Host Response to Meloidodera spp. (Heteroderidae)

Host responses to Meloidodera floridensis Chitwood et al., 1956, M. charis Hooper, 1960, and M. belli Wouts, 1973 were examined on loblolly pine, peony, and sage, respectively, with light, scanning, and transmission electron microscopy. In each case the nematodes induce a single uninucleate giant cell. The giant cell is initiated in the pericycle and expands as it matures. The mature giant cell induced by M. floridensis is surrounded by vascular parenchyma, whereas that caused by M. charts and M. belli coutacts xylem and phloem. The cell wall of giant cells induced by all three Meloidodera spp. is generally thicker than that of surrounding cells, with the thickest part adjacent to the lip region of the nematode. The thinner portion of the wall includes numerous pit fields with plasmodesmata, but wall ingrowths were not detected in a thorough examination of the entire wall. The nucleus of a giant cell induced by M. goridensis is highly irregular in shape with deep invaginations, whereas those caused by M. charis and M. belli include a cluster of apparently interconnected nuclear units. Organelles, including mitochondria, endoplasmic reticulum, and plastids of giant cells caused by Meloidodera, are typical of those reported in host responses of other Heteroderidae. The formation of a single uninucleate giant cell by Meloidodera, Cryphodera, Hylonerna, and Sarisodera, but a syncytium by Atalodera and Heterodera sensu lato, might be considered in conjunction with additional characters to determine the most parsimonious pattern of phylogeny of Heteroderidae.     

Fine structure of body wall cuticle of females of Meloidodera charis,Atalodera lonicerae,and Sarisodera hydrophila (Heteroderidae)

The body wall cuticle of adult females of Meloidodera charis, Atolodera lonicerae, and Sarisodera hydrophila is examined by transmission electron and light microscopy for comparison with Heterodera schachtii and previous observations of additional species of Heterodera, Globodera, and Punctodera. The cuticle of M. charis is least complex, consisting of layers A, B, C (with A outermost), and varies in overall thickness from 3 to 8 μm. As in other species, the cuticle is thickest in mature specimens. The cuticle of A. lonicerae is 6-9 μm thick; unlike M. charis it has an innermost layer, D, in addition to A, B, and C. The cuticle of S. hydrophila varies from 14 to 30 μm thick and includes a D layer similar to A. lonicerae; layer C is subdivided into additional zones relative to other heteroderids, and the external portion of the cuticle is infused with an electron-dense material. The presence of a D layer in A. lonicerae and S. hydrophila is a character state which is shared with Globodera spp. and Punctodera sp. The electron-dense material in the outer layers of S. hydrophila also occurs in Globodera spp. and Punctodera sp. On the other hand, H. schachtii resembles other Heterodera spp. as well as M. charis by the absence of a D layer and lack of electron-dense material in the outer layers. The pattern of occurrence of shared character states, including those of the cuticle, may be useful for phylogenetic analysis of Heteroderidae.     

Gall-forming root-knot nematodes hijack key plant cellular functions to induce multinucleate and hypertrophied feeding cells

Among plant-parasitic nematodes, the root-knot nematodes (RKNs) of the Meloidogyne spp. are the most economically important genus. RKN are root parasitic worms able to infect nearly all crop species and have a wide geographic distribution. During infection, RKNs establish and maintain an intimate relationship with the host plant. This includes the creation of a specialized nutritional structure composed of multinucleate and hypertrophied giant cells, which result from the redifferentiation of vascular root cells. Giant cells constitute the sole source of nutrients for the nematode and are essential for growth and reproduction. Hyperplasia of surrounding root cells leads to the formation of the gall or root-knot, an easily recognized symptom of plant infection by RKNs. Secreted effectors produced in nematode salivary glands and injected into plant cells through a specialized feeding structure called the stylet play a critical role in the formation of giant cells. Here, we describe the complex interactions between RKNs and their host plants. We highlight progress in understanding host plant responses, focusing on how RKNs manipulate key plant processes and functions, including cell cycle, defence, hormones, cellular scaffold, metabolism and transport.     

Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection

Root-knot nematodes are biotrophic parasites that invade the root apex of host plants and migrate towards the vascular cylinder where they induce the differentiation of root cells into hypertrophied multinucleated giant cells. Giant cells are part of the permanent feeding site required for nematode development into the adult stage. To date, a repertoire of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection has been identified. However, the precise role of these candidate effectors during root invasion or during giant cell induction and maintenance remains largely unknown. Primarily, the identification of the destination of nematode effectors within plant cell compartment(s) is crucial to decipher their actual functions. We analyzed the fine localization in root tissues of five nematode effectors throughout the migratory and sedentary phases of parasitism using an adapted immunocytochemical method that preserves host and pathogen tissues. We showed that secretion of effectors from the amphids or the oesophageal glands is tightly regulated during the course of infection. The analyzed effectors accumulated in the root tissues along the nematode migratory path and along the cell wall of giant cells, showing the apoplasm as an important destination compartment for these effectors during migration and feeding cell formation.Key words: plant pathogen, effector, immunocytochemistry, root-knot nematode, secretion, plant apoplasm     

Host-Parasite Relationships of Atalodera spp. (Heteroderidae)

Atalodera ucri, Wouts and Sher, 1971, and A. lonicerae, (Wouts, 1973) Luc et al., 1978, induce similar multinucleate syncytia in roots of golden bush and honeysuckle, respectively. The syncytium is initiated in the cortex; as it expands, it includes several partially delimited syncytial units and distorts vascular tissue. Outer walls of the syncytium are relatively smooth and thickest near the feeding site of the nematode; inner walls are interrupted by perforations which enlarge as syncytial units increase in size. The cytoplasm of the syncytium is granular and includes numerous plastids, mitochondria, vacuoles, Golgi, and a complex network of membranes. Nuclei are greatly enlarged and amoeboid in shape. Although more than one nucleus sometimes occur in a given syncytial unit, no mitotic activity was observed. Syncytia induced by species of Atalodera chiefly differ from those of Heterodera sensu lato by the absence of cell wall ingrowths; wall ingrowths increase solute transport and characterize transfer cells. In syncytia of Atalodera spp., a high incidence of pits and pit fields in walls adjacent to vasctdar elements suggests that in this case plasmodesmata provide the pathway for increased entry of sohttes. The formation of a syncytium by species of Atalodera and Heterodera sensu lato, but a single uninucleate giant cell by Sarisodera and Hylonema, indicates a pattern of host responses that may be useful, with other characters, for phylogenetic inference for Heteroderidae.     

Fine Structure of the Esophagus of Males of Sarisodera hydrophila (Heteroderoidea)

The fine structure of the esophagus, including procorpus, metacorpus, isthmus, gland lobe, and esophago-intestinal junction, is examined in males of Sarisodera hydrophila. A cuticle-lined lumen extends most of the length of the esophagus, broadens to form a pump chamber in the metacorpus, and posteriorly is continuous with junctional complexes among four esophago-intestinal cells. These four cells are partially enveloped by the gland lobe which basically consists of three gland cells, one dorsal and two subventral. Each gland cell has an anterior process which opens into the lumen of the esophagus through a cuticle-lined duct. The dorsal gland joins the lumen in the anterior portion of the procorpus, whereas ducts of the subventral glands terminate at the base of the metacorpus pump chamber. The subventral glands are predominant in the posterior portion of the gland lobe and are partially ensheathed by a narrow portion of the dorsal gland which extends to within 5 μm of the posterior terminus of the gland lobe. Contents of the dorsal gland include primarily electron dense granules, although rough endoplasmic reticulum (RER) is predominant posteriorly. Secretory granules within the subventral glands vary in morphology and are evenly distributed throughout the two ceils among other organelles, including RER and a large Golgi apparatus. Innervation of the esophagus includes nerve processes which originate from several perikaryons (cell bodies) located in the anterior portion of the gland lobe. The esophagus of males of S. hydrophila is compared with that of other Heteroderoidea, Heterodera glycines and Meloidogyne incognita.     

Biological Relationship of Rotyleinchulus borealis on Several Plant Cultivars

The embryogenic development of Rolylenchulus borealis, at 24-26 C, was completed on corn, in 12-15 days, and the life-cycle of the nematode from egg to egg required 35-40 days at 20-25 C. Juveniles remained in the soil as preinfective stages for 17-19 days before becoming adults. Only immature vermiform and swollen egg-laying females were found attached to corn roots. Eggs were laid in a gelatinous matrix on the root surface; the number of eggs per egg mass was 45 ± 28 on corn roots. Bean, green pea, potato, sorghum, and sweet potato were also found to be hosts of R. borealis. The nematode established a permanent feeding site on corn root in an endodermal cell that became hypertrophied. Pericyclic cells close to the feeding site showed granular cytoplasm and nuclei with hypertrophied nucleoli. A cell wall ingrowth was also noted around the area of stylet penetration into the endodermal cell.     

Cytoskeleton reorganization,a key process in root-knot nematode-induced giant cell ontogenesis

Root-knot nematodes are plant parasitic worms that establish and maintain an intimate relationship with their host plants. RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. Major rearrangements of the cytoskeleton occur during giant cell formation. We characterized the first plant candidate genes implicated in giant cell actin and microtubule cytoskeleton reorganization. We showed previously that formins may regulate giant cell isotropic growth by controlling the assembly of actin cables. Recently we demonstrated that a Microtubule-Associated Protein, MAP65-3, is essential for giant cell development. In the absence of functional MAP65-3, giant cells started to develop but failed to fully differentiate and were eventually destroyed. In developing giant cells, MAP65-3 was associated with a novel kind of cell plate—the giant cell mini cell plate—that separates daughter nuclei. Despite karyokinesis occurs without cell division in giant cell, we demonstrated that cytokinesis is initiated and required for successful pathogen growth and development.Key words: cytoskeleton, microfilament, formin, microtubule, microtubule-associated protein, giant cells, nematodeRoot-knot nematodes (RKN) Meloidogyne spp. is one of the most damaging plant pathogen worldwide.¹ Their potential host range encompasses more than 2,000 plant species. During a compatible interaction, these obligate biotrophic pathogen have evolved an ability to manipulate host functions to their own benefit.^2–6 At the onset of parasitism, the infective second stage juveniles (J2) penetrate the root tip and migrate intercellularly to reach the root vascular cylinder. Each J2 then induces the redifferentiation of five to seven parenchymatic root cells into hypertrophied and multinucleate cells, named giant cells. Giant cells result from synchronous repeated karyokinesis without cell division.⁷ Despite lack of complete cytokinesis, we demonstrated that cytokinesis is initiated and essential for giant cell ontogenesis.⁸ Fully differentiated giant cells reach a final size about four hundred times that of root vascular cells and contain more than a hundred polyploid nuclei. Giant cell nuclei show an increase in DNA, possibly reflecting endoreduplication.⁹ These “feeding” giant cells constitute the exclusive source of nutrients for the nematode until reproduction. Giant cell development is accompanied by division and hypertrophy of surrounding cells, leading to a typical root gall formation, the primary visible symptom of infection.     

Root Cortical Cell Spherical Bodies Associated with an Induced Resistance Reaction in Monoxenic Cultures of Meloidogyne incognita

The root-knot nematode Meloidogyne incognita was monoxenically cultured on excised roots of soybean cv. Pickett and tomato cv. Rutgers in agar media containing either 0 to 1,600 μg/ml ammonium nitrate or 0 to 100 μg/ml urea. Observations with scanning and transmission electron microscopy indicated that an elevated concentration of ammonium nitrate or urea inhibited giant cell formation and suppressed nematode development in the infected soybean roots. In the tomato roots, concentrations of ammonium nitrate above 400 μg/ml or urea above 25 μg/ml inhibited giant cell formation and nematode development. Coincident with the nitrogen concentrations that suppressed giant cell formation was the appearance of electron-dense spherical bodies in the cortical parenchyma cells of both the soybean and tomato roots. These bodies, which were 1-4 μm in diameter, appeared to form in the cytoplasm and migrate to the cell vacuole.     

Dolichodorus aestuarius n. sp. (Nematode: Dolichodoridae)

Dolichodorus aestuarius n. sp. from an estuarine habitat near Cedar Key, Florida is described. This nematode has a stylet range of 62-76 μm in females and 60-72 μm in males. The stylet is shorter than those of all described species except D. brevistilus. The probable host plant is Juncus roemerianus.     

Meloidogyne paranaensis n. sp. (Nemata: Meloidogynidae), a Root-Knot Nematode Parasitizing Coffee in Brazil

A root-knot nematode parasitizing coffee in Paran  State, Brazil, is described as Meloidogyne paranaensis n. sp. The suggested common name is Paraná coffee root-knot nematode. The perineal pattern is similar to that of M. incognita; the labial disc and medial lips of the female are fused and asymmetric and rectangular; the lateral lips are small, triangular, and fused laterally with the head region. The female stylet is 15.0-17.5 μm long, with broad, distinctly set-off knobs; the distance from the dorsal esophageal gland orifice (DGO) to the stylet base is 4.2-5.5 μm. Males have a high, round head cap continuous with the body contour. The labial disc is fused with the medial lips to form an elongate lip structure. The head region is frequently marked by an incomplete annulation. The stylet is robust, 20-27 μm long, usually with round to transversely elongate knobs, sometimes with one or two projections protruding from the shaft. The stylet length of second-stage juveniles is 13-14 μm, the distance of the DGO to the stylet base is 4.0-4.5 μm, and the tail length is 48-51 μm. Biochemically, the esterase (F₁) and malate dehydrogenase (N₁) phenotypes are the most useful characters to differentiate M. paranaensis from other species. However, the esterase phenotype appears similar to that of M. konaensis. Reproduction is by mitotic parthenogenesis, 3n = 50-52. In differential host tests, tobacco, watermelon, and tomato were good hosts, whereas cotton, pepper, and peanut were nonhosts.     

Meloidogyne lusitanica n. sp. (Nematoda: Meloidogynidae), a Root-knot Nematode Parasitizing Olive Tree (Olea europaea L.)

A root-knot nematode from Portugal, Meloidogyne lusitanica n. sp., is described and illustrated from specimens obtained from olive trees (Olea europaea L.). Females of the new species have a characteristic perineal pattern with medium to high trapezoidal dorsal arch with distinct punctuations in the tail terminus area. The excretory pore is located posterior to the stylet, about 1.5-2.5 stylet lengths from the anterior end. The stylet is 17.1 μm long with pear-shaped knobs. Males have a rounded, posteriorly sloping head cap and head region not annulated. The robust stylet, 24.5 μ long, has large, elongate knobs. Mean length of the second-stage juveniles is 449.5 μm, stylet length 14.2 μm, and tail length 44.1 μm. Scanning electron microscope observations provide further details of perineal patterns and head and stylet morphology of females, males, and second-stage juveniles. Meloidogyne lusitanica n. sp. did not reproduce on any of the differential hosts used to separate the four most common Meloidogyne species. The common name "olive root-knot nematode" is proposed for M. lusitanica n. sp.     

Curiouser and Curiouser: The Macrocyclic Lactone,Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms

Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced R_max, no change in EC₅₀). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex effects of macrocyclic lactones that may be exploited in combinations with other anthelmintics.     

Histopathology of Okra and Ridgeseed Spurge Infected with Meloidodera charis

Histological observations of okra Abelomoschus esculentus ''Clemson Spineless'' and ridgeseed spurge Euphorbia glyptosperma (a common weed) infected with Meloidodera charis Hopper, indicated that the juvenile nematode penetrated the roots intercellularly. Within 5 days after plant emergence the nematode positioned its body in the cortical tissue parallel to the vascular system. By 10 days after plant emergence the juvenile had extended its head into the vascular system and initiated giant cell formation, generally in protophloem tissue. Giant cells were one celled and usually multi-nucleate. Eggs were observed in the female body 30 days after plants emerged and juveniles were found within the female body by 40 days. Nematode development progressed equally in the root system of either host plant. Generally, throughout the nematode''s life cycle its entire body remained inside the cortical tissue of okra. In ridgeseed spurge, however, the posterior portion of the female erupted through the host epidermis as early as 15 days after plant emergence; only the head and neck remained embedded in the host. The nematode caused extensive tissue disruption in the cortical and vascular system of both plant species. Corn, Zea mays, was another host of the nematode.     

Histological Responses of Four Leguminous Crops Infected with Meloidogyne incognita

Histological responses to Meloidogyne incognita infection in Rhizobium nodules of clover, horsebean, lupine, and pea were investigated. The formation of giant cells in vascular bundles of nodules and roots, and the basal connection of the nodule, were usually associated with abnormal xylem and/or deformed xylem strands. However, giant cells did not disturb or prevent the development of nodular tissues. Areas in which galls formed, wall thickness of giant cells, and number of giant cells around the nematode head varied with plant species. Ranking by gall size and giant-cell wall thickness was horsebean > lupine and pea > clover. The multinucleate condition in giant cells resulted from repeated mitoses without subsequent cytokinesis. The resulting nuclei agglomerated in irregularly shaped masses in some giant cells.     

An ultrastructural study of the response of Blatella germanica (Orthoptera: Blattidae) to the nematode Abbreviata caucasica (Spirurida: Physalopteridae)

An ultrastructural study of the response of Blatella germanica (Orthoptera: Blattidae) to the nematode Abbreviata caucasica (Spirurida: Physalopteridea). International Journal for Parasitology4: 133–138. This study investigates the response of the roach, Blatella germanica L. to the invading spirurid nematode, Abbreviata caucasica v. Linstow. Soon after the first stage nematodes entered the epithelial cells of the colon wall, the surrounding host cells broke down into syncytial giant cells. Large polychromatic epithelial cell nuclei occurred throughout the giant cells and the nematodes moved freely within the cytoplasmic matrix. These giant cells were in turn surrounded by blood cells responding to the disruption. The nematodes developed to the infective third stage juveniles within the giant cells and ingested the syncytial cytoplasm. After reaching the third stage, the parasites remained in a quiescent state within the vacuolated cell which was surrounded by a double tissue layer.Evidence indicated that successful development of the parasite was dependent on the disrupted epithelial cells forming a giant syncytial cell which protected and supplied nourishment to the parasite.     

Factor Affecting Sex Ratios of a Mermithid Parasite of Mosquitoes

The ratio of male to female Reesimermis nielseni Tsai and Grundmann, a nematode parasite of mosquito larvae, increased as the number of parasites per host increased. Hosts with a single nematode produced 9% males compared with essentially 100% males in hosts with more than 7 parasites; hosts with 3 nematodes produced about equal numbers of males and females. Males of R. nielseni generally emerged before females because of the earlier death of multiple-infected mosquitoes. The species of the host mosquito influenced the sex ratio, but the size of a specific host at the time of invasion did not. Host diet also had a noticeable influence on the sex ratio of the nematode: singly infected hosts from a starved population produced 92% males compared with 13% in the normally fed group. The importance of these factors in the mass rearing of R. nielseni is discussed.     

Optimization of Inoculation for In Vivo Production of Entomopathogenic Nematodes

Entomopathogenic nematodes are potent biopesticides that can be mass-produced by in vitro or in vivo methods. For in vivo production, consistently high infection rates are critical to efficiency of the process. Our objective was to optimize in vivo inoculation of Steinernema carpocapsae and Heterorhabditis bacteriophora in Galleria mellonella and Tenebrio molitor by determining effects of inoculation method, nematode concentration, and host density. We found immersing hosts in a nematode suspension to be approximately four times more efficient in time than pipeting inoculum onto the hosts. The number of hosts exhibiting signs of nematode infection increased with nematode concentration and decreased with host density per unit area. This is the first report indicating an effect of host density on inoculation efficiency. We did not detect an effect of nematode inoculum concentration on nematode yield per host or per gram of host. Yield was affected by host density in one of the four nematode-host combinations (S. carpocapsae and T. molitor). We conclude that optimization of inoculation parameters is a necessary component of developing an in vivo production system for entomopathogenic nematodes.     

Tripius gyraloura n. sp. (Aphelenchoidea: Sphaerulariidae) parasitic in the gall midge Lasioptera donacis Coutin (Diptera: Cecidomyiidae)

A new nematode, Tripius gyraloura n. sp., is described from the arundo gall midge, Lasioptera donacis Coutin (Diptera: Cecidomyiidae). This gall midge is being considered as a biological control agent for use in North America against the introduced giant reed Arundo donax (L.) (Poaceae: Cyperales). Thus the present study was initiated to investigate a nematode parasite that was unknown at the time studies with L. donacis were initiated. The new species has a rapid development in the fly host and the mature parasitic female nematodes evert their uterine cells in the hosts’ hemolymph. Because large numbers of nematodes sterilise the host, eradication of the parasite from laboratory colonies of the midge may be necessary before populations of the fly are released.