Consistent action of two partially effective loci conferring resistance to Globodera pallida Pa2/3 across multiple nematode field populations

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are significant pests of potatoes worldwide. The most effective control methods are crop rotation and the deployment of resistant varieties. Complete resistance to G. rostochiensis based on a single resistance gene has successfully been integrated into many varieties. However, resistance to G. pallida has not been as successful to date, with current varieties only exhibiting partial resistance. Combining partially effective quantitative trait loci (QTLs) for resistance can increase the strength and breadth of the resistance. An additive effect on resistance has previously been demonstrated on combining two QTLs from Solanum tuberosum subsp. andigena (GpaIV^s_adg) and Solanum vernei (Gpa5). However, populations of G. pallida can be quite divergent and it was unclear whether the relative effects of the individual QTLs and the combined additive effect would be consistent across different G. pallida Pa2/3 populations. Using a mapping population segregating for both QTLs, the effect of the QTLs individually and combined was examined on four UK‐derived field populations of G. pallida pathotype Pa2/3, and the relative effects of the individual QTLs and the additive effect of the combination found to be consistent across all populations.     

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.     

Evaluation of PCR,IEF and ELISA techniques for the detection and identification of potato cyst nematodes from field soil samples in England and Wales

Effective management of potato cyst nematodes (PCNs) requires simple, rapid and accurate identification and quantification of field populations. Soil samples from a survey of 484 fields in potato rotations in England and Wales were used to compare the identification and quantification of PCNs using IEF, PCR, ELISA and bait plant tests. The cyst counts and bait plant test revealed that 64.3% of field samples contained PCNs. Bait plant tests increased the detection rate of PCNs in field samples by 4–6.4%. This means that some infestations are cryptic and would not normally be detected by standard counts. IEF, PCR and ELISA methods distinguished between Globodera rostochiensis and G pallida and were able to register mixed populations; however they were not in full agreement. All methods suggested that G pallida is the dominant species in the field samples tested. The PCR results indicated that 66% of field samples contained pure G pallida, 8% contained pure G rostochiensis and 26% contained mixtures of the two species. Estimates of the relative process times taken per sample in the PCR, IEF and ELISA techniques are given. © 2001 Society of Chemical Industry     

Interactions Between the Potato Cyst Nematode Globodera rostochiensis and Diseases Caused by Rhizoctonia solani AG3 in Potatoes Under Field Conditions

Findings from 2 years of field experiments investigating the relationship between Globodera rostochiensis and Rhizoctonia solani on unique field sites are reported. In 2000, a field experiment was positioned on land that had previously been used for experimental work investigating integrated potato cyst nematode (PCN) management methods. This study had produced an ‘untypical’ mosaic of PCN population densities ranging from 5 to 221 eggs g⁻¹ soil. In 2001, the field experiment was conducted on a different field site and overlaid on a focus of G. rostochiensis population densities ranging from 11 to 108 eggs g⁻¹ soil. In each experiment, potatoes (cv. Désirée) were grown in plots with similar population densities of G. rostochiensis that were either uninoculated or inoculated with R. solani. A series of potato plant harvests were undertaken to investigate the effects of nematode infestation on the incidence and severity of R. solani diseases and the associated development of plants. In both experiments, a clear relationship was found between the density of G. rostochiensis juveniles present in potato roots and the incidence of stolons infected by R. solani, 6 weeks after planting. For the first time this interaction has been determined under field conditions. The results of the study suggest that the interaction between nematode and fungus is indirect and possible mechanisms are discussed.     

Managing potato cyst nematodes to minimize nematicide use1

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are widespread in the ware potato-growing areas of the UK. Traditionally they were controlled by rotation but more intensive production methods have increased the PCN threat, especially from G. pallida. G. rosrochiensis has become less important since cultivars fully resistant to it were introduced but, where both species are present in a mixture, the G. pallida portion comes to dominate. It is possible to control G. pallida as effectively as G. rostochiensis if cultivars partially resistant to G. pallida are grown with nematicide treatment. Where nematicide is not used, control of G. pallida is more variable. In some years, some cultivars achieve good control but the results are not consistent. In general, G. pallida has increased on untreated plots about 5-fold in trials in the last 3 years.     

Morphological and molecular identification of potato and cereal cyst nematode isolates from Algeria and their phylogenetic relationships with other populations from distant theirgeographical areas

A nematode survey conducted in 2013 in Algeria, revealed that potato cyst nematodes (PCN) and cereal cyst nematodes (CCN) are widely distributed in several potato and cereal growing regions of the country. Sixteen PCN populations from five localities and five CCN populations from four of these localities were collected and characterized at the morphological and molecular levels. The PCN populations were identified as Globodera rostochiensis and G. pallida occurring separately or in mixed populations. Two species of CCN were detected. Heterodera avenae was found in four localities, whereas H. hordecalis only in one locality in association with H. avenae. The morphological and morphometric identification of PCN and CCN was confirmed by diagnostic ITS-RFLP profiles and sequencing. Phylogenetic analysis of the ITS, D2-D3 expansion domains of the 28S rRNA gene and 18S rRNA gene was made for PCN and CCN populations. Globodera pallida and G. rostochiensis from Algeria show great similarity with European and South American populations. Because of the high divergence among Algerian populations of G. pallida and G. rostochiensis it can be assumed that they were multi-introduced in Algeria. The most divergent population of G. pallida, that formed a well-separated group with some populations from Chile and Peru, suggests a later or independent introduction of this population into Algeria. Heterodera avenae and H. hordecalis formed a well-supported cluster with the corresponding populations.     

The Basis of Predictive Modelling for Estimating Yield Loss and Planning Potato Cyst Nematode Management

The basis of modelling yield loss and population dynamics relations of potato cyst nematodes is that both are strongly density-dependent. Potato cyst nematodes (PCN; Globodera pallida and G. rostochiensis) are particularly suitable for analysing such relationships because they have only one generation per year, potato is their only field host, the juvenile nematodes within the egg are very durable (up to 20 years persistence), and they hatch mainly in response to specific chemicals exuded from host roots. Small populations increase the most, up to 50-fold, when a potato crop is grown. Multiplication rates decrease as the population density increases because damage decreases root system size and increases competition so that very large populations may actually be decreased when potatoes are grown. The newly formed eggs have a ‘half-life’ of c.two years when non-host, rotational crops are being grown. Control is achieved largely by the use of rotation, the application of nematicidal chemicals, and growing resistant cultivars. As rotations are shortened, so PCN populations will be increased, and crop damage becomes more likely. Thresholds for damage vary with both soil type (greatest on sandy soils) and potato cultivar. Cultivars differ in their tolerance of PCN damage depending on how vigorously they grow, on their root sensitivity to damage from the PCN juveniles which invade close to root tips, on cultivar resistance which decreases the parasitic effect by reducing the numbers of developing PCN females, and on various environmental factors such as the amounts of fertiliser applied. The dependence of the yield-loss relationship on population density, soil type and cultivar effects has been described in a simple equation and assessed using field-trial data. Effects on yield are described in proportion to the PCN-free yield but the addition of information on expected yield (in tonnes ha⁻¹) in the absence of PCN renders this equation predictive. Nematicides are widely applied to infestations of potato cyst nematodes, both to prevent the crop from being damaged and to prevent population increase which could hazard the next potato crop in the rotation, but they are generally more effective at preventing the former than the latter. A complex equation has also been developed to model the population dynamics of PCN. This equation incorporates a factor for host-crop growth and tolerance (from the yield-loss equation) and also the effects of host resistance. This latter is particularly relevant to G. pallida, where all the resistance currently available is determined by minor genes and hence is ‘quantitative’ or ‘partial’. Effects of rotation and of nematicides can also be incorporated into this model. To provide a realistic prediction also requires accurate information on PCN population densities, species composition and distributions, and rates of PCN population decline between potato crops.     

The use of isoelectric focusing as a tool in the identification and management of potato cyst nematode populations1

Isoelectric focusing on thin layers of polyacrylamide or agarose gels may be used to separate proteins from the potato cyst nematodes Globodera rostochiensis and G. pallida. General protein patterns may be used to identify the two species, even from single cysts. Densitometry of species-specific protein bands may be used to assess the proportions of G. rostochiensis and G. pallida in potato cyst nematode samples. Staining for the enzymes phosphoglucomutase and phosphoglucose isomerase also revealed species-specific patterns, and variation among G. pallida populations was observed. Thus calculations of coefficients of similarity based on six enzymes, phosphoglucose isomerase, phosphoglucomutase, glycerol-3-phosphate dehydrogenase, enolase, hexokinase and malate dehydrogenase and the construction of a dendrogram for several Northern Ireland populations indicated clear separations between G. pallida (Pa1), G. pallida (Pa3) and G. rostochiensis (Ro1). The use of the technique of isoelectric focusing as a routine research and advisory tool in nematology, and as a means of further understanding the genetic basis of pathotype schemes, is discussed.     

Hsp90 gene, an additional target for discrimination between the potato cyst nematodes, Globodera rostochiensis and G. pallida, and the related species, G. tabacum tabacum

The heat-shock gene, Hsp90, was targeted as a new variable genomic region to supplement other DNA-based tests for identification and discrimination of Globodera pallida, G. rostochiensis and G. tabacum tabacum. Populations of the potato cyst nematodes, G. pallida and G. rostochiensis (PCN), originating from Canada, France, Belgium and USA, together with two populations of G. tabacum tabacum from the USA and France were used for the amplification of a fragment of the Hsp90 gene. General and specific primers and probes for each species were derived from the consensus and non-consensus regions of the aligned sequences, respectively. A triplex conventional PCR assay, using a general forward and reverse or three specific reverse primers, as well as a real-time PCR using general primers and specific TaqMan probes, were developed. Melting curve analysis and restriction fragment polymorphisms using high resolution electrophoresis were explored for identifying PCR amplicons that characterized and discriminated the three Globodera species in both pure and mixed samples. Results from the different molecular assay strategies confirmed the usefulness of Hsp90 as a new additional gene target and showed that several different test options could be used for discrimination of PCN.     

Identification of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> and <Emphasis Type="Italic">G. pallida</Emphasis> in the Ukraine by PCR

Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples from the Ukraine. The results show the occurrence of Globodera pallida in the Uzhhorod region (Zakarpatska oblast), where only G. rostochiensis had been previously reported. In the mixed potato cyst nematode (PCN) populations, G. pallida was less prevalent (2–5%) than G. rostochiensis (95–98%). A phylogenetic analysis based on ribosomal DNA internal transcribed spacer sequences showed that the Ukrainian population of G. pallida had >99% sequence identity with other G. pallida pa2/3 isolates from Europe. This study has demonstrated that polymerase chain reaction-mediated amplification of specific regions of the potato cyst nematode genome is not only highly effective as a species diagnostic tool but is also a sensitive method which can be used for taxonomic purposes with cyst collections which vary in age.     

Spatial analysis of blackleg‐affected seed potato crops in Scotland

Potato blackleg, caused by Pectobacterium and Dickeya species, is one of the most significant bacterial diseases affecting potato production globally. Although it is generally accepted to be a seedborne disease, the processes underlying the spread of disease largely remain unknown. Spatial point pattern analysis was applied to blackleg occurrence in seed potato crops in Scotland during the period of 2010–2013 (approximately 8000 blackleg‐affected crops), to assess whether its distribution was random, regular or aggregated, and the spatial scales at which these patterns occurred. Blackleg‐affected crops derived from mother stocks with symptoms were omitted from the analyses in order to examine the statistical evidence for horizontal transmission of blackleg. The pair correlation function was used to test for global spatial autocorrelation, and results indicated significant (P < 0·05) clustering of incidence at a wide range of spatial scales. Strength of clustering (degree of aggregation) among blackleg‐affected crops was notably larger at spatial scales of 25 km or less. A hot‐ and coldspot analysis was performed to test for local spatial autocorrelation, and statistically significant clusters of high and low values of disease were found across the country. These analyses provide the first quantitative evidence of localized and large‐scale spatial clustering of potato blackleg. Understanding the mode(s) of inoculum dispersal will be important for developing new management strategies that minimize host–pathogen contacts in potato and numerous other crops affected by pathogenic Pectobacterium and Dickeya species.     

Selection of nematodes by resistant plants has implications for local adaptation and cross‐virulence

The variability of resistance durability in different potato genotypes harbouring the same resistance QTL but differing by their genetic background was explored. The indirect consequences of the resistance adaptation in terms of local (i.e. genotype‐specific) adaptation and cross‐virulence was also investigated. Following the virulence of the potato cyst nematode Globodera pallida in a long‐term experimental evolution protocol, the results showed that nematode populations were able to adapt to the resistance of four potato genotypes carrying the QTL GpaV from Solanum vernei, and that the plant genetic background has an impact upon the durability of resistance. The pattern of local adaptation observed here indicates that divergent selection has occurred during the experimental evolution performed from the same initial nematode population, and revealed a trade‐off between the adaptation to a resistant potato genotype and the adaptation to another resistant genotype differing in its genetic background. In terms of cross‐virulence between potato genotypes derived from different resistance sources (S. sparsipilum and S. spegazzinii), this study shows that the adaptation to resistance QTL GpaV_vrn does not necessarily allow the adaptation to collinear GpaV loci. The results presented here could be useful for predicting evolution of nematode populations in natural agro‐ecosystems and identifying durable strategies for resistance deployment.     

Differential hatching of the potato cyst nematodes Globodera rostochiensis and G. pallida in root diffusates and water of differing ionic composition

The hatching differences ofGlobodera rostochiensis andG. pallida were assessed in potato root diffusate (PRD) of cv. Bintje, cv. Elkana and clone ZB35-29.G. pallida hatched better in the PRDs thanG. rostochiensis. It was shown that the experimental test conditions strongly influenced the hatching results. The water type used in the hatching tests had a significant discriminating effect on the species;G. rostochiensis reached hatch percentages of 60 to 90% in demineralized and tap water, whereasG. pallida never exceeded the 15%. These differences were independent of the various batches that were used or the different years the tests were carried out. Silver sand percolate had a inhibiting effect on the hatching of both nematode species. Boron and a high electrical conductivity may be responsible for this. The results are discussed from an ecological point of view as well as for research consequences.     

Potato cyst nematodes in the Canary Islands: an epidemiologic model for the Mediterranean region1

This work describes studies done on the Canary Islands in order to support the elaboration of quarantine and control legislation for potato cyst nematodes which take into account the special agroecological characteristics of the Mediterranean region. The interest of the islands for epidemiological studies is based on the occurrence of the oldest potato crops in the Old World and the use of several local cultivars and traditional management cropping systems. Also, the environmental conditions allow field experiments to be performed throughout the year. The phytonematological problems of the potato crop on the Canary Islands is reported and the absence of Ditylenchus destructor, D. dipsaci, Nacobbus aberrans, Rotylenchulus reniformis and trichodorids pointed out. The cyst nematodes, Globodera rostochiensis and G. pallida were found to cause severe damage to the crop and were studied in detail by field trials at Tenerife. Continuous growing of the cv. Cara, with the H1 gene conferring resistance to G. rostochiensis (pathotypes Ro1 and Ro4), exerted selection pressure for G. pallida. Under the edaphic and climatic conditions of Tenerife, nematicide application was found to be inefficient for controlling potato cyst nematodes. Alternative control techniques based on traditional management systems and on the use of environmentally adverse conditions were investigated.     

Plant parasitic nematodes as pests of potatoes in Poland1

Only two plant parasitic nematode species have practical importance in potato crops in Poland. They are Ditylenchus destructor and Globodera rostochiensis pathotype Ro1. Control of D. destructor is done permanently by the elimination of infested seed potatoes and sowing of cereals in the infested fields. Damage to potatoes is observed rather seldom. G. rostochiensis is widely distributed throughout Poland. It is controlled by non-host crops and by resistant potato cultivars. Chemical control of potato nematodes is not put into practice in Poland.     

Identification and management of virulence genes in potato cyst nematodes

Efficient and accurate diagnostic assays are essential for the design and evaluation of control measures of the potato cyst nematodesGlobodera rostochiensis andG. pallida by means of resistance. The hybridoma technology and the polymerase chain reaction (PCR) offer in potential various possibilities to design such diagnostic tests for routine purposes. We set out to devise a refined advisory system based on biochemical assays by using the following stepwise approach.In the early 80's a research program was started to develop an immunoassay to differentiate the two sibling species of potato cyst nematodes. Species specific monoclonal antibodies were raised against nematode proteins which are thermostable, abundant and homologous, and which enable reliable species identification using single eggs.     

A biotechnological strategy involving monoclonal antibodies for improvement of potato farming by identification and quantification of potato cyst nematodes in soil samples1

The two closely related nematode species Globodera rostochiensis and G. pallida are one of the major problems encountered in potato cultivation. There is a spectrum of potato plant genes known, which confer resistance to these species and their pathotypes. Potato growing in The Netherlands has to follow strict rules to control spread of the pests. Since distinction between the two nematode species is difficult, a rapid and reliable identification method is needed to allow better use of existing and forthcoming resistant potato cultivars. The aims of this project were: (1) identification and partial purification of species-specific proteins from the nematodes, (2) production of species-specific monoclonal antibodies, and (3) development of a screening test for qualitative and quantitative determination of Globodera spp. in soil samples.     

Quantification of Globodera rostochiensis and G. pallida in mixed populations using species-specific thermostable proteins

A method has been developed to quantify species ratios in mixed populations. The method is based on the separation of species-specific thermostable proteins by SDS-PAGE. Densitometric analyses of the 17 kD protein ofGlobodera pallida and the 18 kD protein ofG. rostochiensis revealed a high correlation (R ²=0.93) with the species ratio in the mixed samples. Within the limits of 10 to 90% of each species, one can estimate with 95% reliability the species composition with 3 to 6% deviation.Samenvatting Een methode is ontwikkeld om de samenstelling van soortenmengsels vanGlobodera rostochiensis enG. pallida te kwantificeren. Bij deze methode wordt gebruik gemaakt van de soort-specifieke thermostabiele eiwitten die met behulp van SDS-PAGE gescheiden worden. De kleurintensiteit van het 17 kD eiwit vanG. pallida en het 18 kD eiwit vanG. rostochiensis is per gel-laan bepaald m.b.v. een densitometer en heeft een lineair verband met de soortsverhouding in de mengsels (R ²=0.93). Binnen het bereik van 10 tot 90% van elke soort kan men met deze ijklijn met 95% betrouwbaarheid de soortsamenstelling bepalen op 3 tot 6% nauwkeurig.     

Modifications in the potato rhizosphere during infestations of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> and subsequent effects on the growth of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Two controlled environment experiments were conducted to explore the hypothesis that invasion and damage caused to potato roots by the potato cyst nematode Globodera rostochiensis might result in quantitative or qualitative changes in the release of root exudates to subsequently affect the growth of Rhizoctonia solani (AG3) in the potato rhizosphere. The growth of five R. solani isolates was compared on media amended either with root exudates from G. rostochiensis-infested or uninfested potato (cv. Désirée) plants at different time intervals after the introduction of the nematodes. In Experiment 1, the growth of R. solani was higher on medium amended with potato root exudates from G. rostochiensis-infested compared to uninfested plants, collected 4, 6, 8 and 12 days after the G. rostochiensis treatments were administered. Similarly, in Experiment 2, R. solani isolates grew faster on medium amended with potato root exudates from G. rostochiensis-infested than uninfested plants. This trend was particularly pronounced at the 12-day collection. At this time, 49% of the G. rostochiensis juveniles in roots were found to belong to the juvenile moults J2 and J3, indicating that root exudates were modified during the earlier stages of juvenile invasion. Carbohydrate analysis of root exudates indicated significantly higher levels of sucrose in root exudates from G. rostochiensis-infested than uninfested plants, whereas no significant differences were found in total nitrogen content. The results are discussed to help elucidate the mechanism behind the disease complex found between G. rostochiensis and R. solani in previous field research.     

SSR assessment of Phytophthora infestans populations on tomato and potato in British gardens demonstrates high diversity but no evidence for host specialization

Phytophthora infestans populations can differ in composition as a result of host specialization on tomato and potato hosts. In Great Britain many amateur gardeners grow outdoor tomatoes but there is little or no commercial tomato production outdoors. This study analysed isolates of P. infestans from British gardens with 12 multiplexed simple sequence repeat markers that are used to monitor the disease on commercial potato crops. Samples of P. infestans from tomato hosts were collected in 3 years and from potato in 1 year from across Great Britain. Seven previously unreported clonal lineages were detected in garden populations and higher frequencies of unique clonal lineages (28–40%) were present compared with populations from British commercial potato crops reported elsewhere. Garden populations had a lower proportion (11–48% less) of the most common lineages (13_A2 and 6_A1) that together made up at least 86% of the commercial potato populations during the sampling period. Host species accounted for only 2·0% of molecular variance detected between garden potato‐ and tomato‐hosted samples. No significant difference in clonal lineage composition was found between host species in Great Britain and this could be due to the whole P. infestans population overwintering on potato. British garden populations on both hosts were much more diverse than those on commercial potato crops; this finding may be influenced by less frequent fungicide use by gardeners and a higher diversity of unsprayed susceptible potato cultivars, enabling metalaxyl‐sensitive and less aggressive genotypes to survive in gardens.