Abstract
Data: REACCHPNA Biotics - Root Lesion Nematode 2011 Soil sample collection. Two field surveys for P. neglectus, P. thornei and H. avenae were conducted by collecting soil samples from Washington State University Extension Cereal Variety Trials and from growers fields during mid May to mid June in 2010 and 2011. Ninety soil samples were collected in 2010 from spring wheat, winter wheat, spring legumes and spring barley and 84 samples were collected in 2011. Survey sites were located in Whitman, Adams, Walla Walla, Spokane, Lincoln, Grant, Columbia, Douglas, and Franklin counties, representing six major PNW agronomic zones described by Douglas et al. (1992). Root zone soil from 0 to 25 cm depth was collected from major dryland crops including winter wheat, spring wheat, spring barley, spring chickpea, spring lentil, and alfalfa. The root zone soil along with plants was dug in V-shape by shovel and collected in polythene bags. Three soil samples in each field were taken from 100, 25 and 25 paces away from the border of the field in zig-zag manner (Fig. 1). Those three sub samples were mixed in one bag and taken as one sample from each respective field. The collected soil samples were then kept in a cooler and stored at 4oC until being were sent to Western Laboratories, Parma, Idaho for analysis. Similarly, in 2010 two additional field surveys for H. avenae were conducted by collecting soil samples from Whitman County where H. avenae distribution was suspected. A survey was done on the 27th of July 2010 when spring wheat was at heading stage. Another survey was done on the 27th of September 2010 after wheat harvesting. In 2011, the large general survey was repeated in the same way. An additional survey for cereal cyst nematode was conducted on the 1st August, 2011. Nematode extraction. All collected soil samples from field surveys were sent to Western Laboratories, Parma, ID to extract P. neglectus and P. thornei and other plant parasitic nematodes. A modified elutriation method (Ingham 1994) was used to extract nematodes from 250 cc (cubic centimeters) soil for each sample. The measured 250 cc soil was placed on a 35-mesh (500 microns) sieve and washed into a modified Ostenbrink elutritator. Soil on the sieve was drained using the water nozzle above it. The water spray strikes soil particles and helps to dislodge the nematodes in the soil suspension. The suspension in the funnel was drained through a set of 4 sieves. The upper two were 400-mesh (38 microns) and lower two were 450-mesh (32 microns). Sieves were washed immediately with a small amount of water and collected in a cup. The collected water settled for about 2 hours in the same cup. Then water was siphoned off very gently from the top and the nematode suspension at the bottom of cup was saved. The nematode suspension was then transferred to a 50 ml centrifuge tube and centrifuged at 1800 g for 5 minutes. After centrifuging, the supernatant was poured off and the pellet was retained in the tube. The pellet was resuspended in a magnesium sulfate solution and adjusted to specific gravity of 1.18. The suspension was then centrifuged at 1800 g for 4 minutes to bring nematodes to the top layer in the magnesium sulfate solution. Nematodes suspended in the top layer were transferred from Whitman County where H. avenae distribution was suspected. A survey was done on the 27th of July 2010 when spring wheat was at heading stage. Another survey was done on the 27th of September 2010 after wheat harvesting. In 2011, the large general survey was repeated in the same way. An additional survey for cereal cyst nematode was conducted on the 1st August, 2011. Nematode extraction. All collected soil samples from field surveys were sent to Western Laboratories, Parma, ID to extract P. neglectus and P. thornei and other plant parasitic nematodes. A modified elutriation method (Ingham 1994) was used to extract nematodes from 250 cc (cubic centimeters) soil for each sample. The measured 250 cc soil was placed on a 35-mesh (500 microns) sieve and washed into a modified Ostenbrink elutritator. Soil on the sieve was drained using the water nozzle above it. The water spray strikes soil particles and helps to dislodge the nematodes in the soil suspension. The suspension in the funnel was drained through a set of 4 sieves. The upper two were 400-mesh (38 microns) and lower two were 450-mesh (32 microns). Sieves were washed immediately with a small amount of water and collected in a cup. The collected water settled for about 2 hours in the same cup. Then water was siphoned off very gently from the top and the nematode suspension at the bottom of cup was saved. The nematode suspension was then transferred to a 50 ml centrifuge tube and centrifuged at 1800 g for 5 minutes. After centrifuging, the supernatant was poured off and the pellet was retained in the tube. The pellet was resuspended in a magnesium sulfate solution and adjusted to specific gravity of 1.18. The suspension was then centrifuged at 1800 g for 4 minutes to bring nematodes to the top layer in the magnesium sulfate solution. Nematodes suspended in the top layer were transferred through a 635-mesh (20 micron) sieve, the sieve was washed with a small amount of water and the nematodes were collected in a 20 ml/50 ml falcon tube. The nematodes were counted under a microscope and reported as the number of nematodes per 500 cc of soil. Extraction of H. avenae. For the region-wide surveys, H. avenae cysts were extracted by Western Labs using a coarse sieve of 500 micron at the top and a fine sieve of 125 micron at the bottom with a sieving method. Cysts collected from the fine sieve were crushed using a drill-like pestle by stirring at 7500 rpm for 3 minutes. Water suspension containing H. avenae eggs and juveniles were centrifuged first at 3200 rpm for 5 minutes and later using MgSO4 at same speed for 4 minutes. Floated eggs and juveniles were transferred through a 20-micron sieve and finally collected in a 50 ml tube, counted and reported as the number of eggs and juveniles per 500 cc of soil. For the supplemental surveys of H. avenae in Whitman county, cysts were extracted in the facilities of the USDA Root Disease and Biological Control Research Unit by sieving from air-dried soils collected on 27th of July and 27th of September, 2010 (Shurtleff and Averre III 2000) and using the Fenwick can method (Fenwick 1940). Cysts were drained off in 125 mm diameter Whatman filter paper and handpicked with a dissecting needle under a stereomicroscope. Cysts were squashed and second stage juveniles and eggs were counted under a compound microscope. The numbers of juveniles and eggs were recorded as nematode density per 500 cc of air-dried soil. Geographical Positioning System (GPS) coordinates of survey sites. GPS latitude and longitude coordinates in degrees, minutes, and seconds were recorded for every collection site and converted to decimal degrees. Nematode populations for both 2010 and 2011 were plotted in different agrocliamtic zones of eastern Washington described by Douglas et al. (1992) using ArcMap version 9.3 (ESRI, Redlands, CA).