An Essay On Plant Pathogenic Nematodes In Soil

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
and are significant because they save importers and exporters from suffering millions of dollars in product deterioration and other losses, while shipment of crops was unable to be unloaded. For example, more recently in 1997, a devastating seed gall nematode (Afrina wevelli) was identified at several occasions intercepted by APHIS from Eragrostis sp. seed galls from South Africa. 

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, Fuchsiaexcorticata 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. 


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.

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) 


The development of disease in cultivated crops has long been known to depend on the complex interrelationship between host, pathogen and prevailing environmental conditions. In the case of soilborne pathogens, further opportunities exist for interactions with other microorganisms occupying the same ecological niche. The significant role of nematodes in the development of diseases caused by soilborne pathogens has been demonstrated in many crops throughout the world (Table 1). In many cases, such nematode–fungus disease complexes involve root-knot nematodes (Meloidogyne spp.), although several other endoparasitic (Globodera spp., Heterodera spp., Rotylenchulus spp., Pratylenchus spp.) and ectoparasitic (Xiphinema spp., Longidorus spp.) nematodes have been associated with diseases caused by soilborne fungal pathogens. While nematode–fungus complexes have been reviewed previously (Pitcher, 1965; Powell, 1971; Mai & Abawi, 1987; Evans & Haydock, 1993), this review (i) discusses the mechanisms underlying synergistic interactions; (ii) identifies biotic and abiotic factors affecting their progress; and (iii) outlines potential approaches for the resolution and management of nematode–soilborne pathogen complexes.

The natural soil environment harbours a multitude of microorganisms. As many as 106–108 bacterial cells, 106–107 actinomycete cells, 5 × 104–106 fungal colony-forming units (CFU), 105–106 protozoa and 104–5 × 105 algae were estimated to be present in a gram of field soil taken from the surface (Gottlieb, 1976), while Richards (1976) found c. 1 × 107 nematodes in an area of 1 m2 of fertile soil. Although many of these organisms are saprophytic, having little, if any effect on cultivated crops, the moist soil environment is favourable for the activities of plant-parasitic nematodes (PPN) and for the growth and multiplication of pathogenic fungi. It is of no surprise, therefore, that a variety of interrelationships between these organisms have been demonstrated.

It has long been understood that the development of disease symptoms is not solely determined by the pathogen responsible, but is dependent on the complex interrelationship between host, pathogen and prevailing environmental conditions. In addition, in nature plants are rarely, if ever, subject to the influence of only one potential pathogen. This is especially true of soilborne pathogens, where there is tremendous scope for interaction with other microorganisms occupying the same ecological niche. Disease aetiology and reasons for the multifactorial nature of disease causation are described by Wallace (1978).

Examples of interactions between soil microbes influencing disease development can be seen in PPN–pathogen complexes. A disease complex is produced through a synergistic interaction between two organisms. Synergistic interactions can be summarized as being positive where an association between nematode and pathogen results in plant damage exceeding the sum of individual damage by pest and pathogen (1 + 1 > 2). Conversely, where an association between nematode and fungus results in plant damage less than that expected from the sum of the individual organisms, the interaction may be described as antagonistic (1 + 1 < 2). Where nematodes and fungi are known to interact and are shown to cause plant damage that equates to the sum of individual damage by pest and pathogen, the association may be described as neutral (1 + 1 = 2). Although the former two associations can be readily demonstrated experimentally, the latter can prove difficult to identify, as neutral associations can result in similar plant damage to that seen in additive associations, where nematode and pathogen are known not to interact with one another.

The first recorded case of a nematode–fungus interaction was made by Atkinson (1892), who observed that fusarium wilt of cotton (caused by Fusarium oxysporum f.sp. vasinfectum) was more severe in the presence of root-knot nematodes (Meloidogyne spp.). Further evidence for the interaction between Fusarium spp. and root-knot nematodes in cotton was later provided during field experiments in which ethylene dibromide or 1,3-dichloropropene was used to sterilize soil (Smith, 1948; Newson & Martin, 1953). Where a soil sterilant was used, the incidence of wilted cotton plants was significantly reduced. As the chemicals are regarded as having little fungicidal activity, it was assumed that they indirectly reduced pathogen infection by reducing the population densities of nematodes with which they interact.

Since these early observations, interactions between Meloidogyne spp. and fusarium wilt pathogens has been studied and documented in several host crops, including alfalfa (Griffin, 1986); beans (France & Abawi, 1994); chickpeas (Kumar et al., 1988; Uma Maheswari et al., 1997); tomatoes (Abawi & Barker, 1984; Suleman et al., 1997); cotton (De Vay et al., 1997; Abd-El-Alim et al., 1999); coffee (Bertrand et al., 2000); peas (Siddiqui & Mahmood, 1999); bananas (Jonathan & Rajendran, 1998); and lentils (De et al., 2001).

Ectoparasitic nematodes such as Belonolaimus and Trichodorus spp. are rarely recorded to have a role in synergistic interactions with fungi, probably because their feeding behaviour causes only minor tissue damage to plant roots (Hussey & Grundler, 1998). In comparison, ectoparasites such as Xiphinema (dagger nematode) and Longidorus (needle nematode) have longer stylets for feeding in the vicinity of the vascular cylinder, and are recognized as important vectors of plant viruses (Taylor, 1990; Brown et al., 1998; Ipach et al., 2000).

Compared with those of ectoparasites, the life cycles of endoparasitic nematodes are far more complex and involve closer associations with their plant hosts. This means that plants infested with endoparasites are usually subject to various nematode-induced modifications. These can vary from localized forms of damage caused during invasion and feeding to overall systemic effects such as retarded plant growth. It is these changes which influence infections by soilborne pathogens. The endoparasites Globodera, Heterodera, Meloidogyne, Rotylenchulus and Pratylenchus are the genera most commonly reported to be involved in disease complexes with fungal pathogens. These typically interact with the wilt fungi Fusarium and Verticillium and the root-rot pathogens Pythium, Phytophthora and Rhizoctonia. Root-knot nematodes (Meloidogyne spp.) are perhaps the most recurrently recorded nematodes found in disease complexes with fungi. This is illustrated well by the interaction between wilt disease caused by F. oxysporum f.sp. vasinfectum and Meloidogyne incognita, which has long been a problem in cotton crops (Gossypium hirsutum) and has frequently been documented in the literature (Atkinson, 1892; Garber et al., 1979; Mai & Abawi, 1987; De Vay et al., 1997; Abd-El-Alim et al., 1999). More recently, M. incognita has been found in association with the pathogen Thielaviopsis basicola, which causes black root-rot of cotton (Walker et al., 1998). In cotton, neither of these organisms is considered to cause acute effects individually, and plant mortality rarely occurs in their presence (Walker et al., 1998), yet in combination they have consistently been found to increase seedling mortality, increase root necrosis, suppress early seedling growth and subsequently reduce the percentage of bolls (Walker et al., 1998; Walker et al., 1999; Walker et al., 2000; Wheeler et al., 2000).

On potatoes, the Globodera–Verticillium dahliae and Pratylenchus–Verticillium dahliae disease complexes have become particularly notorious. Early senescence or ‘early dying’ caused by V. dahliae and V. albo-atrum is accentuated by populations of Pratylenchus spp. (Martin et al., 1981; Wheeler et al., 1992; Bowers et al., 1996; Hafez et al., 1999), G. rostochiensis (Evans, 1987), and G. pallida (Hide et al., 1984; Storey & Evans, 1987). In terms of yield, Martin et al. (1982) calculated that 15, 50 and 150 P. penetrans per 100 cm3 soil in combination with V. dahliae would result in 36, 60 and 75% reductions in potato tuber weight, respectively. However, tuber weights were unaffected by the presence of the individual pathogens, except where nematode populations were high (150 P. penetrans per 100 cm3), when a 12% reduction was found. Yield reduction from the Pratylenchus–V. dahliae complex has also been reported elsewhere (Botseas & Rowe, 1994), as have other damaging effects such as the disruption of photosynthesis, stomatal conduction and transpiration (Saeed et al., 1997a; Saeed et al., 1997b). However, the fundamental importance of P. penetrans in potato early dying is its ability to activate low populations of V. dahliae that would otherwise be inconsequential in disease development (Bowers et al., 1996).

Another disease complex involves the soyabean cyst nematode Heterodera glycines and the fungus Fusarium solani. Sudden death syndrome (SDS) caused by F. solani is a major disease of soyabean which, among other symptoms, induces root rot, crown necrosis, interveinal chlorosis, defoliation and abortion of pods (Rupe, 1989; Nakajima et al., 1996). The aetiology of SDS is complicated, and changing abiotic factors such as temperature and moisture (McLean & Lawrence, 1993) are influential in its development. Furthermore, H. glycines is considered by many to be an important player in the incitement of SDS (McLean & Lawrence, 1993; Rupe et al., 1993). In two years of microplot experiments, McLean & Lawrence (1993) found that the incidences of SDS symptoms in plots containing both H. glycines and F. solani were 35 and 18% higher than in plots where the fungus was inoculated alone. Recent research on SDS has focused on identifying genes for dual resistance against both nematode and fungus (Chang et al., 1997; Meksem et al., 1999; Prabhu et al., 1999).

Mechanisms underlying synergistic interactions

Utilization of nematode-induced wounds by soilborne pathogens

Depending on specific life cycles, PPN are able to cause a variety of types of wound on host plant roots while entering or feeding. For example, ectoparasitic nematodes such as Trichodorus spp. and Tylenchorhynchus spp. feed on root epidermal cells, leaving behind simple micropuncture-type wounds. In contrast, endoparasitic nematodes are far more disruptive to their hosts’ roots. The root-lesion nematode Pratylenchus spp. is a migratory endoparasite that travels intracellularly through the cortex of roots by cutting through cell walls with its stylet to create a path. The sedentary endoparasites Meloidogyne spp., Globodera spp. and Heterodera spp. have highly specialized feeding strategies together with elaborate life cycles. Vermiform juvenile nematodes (J2) select penetration sites behind growing root tips (Doncaster & Seymour, 1973) and migrate either intracellularly (Globodera and Heterodera spp.) or intercellularly (Meloidogyne spp.) (Von Mende et al., 1998) to the vascular cylinder, where specialized ‘nurse cell systems’ are initiated (Jones & Northcote, 1972).

Some authors (Bergeson, 1972; Taylor, 1990) have regarded nematode invasion sites and tracts as inconsequential in the aetiology of fungal diseases. However, there are a number of reports which clearly illustrate that nematode damage has a role in the establishment and development of disease caused by soilborne pathogens. Inagaki & Powell (1969) adopted a number of methods to investigate the importance of mechanical wounding by the root-lesion nematode Pratylenchus brachyurus on the development of black shank symptoms (Phytophthora parasitica) in flue-cured tobacco (Nicotiana tabacum cv. Hicks). Plants subjected to an artificial root-wounding treatment produced significantly more severe disease symptoms of black shank. Additionally, plants receiving either (i) simultaneous inoculation of the nematode P. brachyurus and the oomycete P. parasitica, or (ii) introduction of P. brachyurus 1 week prior to P. parasitica, also exhibited elevated disease development in comparison to plants inoculated with the oomycete alone. However, plants inoculated with nematodes 2 or 3 weeks before the introduction of P. parasitica did not favour the rapid development of disease. In a further greenhouse experiment, root samples taken from plants inoculated with P. parasitica alone, P. brachyurus alone, or a combination of both were sectioned, stained and examined. While the development of P. parasitica was not found to differ when in close proximity to the feeding sites of P. brachyurus, colonization by the oomycete was reduced in the necrotic lesions caused by the nematode. This might offer some explanation as to why plants inoculated with P. parasitica 2–3 weeks before inoculation with the fungus developed few black-shank symptoms. Inagaki & Powell (1969) suggested that the simultaneous introduction of nematode and P. parasitica allowed the latter to utilize minute openings created by the migratory action of P. brachyurus in the roots. It seems unlikely, however, that the artificial wounding technique employed would be able to simulate the type of damage caused by invading PPN. In truth, such a treatment could have triggered any number of different processes to produce the disease symptoms seen, processes that are discussed below.

Histological studies appear to be the key to unravelling the association between fungal pathogens and the injuries caused to plants by PPN. This is particularly highlighted in the work of Polychronopoulos et al. (1969), where the invasion process of Heterodera schachtii (beet-cyst nematode) was found to facilitate the infection of sugar beet (Beta vulgaris) by the damping-off fungus Rhizoctonia solani. During their investigation, sugar beet seedlings grown in either nematode-infested or nematode-free soil were exposed to R. solani before being examined microscopically over a series of 12 h intervals for 3 days. On inspection of the seedlings 36 h after inoculation, distinct differences could be seen between the two treatments. When in combination with H. schachtii, the hyphae of R. solani were found to grow vigorously through the epidermis and cortex. Closer examination showed that hyphal colonization frequently followed tracts made by invading nematode juveniles. On the epidermal surfaces of the seedlings, the pathogen was found to produce fewer infection cushions in the presence of nematodes than when it was present alone. The authors suggested that infection cushion synthesis could have been hindered in some way by the invading nematodes. However, nematode invasion sites may provide R. solani with the necessary portals for penetration and entry, consequently reducing the need for developing more sophisticated infection structures such as infection cushions. In a similar way, R. solani is known to exploit natural openings on the outer surfaces of plants such as stomata (Chand et al., 1985) and lenticels on potato tubers (Ramsey, 1917) to invade underlying tissue.

Nematode wounding damage has also been found to be fundamental to several other disease complexes. For example, a recent scanning electron microscope (SEM) study on banana roots (Orion et al., 1999) revealed that the mycelium of an unidentified soilborne fungus was closely and frequently associated with the invasion tracts and lesions created by the spiral nematode Helicotylenchus multicinctus. On potatoes, Storey & Evans (1987) have found that the pathology of the wilt fungus V. dahliae is dependent on the timing of invasion by the cyst nematode G. pallida, and also on the potato cultivar. The fungus was found to enter and use the invasion channels of juvenile G. pallida if it was introduced at the same time as the nematode on cvs Pentland Javelin, Maris Peer and Maris Anchor. On both Maris Peer and Maris Anchor, lignified tissue developed following the invasion of the nematodes. If the fungus was introduced 8 days after the nematode, wilt symptoms caused by V. dahliae were less severe than on plants treated with the fungus alone. When sections of root tissue were examined, it could be seen that the lignified tissue had sealed the nematode penetration sites. In fact, the living tissue was partially protected by areas of woody tissue where this response had taken place. This response was not produced by cv. Pentland Javelin after nematode invasion, and V. dahliae was still able to colonize roots to a larger extent than on plants where it was introduced alone.

In addition to the cavities caused during PPN invasion, nematodes produce other forms of mechanical damage to plant roots that are open to exploitation by soilborne fungi. Fagbenle & Inskeep (1987) used SEM to study concomitant infections of Meloidogyne hapla and R. solani on peanut (Arachis hypogaea). Eleven weeks after inoculation, the root galls were often found to be split, leaving a rough surface comprised of cortical cells which rapidly became colonized by R. solani. However, it was unclear whether the cracks of the galled roots aided the penetration of R. solani.

In order for female cyst and root-knot nematodes to reproduce, the females/cysts must rupture through the root cortex to allow the vermiform males to fertilize them (Fig. 1). This event often produces a number of cracks and crevices where the swollen female has emerged. Several authors have suggested that these openings might be used by opportunistic pathogens to reach the underlying tissue of roots more easily (Bergeson, 1972; Golden & Van Gundy, 1975; Evans & Haydock, 1993).

The hypothesis that PPN-induced wounds facilitate the invasion process of some fungal pathogens seems the most likely explanation behind a synergistic interaction, although there are relatively few reports that demonstrate this mechanism, and positive quantitative data coupled with convincing histological evidence are required to validate this hypothesis. Future work in this area might benefit from using equipment such as time-lapse and video-enhanced light microscopy together with some form of image analysis system. Video-enhanced light microscopy has previously been used for studying nematodes (Wyss & Zunke, 1992) and fungi (McCabe et al., 1999). The application of image analysis systems could help overcome the problems of quantifying the density of fungal pathogens in regions of nematode damage.

Nematode-induced physiological changes to the host plant

The feeding sites of sedentary endoparasitic nematodes (giant cells or syncytia) are zones of high metabolic activity, having a large number of Golgi apparatus and mitochondria, while the cytoplasm is dense and contains many ribosomes (Jones, 1981). It is therefore no surprise that these nutrient-rich cells should become the substrate for fungal colonization (Meléndez & Powell, 1970; Wajid Khan & Muller, 1982; McLean & Lawrence, 1993; Abdel-Momen & Starr, 1998). Mayol & Bergeson (1970) made preliminary observations of this effect when tomato plants in gnotobiotic culture were treated with M. incognita and a soil suspension taken from around the roots of healthy tomato plants. Disease assessments of these plants 7 and 12 weeks after planting showed extensive necrosis of their root systems. Fungi isolated from the galls of M. incognita were identified as Trichoderma sp., Fusarium sp. and R. solani. Similarly, Negrón & Acosta (1989) found that F. oxysporum f.sp. coffeae caused increased root necrosis and chlorosis on the foliage of coffee plants (cv. Bourbón) if the plants had been inoculated with M. incognita 2 or 4 weeks previously. Sections taken from the roots of plants preinoculated with M. incognita were found to be colonized by F. oxysporum f.sp. coffeae in a uniquely different way from those where the fungus and nematode were either inoculated simultaneously, or where the fungus was inoculated alone. In the former case, the hyphae of F. oxysporum f.sp. coffeae were found to be abundant in the xylem vessels, giant cells and female nematodes. Giant cells, colonized by the fungus were in varying states of disrepair, with depleted or partially depleted contents. In comparison, plants that had been simultaneously inoculated with M. incognita and F. oxysporum f.sp. coffeae had fewer giant cells colonized by the fungus and no hyphae within the xylem. Taylor (1990) suggested that the 3–4-week nematode preinoculation, found to be critical in investigations of nematode–fungus disease complexes (Golden & Van Gundy, 1975; Wajid Khan & Muller, 1982; Negrón & Acosta, 1989), could be linked to syncytial development which, under optimal conditions in a susceptible host, will take 3–4 weeks to reach peak activity.

Perhaps the most comprehensive studies into such localized nematode-induced modifications are those of Golden & Van Gundy (1975). In their field studies with okra and tomato, fumigants were applied to field plots to reduce M. incognita and R. solani (ethylene dibromide and methyl bromide, respectively) and create independent and combined treatments. In untreated plots (plots with both M. incognita and R. solani), R. solani was isolated from the galls of M. incognita a week after gall formation. Two weeks later, numerous black sclerotia were found encrusted to the galls. In contrast, sclerotia were absent from ungalled regions of the roots. Four weeks after gall formation, substantial root decay occurred. Furthermore, histological sections revealed that R. solani had penetrated cells from the sclerotia attached to the gall surfaces. Rhizoctonia solani appeared to have a marked trophic intercellular pattern through the cortex of galled roots towards nematode-induced giant cells. Wajid Khan & Muller (1982) reported similar observations with M. hapla and R. solani on radish. Galls infected by R. solani had abundant sclerotia on their surfaces, while giant cells were colonized extensively by hyphae. Similarly, Abdel-Momen & Starr (1998) found that a reduction in the pod yield of peanut was significantly greater in coinfections of Meloidogyne javanica and R. solani where a concentration of fungal growth was found around the galled regions.

Cyst nematodes also form nutrient-rich syncytia for the purpose of development. Histological studies of H. schachtii-infested sugar beet seedlings exposed to R. solani indicated that syncytia were a more favourable substrate to the fungus than normal cells (Polychronopoulos et al., 1969). The authors also describe how the syncytium appeared to be a suitable ‘food base’ for colonization of other tissues by the fungus. Hyphae were seen to spread from the syncytia to the corticovascular tissue, which had not been invaded by nematodes.

These studies indicate that nematode-infected plant tissue may be actively selected by certain plant pathogens. According to Taylor (1979); Taylor (1990) and Abawi & Chen (1998), syncytia or giant cells contain higher levels of total protein, amino acids, lipids, DNA and sugars which would be beneficial to many fungi. This would support the suggestion that nematode infection enhances the nutritional composition of portions of plants to fungi, but the relationship remains unproven.

As well as these localized effects, some authors (Bowman & Bloom, 1966; Batten & Powell, 1971; Hillocks, 1986) have supported the notion that nematode-induced physiological changes can be systemic. In such cases, the nematode-induced factors or substances beneficial to fungi are hypothesized to be translocatable within the plant (Wajid Khan, 1993). While investigating this process, Bowman & Bloom (1966) and Hillocks (1986) employed a ‘split-root’ technique whereby the root system of the plant of interest was bisected into two separate containers, and one half of the root system was infested with the interacting nematode species while the other was inoculated with the interacting fungal pathogen. Bowman & Bloom (1966) infested one half of a tomato root system with nematodes (M. incognita) and the other half with F. oxysporum f.sp. lycopersici. Their results revealed that disease development on plants was dependent on being exposed to both M. incognita and the fungus. Regardless of the results, these split-root experiments do not identify systemic physiological modifications or any other mode of interaction. They simply indicate that an interaction might exist. Indeed, it could equally be concluded that the effects seen were a result of nematode occupation causing a loss of resistance or plant stress. Further studies involving critical biochemical analysis of plant material taken from plants either infested or uninfested with nematodes would significantly increase understanding of nematode-induced systemic change. For example, the nutritional quality of plants infested with PPN may prove more favourable to fungal pathogens. By determining the nutritional requirements of the fungus and quantifying these metabolites in nematode-infested and uninfested plants, a clearer picture could be obtained. Equally, PPN infestations may reduce the levels of fungitoxic compounds. Perhaps the sequence of events that leads to systemic induced changes is similar to, or the reverse of, that described in plants with systemic acquired resistance (SAR). Unfortunately, there has been little, if any work to address this question.

Modifications within the rhizosphere

The release of plant root exudates is considered an important factor in the attraction of both soilborne fungi (Flentje, 1957; Reddy, 1980; Grayston et al., 1997) and PPN (Klinger, 1965; Clarke & Hennessy, 1987). There are a number of ways in which PPN might influence the release of root exudates and thus alter the subsequent response of soilborne pathogens. First, the damage inflicted on plant roots during the process of PPN invasion could result in greater volumes of root exudates attractive to fungal invaders. Second, certain potato cultivars have been shown to produce increased numbers of lateral roots in response to invasion by potato cyst nematodes (Evans & Stone, 1977). Such an increase in root surface area may give rise to increased production of root exudates. Finally, PPN infestation may influence the chemical profile of the root exudates released, making them more favourable to fungal pathogens (Bergeson, 1972).

Perhaps the classic examples of this process are those involving root-knot nematodes. The aggregation of fungi, primarily R. solani, around root-knot galls of many plants has drawn attention to changes occurring within the rhizosphere. Golden & Van Gundy (1972) made preliminary observations of this effect during their studies of the M. incognita–R. solani complex of tomato. Tomato roots infested with M. incognita were seen to become more susceptible to fungal attack by R. solani with increasing age. By adopting the cellophane membrane techniques of Kerr (1956) and Flentje (1957), they observed that R. solani would aggregate on cellophane which was directly opposite the galled regions of the roots. In contrast, ungalled areas received only sparse mycelial coverage. In a later publication, Golden & Van Gundy (1975) undertook further studies with semipermeable membranes (cellophane) on tomato and okra infested with M. incognita. Introduction of R. solani (via mycelial plugs) to the external surfaces of the cellophane once again produced sclerotia opposite the galls of M. incognita. Microscopic examination of the sclerotia showed that their formation consisted of irregular branching and interwinding to form loosely constructed, undifferentiated structures. From these studies, the authors concluded that metabolic leakage from the galls of M. incognita could explain the elevated attraction of R. solani.

Van Gundy et al. (1977) undertook an extensive investigation using tomato to evaluate the hypothesis regarding metabolic leakage. First, a technique known as ‘double-root’ was utilized, whereby a secondary root system was induced to allow experimentation on the natural or primary root system. The attraction of R. solani to nematode-infested plants was facilitated by the use of a hydroponic system to remove root leachates. Tomato plants either infested with M. incognita; exposed to R. solani; infested with M. incognita and exposed to R. solani; or left untreated were found to be free of root necrosis after 5 weeks under the hydroponic regime. However, when leachates taken from M. incognita-infested roots were applied to plants exposed to R. solani alone, necrosis developed. Conversely, treatment of R. solani-treated plants with leachate from untreated plants did not result in root necrosis. Furthermore, it was found that if the experiment was repeated in the absence of the hydroponic system, plants would develop root rot only when exposed to both organisms. The results of these studies implied that M. incognita-infested plants were producing some form of attractant for R. solani. When the properties of exudates emanating from the nematode-infested roots were examined, they were found to have elevated levels of 14C metabolites. During the time of sclerotial development, 14–21 days following nematode invasion, the major constituents of the 14C-labelled metabolites were nitrogenous compounds such as amino acids and proteins; such nitrogenous compounds are important in the virulence of R. solani (Weinhold et al., 1972).

Bergeson et al. (1970) recorded a marked increase in the number of propagules of F. oxysporum f.sp. lycopersici found around the galls of M. javanica. Galls produced by M. javanica on tomato roots stimulated the number of spores of F. oxysporum f.sp. lycopersici while reducing the number of cells of actinomycetes antagonistic to Fusarium (Bergeson, 1972).

Reduction of host resistance

In the development of crop species that express resistance to economically important pests and diseases, the significance of nematode–fungus complexes is seldom, if ever reported, yet there are a number of studies that report breakdown of resistance during concomitant infections (Sidhu & Webster, 1977; France & Abawi, 1994; Marley & Hillocks, 1994; Uma Maheswari et al., 1995; Vargas et al., 1996; Uma Maheswari et al., 1997). Bergeson (1972) commented, ‘It is frustrating to the plant breeder to see the fruits of his labour come to naught by the nematode–fungus conspiracy’.

Typically, loss of resistance has been tested with the application of split-root methods as described earlier. Investigators have adopted this type of approach to determine whether the loss of pathogen resistance induced by nematode infestation occurs as a result of the breakdown of a systemic chemical defence system within the host plant. Bowman & Bloom (1966) found that the tomato cvs Rutgers and Homestead, previously resistant to F. oxysporum f.sp. lycopersici, developed symptoms of wilt during split-root experiments with M. incognita. Further studies (Sidhu & Webster, 1977) using root layering and grafting techniques confirmed that a nematode-induced factor could be passed through a resistant scion (a graft from a resistant tomato cultivar) and render it susceptible to F. oxysporum f.sp. lycopersici. In contrast, resistant scions in tomato plants free from M. incognita infestation could block infection by F. oxysporum f.sp. lycopersici. Vargas et al. (1996) observed a similar effect on chilli (Capsicum annuum), where the nematode Nacobbus aberrans caused a loss of resistance to Phytophthora capsici even if the nematode and oomycete were physically separated on split roots. While few reports have addressed how this might occur, Marley & Hillocks (1994) demonstrated that nematode-induced loss of resistance to Fusarium udum in pigeonpea (Cajanus cajun) was associated with reduced levels of the isoflavanoid phytoalexin cajanol. Previously, Marley & Hillocks (1993) determined that the rapid accumulation of cajanol in some pigeonpea cultivars was responsible for conferring resistance to the pathogen. However, cajanol content was 62% lower and resistance was lost during combined infections of F. udum, M. incognita and M. javanica where wilt disease incidence and severity were significantly higher than in plants inoculated with F. udum alone. Although this study clearly shows that nematode infestation reduced a chemical defence mechanism to fusarium wilt in pigeonpea, it is still not known how nematode activity modified the plant. Marley & Hillocks (1994) suggested that either the overall metabolic rate of the plants was reduced, or specific changes were made to the synthesis of isoflavonoids during nematode attack.

There is general agreement among some authors (France & Abawi, 1994; Sugawara et al., 1997) that polygenic resistance is comparatively less stable than monogenic resistance. Francl & Wheeler (1993) state that plants with polygenic resistance to fungal pathogens are frequently found to become susceptible to fungal attack during nematode infestations, whereas plants with a single dominant gene for resistance are rarely affected. This was observed by Abawi & Barker (1984) on tomatoes, where resistance to F. oxysporum f.sp. lycopersici was disrupted by infestations of M. incognita on cultivars with polygenic resistance, but not on those where resistance was expressed by a dominant single I-gene. Transgenic plants involving quantitative trait loci may have a greater capacity for providing durable resistance in the presence of interacting fungal pathogens and PPN.

Wajid Khan (1993) postulated that resistant plants are rendered vulnerable to pathogens via physiological alterations made by the nematode, which have no effect on the gene(s) responsible for encoding resistance. For example, the process of invasion by PPN may provide soilborne pathogens with portals (Powell & Nusbaum, 1960) through a previously impenetrable physical barrier selected for in a plant breeding programme.

Interactions between species of nematodes and fungi can vary considerably over plant species, cultivars and lines, as indicated in studies of disease complexes on multiple crop genotypes (Khan & Husain, 1989; Uma Maheswari et al., 1995; Abd-El-Alim et al., 1999). Consequently, some investigations have been unable to demonstrate resistance loss (Jones et al., 1976; Castillo et al., 1998; Johnson & Santo, 2001), while others have shown the converse with identical combinations of nematode and fungus species. There are also other abiotic factors, such as soil type and temperature, which have been shown to affect interactions (Uma Maheswari et al., 1997) and which may have varied between individual studies on specific disease complexes.

Pathogen-induced changes to the host plant

Just as nematode activity can increase the severity of diseases caused by fungal pathogens, so nematode populations can be elevated during concomitant infections with root-infecting pathogens (Vrain, 1987; Taheri et al., 1994). While there are far fewer reports of such phenomena, several hypotheses have been proposed. For example, Zahid et al. (2002) investigated a large and complex set of interactions between 12 species of root- and stolon-infecting fungi and three species of root-colonizing nematodes on white clover (Trifolium repens), an important forage crop for dairy herds in eastern Australia. Although a good number of interactions were found between the various combinations of nematodes and fungi, the root-knot symptoms of Meloidogyne trifoliophila were commonly found to be increased in treatments containing the stolon-infecting fungus Drechslera halodes. Moreover, the final densities of M. trifoliophila and two other nematode species (Helicotylenchus dihystera and Heterodera trifolii) were significantly increased in plants infected with D. halodes. This case exemplifies the problems of defining disease aetiology where a large number of pests and diseases are present. Previously, Faulkner & Skotland (1965) observed that Pratylenchus minyus reached its reproductive peak at the same time as the maximum expression of wilt disease (V. dahliae f.sp. menthae) on peppermint plants (Mentha piperita). The authors suggested that V. dahliae may produce root growth-promoting substances such as indole-3-acetic acid (as previously recorded for V. albo-atrum; Pegg & Selman, 1959), resulting in an enlarged root system, releasing greater volumes of root exudate and thereby attracting more PPN (Clarke & Hennessy, 1987; Rolfe et al., 2000). Some studies illustrate how plant roots infected with fungal pathogens can be more attractive to PPNs. For example, Nordmeyer & Sikora (1983) considered how the attraction of Heterodera daverti might be affected by Fusarium avenaceum infection in clover (Trifolium subterraneum) seedlings when compared to uninfected plants. In vitro experiments showed that a significantly greater proportion of H. daverti migrated towards diffusates from F. avenaceum-infected clover roots than towards diffusates from healthy plants.

Fungal modifications to PPN host-finding has also been investigated by Edmunds & Mai (1967), who showed that P. penetrans would conglomerate around a CO2 source under in vitro conditions, in agreement with the previous findings of Bird (1959) and Klinger (1965). CO2 measurements taken from alfalfa plants (Medicago sativa) infected with Trichoderma viride and, particularly, F. oxysporum were considerably higher than those found in healthy roots. Elevated levels of CO2 from infected plants may have contributed to the increased attraction of P. penetrans towards alfalfa roots previously seen in earlier experiments (Edmunds & Mai, 1966a; Edmunds & Mai, 1966b). Whether or not CO2 emissions would produce a similar nematode response within the natural soil environment remains open to debate, and subject to further experimentation. Moreover, the subject of nematode orientation is still largely unknown in the field of plant nematology.

Several authors have speculated that nematode penetration is increased in plant roots previously subjected to the enzymes of fungal pathogens (Edmunds & Mai, 1966a; Edmunds & Mai, 1966b; Edmunds & Mai, 1967; Nordmeyer & Sikora, 1983). While the results of Edmunds & Mai (1966b) were largely inconsistent, Nordmeyer & Sikora (1983) reported more convincing findings in their study of H. daverti and F. avenaceum in the roots of clover. By treating clover roots with filtrate from F. avanaceum cultures, a significantly higher number of H. daverti juveniles were recovered than from untreated roots. They also observed that the duration of exposure of clover roots to F. avenaceum was critical to the invasion success of H. daverti, with an optimal exposure time of 450 s. As the fungal enzyme pectinmethylesterase was detected in the filtrates of F. avenaceum, the authors suggested that such a cell wall-degrading enzyme may have enabled H. daverti to penetrate more easily.

Some research has suggested that fungal infections cause a deterioration or breakdown of plant resistance to nematode attack. Hasan (1985) encountered this effect during routine field screening of chilli pepper cultivars and lines which, under controlled greenhouse conditions, had shown promising resistance to M. incognita. In this case, resistance was lost in two out of five previously fully resistant and eight out of 16 previously moderately resistant lines. Furthermore, individual plants subject to resistance loss expressed symptoms of collar rot and damping-off diseases. The disease-inducing soilborne pathogens were isolated and positively identified as R. solani and Pythium aphanidermatum. Subsequently, a greenhouse experiment was devised to test the effect of R. solani and P. aphanidermatum on the resistance of cvs Jawala (resistant) and Longthin Faizibadi (moderately resistant) to M. incognita. On both cultivars, the presence of R. solani or P. aphanidermatum caused a significant increase in the reproductive capacity (number of egg masses and eggs produced) of M. incognita. In terms of resistance, the ratings of Jawala and Longthin Faizibadi were demoted to moderately resistant and susceptible, respectively. The exact mechanism of this phenomenon was not determined, but the activities of pathogen-produced enzymes may have compromised the physical barrier conferred on the resistant chilli lines, or chemical defences, such as the antifeedant proteinase inhibitors described by Lilley et al. (1999), may have been disrupted during nematode infections. New technologies such as proteomics may provide a better understanding of such interactions, but in the meantime the interactions between PPN and soilborne pathogens need to be considered in future plant breeding programmes.

Factors affecting synergistic interactions

As with nearly all investigations in science, many reports on disease complexes contradict one another. While some of these disparities might be explained by experimental procedure and accuracy, there are findings that highlight the specificity of certain disease complexes and the influence of biotic and abiotic factors on them. This is exemplified by studies on the V. dahliae–Pratylenchus complex of potato, where it has been found that the interaction between these organisms varies among different nematode species (Riedel et al., 1985) and populations, as well as fungal genotypes (Botseas & Rowe, 1994). For example, Riedel et al. (1985) and Bowers et al. (1996) observed that potato early dying disease (V. dahliae) was enhanced by populations of P. penetrans but not by P. crenatus or P. scribneri. Furthermore, greenhouse experiments undertaken by Hafez et al. (1999) demonstrated that populations of P. neglectus collected from Ontario, Canada would interact synergistically with V. dahliae, while populations of P. neglectus from Parma, Idaho did not increase disease or yield loss any more than treatment with V. dahliae alone. Restriction analysis of the ITS1 region on rDNA gene from nematodes from the Canada and Idaho populations revealed unique fragments for each population, implying variation within this species. The differences between nematode species and populations/pathotypes in their ability to accentuate V. dahliae wilt are likely to be related to their proficiency as parasites on potato. This is certainly evident from the work of Hafez et al. (1999), where the fecundity of the Canadian population was approximately 50% higher than that of the Idaho population, in which initial populations (Pi) were 5000 and 10 000 nematodes per 5000 cm3 soil. It is also possible that species and pathotypes of Pratylenchus spp. may favour different physical environmental conditions. Indeed, Kimpinski & Willis (1981) recorded differential effects of soil pH and temperature between populations of P. penetrans and P. crenatus.

Fungal genotype can also affect potato early dying complex, as shown by Botseas & Rowe (1994), who found that two pathotypes of V. dahliae vegetative compatibility group 4 (VCG 4) formed different relationships with P. penetrans. In greenhouse and field microplot experiments, the two pathotypes of V. dahliae (VCG 4A and B) exhibited no differences in aggressiveness when inoculated alone. However, plants inoculated with V. dahliae VCG 4A and grown in soil infested with P. penetrans had higher levels of disease severity, lower tuber yields and earlier senescence than plants inoculated with V. dahliae VCG 4B in the presence of P. penetrans. This type of specificity has also been reported by Johnson & Santo (2001), who found that P. penetrans would interact synergistically with V. dahliae VCG 2B but not with VCG 4A on cultivated peppermint and Scotch spearmint. In these findings, the governing factor was host-specific fungal aggressiveness. Although Botseas & Rowe (1994) were unable to detect differences in aggressiveness between V. dahliae

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