昆虫病原线虫rDNA多态性分析

本文对国内外昆虫病原线虫斯氏属和异小杆属的47个品系进行rDNA—ITS PCR—RFLP分析，研究其DNA多态性，并构建了分子系统发育树状图。各品系的ITS区无明显的长度差异，PCR—RFLP分析将47个品系分为斯氏属和异小杆属两大类，两属线虫又分别分为11组和4组。所得结果丰富了ITS PCR—RFLP图谱库，为弄清我国的昆虫病原线虫与国外种类的分子系统发育关系及未定名线虫的鉴定提供重要依据，同时为筛选适合的线虫种类防治害虫奠定基础。     

Biological control potential of Turkish entomopathogenic nematodes against the Mediterranean fruit fly <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The entomopathogenic nematodes Steinernema weiseri, S. feltiae, S. carpocapsae and two strains of Heterorhabditis bacteriophora, isolated from Turkish soils, were evaluated against larvae of the Mediterranean fruit fly (medfly) Ceratitis capitata in plastic cups under laboratory conditions with sandy loam soil and 10% moisture level. At a rate of 100 infective juveniles (IJs)/cm², the last instar larvae of C. capitata were susceptible to the entomopathogenic nematodes: the S. feltiae 09-31 strain recovered from Aydin provided 78% mortality, whereas S. weiseri and S. carpocapsae killed 50% and 56% of the larvae, respectively. Both strains of H. bacteriophora species caused less than 50% mortality. Except for S. feltiae, the majority of infected medflies died as prepupae or pupae within the puparia. More than 90% larval mortality was recorded at 200 and 400 IJs/cm² for S. feltiae. None of the nematode isolates infected the medfly pupae within the puparia. In pot experiments containing soil, S. feltiae caused 96% and 97% mortality at 100 and 200 IJs/cm², respectively. In pot experiments with grass present, more than 94% mortality was obtained in the presence of grass roots.     

Pathogenic variation and molecular characterization of <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from wilted Welsh onion in Japan

Thirty-two isolates of Fusarium species were obtained from wilted Welsh onion (Allium fistulosum) grown on nine farms from six regions in Japan and identified as F. oxysporum (18 isolates), F. verticillioides (7 isolates), and F. solani (7 isolates). The pathogenicity of 32 isolates was tested on five commercial cultivars of Welsh onion and two cultivars of bulb onion in a seedling assay in a greenhouse. The Fusarium isolates varied in the degree of disease severity on the cultivars. Five F. oxysporum isolates (08, 15, 17, 22, and 30) had a higher virulence on the cultivars than the other isolates. The host range of these five isolates was limited to Allium species. Molecular characterization of Fusarium isolates was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA. The 32 isolates were grouped into eight types (four types for F. oxysporum, one for F. verticillioides, and three for F. solani). Restriction patterns of the ITS region were not related to pathogenicity. However, the haplotypes obtained with five enzymes (RsaI, HinfI, HaeIII, ScrFI, and MspI) and the phylogenetic analysis permitted the discernment of the three Fusarium species. The PCR-RFLP analysis should provide a rapid, simple method for differentiating Fusaruim species isolated from wilted Welsh onion in Japan.     

云南省紫茎泽兰根结线虫病病原种类鉴定

为明确云南省紫茎泽兰根结线虫病的病原种类,于2019年2月在云南省澜沧县林下三七种植区采集根部带有明显根结的紫茎泽兰根系进行根结线虫分离,通过观察所分离根结线虫的2龄幼虫、雌成虫、会阴花纹特征对其进行形态学鉴定,并利用序列比对、系统发育树分析、序列特异性扩增区段(sequence characterized amplified region,SCAR)对其进行分子生物学鉴定。结果表明,该病原线虫雌成虫会阴花纹呈圆形至卵圆形,背弓中等高或低平,侧区一侧或两侧延伸形成翼状,尾区有刻点,2龄幼虫、雌成虫形态特征及形态测量指标与北方根结线虫Meloidogyne hapla相似;该病原线虫rDNA的ITS序列和mtDNA的COI序列与NCBI数据库中已登录的北方根结线虫相应序列相似度较高,分别达99.35%和98.05%以上;该病原线虫rDNA的ITS序列、mtDNA的COI序列分别以99%、100%的支持率与北方根结线虫聚为同一分支;利用SCAR特异性引物,该病原线虫均能扩增出大小约1 500 bp的基因特异性条带。综合形态学和分子生物学鉴定结果将云南省紫茎泽兰根结线虫病病原种类鉴定为北方根结线虫。     

Entomopathogenic nematodes (Nematoda: Steinernematidae, heterorhabditidae) as control agents forParahypopta caestrum, a pest in the culture ofAsparagus officinalis

The entomopathogenic nematodes (EPNs)Steinernema feltiae (under the trade name Entonem) andHeterorhabditis bacteriophora (under the trade name Larvanem) were evaluated as potential control agents ofParahypopta caestrum, the major pest of the asparagus crop in Greece. In laboratory experiments the two nematodes provided equal insect suppression, but significant differences were found in the time it took them to kill the larvae.S. feltiae caused high levels of mortality within 24 h and the highest level at 48 h. In contrast,H. bacteriophora required 96 h to achieve the highest level of mortality. In field experiments, the nematodes provided equal insect suppression as compared with the insecticide cadusaphos and the mixture teflubenzuron +Bacillus thuringiensis var.kurstaki. No significant differences were found in the effectiveness of the insecticides used, but there were significant differences between the control and treatments. The findings showed thatS. feltiae andH. bacteriophora could be used to control the insectP. caestrum in asparagus culture. http://www.phytoparasitica.org posting Jan. 10, 2007.     

Native entomopathogenic nematodes isolated from Turkey and their effectiveness on pine processionary moth,Thaumetopoea wilkinsoni Tams

Two native entomopathogenic nematodes were isolated from soil samples in Eastern Mediterranean region of Turkey and characterized based on 28S rDNA region. BLASTN homology and phylogenetic analysis of SK17 and SK-71 isolates indicated 98% and 99% identity to Steinernema affine and Steinernema feltiae, respectively. The results were constructed by neighbour-joining and bootstrap tree methods. Efficacy of S. affine (SK-17 strain) and S. feltiae (SK-71 strain) was tested against the larvae of pine processionary moth, Thaumetopoea wilkinsoni Tams, and remarkable mortality rates were obtained. Both strains caused complete mortality upon application of 500 IJs in foliar tests. However, the same strains caused 30% and 33% mortality at 80 IJs/cm² in soil applications. It was concluded that these native strains could be considered as potential biocontrol agents for reducing the damage caused by T. wilkinsoni larvae.     

Effect of rapid and gradual increase of osmotic stress on survival of entomopathogenic nematodes

The effect of rapid and gradual exposure of entomopathogenic nematodes to osmotic stresses on the induction of a dormant state was determined with the nematodeSteinernema feltiae IS-6 infective juveniles (IJs). Rapid exposure of nematodes to glycerol at concentrations of 24% and 28% (w/w) caused the nematodes to enter a dormant state which was characterized by shrinking and impeded motility of all nematodes within 8 h. However, pre-exposure to gradually increasing glycerol concentrations of 5%, 10% and 18% at 4-h intervals resulted in dormancy after 4 h exposure to 24% glycerol. The total time of exposure to glycerol solution was 16 h in gradual osmotic stress. For nematodes exposed to 24% glycerol solution either rapidly or gradually, recovery occurred after 40 min in distilled water. Infectivity of osmotically stressedS. feltiae IJs was evaluated by two criteria, insect mortality and invasion rate. The assays indicated that infectivity of nematodes desiccated by rapid and gradual osmotic stresses was similar to that of fresh nematodes. Rapid exposure ofS. carpocapsae ‘All’,S. riobravis ‘Texas’,S. glaseri ‘NI’ andHeterorhabditis bacteriophora HP88 IJs to the 24% glycerol solution resulted in dormancy within 8 h. These treatments caused mortality of 48.4% and 11.7% amongS. glaseri Nl andH. bacteriophora HP88 IJs, respectively. Similar effects were observed when these nematode species were exposed to increasing osmotic stress of 5%, 10% and 18% at 6-h intervals. Under these same conditions, mortality ofH. bacteriophora HP88 andS. glaseri Nl IJs was 27.5% and 61.8%, respectively. http://www.phytoparasitica.org posting Sept. 29, 2004.     

Ribotyping of EntomopathogenicVerticittium lecanii in Japan

To estimate the genetic diversity in 30 isolates ofVerticillium lecanii from aphids, whiteflies, mite and black pine in Japan, including two commercialized strains (Mycotal and Vertalec), DNA polymorphisms in ribosomal DNA of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear ribosomal RNA gene of each isolate were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was ~ 580 bp in 27 of the isolates. A 600 bp ITS product was detected in Mycotal and Vertalec. One Japanese isolate produced both the 580 bp and 600 bp products. Enzymatic digestion of the ITS region with Sau3A I,Msp I,Hae III andRsa I revealed RFLPs that consisted of eight haplotypes. Mycotal and Vertalec were specific haplotypes that differed from other isolates. The Japanese isolates had a complex relationship with the original host, but we identified several specific haplotypes common to an aphid origin. Ten distinct IGS haplotypes were detected in the IGS region, some of which were associated with aphid and whitefly origins. These results suggest that the haplotype of rDNA RFLP analysis can be used for studying genetic diversity inV. lecanii.     

Identification of <Emphasis Type="Italic">Ditylenchus</Emphasis> species associated with Fabaceae seeds based on a specific polymerase chain reaction of ribosomal DNA-ITS regions

A technique based on the use of specific primers for polymerase chain reaction (PCR) was developed for the identification of the stem and bulb nematode belonging to the Ditylenchus dipsaci species complex. The internal transcribed spacer region ITS1 and ITS2, the gene 5.8 S and part of genes 18 S and 26 S of twenty populations of the D. dipsaci species complex belonging to both D. dipsaci sensu stricto and Ditylenchus sp. B (corresponding to populations of giant individuals associated to Vicia faba) and three congeneric species were amplified with two universal ribosomal primers. PCR-amplified DNA samples were digested with five restriction enzymes in order to reveal some polymorphism allowing the identification of D. dipsaci populations associated with Fabaceae seeds. The polymorphism among species was confirmed by the sequencing of the PCR products. A primer (DdpS2) was designed in a region conserved in all populations of both D. dipsaci sensu stricto and D. sp. B studied in the present work. The other Anguinidae species (except a few species from Central Asia associated to Astereaceae and D. sp. G associated to Plantago maritima) differ in two to four nucleotides at the 3′ extremity of this region. This sequence portion coincides with a TspEI restriction site. In combination with a primer located in the ribosomal region, this first primer is a good candidate for identification by PCR of populations of the D. dipsaci species complex found in Fabaceae seeds. A second primer (DdpS1) was designed in a similar way and was specific to D. dipsaci sensu stricto. The utility of these two sets of primers is discussed against the background of quarantine regulation.     

甘肃定西地区马铃薯线虫病病原的分离鉴定

为明确甘肃省定西市马铃薯线虫病的病原种群分类地位,采用形态学结合分子生物学的方法对该地区马铃薯上的4个线虫群体进行了鉴定,观察和测量其形态特征值,基于r DNA-ITS序列以UPGMA法构建线虫群体的系统发育树,并按照柯赫氏法则进行了致病性测定。结果表明,4个线虫群体在形态学上与马铃薯腐烂茎线虫Ditylenchus destructor一致,但群体DX27与群体DX11、DX16、DX19雌虫的体长、体长/食道长、体长/尾长值存在极显著差异。利用通用引物TW81/AB28扩增r DNA-ITS序列均获得长度为915 bp的片段;序列比对分析表明,群体DX27与其它3个群体相比在ITS1区的第96~255 bp片段内有25个碱基的差异;系统发育树显示,群体DX27与C型群体聚为1支,群体DX11、DX16、DX19与B型群体聚为1支。根据形态学特征及r DNA-ITS序列分析结果确定该病原线虫为马铃薯腐烂茎线虫,其中群体DX27属于C型,群体DX11、DX16、DX19属于B型。     

Nematicidal Activity of Chrysanthemum coronarium

Organic amendments and green manure are potential alternatives to the harmful chemical control means currently used against plant-parasitic nematodes. In this work, Chrysanthemum coronarium was applied to the soil as a green manure to control the root-knot nematodes Meloidogyne incognita and M. javanica. Chrysanthemum coronarium significantly reduced nematode infection of tomato roots and improved plant-top fresh weight, both in the greenhouse and in microplots. Other green manures, derived from Anthemis pseudocotula, wild chickpea (Cicer pinnatifidum), Geranium spp. and wheat, were not as effective as C. coronarium. Chrysanthemum coronarium, retained its nematicidal activity even when applied as a dried material. Only mature C. coronarium plants, in their flowering stage, exhibited nematode control activity, but the green plant parts were more effective than the flowers. An aqueous extract of C. coronarium exhibited in vitro, nematostatic activity towards M. incognita and M. javanica second-stage juveniles and inhibited their hatching from eggs and egg-masses; its nematostatic activity was expressed also against other phytonematode species such as Heterodera avenae and Pratylenchus mediterraneus, but did not affect the beneficial entomopathogenic nematode Steinernema feltiae.     

Genomic Variation within Monilinia laxa, M. fructigena and M. fructicola, and Application to Species Identification by PCR

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.     

A new plant pathogenic sterile white basidiomycete from Australia

A sterile white fungus was isolated from the healthy looking roots of buffalo grass (Stenotaphrum secundatum) grown on cleared bush land in Perth, Western Australia. The fungal strain was pathogenic on 12 plant species screened under the greenhouse conditions. The clamp connections and dolipore septa indicated that the isolate was a Basidiomycete. Mycelial features, growth rate at different temperatures, as well as pathogenicity patterns of this sterile white basidiomycete (SWB) were distinctly different from those of a strain with a similar morphology, ATCC 28344, previously described as a pathogen in Florida and Georgia (USA). All attempts to induce sporulation failed. The isolates were also compared using the nucleotide sequence analysis of the ribosomal DNA array. Approximately 1 kbp of the 5 end of the large subunit ribosomal RNA gene, complete sequences of the small subunit ribosomal RNA gene and the entire ITS region (including ITS1, ITS2 and 5.8S gene) were sequenced for the purpose. The obtained sequences were compared with the homologous regions of other genera of Agaricales available in GenBank. Relatively low sequence similarities between the American and Australian strains, as well as the phylogenetic analysis of the studied regions has suggested that these two fungi belong to different genera. Interesting results were achieved in the case of the large subunit ribosomal DNA since this region has been widely studied for taxonomy of Basidiomycetes. The Australian strain 3034 appeared to be closely related to the genus Campanella and the American SWB was identified as belonging to the genus Marasmius, possibly to M. graminum. Our data suggest that the Australian strain is a novel pathogen, and is different from the American SWB isolates described to date.     

First record of the invasive mite<Emphasis Type="Italic">Tetranychus evansi</Emphasis> in Greece

The red spider miteTetranychus evansi (Acari: Tetranychidae) Baker and Pritchard was recorded for the first time in Greece, in the area of Tympaki (south-central Crete) onSolanum nigrum. T. evansi is a pest of crops of the family Solanaceae, which are grown extensively in Crete. The species identification was based on both morphological and molecular data. The second internal transcribed spacer was PCR amplified and sequenced in samples from Crete. Sequences were compared with the sequence ofT. evansi from Brazil and with the ITS2 sequences (retrieved from GenBank) of the two closely related tetranychid species most commonly found in Greece,Tetranychus turkestan andTetranychus urticae.     

Note: Extrapolating the use of an entomopathogenic nematode and fungus as control agents forFrankliniella occidentalis toThrips palmi

Little is known regarding non-chemical control measures againstThrips palmi Karny. Since entomopathogenic fungi and nematodes have been found to be active against thrips species such asFrankliniella occidentalis Pergande, comparative bioassays were conducted to determine the extent to which they also show activity againstT. palmi. Significant mortality of the larvae of the species was recorded following treatment withLecanicillium muscarium (Petch) Zare and Gams, and addition of the wetting agent Agral enhanced pathogenicity toT. palmi. T. palmi pupae were not affected byS. feltiae. The potential for use of these agents againstT. palmi in the field is discussed in the light of these results. http://www.phytoparasitica.org posting Aug. 28, 2005.     

斯氏线虫对黄曲条跳甲田间种群的控制作用

在广州,深圳等多个地点的田间试验结果表明,土壤施用斯氏线虫(Steinernema carpo-capsae)Agriotis 品系的侵染期线虫悬浮液来控制黄曲条跳甲(Phyllotreta striolata)幼虫的种群数量,当线虫剂量为100万条/m~2时,线虫的寄生率为40%—70%,有效虫口密度下降38%～84%;以50万条/m~2剂量的线虫与低浓度(1∶1000)的敌百虫混合施用,对跳甲的作用效果和前一种处理所取得的效果相接近。线虫施进土壤后15—20天内对跳甲幼虫尤其是三龄幼虫种群数量控制作用较好,超过这段时间其作用明显下降。因此,在蔬菜移栽至收割期间,施用二至三次100万条/m~2剂量的线虫悬液或50万条/m~2剂量的线虫与1000倍的敌百虫混合液,对黄曲条跳甲可取得较好的防治效果。     

Association between entomopathogenic nematodes and non-synthetic insecticides for improved control of Nasutitermes spp. (Isoptera: Termitidae) in sugarcane plantations

The aim of this study was to evaluate the in vitro efficacy of the combination of entomopathogenic nematodes and non-synthetic insecticides in the control of Nasutitermes spp. (Isoptera: Termitidae). Bioassays were performed upon insect collection from areas with the high incidence of this pest. The experimental arena for insecticide assays consisted of 80-mL plastic containers with a screened cover. We tested six insecticides and the nematode species Heterorhabditidis bacteriophora RS58, Steinernema glaseri RS38, and Heterorhabditis sp. isolates AL39, AL40, AL41, AL42, AL43, AL44, AL46, and AL47, all of which were grown in last-instar larvae of Galleria mellonella L. (Lepidoptera: Pyralidae). The insecticides Codipirol®, Pure Neem Oil®, and Pyroligneous Acid Extract® caused high mortality in adults of Nasutitermes. The nematode Heterorhabditis sp. AL40 presented a median lethal concentration of 7976 infectious juveniles per adult of Nasutitermes sp. In addition, the mixture of these three non-synthetic products at a concentration of 3% caused complete mortality in Heterorhabditis sp. AL40 and S. glaseri RS38. Future trials should be encouraged in order to evaluate the field efficiency of non-synthetic insecticides mixed or not with entomopathogenic nematodes.     

Intraspecific Variation in Phytophthora citrophthora from Citrus Trees in Eastern Corsica

Isolates of Phytophthora pathogenic to citrus crops on Eastern Corsica and associated with gummosis were identified by PCR-RFLP of internal transcribed spacers (ITS) sequences and characterized by the random amplified microsatellites (RAMS) technique. A sample of 114 isolates collected from diseased trunks and fruits, and from soil, were overwhelmingly Phytophthora citrophthora. Further analysis indicated that the P. citrophthora population was not homogeneous in citrus groves. There were two groups, with a few (4%) atypical isolates in two marginal groups. The major groups have been re-examined in the light of mating behaviour, RFLPs of mitochondrial DNA and sequence comparisons of ITS regions of rDNA. They were found distinct with all these criteria and perhaps constitute distinct taxa. The results indicate that important modifications occurred in the population structure of P. citrophthora over time in Corsican groves. These changes may have impact on the recent outbreaks of gummosis.     

Evaluation of entomopathogenic nematodes for biocontrol of the european corn borer,Ostrinia nubilalis, on sweet corn in Israel

The potential of entomopathogenic nematodes for biological control of the European corn borer (ECB),Ostrinia nubilalis (Hübner), was evaluated under laboratory, screenhouse and field conditions. The ‘All’ and ‘Mexican’ strains ofSteinernema carpocapsae (Weiser) and the ‘HP88’ strain ofHeterorhabditis bacteriophora Poinar were compared in both dose response assays (5, 50 and 500 infective juveniles [IJ] per petri dish containing five 5th-instar ECB eggs; 72 h of incubation) and exposure time assays (3, 6 and 9 h of incubation). In the dose response assays the highest rates of ECB killing resulted from infestation with the Mexican strain ofS. carpocapsae. In the exposure time assays there were no significant differences between the killing rates of the three nematode strains. Sweet corn plants(Zea mays var.saccharata) grown in a screenhouse, were infested with ECB neonates and 4 days later sprayed with a suspension of the Mexican strain ofS. carpocapsae (50,000 IJ per plant). The number of ECB larvae found on treated corn plants after one week was significantly (P=0.05) lower (3- to 5-fold) than the number found on untreated plants. Similar treatment in the field significantly reduced the rate of economic ear damage from 20% to 5%. Contribution from the Agricultural Research Organization. No. 2260-E, 1997 series     

Identification of plant-parasitic nematodes by PCR amplification of DNA fragments1

A study concerning the detection and characterization of a DNA fragment from plant-parasitic nematodes to be used as a molecular marker for the identification of different nematodes is described. A fragment of DNA, which is known to consist of a variable region flanked by two conserved regions, has been studied by using PCR amplification. A portion of about 600 nucleotides at the 5’end of the larger rRNA gene has been amplified in different nematodes, using heterologous primers which hybridize with the conserved regions. The results obtained clearly indicate that the same primers can be used for the amplification of this segment in nematodes of different species and of different genera: Meloidogyne artiellia, M. incognita, Xiphinema index, X. diversicaudatum and Globodera pallida. The amplified regions have been partially sequenced. The nucleotide sequences have been analysed by comparison with the published sequence of Caenorhabditis elegans.