© 2012 Plant Management Network.
Estimating Population Densities of Root Lesion Nematodes, Pratylenchus spp., from Soil Samples Using Dual Active and Passive Assays
Ann E. MacGuidwin and Breann E. Bender, Department of Plant Pathology, University of Wisconsin, Madison, WI 53706
MacGuidwin A. E., and Bender, B. E. 2012. Estimating population densities of root lesion nematodes, Pratylenchus spp., from soil samples using dual active and passive assays. Online. Plant Health Progress doi:10.1094/PHP-2012-1120-01-RS.
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.
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 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-µm-pore 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-µm-pore 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.
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.
Table 1. Mean population density (+ standard error) of Pratylenchus spp. and the percentage of nematodes recovered from root fragments in multiple bulked samples (16 to 99) collected before or at the time of planting experiments in field sites with Plainfield loamy sand soil. Samples were assayed from soil and root fragments using the dual extraction method.
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 third- and 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 estimates of root-sheltered nematodes was below 30% for half of the data sets, but it was as high as 142% for some. The regression of the CV against Julian day showed a weak but significant (P = 0.03) positive linear increase in variability as the day of sampling became later (adjusted R² = 13%). The high level of variability for the location of nematodes in soil versus root habitats and the change in variability over time indicate the need to always assay both habitats.
Dual Assay Results from Soil Samples Submitted for Nematode Diagnosis
Our lab began assaying soil samples for the public in 1985 using the methods described here. Data from 853 samples submitted during 1999 to 2004, 2007, and 2010 to 2011 were analyzed to determine the value of assaying the root fragments contained within soil samples for Pratylenchus spp. (Tables 2, 3, and 4). The methods for sample collection were unknown, but all samples were a bulked representation of multiple sites within a field. Nematodes were extracted from the samples within seven days of submission. Samples with zero or one nematodes were included in calculations of the mean and excluded in estimates of the median or the percentage of the populations sheltered within roots. Ninety-five percent of the samples submitted before 15 June came from fields planted with corn, declining to 60% for samples submitted after 1 August. Other crops sampled included vegetable, fruit, grain, and specialty crops such as ginseng and mint. Diagnostic service using different assay methods was provided to growers in 2005, 2006, 2008, and 2009 from the University of Wisconsin Plant Pathology Diagnostics Clinic, a unit independent from our nematode assay service, so those data were not included.
Table 2. Population estimates of Pratylenchus spp. based on active root or passive soil assays only or both together (dual assay) for samples submitted to the UW Nematode Diagnostic Service from 10 March to 15 June for nine years. Estimates of the mean include samples where no nematodes were detected. Estimates of the median or the percent recovered by the root assay are based on samples with nematodes present.
Table 3. Population estimates of Pratylenchus spp. based on active root or passive soil assays only or both together (dual assay) for samples submitted to the UW Nematode Diagnostic Service from 16 June to 31 July for nine years. Estimates of the mean include samples where no root lesion nematodes were detected. Estimates of the median or the percent recovered by the root assay are based on samples with nematodes present.
Table 4. Population estimates of Pratylenchus spp. based on active root or passive soil assays only or both together (dual assay) for samples submitted to the UW Nematode Diagnostic Service from 1 August to 31 December for nine years. Estimates of the mean include samples where no root lesion nematodes were detected. Estimates of the median or the percent recovered by the root assay are based on samples with nematodes present.
The incidence of Pratylenchus spp. was greater when both assays were considered as compared to either assay alone. For samples submitted before 15 June (Julian day 166), the passive soil assay alone underestimated the incidence of root lesion nematodes by 13% and the active root incubation assay by 8% (Table 2). For samples submitted between 16 June and 31 July, estimates of incidence were similar (93% versus 92%) for the two assays (Table 3). For samples submitted after 1 August, incidence based on the soil assay only was 5% greater than the root assay and 8% less than the estimate based on the sum of the two assays (Table 4).
Estimates of the population density of Pratylenchus spp. per volume of soil (100 cm³) were greatly improved when nematodes were extracted from both soil and roots using our dual assay. As for the research samples, many of the root pieces filtered from soil were from crops planted in past years as indicated by their size, color, and texture. All samples contained root fragments, but not all root assays were positive for nematodes. On average, however, 59%, 49%, and 56% of all nematodes recovered from soil came from the active root assay for samples submitted early (Table 2), mid-season (Table 3), and late to post season (Table 4), respectively. Including the root fraction for the dual assay consistently increased estimates of root lesion population density as compared to the soil assay alone, regardless of whether the data were grouped by year or time of submission (Tables 2, 3, and 4). As was the case with the research samples, there was a high degree of variability for the distribution of nematodes between soil and roots within the samples, with coefficients of variation greater than 50% for more than half of the 27 clinic sample cohorts.
Utility of the Dual Assay for Nematodes Other Than Root Lesion
Passive sieving/centrifugal flotation assays are appropriate for nematodes that remain in the soil and feed on plants as ectoparasites, but some large genera can be damaged during the process. The incubation assay of our dual procedure recovered ectoparasites too large to pass through a sieve with 250-µm openings. Some juvenile and all adults of the corn needle nematode, Longidorus breviannulatus, were recovered from the active incubation assay. Clinic samples positive for the corn needle nematode were detected in six of the nine years reported here in the time periods 1 March to 15 June, and 16 June to 30 July, and in four of nine years for the time period 1 August to 31 December. The specimens were in excellent condition as compared to the juveniles recovered using the passive sieving/centrifugal flotation assay. The incubation assay was also useful for dagger nematodes; 61% (± 2.4 SE) of the dagger nematodes detected were from the incubation assay for the combined clinic data sets. Smaller ectoparasites were recovered in low numbers from the incubation assay. Recovery of spiral nematodes (Helicotylenchus spp.), about the same size of root lesion nematodes, was very low from the incubation assay (mean = 2.5% ± 0.5 SE).
Dual Assay and the Importance of Past and Current Crops for Estimating Population Densities of Root Lesion Nematodes
More than half the root lesion nematodes our dual assay recovered from soil were in root fragments regardless of the time of year or method of sample collection. Our data show that extraction methods that do not account for root-sheltered nematodes underestimate nematode population densities. Dead root fragments housing viable nematodes were present in samples year-round, but were particularly important in early season samples before the annual crop was established. Incubation methods for soil such as the Baermann Funnel (1) and the Whitehead and Hemming tray (15) assay roots, but are limited to relatively small soil volumes, do not recover immobile specimens, and donít allow for separate tallies of nematodes in root versus soil habitats. A dual method overcomes these limitations and provides the means to census root lesion nematodes using the same soil units (soil volume or weight) year round. Counts of nematodes from the active root assay can be scaled to root weight or length units if the root fragments are retrieved after incubation and measured. Due to high variability in the distribution of roots within soil and nematodes within roots, it does not appear that merely doubling counts of nematodes from soil assays is sufficient for estimating the size of root lesion nematode populations. Whatever extraction procedures are used, an important guiding principle for root lesion nematodes is that populations of this versatile pest are always distributed among both soil and root habitats.
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