|Plant Parasitic Nematodes|
Plant-parasitic nematodes are recognized as one of the greatest threat to crops throughout the world. Nematodes alone or in combination with other soil microorganisms have been found to attack almost every part of the plant including roots, stems, leaves, fruits and seeds. Although worldwide recognition of nematodes as important casual agents of plant diseases did not occur until the middle of this century, nematodes were studied more than 100 years earlier.
The purpose of this educational material is to give some useful information to all PPQ Inspectors about the major plant parasitic nematodes of economic importance, the importance of nematode identifications to agriculture, the nematode problems and diseases on major crops, the nematode disease symptoms and diagnosis, the examination and collection of soil and plant material, and the extraction of nematodes from soil, in order to assist and make them aware of what they should be looking for while dealing with any nematode interceptions of quarantine importance and the kind of samples they should be receiving or sending for identification purposes. The brief information provided covers some of the major food and cash crops throughout the world and can be used by Animal and Plant Health Inspection Service (APHIS) Inspectors with little practical experience of nematodes.
Nematodes are microscopic worms that cause eighty billion dollars of crop loss in the world each year. All crops are damaged by at least one species of nematode. Nematodes constitute one of the most important and abundant groups within the animal kingdom and are highly capable of surviving in any environment. In general, nematodes have slender, cylindrical, non-segmented bodies tapering towards the head and tail, but females of some of the plant-parasitic species assume varying forms, such as pear, lemon or kidney shapes. They are called plant-parasitic because of the nutrients they get from plants and have a needle-like structure called stylet, which is used to pierce plant cells to get food. The economic consequences of crop loss due to nematode-borne disease are many and varied. These involve reduction in quality and quantity of crop yield.
The science of Nematology began in the 17th century when the compound microscope was discovered. One of the earliest reports of observation of plant-parasitic nematodes was in 1743 when Needham observed nematodes in wheat galls commonly known as cockles, causing considerable loss to wheat crops. In 1855, a root-knot nematode causing galls on cucumber roots was discovered. Later, the sugar-beet cyst nematode was found to cause widespread damage to sugar beets. The discovery of the destructive potato cyst nematode in Long Island, the demonstration that the burrowing nematode causes spreading decline of citrus, the severe crop loss due to root-knot and lesion nematodes in California, the discovery of soybean cyst nematode as a serious pest of soybeans in the United States and many other countries, and the damage due to the stem and bulb and the pine wood nematodes resulted in increased interest in and support of plant nematology. Cyst and root-knot nematodes are the two most widespread, economically important plant-parasitic nematodes and cause serious losses on all crops.
The identification of new or potentially harmful species of nematodes is important to the success of agriculture and aids in the development and evaluation of quarantine or regulatory procedures to minimize their spread. As world travel and the transportation of plant material increases, the need to monitor the movement of destructive nematodes increases. Correct species identification is basic to efficient nematode control and successful plant quarantine operations. Preventive regulatory programs have prohibited the introduction of parasitic nematodes to agricultural areas efficiently and have proved cost-effective to limit future crop losses. In 1941, for example when the golden cyst nematode of potatoes was discovered in Long Island, New York, the immediate implementation of a federal quarantine on this serious pest of potatoes helped prevent the spread of this species in the United States, thereby saving annually millions of dollars in crop loss due to this exotic pest. Also, in 1993, pea cyst nematode was found in commercial pea fields in Washington. Scientists are working to develop strategies to control the spread of this devastating pest of peas.
Quick and accurate nematode identifications are very important in the release of shipments of various domestic and foreign plant material and wood products detained at various ports by Animal and Plant Health Inspection Service (APHIS) inspectors. The results of identifications are used by APHIS personnel for taking appropriate regulatory actions beneficial to the public
There are several threatening exotic nematodes that are not currently in the United States. Some of them represent a greater threat than the others. Following is a list of some of these nematodes:
Potato-cyst nematode (Globodera pallida), confined to Solanaceae, chiefly tuberous Solanum species; G. zelandica, restricted to New Zealand native tree fuchsia, Fuchsia excorticata and other native legume plant; rice cyst nematode (Heterodera oryzae), is restricted to rice fields of Ivory Coast; pigeon-pea cyst nematode (H. cajani), on pigeon-pea (Cajanus cajani) in India; H. mothi, on roots of Cyprus rotundus in India; lucerne cyst nematode (H. medicaginis), on lucerne (Medicago sativa) in the Soviet Union; H. mediterranea, on roots of woody plant, lentisc (Pistacia lentiscus) in Italy; H. pakistanensis, on roots of wheat (Triticum aestivum) in Pakistan; H. graminis, from grass (Cynodon dactylon) in Australia; H. sorghi, on roots of sorghum (Sorghum vulgare) in India; H. raskii, from roots of bulb grass (Cyperus bulbosus) in India; H. spinicauda, from soil around roots of the reed (Phragnutis australis) in the Netherlands. Meloidogyne coffeicola, on roots of coffee (Coffea arabica) in Brazil. M. salasi, on rice (Oryza sativa) in Costa Rica and Panama; M. suginamiensis, from roots of mulberry (Morus alba) in Japan. M. sewelli, roots of spike-rush (Eleocharis acicularis) in Canada; M. microcephala, from roots of tobacco (Nicotiana tabacum); M. arabicida, from coffee (Coffeae arabica) in Costa Rica; M. lini, from roots of rice in China; M. kongi, from roots of citrus in China; M. jianyangensis, from roots of mandarin orange in China; M. vandervegtei, from unidentified woody plant in South Africa; M. hispanica, from roots of peach rootstock, Prunus persica silvestris in Spain; M. brevicauda, from roots of tea (Camellia sinensis) in Sri Lanka; rice stem nematode (Ditylenchus angustus), in rice-growing areas of Bangladesh, Vietnam and other areas of Asia; seed gall nematode (Afrina wevelli), from galls from amongst seed of Eragrostis curvulva from South Africa; Afenestrata africana, parasites of grasses (Panicum maximum) in Ivory Coast; cystoid nematode (Thecavermiculatus andinus), from roots of Oxalis tuberosa in Peru.
Following are some of the major food crops with important nematode diseases:
Several nematode species are associated with citrus, and the most devastating one is the citrus nematode (Tylenchulus semipenetrans) which occurs in all the citrus producing regions of the world. This nematode infests from 50-90% of all the citrus producing regions of the world and causes the disease "slow decline" of citrus. Another nematode damaging citrus is the burrowing nematode (Radopholus similis), which causes a severe spreading decline disease of citrus. In addition, lesion, root-knot, sting, dagger, stubby root and other ecto-parasitic nematodes can damage citrus.
The inspectors should look for the adult females of citrus nematode in the heavily infected feeder roots of citrus which are found attached to the rootlets which often appear dark and thicker than the healthy roots due to soil particles that adhere to gelatinous egg masses produced by the females on the root surface. From soil and root washings around citrus trees second-stage larvae and males of this nematode are commonly encountered. Also, adults of vermiform migratory endoparasites of burrowing nematode can be recovered from the roots of citrus.
Several nematodes are associated with coconut, but the most destructive disease affecting the crop is red ring disease, caused by the red ring nematode (Rhadinaphelenchus cocophilus), and on some islands, threatens the existence of this crop. Another nematode causing damage is the burrowing nematode.
The inspectors should look for the red ring nematode in the roots, trunk and stem tissue of coconut palms causing lesions and the characteristic orange to red ring appears about 3 cm wide and 2.5 cm beneath the stem surface. The nematode invades through root tissue, stem and leaves. The palm weevil (Rhynchophorus palmarum) is the main vector of nematodes from diseased to healthy trees.
Lesion nematodes and corn cyst nematode are considered as economically important nematodes which cause severe damage to corn. The corn cyst nematode (Heterodera zeae) is widespread in India. It also occurs in Egypt, Pakistan and in Maryland, USA.
The inspectors should look for white or yellow stage females or brown cysts of corn cyst nematode on roots of corn (Zea mays). Soil analysis for extraction of cysts, juveniles and males is also recommended.
The two most important root diseases of cotton are root-knot caused by the root-knot nematode, and Fusarium wilt, caused by the fungus Fusarium oxysporum. Infection by root-knot nematodes increases the incidence and severity of Fusarium wilt. Other species which are pathogenic on cotton are reniform, lesion, sting, lance and dagger nematodes. The sting nematode is an aggressive pest of cotton but fortunately is restricted to soils with greater than 85% sand content. Lance nematodes mostly feed in the cortical region of cotton roots causing cell damage and necrosis.
The inspectors should look for the root knots caused by the root-knot nematodes on cotton roots. The other species which are pathogenic on cotton and should be looked for are reniform nematode (Rotylenchulus reniformis), lesion nematode (Pratylenchus brachyurus) and sting nematode (Belonolaimus longicaudatus).
Several plant-parasitic nematodes are associated with legume crops. The pea cyst nematode is an important parasite of peas and broad beans in many countries. The stem nematode is another important nematode on broad beans. Root-knot, cyst and reniform nematodes are the major nematode pests of chickpea and cowpea.
The inspectors should look for roots of peas with white females of pea cyst nematode (Heterodera goettingiana) and for pigeonpea cyst nematode on pigeonpea (Cajanus cajan).
Nematodes damage peanuts in all production regions of the world. The annual loss caused by nematodes to peanuts has been estimated at 12%. The nematodes that attack peanuts and cause damage are root-knot, lesion, sting, ring, stunt and potato-rot nematodes.
The inspectors should look for galls on peanut pods, pegs and roots caused by peanut root-knot nematode (M. arenaria), javanese root-knot nematode (M. javanica) and northern root-knot nematode (M. hapla). M. arenaria is a major disease of peanuts in several countries. Also, lesion nematode (P. brachyurus) is the major lesion nematode parasitizing peanuts.
The most important nematodes threat to potato production is undoubtedly caused by the potato cyst nematodes, which cause severe damage wherever they are present. Two species of cyst nematodes infect potatoes- Globodera rostochiensis (the "golden" nematode) and G. pallida. The golden nematode is found in several countries: Western Hemisphere- Newfoundland and British Columbia (only Vancouver Island) in Canada; New York State (Long Island and Steuben, Wayne, and Orleans counties) in USA; Bolivia, Chile, Colombia, Ecuador, Peru, Panama, Venezuela, and Mexico. World- Algeria, Austria, Belgium, Canary Island, Cyprus, Czechoslovakia, Denmark, Egypt, England, Estonia, Finland, Faroe Islands, France, Germany, Guernsey Island, Greece, holland, Iceland, Italy, India, Ireland, Israel, Japan, Jersey Island, Lapland (Finish), Lebanon, Latvia, Lithuanian republic, Luxembourg, Northern Ireland, New Zealand, Norway, Pakistan, Poland, Portugal, Scotland, South Africa, Spain, Sweden, Switzerland, Tunisia, United Kingdom, USSR, Wales, and Yugoslavia. G. pallida is found in several countries: Western Hemisphere- Newfoundland in Canada, Peru, Bolivia, Colombia, and Ecuador. World- Algeria, Canary Islands, Channel Islands, Faroe Islands, France, Germany, Holland, Iceland, India, Italy, New Zealand, Norway, Peru, Spain, Sweden, Switzerland, United Kingdom, USSR, and Venezuela.
These are the two species which APHIS Inspectors must look for and that G. pallida is a very important pest to keep out. The golden nematode disease is known to occur in several countries, especially in cooler areas of subtropical and tropical regions, as well as temperate regions of the world. Yield loss of as high as 80% have been reported in some potato growing areas of the tropics where infestation levels due to golden nematode are high. Other major nematode parasites of potatoes are root-knot (Meloidogyne), false root-knot (Nacobbus), bulb & stem (Ditylenchus dipsaci), potato-rot (Ditylenchus destructor) and lesion nematodes (Pratylenchus). The Potato rot or tuber nematode and potato stem nematode are reported from temperate climates and also occur in North & South America Potato stem nematode is a parasite of foliage and attacks leaves, petioles and also injures tubers. The Potato rot nematode mainly damages tubers and is a major pest of quarantine importance and import concern. The Columbia root-knot nematode (M. chitwoodi), decreases the quality of potato tubers by causing brown spots on the surface, rendering tubers unacceptable for either processing or fresh market sale. Many other nematodes associated with potato include: sting, dagger, reniform, burrowing and pin nematodes; most of them are of minor importance.
The inspectors should look for potato cyst nematode with small immature females of white or yellow stages or brown cysts if the plant roots are examined at the flowering stage. Soil analysis for extraction of cysts is also recommended for the presence of nematodes and if found, follow the golden nematode quarantine regulations that are designed to protect uninfested fields from becoming infested by this nematode. Also, galls on the roots of potato and swellings on surface of potato tubers caused by root-knot nematode and the bead-like galls on roots caused by false root-knot nematode are easily recognizable. Potato rot nematode symptoms on tuber surface is marked by sunken, dark-colored pits or skin cracks, while as potato stem nematode, mainly a parasite of foliage, also attacks tubers causing conical pits with skin splitting. Root-lesion nematodes cause lesions on both the roots and on tubers.
Rice is the most important crop in the world, predominantly in Asia, where more than 90% of the world's rice is grown and consumed. Many plant-parasitic nematodes are associated with rice, and can be divided into two groups depending on their parasitic habits: the foliar parasites, feeding on stems, leaves and panicles; and the root parasites. Some of the nematode pathogens of economical importance are the white tip nematode (Aphelenchoides besseyi); the rice stem nematode (Ditylenchus angustus); the root nematode, Hirschmanniella spp. and the rice cyst nematode (Heterodera oryzae). The white tip nematode is seed borne and occurs in many rice growing areas. Earlier symptoms are emergence of chlorotic tips of new leaves with a white splash pattern. The grain is small and distorted and kernel may be discolored and cracked. The rice stem nematode is the cause of "ufra" disease in several countries, mainly in deep water rice areas. A root-knot nematode (M. graminicola) damages rice in several countries including the United States. Four cyst-nematode species infect rice roots (H. oryzae, H. oryzicola, H. elachista and H. sacchari), and the infected roots turn brown to black. Lemon shaped white females and brown cysts can be seen on infected roots. Also, lesion nematode species can cause severe damage to rice.
The inspectors should look for symptoms of 'white tip' on rice leaf and necrotic lesions on rice seed caused by white tip nematode. The dispersal method of this nematode is the infected seed and is widely distributed because of its dissemination in seed. Also, root galls on rice seedlings for the rice-knot nematode and the cysts and white females emerging from roots of rice due to rice cyst nematode and the various other symptoms given above caused by other economically important nematodes on rice should be looked.
Over 75 percent of the world production of soybean is grown in the United States. The soybean cyst nematode is the most serious pest of soybean throughout the world. At $220.50 per t ($6/bu), the estimated value of the 1994 harvested world soybean crop in U.S. dollars was $30.39 x 109, and the estimated reduction of soybean yield due to soybean cyst nematode for United States during 1994 was 1,990,000 metric tones. Soybean production is not economically possible without the effective control measures. Of the greater than 50 species of nematodes reported from soybeans, the soybean cyst and root-knot nematodes have received the greatest emphasis in breeding programs. Some other plant-parasitic nematodes which attack soybeans are reniform, sting, lesion and lance nematodes.
The inspectors should look for the soybean cyst and root-knot nematodes, major pest of economic importance on soybeans and also for species of root lesion and lance nematode.
The sugar-beet cyst nematode is the most devastating pest of all the sugar-beet growing areas of the world. The nematode causes less sugar production per area of land. It favors temperate regions and also tolerates broad range of climates; it is widespread in Europe, the United States, Canada, parts of the Middle East and USSR, western and southern Africa, Australia, Chile, and Mexico. The inspectors should look for the sugar-beet cyst nematode in sugar-beet.
Root-knot and root-lesion nematodes are the most important nematode pests of tobacco. The tobacco cyst nematode has been an important in shade-grown tobacco production in Connecticut, USA since 1951. It also occurs in Virginia and Massachusetts in USA, and also reported from many other parts of the world. The other nematodes that frequently parasitize tobacco are, stem and bulb, stunt, spiral and reniform nematodes.
The inspectors should look for the root galls caused by the root-knot nematode attack on tobacco roots. Also, several species of root lesion nematodes and the tobacco cyst nematode are other serious pests of economic importance.
Due to root-knot nematodes, it is often impossible to grow important vegetables like tomato in the tropics and semi-tropics. The disease complex caused by root-knot nematodes and bacterial or fungal wilt organisms is one of the most lethal known. Other plant-parasitic nematodes such as reniform, stubby root, sugar-beet cyst, false root-knot, sting and stunt nematodes are serious pests of vegetables.
The inspectors should look for the presence of galls on the root system of vegetables for the root-knot nematode. The reniform nematode, is the other most important nematode affecting vegetables. Potato cyst nematode infects and damages tomato and eggplant. Stem nematode severely damages onion and garlic. Also, several species of stunt nematodes are often found associated with vegetables.
Wheat and other Cereals
Several plant-parasitic nematodes have been associated with wheat and barley and the most economically important ones are: cereal cyst nematode, Heterodera avenae, the seed gall or ear-cockle nematode, Anguina tritici, root-knot and the lesion nematodes. The cereal cyst nematode is present in many countries. The seed gall nematode, cause of the "ear-cockle" disease of wheat and barley was the first described plant-parasitic nematode in the literature. If the wheat galls are kept in a dry condition, the nematode larvae within may remain viable for more than 25 years. From a single gall up to 90,000 nematodes have been counted. Stunt, root-knot and lesion nematodes have been reported as major pests of sorghum.
The inspectors should look for the typical gall-forming endoparasites of seeds, stems, and leaves of cereals, and other plants. Wheat gall nematode is the most economically important nematode parasite which has been disseminated through infested seed to many wheat-producing regions of the world. Nematode galls contaminated with wheat seeds should be checked and the galls or seeds if kept in water for overnight, facilitates the release of live juveniles. Also, the white females and brown cysts of cereal cyst nematode on roots of wheat and barley and juveniles and males in the soil around their roots can be detected in large numbers.
The importance of nematodes in world agriculture can be judged by weather or not their damage is catastrophic to major crops. Several plant-parasitic nematodes are responsible for this kind of damage. Some of the important ones are:
Heterodera (Cyst nematodes): Female swollen or obese, lemon shaped, 300-600 um in diameter with a distinct neck. Females produce several hundred eggs, and after death, the female cuticle forms a protective cyst. Eggs retained within the cyst. Cysts are either partially enclosed in root tissue or in the soil. It is called a cyst nematode because the greatly swollen, egg-filled adult female is referred to as the "cyst stage". Male vermiform (i e, wormlike) found in soil. Juveniles vermiform 450-600 um long. The genus has world-wide distribution, but not individual species.
Major species: H. glycines, H. avenae, H. schachtii, H. trifolii, H. gottingiana, H. cajani, H. zeae
Globodera (Cyst nematodes): Similar to Heterodera but the cyst is globuse. Species confined to the cooler places. Major species: G. rostochiensis, G. pallida, G. tabacum.
Meloidogyne (Root-knot nematodes): Female embedded in root tissue, globose, 0.5-0.7 mm in diameter with slender neck. Male vermiform 1-2 mm long, free living in soil. Juveniles slender, vermiform about 450 um long. Most of the females are within the galls on the roots. World-wide distribution.
Major species: M. arenaria, M. incognita, M. javanica, M. hapla, M. chitwoodi.
Ditylenchus (Stem and bulb nematode): Slender vermiform nematodes. Ectoparasites of plant stems, leaves and within the tissues. Potato rot nematode (D. destructor) is one of the 5 nematodes, listed on the EPPO quarantine list A-2 (Zero tolerance required in countries in which the pests are imported by reasons of prevailing ecological conditions.
Major species: D. destructor, D. dipsaci, D. angustus.
Anguina (Seed gall nematodes): Typical gall forming endoparasites of seeds, stems and leaves of cereals, grasses and other plants. Adult stages are found only in plant galls, juveniles are found in galls, plant tissues or soil. As the galls matures and dies, the infective juveniles can survive many years in a quiescent state. Major species: A. tritici, A. agrostis, Afrina/Anguina wevelli.
Pratylenchus (Lesion nematodes): Are an important group of migratory endoparasites and ectoparasites of roots. They cause serious damage to many economic plants world-wide. They are small nematodes (less than 1mm long). Major species: P. penetrans, P. brachyurus, P. coffeae, P. zeae, P. goodeyi, P. thornei, P. vulnus.
Radopholus (Burrowing nematodes): These small nematodes (less than 1mm long) constitute an important group of endoparasitic nematodes of plant roots and tubers. The major species is R. similis with two host races that differ in parasitism of citrus.
Hirschmanniella (Root nematodes): Medium size to long, slender migratory endoparasites, many on roots(1-4 mm). H. oryzae is a major pest of rice in several countries. Major species: H. oryzae, H. mucronata, H. spinicauda.
Hoplolaimus (Lance nematodes): Are an important group of basically migratory ectoparasites which feed on roots of many kinds of fruits and other economic plants world-wide. Medium length (1-2mm). Major species: H. columbus, H. seinhorsti, H. indicus.
Rotylenchulus (Reniform nematodes): Immature females establish permanent feeding sites in roots, become semi-swollen, and protrude from roots. They are 0.23-0.64 mm long and have a kidney shaped body. Males are vermiform. Eggs are laid in gelatinous matrix.
The major species is: R. reniformis which found in both tropical and warm temperate soils.
Tylenchulus (Citrus nematode): Immature females are in soil and are vermiform. Mature female anterior part is embedded in root tissues, the slender posterior part protrudes from roots and is swollen. Males and juveniles are vermiform and slender. The major species is: T. semipenetrans, which is found everywhere in citrus growing areas.
Helicotylenchus (Spiral nematodes): Small to medium sized nematodes(0.4-1.2mm), usually in spiral shape. Ectoparasitic, semi-endoparasitc or endoparasitic nematodes of roots. The most damaging species is H. multicinctus. Major species: H. multicinctus, H. mucronatus, H. dihystera, H. pseudorobustus.
Criconemella (Ring nematodes): Migratory ectoparasites. Females are 0.2-1mm long, stout with prominent retrorse annules. Males are slender and short; juveniles are like females with annules. Major species: C. xenoplax, C. axestis, C. spharocephalum.
Xiphinema, Longidorus, Trichodorus & Paratrichodorus (Dagger, needle and stubby root nematodes): Slender, virus transmitting nematodes 0.8-5mm long. Ectoparasites on roots of Perennial and woody plants. World-wide distribution.
Major species: X. americanum, X. elongatum, L. africanus, P. minor.
Aphelenchs (Bud and Leaf & Pine wood nematodes): They have a world-wide distribution. A. fragariae & A. besseyi feed on and damage strawberry plants; the later species also damages rice. A. ritzemabosi causes necrosis on leaves of chrysanthemums and other ornamentals. Pine wood nematode (Bursaphelenchus xylophilus) has been implicated in a serious disease of pine trees (pine wilt), which has devastated pine forests in Japan and occurs in North America on various pines. More recently, in 1997 white pine trees in Maryland were devastated due to the heavy infestation of this nematode. This is a serious quarantine pest and all pine wood chips or wood products for import and export purposes need to be checked for this nematode.
The symptoms of nematode disease are commonly those of root impairment, such as growth reduction, increased wilting, mineral-deficiency symptoms, decreased winter-hardiness, and dieback in perennials. However, some nematode disease symptoms are easily recognized in plants:
Certain species of seed gall nematodes (Anguina spp.) transform floral parts, producing characteristic galls in place of normal seeds. Other species of Anguina produce galls and distortion in leaves and stem. The stem nematode causes swelling and distortion of stems and leaves. Bud and leaf nematode causes foliar discoloration. For the most part, however, the above ground symptoms of nematode infection are indirect and are rather nondescript, such as reduction in vigor, stunting, yield decline, or chlorosis.
The galling caused by root-knot nematode is easily recognized but can be confused with the more apical root-galling caused by certain sheath nematodes or with the bending or and apical galling caused by dagger nematodes. Lesion nematodes produce characteristic lesions in the root cortex of plants. Female cyst nematodes can be seen on the roots of host plants if the soil is carefully removed from the roots. Care must be taken to discriminate cysts from legume nodules, however. Soil clings to a gelatinous matrix secreted by the citrus nematode, causing infected citrus roots to appear dirtier than uninfected ones.
The collection of soil may be made with a variety of augers, with tubes, or simply with a
shovel. Moist soil, preferably in the vicinity of plant roots, rather than dry surface soil should be sampled. Each sample should contain feeder roots whenever possible and should include numerous sub-samples from a field. To avoid drying, plastic bags are preferable to other containers for soil and plant samples. Storage of samples at cool temperatures is essential.
Endoparasitic nematodes can usually be seen by examining small amounts of plant tissue with a stereoscopic microscope at magnifications from 15x50x using transmitted/or incident light. Roots should be gently washed to remove as much soil as possible. Small pieces of plant parts, such as roots, stem, leaves, buds and seeds, may be examined in clear water by tearing the tissue apart with dissecting needles. Endoparasitic nematodes if present, will float out and can be collected with a handling needle and fixed in 3% formaldehyde and sent for species identification in a vial. Immature females may be seen on the outside of roots. The white, yellow or golden flask shaped females may be seen attached to the surface of the roots. The brown leathery, flask shaped cysts are the swollen, egg-containing bodies of the females which have matured and fallen into the soil from the roots and are approximately the size of a pinhead, visible to the naked eye.
Place a soil sample (approximately 250 ml) into bucket. Stir with hand and break up clumps of soil while adding tap water to bucket (approximately 3/4 full). When solution is uniform, wait 30 seconds for some of the heavy sediment to settle, lift bucket gently and pour through the stacked sieves (20, 60, 325) at one time (leaving the settled sediment in the bottom of the bucket). Discard the 20 mesh sieve. Collect cysts and large eel-shaped forms from 60 mesh sieve by back washing into a beaker. Collect other eel-shaped forms and larvae from 325 mesh sieve by backwash into a separate beaker. After the beakers have settled for about an hour, decant the water from the top (sometimes dry old cysts can be seen floating on top) and add 3% formaldehyde solution to the rest of the material. Let settle down again, decant and place the rest in a vial for further study and identification purposes.
20 mesh - (to collect debris- Discard)
60 mesh - (catches cysts, most females, and sometimes large males and eel-shaped forms)
325 mesh - (catches larvae, males and other eel-shaped forms)