Resolution of natural hatching factors for golden potato cyst nematode, Globodera rostochiensis

The hatching responses of Globodera rostochiensis (golden potato cyst nematode) to purified and partially-purified preparations of natural (including the potato glycoalkaloids solanine and α-chaconine) and artificial hatching factors (HFs) were bimodal. At least 10 HFs, mostly anionic, were resolved from potato root leachate by a combination of gel permeation and ion-exchange chromatography. Whereas potato roots were the principal source of HFs, haulm leachate also contained such chemicals. Root leachate from aseptically-grown potato plants lacked several HFs which were present in conventionally-produced leachate.     

A technique to assess the efficacy of non-volatile nematicides against the potato cyst nematode Globodera rostochiensis

The assessment of larval emergence from cysts, second stage larval behaviour in soil, invasion of roots, development within roots and the final population provides a technique which can be used for evaluating the efficacy of any non-volatile organophosphate or carbamate nematicide.
Oxamyl delayed emergence from cysts, disorientated second stage larvae in soil, delayed and reduced invasion of roots, reduced the number of eggs in the final population and markedly reduced the final population of cysts.     

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.     

A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis

The resistance gene H1 confers resistance to the potato cyst nematode Globodera rostochiensis and is located at the distal end of the long arm of chromosome V of potato. For marker enrichment of the H1 locus, a bulked segregant analysis (BSA) was carried out using 704 AFLP primer combinations. A second source of markers tightly linked to H1 is the ultra-high-density (UHD) genetic map of the potato cross SH × RH. This map has been produced with 387 AFLP primer combinations and consists of 10,365 AFLP markers in 1,118 bins (). Comparing these two methods revealed that BSA resulted in one marker/cM and the UHD map in four markers/cM in the H1 interval. Subsequently, a high-resolution genetic map of the H1 locus has been developed using a segregating F₁ SH × RH population consisting of 1,209 genotypes. Two PCR-based markers were designed at either side of the H1 gene to screen the 1,209 genotypes for recombination events. In the high-resolution genetic map, two of the four co-segregating AFLP markers could be separated from the H1 gene. Marker EM1 is located at a distance of 0.2 cM, and marker EM14 is located at a distance of 0.8 cM. The other two co-segregating markers CM1 (in coupling) and EM15 (in repulsion) could not be separated from the H1 gene.Communicated by J.G. Wenzel     

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.     

Mapping of resistance to the potato cyst nematode Globodera rostochiensis from the wild potato species Solanum vernei

A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F₁ population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.Abbreviations BSA bulked segregant analysis - RAPD random-amplified polymorphic DNA - RFLP restriction fragment length polymorphism - SCAR sequence-characterized amplified region     

Early responses of resistant and susceptible potato roots during invasion by the potato cyst nematode Globodera rostochiensis

Signals from roots of resistant (cv. Maris Piper) and susceptible (cv. Désirée) potato cultivars during invasion by second stage juveniles (J2s) of the potato cyst nematode, Globodera rostochiensis, were investigated. Novel experimental chambers enabled the recording of electrophysiological responses from roots during nematode invasion. The root cell membrane potentials were maintained throughout the 3 d required to assess invasion and feeding site development. The steady-state resting membrane potentials of Désirée were more negative than those of Maris Piper on day 1, but the reverse on day 3. After 5 d there was no difference between the two cultivars. Intracellular microelectrodes detected marked spike activity in roots after the application of J2s and there were distinct and reproducible differences between the two cultivars, with the response from Désirée being much greater than that from Maris Piper. The responses to mechanical stimulation of roots by blunt micropipettes and sharp electrodes were consistent and similar in both cultivars to the responses in Maris Piper obtained after nematode invasion, but could not account for the marked response found in Désirée. Exogenous application of exoenzymes, used to mimic nematode chemical secretions, resulted in a distinct depolarization pattern that, although similar in both cultivars, was different from patterns obtained during nematode invasion or mechanical stimulation. The pH of homogenates prepared from roots of both cultivars was measured and a Ca2+ channel blocker was used to assess the role of Ca2+ in nematode invasion. The results indicated a role for Ca2+ in the signalling events that occur during nematode invasion.     

Nematicidal effect of Lepidium sativum on activity and reproduction of potato cyst nematode Globodera rostochiensis in soil

Abstract

In Iran, potato cyst nematode (Globodera rostochiensis) jeopardizes the traditionally high yields of potatoes in Hamadan Province in the west of Iran. Biofumigation is an eco-friendly method for integrated management of plant parasitic nematodes. In the laboratory, water extracts of water cress, fenugreek and dill similarly reduced viability of second stage juveniles after 3?h of exposure, and decreased hatching of encysted eggs to less than 1%. Pre-treatment and combined tests similarly decreased hatch. The nematicidal efficiency of top green manure of Lepidium sativum on the survival of nematode was tested on a susceptible cv in microplots. The weights of biofumigated plants increased. Anti-hatching properties of water cress applied as a biofumigant reduced hatch by average of 56%. Reproduction rates were lowered to below one, and final populations of cysts and their egg contents were reduced by nearly 60% in treated soil. Biofumigation at a 1% amendment rate was sufficient to bring about these results, which were comparable with those achieved with 2 and 3% rates. Nematicidal isothiocyanates released after incorporating glucosinolate-containing brassica plants are fully biodegradable and less toxic than their synthetic equivalents, and their use is considered a safer alternative to soil fumigants such as methyl bromide.     

Water relations during desiccation of cysts of the potato-cyst nematode Globodera rostochiensis

The loss during desiccation of osmotically active water (OAW), which freezes during cooling to -45 degrees C, and osmotically inactive water (OIW), which remains unfrozen, from the cysts of the potato cyst nematode, Globodera rostochiensis, was determined using differential scanning calorimetry. Exotherms and endotherms associated with non-egg compartments were not detected after 5 min desiccation at 50% relative humidity and 20 degrees C. The pattern of water loss from the cysts indicates that water is lost from compartments outside the eggs first, that nearly all the non-egg water is OAW and that the OIW content of the cyst is contained within the eggs. Water is lost from the eggs only after the OAW content outside the eggs falls below that within the eggs. Both OAW and OIW are lost from the eggs during desiccation but the eggs retain a small amount of OIW. Other animals which survive some desiccation but which are not anhydrobiotic will tolerate the loss of OAW but not the loss of their OIW. Anhydrobiotic animals can survive the loss of both their OAW and a substantial proportion of their OIW.     

Origin, distribution and 3D-modeling of Gr-EXPB1, an expansin from the potato cyst nematode Globodera rostochiensis

Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modeling predicted that Gr-EXPB1 domain 1 (D1) has a flat beta-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.     

Structural and functional characterization of a novel, host penetration-related pectate lyase from the potato cyst nematode Globodera rostochiensis

The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene ( Gr-pel2 , 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40–42% identity and 58–60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca²⁺ is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes.     

Identification of RFLP markers linked to the H1 gene conferring resistance to the potato cyst nematode Globodera rostochiensis.

The H1 gene from Solanum tuberosum ssp. andigena confers high levels of resistance to the potato cyst nematode Globodera rostochiensis and is used extensively in potato breeding. Using a dihaploid segregating population, a search was conducted for linkage between this gene and markers on the potato/tomato RFLP map. A total of 60 RFLP markers covering the entire genome were screened on bulk resistant and susceptible segregants. Linkage was indicated for eight markers on chromosome 5. Individual plant analysis placed the closest marker, CD78, at a maximum map distance of 2.7 cM from H1. A molecular marker for the H1 should be useful both as a correlative screening tool for incorporation of resistance into new cultivars and as starting point for map-based cloning of this important gene.     

A multipartite mitochondrial genome in the potato cyst nematode Globodera pallida

The mitochondrial genome (mtDNA) of the plant parasitic nematode Globodera pallida exists as a population of small, circular DNAs that, taken individually, are of insufficient length to encode the typical metazoan mitochondrial gene complement. As far as we are aware, this unusual structural organization is unique among higher metazoans, although interesting comparisons can be made with the multipartite mitochondrial genome organizations of plants and fungi. The variation in frequency between populations displayed by some components of the mtDNA is likely to have major implications for the way in which mtDNA can be used in population and evolutionary genetic studies of G. pallida.     

Detection of hatching inhibitors and hatching factor stimulants for golden potato cyst nematode, Globodera rostochiensis, in potato root leachate

In a comparison of four potato varieties, in-soil hatch of the golden potato cyst nematode (Globodera rostochiensis) was positively correlated to in vitro hatch in response to potato root leachate (PRL). The in-soil hatch of cysts of G. rostochiensis to two of the four varieties was significantly less than that of the control (cysts in gravel without potato plants) in the first 2 wk after plant emergence, suggesting the production of hatching inhibitors (HIs) by young potato plants. The hatching factor: hatching inhibitor ratio of PRL was positively associated with the net hatching activity of the PRL. Four zones of HI activity were resolved following gel permeation chromatography of PRL on Sephadex G-10. Hatch-inactive chemicals, which stimulated the activity of hatching factors (HFs) in PRL (hatching factor stimulants, HSs), were also isolated from PRL, hatch levels induced by individual HFs responding differently to the same HS preparation. The complex interactions between individual HFs and other hatching chemicals in PRL was illustrated when addition of the hatch-active potato glycoalkaloid α-solanine caused both inhibition and stimulation of PRL-induced hatch, depending on the α-solanine concentration.     

Changes in relative abundance of two potato cyst nematode species Globodera rostochiensis and G. pallida in one generation

Changes in relative abundance of the two potato cyst nematode species Globodera rostochiensis and Globodera pallida were studied during the 1983/84 season at two different population levels in small pots in the glasshouse and at a single population density on plants grown outdoors in 2 litre terylene cloth bags. In both environments G. rostochiensis was the more successful species. Although the ratio of the two species changed and G. pallida was at a lower level at the end of the experiment it was never eliminated. However, when the number of G. pallida in the mixture was small it did better than expected and demonstrated a frequency dependent response.     

Potato cyst nematodes Globodera rostochiensis and G. pallida

TaxonomyPhylum Nematoda; class Chromadorea; order Rhabditida; suborder Tylenchina; infraorder Tylenchomorpha; superfamily Tylenchoidea; family Heteroderidae; subfamily Heteroderinae; Genus Globodera.BiologyPotato cyst nematodes (PCN) are biotrophic, sedentary endoparasitic nematodes. Invasive (second) stage juveniles (J2) hatch from eggs in response to the presence of host root exudates and subsequently locate and invade the host. The nematodes induce the formation of a large, multinucleate syncytium in host roots, formed by fusion of up to 300 root cell protoplasts. The nematodes rely on this single syncytium for the nutrients required to develop through a further three moults to the adult male or female stage. This extended period of biotrophy—between 4 and 6 weeks in total—is almost unparalleled in plant–pathogen interactions. Females remain at the root while adult males revert to the vermiform body plan of the J2 and leave the root to locate and fertilize the female nematodes. The female body forms a cyst that contains the next generation of eggs.Host rangeThe host range of PCN is limited to plants of the Solanaceae family. While the most economically important hosts are potato (Solanum tuberosum), tomato (Solanum lycopersicum), and aubergine (Solanum melongena), over 170 species of Solanaceae are thought to be potential hosts for PCN (Sullivan et al., 2007).Disease symptomsSymptoms are similar to those associated with nutrient deficiency, such as stunted growth, yellowing of leaves and reduced yields. This absence of specific symptoms reduces awareness of the disease among growers.Disease controlResistance genes (where available in suitable cultivars), application of nematicides, crop rotation. Great effort is put into reducing the spread of PCN through quarantine measures and use of certified seed stocks.Useful websitesGenomic information for PCN is accessible through WormBase ParaSite.     

Genetic variability and differentiation of three Russian populations of yellow potato cyst nematode Globodera rostochiensis as revealed by nuclear markers

Genetic variability of yellow potato cyst nematode G. rostochiensis from three Russian populations (Karelia, Vladimir oblast, and Moscow oblast) was investigated using two types of nuclear markers. Using RAPD markers identified with the help of six random primers (P-29, OPA-10, OPT-14, OPA-11, OPB-11, and OPH-20), it was possible to distinguish Karelian population from the group consisting of the populations from two adjacent regions (Moscow oblast and Vladimir oblast). Based on the combined matrix, containing 294 RAPD fragments, dendrogram of genetic differences was constructed, and the indices of genetic divergence and partition (P, H, and G(st)), as well as the gene flow indices N(m) between the nematode samples examined, were calculated. The dendrogram structure, genetic diversity indices, and variations of genetic distances between single individuals in each population from Karelia and Central Russia pointed to genetic isolation and higher genetic diversity of the nematodes from Karelia. Based on polymorphism of rDNA first intergenic spacer ITS1, attribution of all populations examined to the species G. rostochiensis was proved. Small variations of the ITS1 sequence in different geographic populations of nematodes from different regions of the species world range did not allow isolation of separate groups within the species. Possible factors (including interregional transportations of seed potato) affecting nematode population structure in Russia are discussed.     

Screening for quantitative resistance to the white potato cyst nematode Globodera pallida

The resistance to Globodera pallida of potatoes bred from Solanum uernei was assessed rapidly in pot tests by counting cysts on the exterior of the root ball. Assessments of resistance made in this way in three consecutive years were reproducible on a ranking basis. There was also a significant agreement in the ranked results of pot and field tests. A more accurate assessment of nematode multiplication rates in the field could be provided by laboratory tests in which temperature, moisture and initial nematode numbers were strictly controlled.     

Control of potato cyst nematode (Globodera rostochiensis) by nematicides and a resistant potato cultivar at four sites in Scotland

The control of potato cyst nematode (Globodera rostochiensis) by the oxime-carbamates aldicarb and oxamyl was tested in four fields in Scotland. Dazomet was tested in three of these fields and carbofuran in one. In untreated plots in the three most heavily infested fields Maris Piper (resistant) yielded better than Pentland Crown (non-resistant). All nematicides increased the yields of both potato cultivars but had a greater effect on the yield of Pentland Crown. Dazomet increased yields of tubers most. Heavy nematode infestation reduced yield of tubers more in a sandy soil than in two sandy loams. In a field with few potato cyst nematodes nematicides did not significantly affect tuber yields. Although the nematicides greatly increased yields, they were not completely effective in controlling potato cyst nematodes. In treated plots in the lightly infested field, there were more nematode eggs following a crop of Pentland Crown than before. In contrast, Maris Piper markedly decreased post-cropping populations and except at one site, where dazomet further decreased nematode numbers, combining nematicides with the resistant cultivar failed to decrease nematode numbers further. Nematicides decreased the numbers of larvae invading potato roots by up to 95%, oxamyl at 5–6 kg/ha being consistently the best treatment.