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.     

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.     

The relationship between glycoalkaloids and resistance to the white potato cyst nematode, Globodera pallida in potato clones derived from Solanum vernei

No relationship was found between the degree of resistance to Globodera pallida and total glycoalkaloid content of the roots or tubers of a number of potato clones derived from Solanum vernei × S. tuberosum. Nematode infestation of the roots did not lead to increases in the glycoalkaloid content of susceptible or resistant potatoes.     

The effect of the potato cyst nematode Globodera pallida on in vitro root growth of potato genotypes, differing in tolerance

Roots of eighteen potato genotypes, differing in tolerance of G. pallida, were grown from tuberpieces on agar in Petri dishes. Juveniles of G. pallida were inoculated directly onto root tips. Root length was measured at various times after inoculation. Inoculation reduced root growth within one day. At later stages, genotypes differed strongly in growth of inoculated roots. Between four and seven days after inoculation, growth of inoculated roots was not significantly correlated with growth of untreated roots, and was only poorly correlated with tolerance assessed in the greenhouse or in the field. However, multiple regression analysis revealed that the tolerance of the tested genotypes was associated with both the rate at which they induced hatching and the growth of roots after inoculation. The combination of these two variables accounted for high percentages explained variance.     

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

Background

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.

Results

The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.

Conclusion

This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-923) contains supplementary material, which is available to authorized users.     

Management of the potato cyst nematode (Globodera pallida) with bio-fumigants/stimulants

Field trials evaluated the effect of four plant-based bio-fumigants/stimulants on population levels of G. pallida and the resulting potato yields and quality. Three formulations contained seaweed biostimulants (Algifol, Nutridip and Metastim) and one bio-fumigant containing mustard and chilli pepper extracts (Dazitol). These were compared with the fumigant nematicide Nemathorin and untreated control plots. The effect of G. pallida on growing potato crops was assessed by recording haulm characteristics which indicated that the nematicide treatment gave most protection. Levels of PCN juveniles and migratory nematodes were assessed during the trial. Plots treated with Nemathorin and Dazitol had fewest PCN, whilst the highest number of migratory nematodes occurred in fallow plots. Sixteen weeks after planting the nematicide treatment produced highest yield and tuber numbers. Dazitol treatment produced a lower yield but the largest tubers.     

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.     

Control of potato cyst nematode (Globodera pallida) by host plant resistance and nematicide

Seven trials conducted over four years on sites naturally infested with the white potato cyst nematode established that potato clones bred for resistance to Globodera pallida allowed significantly less nematode multiplication than conventional cultivars under field conditions. Nematode multiplication was inversely related to initial infestation level. The nematicide, aldicarb, significantly reduced nematode multiplication. However, nematode multiplication on nematicide treated susceptible cultivars was greater than on untreated partially resistant clones, indicating that resistance may offer more effective control of G. pallida than chemical treatment. Integration of host plant resistance and nematicide treatment is discussed.     

The effect of partially resistant potato clones and aldicarb on the multiplication of the potato cyst nematode Globodera pallida in field trials

Potato cultivars with different degrees of resistance to Globodera pallida (PCN) were trialled at three infested sites in 1983, 1984 and 1985. These trials were primarily intended to assess the effects of PCN damage on the yield of tubers and consequently the sites chosen had high initial population densities of G. pallida. The population density of G. pallida was determined from samples taken both pre-planting and post-harvest and results showed that there were significant differences between the clones in the nematode multiplication rates. Although actual rates of multiplication varied between sites the relative differences between clones were maintained across sites. Under the susceptible control cultivars the populations generally increased even in the presence of nematicide. The partially resistant clones gave best control, as measured by nematode multiplication, at the sites with the highest initial population densities but gave good control at all sites when treated with nematicide irrespective of initial population density. The role of partial resistance in integrated control systems and the effectiveness of nematicides against G. pallida is discussed.     

Mapping QTLs for resistance to the cyst nematode Globodera pallida derived from the wild potato species Solanum vernei

Resistance to the potato cyst nematode (PCN) species Globodera pallida, derived from the wild diploid potato species Solanum vernei, has been investigated. This source of resistance, which is effective against all of the major pathotypes of G. pallida and Globodera rostochiensis, has been assumed to be due to several genetic factors, but it has proved difficult to deploy effectively in breeding strategies for potato cultivars. Diploid and tetraploid potato populations segregating for 'vernei' resistance were analysed. At the tetraploid level, a bulk segregant analysis (BSA) approach was employed and detected AFLP markers linked to a resistance QTL on potato linkage group V. Conventional linkage analysis of a diploid population identified QTL on linkage groups V and IX. A marker linked to a QTL on linkage group V has been converted to a single-locus PCR-based marker, which can be used to detect the presence of the QTL in diploid and tetraploid potato germplasm. Moreover, there is evidence that one of the AFLPs detected by BSA appears to be specific to an introgressed segment of DNA from S. vernei. These results are compared with those obtained from other studies on resistance to the PCN species G. pallida.     

The effect of pyramiding two potato cyst nematode resistance loci to Globodera pallida Pa2/3 in potato

Globodera pallida is a major nematode pest causing severe constraints in many potato production regions worldwide. The most prevalent G. pallida pathotypes are Pa2 and Pa3, which exist in mixed populations referred to as Pa2/3. Due to heterogeneity for avirulence genes within these nematode populations, so far, breeders have failed to identify a single resistance gene source which offers complete resistance. Currently, there are two quantitative trait loci (QTL) available, $ GpaIV_{adg}^{s} $ and Gpa5, conferring partial levels of resistance to G. pallida pathotype Pa2/3. The objective of this research was to pyramid $ GpaIV_{adg}^{s} $ and Gpa5 through marker-assisted selection to investigate whether or not pyramiding provides increased resistance to G. pallida Pa2/3. We developed a population segregating for both resistance QTLs and, using diagnostic genetic markers (Contig237 and HC), we identified groups of individuals from this population containing each individual QTL, both QTLs simultaneously, and neither QTL. These individuals were assessed for their resistance levels against G. pallida Pa2/3 population Chavornay. We demonstrated that individuals carrying both QTLs showed a significant reduction in the number of cysts formed in comparison to genotypes carrying $ GpaIV_{adg}^{s} $ or Gpa5, indicating an additive effect. Overall these results show that MAS-based pyramiding of these QTLs is an effective strategy for breeding cultivars exhibiting very high levels of resistance to G. pallida pathotype Pa2/3.     

The effect of Solanum tuberosum×S. vernei hybrids on hatching of the potato cyst nematode Globodera pallida

The hatching activity of partially resistant Solanum tuberosum×S. vernei clones and susceptible cultivars was investigated in a series of tests with G. pallida conducted in pots in the glasshouse. Differences in hatching were apparent, with the partially resistant clones generally stimulating a lower total hatch than the S. tuberosum cultivars. It is concluded that reduced hatching activity can make a small contribution to overall resistance and perhaps also to the tolerance of a clone to G. pallida.     

Paternal leakage of mitochondrial DNA in experimental crosses of populations of the potato cyst nematode Globodera pallida

Animal mtDNA is typically assumed to be maternally inherited. Paternal mtDNA has been shown to be excluded from entering the egg or eliminated post-fertilization in several animals. However, in the contact zones of hybridizing species and populations, the reproductive barriers between hybridizing organisms may not be as efficient at preventing paternal mtDNA inheritance, resulting in paternal leakage. We assessed paternal mtDNA leakage in experimental crosses of populations of a cyst-forming nematode, Globodera pallida. A UK population, Lindley, was crossed with two South American populations, P5A and P4A. Hybridization of these populations was supported by evidence of nuclear DNA from both the maternal and paternal populations in the progeny. To assess paternal mtDNA leakage, a ~3.4?kb non-coding mtDNA region was analyzed in the parental populations and in the progeny. Paternal mtDNA was evident in the progeny of both crosses involving populations P5A and P4A. Further, paternal mtDNA replaced the maternal mtDNA in 22 and 40?% of the hybrid cysts from these crosses, respectively. These results indicate that under appropriate conditions, paternal leakage occurs in the mtDNA of parasitic nematodes, and supports the hypothesis that hybrid zones facilitate paternal leakage. Thus, assumptions of strictly maternal mtDNA inheritance may be frequently violated, particularly when divergent populations interbreed.     

Effect of the trap crop Solanum sisymbriifolium and two biocontrol fungi on reproduction of the potato cyst nematode,Globodera pallida

The potato cyst nematode, Globodera pallida, is one of the most important pests of potato worldwide. Owing to regulatory considerations and potential environmental impact, control options for this nematode are becoming increasingly limited. Solanum sisymbriifolium and biological control agents offer viable alternative options for controlling G. pallida. Therefore, experiments were conducted to determine the effect of the nematode trap crop S. sisymbriifolium, alone or in combination with the biocontrol agents Trichoderma harzianum or Plectosphaerella cucumerina, on population decline of G. pallida. Experiments were conducted for three different ‘cropping systems’: potato (Solanum tuberosum), S. sisymbriifolium, or soil only (fallow), each followed by a potato crop. Soil was amended with P. cucumerina, T. harzianum or left unamended, and then infested with nematodes at a rate of five eggs g^?1 of soil. After 16 weeks in the greenhouse, plants were removed and the soil containing cysts was refrigerated at 4°C for 8 weeks, and then planted to potato. Cysts of G. pallida were counted after an additional 16‐week period. The P_f/P_i of G. pallida was significantly reduced by 99% in potato following S. sisymbriifolium compared to both the potato‐following‐fallow and the potato‐following‐potato treatments. Amendment of soil with T. harzianum significantly reduced P_f/P_i of G. pallida by 42–47% in the potato‐following‐potato but not in either the potato‐after‐fallow nor in the potato‐after‐S. sisymbriifolium cycles which supports evidence that the plant species may play a role in the biocontrol activity of this fungus. Addition of the fungus P. cucumerina resulted in a 64% decrease in P_f/P_i in the potato‐following‐fallow in one experiment, and an 88% decrease in P_f/P_i in potato‐following‐potato but the decrease in P_f/P_i was not consistent over all experiments. However, both biocontrol fungi resulted in lower numbers of progeny cysts after an initial 16‐week incubation with potato. To look at the effect of varied population density of the nematode on efficacy of S. sisymbriifolium to reduce G. pallida populations, potato, S. sisymbriifolium, or barley were planted into soil infested with G. pallida at rates of 5, 20 or 40 eggs g^?1 soil applied as cysts (20, 80 or 160 cysts pot^?1). After 16 weeks, numbers of cysts produced in each treatment were determined for each infestation rate. No new cysts were recovered from either S. sisymbriifolium or barley treatments, confirming that neither plant is a host for G. pallida. High numbers of cysts were recovered with potato. Soil from each treatment (containing original cysts and newly‐formed cysts when present) were then planted with potato. After an additional 16 weeks, few cysts were found in the potato‐after‐ S. sisymbriifolium treatments regardless of initial infestation rate. When potato followed barley, numbers of cysts were similar to those found after a single cycle of potato, indicating that the barley crop had no effect on the survival of initial inoculum. Overall, these results suggest that S. sisymbriifolium has potential to significantly reduce G. pallida populations, and also that the cropping system (i.e. the sequence of non‐host and host plants) may play a significant role in the efficacy of fungal biological control agents.     

Identification and functional characterization of effectors in expressed sequence tags from various life cycle stages of the potato cyst nematode Globodera pallida

In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida . We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.     

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.     

Enhancement of the efficacy of a carbamate nematicide against the potato cyst nematode, Globodera pallida, through mycorrhization in commercial potato fields

Two experiments were conducted over 2 years in commercial potato fields in Shropshire, UK, to evaluate the compatibility of the nematicide aldicarb with commercial inocula of arbuscular mycorrhizal fungi (AMF) in the control of the potato cyst nematode Globodera pallida. The AMF used were Vaminoc (mixed-AMF inoculum), Glomus intraradices (BioRize BB-E) and G. mosseae (isolate BEG 12). In the absence of AMF, the in-soil hatch of G. pallida increased 30% (P < 0.01) from wk-2 to wk-4 after planting. Inoculation of physiologically-aged potato (cv. Golden Wonder) tubers with AMF eliminated this delay in G. pallida hatch by stimulating a mean increase of 32% (P < 0.01) in hatch within 2 wk after planting. In the aldicarb-treated plots in Experiment 1, G. pallida multiplication rate was 38% lower (P < 0.05) in roots of AMF-inoculated than noninoculated plants, but in Experiment 2, this effect was slightly lower (P = 0.07). In these plots, the single AMF inocula showed also a weak trend (P = 0.10) towards greater tuber yields relative to their noninoculated counterparts. Mycorrhization therefore appears to enhance the efficacy of carbamate nematicides against G. pallida and consequently more research is proposed to validate these findings and fully explore the potential of this model.     

The assessment of the tolerance of partially resistant potato clones to damage by the potato cyst nematode Globodera pallida at different sites and in different years

A range of potato cultivars and clones was grown at three sites infested with Globodera pallida in each of four years (1983 – 1986). Comparison of yields from nematicide treated plots with those of untreated plots provided estimates of yield losses due to G. pallida. The proportional yield losses were calculated for each genotype at each site as a measure of nematode tolerance. There were significant differences between genotypes with regard to tolerance of damage by G. pallida. There were also significant interactions between the different genotypes and the sites and years, but the interactions between years and genotypes represented only a small proportion of the observed variation. The interactions between sites and years indicate that not all of the genotypes behaved consistently between sites and years. The extent of yield losses caused by the nematodes varied significantly between sites. The implications of these results are discussed in relation to potato breeding programmes and assessing resistance and tolerance to G. pallida.