Sampling Procedures for Nematodes

The purpose of this short guide is to introduce the principles behind nematode sampling, extraction, fixation etc. to those with little or no experience of nematological techniques. Only basic techniques are included and these can be performed in almost any situation with the simplest of equipment.

It is important to realize that in order to obtain reproduceable and accurate results in nematode sampling and extraction, the same method(s) should be followed every time with scrupulous attention to detail. Failure to do so can result in misleading assessments in both the numbers and type of nematodes present.

Sampling Procedures

Deciding upon the number of samples

The main objective in sampling is to take enough samples to provide a representative picture of a given situation. The major limiting factor in taking a lot of samples is the bulk of soil involved and the time and space required for processing. Consequently a balance must be struck.

Nematodes are not usually evenly dispersed over a field or experimental plot; they tend to form groups or aggregations where conditions are most favourable. As a result, the sampling procedure should allow for this 'lumping' by taking a number of random samples. Clearly the more samples taken the more accurate is the assessment but in practice the quantity of soil involved soon gets out of hand.

The number of samples taken to represent a given situation will vary depending on the prevailing conditions, but usually a number of individual samples will be taken and bulked to form the sample proper. The material in the bulked sample can then be thoroughly mixed and a smaller sample, or sub-sample, taken to assess the nematode population.

Taking a sample

Sampling of soil that is very wet or very dry should in most cases be avoided. The soil for the sample should be taken at least 5-10 cm below the surface as it is here, in the root zone, that the nematodes will congregate. If a crop shows patches of poor growth then separate samples should be taken from the badly affected and normal areas so that a comparison can be made. Individual sample size should be about 250-300 g. After the samples have been bulked and thoroughly mixed, a sub-sample of the same weight can be taken and analyzed.

If at all possible, roots should be either included in the sample or taken separately - about 25-100 g, taken at random, should be sufficient, the lower weight being suitable for vegetables or citrus whilst the higher weight being more applicable to plants with large roots such as banana.

If stems and/or leaves appear to be attacked by nematodes, affected material can be removed and placed in polythene bags. Such samples should be kept separate from soil and/or root samples.

Care of samples

Samples should be placed in strong polythene bags and immediately labelled by means of a pencil-written paper or plastic label placed inside the bag. Data to be recorded are crop, date of sample, locality plus any reference number such as plot identification in the case of trials. Samples should be kept cool - do not leave in the sun or in a closed vehicle left in the sun - and should be treated with care and processed or despatched for analysis as soon as possible. If immediate despatch or processing is impossible then samples can be stored in a refrigerator at 4-8deg.C for several days without severe deterioration or alteration in relative composition of the nematode population. All samples sent abroad should be via airmail and include the necessary documentation.

Equipment

Digging implement eg. fork, hoe or spade, polythene bags, labels, pencil, knife (for cutting roots), notebook (for more detailed visual assessment and/or crop history).

Processing of Soil

Preparation of sample

Ideally the sample should be processed as soon as possible after collection. The soil should be passed through a coarse sieve (mesh openings of 10 mm or 1/4 inch) to remove any lumps, stones, roots, etc. Place the roots to one side and discard any stones or hard lumps of soil. The sifted soil should be well mixed to obtain a uniform distribution of nematodes. The required volume of soil for extraction can now be taken from the sample by loosely filling a graduated container such as a beaker to the appropriate mark. The actual volume of soil will depend on the extraction method used and can vary from as little as 25 ml to 200 ml. If possible a figure of 100 ml should be aimed for as the best all-round quantity.

Extraction

There are two basic techniques - active, where the nematodes move from the soil under their own power, and passive, where they are separated from the soil by physical means such as elutriation or sieving.

Active: nematodes move from the soil into a dish of water, the soil being separated from the water by tissue supported by various devices. The basic requirements involve a water-containing vessel into which is placed another vessel, the extraction filter, containing the soil sample. The two vessels can be adapted from a wide variety of other containers, e.g. photographic developing trays, aluminium pie-pans, plastic tubs and cartons, etc.

The extraction filter is made from a container with most of the bottom cut out. Into this is placed a sheet of coarse mesh (such as plastic mosquito netting) which supports a double layer of facial tissue (eg. Kleenex) upon which the soil sample is spread. The completed extraction filter is then placed in the other vessel and sufficient water carefully added so that the soil is moist. The extraction should be left for 24 hours after which the extraction filter is carefully removed and the water/nematode suspension poured off into a beaker. Fig.1 portrays several different modifications of the basic technique

Passive: there are two major methods - elutriation and sieving. The former involves separating nematodes from soil by an upcurrent of water, the strength of which is such that nematodes are held in suspension whilst the heavier soil particles sink. All methods involve fairly complicated apparatus and depend upon a plentiful supply of water at the correct pressure. This limits their usefulness.

Sieving involves pouring a suspension of soil in water through a bank of suitable sieves. for most plant parasitic nematodes sieve meshes of 75 um and 53 um are adequate and ideally there should, for maximum retention of nematodes, be two or three 53 um sieves in a bank with one or two 75 um sieves on top. A known volume of soil (200-400 ml) is placed in a 2 gallon bucket and water added nearly to the top. Any large lumps of soil can be gently crumbled with the fingers and then the suspension is vigorously stirred for about 10 seconds and left for 30 seconds before being poured through the bank of previously moistened sieves. The material trapped on each sieve is then washed into a beaker with a gentle jet of water applied to the back of each sieve. The whole process can be repeated with the slurry left in the original bucket and the nematode suspension can either be examined directly or cleaned-up by using one of the active methods outlined above.

Immersion sieving is a variety of the standard sieving technique and is used for extracting long and delicate nematodes such as the longidorids. The set-up is shown in Fig.2 and involves a 90 um, or preferably 120 um sieve, because of the much larger nematodes. A soil suspension is prepared as for the sieving technique and then poured through the 120 um sieve, the mesh of which is submerged by the water in the bowl. After the suspension has been poured through, the sieve is gently washed with a jet of water to remove as much soil as possible. The sieve is then removed from the bowl, and the residue washed off as before.

Concentrating

The nematode suspension can be left undisturbed for several hours in which time the nematodes should sink to the bottom (NB there are some nematodes, such as Aphelenchoides besseyi or Ditylenchus angustus, which can actively swim and tend not to settle unless the suspension is cooled in a 'fridge). The surplus water can either be carefully decanted or siphoned off leaving the nematodes in a comparatively small volume of water.

Extraction of Nematodes from Plant Tissues

Nematodes can be found in all types of plant tissues (roots, tubers, corms, rhizomes, seeds, stem and leaves) and various methods are available to extract nematodes from the tissues or examine them directly in situ. After sampling, roots etc. can be stored for some time in plastic bags if kept moist (but not wet) and cool; foliage tends to rot very quickly and has to be processed soon after sampling.

Extraction of live nematodes from tissues

The methods described below can be used for the extraction of migratory endoparasites and semi-endoparasites such as Pratylenchus, Radopholus, Helicotylenchus, Scutellonema, Hirschmanniella, Hoplolaimus, Rotylenchus etc. It is also applicable for the extraction of hatched juveniles of sedentary endoparasites such as Meloidogyne, Nacobbus, Tylenchulus etc. The plant material is carefully washed free of soil and/or debris before extracting.

  1. Root incubation: Whole or cut roots are placed in a closed contained (e.g. beaker with Petri dish on top, conical flask or even a plastic bag) and kept moist by applying a fine spray of water 3 or 4 times a day. Nematodes migrate out of the roots, or hatch from eggs and collect in the shallow layer of water in the base of the container. The water is poured off every 24 hours and examined for nematodes. Extraction can continue for 7 days or longer. It is also possible to keep infested complete plants growing in this way for long periods whilst continuously extracting nematodes.
  2. Chopped plant material: Most endoparasitic nematodes will move out of plant material if it is first chopped into 1 cm lengths or portions. These are placed on the tissue of a Baermann funnel or on a fine sieve, etc. and kept in a Petri dish or similar container. This is left for up to 72 hours, but can be examined much sooner.
  3. Macerated plant material: Roots are cut into short lengths and placed in an electric mixer with a quantity of water. Maceration for 5 secs at half speed and 10 secs at full speed is normally sufficient to break open the plant material without damaging the nematodes, but this will depend on the type of plant. The macerated material is poured onto the tissue of a Baermann funnel or a fine sieve in a Petri dish etc. and left as above.
  4. Mistifier technique: This requires more elaborate apparatus. A continuous fine mist of water is sprayed onto chopped plant material suspended on a sieve over a funnel. The emerging nematodes are washed into the funnel and collected in a container where they settle to the bottom and excess water overflows.
  5. Teasing plant tissues: Immediate extraction of nematodes from plant material is possible by teasing apart the roots, etc. in a shallow dish of water. Emerging nematodes can observed directly. If the material is left for a longer period of time more nematodes will be observed. It is also possible to observe sedentary endoparasites by this method.
  6. Use of hydrogen peroxide in the extraction of endoparasitic nematodes from roots (Gowen, S.R. & Edmunds, J.E., 1973): This method, which has been shown to be most effective, consists of maceration of roots, washing through a bank of sieves and then incubation of the remaining root material in dilute (1%) hydrogen peroxide (H2O2) and some liquid detergent (1 ml/l) for about 2 days. The recovery of nematodes from chopped rather than macerated roots is reduced, possibly because of oxygen starvation or exhaustion of the nematodes. Washing after maceration removes inactive and damaged nematodes that would not be recovered by incubation; it also washes away any phenolic compounds released by the roots (e.g. bananas) which may inactivate nematodes during incubation. For the incubation period H2O2 is used (a) to provide oxygen for the nematodes, and (b) to possibly oxidize any further phenols released by the roots. Liquid detergent ensures that all surfaces of the macerated root tissue are thoroughly wetted.
  7. Extraction of Meloidogyne eggs and juveniles (Gooris, J. & D'Herde, C.J., 1972): Juveniles and eggs of Meloidogyne can be extracted from roots and soil using a variation of the sucrose centrifugation technique, in which the mineral and organic fractions of the soil are separated and treated somewhat differently. The organic fraction is macerated in a blender before centrifugation to release eggs and juveniles from the roots. Maceration of samples with a high mineral content damages a large proportion of nematodes which are not then recovered.
  8. Seeds: Infested seeds can be left in a shallow dish of water and nematodes will emerge over a period of 72 hours. Seeds can be separated from nematode suspension by placing on a small sieve in a Petri dish, etc. With most seed-borne nematodes, e.g. Aphelenchoides besseyi of rice, extraction from seeds is more efficient if seeds are first soaked and the glumes partially removed with a needle and forceps before extracting.

Staining plant tissues

  1. Direct observation: Nematodes in plant material can be stained as follows - wash roots and immerse for 3 minutes in a boiling solution of equal parts lactic acid, glycerol and distilled water + 0.05% acid fuchsin or cotton blue. Clear in a 50:50 mixture of glycerol and distilled water. Nematodes should be stained bright pink or blue, depending on the stain used.
  2. Staining and maceration: Plant material is stained as descibed above prior to macerating. This method enables nematodes that are embedded deep in plant tissues to be observed, but large nematodes such as Hoplolaimus or Hirschmanniella can easily be damaged.

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