Sampling Procedure to Diagnose Nematode Infestations
Saad L. Hafez
Nematodes are minute, worm-like animals that may damage plants but are often difficult or impossible to detect with the unaided eye and are sometimes called the "unseen enemy." The word "nematode" when literally translated means "thread-like." Other names commonly used include "roundworm," "eelworm," or "nema."
Plant parasitic nematodes require the presence of living plants for reproduction and long-term survival. No plant or plant part is totally free from nematode attack, and annual economic losses in the United States have been estimated at 10 percent of the total agricultural crop values. Worldwide, over 2,000 plant parasitic nematode species have been identified and they occupy every available niche provided by plants. When nematodes feed on plant roots the uptake of water and nutrients is reduced, debilitating the entire plant.
Nematode sampling has become increasingly important in modern agriculture as the concepts of Integrated Pest Management (IPM) and Integrated Crop Production are developed and utilized. Scientists concerned with nematode populations have improved methods for their assay; however, data from the best extraction methods are of limited value if the sample is not representative of the area. Effective diagnostic sampling may involve rating plant roots (e.g., galls caused by Meloidogyne spp.), bioassays, or visually assessing above-ground growth for effects of foliar pathogens in addition to collecting soil and root samples for nematode counts.
Primary objectives of nematode sampling include: diagnosis of disease problems, general detection and surveys; providing advice in IPM programs; and fulfilling research needs, e.g., evaluating different management practices.
Sampling for nematodes is an increasingly important component of plant disease diagnosis, especially for high value crops and nursery stock. Without confirmation through sampling, poor plant growth because of nematodes may be misinterpreted as nutrient deficiencies or other maladies. The art and practice of collecting representative root and/or foliage tissue as well as soil samples is a critical component of diagnostic sampling. For example, sampling from adjacent, healthy-appearing plants may be just as important as collecting samples from the most severely affected ones.
Detection and survey
Nematode sampling is the basis for determining the occurrence and distribution of many plant parasitic nematodes. Quarantine or phytosanitary regulations of many countries, or political subunits, require that planting materials be produced on land certified free from nematodes. Soil sampling for certification of widely distributed planting materials requires extreme precision for detection of quarantined pests. Although the objective of detection seems simple, a negative result does not necessarily prove absence of the pest, but only indicates that a nematode population is below the detection level.
The fact that initial numbers of nematodes can be related to the yield of annual crops has enabled nematologists to develop functional advisory programs, even though relationships between nematode numbers and crop damage may be modified by environment. Because of the importance of reliable detection, most sampling for advisory purposes is conducted when population densities are near their maximum levels, often at the end of the growing season after harvest. Sampling at the time of planting, however, theoretically will give a better estimate of the initial nematode problem where population levels are high enough for detection. Follow-up sampling may be necessary with perennials because low, nondetectable populations sometimes increase over time to damaging levels.
Research is conducted to evaluate the effectiveness of different management practices in reducing nematode populations and plant damage. Extra effort in sampling plant tissues and soil is required to obtain accurate results. The accuracy in determining relative numbers and developmental stages of nematodes may be greatly affected by sample handling or extraction. Design and management for sampling may need to be modified for each specific type of study.
Considerations in designing sampling procedures
How to sample for nematodes
Making the proper management decision for a nematode problem depends on the correct diagnosis, which also depends on proper sample collection, handling, labeling, packaging and shipment. Population densities of plant parasitic nematodes vary greatly in time and space. Population densities are often affected by climate, crops grown, weed hosts, chemicals used, and other factors that may lead to patchy distributions. Patchy or aggregated nematode distributions and changes in the species composition of the nematode communities over time pose major sampling problems. Collection of representative soil samples is the primary component of sampling for diagnosis.
The following recommendations are presented as general guidelines and may require modification for specific field situations.
Time of sampling
The distribution of nematodes is seldom uniform or constant and changes may occur rapidly. Most of the time nematode distribution is patchy. For these reasons, the field to be sampled should be mapped into subdivisions. Any observable variation in previous crop growth, soil texture, moisture and draining patterns, or cropping history will constitute a subdivision. An effective sampling map may then be constructed.
The sampling pattern is based on the sampling map and should be designed to obtain a reliable representative sample with as little sampling error as possible. An example of a recommended soil sampling pattern for fallow ground or an established crop is shown in Figure 1.
Number, size, and cost of samples
Sample size depends on the area and crop being sampled, the crop's value and any supplementary information to be gathered during sampling. Sample size is measured in terms of the number of cores of soil constituting the sample. The precision of the resulting nematode estimates can be improved by increasing the number of soil cores in the sample. This is also less expensive than increasing the actual number of samples. A useful rule-of-thumb for estimating cost is to consider allocating 1 percent of the crop's total production expense on nematode sampling.
A good approach in sampling is to bulk soil cores in a clean bucket, mix thoroughly, and submit one quart of the mixture for processing. One sample may be used to represent up to five acres of a uniform field subdivision as determined by mapping. Each sample should contain at least 20 individual cores, or if representing less than five acres, at least four cores per acre. Place the sample in a sturdy, moisture-retaining bag and clearly identify with a tag attached to the outside of the bag. Tags or labels inside the bags may get wet and discolor easily.
Where to take samples
Soil samples should be taken from the plant root zone. If the crop has not been planted, take samples to fit the intended crop's root zone. Plant samples should be taken from the plant parts showing symptoms, if present. Soil or plant samples should be taken from problem areas showing symptoms and from unaffected areas for comparison.
Before planting annual crops, sample cores from a fallow field should be taken by first removing the upper two inches of soil, and then sampling to a depth of 15 to 20 inches. Deeper cores, up to 30 inches, should be taken after prolonged fallow, dry or freezing conditions.
Equipment for collecting soil samples for nematode assays includes shovels, soil augers or tubes, and motorized samplers. The typical cylindrical tube sampler designed by soil scientists serves as the standard equipment. Representative sampling tools are shown in Figure 2.
Include name, location, soil type and texture, observable symptoms (yellowing, necrosis, root rotting, galling, wilting, etc.), cropping history (past several years, current, anticipated), and date of last treatment with a nematicide. This information is invaluable for diagnosis and identification of nematode problems. Refer to the Nematode Diagnostic Laboratory Check Sheet (Figure 3) for examples of pertinent information.
Storage and delivery
Even the most carefully taken samples may yield inferior results if not stored and delivered properly. Keep the sample cool, ideally at 50 to 55° F. Do not leave the sample in direct sunlight, car trunk or other areas that may heat excessively. An insulated cooler is convenient for sample protection. Deliver or mail the sample immediately to the processing laboratory. Use First Class, UPS, Greyhound or other express delivery and pack well in a sturdy cardboard box or coffee can. Remember--Samples must be clearly labeled and accompanied by complete background information. Diagnostic services are available at the University of Idaho, Parma Research and Extension Center, Parma, ID 83660. Telephone: (208) 722-6701.