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Techniques and methodologies for nematode disease diagnosis and nematode identification

M.R. Siddiqi

Surveying, collecting, fixing and mounting nematodes
Detecting nematode diseases
Evolutionary concept and phylogenetic relationships
Morpho-anatomical methods
Morphometric and morpho-anatomical characters
Biological, biochemical and cytogenetic methods
Nematode identification expertise, equipment and aids, literature and nematode reference collections


Most plant-parasitic nematodes occur in soil around roots and are ectoparasitic, but many endoparasitic species are found abundantly in rhizosphere soil. Some plant-parasitic species are not important economically since they do not cause significant damage to plants. When they do cause noticeable damage they are considered pathogenic. Soil-inhabiting ectoparasitic forms become important when their population inflates to reach the so-called economic threshold. For any control programme, whether chemical, biological, physical or cultural, accurate nematode identification is of the utmost importance. Even determining pathotype or race may become important in a particular group, e.g. Heteroderidae. It is also important to know the hosts of a species and how important it is pathologically to plant growth and yield.

Major nematode parasites are polyphagous and invade many plant hosts, multiply quickly to have several generations per year and have easy means for spread and dispersal. Examples are seen in many root parasites and some parasites of above-ground plant parts (Aphelenchoides ritzemabosi, A. besseyi, Ditylenchus dipsaci). Seed-borne pathogenic nematodes (Anguina tritici, Aphelenchoides arachidis, Ditylenchus dipsaci) have developed special adaptive mechanisms, such as the ability to withstand desiccation, for their survival and dispersal. Species like Rhadinaphelenchus cocophilus, Bursaphelenchus xylophilus, Deladenus spp., Fergusobia spp., use insects as vectors/hosts. Host specificity is shown only by some groups of nematodes, e.g. Heteroderidae, Meloidogynidae, Pratylenchidae, Neotylenchidae. Such host-specific nematodes are supposed to have co-evolved along with their hosts. Co-evolution and zoo-geographical distribution of plant nematodes can throw some light on their origin, evolution and radiation.

Besides their normal mode of transmission, parasites, pests and pathogens are consistently being moved about by people. Human activities and cargo traffic by land, sea and air have tremendously increased the chances of dispersal of plant nematodes. Exotic pests are constantly being introduced into clean areas. The nematodes are brought in with exotic plants, imported grain, vegetable and fruit produce and in soil attached to packing material, machinery, tyres of motor vehicles, tools and even shoes. Live third-stage juveniles, which is the dispersal stage of the pinewood nematode Bursaphelenchus xylophilus, were recovered in Finland from pine boards used in a packing case imported from Canada (Tomminen, 1991). Dispersal through wind, rain and floodwater respects no national boundaries.

The potato cyst nematode (Heterodera rostochiensis) possibly originated in the Andean region of South America and was imported into the United Kingdom during the Irish famine of 1845/46 on potatoes brought for resistance against Phytophthora infestans, the causal agent of the Irish famine. From there it was distributed to mainland Europe, India and Australia. Similarly, Heterodera avenae on wheat was distributed by people. Many temperate nematodes are flourishing in high altitudes in the tropics because they were transported there with planting material. Application of detection and identification techniques are basic to checking for plant nematode introduction, spread and distribution.

Knowledge of hosts and geographical distribution of important species is essential. For example, false root-knot nematode, Nacobbus spp. and Monotrichodorus spp., occur only in the New World, although the former may occur under glass in Europe. Heterodera zeae was found on Zea mays and described in India. Later it was discovered in Maryland, United States, on an indigenous monocot by a stream. It was immediately put under quarantine and it was thought that it had come from India. Since it was found on an indigenous plant in the United States, it must be indigenous to that country. Very likely it originated in Mexico and co-evolved with maize and other monocots and was distributed with maize by humans.

Surveying, collecting, fixing and mounting nematodes

Surveying large areas for the presence or absence of plant nematodes is important but difficult. Systematic surveys are conducted using statistical designs to obtain a reliable estimate of nematode abundance. Fields and patches in a large area should be selected at random and sampling procedure should be based on standardized sampling techniques. Nematode population assays should be made to determine both the qualitative and quantitative occurrences of important species and to relate their population levels to crop damage. Non-agricultural areas adjoining cultivated land and non-crop plants in and outside that area should also be surveyed to identify potential pest problems. This will help an understanding of the alternative hosts and aspects of the biology of nematode species. Biological data including damage symptoms, life cycles, population trends and agroclimatic information on soil types, climatic conditions, cropping sequences and the times of sampling are also important in surveys because populations are dependent on them. This knowledge is of utmost importance when control measures are contemplated.

Surveys should have backing from diagnostic and identification centres to obtain quick and reliable identifications. Fields and large areas for surveys must be selected at random and statistical sampling designs must be used to obtain quick and reliable data on nematode prevalence. Periodic large-scale surveys of important species are essential to assess and monitor nematode populations. They should be repeated every two to four years to determine the relative abundance of species and new introductions, and there should be a system of coordinating various surveys to have a clear picture of the host and distribution of important species within an area. Coordination of surveys and pooling of data at an appropriate centre for analyses are important.

About 250 to 300 ml of soil from around roots is collected in a polythene bag and data on host, locality, soil type, etc. are tagged. Care should be taken to ensure that soil samples are moist during transit. About 100 g of roots including sound, partially attacked and well-attacked parts, and a similar quantity of shoots showing apparent damage, are collected and stored in polythene bags. Soil from roots of adjoining grasses and weeds should not be allowed to get mixed with the samples. Auger samplers are used to obtain soil cores for population studies at different depths. Care must be taken that soil is not mixed with soil from above or below the segment to be analysed.

Samples should be processed soon after collection. They may be stored at about 4°C for later extraction. Extraction methods must be directed towards isolating all stages of nematode development. Variously modified Baermann techniques and the Cobb sieving and gravity method are used for obtaining quick and reliable results (Siddiqi, 1986). Their aims are to recover actively migrating nematodes, and their drawbacks include missing out sedentary nematodes. Many nematodes lie dormant in the soil or plant tissue and need soaking in water for several hours before extraction. A simple bucket-sieving method for isolating nematodes from soil samples of about 300 ml, using a large bucket and a 45 to 53m m aperture sieve, is described in Siddiqi (1986). The nematodes thus extracted are separated from debris and mineral particles by pouring the suspension on to a wire mesh covered with tissue paper and submerged in water in a trough. The active nematodes pass through the filter paper into clean water and can be concentrated by decantation or centrifugation. Soil and plant material can be treated similarly to extract live, motile nematodes. The centrifugal sugar-flotation technique and elutriators are good for processing several samples and obtaining all stages of active or inactive nematodes.

Nematodes may be killed by pouring hot water (85° to 95 °C) over them. In doing so, nematodes should be in the smallest possible quantity of water so that they are exposed to instant heat. They may be fixed in 3 to 5 percent formaldehyde solution and preserved in the same medium. Nematode structures are best studied when examined in water, fixative or lactophenol.

For making permanent mounts, the nematodes are transferred to glycerine and mounted in the same medium. Several methods are described in Siddiqi (1986) for making permanent nematode slides. Siddiqi (1964) described a quick method of transferring nematodes to glycerine by processing through warm lactophenol. The nematodes are transferred to warm lactophenol held in a cavity slide or watch glass. They will first shrink but later stretch out to their normal shape within two to three minutes. If they do not do so and remain shrunk, then more heat should be applied until they are fully stretched. Then the slide can be left at room temperature overnight. The slide is again warmed and a few drops of a mixture of 75 percent glycerine and 25 percent lactophenol are poured on to the nematodes held in thick, concentrated lactophenol. The slide is kept warm for five to ten minutes. Finally the nematodes are transferred to glycerine. Sufficient time should be allowed for the glycerine to penetrate the nematodes, which then can be mounted in pure dehydrated glycerine.

For mounting, nematodes already in glycerine are transferred to a small drop of glycerine placed on a clean glass slide and surrounded by glass fibre pieces for support. Three small lumps of paraffin wax (melting point 55° to 60°C) are put around but separated from the glycerine and a 19 mm diameter cover slip is placed over the wax lumps. The slide is then heated until the wax melts and fills the space between the cover slip and the slide. Finally the slide is sealed with glyceel.

Detecting nematode diseases

Detection and identification of nematodes is the first step in controlling them and checking their spread. This may result in economic savings of great magnitude. For example, Radopholus similis was recognized as the causal agent of the spreading decline of citrus in Florida in 1953. Since then, regulatory agencies in neighbouring states have imposed stringent measures to control its entry and spread. A constant vigil has saved the citrus industry of other states of the United States from being devastated by this disease. Heterodera glycine of soybean (Glycine max), occurs in the Far East and United States. It has been detected and identified in Egypt. Unless strict quarantine measures are applied, the nematode will soon spread in the countries of the Near East.

Nematode diseases usually go unnoticed as infected plants are rarely killed. A particular crop may show only patches of damage where the nematode populations are much larger than in the rest of the field. At times, entire fields may be infested and overall health of the plants may appear poor. The yield from such infested plants is considerably reduced. Farmers usually blame unfavourable soil or climatic conditions, but if the nematode disease is controlled, for example by the application of nematicides, yield is dramatically increased.

General above-ground symptoms of nematode infection include unthrifty reduced growth, severe stunting, chlorosis and wilting of leaves and reduced quality and quantity of produce. Root damage includes swelling and knotting; shrivelling, splitting and cracking; brown necrotic lesions; and stubby, broken or girded rootlets.

Root symptoms are produced by three kinds of reaction:

· A hyperplastic reaction results in hypertrophy and hyperplasia in which both cell number and cell size are increased. Simple hyperplastic galling of root tips is caused by the feeding of Xiphinema, Hemicycliophora and Belonolaimus spp. Feeding by Meloidogyne spp. results in severe hypertrophy and hyperplasia of cells; multinucleate giant cells are formed at the feeding site in the cortex, endodermis and pericycle.

· A hypoplastic reaction that inhibits growth in meristematic tissue and causes stunted and stubby roots is caused by the feeding of tylenchorhynchids and trichodorids, respectively.

· A necrotic reaction is caused by nematode feeding, their movement within the tissues and the presence of micro-organisms. Browning and shrivelling of feeder roots is caused by ectoparasitic nematodes. Pratylenchus spp. and Radopholus spp. cause extensive necrotic lesions in roots by their feeding and movement. The necrotic reaction is caused by enzyme secretion into plant tissue and tissue response, as well as by the accumulation of phenolic compounds in the infected area of the root. Secondary invasion by bacteria and fungi aggravates damage.

Diseases may be caused by individual nematode species, or by a combination of several species or nematodes interacting with other pathogens to produce disease complexes. The disease complexes produced by the interaction of nematodes with pathogenic bacteria or fungi are more damaging to plants than these pathogens acting alone. Some nematodes are vectors of plant viruses. Xiphinema index was first shown to be able to transmit the fanleaf virus from diseased to healthy grape-vines. Now we know of several longidorids and trichodorids which transmit the soil-borne viruses of plants.

Nematodes have evolved various types of association with plants. The parasitic ones may be ecto- or endoparasites; they may be migratory or sedentary parasites. Many are host-specific but most are polyphagous. Siddiqi (1983) gave a detailed analysis of the ecological adaptations of plant-parasitic nematodes.

Migratory parasites of above-ground plant parts

Aphelenchoides besseyi, the causal agent of white tip disease of rice, is a migratory ectoparasite and is seed-borne. Infested seeds, when soaked in water for 24 hours, yield motile nematodes. The nematode also attacks above-ground parts of strawberries causing summer dwarf or crimp disease.

The bud and leaf nematodes (Aphelenchoides ritzemabosi and A. fragariae) feed ectoparasitically on buds and parenchymatous cells of the leaf mesophyll. A. ritzemabosi parasitizes leaves of chrysanthemum and other ornamentals. The symptoms produced include spots or blotches on leaves, distorted tissue and stunted and deformed buds. Soaking the infested plant tissues in water releases the live nematodes. Red ring disease of coconut is caused by Rhadinaphelenchus cocophilus in the Caribbean and northern parts of South America. It is transmitted to new trees by the palm weevil, Rhynchophorus palmarum. Bursaphelenchus lignicolus causes a destructive disease of pine trees in Japan. It infests axial and radial resin canals and is pathogenic causing the death of pine trees. It is transmitted by the cerambycid beetle, Monochasmus alternatus.

Stem and bulb nematode Ditylenchus dipsaci was first described on teasel heads in Europe. It attacks stems of pea, beans, alfalfa etc. and bulbs such as onion (causing bloat disease), daffodil and narcissus. In the Sudan and the Syrian Arab Republic, the infested bean and onion fields exhibit patchy areas and larger populations occur in wet rather than in dry areas. Heavily attacked onion and narcissus bulbs show rings of decayed tissue when they are cut. When infested stem or bulb tissues are macerated in water, hundreds of nematodes in various stages of development are liberated. In a diseased bulb, stem or seed, heavily infested with D. dipsaci, the pre-adult juveniles aggregate in thousands and roll up to form what is known as nematode wool which enables them to survive desiccation and other unfavourable conditions.

Ditylenchus angustus feeds ectoparasitically on rice stems, leaves and inflorescences producing various types of malformation of tissue. It causes Ufra disease of rice in Bangladesh and is known to occur in Myanmar, India and Madagascar. D. drepanocercus infests leaves of an evergreen tree, Evodia roxburghiana, in India.

Sedentary parasites of above-ground plant parts

Members of the family Anguinidae (Subanguina, Anguina, Cynipanguina, Nothanguina, Orrina, Pterotylenchus) produce galls on above-ground parts of plants; only one species of anguinids, Subanguina radicicola, produces galls on roots. Anguina tritici and A. agrostis form seed galls on wheat and grasses, respectively. A. tritici causes ear cockle disease of wheat resulting in wheat grains becoming deformed, brown or black galls that transmit the nematodes when sown along with healthy grains. Tundu disease of wheat is caused by the interaction of A. tritici with Corynebacterium tritici.

Anguina funesta produces seed galls on rye grass in Australia and acts as a vector of the toxin-producing bacterium, Corynebacterium rathayi. About 1 000 bacteria-infested galls are lethal to a sheep. Leaf galls are produced by Subanguina, Cynipanguina, Nothanguina, Orrina and stem galls are produced by Ditylenchus, Pterotylenchus etc. Orrina phyllobia is a parasite of silverleaf nightshade (Solanum elaeagnifolium) and can be used as a biocontrol agent for this perennial weed that infests cotton fields in southwestern United States.

Fergusobia spp., of the family Neotylenchidae, produce aerial shoot galls on Eucalyptus spp. in Australia and Syzygium cumini in India. They show dual parasitism of trees and agromyzid flies (Fergusonina spp.). Teasing out galls in water releases all stages of nematode development.

Migratory ectoparasites of roots

Most plant-parasitic nematode species are ectoparasitic. All plant-parasitic members of Dorylaimida, Triplonchida and Criconematina are ectoparasitic. In Criconematina, the male has a degenerate stylet and oesophagus and cannot feed. Migratory ectoparasitic nematodes may have small stylets (Tylenchidae, Merlinius) to feed on root hairs and epidermal cells, or long stylets (Hemicycliophora, Amplimerlinius) to feed on deeper tissues. Long and strong stylet-bearing Hoplolaimus galeatus and Belonolaimus spp. are pests of lawn and turf grasses in Florida. They produce yellow patches in which the root system is considerably reduced in size as a result of the feeding of nematodes. The ectoparasites, Xiphinema and Tylenchorhynchus, kill epidermal cells by feeding and cause discoloration and superficial necrosis. Stubby root and docking disorder in crops are caused by trichodorid nematodes.

Species of Xiphinema and Longidorus are known to transmit soil-borne NEPO viruses of plants. Xiphinema index and X. diversicaudatum transmit fanleaf virus of grapevines and arabis mosaic virus, respectively. Longidorus elongatus is a vector of raspberry ringspot virus and tobacco black ring virus. Trichodorus and Paratrichodorus transmit TOBRA viruses of plants. Plants showing virus disease symptoms should be sampled for these nematodes. The ectoparasitic nematodes are generally polyphagous and survive in soil without a host for many years. They are easily extracted from soil by Baermann funnel, sieving or flotation techniques.

Sedentary ectoparasites of underground plant parts

Sedentary obese females of the ectoparasitic genera Tylenchulus, Sphaeronema, Rotylenchulus, etc. are detected only by examining the roots. The author's report of the finding of Tylenchulus semipenetrans and Rotylenchulus reniformis in India in 1961 was the result of collecting the motile stages of these species by a sieving method using 250-mesh sieves. The immature female stage and the male of the latter species gave rise to the idea of creating a new genus for them since they were unique to Hoplolaimidae. The author had not seen the kidney-shaped mature female, but the timely warning of Dr M.W. Alien of California University prevented a synonym of Rotylenchulus being proposed, as authors of Leiperotylenchus and Spyrotylenchus had done before. For identification of these nematodes, therefore, all stages of development should be examined.

Tylenchulus semipenetrans causes slow decline of citrus and occurs in all areas of citrus culture. The female's anterior end lies deep in the root reaching the stele region where it feeds, while its body, most of which remains outside the root, swells. The root tissues respond to nematode feeding by producing nurse cells near the head of the nematode, thus avoiding destruction and decay. The nematodes may aggregate and form colonies on roots and lay eggs in a protective gelatinous material. Juveniles of T. semipenetrans remain viable in soil for several years after the citrus trees are removed. Recovery of these juveniles from the rhizosphere soil of plants that might have grown there is therefore not a proof of such plants being the hosts of this nematode.

Migratory endoparasites of underground plant parts

Pratylenchus and Radopholus spp. live, feed and multiply within the root and underground stem tissue. Pratylenchus spp. cause necrotic lesions in the epidermis and cortical parenchyma that may extend to the endodermis and stele. The lesions eventually girdle the rootlets and interrupt the water and nutrient supply of infected plants. Often such lesions are invaded by rot-causing bacteria and fungi, completely destroying the rootlets. Radopholus similis similis is a serious pest of banana, coconut, tea and black pepper. It causes severe root rot that, in the case of banana, results in black head or toppling disease, and in black pepper the yellows disease. R. similis citrophilus causes the spreading decline of citrus in Florida. The diseased trees show wilting of leaves, dieback of twigs and general unthriftiness, low fruit yield and a poor root system in which the feeder roots are shortened and reduced to less than half the normal size and number.

Scutellonema bradys infests yam tubers and even multiplies in stored yams causing severe damage. Hirschmanniella oryzae, H. mucronata and H. spinicauda parasitize rice roots in many tropical and subtropical countries. The nematodes feed and move about intercellularly in air channels between the radial lamellae and the parenchyma of the roots. Hirschmanniella miticausa parasitizes taro (Colocasia esculenta) causing mitimiti disease in the southern highlands of Papua New Guinea. Aphelenchoides arachidis infests groundnut pod shells and seed testa in northern Nigeria, is resistant to desiccation and is seed-borne.

Sedentary endoparasites of underground plant parts

The root-knot nematodes, Meloidogyne spp., are obligate endoparasites and are very damaging plant pests particularly in the tropics. They are easily detected due to the galls or knots which they incite, usually on the roots, but occasionally on stems. Pearly white females, egg masses attached to the posterior of females and second-stage juveniles are recovered when a gall is dissected in water. Meloidogyne species found in marine habitats, e.g. M. mersa on Sonneratia alba roots in Brunei Darussalam mangrove swamps, burst in ordinary tap water and should be dissected out from the galls in saline or sea water (Siddiqi and Booth, 1991). Galls are also produced on tomato stems, potato tubers and ginger and banana rhizomes as well as roots.

Meloidogyne species identification is mainly based on the characters of second-stage juveniles and the perineal patterns of the adult female. Root galling is not enough evidence for the presence of a Meloidogyne species. Juveniles in soil should be the best indicator of this nematode. Sometimes Meloidogyne and heteroderid juveniles are found in soil around roots of a crop plant or tree, but repeated searches fail to locate the females. It may be that the females occur on roots of some associated weed or grass and are not a parasite of the crop being surveyed, or that these juveniles are the survivors of the populations from a previous crop.

The false root-knot nematodes, Nacobbus spp., are similar to Meloidogyne spp. in inducing root galls, but are very different in morphology. Since they occur only in the Americas, there is no problem in confusing them with Meloidogyne spp. in the old world.

Cyst nematodes are also endoparasitic, but the mature females protrude from the root surface as they feed and grow, and when their bodies transform into brown or black cysts they can be easily dislodged from the root. Heterodera and Globodera cysts are lemon-shaped and rounded, respectively. Cysts remain viable in soil free of hosts for up to eight to ten years. The cysts are recovered from the soil by flotation techniques. Cyst nematodes induce syncytia in roots from which they derive nutrition throughout their development. Vascular bundles are damaged by this host reaction and the flow of nutrients and water is disrupted. Because of their host specificity, the introduction of cyst nematodes and their spread can be checked by applying strict quarantine measures. Crop rotation with non-hosts is a good control measure.

Evolutionary concept and phylogenetic relationships

Taxonomy is the science of identifying and naming animals and plants and assigning them to groups in a system of classification. The identification and naming of individual taxon (plural taxa) is called microtaxonomy, while placing them in a hierarchical system of classification is macrotaxonomy. Taxa include any category from subspecies to superfamily. Taxa higher than superfamily, e.g. order or class, are not covered by the International Code of Zoological Nomenclature. Taxa are usually diagnosed on the basis of phenotypic similarity/dissimilarity and not on shared common ancestry (as they should be).

Macrotaxonomy as a science of classification is based on the philosophy of organic evolution. All animals and plants are continuously changing, as are all non-living things in the universe. Some changes are brought about in a short time and are observable, but others take thousands of years and are not seen or sensed. Macrotaxonomy uses various methodologies to understand the mechanism and degree of this long-term change. These methodologies utilize the characters/features of animals/plants to determine their primitive or derived nature by assessing their weighting and homology. Conclusions are then drawn about their relationships for building a hierarchical system of classification.

Relationships are interpreted in terms of descent from a common ancestor. This is not an easy task since interpreting homology increases the amount of speculation. Several approaches have been developed to establish the relationships of taxa and to classify them. Siddiqi (1986) in his book on Tylenchida has discussed at length cladistic, evolutionary and phenetic approaches. However, in classification, many nematologists are not using these approaches at all, but are largely depending on their own concepts and experiences of the groups. It is said that identification work, which is different from phylogenetic work, should not necessarily depend on such approaches, but new species and genera, when proposed, should always be properly diagnosed and the criteria for their differentiation be discussed to show their relationships with existing taxa.

Morpho-anatomical methods

Species identification utilizes characters/features of many kinds including morpho-anatomical, physiological, ecological, ethological, embryological and cytogenetical. By far the most useful and widely used characters are the morpho-anatomical.

A comparative study of morpho-anatomical characters/features in a range of closely related species should be made to evaluate their importance in identification. This would include the study of each character's stability/variability within that group. Hasan (1990) states: "Many species occur in mixture in natural populations. In most cases, due to lack of data on interbreeding, reproductive biology, physiology, ecology and geographical distribution etc., the concept of phenetic species has been employed in nematode taxonomy for diagnosing a taxon. Such a concept has its own importance, however many of these species have been known to exhibit a wide range of intraspecific variations in morphology and dimensions which according to animal taxonomists is the function of geographical isolation, ecological/environmental stresses and genetic inequality."

The biospecies concept involves determining a species on the basis of whether populations interbreed if their members are morphologically similar to each other but different from other such populations. Difficulties arise when either the members of the populations are morphologically similar but do not interbreed, or they do interbreed but are morphologically dissimilar. Ditylenchus dipsaci populations from phlox and others do not interbreed successfully (Ladygina, 1974). Are they then separate species? Globodera rostochiensis and G. pallida differ morphologically, but they may mate freely in laboratory experiments and are expected to do so in the field (Sturhan, 1985). A female could mate with several males and thus the offspring in a cyst may not represent a pure culture.

Interbreeding as a test for species recognition does not apply to parthenogenetic forms, and among plant-parasitic nematodes such forms are quite common. Morpho-anatomical determination of species is therefore of utmost importance in parthenogenetic forms and is the main method of identification. Its application to sibling species, however, becomes difficult. For example, Radopholus similis and R. citrophilus are similar in morpho-anatomical details but not so in physiological and cytogenetic characters. In such sibling species, the concept of subspecies is worth while. The two species may be called Radopholus similis similis and R. similis citrophilus to show their relatedness but to keep them as distinct forms. The occurrence of closely related species sympatrically which maintain their morphological identity gives them a good taxonomic status.

Parthenogenetic species are prone to clone-formation because they lack bisexuality and hence the exchange of genetic material. Such clones differ in small morphological differences and thus naming them as separate species could be dangerous. Variations among large populations from different places should therefore be studied thoroughly. These variations are due to isolation, geographical separation, host effects and several physical and chemical factors. Species will, however, continue to be described on morpho-anatomical characters.

The superspecies concept, although not used in plant nematology, is a good one and includes, under one name, morphologically closely related species, sibling or cryptic species and subspecies, e.g. Globodera tabacum can be a superspecies to include G. tabacum, G. virginiae and G. solanacearum.

Morphometric and morpho-anatomical characters

Since plant nematodes are microscopic, morpho-anatomical characters have to be correctly observed and interpreted. Even taxonomists fail to observe some characters correctly. For example, some Helicotylenchus spp. show a distinct dorsal oesophageal gland but indistinct subventral glands and so they have been described under Rotylenchulus/Orientylus. A solid-appearing anterior region of the stylet conus of Tylenchorhynchus and a conus having an asymmetrical lumen in Histotylenchus can only be studied properly when seen in lateral view. A post-anal intestinal sac was wrongly reported in Tylenchorhynchus stegus and Quinisulcius himalayae possibly because the granules in the lateral hypodermal chords looked similar to those in the intestine. Phasmids were reported in the tail region of the Tylenchidae and of Ditylenchus although they are absent in these groups.

Detailed studies on morpho-anatomical characters including their variability in many populations from many hosts and geographic regions provide the basis for creating new species. Taylor and Jenkins (1957) described Pratylenchus hexincisus sp.n. and P. subpenetrans sp. n. and Fortuner (1970) described Aphelenchoides siddiqii sp. n. because they studied a number of closely related populations. The same applies to the author's description of new species of Monotrichodorus, viz. M. acuparvus, M. parvus and M. proporifer from South America.

Morphometric data. Morphometric characters including the de Manian ratios should not be the sole basis for species differentiation because they are often very variable. Taylor and Jenkins (1957) found L, a, b, c, c' ratios in Pratylenchus spp. as highly variable, but the V ratio showed a greater stability. The V ratio is a good taxonomic character which is used to differentiate species of many genera, e.g. Pratylenchus, Paratylenchus, Rotylenchulus. In several cases, overlapping ranges of many morphometric characters make it very difficult to use them for species differentiation.

Sex and sexual dimorphism. Presence/absence of males and of sexual dimorphism are used as differentiating features. Where males are absent, the female spermatheca is reduced and empty. Sexual dimorphism in body shape is a good character of Heteroderidae and Rotylenchulinae and in the anterior region (cephalic region, stylet and oesophagus) is a characteristic of the Radopholinae as against the Pratylenchinae.

Body size and shape. Body length, width and shape (cylindrical, tapering at ends, kidney-shaped, lemon-shaped etc.) and habitus at death (straight, arcuate, spiral etc.) serve as useful differentiating characters. It should be kept in mind that fixation and processing to glycerine may cause reductions, distortions and artefacts. Therefore, specimens in water or fixative should always be measured and studied.

Cuticle. Cuticle thickness, striation, annulation (both transverse and longitudinal), punctation and ornamentation, and cuticular modification (ridges, spines, scales, alae) serve as useful characters as do the lateral field and their incisures. Thickening of the cuticle at the tail tip is a good character for Trophurus and Paratrophurus. Longitudinal ridges characterize Mulkorhynchus while the presence of five or six lateral incisures are used to diagnose Quinisulcius and the Merliniinae, respectively. Body pores are used as taxonomic characters in Trichodoridae and the excretory pore position is used as a taxonomic character. Amphids, deirids, phasmids and sensory papillae including male supplementary papillae are very useful in the taxonomy of all groups. Bursal ribs on the tail are diagnostic for the Aphelenchidae while the presence of hypoptygma around the cloacal aperture differentiates the Merliniinae from the Telotylenchinae.

Cephalic region. The shape and the degree of separation from the body of the cephalic region is used in species differentiation. Its transverse and longitudinal striation, the formation of a labial disc and the degree of sclerotization of its framework are also important.

Stylet and oesophagus. By far the most diagnostic characters are found in the stylet and oesophagus. Length of the stylet and the relative length of its conus are useful, as are the size and shape of the stylet knobs. Siddiqi (1971, 1986) discussed the importance of the oesophagus in the classification of the Tylenchida. Among the Tylenchina, the families Tylenchidae, Psilenchidae and Dolichodoridae have a basal bulb enclosing the three oesophageal glands, while in the Hoplolaimidae, Heteroderidae and Meloidogynidae the glands lie free in the body cavity extending over the intestine. In the Hoplolaimidae, the glands may extend mostly ventrally or dorsally, but in the Heteroderidae and the Meloidogynidae, they are always ventral to the intestine. The dorsal gland nucleus is larger than those of the subventrals in the Xiphinematinae, while it is smaller than the subventrals in the Longidorinae.

The presence of the orifice of the dorsal oesophageal gland within the median bulb is one of the diagnostic characters of the order Aphelenchida. In the same group, the presence of a distinct isthmus is the characteristic feature of the Aphelenchoidea. The shape and size of the cardia or oesophago-intestinal junction are useful in the Dorylaimida.

Intestine, prerectum, rectum and anus. The number of cells in a cross-section of intestine and the length of the prerectum are useful in the taxonomy of the Dorylaimida. The anus in members of the Aphelenchida is a large, posteriorly directed crescentic slit compared with the small and pore-like structure in members of the Tylenchida. Rectum length in respect to anal body width is also used as a character. A post-anal intestinal sac differentiates between Bitylenchus and Tylenchorhynchus and its length is useful in differentiating species.

Female reproductive system. Reproductive systems can be didelphic, monodelphic, pseudomonodelphic (when one branch is secondarily reduced), prodelphic, opisthodelphic, monoprodelphic and mono-opisthodelphic. The length of the reproductive branch as a percentage of the body length is important in some cases, but in others it is highly variable since it depends on the degree of development of the ovary. The shape and location of the vulva is a good character, as is the presence/absence of lateral vulval membranes and epiptygma. The shape and size of the spermatheca and its position in respect to the genital branch (axial or offset) are good diagnostic characters. When a branch is reduced to a sac, its length and the presence/absence of a reduced ovary serve as good characters, e.g. in Pratylenchus. The length of the mature ovary and the arrangement of oocytes are also important, e.g. in Aphelenchoides.

Male reproductive system. The size and shape of the spicules, gubernaculum and spermatozoa are useful characters. There is a distinct difference in the shapes of the spicula and gubernacula of the Tylenchorhynchinae versus the Merliniinae and the gubernacula of the Pratylenchinae versus Radopholinae. The shape of spermatozoa differs in species of Radopholus. Genital papillae and ventromedian supplements are important characters of trichodorid and dorylaimid nematodes. Copulatory muscles were used by Siddiqi (1974) to differentiate between Trichodorus and Paratrichodorus and the presence of hypoptygma was used by Siddiqi (1970) to differentiate Merlinius from Tylenchorhynchus.

Tail. Tail length and shape are used in taxonomy but they show considerable variation. The shape of the tail tip, although a useful character in many groups, was shown to be variable in some species of Pratylenchus (Taylor and Jenkins, 1957). Nevertheless, a pattern of tail tip shape emerges when a large number of specimens are studied. For example, Pratylenchus panamaensis, P. coffeae and P. loosi have characteristic tail tip shapes. The tail is consistently elongated in the Tylenchidae, hooked in the Halenchidae and short and rounded in females of the Hoplolaiminae and males of the Heteroderidae and the Meloidogynidae.

Juvenile characters. Tail shapes in juveniles of various stages is a good character in the differentiation of Xiphinema species. Presence of spined juveniles in Hemicriconemoides differentiates it from Hemicycliophora. The shape and arrangement of spines in juveniles of this genus are used for species differentiation.

Biological, biochemical and cytogenetic methods

Biological characters, especially host preference, can be used in the identification of some plant-parasitic nematodes. However, the effects of physical, chemical and biological factors on host-parasite relationships are often great. Presence or absence of males is also a differentiating character, but it is known that at least in some species, males may arise as a response to environmental stress.

Biochemical and cytogenetic techniques are used to discover the degree of genetic similarity in a taxonomic group. Such data must be obtained for several species to compare with both in-group and out-group members. A comparative study of such data provides valuable information about characters/features that are the result of common genetic material, as well as those that are unique for a particular member of the group. The uniqueness determines the identity of the taxon. Genetically dependent molecular data can be used concordantly with the morpho-anatomical characters to determine natural groupings and evolutionary trends.

Isozyme phenotypes, particularly esterases, have been used in the identification of Meloidogyne spp. (Esbenshade and Triantaphyllou, 1990). An inventory of esterase/phenotypes is made for a comparative study. Although having great potential, polyclonal (PCA) and monoclonal (MCA) antibodies have been utilized so far only in a limited area for the detection and identification of nematode pests. Immunofluorescence and antisera could provide valuable tools for the detection of nematode diseases and identifying and classifying plant-parasitic nematodes. Biochemical characterization makes identification simple and quick.

Serological techniques using PCA and MCA have been tried and some specific antigens from endoparasitic nematodes have been identified and used, for example in separating Globodera pallida from G. rostochiensis (Schots et al., 1990). Davies and Lander (1992) found a large number of MCA using three Meloidogyne spp., M. incognita, M. javanica and M. arenaria, but none was specific and each cross-reacted with at least one other species. However, three MCA were promising in distinguishing between the three species on ELISA-based and immunoblotting assays.

Cytogenetic techniques to determine chromosome number have been used successfully in the classification of higher taxa and the identification of species and pathotypes of Meloidogyne and Pratylenchidae. All the Criconematoidea have a basic chromosome number n=5. Dolichodoroidea have n=8 and Heteroderidae and Meloidogynidae have n=9 (polyploidy 18) and n=18 (polyploidy 36, 54), respectively. Radopholus similis citrophilus was differentiated from 7?. similis similis in having n=5 compared with n=4 in the latter. Meloidogyne species can be differentiated by determining chromosome number, differential host tests and studying perineal patterns.

Nematode identification expertise, equipment and aids, literature and nematode reference collections

Nematode taxonomists

Experts are needed for the development and implementation of any scientific programme. Nematode taxonomists are few and far between and they have been branded as a dying breed. The importance of plant-parasitic nematodes in agriculture, the role of nematodes in ecosystems and the potential use and need for the preservation of biological diversity in non-agricultural lands, have increased the demand for nematode taxonomists. However, the demand cannot be met unless taxonomic teaching and training programmes are implemented and funds to support and sustain them are found.

In the Near East region, there are good taxonomists and diagnostic centres only in Egypt, the Islamic Republic of Iran and Pakistan. But even in these countries, nematode species are not properly surveyed and catalogued. Further surveys and taxonomic research programmes should therefore be launched. For this, support funds should be obtained from national and international funding agencies and collaborative work between the taxonomists of the region should be encouraged.

Microscopes and computers

For any diagnostic work, a good compound microscope is essential. Fitting interference equipment to the stereoscope compound microscope provides sharp images and reduces eye strain. The use of the scanning electron microscope (SEM) has increased in the study of nematode species. New species descriptions often include SEM photomicrographs that show clearly such characters as the amphids, labial and cephalic papillae, lateral field incisures and genital papillae. However, for routine identification work, SEM is not essential. Diagnostic characters, if based only on SEM observations, will be difficult to use. Nevertheless, Radopholus similis similis and R. similis citrophilus, which are not differentiated by morpho-anatomical characters studied with a light microscope, are readily differentiated by examining the SEM photomicrographs of the cloacal region.

Computers are now being used for recording and analysing identification data. Survey records of parasites, hosts, occurrences, soil types, climatic conditions etc. become so large that manual sorting and analysis take an immense amount of time and labour. Also, it is difficult to edit and update the records. All such records and data can now be stored in computerized filing systems providing databases that can be searched, retrieved and sorted within a few minutes. It also avoids storage problems and the possibility of accidental damage. Databases can easily be copied into duplicate diskettes to avoid accidental loss.

Need for a joint effort in computerizing survey records and developing computer-aided identification

In the Near East region, there is hardly any centre where survey records and nematode collections are computerized. Computers are rarely used in identification work. Esser (1991) produced a computer-ready check- list of plant-parasitic nematode genera and species to be used in identification work. Computer-based identification expert systems are now being developed. A joint effort of 33 taxonomists from 13 countries with support given by the NATO Science Programme and the North Carolina State University, reviewed identification methods and proposed the development and implementation of an expert system for plant-parasitic nematode identification (Fortuner, 1988). Countries in the Near East region should try to fund such a project in one of their countries to serve the needs of the entire region.

A system for networking all information on occurrences of plant-parasitic nematodes, including host lists in the Near East region, is a dire need on which due attention must be focused. Such information will be useful to all the countries in the region in checking the spread of pests, parasites and pathogens.


Availability of literature is the most important requirement of a diagnostic and identification centre. Many countries in the Near East region do not even have sufficient funds to subscribe to the core nematological journals, with the result that the papers produced by their workers are often poor in quality. Monographs, checklists, dichotomous and polytomous keys, taxonomic data sheets etc. are useful tools that make identification work comparatively easy. Esser (1991) used Siddiqi's (1986) book on the Tylenchida extensively in his computerized checklist and remarked: "After 1974, M.R. Siddiqi's 1986 Tyienchida is a very valuable source of data". Esser produced many identification aids as keys and compendia which are widely used as identification tools.

Nematode type and reference collections

The importance of type and reference collections in nematode identification cannot be exaggerated. Identification studies invariably involve comparing test animals with specimens of other species. Basing comparisons on published data alone is dangerous since many species descriptions and even illustrations can be poor and sometimes misleading.

High quality and reliable identifications are subject to good taxonomic expertise, and comparison with actual specimens of related species is essential. Taxonomists should always deposit the type specimens of their new species in recognized nematode collections where they can be properly looked after and made available to other workers, if required. Good type and reference collections are available at the national depositories in India (IARI, New Delhi) and Pakistan (NNRC, Karachi) and in international centres such as the International Institute of Parasitology, St Albans, and Rothamsted Experiment Station, United Kingdom.


Davies, K.G. & Lander, E.B. 1992. Immunological differentiation of root-knot nematodes (Meloidogyne spp.) using monoclonal and polyclonal antibodies. Nematologica, 38: 353-366.

Esbenshade, P.R. & Triantaphyllou, A.C. 1990. Isozyme phenotypes for the identification of Meloidogyne species. J. Nematol., 22: 10-15.

Esser, R.P. 1991. A computer-ready checklist of the genera and species of phytoparasitic nematodes, including a list of mnemonically coded subject categories. Gainesville, Florida, USA: Division of Plant Industry. 185 pp.

Fortuner, R. 1970. On the morphology of Aphelenchoides besseyi Christie, 1942 and A. siddiqii n. sp. (Nematoda, Aphelenchoidea). J. Helminth., 44: 141, 152.

Fortuner, R. 1988. Nematode identification and expert system technology. New York, NY, USA, and London, UK, Plenum Press. 386 pp.

Hasan, A. 1990. Morphological variations and the species concept in nematode taxonomy. In S.K. Saxena, M.W. Khan, A. Rashid & R.M. Khan, eds. Progress in plant nematology, p. 15-36. New Delhi, India, CBS Publishers.

Ladygina, N.M. 1974. Ongenetic-physiological compatibility of various forms of stem nematodes. IV. Crossing of the phlox nematode with other ditylenchids. Parazitologiya, 8: 63-69.

Schots, A., Gommers, F.J., Bakkar, J. & Egberts, E. 1990. Serological differentiation of plant-parasitic nematode species with polyclonal and monoclonal antibodies. J. Nematol., 22: 16-23.

Siddiqi, M.R. 1964. Studies on Discolaimus spp. (Nematoda: Dorylaimidae) from India. Z. zool. Syst. Evolut.-Forsch., 2: 174-184.

Siddiqi, M.R. 1970. On the plant-parasitic nematode genera Merlinius gen. n. and Tylenchorhynchus Cobb and the classification of the families Dolichodoridae and Belonolaimidae n. rank. Proc. Helminth. Soc. Wash., 37: 68-77.

Siddiqi, M.R. 1971. Structure of the oesophagus in the classification of the superfamily Tylenchoidea (Nematoda). Indian J. Nematol., 1: 25-43.

Siddiqi, M.R. 1974. Systematics of the genus Trichodorus Cobb, 1913 (Nematoda: Dorylaimida), with descriptions of three new species. Nematologica, 19: 259-278.

Siddiqi, M.R. 1983. Ecological adaptations of plant-parasitic nematodes. Pak. J. Nematol., 1: 63-77.

Siddiqi, M.R. 1986. Tylenchida parasites of plants and insects. Wallingford, UK, CAB International. 645 pp.

Siddiqi, M.R. & Booth, W. 1991. Meloidogyne (Hypsoperine) mersa sp. n. (Nematoda: Tylenchinae) attacking Sonneratia alba trees in mangrove forest in Brunei Darussalam. Afro-Asian J. Nematol., 2: 212-220.

Sturhan, D. 1985. Species, subspecies, race, and pathotype problems in nematodes. EPPO Bulletin, 15: 139-144.

Taylor, D.P. & Jenkins, W.R. 1957. Variation within the nematode genus Pratylenchus with the descriptions of P. hexincisus, n. sp. and P. sub- penetrans, n. sp. Nematologica, 2: 159-174.

Tomminen, J. 1991. Pinewood nematode, Bursaphelenchus xylophilus, found in packing case wood. Silva Fennica, 25: 109-111.

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