Crecimiento de Bacillus en su hábitat natural

2.3.1 Sampling methods and location

A permit to allow plant material collection was obtained from the Tasmanian state government. Coastal reserves around the state were then surveyed for the presence of CR by A. Pirie, C. Narkowicz and J. Renggli. When plant material was found in suitable quantities (i.e. multiple plants greater than 1 metre in diameter), soil (0-15, 15-30, 30-45 cm) and plant samples were taken. The GPS coordinates, site description and amount of shelter plants had (High = 3, Medium = 2, Low = 1) was recorded. Chlorophyll fluorescence characteristics were measured non-invasively on each plant.

2.3.2 Chlorop

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Chlorophyll fluorescence characteristics (Fo, Fm, and Fv/Fm) were measured in the field using an Opti-Sciences OS-30P chlorophyll fluorometer (Opti-Sciences, New Hampshire, USA). Measurements were conducted on 15 minute dark-adapted samples by using leaf clips equipped with light shutters. For each plant, three laterals were selected. Leaf clips were placed on the first mature leaf at the growing tip and the next three mature leaves. On occasions when new laterals were growing close to the tip the next large main-branch leaf was chosen. During preliminary investigations some plants were found to have extremely small leaves and the leaf clip aperture was reduced to 4 mm by gluing black cardboard "washers" onto the leaf clips. This ensured a proper fit on the leaf and prevented erroneous readings from either excess light exciting the leaf chlorophyll or fluorometer radiation bypassing the leaf surface.

2.3.3 Soil Sampling

For each plant or group of plants (if less than 10 m apart), 500 g of soil was collected from 0- 15, 15-30, and 30-45 cm depth. This was taken back to the laboratory, air dried, passed through a 2 mm screen (Endecotts, London, England) and then stored prior to analysis. Sub- samples were weighed into 50 ml centrifuge tubes, mixed 1:5 (w/v) with distilled water and shaken. Na+ and K+ levels were determined via flame photometry (PFP7, Jenway Pty Ltd, Essex, England), Cl- with an ion specific electrode (ISE) (TPS Ltd, Brisbane, Australia), pH using a pH probe (EZ DO PL 600, M.R.C Ltd, Holon, Israel) and electrical conductivity (EC) using an electrical conductivity meter (labCHEM-C, TPS Ltd, Brisbane, Australia).

2.3.4 Stomata density

The epidermis from two leaves per sample were removed, placed on a microscope slide and stomatal counts were conducted using a Leica DM 500 microscope (Leica Microsystems, Germany). Five locations per leaf were counted with density defined as the number of stomata within the 0.52 mm diameter field of view.

2.3.5 Leaf sap nutrient analysis and osmolarity

Leaf sap nutrient analysis and osmolarity were conducted as described by Cuin et al., (2010). In brief, freeze-thawed leaf samples were crushed to extract the leaf sap. The supernatant then transferred to a second tube, frozen and stored until thawing for analysis. Leaf osmolarity was assessed using a VAPRO vapour pressure osmometer 5520 (Wescor Inc, Logan, Utah, USA). Sap samples were then diluted 100 times and measured for Na+ and K+ concentration using flame photometry (as before).

2.3.6 Flavonoid concentration by HPLC

Leaves were randomly selected, washed under deionised water, dried and ground with a mortar and pestle. The ground plant material was then placed in an Eppendorf tube and centrifuged at 15000 g for 5 minutes. Following this the supernatant was transferred to a HPLC sample vial for analysis.

HPLC analysis was conducted using a Varian Pro-Star 230 solvent delivery system coupled with a Varian 410 auto sampler and Varian 335 diode array detector (DAD); operation of these modules was achieved using Varian Star software (all HPLC modules and software supplied by Varian Inc, Melbourne, Australia). The column was an Apollo 5 µm x 250 mm C-18 (Grace, Deerfield, USA) fitted with a guard column (Phenomenex, Security Guard®, Torrance, USA). Eluent profiles used were those optimised for CR by Renggli (2010) and summarised in Table 2.1. Injection volume was 15 l with the spectrum between 200 nm and 450 nm scanned and wavelengths of 254 nm and 350 nm monitored.

Table 2.1. HPLC conditions for flavonoid and tannin detection.

Time (min) Flow rate (mL\minute) % solvent A % solvent B

initial 0.8 60 40 5.00 0.8 45 55 5.01 0.6 45 55 20.00 0.6 45 55 25.00 0.6 45 55 25.01 0.8 45 55 30.00 0.8 0 100

For each sample, area under the curve (AUC) integration was performed for the 350 nm wavelengths. Peaks showing simultaneous 254 nm and 350 nm absorbance with a retention time of less than 9 minutes were assigned as tannins (or flavonoids of non-pharmaceutical interest), whilst peaks from 9 minutes onwards were deemed to be flavonoids. This assignment was based on previous mass spectroscopy results.

2.3.7 Antioxidant activity assessment

Antioxidant activity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay reviewed by MacDonald-Wicks et al., (2006). The method was slightly modified to enable analysis by a microplate reader and absorbance was measured at 490 nm instead of a wavelength between 515 nm and 528 nm. Briefly, DPPH (Sigma-Aldrich, St Louis, United States of America) was diluted in methanol until a 250 laliquot placed in a flat bottomed 96 well microplate (Greiner Bio-one, Monroe, United States of America) gave an absorbance at 490nm of 1.50 ± 0.05 units. A 12 l aliquot of gallic acid (either 100, 50,

Solvent A was 2% acetic acid in H2O;

Solvent B was 2% acetic acid in methanol. Dual wavelength (254 nm, 350 nm) monitoring and a C18 analytical column were used. A five minute equilibration time occurred between runs.

25, 12.5, 6.25, 3.125 g/ml) or leaf sap (undiluted, diluted 1:2, 1:4, 1:8, 1:16, 1:32) was added to sequential microplate wells, followed by 250l of DPPH solution. The plate was covered with foil and stored in the dark for 30 min. After this time the absorbance at 490 nm was measured on a microplate reader (Model 680, Bio Rad Laboratories Pty Ltd, Sydney, Australia) and extract antioxidant activity converted to gallic acid equivalents.

2.3.8 Glasshouse experiment

Concentration-dependent responses to salinity were studied at the University of Tasmania’s glasshouse complex (Hobart, Tasmania, Australia). The average day/night temperatures were 23/17oC; relative air humidity 55 to 70%; natural day length of approximately 15 hours. Cuttings from donor clones were taken and 5 cuttings were planted in each 14 × 200 mm pot. These were then grown under ambient conditions for 2 weeks until they had established and then treatments were applied. Treatments consisted of distilled water with NaCl added to make concentrations of 0, 50, 100, 200, 300, 400 and 500 mM respectively, with two pots used per treatment. All plants were monitored and watered to saturation every 2-3 days until the completion of the trial at 21 days.

After 21 days, soil was washed out of the pots and plants were individually assessed for root, stem and leaf length; fresh weight; and the number of new leaves produced. The two newest mature leaves from each plant were taken for more in depth analysis including leaf weight, length, width, and height before the photosynthetically active outer leaf was separated from its vacuolated inner core. These segregated sections were frozen and then analysed for osmolarity, antioxidant activity (DPPH), flavonoid content (HPLC), Na+ and K+content (as described earlier). The remaining plant was separated into roots and stems then oven dried (60 oC, 48hrs) prior to assessment of dry matter production.

2.3.9 Statistical Analysis

Multiple regressions were performed in Minitab 15 (Minitab Inc, Sydney, Australia) using site modelled climatic variables obtained from the SILO website (QCCE, 2011) and as individual soil test and plant parameter results.

The means for each plant were subjected to a hierarchical cluster analysis in Minitab 15, using the variables flavonoid and tannin concentration (centroid linking method for measuring Euclidean distance, after transforming using Z-scores to standardise across variables measured in different units), to generate a dendrogram illustrating relationships between plants. Differences between the treatment groups in glasshouse trail were assessed using the statistical program JMP8 (SAS Institute Inc., North Carolina, United States of America)using an ANOVA analysis. Differences between treatments and the control (50mM NaCl) were assessed using Dunnett’s method.