Nematode Strains Research Articles

Heterorhabditis bacteriophora pampean-strain VEli (Nematoda): identification and pathogenicity against the strawberry pest Lobiopa insularis (Coleoptera: Nitidulidae)

The pampean region in the Buenos Aires province of Argentina is a major horticultural area. Biological pesticides to control pest insects available in the market are scarce in this country and nonexistent based on entomonematodes. In this paper, we characterized a native entomopathogenic nematode isolated from soil in the pampas, and evaluated its infectivity and reproductive capacity in the strawberry sap beetle Lobiopa insularis. Morphological and molecular-genetic analysis determined this isolate to be a nematode of Heterorhabditis bacteriophora, thereafter designated as the VEli strain. A 901-bp genomic-DNA fragment was isolated (GenBank Accesion No KJ575524). The new strain was pathogenic against L. insularis. In 200 ml containers with autoclaved clay soil (100 ml), at concentrations of 1,000; 5,000 and 10, 000 IJs, mortality in larvae ranged from 25 to 77 %. At a concentration of 10,000 IJs, infectivity was higher in larvae and pupae (77 ± 20% and 64 ± 25 %, respectively) than in adults (2.2 ± 1.1 %). The lethal concentration required to kill 50 and 90 % of the larvae (LC50 and LC90) over 10 days was 2,840 and 23,743 IJs. Differences were observed in the parasitism levels and emergence time of IJs between concentrations and stages, although not for production. Heterorhabditis bacteriophora strain VEli is the first entomopathogenic nematode found to infect and kill L. insularis, capable of completing its life cycle in all strawberry sap beetle stages. This availability would enable the establishment and subsequent dispersal of the strain in strawberry fields. Finally, this report constitutes the first presentation of data on the morphological, morphometric, and molecular-genetic characterization of a native entomopathogenic nematode strain from the Buenos Aires province, Argentina.

The combined approach of strain discovery and the inbred line technique for improving control of Delia radicum with Heterorhabditis bacteriophora

Entomopathogenic nematodes are potent biocontrol agents but their efficacy can be compromised under unfavorable environmental conditions. For example, cold tolerance can be an essential requirement when utilizing entomopathogenic nematodes in cooler regions. Discovery of new nematode species or strains that are adapted to local conditions is one approach that can be used to enhance efficacy. Once a strain is isolated however, serial culturing can lead to loss of beneficial traits (such as temperature tolerance). Trait deterioration can be eliminated or reduced by creating homozygous inbred lines. In a novel approach to strain improvement, we combined strain discovery (to obtain superior traits) with the inbred line technique (to stabilize traits). The efficacy of ten homozygous inbred lines from a Heterorhabditis bacteriophora strain isolated in Ontario, Canada was determined against insect larvae, under low temperature conditions. To assess the impact of serial culturing on nematode performance, two wild-type parent populations (the original ‘non-cultured’ parent population, and a repeatedly ‘sub-cultured’ population) were compared with the ten inbred lines. The first experiment evaluated efficacy against Galleria mellonella under five constant temperature regimes from 8 to 24 °C. At 16 °C, eight inbred lines and the ‘non-cultured’ parents demonstrated efficacy against G. mellonella. At the warmest temperatures, all nematode treatments were effective. In a subsequent experiment, the efficacy of the nematode lines and two commercial H. bacteriophora strains was evaluated at 16 °C against the cabbage maggot, Delia radicum. All treatments except one of the inbred lines caused higher levels of infection than the control. The majority of the inbred lines and the ‘non-cultured’ parental line exhibited superior efficacy compared with the ‘sub-cultured’ population and both commercial strains. This study demonstrates the utility of combining strain discovery with the inbred line approach for improved biological control.

Entomopathogenic nematodes as potential biological control agents of Popillia japonica (Coleoptera, Scarabaeidae) in Piedmont Region (Italy)

AbstractIn 2014, the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) was first spotted in northern Italy in the Nature Park of the Ticino Valley, its first detection in continental Europe. This polyphagous invasive species has the potential to cause serious losses to horticulture and agriculture. Particularly for its management in a Nature Park, environmentally friendly strategies are necessary. To develop baseline data for a biological control approach to the Italian outbreak of P. japonica, we conducted laboratory and field experiments testing several indigenous and commercial strains of the entomopathogenic nematode (EPN) species Heterorhabditis bacteriophora and Steinernema carpocapsae against P. japonica larvae. In the laboratory, strains of H. bacteriophora caused greater mortality (ranging from 57% to 100%) than those of S. carpocapsae (3% to 77%). In micro‐plot field tests carried out at three different times, the most virulent indigenous EPN strain, H. bacteriophora ItH‐LU1, showed again the best results ranging from 44% to 93% against young larvae. Finally, in a large‐plot field trial, the commercial H. bacteriophora product provided 46% larval mortality. This study shows that H. bacteriophora strains have good potential as biological control agents of larvae of the invasive P. japonica in northern Italy.

The Combined Effect of Entomopathogenic Nematodes on some Biological Aspects of Cotton Pests Spodoptera littoralis (Boisd) and Agrotis ipsilon (Huf.).

Two species of the Entomopathogenic nematodes, Steinernema carpocapsae and Steinernema scapterisici were studied against cotton leaf worm Spodoptera littoralis (Boisd) and black cutworm Agrotis ipsilon (Hufn.).Under laboratory conditions, by using different nematodes strains at Lc50 values were 3, 6, 7 juveniles IJ/ml of S. scapterisici , S.carpocapsae , combination of both strains and using water for control .The results investigated that effect of all treatments on some biological aspects of target pests such as, larval duration of S. littoralis were recorded that, 11.5, 11.5,11 and 11.8 days, after treatments with S.carpocapsae , S. scapterisici, combination and control, respectively. Furthermore A.ipsilon showed that highly tolerance more than cotton leaf worm,also the percentages of larval mortality were 32 % for S.carpocapsae ,44 % for S. scapterisici, 26% for combination of both strains and no larvae mortality on control treatments. Also there is no significant differences between pupal duration of both target pests while, the results indicated that, significant differences between pupal weight (mg) of S. littoralis and A. ipsilon. No emergency of S.littoralis moths after treatment with strain S.carpocapsae and this percentage increased to 66 % with S. scapterisici, however the combination of both strains affected the emergency of moths to reach 33% compared with control 84%. As regard the 2nd insect pest A.ipsilon showed that highly emergency of moths 92 % for control treatment with water only and 50 % when treated with S. scapterisici and combination of both strains while decreased to 33% for S.carpocapsae. Subsequently, all the biological aspects of adults of both pests as ovi-position, fecundity (Eggs/ female), fertility and also the intermediated shapes were affected mostly by S. scapterisici, these findings might be taken into consideration in evaluating the success of the Entomopathogenic nematodes as a pathogenic insecticides for S.littoralis and A. ipsilon as an economic pests of cotton plants and opens new approaches to develop highly efficient combined biological products on the basis of entomopathogenic nematodes as a bio-agents.

Quantification of resistant alleles in the \u03b2-tubulin gene of field strains of gastrointestinal nematodes and their relation with the faecal egg count reduction test

BackgroundBenzimidazole (BZ) resistance in gastrointestinal nematodes is associated with a single nucleotide polymorphism (SNP) at codons 167, 198 and 200 in the isotype 1 of beta-tubulin gene although in some species these SNPs have also been associated with resistance to macrocyclic lactones. In the present study we compared the levels of resistance in Teladorsagia circumcincta and Trichostrongylus colubriformis by means of the faecal egg reduction test (FECRT) and the percentage of resistant alleles obtained after pyrosequencing. The study was conducted in 10 naturally infected sheep flocks. Each flock was divided into three groups: i) group treated with albendazole (ABZ); ii) group treated with ivermectin (IVM); iii) untreated group. The number of eggs excreted per gram of faeces was estimated at day 0 and 14 post-treatment.ResultsResistance to ABZ was observed in 12.5% (1/8) of the flocks and to IVM in 44.4% (4/9) of them. One flock was resistant to both drugs according to FECRT. Coprocultures were performed at the same dates to collect L3 for DNA extraction from pooled larvae and to determine the resistant allele frequencies by pyrosequencing analysis. In T. circumcincta, SNPs were not found at any of the three codons before treatment; after the administration of ABZ, SNPs were present only in two different flocks, one of them with a frequency of 23.8% at SNP 167, and the other 13.2% % at SNP 198. In relation to T. colubriformis, we found the SNP200 before treatment in 33.3% (3/9) of the flocks with values between 48.5 and 87.8%. After treatment with ABZ and IVM, the prevalence of this SNP increased to 75 and 100% of the flocks, with a mean frequency of 95.1% and 82.6%, respectively.ConclusionThe frequencies observed for SNP200 in T. colubriformis indicate that the presence of resistance is more common than revealed by the FECRT.

Journal Digest

Journal Digest

The pathogenicity and reproduction capability of four entomopathogenic nematode strains on house cricket (Acheta domesticus) in the laboratory condition

It is first time in Vietnam the pathogenicity evaluation and reproduction capacity of four indigenous strains of entomopathogenic nematodes, viz. S-PQ16, S-QTr, H-KT3987 and H-CB3452 on house cricket (Acheta domesticus) were conducted in the labolatory condition. The lethal concentrations for the mortality 50% insect host (LC50) of S-PQ16, S-QTr, H-KT3987 and H-CB3452 strains were 99, 116, 124 and 100 IJs per one cricket, respectively. These lethal concentrations might be relative low and small LC50 values meaning that the high virulence of the epn strains to house cricket. The reproduction capacity of four epn strains in Acheta domesticus was also with high mortality in comparison with other epn strains in Vietnam and in the world. Of these two Heterorhabditis strains, e.g. H-KT3987 and H-CB3452 128.3×103 IJs and two Heterorhabditis strains with 119.6×103 IJs that were higher in comparison with their yields on Galleria mellonella and on some other insect pests. With low lethal concentration and high reproduction capacity these four epn strains to be adapted potential agents for biocontrol of house cricket. In addition, with the high reproduction capacity of epn strain house cricket can used as insect material for epn culturing in vivo in order to restore the toxic actives of the epn strains. In addition, with the available resources and cheap price, the house crickets would be used as a good alternative option for of G. mellonella in maintaining of epn strains for biocontrol of insect pests in Vietnam. Citation: Do Tuan Anh, Nguyen Ngoc Chau, 2017. The pathogenicity and reproduction capability of four entomopathogenic nematode strains on house cricket (Acheta domesticus) in the laboratory condition. Tap chi Sinh hoc, 39(1): 24-31. DOI: 10.15625/0866-7160/v39n1.8336. *Corresponding author: nguyengochau.iebr@gmail.com. Received 16 May 2016, accepted 20 March 2017

Field Monitoring and Efficacy of Entomopathogenic Nematodes Against Chamomile Stem Weevil Microplontus rugulosus and Chamomile Smooth Beetle Olibrus aeneus Under Greenhouse Conditions

Chamomile Matricaria recutita (L.) is an ancient healing plant; it is used for a sore stomach, a mild laxative, anti-inflammatory and a gentle sleep aid. Chamomile plants are infested by chamomile stem-weevil Microplontus rugulosus (Col., Curculionidae) and chamomile smooth beetle Olibrus aeneus (Col., Phalacridae), and can lead to a high reduction in chamomile yield. This work aimed to survey M. rugulosus and O. aeneus by different monitoring methods as well as to manage them by using entomopathogenic nematodes. Sweep nets, water traps, and dissecting the chamomile stems and flowers were used to monitor for M. rugulosus and O. aeneus. Chamomile insects were collected from naturally infested plants moved from the field to the greenhouse. Management of chamomile insects was conducted using three strains of entomopathogenic nematodes; while the control plants were sprayed only with water. Each strain was replicated three times and each replicate included 50 chamomile plants (combined total was 150 for each strain). Three treatment dates were carried out in May, June and July; the first one was mainly for controlling stem weevil, while the other two dates were for smooth beetle. The results showed that 70–80 % of stems had damage and symptoms of stem weevils. Thirty percents from the investigated plants were larvae free of smooth beetle; whiles 70 % was infested with different classifications (e. g. 1, 2, 3, 4 or more than 4 larvae/blooms). The results confirm that smooth beetle infestation and larval numbers were higher in May and June than July. Steinernema carpocapsae had more efficiency than S. feltiae and Heterorhabditis bacteriophora in controlling both insects. Entomopathogenic nematodes could be one of the effective management strategies to control these insects in the chamomile fields.

Acetylcholinesterases from entomopathogenic nematode Heterorhabditid bacteriophora: Susceptibility to insecticides and immunological characteristics

Acetylcholinesterases (AChEs) from the infective juveniles (IJs) of entomopathogenic nematode (EPN) have been investigated with respect to their susceptibility to insecticides and immunological characteristics, aiming at nominating the most compatible insecticide(s) to be used in conjunction with the most insecticide-tolerant EPN strain before incorporation in integrated pest management (IPM) programs. The inhibition kinetics of two purified AChE isoenzymes, AChEAII and AChEBI isolated from Heterorhabditid bacteriophora EM2 strain, by different insecticides revealed that the insensitivity to inhibition by such insecticides could be arranged in a descending order as; methomyl>carbofuran>acetamiprid>oxamyl>malathion. Except for malathion, the insecticides competitively inhibited AChEs with Ki values ranging from 0.1 to 15mM and IC50 values from 1.25 to 23mM. The two AChE isoforms are several folds less sensitive to inhibition by methomyl and carbofuran compared to those previously reported for other insect species. AChEBI was used as an immunogen to raise anti-AChEBI antisera in rabbits. The prepared antisera cross-reacted with AChEs of five different heterorhabditid nematode strains implying the presence of common epitopes shared along all the examined strains. Such studies could aid in the rational selection of the compatible insecticide(s) and the prepared polyclonal anti-AChE antisera would be a valuable immunodiagnostic tool for evaluating the most insecticide-tolerant EPN strain(s) in IPM programs.

The use of entomopathogenic nematodes in the control of stored-product insects

This review highlights the latest findings regarding the use of entomopathogenic nematodes against post-harvest insects. Due to their requirements for moisture, entomopathogenic nematodes had received, until lately, limited attention for application to the dry storage environment. Recent improvements of their formulation and application approaches, as well as the discovery of new, more virulent strains have enhanced the efficacy of entomopathogenic nematodes and renewed interest in using entomopathogenic nematodes in the stored-product environment. The nematode species tested against storage insects belong exclusively to the genera Steinernema and Heterhorhabditis. The virulence of entomopathogenic nematodes against post-harvest insects varies depending on the nematode species and strain. Usually, only a single-nematode species or strain is not equally effective against all major stored-product insect pests; therefore, one nematode strain or species cannot be suitable for controlling all species present in the storage environment, where several insect species coexist. The successful application of entomopathogenic nematodes in storage environments for controlling post-harvest insects is highly dependent on several biotic and abiotic factors, such as the host life stage, temperature, and relative humidity. Entomopathogenic nematodes have still to overcome substantial hurdles to become a reliable alternative for commercial applications in warehouses and storage facilities. However, the use of innovative, enhanced methods of formulation and application, such as encapsulation or bait traps, could boost the exploitation of entomopathogenic nematodes in storage facilities against post-harvest insects.

Laboratory and field evaluation of entomopathogenic nematodes for control of Agriotes obscurus (L.) (Coleoptera: Elateridae)

AbstractThe susceptibility of the dusky wireworm, Agriotes obscurus (L.) (Coleoptera: Elateridae), to different species and strains of entomopathogenic nematodes was tested in a virulence assay in the laboratory. Larvae were exposed to different nematode doses of 50 and 100 IJs/cm2. At a dose of 50 IJs/cm2, only a commercial strain Heterorhabditis bacteriophora Poinar and the native strain Steinernema carpocapsae (Weiser) B14 caused increased mortality compared with the control (11.1% and 13.3% mortality, respectively). At the higher dose tested, all strains (except Steinernema sp. D122) were virulent to A. obscurus larvae. Steinernema carpocapsae B14 caused higher mortality of wireworm (75.6%) and was used for the assay conducted in cages, with a dose of 100 IJs/cm2, in field conditions. The results showed that S. carpocapsae B14 controlled 48.3% of A. obscurus larvae, demonstrating that some entomopathogenic nematodes have the potential to control larvae of A. obscurus. However, further work is needed to improve their efficacy.

Field Efficacy of Steinernema Carpocapsae Against Lima Bean Pod Borer, Etiella zynckenella Treitschke, Attacking Groundnut

Lima bean pod borer (LBPB), Etiella zynckenella Trietschke, is major insect pest of soybean in Indonesia. Currently it was also reported attacking groundnut in some parts of Sumatera causing serious yield loss. Insecticide treatment is not only ecologically unsafe, but also is not practical due to concealed behavior of the larvae within pods below soil surface. Systemic insecticide might be so but unsafe as it might leave unaccepted residue in harvested pods. It is clear that safer more effective technology such as biological control is badly needed. A Field study was conducted at the University of Bengkulu Research Station in 2012-2013. The objectives was to determine the effectiveness of non local strain of entomopathoegen nematode (EPN), Sterinenema carpocapsae , to control LBPB attacking groundnut. Groundnut seeds were sewed in plots which were later treated with S. carpocasae produce by Biological Control Laboratory University of Jember at the dose of 500.000 JI m -2 . EPN was sprayed upon plants and soil surface twice at plant age of 45 and 65 days after sewing (DAS). There were insecticide treatment and control plots as comparison. Observations of damaged pods and the presence of LBPB larvae were done at plant age of 63, 77, 90 DAS and at the haevesting date. Data were subjected to analysis of variance and the means of damaged pods were separated with least significant different (LSD). The result revealed that in two years study, damaged pods consistently tended to be less in plots treated with S. carpocasae or insecticide than those in controls, indicating that it might be effective against PLPB. The lack of significant different between EPN treated and control plots might be as a result of low percentages of EPN larval survival due shipment problem (i.e. inappropriate packaging and long transportation that toke several days) or it might be that the strain was less adapted yet to local conditions. I speculate that local strain of EPN might be more effective to control LBPB attacking groundnut.

Deletion of Phytochelatin Synthase Modulates the Metal Accumulation Pattern of Cadmium Exposed C. elegans.

Environmental metal pollution is a growing health risk to flora and fauna. It is therefore important to fully elucidate metal detoxification pathways. Phytochelatin synthase (PCS), an enzyme involved in the biosynthesis of phytochelatins (PCs), plays an important role in cadmium detoxification. The PCS and PCs are however not restricted to plants, but are also present in some lower metazoans. The model nematode Caenorhabditis elegans, for example, contains a fully functional phytochelatin synthase and phytochelatin pathway. By means of a transgenic nematode strain expressing a pcs-1 promoter-tagged GFP (pcs-1::GFP) and a pcs-1 specific qPCR assay, further evidence is presented that the expression of the C. elegans phytochelatin synthase gene (pcs-1) is transcriptionally non-responsive to a chronic (48 h) insult of high levels of zinc (500 μM) or acute (3 h) exposures to high levels of cadmium (300 μM). However, the accumulation of cadmium, but not zinc, is dependent on the pcs-1 status of the nematode. Synchrotron based X-ray fluorescence imaging uncovered that the cadmium body burden increased significantly in the pcs-1(tm1748) knockout allele. Taken together, this suggests that whilst the transcription of pcs-1 may not be mediated by an exposure zinc or cadmium, it is nevertheless an integral part of the cadmium detoxification pathway in C. elegans.

Virulence of entomopathogenic nematodes and their symbiotic bacteria against the hazelnut weevil Curculio nucum

AbstractThe virulence of different entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae, isolates from Catalonia (NE Iberian Peninsula), and their symbiotic bacteria was assessed with regard to the larvae and adults of the hazelnut weevil, Curculio nucum L. (Coleoptera: Curculionidae). The nematode strains screened included one Steinernema affine, five Steinernema feltiae, one Steinernema carpocapsae, one Steinernema sp. (a new species not yet described) and one Heterorhabditis bacteriophora. The pathogenicity of all the strains of nematodes was tested on larvae and only four of them on adults of the hazelnut weevil. Larval mortality ranged from 10% with S. affine to 79% with Steinernema sp. Adult mortality was higher in S. carpocapsae, achieving 100% adult weevil mortality. The pathogenicity of the symbiotic bacteria Xenorhabdus bovienii, X. kozodoii, X. nematophila and Photorhabdus luminescens was studied in larvae and adults of C. nucum. In the larvae, X. kozodoii showed a LT50 of 22.7 h, and in the adults, it was 20.5 h. All nematodes species except S. affine tested against larvae showed great potential to control the insect, whereas S. carpocapsae was the most effective for controlling adults.

Entomopathogenic nematodes for the control of the codling moth (Cydia pomonella L.) in field and laboratory trials.

Three commercially available entomopathogenic nematode (EPN) strains (Steinernema feltiae and Heterorhabditis bacteriophora Hb1 and Hb2) and two local species (S. jeffreyense and S. yirgalemense) were evaluated for the control of the codling moth (Cydia pomonella). In field spray trials, the use of S. jeffreyense resulted in the most effective control (67%), followed by H. bacteriophora (Hb1) (42%) and S. yirgalemense (41%). Laboratory bioassays using spray application in simulated field conditions indicate S. feltiae to be the most virulent (67%), followed by S. yirgalemense (58%). A laboratory comparison of the infection and penetration rate of the different strains showed that, at 14°C, all EPN strains resulted in slower codling moth mortality than they did at 25°C. After 48h, 98% mortality was recorded for all species involved. However, the washed codling moth larvae, cool-treated (at 14°C) with S. feltiae or S. yirgalemense, resulted in 100% mortality 24h later at room temperature, whereas codling moth larvae treated with the two H. bacteriophora strains resulted in 68% and 54% control, respectively. At 14°C, S. feltiae had the highest average penetration rate of 20 IJs/larva, followed by S. yirgalemense, with 14 IJs/larva. At 25°C, S. yirgalemense had the highest penetration rate, with 39 IJs/larva, followed by S. feltiae, with 9 IJs/larva. This study highlights the biocontrol potential of S. jeffreyense, as well as confirming that S. feltiae is a cold-active nematode, whereas the other three EPN isolates tested prefer warmer temperatures.

Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans.

Many genes play essential roles in development and fertility; their disruption leads to growth arrest or sterility. Genetic balancers have been widely used to study essential genes in many organisms. However, it is technically challenging and laborious to generate and maintain the loss-of-function mutations of essential genes. The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. Here, we have developed a method to induce chromosomal translocations and produce genetic balancers using the CRISPR/Cas9 technology and have applied this approach to edit essential genes in Caenorhabditis elegans. The co-injection of dual small guide RNA targeting genes on different chromosomes resulted in reciprocal translocation between nonhomologous chromosomes. These animals with chromosomal translocations were subsequently crossed with animals that contain normal sets of chromosomes. The F1 progeny were subjected to a second round of Cas9-mediated gene editing. Through this method, we successfully produced nematode strains with specified chromosomal translocations and generated a number of loss-of-function alleles of two essential genes (csr-1 and mes-6). Therefore, our method provides an easy and efficient approach to generate and maintain loss-of-function alleles of essential genes with detailed genetic background information.

Enhanced toxicity of silver nanoparticles in transgenic Caenorhabditis elegans expressing amyloidogenic proteins

The increasing number of applications of silver nanoparticles (AgNP) prompted us to assess their toxicity in vivo. We have investigated their effects on wild type and transgenic Caenorhabditis elegans (C. elegans) strains expressing two prototypic amyloidogenic proteins: β2-microglobulin and Aβ peptide3–42. The use of C. elegans allowed us to highlight AgNP toxicity in the early phase of the worm’s life cycle (LC50 survival, 0.9 µg/ml). A comparative analysis of LC50 values revealed that our nematode strains were more sensitive to assess AgNP toxicity than the cell lines, classically used in toxicity tests. Movement and superoxide production in the adult population were significantly affected by exposure to AgNP; the transgenic strains were more affected than the wild type worms. Our screening approach could be applied to other types of nanomaterials that can enter the body and express any nanostructure-related bioactivities. We propose that C. elegans reproducing the molecular events associated with protein misfolding diseases, e.g. Alzheimer’s disease and systemic amyloidosis, may help to investigate the specific toxicity of a range of potentially harmful molecules. Our study suggests that transgenic C. elegans may be used to predict the effect of chemicals in a “fragile population”, where an underlying pathologic state may amplify their toxicity.

Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains.

Effect of Colombian Strains ofSteinernema colombiense(Rhabditida: Steinernematidae) andHeterorhabditis bacteriophora(Rhabditida: Heterorhabditidae) AgainstEurhizococcus colombianus(Hemiptera: Margarodidae) andAeneolamiasp. (Hemiptera: Cercopidae)

Summary Two entomopathogenic nematodes, Steinernema colombiense sp. nov. (strain SNIO198) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (strain Fresno HNI0100) (Rhabditida: Heterorhabditidae), were isolated from the Colombian Andes and cultured in the laboratory. In semi-field tests against the ground pearl Eurhizococcus colombianus Jakubski (Hemiptera: Margarodidae) on blackberry, neither species was pathogenic to crawlers and only S. colombiense was moderately infective against adults at 105 IJ/ml. Higher activity for both nematodes was observed against immature spittlebugs Aeneolamia sp. (Hemiptera: Cercopidae) infesting kikuyu grass, with H. bacteriophora and S. colombiense killing 100% and 75% of insects, respectively, in our tests. Our results demonstrate the potential use of native strains of entomopathogenic nematodes as biological control agents in Colombia.

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that "simple" organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N'-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a "bisecting" β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.