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).