Distribution and genetic diversity of root‐knot nematodes (Meloidogyne spp.) in potatoes from South Africa

A molecular‐based assay was employed to analyse and accurately identify various root‐knot nematodes (Meloidogyne spp.) parasitizing potatoes (Solanum tuberosum) in South Africa. Using the intergenic region (IGS) and the 28S D2–D3 expansion segments within the ribosomal DNA (rDNA), together with the region between the cytochrome oxidase subunit II (COII) and the 16S rRNA gene of the mtDNA, 78 composite potato tubers collected from seven major potato growing provinces were analysed and all Meloidogyne species present were identified. During this study, M. incognita, M. arenaria, M. javanica, M. hapla, M. chitwoodi and M. enterolobii were identified. The three tropical species M. javanica, M. incognita and M. arenaria were identified as the most prevalent species, occurring in almost every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu‐Natal, respectively, while M. chitwoodi was isolated from two growers located within the Free State. Results presented here form part of the first comprehensive surveillance study of root‐knot nematodes to be carried out on potatoes in South Africa using a molecular‐based approach. The three genes were able to distinguish various Meloidogyne populations from one another, providing a reliable and robust method for future use in diagnostics within the potato industry for these phytoparasites.     

象耳豆根结线虫果胶酸裂解酶基因的克隆及其RNAi 效应分析

 从象耳豆根结线虫中首次克隆了果胶酸裂解酶基因Me-pel-1,该基因cDNA 的开放阅读框(ORF)长813 bp,编码270 个氨基酸,具有果胶酸裂解酶第3 家族的4 个保守域特征。ME-PEL-1 与南方根结线虫的果胶酸裂解酶MI-PEL-1 和爪哇根结线虫的果胶酸裂解酶MJ-PEL-1 相似性最高,且系统进化树显示ME-PEL-1 也与它们最为接近。利用RNAi 技术对象耳豆根结线虫2 龄幼虫的Me-pel-1 进行沉默,结果发现Me-pel-1 基因被干扰后,象耳豆根结线虫2 龄幼虫对番茄的侵染率显著降低。该结果表明Me-pel-1 基因在象耳豆根结线虫侵染寄主的过程中发挥重要作用。     

湖南省辣椒上首次发现象耳豆根结线虫

应用线虫比较形态学、同工酶技术和IGS-PCR技术,对采自湖南省永州地区辣椒上的根结线虫进行种类鉴定。结果表明,该病原线虫为象耳豆根结线虫(Meloidogyne enterolobii),这是首次在湖南地区发现象耳豆根结线虫。     

Virulence of Meloidogyne incognita populations and Meloidogyne enterolobii on resistant cucurbitaceous and solanaceous plant genotypes

Journal of Plant Diseases and Protection - Root-knot nematodes (Meloidogyne spp.) cause extensive damage to tomato and cucurbit crops in protected cultivation systems. An environmentally benign...     

输华马铃薯上哥伦比亚根结线虫风险分析

依据国际植物检疫措施标准(ISPM)规定的有害生物风险分析程序(PRAS),利用多指标综合评估方法,通过对哥伦比亚根结线虫(Meloidogyne chitwoodi)的生物学特性、地理分布、寄主、经济意义和危害性等方面进行分析,结合我国进口马铃薯种薯和马铃薯种植情况,确定了其在中国具有传入、定殖和扩散的可能性,对各指标值进行赋值运算,获得总指标值R为2.25,符合高风险的检疫性有害生物标准,因此建议将其列入输华马铃薯检疫性有害生物名单,实施风险管理。本文提出了风险管理措施方案。     

3种药剂对象耳豆根结线虫卵及2龄幼虫的影响

为了筛选对象耳豆根结线虫Meloidogyne enterolobii防治效果好的药剂,本文采用浸虫法分别测试了 3％阿维菌素微乳剂、41.70％氟吡菌酰胺悬浮剂及20％噻唑膦水乳剂不同浓度、不同处理时间对象耳豆根结线虫2龄幼虫的致死作用,以及对卵孵化和卵胚胎发育的影响.结果表明,3％阿维菌素微乳剂对2龄幼虫致死作用最...     

温度和初始种群密度对象耳豆根结线虫侵染番茄的影响

通过室内接种方法,研究了不同温度和接种密度对象耳豆根结线虫侵染番茄能力的影响。研究结果表明,28~30 ℃是象耳豆根结线虫侵染番茄的最适温度。随着温度的下降,其侵染能力明显降低。当象耳豆根结线虫2龄幼虫接种密度大于10条/100 cm3土壤时,番茄的生长会受到显著的抑制,随着线虫接种密度的增加,根结指数显著增加、线虫对番茄的危害也逐渐加重。研究结果将为广东省蔬菜产区制定象耳豆根结线虫防治指标提供理论依据。     

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.     

菜豆普通花叶病毒RT-PCR承检测及3'末端序列分析

根据哈尔滨地区豇豆感病植株的症状,初步鉴定感染豇豆的病毒为菜豆普通花叶病毒(Bean common mosaic virus,BCMV).利用马铃薯Y病毒属通用引物扩增出病毒基因组3'末端序列,经BLAST检索表明该病毒为BCMV,将该序列与GenBank上的21个BCMV分离物的3'末端序列进行比较,显示其核苷酸序列与其他分离物的序列同源性为91.7%～97.3%.系统进化分析显示不同分离物可聚为5个类群,并显示出一定的寄主相关性.哈尔滨分离物BCMV-X与2个浙江分离物、1个澳大利亚分离物聚为一支,且该4株分离物的寄主均为豇豆.RNA二级结构分析显示BCMV基因组3'末端非编码区形成4个茎环结构,不同分离物的序列变化并未引起茎环结构的明显变化.     

A diagnostic assay based on variable intergenic region distinguishes between Leishmania donovani and Leishmania infantum

We have developed a PCR assay that in a single reaction distinguishes between Leishmania infantum and Leishmania donovani strains on the basis of different size of the amplicon. The targeted intergenic region between putative biopterin transporter and nucleotide binding protein on chromosome 35 is highly variable, species-specific and can be amplified from clinical samples. Based on the assay, five tested Leishmania archibaldi and L. infantun strains from the Sudan and Ethiopia clearly belong to L. donovani, which is in accordance with a recent multifactorial analysis of these strains. The nucleotide sequence reported in this paper has been submitted to the GenBankTM with accession number EU068004.     

Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA

ABSTRACT Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.     

Detection of the root-knot nematode Meloidogyne ethiopica in Greece

Root-knot nematodes (RKN), Meloidogyne spp., are very significant in agriculture because they can be found almost everywhere and have a wide host range. In summer 2009, two soil samples from maize and kiwi crops, from the area of Kavalla in North Greece, were analyzed for the presence of nematodes. RKN were detected in both samples and identified as M. ethiopica on the basis of biochemical (esterases) and sequence data (18S rDNA). Meloidogyne ethiopica poses a significant threat to farmers in Greece and the establishment and spread of this species has to be controlled. This is the first report of M. ethiopica in Greece and the second report of this species for Europe.     

基于DNA条形码对粘虫四种近缘种的鉴定与亲缘关系分析

为开发快速、准确的粘虫Myth imna separata (Walker)近源种鉴定技术,通过线粒体COI基因DNA条形码技术对在野外采集的4种粘虫近缘种——白点粘夜蛾Leucania loreyi、淡脉粘夜蛾L.roseilinea、瘠粘夜蛾 L.pallidior和虚研夜蛾Aletia pseudaletiana的COI基因片段进行序列分析,并对4种粘虫近缘种同粘虫和GenBank中其它6种粘虫近缘种之间的亲缘关系进行了分析.结果表明,克隆得到白点粘夜蛾、淡脉粘夜蛾、瘠粘夜蛾和虚研夜蛾大小为658 bp的COI基因片段,与粘虫COI基因片段的核苷酸序列同源性依次为93.31％、92.86％、91.95％和90.88％,遗传距离分别为0.067、0.071、0.081和0.091;所编码的219个氨基酸序列同源性依次为100.00％、100.00％、99.09％和99.09％.与其它6种粘虫近缘种的COI基因片段序列分析中,白点粘夜蛾和淡脉粘夜蛾与粘虫、一点粘虫Mythimna unipuncta、仿劳粘夜蛾L.insueta、白脉粘夜蛾L.venalba聚为1个亚群,亲缘关系较近;瘠粘夜蛾和虚研夜蛾与白杖研夜蛾A.lalbum、角线研夜蛾A.conigera、秘夜蛾M.turca聚为1个亚群,亲缘关系较近.表明4种近缘种与粘虫的亲缘关系由远到近依次为白点粘夜蛾、淡脉粘夜蛾、粘瘠粘夜蛾和虚研夜蛾.     

Evaluation of loop‐mediated isothermal amplification (LAMP) assays based on 5S rDNA‐IGS2 regions for detecting Meloidogyne enterolobii

A loop‐mediated isothermal amplification (LAMP) assay for detection of Meloidogyne enterolobii (Me‐LAMP) was developed based on the sequences of the 5S ribosomal DNA (5S rDNA) and intergenic spacer 2 (IGS2) segment. The LAMP amplification was achieved at 65°C isothermal conditions within 1–1·5 h. Its amplicons were confirmed using gel electrophoresis, SacI enzyme analysis, lateral flow dipstick (LFD) assay, and visual inspection through SYBR Green I and calcein staining. The results demonstrated that the Me‐LAMP was able to specifically detect M. enterolobii populations from different geographical origins, with a detection limit of about 10 fg M. enterolobii genomic DNA, which was 10–100 times more sensitive than conventional PCR. In addition, the applicability of LAMP to field detection was confirmed following its successful performance in detecting the pest on root and soil samples. The Me‐LAMP assay possessed the characteristics of simplicity, sensitivity and specificity, and is a promising and practical molecular tool for M. enterolobii diagnosis in pest quarantine and field surveys.     

根结线虫种群的线粒体DNA分析

 在同工酶和形态学鉴定的基础上,利用引物#C2F3和#1108对42个根结线虫种群线粒体DNA (mtDNA)中的COⅡ和LrRNA间区域进行特异性PCR扩增,35个种群的扩增产物约为1.7kb,其中29个是南方根结线虫,6个是爪哇根结线虫;3个花生根结线虫种群的扩增产物约为1.1kb;1个种群的扩增产物约为0.7kb,为根结线虫属在中国的新记录种;3个北方根结线虫种群的扩增产物约为0.5kb。用单条2龄幼虫提取物作模板得到的结果与大量提取DNA作模板的结果相同。为了区分产生相同大小片段的南方根结线虫和爪哇根结线虫,用限制性内切酶HinfⅠ对扩增产物进行酶切,结果表明:所有供试的南方根结线虫都可以被HinfⅠ酶切,且产生约1.3和0.4kb的2个限制性片段;但供试的爪哇根结线虫种群不能被酶切。由此表明,利用mtDNA PCR及酶切实验可以作为快速而准确地鉴定常见根结线虫的方法。     

Phylogenetic analysis of the citrus Huanglongbing (HLB) bacterium based on the sequences of 16S rDNA and 16S/23S rDNA intergenic regions among isolates in China

Phylogenetic analysis of Chinese isolates of the citrus Huanglongbing (HLB) bacterium based on the 16S rDNA and 16S/23S rDNA intergenic regions sequences was carried out. Nine HLB samples collected from different hosts with different symptoms in seven Chinese provinces, were subjected to PCR for amplifying and sequencing the 16S rDNA. The identity level among Chinese isolates was 98.5% to 100% and was the same with the Indian HLB isolate ‘Poona’ (GenBank accession number: L22532). By contrast, identity values were 97.5% to 97.8% with Candidatus Liberibacter africanus strain ‘Nelspruit’ (L22533), 96.3% to 97.3% with Ca. L. africanus subsp. ‘Capensis’ (AF137368), 95.3% to 96.5% with the Ca. Liberibacter sp. ‘LSg2’ (AY919312), and 94.9% to 96.0% with a strain of Ca. L. americanus from Brazil (São Paulo State; AY742824). A phylogenetic tree constructed with 16S rDNA sequences showed that all Chinese isolates belong to Ca. L. asiaticum. Analysis of the 16S/23S rDNA intergenic region was conducted on 18 HLB-diseased citrus samples with different symptoms, collected in seven provinces. These isolates showed no obvious variation and had an identity level >99.0% with one another. Sequence analysis of 16S/23S rDNA intergenic region and the relative phylogenetic tree showed that the Chinese isolates are very close to Ca. L. asiaticus, and distinct from Ca. L. africanus and Ca. L. americanus. These results suggest that the Chinese HLB isolates belong to the species Candidatus Liberibacter asiaticus. This is the first report on the classification of HLB isolates from China based on molecular investigations.