植物源提取物防治根结线虫的分子研究

针对内生真菌的生物防治,重点阐述了中草药提取物的杀虫活性、植物抗线虫基因的发现以及植物防御系统激发的最新研究.植物源提取物作为潜在的线虫杀虫剂,为生物源农药开发和利用提供新的思路.同时,对于今后线虫杀虫剂开发中存在的问题及解决方案进行了探讨,就其发展方向提出了建议.     

象耳豆根结线虫的PCR鉴定和检测方法

 象耳豆根结线虫是一种在中国具有潜在经济重要性的农作物病原物。为提供有助于控制象耳豆根结线虫传播扩散的方法,研制了该线虫的快速PCR鉴定和检测法。该方法PCR引物的扩增目标为rDNA-IGS2区域,其设计依据象耳豆根结线虫与南方、爪哇、花生和北方根结线虫在该区域核酸序列的差异。通过对6种近似根结线虫的不同地理群体及自然土壤线虫群体的测试,验证了设计的PCR引物针对象耳豆根结线虫的特异性和可靠性。本方法具有快速灵敏的特点,可用于象耳豆根结线虫单条线虫的直接鉴定以及混合土壤线虫群体中象耳豆根结线虫的检测。     

Nuclear and Mitochondrial DNA Polymorphisms in Three Mitotic Parthenogenetic Meloidogyne Spp.

In order to expand our understanding of the genetics of root-knot nematodes, variation in nuclear DNA and mitochondrial DNA in Meloidogyne incognita, M. arenaria and M. javanica was investigated. Despite the obligate mitotic parthenogenetic mode of reproduction, a large number of AFLP polymorphisms were observed among all 16 populations studied. Both UPGMA and principle coordinate analyses revealed three distinct groups that corresponded with the respective species identities of the 16 populations. M. incognita was genetically most distinct. Amplification of 63-bp tandem repeats (TR) in mtDNA from single individuals enabled the calculation of diversity measures at three hierarchical levels: within individuals, among individuals of a single population and among populations. For all three species, the highest diversity was observed within individuals explaining 43–65% of the total diversity. Many individuals contained more than one mtDNA size variant. M. incognita harboured the most heteroplasmic individuals and was the most homogenous at the population level. Only 13% of the total diversity was observed among populations, while this figure was 35% for M. arenaria. Both TR and AFLP data showed that M. arenaria is the most heterogeneous species. The comparison of the genetic distances based on AFLPs and mtDNA size variants revealed a significant correlation for the six M. arenaria populations, whereas no consistent correlation was observed for the populations of the other two species.     

Efficacy of Different Control Methods Applied Separately and in Combination in Managing Root-knot Nematodes (Meloidogyne spp.) in Common Beans

Investigations on the efficacy of various methods of managing root-knot nematodes in microplots and under field conditions revealed that soil solarization, Furadan 5G and Tagetes erecta applied separately or in combination with other control methods, were the most effective in reducing the numbers of three root-knot nematodes, and root gall and egg mass indices. These management methods also resulted in significant increases (P0.05) in number of pods per plant, number of seeds per pod and 100-seed weight, and increased seed yield by up to 96.7 percent. Farmyard manure and Crotalaria ochroleuca were the least effective treatments. The use of T. erecta was the most economical root-knot nematode control method.     

Occurrence of Resistance-breaking Populations of Root-knot Nematodes on Tomato in Greece

Nine populations of Meloidogyne spp. from Greece have been identified as M. javanica or M. incognita using either isozyme phenotypes or the sequence characterized amplified region-polymerase chain reaction (SCAR-PCR) technique. Virulence against the Mi resistance gene was assayed by pot experiments in controlled conditions and revealed the ability of five populations of M. javanica and one population of M. incognita to reproduce on tomato cultivars containing that gene. A resistance-breaking population of M. incognita is reported for the first time in the country; the M. javanica populations constitute new records for the Greek mainland.     

功能型复合微生物菌剂防治黄瓜根结线虫的研究

根结线虫是一类重要的土传性病原物,对露地和温室蔬菜造成严重的经济损失。本研究通过平板亲和性试验,构建出一组(淡紫拟青霉、红灰链霉菌和苍白杆菌各1株)生物兼容性好、作用方式互补的功能型复合微生物菌群;以各功能菌株发酵菌体为活性成分,研制出一种防治黄瓜根结线虫的功能型复合微生物菌剂;对该复合菌剂对黄瓜根结线虫的防效及植株生物量和产量的影响进行了评价,大田试验结果表明,施用该复合菌剂的土壤线虫扩繁指数最低;移栽后38和105 d,该复合菌剂处理的根结级数分别为2.0和4.6,对病害防效为56.5%和42.5%,效果优于10%噻唑膦处理;施用该复合菌剂可以促进植株生长、增加黄瓜产量。本研究为蔬菜根结线虫病的生物防治提供了新思路。     

Specific Diagnosis of Two Root-Knot Nematodes, Meloidogyne chitwoodi and M. fallax, with Satellite DNA Probes

ABSTRACT Meloidogyne chitwoodi and M. fallax are serious pests of potato, and both species have been recently designated as quarantine organisms in the European Community and in Canada. The sympatric and less damaging species M. hapla is often found associated with both of them under temperate climates. Here, we describe the use of satellite DNA (satDNA) sequences previously isolated from these three root-knot nematode species for the development of specific diagnostic procedures. In dot-blot experiments, it was unambiguously possible to separate M. chitwoodi and M. fallax from M. hapla using satDNA monomers as probes. In squash-blot experiments, satDNAs allowed discrimination between single individuals of M. chitwoodi or M. fallax from M. hapla, even within root tissues, without the need for DNA purification. The same results were obtained with radioactive or digoxigenin-labeled probes with no loss of sensitivity in detection. M. fallax and M. chitwoodi could not be distinguished. From this study, it is concluded that such cloned satDNA sequences may constitute a powerful tool for the identification and management of Meloidogyne spp. populations in the field and for the implementation of quarantine regulations against these pests.     

Yield Responses in Relation to the Chemical Control of Root-knot Nematodes in Southern Italy

Nematicide trials have been conducted in Southern Italy to determine the yield response of tobacco, tomato and egg-plant. The nematode species involved were mainly Meloidogyne incognita (Kofoid & White) Chitwood and M. arenaria (Neal) Chitwood. Many nematicides have an impact also on other soil pathogens or even on insects, so that the yied increase observed in the field has to be considered as a combined effect. On tobacco, most of the nematicides gave positive results, but it appeared that there might be important differences according to the variety considered. In certain tomato fields, which had not been specially attacked by Meloidogyne, the positive effects seemed to be also due to the insecticidal or fungicidal effect of the chemicals. No statistically significant yield increases were, however, obtained on treated egg-plant fields.     

Phosphite Inhibits Development of the Nematodes Heterodera avenae and Meloidogyne marylandi in Cereals

ABSTRACT Phosphonic acid (H(3)PO(3)) solutions were applied to wheat or to bristle oat as soil drenches before inoculation with juveniles of the sedentary, endoparasitic nematodes Heterodera avenae or Meloidogyne marylandi. All the solutions, which were pH adjusted and added at levels as low as 0.63 mg of phosphite (HPO(3)(2-)) per plant, reduced the numbers of H. avenae females and M. marylandi egg masses. Phosphate (PO(4)(3-)), applied as potassium phosphate at the same concentrations, did not reduce the number of female nematodes on the wheat. Addition of phosphate to the phosphite solutions did not change the inhibitory effect of phosphite on H. avenae, but it reduced phosphite's effect on M. marylandi. Phosphite also reduced the number of H. avenae females when applied as many as 20 days after addition of nematodes. The phosphite treatment did not prevent M. marylandi juveniles from penetrating wheat roots or inducing giant cells. However, phosphite inhibited giant cell development: 14 days after inoculation, the giant cells in the phosphite-treated wheat were almost completely vacuolated, whereas those in untreated wheat contained dense cytoplasm.     

南方、爪哇和花生根结线虫的快速灵敏的PCR鉴定方法

 为了研制南方、爪哇和花生根结线虫快速灵敏的检测和鉴定方法,分别分离了4个南方根结线虫和3个爪哇根结线虫特异性的随机扩增多态性DNA (RAPD)片段。在这些RAPD标记DNA序列的基础上,设计了多对SCAR PCR引物,并用源于国内外的南方、爪哇、花生、北方和象耳豆根结线虫群体验证其扩增特异性和灵敏度。最终确定了3对高效扩增的SCAR引物,它们组合使用可以可靠灵敏地鉴定南方、爪哇和花生根结线虫。3对引物的扩增灵敏度达1/3条的二龄幼虫、雄虫或雌虫,这表明本研究研制的PCR鉴定法可用于生产实践中土样和根样中3种根结线虫快速灵敏的鉴定。     

南方、花生和爪哇根结线虫PCR检测方法

为快速、准确、稳定的鉴定南方、花生和爪哇根结线虫,利用已报道的南方和爪哇根结线虫的两对特异性引物,结合本研究设计的花生根结线虫特异性引物,通过优化PCR反应体系,建立了3种根结线虫的PCR检测方法。结果表明,该方法能够特异性扩增以上3种根结线虫,特征片段长度分别为399、335和670 bp,灵敏度达到单条2龄幼虫的水平。研究结果将为以上3种根结线虫的快速鉴定提供技术支持。     

低钾逆境下根结线虫感染所诱导的番茄根巨型细胞的组织病理学反应

 Comparison of histopathological response and quantitative measurement of giant cell(GC) induced by Meloidogyne javanica in tomato root were studied under potassium-deficient(0.2 mmol/L K⁺) and replete conditions(control,6.0 mmol/L K⁺).K⁺-deficient stress did not impede the formation and maintenance of GC.The mean number of GC per feeding site as well as the mean diameter of GC did not differ between the treatments.However,the thickness of cell wall including components resulted from the accumulated polysaccharide and the length of cell-wall ingrowth increased 5-25 d after inoculation in K⁺-deficient as compared with K⁺-replete conditions.An increase of cell-wall ingrowth suggested a kind of compensational response to the potassium stress.     

Activity of Hydroxamic Acids from Secale cereale Against the Plant-Parasitic Nematodes Meloidogyne incognita and Xiphinema americanum

ABSTRACT Cyclic hydroxamic acids are secondary metabolites found in the family Poaceae and have been implicated in the allelopathy of rye (Secale cereale). The toxicity of these compounds against plant-parasitic nematodes is unknown. DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one), DIMBOA (2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one), and their degradation products BOA (benzoxazolin-2(3H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) were screened in vitro against Meloidogyne incognita second-stage juveniles (J2) and eggs and mixed-stages of Xiphinema americanum. Xiphinema americanum was more sensitive to DIBOA and DIMBOA than M. incognita J2, with a maximum apparent mortality of 96 and 92% compared to 73 and 72% at 90 mug/ml. Eggs of M. incognita were less sensitive to the hydroxamic acids than J2; only DIBOA resulted in a 50% reduction in egg hatch, with a lethal concentration (LC(50)) of 74 mug/ml compared to 21 mug/ml for J2. When M. incognita J2 were exposed to DIBOA for 48 h and the compound was removed and replaced with water, the LC(50) value increased from 21.0 to 40.7 mug/ml. MBOA was not toxic to X. americanum or M. incognita eggs, but was toxic to M. incognita J2, with LC(50) values of 44 and 20 mug/ml before and after the compound was removed and replaced with water. BOA was the least toxic hydroxamic acid tested; it did not reduce M. incognita egg hatch after 1 week of exposure or increase X. americanum mortality after 24 h of exposure. While in vitro studies provide a valuable starting point in determining the toxicity of the chemical component of rye, the relevance of the data to soil remains to be determined.     

Evaluation of Natural Chemical Compounds Against Root-lesion and Root-knot Nematodes and Side-effects on the Infectivity of Arbuscular Mycorrhizal Fungi

The survival of two species of plant parasitic nematodes: the root-lesion nematode Pratylenchus brachyurus, and the root-knot nematode Meloidogyne javanica, was evaluated in saturated atmospheres of 12 natural chemical compounds. The infectivity of two isolates of arbuscular mycorrhizal fungi: Glomus mosseae and Glomus intraradices, under identical experimental conditions, was also determined. All the compounds tested exerted a highly significant control against M. javanica and among them, benzaldehyde, salicilaldehyde, borneol, p-anisaldehyde and cinnamaldehyde caused a mortality rate above 50% over P. brachyurus. The infectivity of G. intraradices was inhibited by cinnamaldehyde, salicilaldehyde, thymol, carvacrol, p-anisaldehyde, and benzaldehyde, while only cinnamaldehyde and thymol significantly inhibited mycorrhizal colonization by G. mosseae.     

Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes

ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.     

Incidence of Viruses Infecting Allium Spp. in Greece

A survey identified viruses infecting garlic, leek and onion crops and wild Allium species in Greece. Virus identification was based on ELISA, immunoelectron microscopy, and occasionally on RT-PCR. Samples of cultivated Allium species were collected from five districts, whereas samples of twenty-seven wild Allium species were also collected from all over Greece. Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV) were identified in 98.5% and 83.7% of all samples, respectively, and were found in all regions. Allexiviruses were also detected in all regions and their incidence ranged from 62.5% to 70.5% (depending on region and type of allexivirus). Garlic common latent virus (GCLV) was detected in samples from Arcadia (97.6%) and Evia (18.0%) and in one field in Larissa (23.0%). Shallot latent virus (SLV) was found only in two areas (Evros and Theva) and in fields planted with imported propagative material, from Iran and China. The incidence of virus-like symptoms in leek crops ranged from 10.0% to 90.0% in different regions and fields and all symptomatic plants were found to be infected by LYSV. Onion yellow dwarf virus was only found in seven symptomatic onion samples from southern Greece. Allium ampeloprasum spp. ampeloprasum and Allium flavum, were the only wild Allium species found to be infected with LYSV. Finally Turnip mosaic virus (TuMV) was found in A. sphaerocephalon, A. guttatum, A. subhirsutum, and A. neapolitanum.     

我国植物青枯菌菌株的遗传多样性和组群划分

 采用15条随机引物对我国11个省(市、区)6种不同寄主植物的43个青枯菌代表性菌株和4个国外青枯菌菌株,进行了PCR扩增.引物OPB11、OPA15、OPE1和OPZ10对上述所有菌株扩增获得了相似的产物电泳图谱,分别具1~5条谱带不等;引物OPB7、OPA10和OPF1对马铃薯菌株获得了相同的产物图谱,但对其它寄主菌株的产物间有明显差别;引物OPA14、OPC,6、OPG14、OPF5、OPK14、OPK20和OPK17对于不同菌株的扩增产物多态性很强.供试菌株被聚类为2个组群,即组群A和组群B.组群A中又可分为7个亚组(A1、A2、A3、A4、A5、A6、A7),其中A1含有2个类型(A1-1、A1-2);组群B中也可分为2个亚组(B1、B2),其中B1含有3个类型(B1-1、B1-2、B1-3),B2也含有3个类型(B2-1、B2-2、B2-3).RAPD组群A中包含了27个来自我国不同地区的马铃薯菌株,主要是3号小种、生化变种2;组群B中含有20个来自不同地区、不同寄主的菌株,分属于其它不同的小种和生化变种.研究结果表明,我国青枯菌菌株RAPD组群的划分与菌株的地理来源关系不大,而与寄主来源有明显相关性.此外,通过对我国青枯菌菌株组群进行同源性PCR分析表明,来源自马铃薯的3号小种菌株属于美洲分支"Americanmm",而来自其它寄主的青枯菌1号、5号小种菌株属于亚洲分支"Asiaticum",与本研究RAPD组群A和组群B的划分是一致的.     

Application of a putative fatty-acid binding protein to discriminate serologically the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from other Meloidogyne species

Two major proteins, Mcf-A67 and Mcf-B66, were identified by mini two-dimensional polyacrylamide gel electrophoresis in order to distinguish the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from eight other species. These quarantine proteinic markers have been microsequenced after enzymatic digestion. The internal amino acid sequences exhibit similarities to members of a family of low molecular weight intracellular lipid-binding proteins. Moreover, to explore a simple, rapid, and inexpensive way to identify the two quarantine nematodes, dot blot hybridizations were performed using an antiserum (A67) produced from the longest amino-acid sequence of the protein Mcf-A67. Although several proteins stained on the M. chitwoodi and M. fallax western blot membranes, the two nematodes were easily distinguished from other root-knot nematodes, on dot blot assays with soluble proteins extracted from a single female. Because of its specificity and sensitivity, the use of the A67 antiserum to improve the diagnosis of the two European quarantine root-knot nematodes is discussed.