Trichodorid Species Research Articles

Distribution of trichodorid species in mainland China with description of Trichodorus hangzhouensis sp. nov. (Nematoda, Triplonchida).

Seven trichodorid species including a new one (Trichodorus hangzhouensissp. nov., T. nanjingensis, T. pakistanensis, T. cedarus, Paratrichodorus porosus, Nanidorus renifer and N. minor) were recovered from the rhizosphere of different hosts in 13 provinces of China. Each of the recovered species was characterized based on morphology and molecular data using rRNA gene sequences. Trichodorus hangzhouensissp. nov. is characterized by its males having medium-sized onchiostyle (46–49 µm) and three ventromedian cervical papillae (CP) anterior to the secretory-excretory (S-E) pore, CP1 located opposite the anterior part of isthmus, S-E pore opposite the isthmus or anterior end of pharyngeal bulb, spicules slightly ventrally curved, relatively small, 33.2 (32.0–34.5) µm long, wider slightly marked capitulum, lamina partially striated without bristles at striation; and females having rounded triangular sclerotized vaginal pieces with tips directed towards vulva, 1.5–2.0 µm sized, at about 1 µm apart, vulva pore-like in ventral view. Phylogenetic analysis based on D2-D3 28S rRNA gene sequences differentiated the new species among Trichodorus species from Europe, Asia and USA which formed a large clade. A review of the distribution of Trichodorus, Nanidorus and Paratrichodorus species revealed that T. cedarus, T. nanjingensis, T. pakistanensis and P. porosus are the most widespread species recorded from different provinces of China. This is the first extensive study of trichodorid species occurring in China.

Dynamic biospeckle analysis, a new tool for the fast screening of plant nematicide selectivity

BackgroundPlant feeding, free-living nematodes cause extensive damage to plant roots by direct feeding and, in the case of some trichodorid and longidorid species, through the transmission of viruses. Developing more environmentally friendly, target-specific nematicides is currently impeded by slow and laborious methods of toxicity testing. Here, we developed a bioactivity assay based on the dynamics of light ‘speckle’ generated by living cells and we demonstrate its application by assessing chemicals’ toxicity to different nematode trophic groups.ResultsFree-living nematode populations extracted from soil were exposed to methanol and phenyl isothiocyanate (PEITC). Biospeckle analysis revealed differing behavioural responses as a function of nematode feeding groups. Trichodorus nematodes were less sensitive than were bacterial feeding nematodes or non-trichodorid plant feeding nematodes. Following 24 h of exposure to PEITC, bioactivity significantly decreased for plant and bacterial feeders but not for Trichodorus nematodes. Decreases in movement for plant and bacterial feeders in the presence of PEITC also led to measurable changes to the morphology of biospeckle patterns.ConclusionsBiospeckle analysis can be used to accelerate the screening of nematode bioactivity, thereby providing a fast way of testing the specificity of potential nematicidal compounds. With nematodes’ distinctive movement and activity levels being visible in the biospeckle pattern, the technique has potential to screen the behavioural responses of diverse trophic nematode communities. The method discriminates both behavioural responses, morphological traits and activity levels and hence could be used to assess the specificity of nematicidal compounds.

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 analysis of Paratrichodorus teres (Hooper 1962) (Nematoda: Trichodoridae): a groundwork for discussion on the phylogeny and pathogenicity of Paratrichodorus species

Due to its ability to transmit plant viruses, Paratrichodorus teres (Hooper in Nematologica, 7, 273–280, 1962) is recognized as an economically important trichodorid species. Morphological and molecular analyses (18S and 28S rDNA) were performed, and 10 new plant hosts are reported for Polish P. teres populations. Major morphological features and the measurements obtained for the investigated specimens were within the wide ranges indicated for this species. However, a more detailed comparative analysis of Polish and Iranian P. teres showed significant morphological differences, particularly, in the shape and the structure of the walls of pars proximalis vaginae and the shape of the rectum. Phylogenetic study based on the 18S rDNA data suggests positioning of the Polish P. teres sequences within a cluster of sequences originating from the Netherlands. A comparison of the 28S rDNA fragment from Polish populations with the only P. teres 28S rDNA sequence available (from Iran) in GenBank revealed a sequence variability of 9.3%. The variation across these two representatives was higher than in the case of many other pairs of Trichodoridae species. The results obtained on the Polish P. teres specimens are discussed in the framework of the species taxonomy and phylogenetic relationships.

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).

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.

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.

First Report of the Stubby-Root Nematode, Paratrichodorus teres, from Potato in the Columbia Basin of Washington State

Stubby-root nematodes of the genus Paratrichodorus are migratory ectoparasites that cause stunted and deformed roots. They also transmit Tobacco rattle virus, the causal agent of corky ringspot disease in potato. Thus, they are important to the potato industry in the Pacific Northwest, including the Columbia Basin of Washington state, which is one of the largest potato-producing regions in the United States. Stubby-root nematode species are difficult to identify because the morphological differences between the species are very small (4). Paratrichodorus teres is a morphologically similar species and has been reported mainly from temperate regions within Europe. P. teres has also been reported in potato fields in eastern Oregon (3). The morphological similarity between P. allius and P. teres creates problems for routine identification because trichodorid species have a conservative morphology and are separated by the following primary characteristics: females, type of genital system and vulva position; males, number of ventromedian precloacal supplements and cervical papillae and habitus (2). As with most soil nematodes, females are more prevalent than the males. Unfortunately, in the generic key (2), female P. allius and P. teres belong to subgroup 1-12 and are separated by a secondary characteristic, vaginal shape that could be affected by fixation or handling. Male P. allius and P. teres are separated by the former species having one fewer ventromedian precloacal supplement. Thus, the morphological similarity between P. allius and P. teres creates problems for routine identification. This might be one of the reasons that P. teres has not been diagnosed in Washington until now. Using nematode morphological diagnostic keys (1,2) and comparing 18S rDNA sequence data obtained from specimens with those listed on public sequence databases, e.g., NCBI GenBank, stubby-root nematodes from two potato fields close to Moses Lake, Grant County, WA, were diagnosed as P. teres. The two adjacent potato fields are irrigated and are situated approximately 100 miles from the eastern Oregon site where P. teres was first described (3). Soil samples were collected from several Columbia Basin potato fields that had corky ringspot disease and stubby-root nematodes. Corky ringspot disease is an emerging disease in Washington, and the presence of a new viruliferous nematode species might be of concern to the potato industry.

First Report of the Stubby-Root Nematode Paratrichodorus teres Associated with Artichoke (Cynara scolymus) in Greece.

The polyphagous stubby-root nematode species, Paratrichodorus teres (Hooper) Siddiqi, was first described from soil under lettuce near Norwich, UK and subsequently reported from South Africa and the United States, but predominantly from temperate regions within Europe (4). P. teres is one of 13 economically important trichodorid species known to be vectors of Tobacco rattle virus (TRV) (4). Artichokes planted during 2000 in a field located in the Kandia area of the Argolis Region, Greece (37°32'N, 22°56'E) exhibited symptoms of a virus infection. Sampling was done to ascertain the presence of Longidorus fasciatus, a vector of artichoke Italian latent nepovirus known to occur in the area (1,4). In addition to L. fasciatus, an unknown trichodorid species and Tylenchorhyncus sp. were recovered from the root zone of artichoke at a number of sites within the field. Measurements and morphological examination of the female (n = 13, body length = 741.7 ± 25.5 μm, onchiostyle = 43.7 ± 0.8 μm, and position of vulva from anterior region relative to total body length V% = 53.8 ± 0.4 μm) and male (n =1, body length = 720.5 μm, onchiostyle = 43.5 μm, spicule length = 51.7 μm, and number of ventromedian precloacal supplements = 3) trichodorids isolated from soil samples conformed to the original description of P. teres and the generic polytomous key (2). Furthermore, morphological identification was supported by molecular data. DNA was extracted from seven individual trichodorids, each of which were placed into separate 0.5-ml micro-centrifuge tubes containing 20 μl of 0.25 M NaOH and incubated at 25°C overnight. Thereafter, samples were incubated at 99°C for 3 min and 10 μl of 0.25 M HCl, 5 μl of 0.5 M Tris-HCl, (pH 8.0) and 5 μl of 2% Triton X-100 were added to each tube. Samples were incubated at 99°C for a further 3 min and stored at -20°C. Template DNA was amplified using polymerase chain reaction with primers specific for 18S rDNA and sequenced (3). The resultant consensus sequence had 99.8% homology to P. teres populations isolated from Portugal and good homology (95 to 98%) with five other Paratrichodorus spp. listed on public sequence databases, e.g., NCBI GenBank. This constitutes a new geographic record and a possible association of P. teres on artichoke.

Paratrichodorus divergens sp. n., a new potential virus vector of tobacco rattle virus and additional observations on P. hispanus Roca & Arias, 1986 from Portugal (Nematoda: Trichodoridae)

AbstractDuring a survey of trichodorids in continental Portugal, a new trichodorid species, Paratrichodorus divergens sp. n., was found. It is described and illustrated with specimens from the type locality together with additional morphometric data and photographs of specimens obtained from soil samples collected in seven other localities. The species is characterised in the female by distinct drop-like to triangular oblique vaginal sclerotisations diverging outwards and sperm cells usually distributed all through the uteri, and in the male by thin, almost straight, striated spicules, the two posteriormost precloacal supplements being located relatively close to one another and usually opposite the distal third of the retracted spicules, presence of a slightly bilobed cloacal lip and sperm cells with a sausage-shaped nucleus. Paratrichodorus divergens sp. n. most closely resembles P. hispanus Roca & Arias, 1986 with which it often occurs in mixed populations. Additional information is also provided for P. hispanus. Coding of the features of the new species is given following the identification key of Decraemer and Baujard.

Molecular characterisation of the ribosomal DNA of Paratrichodorus macrostylus, P. pachydermus, Trichodorus primitivus and T. similis (Nematoda: Trichodoridae)

AbstractTrichodorids are plant-ectoparasitic nematodes, causing economically important diseases in many crops. In this study, we characterised ribosomal DNA (rDNA) of four trichodorid species from two different genera (i.e., Paratrichodorus macrostylus, P. pachydermus, Trichodorus primitivus and T. similis). Sequence data from the 18S and 5.8S genes showed high similarity; in contrast, sequence data from the internal transcribed spacer regions (ITS) were highly variable. Sequence divergence between the two genera included many insertions and deletions. Trichodorus primitivus and T. similis ITS regions of 1537 and 1303 bp, respectively, are the longest reported to date in the Phylum Nematoda. Further exploration of the sequence data indicated perfect and non-homologous repeats which were confined to the middle of the ITS regions.

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.

Description of Trichodorus gilanensis n. sp. from a forest park in Iran and observations on Paratrichodorus tunisiensis (Siddiqi, 1963) Siddiqi, 1974 (Nematoda: Diphtherophorina)

AbstractA new trichodorid species, Trichodorus gilanensis n. sp. is described from a forest park in northern Iran. The new species is characterised in the male by three ventromedian cervical papillae with the anterior two at the level of the onchiostyle region, the posterior-most ventromedian procloacal supplement (SP1) at the level of the retracted spicule head, spicules smooth and manubrium continuous with shaft, and in the female by large triangular vaginal sclerotised pieces close to one another in lateral view, a barrel-shaped vagina, a transverse slit-like vulva in ventral view and a slightly swollen tail region. Three other trichodorid species found, T. primitivus, Paratrichodorus porosus and P.tunisiensis represent new records for Iran.

Ecology and Pathogenicity of Four Trichodorid Species From the Semi-Arid Region of West Africa

Laboratory studies showed that trichodorids from the semi-arid region of West Africa need relatively low soil temperature levels for reproduction. The nematodes are not able to enter anhydrobiosis. Their localization at depth is probably a response to desiccation of the soil during the dry season and to high soil temperatures in the upper layers of the soil during the rainy season. Laboratory experiments showed that P. minor is pathogenic to sorghum, cowpea and eggplant, P. nanus to millet and sorghum, and P. rhodesiensis to peanut and sorghum. In each case the weight of shoots and roots was reduced by the nematodes but otherwise there were no obvious disease symptoms.

Trichodorid Nematodes From Australia With Description of Two New Species From Native Vegetation.

During a survey of plant parasitic nematodes associated with native vegetation in Australia, two new species of Paratrichodorus were found. Paratrichodorus orrae n.sp. was collected from Eucalyptus woodland and P. queenslandensis n.sp, from tropical rainforest, both closely resembling P. grandis. Paratrichodorus lobatus and the closely related P. teres are studied and the six trichodorid species recorded in Australia are listed.

Trichodoridae (Nematoda) From Brazil

Soil samples collected in Bahia State, Brazil, yielded Paratrichodorus minor and five monodelphic trichodorid species. Four of them are described as new species belonging to the genus Allotrichodorus. The females of the new species can mainly be distinguished by the shape of the vaginal sclerotizations. The males of A. sharmai n. sp. can be distinguished by the presence of one ventromedian cervical papilla, the absence of a capitular extension of the spicules and by the spicule length. The males of A. brasiliensis n. sp. can be distinguished by the absence of a ventromedian cervical papilla, the absence of a capitular extension of the spicules, onchiostyle and spicule length. The males of A. longispiculis n. sp. can be distinguished by the presence of one ventral cervical papilla, the absence of a capitular extension of the spicules and by the long distally arcuate spicules. The males of A. loofi n. sp. can be distinguished by the presence of one ventral cervical papilla, the absence of a capitular extension of the spicules and by the short distally curved spicules. The reliability of the morphological characters used in the literature to distinguish Monotrichodorus and Allotrichodorus is discussed together with the taxonomic position of A. westindicus (Rodriguez-Montessoro, Sher & Siddiqi, 1978) n. comb. An emended diagnosis of the genus Allotrichodorus is given and also a key to the females and males.