Molecular characterization of a morphologically unusual potato cyst nematode*

During routine screening of soil samples for potato cyst nematodes, a morphologically variant cyst was detected. The internal transcribed spacer regions of the ribosomal DNA repeat unit from individual juveniles hatched from the cyst were amplified by PCR. The resulting products were digested with a range of restriction enzymes and their RFLP profiles compared with standard samples of Globodera rostochiensis and Globodera pallida . Most of the RFLP profiles yielded patterns similar to those of G. pallida although some restriction enzymes gave hybrid patterns showing elements of both G. rostochiensis and G. pallida . Clones of individual ITS regions from a single nematode from the variant cyst displayed either a G. pallida or G. rostochiensis pattern.     

Distribution and frequency of occurrence of potato cyst nematode pathotypes in Northern Ireland

The distribution and frequency of occurrence of potato cyst nematodes, Globodera rostochiensis and G. pallida , were examined in Northern Ireland. Globodera rostochiensis (pathotype Rol) was the most abundant species, while Pal and Pa3 pathotype reactions could be discerned within G. pallida. Unlike other parts of the UK, species proportions appear not to have changed over the last 20 years, probably as a result of the legislative control currently practised combined with the limited use of Rol-resistant cultivars within the country. Differences in the geographical distributions of the three pathotypes were observed, and these are discussed in relation to original potato cyst nematode introductions and subsequent agricultural practices.     

警惕检疫性有害生物马铃薯孢囊线虫(Globodera rostochiensis和G. pallida)入侵我国

马铃薯孢囊线虫包括马铃薯金线虫Globodera rostochiensis和马铃薯白线虫G. pallida, 是马铃薯生产上危害最为严重的植物寄生线虫, 一般造成30%的产量损失, 在热带发病严重地区, 产量损失高达80%~90%, 甚至绝收?由于其危害严重性, 包括我国在内的100多个国家将其列为重要检疫性有害生物?我国目前尚无马铃薯金线虫和白线虫的发生报道, 但随着贸易全球化, 马铃薯孢囊线虫传入我国的风险日趋增高?本文主要对马铃薯孢囊线虫的发生分布?危害症状?经济损失?生物学特性?传入我国的潜在风险和预防控制措施进行综述, 旨在为防止马铃薯孢囊线虫入侵我国提供参考?     

The potential value of British wild Solanum spp. as trap crops for potato cyst–nematodes, Globodera rostochiensis and G. pallida

Two forms of Solanum nigrum subsp. nigrum, two forms of S. nigrum subsp. schultesii, S. americanum, S. chenopodioides, S. sarrachoides, S. scabrum, S. villosum subsp. villosum and S. villosum subsp. puniceum were grown in pots for over 4 months to measure their effect on numbers of potato cyst–nematodes, Globodera rostochiensis and G. pallida, in the soil. No Solanum tested significantly reduced numbers of G. rostochiensis or G. pallida. Solanum americanum, S. sarrachoides and S. villosum puniceum increased numbers of G. pallida slightly.     

Morphological and molecular characterization of Globodera populations from Oregon and Idaho

An unusual population of cyst nematode was found in soils collected from a Powell Butte, OR field with a cropping history including potato, wheat, other crops, and significant weed presence. These nematodes could not be placed with certainty into any known species and exhibited some unique morphological features in some specimens. Compared with Globodera pallida, the cyst body length was slightly longer and the second-stage juvenile stylet length was slightly shorter. In some individuals, the J2 stylet knob height was greater and the tail annules were more prominent than in G. pallida, and the tail abruptly narrowed, with a slight constriction near the posterior third of the hyaline terminus. Compared with G. rostochiensis, the hyaline tail terminus had a larger number of refractive bodies, and cysts of this population had a smaller Granek's ratio and fewer cuticular ridges between the anus and vulva. In some individuals, the tail termini of second-stage juveniles were more bluntly pointed, and the stylet knobs were more anteriorly directed with greater height. Unlike G. tabacum, the cyst wall often lacked a network-like pattern and, in some individuals, the juvenile tail terminus distinctly narrowed after a constriction. Molecularly, the population was distinct from G. pallida, G. rostochiensis, and G. tabacum. Multiplex polymerase chain reaction of the internal transcribed spacer (ITS) rDNA region gave results similar to G. tabacum; however, ITS restriction fragment length polymorphism patterns were observed to have individual bands in common with G. rostochiensis and G. pallida. Phylogenetic analysis based on ITS1 and -2 rDNA sequences showed greatest similarity to populations from Argentina and Chile; together, they form a moderately supported clade, distinct from G. rostochiensis, G. tabacum, G. "mexicana," European type G. pallida, and several G. pallida populations from South America.     

马铃薯金线虫的分子检测技术

本研究通过利用随机引物OPK-4对马铃薯金线虫和白线虫及甜菜胞囊线虫进行RAPD-PCR,供试马铃薯金线中5个群体能产生630bp的特异性的片段,将特异性片段进行测序后发现5个不同来源的马铃薯金线虫产生的特异性片段序列完全一致。根据测定的DNA序列设计出特异性探针,可有效地用于马铃薯金线虫的分子检测。     

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.     

A Model for Infestation Foci of Potato Cyst Nematodes Globodera rostochiensis and G. pallida

ABSTRACT Infestation foci of potato cyst nematodes (Globodera rostochiensis and G. pallida) in the provinces of Zeeland, Friesland, Groningen, and Drente of the Netherlands were sampled to validate a model describing infestation foci based on data from Flevoland. Eighty-two fields were presampled to locate infestation foci using a coarse sampling grid (8 x 3 m). Parts of 37 fields containing one or more foci were sampled intensively by extracting at least 1.5 kg of soil per square meter (1.33 x 0.75 m). Forty foci were analyzed for spatial distribution characteristics of cysts using multiple regression with generalized linear models and classical linear models. The results showed that the data from all investigated cropping areas fit well to an exponential model with two parameters, the length and width gradient parameters. Significant differences in these parameter values between cropping areas could not be demonstrated. As both parameters follow a normal distribution, the probability of any combination can be described by a bivariate normal distribution. Gradient parameters were correlated, but significant correlations between these parameters and certain variables such as the nematode species involved, the time interval between sampling and the last potato crop, soil type, cropping frequency, and cyst density in the focus center could not be demonstrated.     

Problems of potato cyst nematode pathotypes in the Czech Republic

During 1983–1989 about 1,200 infested plots in the Czech republic were investigated for the occurrence of new pathotypes of the potato cyst nematodes (PCN) Globoderu rostochiensis and G. pallida. Only eight populations of the pest were suspected of containing another pathotype than Ro1. Glasshouse pot trials were performed with those eight natural populations, two populations of Ro1, 10 potato cultivars resistant to Ro1 and 10 cultivars resistant to more pathotypes, G. pallida included. The multiplication rates (Pf/Pi) showed that four of the PCN populations were probably mixtures, with pathotype Ro1 prevailing. Three populations were suspected of containing G. pallida , but their morphological characters corresponded only with G. rostochiensis. One population corresponded with pathotype Ro5. The extent of the occurrence of pathotypes or virulence groups different from Ro1 can be estimated at 0.3–0.4% of infested plots.     

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....     

A selective survey of potato cyst-nematode pathotypes in Great Britain

One hundred and forty-four samples of potato cyst-nematodes, the majority chosen to contain G. pallida , from England, Wales and Scotland were identified to pathotype. Where G. rostochiensis was present it proved to be pathotype Rol, there being no indication of other pathotypes of this species. Of G. pallida , a minority of samples incorporated pathotype Pa 1; many were pathotype Pa3 but distinction between Pa2 and Pa3 was unsatisfactory in many more. It is concluded that recognition of these latter two pathotypes is of little value. The proposed pathotype New Leake could not be recognized.     

Inhibition of egg hatch of the potato cyst nematode Globodera rostochiensis by chitinase-producing bacteria

Plant-parasitic nematodes are major agronomic pests. Purified commercial chitinase inhibited egg hatch of the potato cyst nematode, Globodera rostochiensis (Ro1) in vitro by up to 70% when compared with an untreated control. A screening strategy was devised to isolate chitinase-producing bacteria from a soil with no documented history of damage due to potato cyst nematodes in the last 30 years and that was cropped with potato cv. Kerr's Pink. Only 137 of 3,200 bacterial isolates tested for chitinase production on chitin agar plates were chitinase-positive (i.e. about 4%). All the chitinase-producing bacteria tested in vitro could reduce the hatch of G. rostochiensis eggs, some by up to 90% compared with the controls. One of these strains, M1-12, was identified as Stenotrophomonas maltophilia and a second strain UP1 was classified as a Chromobacterium sp. based on morphological and biochemical tests. The inoculum level and the incubation time influenced the degree of inhibition of egg hatch of G. rostochiensis by M1-12 and UP1 in vitro. An initial cell density of 10⁶ CFU ml^-1 or greater and an incubation time of two weeks was needed to inhibit egg hatch. The longer UP1 was allowed to act on the eggs of G. rostochiensis the greater the level of inhibition. Strains M1-12 and UP1 also reduced the ability of G. rostochiensis to hatch in soil microcosms planted with potato seed tubers cv. D'sir'e. The inhibition of egg hatch of G. rostochiensis by chitinase-producing bacteria is suggested as a biocontrol strategy for the defence of potato crops from potato cyst nematodes.     

Development and Evaluation of Sampling Methods for Fields with Infestation Foci of Potato Cyst Nematodes (Globodera rostochiensis and G. pallida)

ABSTRACT A computer program, SAMPLE, was developed to evaluate existing and create new sampling methods for the detection of infestation foci of the potato cyst nematode (Globodera rostochiensis and G. pallida). By combining a model for the medium-scale distribution of cysts that provides the expected population densities at each position within the focus and a model for the small-scale distribution within square meters, sampling procedures can be simulated. The importance of the parameters of the two distribution models-the length and width gradient for the medium-scale distribution and the aggregation factor k of the negative binomial distribution for the small-scale distribution-was investigated by sensitivity analysis. Parameter k proved to be less important when calculating the average detection probability of a focus than were the length and width gradient parameters. Several existing versions of the statutory sampling method used in the Netherlands were tested for their performance on a standard infestation focus with a central population density of 50 cysts per kg of soil, which is small enough to use resistant potato cultivars as a control measure without noticeable yield reductions in a 1:3 year cropping frequency with potato. These methods did not perform with the desired average detection probability, set at 90%; therefore, SAMPLE was used to develop several new sampling methods for focus detection. SAMPLE is a tool to develop sampling methods on demand for every possible combination of characteristics required for use by seed and ware potato growers (recommendations for optimum control measures leading to maximum returns and integrated pest management) and by governments (legislation, quarantine, and export protection).     

The Effects of Founding Events and Agricultural Practices on the Genetic Structure of Three Metapopulations of Globodera pallida

To assess the genetic constitution of the Globodera pallida populations in the Netherlands and the effects of agricultural practices, three geographically separated metapopulations, in total consisting of 226 local populations, were analyzed by two-dimensional gel electrophoresis (2-DGE) of total proteins. This technique allows the accurate assessment of allele frequencies in homogenates of mixtures of individuals. Based on the estimated average heterozygosity, the average proportion of polymorphic loci and the average number of alleles per locus, the genetic diversity among 226 local G. pallida populations was small. The small genetic basis of G. pallida in the Netherlands will facilitate the identification of Solanum genotypes with a broad and durable resistance to G. pallida. Instead of clusters of local populations with unique alleles, a continuous range of allele frequencies was observed. Analysis of the three metapopulations by the Shannon-Weaver index and Nei's G(ST) revealed that the metapopulation from a region with sandy-loam soils was clearly distinguishable from the remaining two; the local populations within this metapopulation were more similar and the genetic diversity within the individual local populations was significantly higher than the local populations from the two remaining regions. These regions are characterized by wider crop rotation schemes and a very limited use of nematicides. The less intensive cultivation of potatoes in these regions with sandy-clay soils resulted in relatively little variation within and more variation between local nematode populations. To our knowledge, the effects of agricultural practices on the genetic constitution of potato cyst nematode populations have not been pinpointed before.     

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.     

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.     

Durability of Resistance to Globodera pallida I. Changes in Pathogenicity, Virulence, and Aggressiveness During Reproduction on Partially Resistant Potato Cultivars

ABSTRACT We studied changes in pathogenicity, virulence, and aggressiveness of Globodera pallida populations over time. As a measure for pathogenicity, the reproduction factor on a partially resistant host was used; for aggressiveness, the reproduction factor on a susceptible reference host was used; and, for virulence, the ratio pathogenicity/aggressiveness was used. The G. pallida populations were reared in a glasshouse for four generations on potato cultivars with different levels of resistance. The cultivar Elkana did not increase pathogenicity significantly, but the more resistant cultivars Karakter and Darwina did. This increase in pathogenicity was caused by an increase in virulence, whereas aggressiveness generally was not altered significantly. The increase in virulence appeared to be caused by an enhanced ability of eggs to develop into cysts, and not by an increase in egg production per new cyst. The observed changes in virulence could be predicted reasonably well by a simple numerical model. The rate of selection depended strongly on the nematode population. Rearing a mix of two different populations on a susceptible host decreased the virulence strongly, as predicted by the Hardy-Weinberg equilibrium, but increased the aggressiveness because of heterosis.     

应用TaqMan探针进行马铃薯金线虫实时荧光PCR检测技术研究

马铃薯孢囊线虫是全球性的植物检疫性线虫,亦是我国重点关注的有害生物。针对马铃薯金线虫ITS序列,设计了引物和TaqMan 探针,使用15个马铃薯孢囊线虫群体和4个其他孢囊线虫样品进行验证,可高度灵敏地检测单个马铃薯金线虫的孢囊或幼虫,并可进行定量;最高检测灵敏度达到10fg;同时开展了混合样品和未知样品的检测,证明了引物的专化性和TaqMan探针的特异性。该检测方法可自动化检测马铃薯金线虫并进行定量,适合进行标准化的常规检测。     

The demarcation of Globodera rostochiensis and Globodera pallida infestations in fields for seed potatoes, using a Monte Carlo approach

A Monte Carlo approach was utilized to calculate the size of the demarcation unit, necessary to cover infestations with Globodera rostochiensis and Globodera pallida (PCN), detected with the new EU-sampling method. Results from 90 sampled fields were used to obtain frequency distributions of the central population density (CPD), the length and width gradients of infestation foci and the distance between 'primary' and 'secondary' foci within one field section in the direction of cultivation. With a simulation model written in R, a free software environment for statistical computing, the dimensions of the demarcation unit were calculated, based on the dimensions of the sampling unit, the position of the 'primary' infestation within the sampling unit and the size of the infestation focus. It is concluded that a demarcation of 90% of possible foci positions and dimension, analogous to the specified detection probability of the EU sampling method, provides sufficient security to demarcate any infestation. With the preferred sampling unit of 11 m width and 300 m length (1/3 ha sampled), used in The Netherlands, only 0.61% of the cyst numbers will be situated outside the demarcated unit. These cyst numbers compare to 0.17% of total amount of cysts in the EU infestation model: 1 focus with a CPD of 100 cysts kg⁻¹ and three foci with CPDs of 50 cysts kg⁻¹. The required demarcation area has a width of 36 m and a (maximum) length of 402 m and covers an unit of 1.5 ha. The methodology described can be applied to all proposed sampling methods and sampling unit dimensions.