4种常见根结线虫基因组DNA的RAPD分析

 用120个随机引物对4种常见根结线虫10个小种和类型进行了全基因组随机扩增DNA多态性(RAPD)分析,筛选出的11个适宜引物共扩增出91条RAPD谱带,86条是多态性谱带,占总谱带的94.5%;OPL12、OPK01对4种根结线虫种及其小种扩增的谱型有明显的特异性。聚类分析显示在种间水平上4种根结线虫中花生根结线虫和爪哇根结线虫亲缘关系最近,遗传距离为0.532,北方根结线虫与另外3种根结线虫的亲缘关系最远,平均遗传距离为0.786;种下水平上同种根结线虫的不同小种和类型间存在不同程度的遗传差异,南方根结线虫4个生理小种间,花生根结线虫2个生理小种间亲缘关系较近,爪哇根结线虫2个酯酶谱带类型间,北方根结线虫2个细胞生物学小种间遗传差异较大。在RAPD技术的基础上探索根结线虫分类鉴定的分子方法有着良好的前景。     

佛手山药线虫病害发生调查与病原鉴定

湖北省武穴市佛手山药是“农产品地理标志产品”, 近年佛手山药线虫病害严重, 为此在病源地采集山药土样进行了病害发生调查及病原鉴定?通过形态特征观察?形态特征测计值比较, 以及基于rDNA 28S D2/D3区序列构建短体线虫属线虫群体系统进化树并进行SCAR验证, 对引起佛手山药线虫病的病原线虫进行种类鉴定?结果表明:危害佛手山药的病原线虫的群体特征与咖啡短体线虫Pratylenchus coffeae相似?系统进化树显示:该病原群体与中国香港群体(HQ688677.1)聚类在一个分支上, 置信度达99%?因此, 将该群体线虫鉴定为咖啡短体线虫Pratylenchus coffeae?此外田间调查表明:线虫主要集中分布于10~20 cm深度的土层中, 线虫密度随土壤深度的增加而呈递减趋势, 且连作1年地较连作2年地的线虫数量少, 施用阿维菌素的地块较未施用的线虫数量少?上述结果表明, 在佛手山药生产中实行轮作和科学选择药剂防治, 避免连作, 可降低该线虫发生量?     

Ultrastructure, Morphology, and Sporogenesis of Pasteuria penetrans

ABSTRACT Pasteuria penetrans is a bacterial parasite of root-knot nematodes that shows great potential as a biocontrol agent. Scanning and transmission electron microscopy were used to study the ultrastructure, morphology, and sporogenesis of four isolates of P. penetrans. The effects of different Meloidogyne spp. and tobacco cultivars on sporangium size and morphology of endospores attached to the cuticle of second-stage juveniles (J2) of root-knot nematodes also were investigated. The P. penetrans isolates and their origins were P-20 from M. arenaria race 1 in Levy County, FL; P-100 from Meloidogyne sp. in Pasco County, FL; B-4 from Pratylenchus scribneri in Seminole County, FL; and P-120 from Meloidogyne spp. in Alachua County, FL. Sporangia of the four isolates were identical morphologically and similar in their dimensions, ranging from 2.39 to 3.42 mum in diameter and from 1.38 to 2.38 mum in height. Different Meloidogyne spp. and tobacco cultivars had no effect on sporangium size. Endospores attached to J2 were visualized in three forms: endospores retaining the sporangium wall, endospores covered with a thin exosporium, and endospores without covering. Sporogenesis of P. penetrans was similar to that of other gram-positive bacteria and generally matched the seven-stage scheme reported for Bacillus thuringiensis.     

四种短体线虫的形态和分子生物学鉴定

 采用形态学和分子生物学方法对来自荷兰的藏橐吾和铃兰、泰国的高山榕、缅甸的香蕉等种苗(球)分别鉴定出玻利维亚短体线虫Pratylenchus bolivianus、铃兰短体线虫P. convallariae、咖啡短体线虫P. coffeae和斯佩奇短体线虫 P. speijeri。对这4种线虫的形态特征进行较为详细的描述后,认为头环、口针、侧区、生殖系统和尾形等形态特征是种类鉴定的重要依据;进一步利用引物28S-D2A/28S-D3Br扩增测序得到上述4种线虫的28S rRNA基因D2/D3区序列,序列分析发现玻利维亚短体线虫从欧洲到美洲的不同种群之间的遗传距离变异较小,仅为0~0.007;铃兰短体线虫同一种群不同个体之间的遗传变异较大,为0.006~0.029;咖啡短体线虫不同种群之间的平均遗传距离为0.013;斯佩奇短体线虫不同种群之间的平均遗传距离为0.010;咖啡短体线虫P. coffeae和斯佩奇短体线虫P. speijeri亲缘关系很近,二者种间平均遗传距离仅为0.026。本研究再次证明线虫28S rRNA基因D2/D3区基因序列可作为短体线虫种间鉴定的依据。     

Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root-knot nematode (Meloidogyne spp.)

A molecular protocol is presented for distinguishing seven of the most common and economically important Meloidogyne spp. DNA was extracted from individual second-stage juvenile (J2) nematodes of Meloidogyne spp. and amplified by PCR (polymerase chain reaction). Fifteen PCRs including amplification of rDNA, specific SCAR (sequence characterized amplified region) and RAPD (random amplified polymorphic DNA) fragments were possible from the extracted DNA. This enabled a molecular diagnostic key for M. incognita , M. javanica , M. arenaria , M. mayaguensis , M. hapla , M. chitwoodi and M. fallax to be designed. The key unifies published methods into a single logical schematic using primer combinations that were previously validated and shown to work reliably and specifically. The protocol can be used with single juvenile or adult nematodes and the schematic can readily be expanded to accommodate more species. The use of RAPD amplification to assist with identification of samples which do not yield diagnostic amplification products after the first three steps of the molecular key is also described.     

河南省温县铁棍山药根腐线虫种类鉴定

为明确河南省温县铁棍山药病原线虫种类,在温县铁棍山药种植区采集有明显症状的块茎进行线虫分离,观察线虫的雌、雄成虫的形态特征,并扩增rDNA的28S D2-D3序列对其进行分子生物学鉴定。结果表明,从山药块茎中分离出的根腐线虫形态学特征及测量指标与咖啡短体线虫Pratylenchus coffeae基本一致,其28S D2-D3序列与P.coffeae相似性达99.87%～100%。本研究明确了河南省温县地区铁棍山药根腐线虫为咖啡短体线虫,为山药线虫病的鉴别及防控提供了科学依据。     

四种短体线虫的形态和分子生物学鉴定

采用形态学和分子生物学方法对来自荷兰的藏橐吾和铃兰、泰国的高山榕、缅甸的香蕉等种苗(球)分别鉴定出玻利维亚短体线虫Pratylenchus bolivianus、铃兰短体线虫P.convallariae、咖啡短体线虫P.coffeae和斯佩奇短体线虫P.speijeri。对这4种线虫的形态特征进行较为详细的描述后,认为头环、口针、侧区、生殖系统和尾形等形态特征是种类鉴定的重要依据;进一步利用引物28S-D2A/28S-D3Br扩增测序得到上述4种线虫的28S rRNA基因D2/D3区序列,序列分析发现玻利维亚短体线虫从欧洲到美洲的不同种群之间的遗传距离变异较小,仅为0～0.007;铃兰短体线虫同一种群不同个体之间的遗传变异较大,为0.006～0.029;咖啡短体线虫不同种群之间的平均遗传距离为0.013;斯佩奇短体线虫不同种群之间的平均遗传距离为0.010;咖啡短体线虫P.coffeae和斯佩奇短体线虫P.speijeri亲缘关系很近,二者种间平均遗传距离仅为0.026。本研究再次证明线虫28S rRNA基因D2/D3区基因序列可作为短体线虫种间鉴定的依据。     

Protection of olive planting stocks against parasitism of root-knot nematodes by arbuscular mycorrhizal fungi

The effects were investigated, under controlled conditions, of single and joint inoculation of olive planting stocks cvs Arbequina and Picual with the arbuscular mycorrhizal fungi (AMF) Glomus intraradices , Glomus mosseae or Glomus viscosum , and the root-knot nematodes Meloidogyne incognita and Meloidogyne javanica , on plant performance and nematode infection. Establishment of the fungal symbiosis significantly increased growth of olive plants by 88·9% within a range of 11·9–214·0%, irrespective of olive cultivar, plant age and infection by M. incognita or M. javanica . In plants free from AMF, infection by Meloidogyne spp. significantly reduced the plant main stem diameter by 22·8–38·6%, irrespective of cultivar and plant age. Establishment of AMF in olive plants significantly reduced severity of root galling by 6·3–36·8% as well as reproduction of both Meloidogyne spp. by 11·8–35·7%, indicating a protective effect against parasitism by root-knot nematodes. Infection by the nematodes influenced root colonization by AMF, but the net effect depended on the AMF isolate–olive cultivar combination. It is concluded that prior inoculation of olive plants with AMF may contribute to improving the health status and vigour of cvs Arbequina and Picual planting stocks during nursery propagation.     

Effects of root- and stolon-infecting fungi on root-colonizing nematodes of white clover

The effects on white clover ( Trifolium repens ) of different combinations of the nematodes Meloidogyne trifoliophila , Helicotylenchus dihystera and Heterodera trifolii and nine stolon-infecting and three root-infecting fungi were studied in a glasshouse experiment. The presence of the fungus Phytophthora megasperma alone increased ( P  < 0·001) root-rot severity and reduced ( P  < 0·001) plant growth. Other species combinations, such as Phoma nebulosa and Alternaria alternata , interacted and increased root-rot severity. Combinations of P. megasperma with Pythium irregulare , and P. nebulosa with Phoma medicaginis or A. alternata , increased M. trifoliophila populations. Several other fungi ( P. irregulare , P. nebulosa , Colletotrichum coccodes , Macrophomina phaseolina , P. medicaginis and Phoma sp.) interacted with the nematode M. trifoliophila causing severe root-knot symptoms. The results indicated that fungi and nematodes interacted to cause root and stolon rot and reduced yields, and that poor persistence of white clover in pastures is likely to be a problem with a complex etiology.     

海南岛葫芦科蔬菜根结线虫危害性调查与种类鉴定初报

对海南11个市县的葫芦科蔬菜进行根结线虫危害性调查,结果显示,大部分地区发病率在50%以上;其中琼中腰子、澄迈长安和琼中营根主产菜区的病情指数分别达到了47.12、47.14和40,已造成严重损失。采自海南11个县市5种葫芦科蔬菜24个根结线虫种群经单卵块纯化培养,运用形态学和同工酶的方法对其进行种类鉴定,结果表明,南方根结线虫占总数的79.17%,爪哇根结线虫占总数的12.5%,根结线虫疑似新种或新纪录种占总数的8.3%。南方根结线虫是危害海南葫芦科蔬菜的优势种。     

山西省窖藏薯类块茎中2种短体线虫的种类鉴定

本文采用传统形态学与分子生物学相结合的方法,对采自山西省窖藏马铃薯和薯蓣块茎中的短体线虫进行了种类鉴定.结果 表明,从马铃薯块茎中分离出的短体线虫形态学特征与斯克里布纳短体线虫Pratylenchus scrib-neri一致,其SSU序列与P.scribneri美国群体相似性达99.8％.从薯蓣块茎中分离出的短体线虫...     

Disease complex in coffee involving Meloidogyne arabicida and Fusarium oxysporum

Coffee corky-root disease, also called corchosis, was first detected in 1974 in a small area of Costa Rica where the root-knot nematode Meloidogyne arabicida is the dominant species. An epidemiological study revealed a constant association between Meloidogyne spp. and Fusarium sp. in cases of corky root. No corky root appears to have been reported in association with Meloidogyne exigua , which is the prevalent root-knot nematode on coffee in Costa Rica. Fusarium spp. are often cited as components of disease complexes in association with nematodes. Combined inoculations using M. arabicida or M. exigua with Fusarium oxysporum under controlled conditions showed that only the combination with M. arabicida produced corky-root symptoms on Coffea arabica cvs Caturra or Catuai. Fusarium oxysporum alone was nonpathogenic. Meloidogyne exigua or M. arabicida alone caused galls and reduction in shoot height, but no corky-root symptoms. When cultivars susceptible and resistant to M. arabicida were studied under field conditions for 5 years, all the susceptible cultivars exhibited corky-root symptoms on 40–80% of their root systems. Cultivars that were resistant to M. arabicida but not to M. exigua showed no corky root. These observations lead to the conclusion that corky-root disease has a complex etiology, and emphasize the dominant role of M. arabicida as a predisposing agent to subsequent invasion by F. oxysporum . Consequently, genetic resistance to M. arabicida appears to provide an effective strategy against the disease.     

Variation in resistance to the root-knot nematode Meloidogyne incognita in tomato genotypes bearing the Mi gene

Root-knot nematodes ( Meloidogyne spp.) are among the main pathogens of tomato ( Lycopersicon esculentum ) worldwide. Plant resistance is currently the method of choice for controlling these pests and all the commercially available resistant cultivars carry the dominant Mi gene, which confers resistance to the three main species Meloidogyne arenaria , M. incognita and M. javanica . However the emergence of virulent biotypes able to overcome the tomato resistance gene may constitute a severe limitation to such a control strategy. To date, little was known of the possible influence of the homozygous vs heterozygous allelic state of the Mi locus, or the tomato genetic background, on the expression of the resistance. In order to test both these factors, the resistance was evaluated of a large panel of L. esculentum genotypes (selected from the Vilmorin germplasm stock collection) to seven M. incognita lines avirulent or virulent against the Mi gene. Plant resistance was estimated by counting the egg masses on the root systems after inoculation with second-stage juveniles (J₂). Reproduction of the nematodes was similar or, more often, significantly higher on heterozygous tomato genotypes than on homozygous ones, suggesting a possible dosage effect of the Mi gene. Data also indicated that the tomato genetic background had a major effect on the variations observed in nematode reproduction, especially when tomato genotypes were heterozygous for the Mi gene. These results have important consequences in terms of breeding strategies and durability of the resistance conferred by the Mi gene.     

Detection and analysis of inter- and intraspecific diversity of Pratylenchus spp. using isozyme markers

The root-lesion nematodes of the genus Pratylenchus are an economically important group of plant parasitic nematodes that show high similarity among sibling species. Isozyme patterns obtained by isoelectrofocusing (IEF) were used to differentiate and establish genetic relatedness among Pratylenchus species. A total of 40 populations comprising 9 Pratylenchus species and Radopholus similis from broad host and geographic origins was examined to compare isozyme patterns of esterase (EST), hexoquinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Of these systems, only EST, MDH, PGI and PGM were useful for differentiation of P. vulnus , P. goodeyi , P. penetrans , P. scribneri , P. thornei and R. similis populations. The greatest intraspecific diversity was found within P. coffeae based on the isozyme patterns for MDH, PGI and PGM. Intraspecific variability was also detected among R. similis populations, which showed two isozyme patterns in EST and PGI systems. Less intraspecific variation was found within the P. penetrans group. The P. goodeyi population from Cameroon differed from the other populations in this specific group in its MDH, PGI and PGM phenotypes. Highly similar banding patterns of EST, MDH and PGI activity were found among the P. scribneri populations and the one population of P. agilis . A cluster analysis of the 40 populations, generated from the four enzyme banding patterns, produced groupings that broadly matched the previous classification into specific groups, reflecting intraspecific variability in some cases. The results confirm the potential use of isozyme patterns as markers for these nematode species and their value for diagnostic application.     

Suitability of weed species prevailing in Spanish vineyards as hosts for root-knot nematodes

Commercial vineyards in southern Spain were surveyed and sampled during October to December 2004 to determine the extent to which common weeds present were suitable hosts of root-knot nematodes infesting soils of those vineyards. Seven weed species commonly growing in grapevine soils in southern Spain were found infected by either Meloidogyne incognita or M. javanica: Amaranthus retroflexus (redroot pigweed), Anchusa azurea (ox-tongue), Chenopodium album (goosefoot), Erodium moschatum (musk stork’s bill), Malva rotundifolia (low mallow), Sinapis alba (white mustard), and Solanum nigrum (black nightshade). The host suitability of the weeds to root-knot nematodes was evaluated on the basis of root galling severity and nematode population densities in soil and roots. Also, the host–parasite relationship in these naturally Meloidogyne-infected weeds was examined. All the weed species in the study were considered suitable hosts for M. incognita and M. javanica because: (a) high Meloidogyne spp. populations occurred in roots and surrounding soil of the weed species; (b) the severity of root galling was high, and (c) well-established permanent feeding sites were observed in the histopathological studies of infected root tissues. In addition, this study presents the first reports of S. alba and A. azurea as hosts for M. incognita, and of E. moschatum as a new host for M. javanica, thus increasing the list of reported weed hosts for Meloidogyne spp. These results indicate that noticeable population densities of M. incognita and M. javanica can be maintained or increased in these weeds, at population levels higher than those previously reported for the same nematodes infecting grapevine roots. The weeds infesting vineyards thus represent an important source of inoculum of Meloidogyne spp., and furthermore may act as reservoirs of these nematodes which can be disseminated within or among vineyards by agricultural operations.     

安徽省潜山县栝蒌根结线虫发生危害及大田药效试验

为了有效防治栝蒌根结线虫，调查了安徽省潜山县栝蒌根结线虫及其发生，并分析了栝蒌根围土壤线虫的组成。通过线虫形态学鉴定，认为潜山栝蒌上的根结线虫为Meloidogyne incognita。栝萎根围土壤中有口针线虫占调查总量的50．7％，根结线虫2龄幼虫（J2）占31．8％。对当年栝萎J2在土壤表、中、下层的分布和年发生调查结果表明，J2在各层的分布量因月份不同而不同，并分别于3月初到9月中旬问出现2次虫口高峰；防治试验结果表明，除有机肥外，各试验小区在施药后49天J2虫口相对减退率在89．6％以上；施药后124天，克线丹和1．8％阿维菌素处理的小区增果率为250％～480％，收到很好的防效。     

Comparative host suitability to Meloidogyne spp. and Pratylenchus vulnus in Myrobalan plum (Prunus cerasifera)

Nine Myrobalan plum clones ( Prunus cerasifera ) obtained from in vitro propagation were tested for host suitability to four root-knot nematode (RKN) isolates ( Meloidogyne incognita (2), M. javanica (1), and M. hispanica (1)) and one root-lesion nematode (RLN), Pratylenchus vulnus , isolate. The clones are characterized according to the Ma major resistance genes that control resistance to M. arenaria , M. incognita and M. javanica and are categorized into five resistant (R) and four host (H) clones to these RKN species. As expected, the clones ranged, regardless of the RKN species or isolate, into the same distinct R (P.2175, P.1079, P.2980, H.17 and H.21) and H (P.2794, H.18, H.23 and P.16.5) groups for RKN resistance. The P. vulnus tests showed that all the clones had higher final populations than initial populations ( P _f :  P _i > 1) and that P _f :  P _i ratios were equivalent in each RKN resistance group (R or H). Consequently, resistant alleles of RKN Ma genes in Myrobalan plum have no major effect on RLN multiplication. Our data must be taken into account for breeding strategies aimed at incorporating multiple resistance to both RKN and RLN in Prunus spp.     

甘肃省白银市保护地蔬菜根结线虫病病原鉴定

2012年5月-7月在甘肃省白银市采集了番茄、黄瓜、豇豆等几种保护地蔬菜的25个根结线虫群体.直接解剖获得雌虫,通过雌虫会阴花纹观察,结合酯酶和苹果酸脱氢酶(MDH-EST)测定,鉴定出所收集的25个群体中有16个为南方根结线虫[Meloidogyne incognita (Kofoid&White)],9个为爪哇根结线虫[M.javanica (Treub)].南方根结线虫为优势种群,本次试验也是首次在甘肃省发现爪哇根结线虫.     

南海口岸进境林木种苗线虫检疫情况分析

本文汇总了2008～2010年南海口岸进境林木种苗的线虫截获情况,共截获15科26属超过40种植物寄生线虫,其中检疫性线虫有7种,分别为短体线虫(非中国种)Pratylenchus Filipjev(non-Chinese species)、毛刺线虫属(传毒种类)Trichodorus Cobb(The species transmit viruses)、根结线虫(非中国种)Meloidogyne Goeldi(non-Chinese species)等;根据携带线虫状况分析了检疫性线虫的截获情况,提出了防止危险性线虫入境、扩散措施,为口岸防止线虫入境提供借鉴。     

我国南方地区主要根结线虫DNA变异的RAPD分析

 本试验用5组随机引物对来自我国南方地区的30个根结线虫种群进行RAPD分析,并从中筛选出多态性较好的引物12个。共扩增出179条DNA多态带,各供试种群间存在着丰富的遗传多态性。扩增结果表现出种间差异大于种内差异的共同趋势,这表明上述12个引物能够较客观地反映种群间亲缘关系的远近。北方根结线虫与另外3种线虫(南方根结线虫、爪哇根结线虫、花生根结线虫)的亲缘关系最远;在3种主要根结线虫中,爪哇根结线虫与南方根结线虫的亲缘关系相对较近。基于种群间的相似系数分析和应用UPGMA法构建的聚类树状图,显示出不同的根结线虫在较低的相似性系数范围聚类,而绝大多数种内的不同种群均以较高的相似性系数聚在一起,这与形态分类基本一致,反映了形态学分类的分子遗传本质,同时也表明了应用RAPD技术进行根结线虫亲缘关系分析和种类鉴定具有合理性和可行性。本文还对RAPD方法对南方根结线虫小种鉴定的可能性进行了初步探讨。