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。     

广州芳村盆景线虫种类调查和根结线虫鉴定

本文通过对广州芳材６个盆场１０种分影的调查,初步鉴定了Ａｐｈｅｌｅｎｃｈｏｉｄｅｓ,Ｃｒｉｃｏｎｅｍｏｉｄｅｓ,Ｈｅｌｉｃｏｔｙｌｅｎｃｈｕｓ,Ｈｏｐｌｏｌａｉｍｕｓ,Ｌｏｎｇｉｄｏｒｕｓ,Ｍｅｌｏｉｄｏｇｙｎｅ,Ｐａｒａｔｙｌｅｎｃｈｕｓ,Ｐｒａｔｙｌｅｎｃｈｕｓ,ｓｉｌｅｎｃｈｕｓ,Ｔｒｉｃｈｏｄｏｒｕｓ,Ｔｒｏｐｈｕｒｕｓ,Ｔｙｌｅｎｃｈｏｒｈｙｎｃｈｕｓ,Ｔｙｌｅｎｃｈｕｓ,Ｘｉｐｈｉｎｅ     

湖南柑橘根结线虫种类鉴定及特异性PCR检测

根结线虫病是严重危害湖南柑橘生产的主要病害,快速准确地进行病原线虫鉴定,从而制定针对性的防治措施,对柑橘产业的稳定发展至关重要。运用形态学和分子生物学技术对湖南永州地区柑橘根结线虫进行病原种类鉴定,确定其为番禺根结线虫Meloidogyne panyuensis。通过引物设计与筛选,建立了番禺根结线虫特异性PCR检测技术。结果表明,该检测方法特异性好,灵敏度高,操作简单,能有效地从多种根结线虫中特异性检测出番禺根结线虫,为柑橘病原线虫的快速检测鉴定提供技术支撑。     

广州芳村盆景线虫种类初步调查和根结线虫鉴定

本文通过对广州芳村六个盆景场十种盆景的调查,初步鉴定了Ａｐｈｅ ｌｅｎｃｈｏｉｄｓ,Ｃｒｉｃｏｎｅｍｏｉｄｅｓ,Ｈｅｌｉｃｏｔｙ ｌｅｎｃｈｕｓ,Ｈｏｐｌｏｌａｉｍｕｓ,Ｌｏｎｇｉｄｏｒｕｓ,Ｍｅｌｏｉｄｇｙｎｅ,Ｐａｒａｔｙ ｌｅｎｃｈｕｓ,Ｐｒａｔｙ ｌｅｎｃｈｕｓ,Ｐｓｉｌｅｎｃｈｕｓ,Ｔｒｉｃｈｏｄｏｒｕｓ,Ｔｒｏｐｈｕｒｓ,Ｔｙｌｅｎｏｒｈｙｎｃｈｕｓ,Ｔｙｌｅｎｃｈｕｓ,Ｘｉｐｈｉｎｅ     

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。     

毒杀南方根结线虫的木霉种类鉴定及活性研究

木霉是国际上研究与应用非常普遍的生防真菌之一,具有重要的科研价值和广阔的应用前景。本研究采用形态学鉴定、ITS和β-tubulin序列分析法对4株具有杀线虫活性的木霉菌株进行种类鉴定,结果显示FT311和FT312为绿色木霉(Trichoderma viride),FT1937和FT85为哈茨木霉(T.harzianum)。利用贝氏皿浸没法测定,4株木霉发酵滤液均能显著降低南方根结线虫卵的孵化和2龄幼虫(J2)的活性,其中FT1937和FT85对卵孵化抑制率和J2致死率均达到85%~92%,而FT311能够达到80%~85%,但FT312毒性较低,仅达40%~60%;2株哈茨木霉的活性好于2株绿色木霉;同时观察到发酵液对线虫卵和幼虫体壁有消解作用、抑制和延迟卵孵化,并使幼虫体内空泡化。本研究为根结线虫病害的生防防治提供了更多的木霉资源信息。     

用PhastSystem电泳仪快速鉴定根结线虫种类

 应用瑞典Pharmacia Biotech.公司开发的全自动快速水平电泳仪(PhastSystem)进行酯酶和苹果酸脱氢酶电泳,对云南省烟草和观赏植物一串红上的根结线虫进行快速鉴定,得出4种根结线虫:花生根结线虫(Meloidogyne arenaria)、爪哇根结线虫(M.javanica)、南方根结线虫(M.incognita)和北方根结线虫(M.hapla)。文章提供电泳方法,认为PhastSystem电泳仪是根结线虫种类快速鉴定的理想设备。     

福建省毛豆根结线虫种类鉴定及初侵染来源

福建省漳州市一基地毛豆(品种:‘毛豆64’)苗期发生严重根结线虫病, 发病株率为78%。为明确其病原的种类及其初侵染来源, 通过形态学、分子生物学方法进行了种类鉴定。研究结果表明, 该线虫雌虫、雄虫、2龄幼虫(J2)的形态学特征与测量值和拟禾本科根结线虫Meloidogyne graminicola相吻合;基于线虫rDNA-ITS序列和28S rDNA D2D3序列构建的系统发育树进一步确定其为拟禾本科根结线虫;室内接种试验确定了其致病性。这是首次在福建省毛豆上发现拟禾本科根结线虫。在毛豆基地畦地残留的前作水稻稻茬根内和根际土壤中均分离到大量J2s, 经特异性引物扩增确定为拟禾本科根结线虫, 研究认为前作水稻根系受拟禾本科根结线虫侵染, 在灌溉淹水状态下线虫2龄幼虫和卵在根内与土壤中长期存活, 成为轮作毛豆根结线虫病的初侵染源。     

2个根结线虫种群鉴定及致病性研究

采用雌虫会阴花纹形态观察比较、EST同工酶图谱分析以及与mt DNA-PCR分子技术相结合的方法,将来自海巴戟的2个根结线虫种群万宁种群和定安种群鉴定为花生根结线虫,酯酶分别为A2、A1;通过盆栽接种测定了这2个根结线虫种群对32种农作物的致病性。试验结果表明,定安种群的致病性比万宁种群强,且寄主范围比万宁种群广。     

Identification of Meloidogyne chitwoodi, M. fallax and M. hapla Based on SCAR-PCR: A Powerful Way of Enabling Reliable Identification of Populations or Individuals that Share Common Traits

This study describes the development of species-specific pairs of PCR primers for the root-knot nematodes Meloidogyne chitwoodi, M. fallax and M. hapla that amplify species-specific RAPD fragments. After sequencing the fragments, longer primers were designed to complement the terminal sequences of the polymorphic DNA fragments. The resulting pairs of primers were used to generate the sequence-characterized amplified regions (SCARs). Using the developed pairs of SCAR primers, SCAR fragments of M. chitwoodi, M. fallax or M. hapla were easily amplified from DNA extracts from juveniles, egg masses, females of the particular nematode species investigated, either present alone, in a mixture with other nematode species or in infested plant material. A specially designed multiplex assay using three pairs of SCAR primers enabled the identification of multiple species in a mixture in a single PCR step. Single juveniles were easily identified by applying this multiplex assay followed by a subsequent multiplex PCR using three pairs of nested primers. The SCAR-PCR-based assays described have potential to be optimized for routine practical diagnostic tests. The usefulness of converting RAPD markers into SCAR markers is discussed.     

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.     

Differential reproduction of Meloidogyne incognita and M. javanica in watermelon cultivars and cucurbit rootstocks

The suitability of watermelon cultivars and cucurbit rootstocks as hosts to Meloidogyne incognita and M. javanica was determined in pot and field experiments. Meloidogyne incognita showed higher reproduction than did M. javanica on watermelon and cucurbit rootstocks. The watermelon cultivars did not differ in host status when challenged with these two species and supported lower nematode reproduction than the cucurbit rootstocks. Rootstocks Lagenaria siceraria cv. Pelops and Cucurbita pepo AK15 supported lower reproduction than did the squash hybrid rootstocks (C. maxima × C. moschata). Egg production increased (P < 0·05) with a rising initial inoculum level (Pi) in the non‐grafted Sugar Baby but the reproduction factor Rf (eggs per plant/Pi) was similar at two Pi levels. The total egg production in the plants grafted onto squash hybrids RS841 and Titan was greater (P < 0·05) at the higher Pi, but the Rf values were lower. The development of field‐grown non‐grafted watermelon plants was significantly stunted in plots where nematodes were detected at planting. However, no differences were observed in plots with grafted plants. In plots with nematodes, non‐grafted and Titan‐grafted plants had similar yields that were higher than that of RS841‐grafted plants. In the commercial plastic houses with grafted watermelon, the average Rf value was 42‐fold, confirming the high susceptibility of squash hybrids as rootstocks for grafted watermelon. The Titan–Sugar Baby combination was tolerant to M. javanica.     

Host suitability of Solanum torvum cultivars to Meloidogyne incognita and M. javanica and population dynamics

Several experiments were carried out to assess the performance of commercial Solanum torvum cultivars against the root knot nematodes Meloidogyne incognita and M. javanica in Spain. The response of S. torvum rootstock cultivars Brutus, Espina, Salutamu and Torpedo against M. incognita and Mi-1.2 (a)virulent M. javanica isolates was determined in pot experiments, and of ‘Brutus’ to an N-virulent isolate of M. incognita, compared with that of the eggplant S. melongena ‘Cristal’. The relationship between the initial and final population densities of M. javanica on ungrafted and grafted ‘Cristal’ onto the S. torvum ‘Brutus’ was assessed, together with the effect on dry shoot biomass. Finally, the population growth rate and the resistance level of the four S. torvum cultivars against M. incognita was assessed under plastic greenhouse conditions in two cropping seasons. All S. torvum rootstocks responded as resistant to the M. incognita isolates and from highly resistant to susceptible against M. javanica isolates. The maximum multiplication rates of M. javanica on the ungrafted or grafted eggplant were 270 and 49, respectively, and the equilibrium densities were 1318 and 2056 eggs and J2 per 100 cm³ soil, respectively. The tolerance of the ungrafted eggplant was 10.9 J2 per 100 cm³ soil, and the minimum relative dry shoot biomass was 0.76. The population growth rate of M. incognita on eggplant cv. Cristal differed from that of the S. torvum cultivars in both cropping seasons. These results suggest that S. torvum is a valuable rootstock for managing the two Meloidogyne species irrespective of the (a)virulence status.     

A Molecular Marker Correlated with Selected Virulence Against the Tomato Resistance Gene Mi in Meloidogyne incognita, M. javanica, and M. arenaria

ABSTRACT Root-knot nematodes of the genus Meloidogyne are economically important pathogens of a wide range of crops. The tomato resistance gene Mi typically confers resistance to the three major species, M. incognita, M. javanica, and M. arenaria. However, virulent populations completely overcoming the Mi resistance still occur. In an attempt to develop molecular markers for virulence against Mi and gain insights into the genetic relationships among virulent populations of different species and origins, random amplified polymorphic DNA (RAPD) analyses of laboratory-selected virulent, field virulent, and avirulent populations of M. incognita, M. javanica, and M. arenaria were carried out. A RAPD marker, specific for selected virulent populations, was identified, and subsequently, converted to a sequence characterized amplified region (SCAR). Sequence characterization of the SCAR locus showed that alleles from laboratory- and field-selected virulent populations were highly similar to each other and clearly different from alleles from natural virulent and avirulent populations. This result suggests that the genetic mechanism for virulence against Mi may be similar among selected virulent populations of the three Meloidogyne spp., but different between selected and natural virulent populations. Based on the nucleotide polymorphisms at the SCAR locus, codominant and dominant polymerase chain reaction-based markers were developed enabling rapid diagnosis of selected virulent genotypes in M. incognita, M. javanica, and M. arenaria.     

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.     

Host suitability and response of different vegetable genotypes to Meloidogyne incognita race 2 and Meloidogyne javanica in South Africa

The host suitability of 20 locally available genotypes of Capsicum, 10 of Daucus carota, 7 of Beta vulgaris- and 3 of Spinacea oleracea were assessed in separate greenhouse studies for resistance to Meloidogyne incognita race 2 and M. javanica, respectively. Substantial variation existed amongst the vegetable genotypes in the greenhouse screenings with regard to their host status to the respective root-knot nematode species. None of the genotypes of D. carota, B. vulgaris and S. oleracea showed resistance to the nematode species tested. However, resistance to M. incognita race 2 was identified in Capsicum genotype “Tobasco”, which was subsequently verified in a follow-up microplot trial using a range of initial population densities together with a susceptible Capsicum genotype “Paprika”. Reproduction factors of the nematodes were used as the main criterion to evaluate resistance. In the microplot trial, genotype “Tobasco” showed resistance at the lower inoculum levels but not at the higher nematode population levels. The need exists for more frequent and extensive screenings of the various vegetable genotypes in order to provide small-scale farmers with better options for improved and sustainable yields.     

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.     

Evaluating Pochonia chlamydosporia in a double-cropping system of lettuce and tomato in plastic houses infested with Meloidogyne javanica

The effect of Pochonia chlamydosporia , a facultative fungal parasite of nematode eggs, alone or in combination with oxamyl was evaluated in a double-cropping system of lettuce and tomato in unheated plastic houses infested with Meloidogyne javanica at two sites for two consecutive growing seasons. An additional treatment of methyl bromide fumigation was included to compare crop yield in nematode-free vs. nematode-infested soil. Final population densities, reproductive rate, root gall rating, and egg production were determined after each crop. Pochonia chlamydosporia was isolated from nematode eggs up to nine months after application to soil. The fungus survived in the rhizosphere for the entire growing season at one site, but only at low densities. Final population densities of M. javanica decreased after cultivation of lettuce and increased after tomato, and this pattern of population fluctuation was unaffected by treatment, experiment or site. The reproductive rate on lettuce was equal to or below 1, and it was similar among treatments in both experiments at both sites. Eggs were not found on lettuce roots. On tomato, the reproductive rate in the fungus + oxamyl treatment was significantly lower ( P  < 0·05) than other treatments in experiment 1 at both sites. Fungus + oxamyl consistently reduced root gall ratings on tomato in all cases, but numbers of eggs per g root varied depending on treatment. Methyl bromide-treated plots remained free of M. javanica at the end of the 2-year study.