite, to ensure flowability (Yates Australia, Padstow, NSW,
Australia). Soluble Mn was applied to the plants in a randomised block
design. The applied treatments were the control (T1 ), and soils
with final dosed Mn2+ concentrations of 200 µg
g-1 (T2 ), 500 µg g-1(T3 ) and 1000 µg g-1 (T4 ) replicated
three times yielding a total of 12 experimental groups. Each treatment
was administered monthly as aqueous
MnSO4.H2O solutions for a period of 12
months; a similar volume of water was added to the control each time.
The individual pots were placed on saucers and hand watered daily to
field capacity to prevent loss of treatment solutions.
Field sampling of G. acmenoides – Gossia
acmenoides was sampled at a field site within the Amamoor State Forest
in Queensland, Australia (26°20’41.0”S 152°37’7.0”E). The geology of
this subtropical area is predominantly volcanic rock (andesite)
overlaying variably silicified shale or tuffs containing Mn-rich
(~30–50 wt%) minerals such as bixbyite and pyrolusite.
Krasnozem soils derived from this parent rock contain Mn to levels as
high as 40 wt% (Isbell, 1994). Old and young G. acmenoidesleaves (10–20 each) were harvested for total mineral nutrient analysis,
while small branchlets with old and young leaves were detached and
stored fresh for XRF analyses. Soil samples were collected from beneath
the trees (<10 cm depth) at three different points free of
surface litter.
Chemical analysis of soil and plant samples – After harvestingG. bidwillii , soils were extracted in each pot and emptied into
respective plastic bags. Soils on which G. acmenoides was growing
were also collected as described above. All soils were oven dried at 60º
C and later sieved using the 2mm sieve. Soil pH was obtained in a 1 to
2.5 soil to water mixture after 2 hr shaking. Exchangeable trace
elements were extracted in 0.1 M
Sr(NO3)2 at a soil:solution ratio of 1:4
(10 gram soil with 40 mL solution) and 2 hr shaking time was adapted
from Kukier and Chaney (2001). As a means of estimating potentially
phytoavailable trace elements, the DTPA-extractant was used according to
Dai et al. (2004) which was adapted from the original method by
Lindsay and Norvell (1978), with the following modifications: excluding
TEA, adjusted at pH 5.3, 5 g soil with 25 mL extractant, and extraction
time of one hr.
Plant material samples were oven dried at 60°C for three days and then
weighed, ground to fine powder and (300 mg) digested using 4 mL
HNO3 (70%) in a microwave oven (Milestone Start D) for
a 45-minute programme. Digests were then diluted to 45 mL with ultrapure
water (Millipore 18.2 MΩ·cm at 25°C) for analysis with Inductively
coupled plasma atomic emission spectroscopy (ICP-AES) using a Thermo
Scientific iCAP 7400 instrument for macro-elements (Al, Na, Mg, K, P,
Ca) and trace-elements (Fe, Ni, Mn, Co, Zn) in radial and axial modes,
depending on the element and expected analyte concentration. In-line
internal standardization using yttrium was used to compensate for
matrix-based interferences. Quality controls included matrix blanks,
certified reference material (Sigma-Aldrich Periodic Table mix 1 for ICP
TraceCERT®, 33 elements, 10 mg L-1 in
HNO3) and Standard Reference Material (NIST Apple 1515
digested with HNO3).
Laboratory µXRFelemental
mapping – Live samples (a whole branch) from Gossia bidwilliioriginating from the Mn1000 treatment and G. acmenoides collected
from Amamoor, Queensland were used for the microXRF scanning. The UQ
microXRF facility contains a modified IXRF ATLAS X system, mounting two
50W X-ray sources fitted with polycapillary focussing optics: XOS
microfocus Mo-target tube producing 17.4 keV X-rays (flux of 2.2 ×
108 ph s-1) focussing to 25 μm and a
Rh-target tube producing 20.2 keV (flux of 1.0 × 107ph s-1) focussing to 5 μm. The system is fitted with
two silicon drift detectors of 150 mm2. Typical energy
resolution is <145 eV with a maximum input count rates of 2 M
counts per second. The motion stage can address areas up to 300 × 300
mm. Measurements were conducted at atmospheric temperature
(~20°C), using the Mo 25 μm X-ray source at a 40 kV,
1000 uA, with a rise time of 0.25 µs and a per-pixel dwell of 100 ms.
The hydrated foliar samples were mounted between two sheets of 4 μm
Ultralene thin film in a tight sandwich to limit evaporation and
analysed within 10 minutes after excision. The mounted samples between
Ultralene thin film were stretched over a Perspex frame magnetically
attached to the x-y motion stage at
atmospheric
temperature (~20°C). The possibility of
radiation-induced damage in μ-XRF analysis (especially in fresh hydrated
samples) is an important consideration, but such damage was not observed
because the source produced a flux of 2.2 × 108photons s-1 in a 25 μm beam spot, at a maximum dwell
of 100 ms this results in a deposited radiation dose of just 6.6 Gy.
Data processing and statistical analysis – The XRF spectra on
the UQ microXRF facility were acquired in mapping mode using the
instrument control package, Iridium (IXRF systems) from the sum of
counts at the position of the principal peak for each element. These
were each exported into ImageJ as greyscale 8-bit TIFF files, internally
normalised such that each image covered the full dynamic range and
displayed using ImageJ’s “Fire” lookup table.
The concentrations of Mn presented as boxplots were performed using R
version 3.6.1 (2019-07-05). Concentrations of elements presented in
Tables as mean ± standard error were conducted using One-Way ANOVA and
means compared with Tukey’s honestly significant difference (HSD) Post
Hoc Test in the IBM SPSS Statistics 27 software package (IBM, New York,
USA). Values with different small letters are significantly different
(p <0.05).