Sampling was done by using a clean plastic scoop and collecting soil to a 10-cm depth. The soil was scooped onto a 2mm screen and then sifted into a clean metal pan. After sifting, the rocks were discarded and the screen was removed. 100mL of the soil was then poured through a funnel into a 500mL Nalgene bottle containing 100mL of 1.25M sugar solution. The bottle was tightly capped for transportation back to the lab.  

 

The soils were processed within 3 days of collection from the field. Until the n, the soils were stored in a 4_C refrigerator.  To extract the nematodes, first the bottles were filled with an additional 100 mL of 1.25M sugar solution. Then, a long spatula was used to stir the soil so that it was suspended in the sugar (30 seconds of stirring). Next, the sample was poured onto a set of No. 40 over No. 400 U.S.A. standard testing sieves that had been pre-wetted with 1.25M sugar solution. The top screen was rinsed with 1.25M sugar solution and then removed.  The bottom screen was rinsed with 1.25M sugar solution that was squirted only on the front of the screen at an angle. Using this rinsing method, the soil was carefully worked to the bottom wedge of the screen, and the sugar volume was reduced by gently tapping the screen and letting more solution pass through.  Next, the sample was rinsed into a 150mL beaker through a funnel, and then the funnel rinsed to catch any residue.

 

Once the sample was collected into the 150mL beaker, the sugar and sediment was pipetted (using an automatic pipette) into a centrifuge tube containing 2M sugar solution. The pipetting was done slowly and at an angle so that the boundary between the two sugar layers was maintained. Then the beaker was rinsed wi th 1.25M sugar solution and the dregs were pipetted into the centrifuge tube as well. When necessary, more than one centrifuge tube was used to collect all the sample.

 

Next, the tubes with sample were centrifuged for five minutes at 1744 RPM. Then, the liquid contents of the tube were poured through a No. 500 sieve. Finally, the contents of the sieve were rinsed into glass centrifuge tubes and stored at 4_C until counting. Samples were washed in to a counting dish and examined under a microscope at x100 or x200 magnification. Nematodes were identified as coiled or straight and counted. Total numbers in each sample were recorded on data sheets. Data were entered in to Excel files, printed, and checked for errors.