Stage-specific Monoclonal Antibodies to the Potato Cyst Nematode Globodera pallida Stone (Behrens)

Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development.     

The distribution and effect of intracellular rickettsia- like micro- organisms infecting second- stage juveniles of the Potato cyst- nematode Globodera rostocbiensis

For the first time rickettsia-like micro-organisms are illustrated infecting most tissues of second-stage juveniles of Globoderu rostochiensis . They were seen only inside host cells, except for the few degenerating forms in the intestinal lumen. They were most common in the non-contractile (sarcoplasmic) region of body wall muscles. A study of the oesophageal glands of the nematode suggested that micro-organisms present within these glands would be unlikely to pass out of them and that the nematode would not act as a vector of these micro-organisms to its host plants. No micro-organisms were found in the genital primordia of the nematodes. Stored, hatched juveniles appeared to contain more microorganisms than newly hatched juveniles, indicating an increase in the intensity of the infection subsequent to hatching. No obvious signs of pathogenicity were observed in the nematodes. Uninfected juveniles apparently live longer than infected ones in viro , probably because infected juveniles exhaust their lipid reserves sooner than uninfected juveniles. The possible effect of the micro-organisms on their nematode hosts and their potential as biological control agents are discussed.     

EVect of the Entomopathogenic Nematodes, Steinernema feltiae and S. carpocapsae , on the Potato Cyst Nematode, Globodera rostochiensis , in Pot Trials

Two pot experiments, one in a glasshouse and the other in an outdoor sand plunge, were conducted to examine the influence of the entomopathogenic nematodes, Steinernema feltiae and S. carpocapsae , on the invasion and development of the potato cyst nematode, Globodera rostochiensis . Of a total of eight diVerent treatments with entomopathogenic nematodes in the glasshouse trial, three reduced the invasion of G. rostochiensis and one reduced the numbers of new cysts that were produced compared with controls. In the outdoor experiment, seven of the 12 treatments gave a reduction in invasion but none resulted in changes in the numbers of cysts found at plant senescence. In general, invasion of G. rostochiensis juveniles was reduced more eVectively by S. carpocapsae than by S. feltiae , and was greatest in the outdoor trial where larger inocula of entomopathogenic nematodes were used. Overall, the results indicated that use of S. feltiae and S. carpocapsae is unlikely to provide a viable control strategy for G. rostochiensis .     

Role of 2,4-Diacetylphloroglucinol in the Interactions of the Biocontrol Pseudomonad Strain F113 with the Potato Cyst Nematode Globodera rostochiensis

The potato cyst nematode Globodera rostochiensis is an important pest of potato (Solanum tuberosum). Pseudomonas fluorescens F113, which produces 2,4-diacetylphloroglucinol (DAPG), was investigated as a potential biocontrol agent against G. rostochiensis. Exposure of nematode cysts to the pseudomonad, under in vitro conditions or in soil microcosms, almost doubled the ability of the eggs to hatch. The percentage of mobile juveniles was reduced threefold following their incubation in the presence of the pseudomonad, both in vitro and in soil. Results obtained with a transposon-induced DAPG-negative biosynthetic mutant of F113 and its complemented derivative with restored DAPG synthesis showed that the ability of strain F113 to produce DAPG was responsible for the increase in hatch ability and the reduction in juvenile mobility. Similar effects on egg hatch ability and juvenile mobility of G. rostochiensis were obtained in vitro by incubating nematode cysts and juveniles, respectively, in the presence of synthetic DAPG. DAPG-producing P. fluorescens F113 is proposed as a potential biocontrol inoculant for the protection of potato crops against the potato cyst nematode.     

Resistance quantitative trait loci originating from Solanum sparsipilum act independently on the sex ratio of Globodera pallida and together for developing a necrotic reaction

Plant resistance to nematodes is related to the ability of the host to reduce the development of nematode juveniles into females. Resistance to the potato cyst nematode (PCN) Globodera pallida, originating from the wild species Solanum sparsipilum, was dissected by a quantitative trait loci (QTL) approach. Two QTL explained 89% of the phenotypic variation. The QTL GpaV(s)spl on chromosome V displayed the major effect on the cyst number (coefficient of determination [R2] = 76.6%). It restricted G. pallida development to 16.2% of juveniles, 81.5% of males, and 2.3% of females. The QTL GpaXI(s)spl on chromosome XI displayed a lower effect on the cyst number (R2 = 12.7%). It restricted G. pallida development to 13.8% of juveniles, 35.4% of males, and 50.8% of females. Clones carrying both QTL restricted the nematode development to 58.1% juveniles, 41.1% of males, and 0.8% of females. We demonstrated that potato clones carrying both QTL showed a strong necrotic reaction in roots infected by nematodes, while no such reaction was observed in clones carrying a single QTL. This result underlines the importance to introgress together GpaV(s)spl and GpaXI(s)spl into potato cultivars, in order to reduce the density of this quarantine pest in soil and to decrease the risk of selecting overcoming G. pallida subpopulations.     

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.     

Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi

RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted beta-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plant-parasitic nematode. Knockout of the beta-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.     

Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.     

Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode Globodera rostochiensis

Perry R. N. and Wharton D. A. 1985. Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode, Globodera rostochiensis. International Journal for Parasitology15: 441–445. Hatched second-stage juveniles of Globodera rostochiensis can survive sub-zero temperatures by supercooling when not in contact with water. When frozen in water free juveniles cannot survive ice seeding across the cuticle and concomitant freezing of their body contents. Unhatched juveniles can survive in water, probably because the egg-shell protects the juvenile from ice seeding across from the medium; in this state juveniles survive by supercooling.     

Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.     

Rapid identification of cyst (Heterodera spp., Globodera spp.) and root-knot (Meloidogyne spp.) nematodes on the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) in cultures and field samples

Cyst and root-knot nematodes show high levels of gross morphological similarity. This presents difficulties for the study of their ecology in natural ecosystems. In this study, cyst and root-knot nematode species, as well as some ectoparasitic nematode species, were identified using the second internal transcribed spacer (ITS2) sequence variation detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). The ITS2 region was sufficiently variable within the taxa investigated to allow species to be separated on the basis of minor sequence variation. The PCR primers used in this study were effective for 12 species with three genera within the Heteroderinae (Globodera pallida, G. rostochiensis, Heterodera arenaria/avenae, H. ciceri, H. daverti, H. hordecalis, H. mani, H. schachtii, H. trifolii, Meloidogyne ardenensis, M. duytsi and M. maritima). However, pathotypes of Globodera pallida and G. rostochiensis could not be distinguished. The method was tested at two coastal dune locations in The Netherlands (one in the lime-poor dunes of the north and one in calcareous dunes of the south) to determine the population structure of cyst nematodes. At each site, cyst nematodes were associated with three plant species: two plant species on the foredune (Elymus farctus and Ammophila arenaria) and one plant species occurring further inland (Calamagrostis epigejos). Two species of cyst nematodes, H. arenaria and H. hordecalis, were found. H. arenaria associated with vigorous A. arenaria and H. hordecalis in association with degenerating A. arenaria and C. epigejos. The field survey demonstrated that in coastal dunes abiotic factors may be the important affecting the distribution of cyst nematodes.     

Functionality of resistance gene Hero, which controls plant root-infecting potato cyst nematodes, in leaves of tomato

The expression of host genomes is modified locally by root endoparasitic nematode secretions to induce the development of complex cellular structures referred as feeding sites. In compatible interactions, the feeding sites provide the environment and nutrients for the completion of the nematode's life cycle, whereas in an incompatible (resistant) interaction, the host immune system triggers a plant cell death programme, often in the form of a hypersensitive reaction, which restricts nematode reproduction. These processes have been studied in great detail in organ tissues normally infected by these nematodes: the roots. Here we show that host leaves can support a similar set of programmed developmental events in the potato cyst nematode Globodera rostochiensis life cycle that are typical of the root-invading nematodes. We also show that a gene-for-gene type specific disease resistance that is effective against potato cyst nematodes (PCN) in roots also operates in leaves: the expression of the resistance (R) gene Hero and members of its gene family in leaves correlates with the elicitation of a hypersensitive response only during the incompatible interaction. These findings, and the ability to isolate RNA from relevant parasitic stages of the nematode, may have significant implications for the identification of nematode factors involved in incompatible interactions.     

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.     

<Emphasis Type="Italic">Myrmeconema neotropicum</Emphasis> n. g., n. sp., a new tetradonematid nematode parasitising South American populations of <Emphasis Type="Italic">Cephalotes atratus</Emphasis> (Hymenoptera: Formicidae), with the discovery of an apparent parasite-induced host morph

A new genus and species of tetradonematid nematode, Myrmeconema neotropicum n. g., n. sp., is described from larval, pupal and adult stages of Cephalotes atratus L. (Hymenoptera: Formicidae) in Peru and Panama. Diagnostic characters of the new genus include: males and females subequal in size; cuticle with minute annulations; six cephalic papillae; stylet present in all stages; stichocytes absent; trophosome degenerate; three penetration glands; gonads paired and opposite; vulva in mid-body region; single spicule; genital papillae absent; adult tails rounded; infective juveniles moult once in egg; and adults of both sexes remain in the host throughout their development. As the female nematodes mature inside the worker ants, the host gasters change colour from black to red.     

A method for estimating the contribution of seed potatoes, machinery and soil tare in field infestations with potato cyst nematodes on a national scale

In order to make a cost benefit analysis for the management of the potato cyst nematodes Globodera rostochiensis and G. pallida, we developed a method to estimate the relative importance of three basic distribution channels of potato cyst nematodes: seed potatoes, machinery and soil tare. The baseline is determined by the area planted with potatoes, the area infested with potato cysts, the proportion of resistant potato cultivars and the distribution of cysts trough different channels. This quantification forms a basis for the evaluation of the effects of different control measures for potato cyst nematode on a national scale. The method can be useful as an example for application in other countries.     

Mechanisms of interrelations in a system "Globodera rostochiensis ‐ host plant”;

In researching the function of the system of Globodera rostochiensis resistant plants, we observed the development of G. rostochiensis (pathotype Ro 1) in glasshouse experiments. For a period of two months, we determined the qualities (the presence of juveniles stages) and quantities (the ratio of ages and the duration of periods of development of the juveniles stages) of parameters of nematodes development on potato wild and cultured species as well as hybrids. Comparative analysis of the rate and specifics of ontogenetic changes in G. rostochensis (Ro 1) during parasitism in the boundaries of specific plant groups, allowed us to assert the following types of resistance to G. rostochiensis: antixenosis, antibiosis, hypersensitive response.     

The Relationship between Temperature and Development in Globodera ellingtonae

A new cyst nematode species, Globodera ellingtonae, was recently described from populations in Oregon and Idaho. This nematode has been shown to reproduce on potato. Because of this nematode’s close relationship to the potato cyst nematodes, G. rostochiensis and G. pallida, an understanding of the risk of its potential spread, including prediction of potential geographical distribution, is required. To determine the development of G. ellingtonae under different temperatures, we conducted growth chamber experiments over a range of temperatures (10.0°C to 26.5°C) and tracked length of time to various developmental stages, including adult females bearing the next generation of eggs. Both the time to peak population densities of G. ellingtonae life stages and their duration in roots generally increased with decreasing temperature. Regression of growth rate to second-stage (J2) and third-stage (J3) juveniles on temperature yielded different base temperatures: 6.3°C and 4.4°C for J2 and J3, respectively. Setting a base temperature of 6°C allowed calculation of the degree-days (DD6) over which different life stages occurred. The largest population densities of J2 were found in roots between 50 and 200 DD6. Population densities of J3 peaked between 200 and 300 DD6. Adult males were detected in soil starting at 300 to 400 DD6 and remained detectable for approximately 500 DD6. By 784 to 884 DD6, half of the eggs in adult females contained vermiform juveniles. Given the similarity in temperature ranges for successful development between G. ellingtonae and G. rostochiensis, G. ellingtonae populations likely could survive in the same geographic range in which G. rostochiensis now occurs.     

Reproduction of Globodera rostochiensis on Transformed Roots of Solanum tuberosum cv. Desiree

Transformed roots of the susceptible potato Solanum tuberosum L. cv. Desiree were inoculated with second-stage juveniles (J2) of Globodera rostochiensis pathotype Rol. Adult males emerged after 3-4 weeks and matings with females occurred. After 8 weeks gentle pressure on the eggs of maturing females released the J2 which were viable. Because this technique enables the production of vigorously growing roots with numerous laterals, it may be suitable for obtaining a high yield of sterile potato cyst nematodes.     

Control of potato cyst-nematode, Globodera rostochiensis, and root-knot nematode, Meloidogyne incognita, by organosphosphorus, carbamate, benzimidazole and other compounds

Control of potato cyst-nematode, Globodera rostochiensis, was examined on potato or tomato in pots and on potato in field plots by various chemicals incorporated into the soil at planting. The most effective nematicides were organophosphorus compounds, generally of the type O,O-diethyl O-phosphoro-thioates or O,O-diethyl phosphorodithioates, carbamates and benzimidazoles. In organic soils, the more lipophilic compounds were less effective, presumably because of sorption onto soil organic matter. Foliar sprays of chemicals, including oxamyl which is known to be translocated to roots, gave poor control of G. rostochiensis. The root-knot nematode, Meloidogyne incognita, on tomato, widely used in screening for nematicidal activity, was controlled by aldicarb or phoxim incorporated into the soil at planting, but not by benomyl or thiabendazole, in contrast to the moderate effectiveness of these latter two chemicals against G. rostochiensis.     

Mechanisms of molecular mimicry of plant CLE peptide ligands by the parasitic nematode Globodera rostochiensis

Nematodes that parasitize plant roots cause huge economic losses and have few mechanisms for control. Many parasitic nematodes infect plants by reprogramming root development to drive the formation of feeding structures. How nematodes take control of plant development is largely unknown. Here, we identify two host factors involved in the function of a receptor ligand mimic, GrCLE1, secreted by the potato cyst nematode Globodera rostochiensis. GrCLE1 is correctly processed to an active form by host plant proteases. Processed GrCLE1 peptides bind directly to the plant CLE receptors CLV2, BAM1, and BAM2. Involvement of these receptors in the ligand-mimicking process is also supported by the fact that the ability of GrCLE1 peptides to alter plant root development in Arabidopsis (Arabidopsis thaliana) is dependent on these receptors. Critically, we also demonstrate that GrCLE1 maturation can be entirely carried out by plant factors and that the availability of CLE processing activity may be essential for successful ligand mimicry.