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.     

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

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.     

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.     

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.     

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.     

马铃薯品种对枯萎病菌的抗性鉴定

通过室内接种试验与田间接种试验,调查评价供试马铃薯品种对枯萎病菌的抗性。结果显示,供试的马铃薯品种中没有免疫品种,脱毒组培苗接种病原菌测定的19个品种中,表现为中抗的品种有2个,占供试品种的10.53%;表现为中感的品种有10个,占供试品种的52.63%;表现为高感的品种7个,占供试品种的36.84%。田间接种病原菌测定的20个品种中,中抗及以上品种有9个,占供试品种的45%,中感及高感品种11个,占供试品种的55%。     

Effect of sample size on cyst recovery by flotation methods: Recommendations for sample processing during EU monitoring of potato cyst nematodes (Globodera spp.)

Under EC Council Directive 2007/33/EC member states of the European Union are recommended to take soil samples ranging from 100 mL to >1500 mL to monitor populations of potato cyst nematodes [PCN] ( Globodera spp.) in targeted fields. This study examines the effects of varying sample size on sample processing and cyst extraction using two widely used laboratory methods. Cyst recovery was stable using the Fenwick can from 100 mL up to the physical limits of the can. Recovery was significantly lower for low density 50 mL samples; however, this was partly due to a statistical artefact since higher numbers of cysts were lost from successively larger samples with constant cyst numbers (i.e. decreasing densities). The Schuiling centrifuge had functional limits of <100 mL and >500 mL. Outside this range, cyst recovery from low density samples was significantly reduced. Cyst recovery from samples of varying size, but with equal numbers of cysts was only constant above 100 mL. However, samples ranging from 200–500 mL were optimal for the Schuiling centrifuge. Cyst extraction efficiency was similar using both methods for samples ranging from 150 mL to 1500 mL, where larger samples were divided into <500 mL portions. However, processing times (i.e. cyst extraction and microscopic examination) and water consumption increased significantly with increasing sample size and were significantly higher when using the Fenwick can.     

Detection of viable potato cyst nematodes (Globodera rostochiensis and G. pallida) by reverse transcription PCR

Journal of Plant Diseases and Protection - Currently plant infectivity assays (bioassays) seem most reliable to determine viability of potato cyst nematodes (Globodera rostochiensis and G....     

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.     

Experimentally evolved populations of the potato cyst nematode Globodera pallida allow the targeting of genomic footprints of selection due to host adaptation

In the current agronomical context of pesticide use reduction, deciphering the genetic bases of pathogen adaptation to plant defences is of major importance to improve durability of resistance. Indeed, knowledge of virulence gene frequencies in pathogen populations could allow the prediction of resistance durability before deployment. Globodera pallida is a major pest of potato crops for which a promising resistance QTL, GpaV_vrn, has been identified in Solanum vernei. An experimental evolution study, in which G. pallida lineages evolved on resistant or susceptible potato genotypes for up to eight generations, previously showed that G. pallida was able to rapidly overcome GpaV_vrn resistance. However, it was not known if enough genetic mixing occurred in these lineages to be able to detect islands of differentiation in a genome scan approach. Here, this question was investigated using 53 polymorphic microsatellite markers distributed along the genome and three different tests based on genetic differentiation and heterozygosity. Eight outlier loci were identified, indicative of genomic regions putatively involved in host adaptation. Several loci were identified by multiple detection methods and/or in two independent adapted lineages. Some candidate genomic regions identified also seemed to be involved in overcoming resistance to nematodes in a plant genotype harbouring the same resistance QTL in a different genetic background. These results validate the feasibility of a genome scan approach on biological material coming from short experimental evolution, and encourage the use of a high coverage genome scan by whole genome resequencing.     

Comparison of the Fenwick can and Schuiling centrifuge methods for the extraction of potato cyst nematodes from soil1

An evaluation of the efficacy of the Schuiling centrifuge and traditional Fenwick can methods of extracting Globodera cysts from infested field soils confirmed the accuracy of the Schuiling centrifuge extraction method. Experience has shown that samples with a high organic matter content may reduce extraction efficiency. Improvements in the mechanical and electrical construction of the present Schuiling apparatus are needed, but once they have been made the choice of extraction method in large-scale statutory and advisory programmes need only depend on considerations such as convenience of use and cost-effectiveness.     

宁夏马铃薯主栽品种对黑痣病的抗性鉴定

为明确宁夏马铃薯主栽品种对黑痣病的抗性水平,利用田间和室内接种相结合的方法对供试马铃薯品种的抗病性进行评价。室内接种试验表明,供试的20份马铃薯材料中没有免疫及高抗品种,接种量与抗性水平呈负相关,其中‘陇薯7号’,‘黑美人’,‘青薯9号’在接种量为5~15g水平上表现为中抗。通过聚类分析,接种量为5g和10g时,以欧式距离13作为最佳聚类分割点,供试品种可分为3个类群。田间接种综合评价分析表明,不同品种间抗性存在显著差异,通过聚类分析,以欧式距离13作为最佳聚类分割点,试验品种可分为4个高感品种、11个中感品种和5个中抗品种。     

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.     

The influence of cyst nematodes and drought on potato growth. 2. Effects on plant water relations under semi-controlled conditions

Potatoes were grown under a permanent rain shelter in mobile containers in soil with and without potato cyst nematodes (Globodera pallida). The plants were either subjected to an early drought stress period from planting until 43 days after planting, to a late drought stress period during tuber bulking or to a drought control. Leaf water potentials, stomatal diffusion resistances for water vapour, transpiration rates, dry matter accumulation and water use efficiencies of the plants were determined periodically. Both drought and nematodes decreased leaf water potential and increased stomatal resistance.Drought led to a higher water use efficiency, cyst nematode infection, however, reduced the water use efficiency at early stages of growth, and increased it at later stages. It is concluded that at least two main growth reducing mechanisms exist of which the relative importance varies with time. Firstly, reduced apparent assimilation rates, which are unrelated to a change in the water balance caused by the initial attack by the cyst nematodes. Secondly, a reduced dry matter accumulation resulting from a decrease of water uptake. Effects of drought and cyst nematode infection on plant growth and water relations were not always additive mainly because infected plants used up less water leading to less drought stress.     

Models of the effects of dose heterogeneity and escape on selection pressure for pesticide resistance

ABSTRACT Models of the intensity of selection for resistance resulting from the application of heterogeneous doses of pesticide are developed. Dose heterogeneity is assumed to arise from two factors. First, a proportion of the population completely escapes treatment. Second, the rest of the population receives a distribution of doses that can be described by a probability distribution that is approximately logistic on a logarithmic scale. Various assumptions about the biology of the target population are explored, including both sexual and asexual reproduction of haploid and diploid organisms with either polygenic or monogenic inheritance of resistance. There are two major conclusions. (i) The presence of escape produces a maximum in the graph of selection intensity against dose or control; below the dose corresponding to the maximum, selection is reduced if dose is reduced, but above the maximum, selection is reduced if dose is increased. Informal arguments suggest it is unlikely that field doses would often be above the maximum dose, so selection would usually be decreased by decreasing dose. (ii) For the same control, selection for resistance is reduced by greater heterogeneity of pesticide dose, even though larger average doses may be needed.     

Methods of testing potato cultivars for resistance to soft rot of tubers caused by Erwinia carotovora subsp. atroseptica

The resistance of eight potato cultivars to tuber soft rot caused by E. carotovora subsp. atroseptica was assessed in 2 years using three different test methods. Similar cultivar resistance rankings were obtained for any one method within a year and between years for two methods (single site, infectivity titration), but not for the third (vacuum infiltration). However, the ranking of cultivars differed for the three methods. Ranking was not affected by inoculating the cortex or the more susceptible medullary tissue, or by assessing rotting in terms of infection frequency or lesion size, but it was affected by oxygen concentration during incubation. Differences among cultivars were greater when inoculated tubers were incubated anaerobically than when incubated with 5% oxygen. There was no relationship between the relative susceptibilities of cultivars to tuber soft rot in storage in January/February and those of mother tubers after planting.