Optimum Conditions and LC-ESI-MS Analysis of Phenolic Rich Extract from Eucalyptus marginata L. under Maceration and Ultrasound-Assisted Extraction Methods Using Response Surface Methodology

Eucalyptus marginata L. has a significant value in traditional medicine and recently has been shown to possess many pharmacological properties in vitro. The main goal of the present study was to optimize the extraction parameters of phenolic compounds from Eucalyptus marginata L. leaves using the extraction technique assisted by ultrasound in comparison with maceration using response surface methodology as a predicted tool. Therefore, total phenolic and flavonoid contents have been optimized, taking into account four variables: extraction time, temperature, liquid-to-solid ratio, and ethanol concentration. The optimum ultrasound-assisted extraction method for total phenolic and total flavonoid contents was obtained by ensuring the following parameters: t = 49.9 min, T = 74.9°C, liquid-to-solid ratio = 39.5 ml/g, and ethanol = 58.48%. The optimum extract has been subjected to LC-ESI-MS analysis. This technique allowed us to identify ten phenolic compounds: four phenolic acids mainly gallic acid (27.77 ± 0.06 µg/g DW) and protocatechuic acid (37.66 ± 0.04 µg/g DW) and six flavonoid compounds such as quercetrin (150.78 ± 0.02 µg/g DW) and hyperoside (39.19 ± 0.03 µg/g DW). These green and efficient procedures should be a promising option to guide industrial design for the production of phenolic-rich plant extracts.

Phosphorus uptake and toxicity is delimited by mycorrhizal symbiosis in P-sensitive Eucalyptus marginata but not in P-tolerant Acacia celastrifolia

Many plant species from regions with ancient, highly-weathered nutrient-depleted soils have specialised adaptations for acquiring P and are sensitive to excess P-supply. Mycorrhizal associations may regulate P-uptake at high external P-concentrations, potentially reducing P-toxicity. We predicted that excess P-application will negatively impact species from the nutrient-depleted jarrah forest of Western Australia and that mycorrhizal inoculation will reduce P-toxicity by regulating P-uptake. For seedlings of the N2-fixing legume Acacia celastrifolia and the tree species Eucalyptus marginata, we measured growth at P-concentrations of 0 to 90 mg kg-1 soil and in relation to inoculation with the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis. Non-inoculated A. celastrifolia maintained leaf P-concentrations at <2 mg g-1 dry mass (DM) across the range of external P-concentrations. However, for non-inoculated E. marginata, as external P-concentrations increased leaf P also increased, reaching >9 mg g-1 DM at 30 mg P kg-1 soil. A. celastrifolia DM increased with increasing external P-concentrations, while E. marginata DM was maximal at 15 mg P kg-1 soil, declining at higher external P concentrations. Neither DM nor leaf P of A. celastrifolia were affected by inoculation with AMF. For E. marginata, even at 90 mg P kg-1 soil, inoculation with AMF resulted in leaf P remaining <1 mg g-1 DM, and DM being maintained. These data strengthen the evidence base that AMF may not only facilitate P-uptake at low external P-concentrations, but are also important for moderating P-uptake at elevated external P-concentrations and maintaining plant P concentrations within a relatively narrow concentration range.

Signatures of natural selection in a foundation tree along Mediterranean climatic gradients

Temperature and precipitation regimes are rapidly changing, resulting in forest dieback and local extinction events, particularly in Mediterranean-type climates. Strategic forest management approaches that enhance forests’ resilience to future climates are urgently required, however adaptation to climates in heterogeneous landscapes with multiple selection pressures may be complex. For widespread trees in Mediterranean-type climates we hypothesized that patterns of local adaptation are associated with climate; precipitation is a stronger factor of adaptation than temperature; functionally related genes show similar signatures of adaptation; and adaptive variants are independently sorting across the landscape. To test our hypotheses, we sampled 28 populations across the geographic and climatic distribution of Eucalyptus marginata (jarrah), in south-west Western Australia, and obtained 13,534 independent single nucleotide polymorphic (SNP) markers across the genome. While overall levels of population differentiation were low (FST=0.04), environmental association analyses found a total of 2,336 unique SNPs potentially associated with five climate variables of temperature and precipitation. Allelic turnover was identified for SNPs associated with temperate seasonality and mean precipitation of the warmest quarter (39.2% and 36.9% deviance explained, respectively), suggesting that both temperature and precipitation are important factors in adaptation. SNPs within similarly function genes, according to gene ontology enrichment analysis, had analogous allelic turnover along climate gradients, while SNPs among temperature and precipitation variables had orthogonal patterns of adaptation. These contrasting patterns of adaptation provide evidence that there may be standing genomic variation adapted to changing climates, providing the substrate needed to promote adaptive management strategies to bolster forest resilience in the future.

Effects of Fruit Maturity Stages on GC-FID Fatty Acid Profiles, Phenolic Contents, and Biological Activities of Eucalyptus marginata L.

The objective of this study was to determine the impact of development stages of Eucalyptus marginata’s fruits on the fatty acid composition as well as on phenolic, flavonoid, and tannin contents of oils. Taking into account fruit maturity stages, vegetable oils have been evaluated for their biological potentials. Fatty acid profiles were quantified using gas chromatography (GC) coupled to a flame ionization detector (FID). The fatty acid profiles of oils obtained from mature fruits showed highest linoleic acid content (49.21%) and Z-vaccenic (C18:1n-7) + oleic (C18:1n-9) acids (22.40%) and a low content of linolenic acid (C18:3) (1.59%). On the other hand, the major saturated fatty acid compound found in the oil of immature Eucalyptus marginata fruits was palmitic acid (C16:0) with about 27%. Based on the Folin–Ciocalteau method, the obtained results revealed a significant difference in the contents of total polyphenols, flavonoids, and tannins according to the stage of fruit maturity ( p < 0.05 ). Furthermore, the detected antimicrobial potentials were related to the fruit maturity stage. While both veg\etable oils extracted from mature and immature Eucalyptus marginata fruits exhibited notable antibacterial activities against the species Staphylococcus aureus, Enterococcus faecalis, Serratia marcescens, and Escherichia coli, only the oils extracted from immature fruits exhibited an antifungal activity against Candida parapsilosis.

Inhibition of Dermatophyte Fungi by Australian Jarrah Honey

Superficial dermatophyte infections, commonly known as tineas, are the most prevalent fungal ailment and are increasing in incidence, leading to an interest in alternative treatments. Many floral honeys possess antimicrobial activity due to high sugar, low pH, and the production of hydrogen peroxide (H2O2) from the activity of the bee-derived enzyme glucose oxidase. Australian jarrah (Eucalyptus marginata) honey produces particularly high levels of H2O2 and has been found to be potently antifungal. This study characterized the activity of jarrah honey on fungal dermatophyte species. Jarrah honey inhibited dermatophytes with minimum inhibitory concentrations (MICs) of 1.5–3.5% (w/v), which increased to ≥25% (w/v) when catalase was added. Microscopic analysis found jarrah honey inhibited the germination of Trichophyton rubrum conidia and scanning electron microscopy of mature T. rubrum hyphae after honey treatment revealed bulging and collapsed regions. When treated hyphae were stained using REDOX fluorophores these did not detect any internal oxidative stress, suggesting jarrah honey acts largely on the hyphal surface. Although H2O2 appears critical for the antifungal activity of jarrah honey and its action on fungal cells, these effects persisted when H2O2 was eliminated and could not be replicated using synthetic honey spiked with H2O2, indicating jarrah honey contains agents that augment antifungal activity.

Development and validation of a new microplate assay that utilises optical density to quantify the antibacterial activity of honeys including Jarrah, Marri and Manuka

The phenol equivalence assay is the current industry-adopted test used to quantify the antibacterial activity of honeys in Australia and New Zealand. Activity is measured based on the diffusion of honey through agar and resulting zone of growth inhibition. Due to differences in the aqueous solubilities of antibacterial compounds found in honeys, this method may not be optimal for quantifying activity. Therefore, a new method was developed based on the existing broth microdilution assay that is widely used for determining minimum inhibitory concentrations (MICs). It utilises the four organisms Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and an optical density endpoint to quantify bacterial growth. Decreases in bacterial growth in the presence of honey, relative to the positive growth control, are then used to derive a single value to represent the overall antibacterial activity of each honey. Antibacterial activity was quantified for a total of 77 honeys using the new method, the phenol equivalence assay and the standard broth microdilution assay. This included 69 honeys with undisclosed floral sources and the comparators Manuka, Jarrah (Eucalyptus marginata), Marri (Corymbia calophylla), artificial and multifloral honey. For the 69 honey samples, phenol equivalence values ranged from 0–48.5 with a mean of 34 (% w/v phenol). Mean MICs, determined as the average of the MICs obtained for each of the four organisms for each honey ranged from 7–24% (w/v honey). Using the new assay, values for the 69 honeys ranged from 368 to 669 activity units, with a mean of 596. These new antibacterial activity values correlated closely with mean MICs (R2 = 0.949) whereas the relationship with phenol equivalence values was weaker (R2 = 0.649). Limit of detection, limit of quantitation, measuring interval, limit of reporting, sensitivity, selectivity, repeatability, reproducibility, and ruggedness were also investigated and showed that the new assay was both robust and reproducible.

Within-Tree Distribution and Survival of the Eucalyptus Longhorned Borer Phoracantha semipunctata (Coleoptera: Cerambycidae) in a Mediterranean-Type Ecosystem

The attack patterns, infestation success and larval development of woodborers within living trees are complex and are largely shaped by host tree characteristics. Following a severe drought in a native eucalypt forest where outbreak densities of a native Australian beetle, the eucalyptus longhorned borer (Phoracantha semipunctata), occurred, a tree dissection study was conducted in Australia. This involved felling 40 trees each of jarrah (Eucalyptus marginata) and marri (Corymbia calophylla) that were cut into 1-m sections and neonate larval galleries, larvae in pupal cells and adult borer emergence were measured and added to give total numbers per tree to determine the within-tree distribution and survival of P. semipunctata. There was a significant impact on larval survival in both species, in contrast, pupal survival remained high. Within-tree distribution of P. semipunctata was directional with borer emergence and incidence of larval galleries both negatively associated with tree section height above the ground and positively associated with section diameter and bark thickness, reaching a maximum towards the base of trees. High incidence and survival in lower thicker tree sections indicate a more conducive environment for larval development, in contrast to poor larval survival in smaller thinner sections at the top of trees. The dependence of larval survival on tree characteristics controlling the within-tree distribution of borer emergence is emphasized, and needs to be considered when estimating the spread of borer populations during outbreaks.