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© 2011 Plant Management Network.
Accepted for publication 15 July 2011. Published 1 September 2011.


Sampling for Plant-parasitic Nematodes in Corn Strip Trials Comparing Nematode Management Products


Gregory L. Tylka, Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011; Timothy C. Todd, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506; Terry L. Niblack, Department of Plant Pathology, Ohio State University, Columbus, OH 43210; Ann E. MacGuidwin, Department of Plant Pathology, University of Wisconsin, Madison, WI 53706; and Tamra Jackson, Department of Plant Pathology, University of Nebraska, Lincoln, NE 68588


Corresponding author: Gregory L. Tylka. gltylka@iastate.edu


Tylka, G. L., Todd, T. C., Niblack, T. L., MacGuidwin, A. E., and Jackson, T. 2011. Sampling for plant-parasitic nematodes in corn strip trials comparing nematode management products. Online. Plant Health Progress doi:10.1094/PHP-2011-0901-01-DG.


Introduction

Protectant seed treatments are a new management option for plant-parasitic nematodes that feed on corn. Avicta from Syngenta Crop Protection became widely available for use on corn in the United States in the 2010 growing season, and Votivo from Bayer CropScience is now available for use on corn in the 2011 growing season.

Many growers and agribusiness personnel working for co-ops, grain elevators, and seed and chemical companies are conducting strip-trial comparisons of nematode seed treatments in growers’ fields. Yield monitors in combines and/or weigh wagons can be used to collect yield data from multiple-row strips that stretch across an entire field. But some growers and agribusiness personnel also want to assess plant-parasitic nematode populations in these strip trials to gauge whether the seed treatments are affecting nematode numbers.

Drawing conclusions about the effects of treatments on numbers of plant-parasitic nematodes in strip trials is problematic because of the natural variability of nematode populations and their densities in the field. Plant-parasitic nematodes are microscopic worms and their population densities can vary greatly over short distances. The variability in nematode population densities in two or more samples collected from the same treatment in a strip trial may be equal to or greater than the differences in nematode numbers in samples collected from two or more different treatments. And, if differences in numbers of nematodes from samples taken from different treatments are detected, one cannot assume that the differences are due to effects of the treatments. The inability to attribute differences in nematode numbers to treatment effects is especially true if only one sample is taken from each treatment. Multiple samples must be collected uniformly, consistently, carefully, and at the proper time in order to have a chance of detecting differences in nematode population densities among treatments.


Host: Corn or maize (Zea mays L.)


Disease

Yield loss caused by damage due to feeding of plant-parasitic nematodes on corn in the United States has been known and recognized since the 1980s (8). The most recent survey of estimated crop losses to corn caused by plant-parasitic nematodes was conducted in 1994, and loss estimates ranged from 0 to 10 percent in the 25 United States states that were surveyed (5).


Pathogens

There are at least 120 different species of plant-parasitic nematodes known to feed on corn worldwide, and more than 60 species occur in North America (8). The common genera of plant-parasitic nematodes that feed on corn are listed in Table 1. Most genera occur only in the soil and feed from outside of the roots (Fig. 1); these nematodes are described as ectoparasites. To determine the population densities, or numbers, of ectoparasitic nematodes, soil samples are collected and nematodes are extracted from the soil, identified, and counted.


Table 1. Basic characteristics of common genera of plant-parasitic nematodes that feed on corn.

Common
name
Scientific name
(genus name)
Feeding
type
Primary
location
during the
growing
season
General
damage
potential
dagger Xiphinema ectoparasite soil moderate
to high
lance Hoplolaimus endoparasite roots low to
moderate
needle Longidorus ectoparasite soil high
pin Paratylenchus ectoparasite soil low
ring Mesocriconema ectoparasite soil low to
moderate
root-knot Meloidogyne endoparasite roots & soil low to
 high*
root-lesion Pratylenchus endoparasite roots moderate
to high
sheath Hemicycliophora ectoparasite soil low
spiral Helicotylenchus ectoparasite soil low to
moderate
sting Belonolaimus ectoparasite soil high
stubby root Paratrichodorus ectoparasite soil moderate
to high
stunt Tylenchorhynchus and Quinisulcius ectoparasite soil low to
moderate

 * Some populations of root-knot nematode are more likely to injure corn and reduce yields in the southern Corn Belt than in the Midwest.



 

Fig. 1. Ectoparasitic stunt nematode feeding on a corn root.

 

A few nematode genera that feed on corn enter and feed completely within the root tissue (Fig. 2). These nematodes, which complete their entire life cycle within the roots, are described as endoparasites and include lance nematode (Hoplolaimus spp.), root-knot nematode (Meloidogyne spp.), and root-lesion nematode (Pratylenchus spp). To assess population densities of endoparasitic lance and root-lesion nematodes, nematodes must be extracted from root tissue and then identified and counted. The numbers of lance and root-lesion nematodes in the soil during the growing season can be deceptively low, while several thousands of nematodes can be present in a single gram of root tissue (8).


 

Fig. 2. Endoparasitic lance nematodes (stained red) feeding within root segments.

 

Symptoms and Signs

Above-ground symptoms of nematode damage to corn include general stunting resulting in uneven plant height in a field, general yellowing of leaf tissue, poor pollination, small ears, and lodging later in the growing season (8). The symptoms that occur below ground are quite variable depending on the nematode species and the environmental conditions present in the field and can include stunting of the root system, slight, general root discoloration, distinct, dark necrotic root lesions, and a proliferation of fibrous roots (8). None of the symptoms are unique enough to provide a conclusive diagnosis of nematode damage in the field, and there are no reliable, apparent signs of the nematodes in the field.


Host Range

All of the nematodes that feed on corn have alternative hosts; none feed exclusively on corn. Most species of plant-parasitic nematodes that parasitize corn in the United States Midwest also parasitize soybean (8).


Geographic Range

In general, plant-parasitic nematodes are ubiquitous in nature, and it is expected to find species feeding on corn anywhere the crop is grown in the world (8). In almost all cases, the presence of a few individuals of a nematode species that feeds on corn does not cause noticeable symptoms or yield loss. Populations of these nematodes must increase to damaging densities before corn yield loss occurs (8).


Pathogen Isolation, Identification, and Quantification

Common methods to extract nematodes from soil are wet sieving/decanting (3) and the Baermann funnel (10). Extracting endoparasitic nematodes from roots involves incubating root fragments in water or an extraction solution (1) for several days and identifying and counting nematodes that exit the root and are present in the extraction solution.

Plant-parasitic nematodes can be identified to the genus level generally using overall body shape, size, stylet shape and size, and several other measures of gross body morphology. It is sufficient to identify the nematodes only to the genus level for most practical purposes, including collecting information from field strip trials. The criteria and methods used to differentiate species of plant-parasitic nematodes vary by genus and include fine microscopic observation of different juvenile and adult life stages of the nematode to measure subtle morphological details. It is beyond the scope of this paper to review or even highlight the characters and techniques used to identify this large and diverse group of nematode genera and species. A useful resource for identifying common genera of plant-parasitic nematodes is Plant-Parasitic Nematodes: a Pictorial Key to Genera by W. F. Mai and P. G. Mullin, which includes a dichotomous key to the common genera (7).

Many land-grant universities in the United States have a plant disease clinic or a nematology laboratory that extracts plant-parasitic nematodes from soil and root samples and identifies and counts the nematodes (Table 2). Some state universities may not have such a facility to process samples for nematodes, in which case samples might need to be sent to an adjacent state university facility, if they are able to accept out-of-state samples. It is best to check in advance of sending samples for nematode analysis to a facility located in another state. There also are several private laboratories that are able to extract and identify plant-parasitic nematodes from samples.

Extracting nematodes from soil and identifying and counting the plant-parasitic nematodes that are present are somewhat complicated and time-consuming activities. One should consult with the laboratory where samples from strip trials would be sent in advance of collecting the samples to determine if the facility is capable of processing a large number of samples in a timely manner. Also, there are no rigid standard procedures for extracting nematodes from soil and roots, and methodology can greatly affect results, so results of samples processed by different laboratories should not be compared directly.

When communicating with the laboratory that will process your samples, it is a good idea to explain to them the purpose of the samples. The laboratory personnel may benefit from knowing whether the samples are from a strip trial or are samples that were collected to diagnose a corn production problem.


How to Collect Samples

• Using a 1-inch-diameter (2.5-cm-diameter) soil probe, collect up to 20 soil cores from each area being sampled. Angle the soil probe in under the seed row (Fig. 3), collecting the soil from within the root zone of growing corn plants. Soil cores should be at least 12 inches long.

• Do not break up or mix the soil cores. Place them in a plastic bag and label the outside of each sample bag with a permanent marker. A standard paper soil sample bag will not be large enough to hold all of the soil cores that comprise a sample.

• To check for endoparasitic nematodes, nematologists in some states recommend collecting several whole plants early in the season, when plants are relatively small (Fig. 4), and several root masses later in the season (Fig. 5). Nematologists in other states extract the endoparasitic nematodes from root fragments recovered from the soil cores and do not recommend collecting separate root samples. Check with the laboratory that will be processing the samples to make certain that they extract, identify, and count endoparasitic nematodes and to determine if separate root samples need to be submitted along with soil cores.

• Protect soil and root samples from physical impact and high temperatures. Nematodes are soft-bodied animals that can be ruptured and destroyed by blunt physical force. Never throw or drop soil sample bags, and be sure to cushion sample bags well with crumpled newspaper in the box if samples are being shipped. Excessive heat also can damage or kill nematodes, and some laboratories use extraction methods that require the nematodes to be alive and mobile. So protect samples from direct sunlight and avoid shipping samples late in the work week because they may sit over the weekend in a delivery truck or warehouse that is not air conditioned. Samples should be sent via overnight delivery or driven to the laboratory that is going to process them.


 

Fig. 3. Collecting a soil core with a soil probe from a row of corn to determine population densities of plant-parasitic nematodes.

 

Fig. 4. Young corn plants collected to test for endoparasitic nematodes in root tissue.


 

Fig. 5. Root mass collected to test for endoparasitic nematodes in roots mid season.

 

When to Sample Strip Trials

The different species of plant-parasitic nematodes that feed on corn vary in how long it takes for them to complete a generation (i.e., generation time). Root-lesion nematodes (Pratylenchus spp.) complete a generation in 21 to 28 days (9); dagger and needle nematodes (Xiphinema spp. and Longidorus spp., respectively) take an entire growing season or more to complete one generation (2,4). The likelihood of detecting a difference in nematode numbers due to effects of seed treatments (or any other management tactic) depends on the specific effect of the treatment on nematodes and what nematodes are present in the field, their generation times, and when the nematode samples are taken. Following are some specific points to consider.

• The new nematode seed treatments are described as providing early season protection of corn roots from nematode feeding, not season-long nematode control. The duration of the root protection into the growing season is not specified or known and may vary according to growing conditions.

• Plant-parasitic nematodes are obligate parasites that must feed on living host roots to develop, mature, and reproduce. It is not known exactly when in the growing season that differences in nematode population densities in soil and roots can be detected as a consequence of seed treatments preventing nematode feeding and reproduction.

• Differences in numbers of ectoparasitic nematodes due to the effects of seed treatments may be apparent early in the season, when the seed treatments are repelling the nematodes or preventing their access to the roots, but not later in the growing season, after the treatments become less effective and/or nematode numbers build back up.

• Many nematodes that feed on corn may be deep in the soil profile, and some, including needle nematodes, can migrate down as the growing season progresses (6). These nematodes are missing or underrepresented in the typical 6- to 8-inch-deep soil cores collected in other crops, such as soybean. As we mentioned earlier, cores should be taken at least 12 inches deep, and even up to 18 inches deep (if possible) later in the season as nematodes have migrated downward. Even so, soil samples collected at mid to late season may yield nematode population densities that are deceptively low.

• The current nematode seed treatments are not believed to enter the roots. If the seed treatments’ active ingredients only occur outside of the roots, there is no reason to believe the treatments would affect feeding and reproduction of endoparasitic nematodes once they have successfully entered the roots. So the numbers of endoparasitic nematodes living inside the roots in the first several weeks of the growing season may be a better indication of the protection provided by the seed treatments than nematode numbers from roots collected later in the season, once the nematodes that were able to penetrate into roots have a chance to multiply.

• If nematodes are prevented from feeding on roots, the numbers of nematodes in soil and/or roots should eventually decline because of lack reproduction and eventual starvation. But if samples are collected before enough time has passed for the nematodes to complete a generation, their failure to multiply would not be reflected in numbers of nematodes in a sample. It is not likely that there will be differences in nematode numbers attributable to treatments in early or mid-season samples if the predominant nematodes in the field are those that take an entire growing season to complete one generation.


Where to Sample in Strip Trials

It is difficult to determine where to collect samples from strip trials composed of strips that are 20 to 40 rows wide and stretch across an entire field. And, it is not practical for growers or agronomists to plant multiple strips of randomly arranged treatments in a field. Instead, planters often are loaded with two or three different types of seeds (treated with different compounds or untreated) and then strips are planted as the tractor and planter moves in a back and forth or serpentine pattern planting the field. Following are some guidelines for creating specific sampling areas or “plots” in strip trials.

• Create designated sampling areas akin to “plots” in a strip trial by marking the center four or eight rows of a 25- to 50-ft-long (7.5- to 15-m-long) area of each 8- to 12-row-wide strip using flags and global positioning system (GPS) coordinates. Marking sampling areas in this manner will allow repeated sampling from the same areas throughout the growing season (if that is desired). Having GPS coordinates of the sampling areas also will allow comparison of nematode population densities to yields in those areas, if a georeferenced yield map of the field is available and yield data can be extracted from maps for the specific areas that were sampled for nematodes. The georeferenced nematode data will be useful in future years, too, as areas of damaging nematode populations ("hot spots") tend to occur in the same places year after year.

• Collect numerous soil cores and, if recommended by the laboratory that will process the samples, a few plants or root masses from several sets of sampling areas or “plots” in a strip trial. Use a shovel to dig plants for root samples so that small roots, which many nematodes prefer, are not lost.

• Collect sets of samples from “plots” that form a straight line (or transect) across the various treatments in the strips. Ideal places to position the transects in the field would include areas suspected of having damaging nematode populations, areas where there currently is or has been unexplained poor corn growth in past seasons, and areas with uniform soil conditions (and possibly uniform nematode populations).

• If there are multiple strips of each treatment being compared in a field, it may be possible to collect samples from “plots” that form just two or three transects across the strips (Fig. 6).

• If each treatment only occurs once in a field, samples should be collected from numerous transects that traverse the strips at different places in the field (Fig. 7).

• Avoid establishing strip trials to compare nematode seed treatments in fields that have had strips trials conducted in them in recent years, especially if the past treatments may have affected nematode numbers. The effects of treatments in past years may persist for more than one year and will confound the data collected from the current study.


   

Fig. 6. Layout of a strip trial with 12-row (30-ft-wide) strips of three treatments (A, B, and C) repeated several times, planted with a 24-row planter. Treatment C would be in the left 6 units, treatment B in the center 12 units, and treatment A in the right 6 units of the planter. The arrangement of treatments shown in the diagram is created when the tractor follows a back-and-forth or serpentine pattern to plant the field, starting in the lower left corner of the field. Areas in white depict border rows of treated seed that would not be used for data collection. Sampling areas or “plots” are created in the strips by designating 12-row-wide by 30-ft-long areas for sampling. Ideally, yield data could also be obtained from the sampling areas or “plots.”

 

 

Fig. 7. Layout of a strip trial with single 12-row (30-ft-wide) strips of three treatments (A, B, and C) planted with a 24-row planter. Treatment C would be in the left 6 units, treatment B in the center 12 units, and treatment A in the right 6 units of the planter. Areas in white depict border rows of treated seed that would not be used for data collection. Sampling areas or “plots” are created in the strips by designating 12-row-wide by 30-ft-long areas for sampling. Ideally, yield data could also be obtained from the sampling areas or “plots.”

   

Comparing Nematode Numbers Among Strips

Populations of plant-parasitic nematodes are extremely variable in fields. The genera and numbers of nematodes in a sample can vary greatly from location to location in the field, from time to time throughout the growing season, and from season to season.

Nematode numbers from samples collected from strips in a field will be highly variable, even when consistent sampling methods are used. So how would one know if a treatment is affecting nematode numbers? Answering such a question with any degree of confidence is very difficult with data collected from strip trials as described above. Consider the following points:

• To relate yield differences among treatments in strip trials to effects on population densities of plant-parasitic nematodes, trials should be located in fields infested with plant-parasitic nematodes known to be damaging to corn. And ideally, the nematodes present would be at population densities that can cause yield loss. Samples can be taken the fall before a study is initiated or in early spring of the growing season to guide field selection.

• The ability of nematodes to damage corn varies greatly among different nematode genera (Table 1), and most genera, except Belonolaimus and Longidorus, are not believed to be damaging when numbers are low. Little is known about combined effects of several species of nematodes feeding on corn concurrently, which is a very common occurrence.

• To determine whether a treatment is having any consistent effect on nematode numbers, it is critically important to collect many (8 or more) sets of samples across strips of treatments and to compare nematode numbers from all samples. Results from one set of samples collected at one location across strips will not provide useful information.

• The most convincing evidence that a treatment reduced numbers of plant-parasitic nematodes would be if combined numbers of plant-parasitic nematodes, or combined numbers of nematodes with moderate and/or high damage potential (Table 1), were consistently lower in samples collected from treated strips than in samples from untreated strips. But such a result is unlikely because of the natural variability in distribution of plant-parasitic nematodes in fields. More likely, nematode numbers may be lower in samples from some sampling areas or “plots” in treated strips than in untreated strips, but not all.

• Less convincing, but still reasonable evidence of a treatment effect would be if overall average nematode numbers, or average numbers of nematodes with moderate and/or high damage potential, from samples collected from all of the treated strips combined is lower than average numbers from samples collected in the untreated plots combined.

• An additional set of information that might be useful is the numbers of nematode present in the sampling areas or “plots” at the beginning of the season. Results from samples collected at or near the time of planting would provide information on the base population of plant-parasitic nematodes that were present before treatments were imposed or had an effect. This information would help characterize an experiment as being located in a field with low, moderate, or high risk of nematode damage to corn based on the nematode populations that were present. And mid-season nematode population densities may be more meaningful when compared with nematode population densities that were present at the beginning of the season.


Other Important Things to Consider

It is important to keep as many factors as possible consistent across treatment strips except for the treatments being compared. Following are some key points to keep in mind when conducting a strip trial and attempting to determine whether treatments are affecting population densities of plant-parasitic nematodes.

• Ideally, all factors in strips should be kept constant other than the treatments being compared. The same lot of seed of the same corn hybrid should be planted on the same day with the same equipment, and nematode samples should be collected by the same person in exactly the same way from sampling areas in all strips. This is especially important when comparing products from different companies. But it is unlikely that growers or agribusiness personnel will be able to compare products from different companies on a single lot of seed of one hybrid because many seed companies only sell one of the current choices of seed-treatment nematode protectant products. If products are applied to different corn hybrids, the hybrids should be of similar maturity and possess similar defensive traits, such as herbicide and insect resistance, and even then, direct comparisons of results from different corn hybrids should not be made.

• The seed-treatment nematode protectants are being sold in combination with other seed-treatment protectants, not as stand-alone nematode management products. For example, Avicta is sold with Cruiser Extreme 250 as Avicta Complete Corn, a combination of the seed treatment nematicide (active ingredient abamectin), a seed treatment insecticide and three seed treatment fungicides. And Votivo is a biological seed treatment containing the bacterium Bacillus firmus that will be sold with a seed-treatment insecticide as Poncho/Votivo.

If a strip trial is being conducted to determine if seed treatments are controlling plant-parasitic nematodes resulting in increased corn yields, nematode population densities and yields should be compared between strips with and without the nematode protectant seed treatment. For example, the best comparisons would be between Avicta Complete Corn and Cruiser Extreme 250 and between Poncho/Votivo and Poncho because each pair of treatments varies only by the presence or absence of the nematode protectant. Also, the rates of insecticides and fungicides in the seed treatments being compared should be the same among the products being compared. Figuring out and standardizing the rates of these components of seed-treatment products may require some effort because rates of insecticides and fungicides can vary among seed-treatment products within a company, even with products with the same or similar names.

• When comparing yields of corn treated with products from different companies, be sure to use products with similar components in each company’s treatment. For example, as explained above, Avicta Complete Corn has a nematicide, an insecticide, and three fungicides. So the most equivalent comparison treatment would be corn treated with Poncho/Votivo plus several seed-applied fungicides comparable to those in Avicta Complete Corn.

• Other possible treatments to include in strip trials to assess if nematode protectant seed treatments increase yields by reducing nematode numbers are an untreated control and a soil-applied nematicide. It may be difficult to get untreated corn seed to serve as an untreated control in strip trials. Currently, Counter 15G and 20G from AMVAC Chemical Corporation are soil-applied nematicides labeled for use on corn. If including a soil-applied nematicide as a control treatment, the seed should be treated with the same or similar insecticides and fungicides as those present in the nematode seed treatment products in the strip trial.

• It is always best to vary treatments being compared by only one factor to get the most meaningful information from the comparison. The more components that vary in treatments being compared in a field, the less specific information will be obtained about the likely reason that a difference in yield or nematode numbers occurred. For example, increased corn yield associated with use of a seed treatment containing a nematicide, an insecticide, and a fungicide compared to yield of corn from seed treated just with a fungicide may be due to the activity of the nematicide, the insecticide, both pesticides acting additively, or an interaction between the two pesticides. In such a case, there is no way to know for certain the true basis of the yield increase because the treatments varied by more than one component. Still, comparison of yields of corn treated with various products that vary by more than one component can be useful information, but only in regards to yield and not with respect to the basis for any possible yield differences detected.


Collecting Nematode Data from Strip Trials with Other Field Crops

The methods and considerations discussed herein are also applicable for collecting samples to determine nematode population densities from strip trials of other field crops, including soybeans and the soybean cyst nematode (SCN). However, when doing so, great consideration must be given to the biology of the nematodes and the crop being studied and also the specific modes of action of the treatments being compared. For example, collecting samples to determine possible effects of nematode seed treatments that provide early season protection against SCN probably should be taken in the first four to six weeks of the season, when the effects of the treatments would be most apparent and after enough time had passed for the SCN population to have completed a full generation. Population densities from samples collected at the end of the season may not show any treatment effects because nematode numbers may resurge in mid and late season, after the protective effects of the seed treatment have worn off.


Summary

The guidelines presented above are for individuals conducting strip trials to assess the effects of new nematode protectant seed treatments on population densities of plant-parasitic nematodes in the field. The suggestions are meant to facilitate useful, yet practical comparisons, particularly with respect to the effects of the tested treatments on population densities of plant-parasitic nematodes that feed on corn.

As mentioned in the introduction, it is unlikely that strong conclusions can be drawn about effects of treatments compared in a strip trial because of the natural variability in spatial distribution and numbers of plant-parasitic nematodes.

Also, the information obtained from in-field comparisons conducted as suggested above is not a substitute for data from scientifically valid experiments conducted with replicated plots, randomly assigned treatments, intensive sample collection, and appropriate experimental design and statistical methods to discern treatment effects. And one should be mindful of the large influence that environment can have on the interaction of plant-parasitic nematodes with host plants and on the resultant yield loss and the changes in nematode population densities. The effects of seed-treatment nematode protectant products may vary from field to field and year to year because of the large effect of the environment.

It is always best to check with university nematologists or plant pathologists with nematode expertise in your own state for the most accurate information concerning nematodes that feed on corn and how to assess the effects of management strategies on nematode population densities. The guidelines presented herein are generally applicable but local experts may have other advice to improve the effectiveness of the comparisons.


Disclaimers

The pesticides mentioned herein should be used according to their labels and only if they are registered with the Environmental Protection Agency and your state department of agriculture. If a product registration is changed or cancelled, any suggestions herein are no longer recommended. Check with state authorities, chemical company personnel, or university extension staff for the latest information before any pesticides are used. Also, endorsement is not intended of commercial products mentioned herein, nor is criticism implied of similar products not mentioned.


Acknowledgments

Appreciation is expressed to Ray Knake of Bayer CropScience, Daren Mueller of Iowa State University, Palle Pedersen of Syngenta Crop Protection, and Jennifer Riggs of Bayer CropScience for reviewing the manuscript and providing helpful suggestions for improvements.


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