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.     

The influence of cyst nematodes and drought on potato growth

In two experiments in the Wageningen Rhizolab with potato cv. Mentor planted in soils with or without potato cyst nematodes (Globodera pallida, W) the number of roots per cm² was observed two weekly by video camera, in horizontally placed minirhizotrons at depths varying from 5 to 100 cm. In both experiments initial root growth was more rapid in the top soil of the uninfected soil. In the first experiment under optimal water supply, root formation continued longer in the top 30 cm of infested soil leading to twice as many roots at the end of the growing season as in uninfested soil. In the subsoil from 30 to 100 cm, however, root formation was strongly reduced by cyst nematodes leading to an uneven distribution of roots throughout the profile. In the second experiment potato cyst nematodes only increased rooting in the top soil with reduced irrigation. Potato cyst nematodes did not affect the water use efficiency of the crop whereas reduced irrigation increased water use efficiency by about 22%. Without potato cyst nematodes the soil profile was depleted of mineral nitrogen until a depth of 1 m whereas with high initial population densities no nitrogen was taken up in the subsoil between 30 and 100 cm. The spational heterogenity of roots and nitrogen in the soil is an important mechanism of damage. This finding may lead to improved cultural practices and breeding for tolerance.     

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.     

Identification and management of virulence genes in potato cyst nematodes

Field inoculation of leek with zoospores ofPhytophthora prorri resulted in high infection within a short time. Inoculation with infected leaf tissue resulted in a more gradual increase of disease incidence. Inoculation with oospores was relatively unsuccessful. Zoospores were produced in Petri-dishes by treating fast-growing, young mycelium with a diluted soil extract for at least 2 days, followed by a cold treatment in sterile demineralized water. The successful methods can be used for evaluation of resistance or fungicide performance, and for epidemiological experiments.     

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.     

Assessment of partial resistance of potato to, and pathotype and virulence differences in, potato cyst nematodes

Results are reported for several international collaborative experiments which examined methods of assessing degrees of partial resistance in potato and virulence in potato cyst nematode (PCN, Globodera pallida). It was demonstrated that absolute rates of multiplication can be extremely variable on both susceptible and partially resistant clones, even when the same population and test procedures are employed. It was therefore concluded that, on clones with quantitatively inherited resistance (i.e. partially resistant), absolute rates of multiplication cannot be used to separate pathotypes. Expression of these rates as percentages of those on the susceptible controls reduced the absolute differences between tests, but the values obtained were still too variable for statutory use. However, whatever the environment or nematode population used, it was observed that the resistance of the test clones and virulence of the nematode populations were generally ranked in a similar order. The main exceptions to this were: (1) a Petri-dish test where cv. Vantage was less resistant than in canisters or pots and (2) a pot test where cv. Darwina tended to be more interactive with environmental factors than the other test clones. It was also observed that with some populations of PCN in pots the susceptible cv. Bintje was a less good host than cv. Désirée. On the basis of these results it is suggested that certain partially resistant clones should be used as internal references in statutory, recommended list and breeders' assessment tests against which the resistance of the test clones are compared. For international comparability it is necessary that the different centres conducting such tests use the same reference clones and nematode populations, and similar test methods.     

Dispersal of potato cyst nematodes measured using historical and spatial statistical analyses

Rates and modes of dispersal of potato cyst nematodes (PCNs) were investigated. Analysis of records from eight countries suggested that PCNs spread a mean distance of 5.3 km/year radially from the site of first detection, and spread 212 km over ≈40 years before detection. Data from four countries with more detailed histories of invasion were analyzed further, using distance from first detection, distance from previous detection, distance from nearest detection, straight line distance, and road distance. Linear distance from first detection was significantly related to the time since the first detection. Estimated rate of spread was 5.7 km/year, and did not differ statistically between countries. Time between the first detection and estimated introduction date varied between 0 and 20 years, and differed among countries. Road distances from nearest and first detection were statistically significantly related to time, and gave slightly higher estimates for rate of spread of 6.0 and 7.9 km/year, respectively. These results indicate that the original site of introduction of PCNs may act as a source for subsequent spread and that this may occur at a relatively constant rate over time regardless of whether this distance is measured by road or by a straight line. The implications of this constant radial rate of dispersal for biosecurity and pest management are discussed, along with the effects of control strategies.     

A rapid, sensitive, and cost-efficient assay to estimate viability of potato cyst nematodes

Potato cyst nematodes (PCNs) are quarantine organisms, and they belong to the economically most relevant pathogens of potato worldwide. Methodologies to assess the viability of their cysts, which can contain 200 to 500 eggs protected by the hardened cuticle of a dead female, are either time and labor intensive or lack robustness. We present a robust and cost-efficient viability assay based on loss of membrane integrity upon death. This assay uses trehalose, a disaccharide present at a high concentration in the perivitelline fluid of PCN eggs, as a viability marker. Although this assay can detect a single viable egg, the limit of detection for regular field samples was higher, ≈10 viable eggs, due to background signals produced by other soil components. On the basis of 30 nonviable PCN samples from The Netherlands, a threshold level was defined (ΔA(trehalose) = 0.0094) below which the presence of >10 viable eggs is highly unlikely (true for ≈99.7% of the observations). This assay can easily be combined with a subsequent DNA-based species determination. The presence of trehalose is a general phenomenon among cyst nematodes; therefore, this method can probably be used for (for example) soybean, sugar beet, and cereal cyst nematodes as well.     

Foliar calcium concentration of potato and its relation to genotype lateness and tolerance of cyst nematodes

In field trials during three years respectively 18, 22 and 57 potato genotypes were grown on soils moderately or heavily infested with potato cyst nematodes (Globodera pallida) after soils were fumigated or not. Nematode infection increased leaf calcium contents but genotypes that were relatively tolerant of potato cyst nematodes (Globodera pallida) had lower leaf calcium concentrations on a particular sampling date. Tolerance of potato cyst nematodes was closely linked to genotype lateness and may be related to tolerance of drought. When using the method in plant breeding and screening for genotypes with tolerance, plants may be planted in infected or in uninfected soils, without influencing the outcome. The relationship between tolerance and calcium contents was clearest when sampling took place towards the end of the growing season when the variability was at it highest with a good distinction between newly formed leaves in late maturing genotypes and aged leaves in early maturing cultivars.     

Environmental differences and their effect on the assessment of quantitative resistance to potato cyst nematodes1

Experiments assessing the quantitative resistance of potato clones to potato cyst nematodes tend to produce variable results because of genotype-environment interactions. Despite these interactions clones are ranked in the same way even when a major environmental factor such as initial nematode population density is changed. It is also argued that as the reproductive rate is so environmentally sensitive the use of fixed levels of nematode multiplication rate as criteria for classifying clones is inappropriate. It is proposed that standard resistant and partially resistant clones be included in all assessment tests. They would act as delineators of resistance classes and would allow easy comparisons to be made between tests whether conducted in glasshouse or field.     

Co-evolution of potato cyst nematodes and their hosts: implications for pathotypes and resistance1

The co-evolutionary process is believed to have resulted, through interaction of wild populations of potato cyst nematodes and their hosts in geological time, in the resistant hosts now utilized in plant breeding programmes and in nematode populations with genes for resistance-breaking or virulence. It is argued that all such interactions between highly adapted, truly parasitic plant nematodes and their hosts are likely to be governed by gene-for-gene interrelationships. Practical implications of this hypothesis are that only pathotypes (resistance-breaking races) defined against identified resistance genes are scientifically sound and of practical value; that, in the case of potato cyst nematodes, other pathotypes (Ro2, Ro3, Ro5 and Pa2 and Pa3) should be abandoned; and that oligogenically based resistance to potato cyst nematodes, especially important in providing resistance to Globodera pallida, is non-durable. Working definitions of the terms ‘pathotype’, ‘host-race’ and ‘virulence’ are provided.     

The influence of cyst nematodes and drought on potato growth. 3. Effects on carbon isotope fractionation

A good agreement was found between drought induced water-use efficiencies and carbon isotope fractionation of potato plants of the same age. Discrimination of¹³Carbon isotope increased with plant age and with the rank order of the distinguished plant organs. In young plants, nematode infection (contrary to drought) reduced¹³C discrimination but also reduced the water-use efficiency, leading to the conclusion that nematode infection, by slowing down the plant metabolism may have reduced the extrusion of¹³C. Towards senescence both nematode infection and drought reduced¹³C discrimination and increased water-use efficiencies indicating that then nematode caused water stress in the plants.     

The effect of physiological age of potato tubers on female production of potato cyst nematodes (Globodera pallida Pa 2/3)

Fourteen cultivars generally regarded as uniformly susceptible toGlobodera pallida Pa 2/3 were assessed in plastic closed containers for degree of susceptibility and also for root and sprouting characteristics. The material was assessed by scoring degree of sprouting before planting and number of cysts and root growth at the end of experiment at intervals from the time of initial dormancy break through to prolific sprouting. Using covariate analyses, it was found that the degree of root development, particularly in the earlier assessments, significantly influenced the degree of cyst development and so the perceived levels of susceptibility/resistance. Having taken account of root growth characters, significant differences between the cultivars with regard to nematode multiplication were still evident. The implications of the results are discussed in relation to potato breeding and nematological studies.     

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.     

The use of associations of nematode species to aid the detection of small numbers of virus–vector nematodes

Information from a large data bank of the most commonly occurring plant–parasitic nematodes within the British Isles was used to assess the frequency of joint occurrences of pairs of species. Analysis of the results showed that many of the virus–vector species had significant positive relationships with non–vector species, while only a few significant negative relationships were observed. A comprehensive table of joint occurrences enables the likely presence of virus–vector species to be predicted from the presence or absence of another species in a soil sample. This information is particularly valuable when the detection of very small numbers of virus–vector nematodes is required.     

Assessing reproduction of potato cyst nematodes (Globodera rostochiensis and G. pallida) on potato cultivars for National Listing1

To investigate testing procedures for assessing the reproduction of potato cyst nematodes (PCN) on potato cultivars, and to minimize variation in results, trials were done jointly for 3 years at two UK National Listing test centres (DANI in Northern Ireland, DAFS in Scotland). Test clones comprised susceptible, resistant and ‘partially resistant’ cultivars. The factors under investigation included the effect of season, site, location, temperature, PCN population, inoculation method, pot size, hatch rates and methods of assessment of reproduction. Standard procedures are recommended for testing potato cultivars for resistance to PCN.     

The influence of cyst nematodes and drought on potato growth. 1. Effects on plant growth under semi-controlled conditions

Potatoes were grown under a permanent rain shelter in mobile containers in soil with or without potato cyst nematodes (Globodera pallida). The plants were subjected to an early drought stress period starting at planting until 43 days after planting, to a late drought stress period starting at 43 days until senescence at 92 days and to a drought control. Dry matter weight and characteristics of leaves, stems, stolons and roots were determined at periodic harvests. The early drought stress and nematode infection affected many plant organ characteristics in similar ways. Numbers of leaves, specific leaf area, plant height, specific stem weight, leaf area ratio, mean tuber weight and harvest index were reduced by both stress factors at early stages of growth.Later on, interactions between both stress factors which influence the development rate of the plants led to more diverse plant reactions. Plants of all treatments rapidly senesced at about 90 days after planting. Uninfected plants had then depleted the soil nutrient supply whereas the plants grown in the inoculated soil senesced as a result of the potato cyst nematode infection.     

Resistance of potato varieties to field populations of the potato cyst nematode Globodera pallida in the northeastern part of the Netherlands

In the northeastern part of the Netherlands starch potatoes are grown intensively. From 392 fields in that area, soil samples that contained the potato cyst nematodeGlobodera pallida were used to analyze the resistance of 7 varieties of starch potatoes.Differences and similarities in resistance of the varieties to the field populations ofG. pallida could well be explained by their descent from distinct sources of resistance.TheG. pallida-populations that showed a relatively high virulence to the varieties Producent, Elles, and Darwina were not equally distributed over the area, but were confined to a small number of regions.     

Decline of potato cyst nematodes, Globodera rostochiensis and G. pallida, in spring barley microplots

In microplots of nine soils freed from volunteer potatoes and sown for four consecutive years with spring barley, the eggs of five populations of Globodera pallida and four populations of G. rostochiensis declined at annual rates of 12-8-40-5%. One population of G. rostochiensis declined faster than other populations of either species, although average rates of decline did not differ significantly between the two species of Globodera. These results are compared with earlier research and their relevance to control of the nematodes by crop rotation is discussed.     

大豆种质资源对大豆孢囊线虫的抗性评价

 大豆孢囊线虫(Heterodera glycines Ichinohe,SCN)是大豆(Glycine max (L.) Merrill)生产中的毁灭性害虫,种植抗性品种是控制其危害、减少产量损失的最佳途径。本研究通过温室盆栽实验评价了300份大豆种质对大豆孢囊线虫3号和4号生理小种的抗性。分别筛选到高抗和中抗3号生理小种的大豆种质27份和21份;高抗和中抗4号生理小种的大豆种质11份和9份。在所有供试材料中有10份材料同时对大豆孢囊线虫3号和4号生理小种表现高抗。线虫侵染实验表明,抗性材料对大豆孢囊线虫的发育有阻碍作用,并能显著降低最终形成的孢囊数。