Nematode extraction and identification

Nematodes were isolated from 100 g of the mixed fresh soil samples by a combination of Cobb sieving and decanting (Cobb, 1918) and a modified Baermann techniques (Van Bezooijen, 2006). Nematodes were extracted from aqueous soil suspensions using a set of two cotton-propylene filters. Subsamples were removed after extraction for 48 hr at room temperature. The aqueous suspensions containing nematodes were examined under a stereomicroscope, excessive water was removed, and the nematodes were fixed in hot fixative 99:1 solution of 4% formaldehyde: pure glycerol and evaluated on permanent glycerine slides (Southey, 1986). All isolated nematodes were microscopically examined at 100, 200, 400, 600, and 1,000 × magnification, identified from permanent glycerine slides mostly to species level (juveniles were identified to genus level) using an Eclipse 90i Nikon, Japan light microscope, with original species descriptions, and several taxonomic keys: Brzeski (1998), Loof (1999), Siddiqi (2000), Andrássy (2005, 2007, 2009), and Geraert (2008, 2010).

Cysts of Heterodera juveniles were extracted by floatation (Sabová and Valocká, 1980) from 100 g of soil for species identification based on morphological markers and morphometric data for both cysts and juveniles.