2.6. Root colonization
Roots were washed in running tap water, cleaned by soaking in 10% KOH for 4 days, acidified with 1 N HCl for 5 min, and then stained with lacto-phenol cotton blue. The roots were observed under 630X optical microscope (Olympus BH2, Leeds Precision Instrument, Inc.). Estimation of percent colonization was done using the grid line-intersect method (Norris et al., 1994 and Varma and Kharkwal, 2009). Spores were counted under a stereo binocular microscope. Percentage root colonization was calculated with the formula: percent colonization = number of colonized roots × 100/number of roots observed.
2.7. Measurement of egg population density
Plants were harvested 60 days after inoculation of juveniles. The soil was thoroughly mixed, and a subsample of 100 cc soil (weight of approximately 190 g of soil ≈ 100 cc soil) was collected in a 1-liter beaker and soaked in 500 ml water for 30 min. The eggs were isolated and cleaned using the procedure described above. The eggs were then suspended in 50-mL water and the number of nematode eggs in a 200 μL subsample was counted using an inverted microscope in a petri-plate marked with a grid. The total number of eggs per 100 cc soil was calculated with the following formula: total number of eggs = number of eggs counted in sample volume × total volume of egg suspension/sample volume of egg suspension counted.