MOLECULAR BIOLOGY AND DIAGNOSTICS

Survey of Russian conifers for Bursaphelenchus species and their identification using PCR-RFLPs and sequences of ribosomal DNA.

Oleg A. KULINICH1*, Sergei A. SUBBOTIN1, Pavel V. TOULDOUKOV1, Maurice MOENS2, Andrei O. REMEZOV3

1Institute of Parasitology of Russian Academy of Sciences, Leninskii Prospect, 33, Moscow 119071, Russia,*okulinich@hotbox.ru

2Crop Protection Department, Agricultural Research Centre, Burg, Van Gansberghelaan 96, 9820 Merelbeke, Belgium

3Plant Quarantine State Inspection of Altai region, Gorkii str. 4, Barnaul, 656056, Russia

The genus Bursaphelenchus contains nearly 60 species and includes quarantine and other plant-pathogenic nematodes. Eleven Bursaphelenchus species were found in Russia. The data presented here are from a survey of conifers in the Asian part of Russia for pathogenic nematodes of the genus Bursaphelenchus. The analysis of wood samples collected during surveys in 2002 in Irkutsk, Krasnoyarsk, Sverdlovsk, Novosibirsk and Altai regions revealed only the presence of B. mucronatus. Out of 442 wood samples of pine, larch, fir, B. mucronatus was found 42 times, i.e. in Irkutsk (in 20% of the samples), Krasnoyarsk (1%), Sverdlovsk region (31%), Novosibirsk (8%), and Altai (10%). For the 247 conifer wood samples analyzed from the Krasnoyarsk region B.mucronatus was found only three times. The differentiation of the pinewood nematode B. xylophilus and relative species, B. mucronatus and B. fraudulentus, based only on morphological parameters is difficult or sometimes impossible. The genetic diversity of specimens from twelve isolates from all the mentioned regions maintained on Botrytis cinerea was studied. PCR-RFLPs and sequences of ITS-rDNA confirmed the morphological identification and revealed that all studied isolates belong to the European genotype of B. mucronatus. (RFBR grant No. 01-04-49121).

Evidence from SSU rDNA of Anoplostoma rectospiculum (Galtsova, 1976) suggests that Anoplostomatidae is not related to Oncholaimidae

Anna N. PEGOVA1*, Elena D. KRASNOVA1, Vladimir V. ALEOSHIN2

1Pertsov White Sea Biological Station, Department of Biology, Moscow State University, Moscow, 119899, Russia, *Anna_Pegova@mail.ru

2Belozersky Institute of Physico-Chemical Biology, Department of Evolutionary Biochemistry, Moscow State University, Moscow, 119992, Aleshin@genebee.msu.su

Nematodes from the order Enoplida constitute a mainly free-living marine group of species for which there are no morphological synapomorphies currently available for their higher-rank classification. Thus, the small enoplid family Anoplostomatidae was usually classified within the superfamily Oncholaimoidea, but, alternatively, it was also being placed inside the Enoploidea (Lorenzen, 1994). The family possesses some features unique or rare within Enoplida. These are: a spacious toothless buccal cavity surrounded by pharyngeal tissue in the posterior section only, a cephalic capsule without an insertion point for musculature and a copulatory bursa in males. We analysed the position of Anoplostoma rectospiculum in a dataset comprising all enoplid sequences published so far, which belong to three monophyletic superfamilies (Enoploidea, Oncholaimoidea and Tripyloididea). MP, ML and NJ algorithms with bootstrap replicates consistently infer A. rectospiculum as a member of Enoploidea despite long branch leading to this species on trees. This topology is supported by an unique 1-bp insertion shared by A. rectospiculum and other members of Enoploidea. This character can be used for identification of 18S rRNA sequences of Enoploidea. (RFBR grants No. 01-04-48832, 02-04-48958; S.Sc. grant No. 1712.2003.4)

The SSU rRNA gene as a phylogenetic marker for major groups of Nematoda

Leonid Y. ROUSSINE1*, Paul De LEY2, Mark BLAXTER3, Andy VIERSTRAETE4, Vladimir V. ALEOSHIN1

1Belozersky Institute of Physico-Chemical Biology, Department of Evolutionary Biochemistry, Moscow State University, 119992, Moscow, Russia,*roussine@yandex.ru

2Department of Nematology, University of California, Riverside, CA 92521, USA, pdeley@citrus.ucr.edu

3Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh EH9 3JT, UK, mark.blaxter@ed.ac.uk

4University of Ghent, Department of Biology, B-9000 Gent, Belgium, andy.vierstraete@rug.ac.be

SSU rDNA data has been used in universal phylogenetic studies to define major lineages within Nematoda and suggest a new scenario of their relationships. However, since first attempts to recover deeper nematode phylogenies it has become clear that conventional techniques of inference often are not sufficient for this purpose. Intensive molecular phylogenetic analysis of the three major nematode clades demonstrated that relationships within Chromadoria are robustly inferred down to the deepest divergences. The deeper phylogenetic structure of Enoplia, on the contrary, is not robustly recovered even with the most sophisticated algorithms. The similar situation, albeit to a lesser extent, is characteristic of the Dorylaimia clade represented by only a few compact recent taxa. Our analyses suggest that the behaviour of the SSU rRNA gene is fundamentally similar in all nematode taxa. The main cause of bias in reconstructions of enoplian and dorylaimian phylogenies is a marked scarcity of reliable molecular characters to define deeper nodes of the tree, which is most likely due to poor taxon sampling. The current availability of evolutionary descendants of mile-stone divergences in Chromadoria permits the breaking of long inner branches of the tree and thus avoids computational artifacts caused, in particular, by disparities in rates of molecular evolution. A shortage of recent intermediate taxa in Enoplia appears to preclude similar solutions, and may result from their rapid initial radiation with subsequent extinction of greater number of descendant lineages. A similar evolutionary pattern is probably characteristic of Dorylaimia, although the amount of their recent taxa is very few. The latter allowes one to individually assess few competing phylogenies for reliability. (Grants RFBR No. 01-04-48832, 02-04-48958; RRSCCE No. 1712.2003.4; FEBS Collaborative scholarship for Central & Eastern Europe).

The sequence analysis of D2-D3 domain of LSU rDNA of Thelastomatidae (Thelastomatoidea, Oxyurida) from hind gut of cockroaches

Sergei E. SPIRIDONOV1, Elena A. GUZEYEVA2, Sergei V. LUKYANTSEV2

1Institute of Parasitology of RAS, Leninskii pr., 33, Moscow, 117071, Russia, spiridon@rjnem.msk.ru

2Laboratory of Entomology, Department of Plant protection, Tomsk State University, pr. Lenina, 36, Tomsk, 634050, Russia, look@res.tsu.ru

The sequences for 739-788 bp long fragments of LSU rDNA D2-D3 domain were obtained for 6 species of cockroach thelastomatids (Cranifera cranifera, Hammerschmidtiella diesingi, Hammerschmidtiella sp. from Gromphadorhina portentosae cockroaches, Leidynema appendiculata, L. porentosae, and unidentified Thelastomatidae females from Blaptica dubia), and single Oxyuridae from toads, Thelandros sp. (to use as outgroup). The BLAST search in GenBank revealed sequences for Acrobeloides and Cervidellus (Rhabditida) as the closest ones for studied oxyurids. Maximum parsimony analysis revealed moderate support for the monophyly of Thelastomatidae, but 100% bootstrap support for the monophyly of Oxyuridae and Thelastomatidae. The close relationships of Cranifera and Leidynema were strongly supported, with unidentified Thelastomatidae clustering with Cranifera under weak bootstrap support. Total nucleotide differences in 8-10% (60-75 bp) were reported between species (with 8-16 nucleotide autapomorphies for 5 thelastomatids identified up to the species) and in 15-20% (100-150 bp) between genera. Thelastomatids of the same genus can inhabit hind guts of cockroaches belonging to distant clades of Blattoidea evolutionary trees (Kambhampati, 1995; Grandcolas and DíHaese, 2001). Prominent molecular and morphological differences were shown for Leidynema appendiculata and L. portentosae parasitizing cockroaches from separate evolutionary lines (Blattidae vs. Blaberidae). Also D2-D3 sequence of Hammerschmidtiella specimens from Gromphadorhina portentosae (Blaberidae) was found to be significantly different from that of H. diesingi inhabiting Blatta orientalis and Periplaneta americana (Blattidae). Combined molecular and morphological characters support the independence of Hammerschmidtiella species from G. portentosae.

Molecular and morphological characterization of new EPN isolates from Western Siberia

Sergei E. SPIRIDONOV1, Maurice MOENS 2

1Institute of Parasitology of RAS, Leninskii pr., 33, Moscow, 117071, Russia, spiridon@rjnem.msk.ru

2Agricultural Research Center, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium, mmoens@clo.fgov.be

Eleven cultures of entomopathogenic soil nematodes (9 of Steinernematidae and 2 of Heterorhabditidae) were established during the field collection work on the territory of Altai Republic (Gorny Altai), Novosibirsk and Tomsk regions in Western Siberia, Russia. Cultures were established through traditional baiting technique and through direct extraction of infective juveniles from soil samples originated from natural habitats ("black taiga" forest on different altitudes, meadows) and agricultural ecosystems. DNA extraction and PCR amplification of ribosomal genes were done to obtain full sequence of ITS1+5.8S+ITS2 fragment of ribosomal genes for 8 steinernematid and two heterorhabditid isolates. Two isolates of S. feltiae were found in Altai Republic and Tomsk Region (both in agricultural habitats). Analysis of sequence data revealed that S. feltiae culture isolated in Tomsk corresponds to the type one from North-Eastern Russia (Izhevsk), when that from Gorny Altai - to the form of this species more common in South-Western Europe and Asia. Two steinernematid species were isolated in natural habitats of Altai mountains: several cultures of S. kraussei and steinernematids morphologically similar to the S. intermedium, but with differences from this latter in ITS rDNA sequence. In their ITS rDNA sequences S. kraussei cultures from Altai mountains are close to conspecific isolates from Switzerland and Scotland and "intermedium-like" cultures - to morphologically similar isolates from UK (E1), Russia (Zvenigorod), Estonia (E6) and Belgium (Rochefort). Both isolated Heterorhabditis cultures (Gorny Altai and Novosibirsk) belong to the H. megidis, what represents first molecularly-confirmed report of this species from Russia. (RFBR grant No. 02-04-48389).

Preliminary results of ITS rDNA sequence analysis for steinernematid strains (Rhabditida; Steinernematidae) from Russian Far East 

Sergei E. SPIRIDONOV1, Mutsuhiro YOSHIDA2, Maurice MOENS3

1Institute of Parasitology of RAS, Leninskii pr., 33, Moscow, 117071, Russia, spiridon@rjnem.msk.ru

2National Institute for Agro-Environmental Sciences 3-1-3, Kannondai Tsukuba, Ibaraki 305-8604, Japan, mutsuysd@niaes.affrc.go.jp

3Agricultural Research Center, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium, m.moens@clo.fgov.be

Full sequences of the ITS1+5.8S+ITS2 domain of rDNA were obtained for the Sakhalin isolate of Steinernema feltiae, four isolates of S. kraussei from Russian Far East (Sakhalin and Primorski Territory) and one S. feltiae isolate from Hokkaido. Sequences for S. kraussei were obtained through direct sequencing of PCR product; those for S. feltiae were obtained by cloning of the PCR product in Escherichia coli competent cells. The comparison of obtained sequences with ITS rDNA sequences for other S. feltiae and S. kraussei isolates was performed through maximum parsimony analysis. All four S. kraussei isolates clustered together under moderate bootstrap support. They form a strongly supported group of isolates together with S. kraussei isolates from Western Siberia and some S. kraussei from Europe. All these Far Eastern S. kraussei isolates were characterised by the presence of an identical unique insertion in ITS2 rDNA. Several characteristic nucleotide substitutions were also found in the ITS1 rDNA of these strains. One such substitution was also reported for S. kraussei from Northern Europe (North East of Russia and Iceland), when two others were only present in two unusual Steinernema populations of the "kraussei" group from Europe (probably new steinernematid species). Both sequenced S. feltiae isolates clustered under strong bootstrap support with the topotype isolate of S. feltiae from North-East of European Russia, although the characteristic 10 bp insertion in the ITS1 rDNA was absent in these Far Eastern isolates. This insertion is reported for several S. feltiae strains common in Asia, Australia but is rarely reported from Europe.


© Russian Society of Nematologists, Institute of Marine Biology FEB RAS, 2002