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.     

无花果树苗中截获的无花果孢囊线虫形态和分子鉴定

上海口岸从旅客携带进境的树苗中分离到1种孢囊线虫属的2龄幼虫,通过形态特征和分子特征分析,鉴定为无花果孢囊线虫(Heterodera fici Kirjanova,1954)。该2龄幼虫的主要形态特征为:体长409.9~454.4μm;唇环3个;口针发达,长22.6~24.0μm,基部球圆形略向前倾斜;侧线4条;尾长圆锥形,末端细圆,尾部透明区长24.7~30.5μm,约占尾长的1/2。序列分析显示其ITS区序列与GenBank中无花果孢囊线虫的序列相似性为99.48%~99.90%,序列差异为1~5 bp。系统发育分析显示该线虫与无花果孢囊线虫处于一个聚类组内。这是我国口岸首次报道截获无花果孢囊线虫。     

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.     

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.     

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?     

Occurrence of Globodera pallida in Hungary

Golden potato cyst nematode, Globodera rostochiensis , has been known to occur in Hungarian potato fields for more than 20 years. The Hungarian NPPO has been carrying out a country-wide Globodera monitoring programme since 1995 and has inspected 16 000 samples. Until 2000, all records were of G. rostochiensis . However, in 2001, three localities in Pest county were found to be infested with Globodera pallida . This was the first official record of G. pallida in Hungary. The two species have been found together in five fields. Unmixed populations of G. pallida have not been found. Identification was based on morphometric data, as well as on biochemical methods (ELISA technique).     

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。     

实时荧光PCR检测马铃薯白线虫技术研究

马铃薯胞囊线虫是马铃薯上最重要的有害生物之一,也是我国特别关注的重要植物检疫性线虫.针对马铃薯白线虫ITS序列,我们设计了引物和TaqMan探针,使用15种马铃薯胞囊线虫群体和4种其它胞囊线虫样品进行验证,可高度灵敏地检测单个马铃薯白线虫的胞囊或幼虫,最高检测灵敏度达到10fs;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探针特异性.该检测方法可自动化检测马铃薯白线虫并进行定量,适合进行标准化的常规检测.     

Potato root diffusate-induced secretion of soluble, basic proteins originating from the subventral esophageal glands of potato cyst nematodes

ABSTRACT In preparasitic second-stage juveniles (J(2)) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses of 30, 31a/b, 32, 39, and 49 kDa were recognized on Western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J(2) and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J(2) showed that the svp's were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of svp's through the metacorporal pump chamber was confirmed by the presence of svp's in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.     

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.     

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.     

甘肃小麦禾谷孢囊线虫rDNA-ITS和28S rDNA-D2/D3区序列特征及ITS-RFLP分析

禾谷孢囊线虫是危害禾谷类作物的重要病原,严重威胁我国小麦主产区的小麦产量和品质。利用通用引物对甘肃、河南、安徽禾谷孢囊线虫群体28SrDNA-D2/D3区和rDNA-ITS区进行PCR扩增和序列测定,利用UPG-MA方法分析了甘肃省7个种群、河南1个种群、安徽1个种群的禾谷孢囊线虫群体D2/D3区和ITS区的系统发育关系;用9种限制性内切酶对7个甘肃禾谷孢囊线虫群体的rDNA-ITS区进行了RFLP分析。结果表明:中国甘肃的禾谷孢囊线虫rDNA-D2/D3区片段长度约为780bp,rDNA-ITS片段长度约为1040bp。7个甘肃禾谷孢囊线虫群体、1个河南安阳群体、1个安徽蚌埠群体的D2/D3区和新西兰的H.aucklandica群体亲缘关系很近;其ITS区同澳大利亚的H.australis、北京通州的H.avenae(AY148382)的亲缘关系很接近。RFLP分析表明,9种限制性内切酶酶切禾谷孢囊线虫群体的rDNA-ITS共产生了22个酶切片段,不同酶切的RFLP分布型在7个种群间没有差异。甘肃省禾谷孢囊线虫群体的rDNA-ITS区具有高度的保守性,与中国的C型群体相近,但不同于欧洲的A型群体和印度的B型群体。这是首次报道甘肃CCN种群分子特征。     

A New Root-Knot Nematode, Meloidogyne baetica n. sp. (Nematoda: Heteroderidae), Parasitizing Wild Olive in Southern Spain

ABSTRACT High infection rates of wild olive (Olea europaea sp. sylvestris) feeder roots and soil infestation by a new root-knot nematode were found in sandy soil at Vejer de la Frontera (Cádiz), southern Spain. Morphometric traits and analyses of the nematode esterase electrophoretic pattern as well as of the internal transcribed spacer 1 (ITS1)-5.8S gene and D2-D3 fragment of the 28S gene of rDNA showed that specimens differed clearly from known root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected wild olive plants and in olive planting stocks (cvs. Arbequina and Picual) artificially inoculated with the nematode. However, the nematode did not reproduce in artificially inoculated chickpea, pea, and tomato. Because of the ability of this new nematode to infect wild and cultivated olives only, we suggest the common name, "Mediterranean olive root-knot nematode." The species is herein described and illustrated, and named as Meloidogyne baetica n. sp. The new root-knot nematode can be distinguished from other Meloidogyne spp. by (i) the perineal pattern, which is almost similar to that of M. artiellia, characterized by distinct inner striae forming two distinct longitudinal bands, extending throughout the perineum to just below the vulva; (ii) female excretory pore anterior to the level of stylet knobs, excretory pore distance from anterior end/length of stylet ratio extremely small (0.5 to 0.8); and (iii) second-stage juveniles with elongate-conoid tail. Phylogenetic trees derived from maximum parsimony analyses showed that M. baetica is closely related to M. artiellia, the cereal and legume root-knot nematode.     

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.     

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.     

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).     

Species Identification of European Cereal and Grass Cyst Nematodes by Larval Characters1

Second-stage juveniles of heteroderid species often present more reliable diagnostic characters than cysts. For identification of European cereal and grass cyst nematodes, size and position of the phasmids, swelling of the cuticle behind the lip region, expression of the lateral fields, shape and size of the stylet knobs and measurements of, for example, the tail and the hyaline tail portion are useful. A key presented, based on larval characters, allows identification of seven European cereal and grass cyst nematode species and their distinction from other European heteroderids not reproducing on Gramineae. Species identification is often difficult in the Heterodera avenae/H. mani complex and evidence exists that additional species are present within this group.     

甘肃河西地区番茄根结线虫病病原鉴定

利用形态特征对甘肃省河西地区番茄根结线虫病病原线虫进行鉴定,其重要的形态特征如下：雌虫体长L=599.0（510.0～690.0）μm,体宽W=439.0（409.0～473.0）μm,口针长ST=15.4(15.0～16.0)μm,背食道腺开口到口针基部球的距离DGO=3.6(2.0～4.3)μm。雌虫梨形,排泄孔位于口针基球处,会阴花纹背弓明显高,线纹由平滑到波浪状,有些线纹向侧面分叉,但无明显的侧线;2龄幼虫L=376.0（360.0～393.0）μm,W=14.8（14.2～15.2）μm,ST=11.7(11.2～12.5)μm,DGO=3.6(2.1～4.0)μm,头部前端平,口针基部球明显,半月体邻近分泌和排泄孔,尾透明且纤细,尾尖尖圆;雄虫L=1 411.0（532.0～1 720.0）μm,W=32.0（28.0～38.0）μm,ST=24.5(23.0～26.0)μm,DGO=3.2(2.0～4.1)μm,头区不缢缩,有不完全的头环,没有侧唇,口针基球圆形至扁圆形。综合以上雌虫、2龄幼虫和雄虫的形态特征,确定了该地区的番茄根结线虫病病原线虫为南方根结线虫（Meloidogyne incognita）。