An immunocytochemical procedure for protein localization in various nematode life stages combined with plant tissues using methylacrylate-embedded specimens

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access.     

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.     

植物寄生线虫分支酸变位酶基因的研究进展

 植物寄生线虫食道腺中表达的寄生基因编码的分泌蛋白在线虫侵入寄主植物、建立取食位点和抑制寄主的防御反应过程中起重要作用。利用植物寄生线虫食道腺削减cDNA文库及基于同源克隆等方法,鉴定了这些过程中起作用的分支酸变位酶(CM)基因。带有氨基酸末端信号肽的根结线虫CM、孢囊线虫CM与细菌CM的蛋白质序列非常相似。mRNA原位杂交表明,CM基因专门存在于植物寄生线虫的亚腹食道腺中。RT-PCR分析表明,它们的转录丰度在线虫寄生的早期丰度较高,在后期较低或者难以检测到。Southern杂交表明,这些CM基因为多基因家族。CM的蛋白质在专性内寄生线虫中广泛存在,表明这种多功能的酶在控制线虫侵染植物的过程中起重要作用。     

拟短体线虫属一新种

 甘薯拟短体线虫新种(Pratylenchoides batatae n. sp.)根据采自河北省卢龙县前双庙的甘薯根部及根际土壤中的一个拟短体线虫种群进行描述和作图。新种的主要鉴别特征是:唇区有5个唇环,侧区4条侧线并有网格纹,尾呈圆柱形、有24~26个体环,尾端圆、具粗纵纹。食道腺末端覆盖于肠背面,2个亚腹食道腺核均在背食道腺核之后,一个亚腹食道腺核位于食道-肠瓣门前的腹面、另一个位于食道-肠瓣门稍后。近似种为齿尾拟短体线虫(P. crenicauda),该种与新种的主要区别是:唇区3~4个唇环,侧区有6条侧线、无网纹,亚腹食道腺核一个在背食道腺核前、另一个在背食道腺核后。新种为两性种。模式标本保存于福建农林大学植物保护学院植物线虫学实验室。     

寄生在柑桔根上的根结线虫新种——简阳根结线虫

 简阳根结线虫Meloidogyne jianyangensis n.sp.采自四川简阳县柑桔园的柑桔根上。雌虫的会阴花纹近圆形,线纹平滑、纤细,背腹线纹通常连贯。以尾尖处向两侧有脊状放射形条纹,条纹数目不定、在阴肛区的背侧有半圈脊状条纹。在放射状的条纹间,线纹有时不连贯,有如许多小刻点。尾觉器距离很近。雄虫头冠高,唇盘和中唇融合,无侧唇。口针长19.1-26.0(21.8μm)。口针基球和杆部界限明显。背食道腺开口距口针基部2.5-3.5(3.0μm)。侧带处有4条侧线,两边两条间有横纹,中间两条侧线间无横纹。二龄幼虫长387.6-483.3(423.2)μm。头冠高,唇盘和中唇融合成哑铃状。口针长13.0-16.8(15.1)μm。背食道腺开口距口针基部2.1-3.7(2.5)μm。尾部渐细,尾尖钝圆。简阳根结线虫雌虫的主要酯酶带Rf=0.41、0.45和0.48。     

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.     

Presence of Grapevine leafroll-associated virus 3 in primary salivary glands of the mealybug vector Planococcus citri suggests a circulative transmission mechanism

Grapevine leafroll-associated virus 3 (GLRaV-3) is a mealybug-transmissible ampelovirus. Though the transmission mechanism has been described as semipersistent on the basis of temporal parameters, definitive proof of this mechanism has never been provided. In the present study, we carried out preliminary assays to establish the location of the virus in its vector, Planococcus citri. After dissecting the insects, GLRaV-3 was detected by means of IC-RT-PCR in the salivary glands, intestine and Malpighian tubes, but not in the sucking apparatus. Immunogold labelling of the capsid protein revealed the presence of the virus in some cells of the primary salivary glands, but not in the alimentary channel of the stylet, or in the accessory salivary glands. The strong labelling of the electron-dense secretion vesicles in some cells of the primary salivary glands, together with the non-detection of the virus in the sucking apparatus suggests that the transmission mechanism may be different from that previously described. We propose a circulative transmission mechanism based on a specific transportation route for the viral particles from the midgut or hindgut to the salivary glands. As the transmission mechanism is generally a common feature of a viral genus, the existence of a circulative transmission mechanism for other mealybug-transmitted ampeloviruses is expected. Organ by organ analysis of GLRaV-1, another ampelovirus not transmissible by P. citri, showed the absence of the virus in the salivary glands, thus providing further, though indirect, evidence in favour of circulative transmission for this virus genus.     

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。     

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）。     

稻曲病菌SIX效应蛋白参与调控植物免疫

为明确稻曲病菌Ustilaginoidea virens的SIX(secreted in xylem)效应蛋白功能,利用生物信息学软件预测6个SIX1蛋白和1个SIX2蛋白的分泌特性,并通过酵母分泌系统对其分泌特性进行验证,在烟草上通过根癌农杆菌Agrobacterium tumefaciens介导的瞬时表达系统检测SIX蛋白对小鼠促凋亡蛋白BAX、致病疫霉Phytophthora infestans激发子INF1、大豆疫霉Phytophthora sojae病原相关分子模式糖基水解酶XEG1引起烟草细胞坏死的作用,在烟草上表达SIX蛋白后接种辣椒疫霉Phytophthora capsici并观察其对辣椒疫霉侵染的作用。结果显示:所检测的SIX蛋白中有5个含有预测的信号肽,2个被预测为非经典分泌蛋白,但经酵母分泌系统验证均具有分泌功能;大部分SIX蛋白能够抑制BAX、INF1、XEG1引起的烟草细胞坏死,但抑制坏死的情况并不完全一致;大部分含信号肽的SIX蛋白能够促进辣椒疫霉侵染,UV8b＿3638去信号肽突变体也能够促进辣椒疫霉侵染。表明大多数SIX蛋白参与调控植物免疫,且能促进病原...     

Feeding behavior of sedentary plant parasitic nematodes

With the aid of video-enhanced contrast light microscopy the feeding behavior of the cyst nematodeHeterodera schachtii and the root-knot nematodeMeloidogyne incognita has been studied in roots of various cruciferous plants, especially inArabidopsis thaliana, a model plant for molecular studies. The juvenile stage 2 (J2) nematodes ofH. schachtii migrate intracellularly with destructive stylet thrusts before establishing their permanent feeding site (syncytium) within the vascular cylinder. After the selection of the initial syncytial cell (ISC) they undergo a preparation period before they start feeding from the ISC. During all developmental stages feeding occurs in cycles, composed of three distinct phases. In contrast, the J2 ofMeloidogyne incognita reach their permanent feeding site by intercellular migration, first towards the apex of the root, where they turn round, and move towards the differentiating vascular cylinder. A characteristic behavioral pattern, composed of continuous head and stylet movements, interspersed by periods of stylet-tip protrusion and metacorpal bulb pumping is maintained during all phases of parasitism, i.e., root invasion, intercellular migration and feeding from the giant-cells.     

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.     

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.     

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.     

斜纹夜蛾化学感受蛋白SlitCSP2的表达模式定位及配体结合特性分析

研究斜纹夜蛾Spodoptera litura化学感受蛋白SlitCSP2的表达模式、组织定位及与配体结合特性,为揭示SlitCSP2在化学通讯中的功能提供理论依据。利用RT-qPCR检测了SlitCSP2在蛹及雌、雄成虫各组织的基因表达水平;Western blot方法检测了SlitCSP2在幼虫期和雄成虫头部、触角和足部的蛋白表达水平;利用免疫组织化学技术对SlitCSP2在幼虫各龄期的分布情况进行定位分析;利用荧光竞争结合试验检测SlitCSP2与13种非挥发性化合物的结合特性。RT-qPCR结果表明,SlitCSP2在3龄幼虫表达量最高,3～6龄表达量逐渐降低;成虫阶段雄成虫足部及胸部和雌成虫头部及翅的表达量最高,且雄成虫头部、足部和胸部的表达量显著高于雌成虫。Western blot结果显示,SlitCSP2在3龄幼虫头部（去触角）的蛋白表达水平较高。免疫组化结果表明,SlitCSP2在幼虫阶段主要分布于头部、口器和胸足;在雄成虫中,SlitCSP2主要分布于足部前跗节和足部外骨骼内侧。荧光竞争结合试验表明,SlitCSP2与单宁（K_i＝1.77）、花青素（K_i＝2.34）和叶酸（K_i＝4.56）有很强的结合能力。本研究明确了SlitCSP2在幼虫各龄期和成虫各组织的表达特征。根据免疫定位及荧光竞争结合试验结果,我们推测SlitCSP2可能在斜纹夜蛾寄主选择和解毒过程中发挥重要作用。     

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).