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Root lesion nematodes are versatile parasites that move freely between root and soil habitats. Most laboratories conduct separate assays for soil or root tissue, using time-of-year as the selecting factor. We used a dual assay that simultaneously extracts nematodes from soil and root fragments in soil samples to identify the value of soil versus root tests using 920 research samples collected 1 April to 15 May, and 853 clinic samples collected year round. Nematodes were recovered from both soil and root fragments regardless of the time of year or origin of the sample. When the data were summarized by cohort, the mean percentage of nematodes recovered from root fragments was 65% for the research samples, 59% for clinic samples submitted 1 March to 15 June, 56% for clinic samples submitted 16 June to 31 July, and 49% for clinic samples submitted after 1 August. Both the incidence and population density of root lesion nematodes was underestimated if only the soil or only the root fraction was considered, indicating the need for testing methods that consider both habitats. The variability among samples for the distribution of nematodes between root and soil habitats was high, negating the option of running one assay and using a constant scaling factor to account for the other. Introduction Root lesion nematodes, Pratylenchus spp., are a common and important pest of many crops (10,14). Root lesion nematodes have a wide host range causing damage that varies according to nematode species, host plant, and soil conditions, but follows the general relationship that yield loss is proportional to nematode population densities at the time of planting (2,6,11). Measures to reduce population densities must be selected before the crop is planted, so estimates of nematode population densities for advisory purposes must often be made from soil samples when there is no living crop present. Migratory endoparasitic nematodes, like Pratylenchus spp., occupy both soil and root habitats for their entire life cycle. Upon hatching, all life stages feed on root cells. Nematodes remain in the rhizosphere when feeding on epidermal cells and enter completely into roots when feeding on cortical cells (17). In a sandy soil, 20 to 50% of Pratylenchus scribneri associated with potato or corn were recovered from bulk or rhizosphere soil from the time of planting until after harvest (4). Over the winter in the same site, 35 to 67% of the nematodes recovered were sheltered in dead root fragments (5). Methods to extract nematodes from soil and plant material are active or passive as determined by the role of nematode motility in the process. The most common methods for root assay are active, because they rely on egress of nematodes from roots incubated in funnels, trays, or flasks. Nematodes are separated from the soil matrix using passive methods such as decanting, filtering, and centrifugation. There are advantages and disadvantages for every extraction method and none is able to recover 100% of the nematodes present (9,15). Estimates of Pratylenchus spp. passively extracted from soil using filtering and centrifugal flotation methods were not predictive for corn yield in 20 November 2012 Plant Health Progress several studies (8,12,13) so some laboratories consider active root assays to be the only acceptable way to estimate population densities of root lesion nematodes (14). One disadvantage of using root assays for advisory purposes is that it is done after planting, past the point of mitigation measures for that crop. Another disadvantage is that nematode estimates scaled to root weight are not useful for multiple year comparisons in fields rotated with crops that vary for root biomass such as corn and soybean. We developed a protocol that performs active and passive extraction assays on the same soil sample, resulting in a complete census of Pratylenchus spp. from soil and root fragments within the soil. Estimates of the population density of P. penetrans in preplant soil samples processed using this protocol were predictive of yield for potato (6), as well as corn and soybean (A. E. MacGuidwin, unpublished). This method is used routinely for research samples and samples submitted to the University of Wisconsin Nematode Diagnostic Service. The assays are independent activities so we report here data showing the importance of assaying soil and root habitats when estimating population densities of Pratylenchus spp. Dual Extraction Protocol Soil samples were thoroughly mixed and soil cores and aggregates broken manually. A subsample was added to a graduated cylinder containing water until the water was displaced by 100 ml. The sample, now in water, was placed in a pitcher and stirred vigorously to suspend nematodes and light fraction material and then decanted through a 250-μm-pore sieve nested over a 38-μmpore sieve. Material retained on the two sieves was assayed separately. Active incubation assay. Material caught on the 250-μm-pore sieve was placed on a Baermann Funnel (1) with a 120-mm-wide aperture. Each funnel had a circular piece of 1-mm mesh wire screen (9.75-cm diameter) situated about 2.25 cm from the top of the funnel that was covered with a 2-ply facial tissue. Root fragments were present in all samples, but the proportion that appeared to come from living plants varied seasonally. A representative preplant sample is shown in Figure 1. The water level in the funnel was just adequate to cover the particulate matter resting on the tissue, so plastic film was placed on top of the funnel to retard evaporation. Samples were drained into 15-ml test tubes after 48 h. Passive sieving/centrifugal flotation assay. Material caught on the 38-μmpore sieve was centrifuged in water at 3000 RPM (1663 g) for 4 min to pellet the nematodes in the mineral fraction of the soil using methods similar to Jenkins (3). After decanting the water, the centrifuge tubes were then filled with a sucrose solution with a specific gravity of 1.14 and centrifuged again for 4 min to include the nematodes but not the mineral fraction of the sample. The sucrose solution was decanted over a 20-μm-pore sieve and rinsed with tap water to collect nematodes. Fig. 1. Root fragments and other residue caught on the 250-μm sieve from a typical soil sample (Plainfield loamy sand soil) assayed in May before a new crop was planted. 20 November 2012 Plant Health Progress The end product of both assays was a test tube filled with tap water with nematodes settled at the bottom of the tube. Samples were stored at 4°C until they were ready to be counted. Water was carefully drawn from the tube so as not to disturb nematodes and then the content of the tube was placed in a glass counting dish and examined using a stereomicroscope at 40x magnification. All plant parasitic nematodes in each sample were counted by genus. For samples with high abundance, the sample was mixed to evenly disperse the specimens and a portion of the dish was examined and the counts adjusted to represent the entire 100-cm3 sample. Species identification for the research samples was made using morphological features of adult females and males (if present). The extraction efficiency of the two assay methods were similar for adults and varied for different life stages, ranging from 9% for second-stage juveniles to about 40% for fourth-stage juveniles. The passive soil assay was superior for recovering adult P. scribneri (67% ± 11%) than the active root assay (36% ± 8%) for adults (7). Research with P. penetrans showed reproductive females were less likely to exit roots than males (16). Dual Assay Results for Pre-Plant Soil Samples from Research Plots Samples were collected prior to planting to provide estimates of nematode population densities for experiments. Table 1 shows preplant soil data collected over 13 years for root lesion nematodes from 30 research sites in central Wisconsin with a loamy sand soil texture. All sites were infested with P. penetrans, but other species were present in some samples. Multiple plots were sampled for each experiment (range = 16 to 99, total = 920) and each sample consisted of 6 to 10 bulked cores collected to a depth of 15 to 22 cm, purposely avoiding any live weed or volunteer crop plants. Nematodes were extracted from the samples within 10 days of collection. Samples with zero or one nematode were included in calculations of the mean for each experiment and excluded in estimates of the percentage of the populations sheltered within roots. Population densities of root lesion nematodes would have been dramatically underestimated if a passive sieving/centrifugal flotation extraction was the only procedure used. For 82% of the sites, greater than 50% the nematodes recovered were from very small dead root fragments. Averaged across all experiments, 65% of the total nematodes extracted were from the root incubation assay (Table 1). Some samples contained second-stage juveniles presumably hatched during the 48-h incubation, but they were outnumbered significantly by adults and thirdand fourth-stage juveniles. The grand mean for the number of root lesion nematodes extracted per 100 cm3 soil was 221 based on the sum of the active and passive assays, as compared to 57 per 100 cm3 for the passive centrifugation assay alone. No significant differences were detected among multiple crops for experiments studying overwinter survival and there were no apparent trends in the data based on the previous crop. Soil samples from the research plots were collected from 1 April (Julian day 91) to 9 June (Julian day 160) and represented every year except 2004 for the period 1999 to 2011 (Table 1). There was a significant (P = 0.01) linear decline in the percentage of the population recovered from root fragments as compared to soil with advancing Julian day (adjusted R2 = 26%), although roots accounted for nearly half of the nematode population even in June. The coefficient of variation (CV) for the esti

Estimating Population Densities of Root Lesion Nematodes, Pratylenchus spp., from Soil Samples Using Dual Active and Passive Assays