2 GENERAL METHODS
2.1 COLLECTION OF SOIL AND GENERAL HARVEST TECHNIQUES
2.1.1 SOIL COLLECTION AND PROCESSING
Figure 2.1: Site location where the loamy sand (Newport series), clay loam (Worcester series) and sandy loam (Dunnington heath series) were collected.
In all experiments top soil (5!20 cm depth) was either collected from one or three different field sites each with different soil textures. The soils examined included the Newport series, a loamy sand (brown sand) and Worcester series, a clay loam
DUNNINGTON HEATH NEWPORT
WORCESTER WORCESTER
NEWPORT
Chapter 2: General Methods Page 44
(Argillic Pelosol) from the University of Nottingham’s experimental farm site at Bunny, Nottinghamshire (GB Ordnance Survey Grid Reference: SK 587 294 and SK 587 289 respectively) and the Dunnington Heath series, a sandy loam (Stagno Glegic Luvisol) from the University of Nottingham farm site at Sutton Bonington, Leicestershire (GB Ordnance Survey Grid Reference: SK 512 267) (Figure 2.1). This Dunnington Heath (sandy loam) top soil was used within all experiments. Selected soil physical and chemical characteristics of these soils are given in Table 2.1 and Figure 2.2.
0 20 40 60 80 100 120
0.0001 0.001 0.01 0.1 1
Particle Size Diameter (Cm)
Total Volume (%)
Loamy sand Clay loam Sandy loam
Figure 2.2: Particle size distribution of each of the three soils used within the experiment (determined by laser particle analysis).
Note: Red lines indicate the 10 % and 60 % points at which the coefficient of uniformity is calculated (section 2.2.3).
Chapter 2: General Methods Page 45
Table 2.1: Characteristics of soils used in microcosms
Particle size analysis of the samples was undertaken using laser particle analysis (section 2.2.1).
* Organic matter content determined by loss on ignition.
Upon collection, the soil was air dried and sieved to < 2 mm before sealing the processed soil in double plastic bags containing ~ 7!8 kg for sterilisation using gamma radiation (Isotron Ltd. Daventry, UK) unless otherwise stated.
2.1.2 INOCULATION OF SOIL
After soil processing and sterilisation, soil was packed into microcosms and macrocosms of varying sizes, as stated in the appropriate experimental chapters.
Experimental macrocosms were inoculated using the dilution technique (Salonius, 1981; Griffiths et al., 2001). Soil micro! or macrocosms were inoculated using a soil slurry solution, made from fresh field soil (taken from the respective field site where the soil texture was collected from) by diluting it in ¼ strength sterile Ringers solution (where full strength Ringer solution is: 2.25 g NaCl, 0.105 g KCl, 0.12 g CaCl2 and 0.05 g NaHCO3 dissolved in 1 L of sterile de!ionised water (Dickinson Austin and Goodfellow, 1975)). The soil slurry solution was made to differing dilutions depending on the experiments in question. A 10!1 soil suspension was prepared by mixing 100 g of field fresh to 1000 ml ¼ strength sterile Ringers solution, with
Chapter 2: General Methods Page 46
subsequent serial dilutions make to a 10!6 dilution. Generally however either a 101 (low), 106 (high) or no dilution (i.e. just sterilised water) was used to inoculate experimental soil.
Inoculation involved saturating the sterilised air!dried micro! or macrocosms overnight in a specifically diluted soil slurry solution (by placing the micro! or macrocosms in trays containing the inocula) allowing capillary uptake of the solution containing microorganisms throughout the soil. Once the cores were saturated, they were removed from the solution and left to drain for 2 days to reach field capacity and weighed prior to the start of experiments.
Depending on the experimental setup, some macrocosms also underwent planting with Plantago lanceolata (Herbiseed, Twyford, UK) either by transplanting seedlings or by growing P. lanceolata directly from seeds within the macrocosms. P. lanceolata was selected for the experiment due to its known mycotrophy (Šmilauer, 2001) and since AMF colonisation does not affect the lifespan of P. lanceolata roots (Hodge, Robinson and Fitter, 2000).
2.1.3 HARVEST TECHNIQUES
Soil macrocosms were destructively harvested at specific harvest periods after inoculation and plant transplanting or establishment for microbial and structural assessment. At each harvest above ground plant biomass (from treatments containing plants) was determined by removing the plant at the soil level before macrocosm destruction. Soil from each sampled macrocosm was removed gently to prevent destruction of the soil aggregates and damage to roots (where present). All possible
Chapter 2: General Methods Page 47
root material was gently removed from the soil, with subsections removed and stored in 70 % ethanol or frozen at !80 oC for assessment of AMF colonisation, with the remaining root material used to estimate the total below ground plant biomass (after taking into account the weight of the undried subsample). Soil removed from macrocosms was homogenised gently prior to sub!sampling for immediate determination of soil moisture, organic matter content (loss on ignition) and metabolic potential. Additional subsamples were taken for soil biomass and relative abundance determination and stored at !20 oC and !80 oC respectively. A subsample of soil, for assessment of the soil structure i.e. (aggregate size distribution, aggregate stability and water repellency), was also removed and left to air dry.
Additional soil structural analysis was undertaken using X!ray JCT and X!ray CT depending on column size. Separate micro! and macrocosms were specifically used for this assessment for each experiment. In all experiments the same set of macrocosms were scanned to allow changes in soil structure overtime to be assessed.
2.2 ANALYSIS OF SOIL PHYSICAL PROPERTIES
This section focuses on the techniques used to determine soil texture, soil aggregate stability, total porosity and pore size and morphology.
2.2.1 SOIL TEXTURE
Air dried soil was sieved to < 2 mm in size, with 0.5 g weighed into a 50 ml centrifuge tube. Soil organic matter was chemically removed from the soil using 25 ml of hydrogen peroxide (H2O2) overnight. To ensure all organic matter had been
Chapter 2: General Methods Page 48
removed from the soil sample, the centrifuge tube was placed in a 60 oC water bath for 1!1.5 hours with the temperature raised to 90 oC for an additional 1!1.5 hours.
Samples were topped up with 25 ml of deionised water prior to centrifuging at 3500 rpm for four minutes. The remaining solution was decanted off, with an additional 35 ml deionised water added to the sample prior to centrifuging at 3500 rpm for four minutes again. The remaining solution was decanted and 25 ml of calgon (35 g of sodium hexametaphosphate, 7 g sodium carbonate in 1 L of de!ionised water) added before shaking and placing in an ultrasonic bath for 30 minutes. Samples were then analysed in a particle size analyser (Beckman Coulter LS230, Beckman Coulter Inc., High Wycombe, UK).
2.2.2 SOIL MOISTURE AND ORGANIC MATTER CONTENT
Soil moisture at column harvest and organic matter content were determined by oven drying samples and determining the loss on ignition (Rowell, 1994).
Soil samples from the soil columns were placed in weighed crucibles. The water content of soils was determined by drying at 105 oC overnight and using Equation 2.1.
Water content = Mass of fresh soil – Mass of oven!dry soil (Eq. 2.1) Mass of oven!dry soil