Trichodorid Nematodes Research Articles

Transcriptome Sequencing Reveals the Genome Sequence of Pea Early Browning Virus from a 29-Year-Old Faba Bean Sample.

Pea early browning virus (PEBV) is transmitted by soil-inhabiting trichodorid nematodes and via seeds. The transcriptome sequencing method, followed by de novo assembly, revealed the PEBV Libyan isolate LyV66-91 genome. Its RNA1 resembled that of UK isolate SP5 with 93.91% nucleotide identity, and its RNA2 had 63.32% nucleotide identity to that of Dutch isolate E116.

Developing a One-Step Multiplex PCR Assay for Rapid Detection of Four Stubby-Root Nematode Species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus.

Four trichodorid species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus, were found in multiple states in the United States. Traditional diagnosis based on morphology and morphometrics is laborious and requires an experienced taxonomist. Additionally, end-point diagnosis using PCR was only available for P. allius. To increase diagnostic efficiency and reduce costs, a one-step multiplex PCR assay was developed to simultaneously identify these four species using one PCR reaction. Available sequences of 18S ribosomal DNA and internal transcribed spacer 1 (ITS1) region of these species were aligned and five primers were designed. The conserved forward primer located in the 18S region, in combination with the species-specific antisense primer in the ITS1 region, amplified a single distinctive PCR fragment for each species (421/425 bp for P. allius, 190 bp for P. minor, 513 bp for P. porosus, and 353 bp for T. obtusus). In silico analysis with 10 other trichodorid species and experimental analysis using samples with these four species, 20 other plant-parasitic and three non-plant-parasitic nematodes demonstrated high specificity with the primers designed. The multiplex PCR amplified desirable fragments using a set of artificially mixed templates containing one, two, three, or four targeted species. The reliability of multiplex PCR results was demonstrated by using nematode populations isolated from infested fields from diverse geographic regions in eight states. The multiplex PCR-based tool developed in this study for the first time provides a simple, rapid, and cost-friendly assay for accurate diagnosis of the four major trichodorid nematodes in the United States.

Morphological and molecular characterisation of Trichodorus golestanensis (Nematoda: Trichodoridae), a new species from Iran

Several species of trichodorid nematodes cause economically important disease on plants. Trichodorus comprises most of the trichodorid species. Trichodorus golestanensis n. sp. is described from a forest park in northern Iran. The new species belongs to the T. lusitanicus morpho-species group and is characterized in male by three ventromedian cervical papillae of which two at level of onchiostyle region, three ventromedian precloacal supplements, the posterior one just anterior to retracted spicules and ventrally curved spicules with mid-blade indentation provided with a few bristles. Females are distinguished by well developed triangular vaginal sclerotized pieces in lateral optical view, a rhomboid-shaped vagina and a transverse slit-like vulva in ventral view. Molecular analysis using the sequence of D2-D3 expansion segment of 28S rDNA differentiated the new species and confirmed the relationships of T. golestanensis n. sp. with T. andalusicus and T. asturanus. The sequences of D2-D3 regions were also provided for previous trichodorid records from Iran T. gilanensis, P. teres and N. minor together with discussion of their relationship.

Molecular diagnosis of trichodorid species from Portugal

A PCR‐RFLP assay was developed for the identification of trichodorid nematodes belonging to Nanidorus, Paratrichodorus and Trichodorus genera. Using the variability of the 18S SSU rDNA gene, this method provides a new molecular diagnosis tool which allows identification within mixed samples of trichodorid and non‐trichodorid species, differentiation of juveniles, and represents an alternative to the difficult and time consuming phenotyping of similar species. Based on the alignment of previously obtained 18S rDNA nucleotide sequences of trichodorids from Portugal, a pair of selective primers was designed in conserved regions to allow the amplification of a variable region located at the 3′ end of the gene in all known Portuguese species. The 615 bp PCR product showed nucleotide variability enabling the generation of restriction fragment patterns which were consistent among populations of the same species but allowed discrimination of trichodorids at the species level. The proposed protocol was tested and proved effective with 12 trichodorid species from Portugal (N. minor, P. allius, P. anemones, P. divergens, P. hispanus, P. pachydermus, P. porosus, T. beirensis, T. lusitanicus, T. primitivus and two other Trichodorus species, A and B) and six non‐indigenous trichodorid populations (N. minor, P. allius, P. anemones, P. pachydermus, P. porosus and T. primitivus).

Tobraviruses—plant pathogens and tools for biotechnology

The tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), are positive-strand RNA viruses with rod-shaped virus particles that are transmitted between plants by trichodorid nematodes. As a group, these viruses infect many plant species, with TRV having the widest host range. Recent studies have begun to dissect the interaction of TRV with potato, currently the most commercially important crop disease caused by any of the tobraviruses. As well as being successful plant pathogens, these viruses have become widely used as vectors for expression in plants of nonviral proteins or, more frequently, as initiators of virus-induced gene silencing (VIGS). Precisely why tobraviruses should be so effective as VIGS vectors is not known; however, molecular studies of the mode of action of the tobravirus silencing suppressor protein are shedding some light on this process.

Phylogenetic relationships, based on SSU rDNA sequences, among the didelphic genera of the family Trichodoridae from Portugal

Abstract A survey of virus vector trichodorid nematodes was carried out in the central and northern regions of Portugal. Morphobiometric identification showed the presence of trichodorid species previously reported from Portugal, except for Paratrichodorus porosus, which is reported for the first time in Continental Europe. Small subunit ribosomal DNA (SSU rDNA) sequences of ten different species occurring in Portugal were obtained and a phylogenetic analysis based on their alignment was performed to infer relationships among the different Portuguese trichodorid species and three non-indigenous populations (Nanidorus minor, P. allius and P. teres). The resulting phylogenetic tree is in agreement with the currently accepted classification for Trichodoridae, except for Nanidorus, which clusters together with Trichodorus species, while the genera Paratrichodorus and Trichodorus appear as two distinct groups. A better understanding of the generic groupings in the family Trichodoridae was found. Based on the new molecular analyses we herein accept Nanidorus as a valid genus.

First Report of Trichodorus similis from the Czech Republic (Nematoda: Trichodoridae).

Members of the Trichodoridae can cause substantial crop losses directly by feeding on plant roots and indirectly as vectors of tobraviruses; both vector and virus are polyphagous. Although trichodorid nematodes are important pests of agricultural crops, no data are available on the presence or extent of these nematodes in the Czech Republic. In June 2005, three soil samples from the rhizosphere of a Quercus sp. at Cerveny Vrch yielded a population of Trichodorus similis Seinhorst, 1963. Specimens were extracted from soil by a decanting-sieving method, heat killed, and fixed in triethanolamine formalin (TAF), and processed and mounted in anhydrous glycerin. Nematodes were identified by morphological and morphometrical characters (2). Classical identification of these nematodes was further verified by molecular study. A single, male specimen was temporarily mounted in distilled water on a glass slide and relaxed with gentle heat. Measurements and photographs were taken, and the specimen was transferred to a 0.5-ml Eppendorf tube containing 0.25 M NaOH. Total genomic DNA was prepared by a rapid technique (4). Morphometric data of the male specimen used for DNA study are: body length 867 μm; body width 81 μm; onchiostyle length 44 μm; spicule length 36 μm; distance of anterior cervical papilla (CP)1 from anterior end 39 μm, CP1 to CP2 25 μm, CP2 to CP3 22 μm; posterior precloacal supplement (SP1) to cloacal opening 27 μm, distance SP1 to SP2 32 μm, distance SP2 to SP3 39 μm. The following primers were used in the PCR reaction: species-specific sense primer SIMIREV2 (5'-CACTCGTCGGACTCAAACC-3') and universal antisense primer UNIVERSAL (5'-CCCGTCGCTACTACCGATT-3') (1). A single fragment of approximately 452 bp was amplified. The D2 and D3 expansion regions of the large subunit 28S rDNA were amplified using the primer D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3' (3). The region was sequenced after purification of PCR products from the gel slice with a Qiagen gel purification kit (Qiagen Inc., Valencia, CA). The obtained sequence was deposited in Genbank (Accession No. DQ832183). The obtained sequence was compared by BLAST in NCBI and the results showed strong similarities with T. similis (Accession No. AM180730). To our knowledge, this is the first report of T. similis associated with a deciduous forest in the Czech Republic. Taking into account the agricultural importance of trichodorids and tobraviruses as plant pathogens, there is a need for a comprehensive survey of these taxa in the Czech Republic. The damage level threshold is in the case of virus vector species equivalent to a single nematode. Therefore, information on these plant parasites would be useful for developing nematode management strategies.

Real-time PCR detection and quantification of vector trichodorid nematodes and Tobacco rattle virus

This report describes a novel diagnostic method for virus-vector trichodorid nematodes and associated Tobacco rattle virus (TRV) based on a real-time fluorogenic 5' nuclease PCR assay (TaqMan). Two independent primer/probe sets were designed targeting the 18S gene of the ribosomal cistron for the trichodorid species, Paratrichodorus pachydermus and Trichodorus similis. Assays using purified plasmid DNA containing clones of the 18S region and genomic DNA extracted from individuals from both nematode species displayed high specificity as no cros s-reaction was observed between the species or with two non-target trichodorid species Paratrichodorus anemones and Trichodorus primitivus. Relative quantification of target DNA present in unknown samples was performed by comparison of the fluorescence signals of the samples to those obtained from plasmid standard dilutions. Three primer/probe sets were also used to target TRV; one set for RNA1 and the two other sets for RNA2 of specific isolates (TRV-PpK20 and TRV-TpO1). Detection of both trichodorid species and TRV RNA1 and RNA2 from a single sample was achieved and field samples were used to demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular information in relation to risk assessment in the field.

Molecular diagnostics of some trichodorid nematodes and associated Tobacco rattle virus

Several Paratrichodorus and Trichodorus (trichodorid) nematode species are natural vectors of Tobacco rattle virus (TRV) and cause economically important diseases, especially in potato and ornamental bulbous crops. Identification of trichodorid species based on morphological characters is laborious, time‐consuming, and requires the services of highly trained personnel. Molecular diagnostics for trichodorid nematodes, using the ribosomal DNA repeat unit, were successfully developed to distinguish two Paratrichodorus and two Trichodorus species. The complete sequences of the 18S genes and the ITS‐1 regions for these species were obtained and species‐specific primers successfully designed for them. An RT‐PCR assay was developed utilizing isolate‐specific primers that amplify serologically distinguishable strains of TRV in individual trichodorid nematodes. The primers were based on the highly conserved RNA‐1 segment of the bipartite genome and also on different parts of the RNA‐2 segment of the virus genome.

An in vitro examination of the feeding behaviour of Paratrichodorus anemones (Nematoda: Trichodoridae), with comments on the ability of the nematode to acquire and transmit Tobravirus particles

AbstractThe plant-parasitic nematode Paratrichodorus anemones efficiently transmits tobacco rattle virus that causes substantial economic damage to vegetable and ornamental flower-bulb crops. The nematode acquires and transmits virus particles when feeding on plant roots, but previous studies on feeding by trichodorid nematodes suggested that the nematodes destroyed the cells upon which they fed. For successful establishment of virus infection in a plant, virus particles would have to be introduced by the nematode into root cells that would remain fully functional. Consequently, the efficient transmission of the genus Tobravirus by trichodorids could not occur where the majority of attacked root cells become irreversibly damaged and, therefore, unsuitable for establishment of virus infection. Live specimens of P. anemones feeding on Nicotiana tabacum seedlings growing in agar were examined in real time using videoenhanced interference light microscopy to determine how trichodorids acquire and transmit Tobravirus particles. The feeding cycle of P. anemones had four distinct phases: i) root exploration, ii) cell exploration, iii) cell sampling, and iv) cell feeding, followed by a quiescent period. Preceding the initiation of phase iv) an average of four cells, which had been perforated by the nematode onchiostyle, were immediately abandoned and thus remained fully functional. Also, during this phase, about 5% of the perforated cells remained alive and fully functional, providing a suitable environment for the establishment of virus infection. Each feed on a single cell was similar to that previously observed with Trichodorus similis, being in four phases, i.e., cell wall perforation, salivation, ingestion and withdrawal.

EFFECT OF GREEN MANURE CROPS AND ORGANIC AMENDMENTS ON INCIDENCE OF NEMATODE-BORNE TOBACCO RATTLE VIRUS

Tobacco rattle tobravirus (TRV) may infect several ornamental bulb crops and is transmitted by trichodorid nematodes. Paratrichodorus teres, P. pachydermus and Trichodorus similis are the main vectors in the Netherlands. In field experiments the effects of various pre-crops and organic amendments on the TRV Infection Potential of Soils (TRV-IPS) and on disease level in tulip and gladiolus were studied. Organic matter amendment of soil at a rate of 1% dry weight has been shown to reduce the host finding activity of P. teres under laboratory conditions. In a field containing viruliferous P. teres dahlia, italian ryegrass, white mustard and fodder radish were grown or the soil was kept fallow and the resulting TRV-IPS prior to the bulbous test crops was measured by a soil dilution bait test method. The application of organic matter was tested after dahlia as pre-crop. Household waste compost (GFT compost) was applied as a soil mix or planting furrow treatment at 12 tons dry weight per ha for tulip and gladiolus. Spent mushroom compost (Champost) was added as planting furrow treatment at 17 or 12 tons dw/ha, respectively, for tulip and gladiolus. The percentage of TRV diseased plants was determined at flowering in all pre-crop and organic amendment treatments. Champost in the planting furrow and fodder radish as a preceding crop reduced the percentage infection in tulip under favourable conditions for TRV infection. In gladiolus most organic amendments, fodder radish as pre-crop and keeping the soil fallow reduced the TRV infection rate of the plants during the first growing season, but not of the plants grown from the corms in the next year.

Root bacteria from nematicidal plants and their biocontrol potential against trichodorid nematodes in potato

Trichodorid nematodes (Nematoda: Trichodoridae) are vectors of tobacco rattle virus (TRV), one of the causal agents of spraing disease in potato. Root bacteria from nematicidal plants and their control potential against Trichodoridae were the focus of this study. Bacteria isolated from the roots of 12 nematicidal plants and potato were characterized for their production of hydrolytic enzymes, hydrogen cyanide, phenol oxidation ability and antifungal activity towards the potato pathogen Rhizoctonia solani. Based on these functional traits, bacteria isolates were selected and tested in greenhouse conditions on potato (cv. Saturna) for their effect on plant growth, and screened for nematicidal activity against Paratrichodorus pachydermus and Trichodorus primitivus in naturally infested soil. Sixteen bacteria isolates out of 44 reduced nematode densities by 50–100%. Nine selected isolated were further tested by bacterizing potato tubers (cv. King Edward) which were planted in a trichodorid and TRV-infested soil. Four bacterial isolates consistently reduced nematode densities (by 56.7–74.4%) with no visual negative effect on plant growth. These isolates were tentatively identified, partly by fatty acid methyl ester (FAME) analysis as: Stenotrophomonas maltophilia, Bacillus mycoides, Pseudomonas sp., and one unidentified bacterium. The isolates originated from potato, Plantago major, Thymus vulgaris and Asparagus officinalis, respectively. Two Pseudomonas isolates obtained from Zinnia elegans and selected for their strong nematicidal activity in soil screening tests, did not reduce the nematode population when tested on potato. It is concluded that plants releasing nematicidal compounds may harbour nematode-antagonistic bacteria as well.

The application of safranin-O for staining virus-vector trichodorid nematodes for electron and confocal laser scanning microscopy

Abstract Post-fixation with osmium tetroxide (OsO4), prior to examination by electron microscopy, enables nematodes to be located in epoxy-resin blocks, and improves contrast of ultrastructural features. For specimen visibility, alternatives to OsO4 were examined as it can inhibit antigenicity, thus preventing application of serological techniques. Basic fuchsin stain does not affect antigenicity and when applied to trichodorids enabled specimens to be readily located in the block, but had several disadvantages such as causing substantial alteration of the nematode structure. Safranin-O stain enabled Paratrichodorus anemones and P. pachydermus specimens to be located in resin blocks, different parts of the nematode body to be distinguished, and did not affect specimen ultrastructure. Also, with viruliferous specimens it allowed immunogold labelling techniques to be applied for identifying tobacco rattle virus particles at the site of retention in the nematodes. Safranin-O is fluorescent and this feature was used to examine sections from the spicula region of a male P. pachydermus specimen under a confocal laser scanning microscope. La post-fixation au tétroxyde d’osmium (OsO4) avant l’observation en microscopie électronique empêche la localisation des nématodes dans les blocs de résine epoxy et augmente le contraste des caractéristiques ultrastructurales. Pour la localisation des spécimens, des solutions alternatives au OsO4, qui peut inhiber l’antigénicité et donc empêcher l’application de techniques sérologiques, ont été examinées. Le coloration à la fuschine basique n’affecte pas l’antigénicité et permet de situer les spécimens dans les blocs, mais elle présente de nombreux désavantages telle l’altération substantielle des structures du nématode. La safranine-O permet la localisation de Paratrichodorus anemones et de P. pachydermus, les différentes parties du corps du nématode étant alors identifiables, et n’affecte en rien l’ultrastructure du nématode. Chez les spécimens portant des virus, elle permet également l’utilisation de techniques de marquage immunologique à l’or pour identifier les particules du virus du tobacco rattle dans les sites de rétention chez le nématode. La safranine-O est fluorescente et cette particularité a été utilisée pour observer des sections de la région des spicules chez un mâle de P. pachydermus en microscopie confocale à balayage.

Evidence for persistent, symptomless infection of some potato cultivars with tobacco rattle virus

Infection with an M-type (particle producing) isolate of tobacco rattle virus (TRV) was detected in leaves and/or roots of some plants from 11 of 13 potato cultivars grown in soil containing viruliferous trichodorid nematodes. Virus was detected in tubers of 8 of 13 cultivars, although only two (Pentland Dell and Maris Bard) developed spraing symptoms. Six cultivars (Arran Consul, Home Guard, King Edward, Romano, Sante and Wilja) were infected with TRV without developing spraing symptoms. Plants grown from virus-containing, symptomless tubers became systemically infected with M-type TRV and produced symptomless infected daughter tubers. Virus was maintained through three generations of vegetative propagation, and the plants were sources for acquisition of the virus by trichodorid nematodes. Distribution of virus in both spraing-affected and symptomless tubers was erratic. Movement of symptomlessly infected seed tubers may be a means of dissemination of the virus and of its introduction to previously unaffected sites.

Immunological detection and mutational analysis of the RNA2-encoded nematode transmission proteins of pea early browning virus.

Pea early browning virus (PEBV) is transmitted between plants by root-feeding trichodorid nematodes. Mutagenesis studies have implicated two non-structural viral proteins in the transmission process. These two proteins [the 29 kDa ('29K') protein and the 23K protein] were expressed in bacteria and used to raise antibodies. In Western blotting experiments, the antibodies detected both of these virus proteins in leaves and roots of infected Nicotiana bethamiana and N. clevelandii plants. Periodate treatment of proteins transferred to nitrocellulose membranes suggested that the PEBV 23K protein may be glycosylated. A PEBV mutant was constructed lacking the complete 23K coding sequence. The mutant was able systemically to infect Nicotiana spp. but caused striking chlorotic ringspot leaf symptoms and stunting of both leaves and roots. These symptoms were absent in plants doubly-infected with the mutant and wild-type PEBV. The 23K gene deletion mutant was transmitted by nematodes at a much reduced frequency compared to wild-type virus, indicating that the 23K protein is involved in but not essential for vector transmission. Western immuno-blot and ELISA experiments revealed that the reduction in the nematode-transmissibility of PEBV carrying mutations in the 23K gene did not result from interference in the expression of the 29K transmission protein or from gross changes in the titre of virus in the roots of infected plants.

Viral Determinants of Pea Early Browning Virus Seed Transmission in Pea

Pea early browning virus (PEBV) is a member of the genus, Tobravirus. It is transmitted by soil-inhabiting trichodorid nematodes and through seeds from diseased plants. By introducing mutations into the PEBV genome, we have studied the viral determinants of seed transmission in pea. Neither deleting a portion of the genome containing the three nonstructural genes in RNA2 nor the interuption of any of the three genes individually prevented PEBV seed transmission. However, a comparison of two PEBV isolates indicated a minor role for RNA2 or its products. In contrast, the removal of the coding sequence of the 12K gene in RNA1 almost completely abolished viral seed transmission. The virus lacking the 12K gene caused more severe symptoms on leaves and pods, and accumulated to a higher level than the wild-type virus in both types of tissues. However, the 12K deletion mutant accumulated poorly in anthers and carpels, and could not be detected in pollen grains and ovules. These results suggest that the 12K gene is involved in the infection of the gametic cells and hence the seed transmission of PEBV in pea.

The Occurrence and Distribution of Trichodorid Nematodes and Their Associated Tobraviruses in Europe and the Former Soviet Union

Information on the distribution and occurrence of Trichodorus and Paratrichodorus species and their associations with tobraviruses in Europe and the former Soviet Union (FSU) is reviewed. A total of 17 Trichodorus and 13 Paratrichodorus species have been reported and of those, 14 have been reported to transmit virus. Paratrichodorus pachydermus and Trichodorus primitivus are the species most often associated with virus transmission.

Analysis of the occurrence and distribution of tobacco rattle virus in field soil and disease in a subsequent tulip crop

Summary.The occurrence and distribution of tobacco rattle virus (TRV) in field plots was determined by soil bait‐testing and disease incidence in tulips subsequently grown on these plots was studied. The virus occurred in patches, calculated as 1.5 m × 3.6 m. The presence of virus was not correlated with numbers of potential vector trichodorid nematodes. Of three trichodorid nematode species present, only Paratrichodorus teres transmitted TRV which, as with virus isolates obtained in bait‐tests and from infected tulips, reacted in serological tests with an antiserum prepared against a Dutch isolate of pea‐early browning virus (PEBV). Virus prevalence in a subsequent tulip crop was 0.8% and in a sample of tulip plants, virus was recovered only from plants showing virus symptoms. Plots from which TRV was recovered in bait‐tests yielded significantly more virus diseased tulips than plots which tested negative for virus. Growing bait‐plants in field‐plots, as compared with greenhouse tests using soil collected as a series of sub‐samples, resulted in an underestimate of the occurrence of TRV.

Pea early‐browning tobravirus

A disease of pea caused by pea early‐browning virus was first described in the Netherlands, and shortly afterwards was reported from England. The virus has since been reported from several other European countries, and also from North Africa. It can systemically infect crop legume species and the disease is locally important in pea in the Netherlands and England. The virus, a member of the Tobravirus genus, is transmitted through sandy or light‐textured soils by species of trichodorid nematodes, and also through seed of pea and faba bean. Seed transmission is the most probable means of virus spread, especially over long distances, to previously uninfested soils. Aspects of the disease including symptoms, soil and seed transmission, detection, resistance and control, and characteristics of the pathogen, are reviewed.

Transmission of Viruses by Plant Nematodes

ABSTRACf Longidorid and trichodorid nematodes transmit nepo- and tobraviruses, respec­ tively. A specific association exists between vectors and their viruses which is a consequence of the nature, site and mechanism of virus particle retention within the vecto r. It is correlated with the serological properties of the virus coat protein and detennined by the RNA-2 segment of the virus genome and by an inherited character of the vector. The virus coat protein is probably involved in the recognition process between vector and virus but is not the sole determinant for transmission of tobraviruses. Genetic changes made to proteins present in the RNA-2 segment of pea early.browning tobravirus have been used to reveal the probable involvement of several proteins in vector transmission.