Arbuscular mycorrhizal fungal population assessment in soils

Soil samples collected from each treatment plot from both field Sites were sieved through a 2mm sieve to remove large debris. Air dried sub sample (100 g) was taken from each sample and placed in a 500 ml beaker containing 200 ml 0.08 M sodium hexametaphosphate solution to break up clay clumps. The suspension was agitated for 5 mins and left to settle for 15 secs (Smith and Dickson, 1997). The supernatant was decanted through a nest of sieves with reducing mesh sizes from 425µm, 250µm, 125µm to 45µm. This step was repeated with water twice and the debris from the 425µm was discarded. The debris on the remaining sieves, containing the AM spores was washed into 50 ml centrifuge tubes for purification. The spore suspension was centrifuged (J.P. Selecta S.A) at 3000 rpm for 5 mins, after which the supernatant was discarded. The pellet was re-suspended in 60% sucrose solution and centrifuged for another 5 mins. The supernatant containing AM fungal spores was decanted into a 45µm sieve and washed with water to remove sucrose on the spores (Smith and Dickson, 1997).

Spore enumeration

Spores were then washed onto a 9 cm grided filter paper disc (Whatman #1) in a Buchner funnel and vacuum filtered. The filter paper was transferred to clean Petri dish lids and enumerated. AM fungal spore enumeration included both dead and viable spores, although every attempt was made to count only healthy looking spores.

Spores were recorded as representatives of AM fungal species present in 100g of sample (Smith and Dickson, 1997). This was done using a dissecting microscope, Leica S4E.

2.6.2 Root colonisation

Root samples from pot trials, Site 1 and Site 2, were carefully washed and cut into 1-3cm sections. Sections were covered with 5% KOH solution and incubated at 90°C for 45 mins, to remove the cytoplasm and all coloured material from the plant cells.

The KOH solution was discarded and the roots were rinsed well with distilled water.

Roots were covered with a freshly prepared alkaline H2O2 solution (Appendix G) to bleach for 60 mins. The bleaching solution was discarded and the roots were rinsed with water. Roots were acidified in a 0.1M HCl solution overnight to ensure adequate binding of stain to fungal structures. The HCl solution was discarded and roots were covered with Lactoglycerol Trypan Blue (0.05%) stain and incubated for 45 mins at 90°C. The stain was poured off and roots were covered with lactoglycerol destain (Appendix G). Roots were allowed to destain overnight before microscopic examination (Smith and Dickson, 1997). Finally, roots were mounted on microscopic slides and using a compound microscope examined. The percentage root colonisation was calculated using a modified Line Intersect Method (McGonigle et al., 1990). This method involved squashing segments of stained roots on a microscope slide after it is covered with a cover slip. Roots were selected and examined for their entire length, a field of view at a time. One field of view using 40 x magnifications was scored at intersects between an eyepiece micrometer with or without mycorrhizal structures (arbuscules, vesicles or hyphae). The number of mycorrhizal structures in a 100 fields of view was equal to the percentage colonisation.

2.6.3 Glomalin extraction and quantification

Glomalin, a glycoprotein commonly produced by AM fungi was extracted from soil (1g) samples collected from trial plots as described by Wright and Upadhyaya (1996;

1998). The total protein extraction was carried out with 8 ml, 50mM sodium citrate, pH 8.0 at 121˚ C for 60 mins. Samples were centrifuged immediately at 5000g for 15 mins and were repeated twice. Supernatant containing protein was removed and stored at 4˚C for quantification.

Bradford assay

Concentration of glomalin (mg/ml) was determined with the Bradford assay, using Bovine Serum Albumin as a standard (Bradford, 1976). BSA stock solution was prepared by dissolving 16mg of BSA (Sigma Cat no. A3294) in 10 ml triple distilled water. Standard concentrations ranging from 0.1-1.6 to a final volume of 160µl (Appendix H) were prepared using stock solution and water. A volume (5µl) of standard, blank (water) and test samples were put on microtitre plates, after which 250µl of Bradfords reagent (Sigma Cat no. B6916) was added into each wells and left for 15 mins. Spectroscopy readings were taken at 595nm using a Power Wave X Spectrophotometer (Biotek). Readings from the test sample were compared to the standard curve (Appendix H), which is known to exhibit a linear relationship (Bradford, 1976). Concentrations were calculated using the equation y = ax + bc derived from the standard curve (Appendix H), where x gives the value of the unknown concentration.

2.6.4 Most Propable Number

An average most probable number (MPN) was conducted using soil samples from treatment and control plots of each Site. Procedure followed was that of Smith and Dickson (1997). It involved a series of ten-fold soil-soil dilutions, using test soil and pasteurised soil. Each dilution had five replicate plants. A 10^-1 dilution was made by adding 200g of test soil to 1800g of sterilized soil in a plastic bag. Samples were mixed thoroughly and 200g were removed for the next dilution (10^-2). The remaining soil sample was put into torpedo tubes that were pre-washed in 30% sodium hypochloride. The next dilution (10^-2) was made by using the 200g from the previous

dilution and mixing it with 1800g of sterilized soil. 200g from the mixture were also kept for the next dilution. This was repeated until the 10^-4 dilution. Seedlings (Sorghum grain), used as trap plants, were surface sterilised in 30% sodium hypochloride for 20 mins. The seeds were then rinsed thoroughly with water and planted into the tubes starting from the highest dilution. Seeded trays were kept in a plastic tunnel where they were watered daily with UV sterilised water for 4 weeks.

Plant roots were stained (section 2.6.2) and examined for the presence or absence of AM fungal colonisation under a compound microscope. A plus was given when any mycorrhizal structures were found in the entire root system.

MPN infectivity assessment

The MPN readings were calculated using equations based on the probability theory from the number of root systems in dilutions made above i.e. 10^-1- 10^-4 (Smith and Dickson, 1997). From the results, the P values (P1, P2 and P3) were obtained. Where P1 was the number of mycorrhizal plants in the least concentrated dilution that had the greatest colonisation; P2 and P3 was the number of plants with mycorrhizal colonisation in the next two higher dilutions. Using the probability table (Cochran, 1950), the row to which the observed P1 and P2 corresponds to P3 was taken as the MPN of propagules in that compartment. The value was then multiplied by the dilution to give the MPN per 200g of soil.