Nematology, 1999, Vol. 1(2), 195-207
Identi cation of Heterodera avenae group species by morphometrics and rDNA-RFLPs
Sergei A. SUBBOT IN 1 , Lieven WAEYENBERGE 2 , Irina A. M
OLOKANOVA 1 and Maurice M OENS 2
Institute of Parasitology of Russian Academy of Sciences, Leninskii prospect 33, Moscow, 117071, Russia; 2 Agricultural Research Centre, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium
Accepted for publication: 5 July 1998
Summary – Canonical discriminant analysis of four morphometric charactersof juvenilesand restriction enzymes analysis of ribosomal DNA sequences were used to distinguish Heterodera arenaria, H. aucklandica, H. avenae, H. lipjevi, H. hordecalis, H. iri, H. latipons, H. litoralis, H. schachtii and an undescribed species from grasslands. The results of unweighted pair group cluster analysis showed that H. avenae populations formed three groups and H. lipjevi two groups at the 80% level of similarity. Intraspeci c polymorphism was revealed by rDNA-RFLP studies and two types of ITS regions within H. avenae populations can be distinguished. The pattern of restriction bands obtained with BsuRI, PstI and TaqI clearly distinguishedpopulations of H. lipjevi from other species of the H. avenae group. Further enzymes and their combinations distinguished the other species. There are no enzymes which differentiate European populations of H. avenae from H. arenaria. Morphometrics, restriction endonuclease cleavage maps of ITS regions and a dendrogram of putative phylogenetic relations of several cyst-forming nematode species are given. Résumé – Identi cation des espèces du groupe Heterodera avenae par la morphometrie et les rDNA-RFLP – L’analyse canonique discriminante sur quatre caractères morphométriques des juvéniles et l’analyse des enzymes de restriction de l’ADN ribosomal ont été utilisées pour identi er Heterodera arenaria, H. aucklandica, H. avenae, H. lipjevi, H. hordecalis, H. iri, H. latipons, H. litoralis, H. schachtii et une nouvelle espèce originaire de prairies. Les résultats de l’analyse UPGMA ont montré que les populationsd’ H. avenae formaient trois groupes et celles d’H. lipjevi deux groupes à un niveau de similarité de 80%. Le polymorphisme intraspéci que a été révélé par des études de rDNA-RFLP et deux types de régions de l’ITS peuvent être mis en évidence dans les populations d’ H. avenae. Les modèles de bandes de restriction obtenus avec BsuRI, PstI et TaqI ont identi é clairement les populations d’H. lipjevi des autres espèces du groupe H. avenae. D’autres enzymes et leurs combinaisons ont identi é les autres espèces. Aucun enzyme n’a différencié les populations européennes d’H. avenae de H. arenaria. Les caractères morphométriques, les cartes de clivage des régions des ITS par l’endonucléase de restriction et un dendogramme des relations phylogénétiques supposées sont donnés. Keywords: canonical discriminant analysis,cyst nematodes, Heterodera avenae, H. lipjevi, identi cation, ITS-rDNA, morphometrics, RFLP.
The Heterodera avenae group sensu lato presently contains eleven valid species: H. arenaria, H. aucklandica, H. avenae, H. bifenestra, H. lipjevi, H. hordecalis, H. iri, H. latipons, H. mani, H. spinicauda and H. turcomanica and several undescribed species (Sturhan & Wouts, 1995; Wouts & Sturhan, 1995; Robinson et al., 1996). Comparative morphology and morphometrics (Stone & Hill, 1982; Valdeolivas & Romero, 1990; Subbotin et al., 1996), protein electrophoresis (Rumpenhorst, 1985; Ferris et al., 1989, 1994; Bossis & Rivoal, 1996; Sturhan & Rumpenhorst, 1996; Subbotin et al., 1996) and random ampli ed polymorphic DNA (López-Bra?a et al., 1996) allow separation of several species.
c ? Koninklijke Brill NV, Leiden, 1999
The analysis of coding and non-coding regions of ribosomal DNA has become a popular tool for species and subspecies identi cation of several nematode species (Vrain et al., 1992; Wendt et al., 1993; Powers et al., 1997). ITS regions were found to be useful to differentiate species within the H. avenae group (Ferris et al., 1994; Bekal et al., 1997). This article presents a comparative analysis of the morphometrics of juveniles and cysts and of the ITS regions, including the 5.8S rDNA gene plus anking areas of the 18S and 26S genes, of several populations of seven species of the H. avenae group.
S.A. Subbotin et al.
Materials and methods
N EMATODE SPECIES AND POPULATIONS Twenty-four populations belonging to seven valid species of the H. avenae group and three populations of unknown species from the same group were studied (Table 1). To these were added one populationof H. schachtii and one of H. litoralis. Cysts were isolated from soil by routine methods of sieving and otation. Some cyst populations were allowed to dry, others were used freshly extracted from the soil. A minority was transferred to slides. Second-stage juveniles (J2s) were isolated from cysts. L IGHT MICROSCOPY J2s were killed by gentle heat, xed in TAF and embedded in glycerol as permanent slides following Seinhorst (1959). Cyst vulval cones were mounted in glycerinegelatine. The specimens were examined and measured with a JENAVAL light microscope. Four morphometrical characters of the J2s (body length, stylet length, tail length and hyaline part of tail length) and two J2 morphological characters (stylet knob shape and shape of tail terminus) along with four morphometrical characters of the cysts (fenestra length, mean semifenestral width, vulval bridge width and vulval slit length) were studied. All these characters are considered taxonomically important for this group (Wouts & Weischer, 1977; Sturhan, 1982; Wouts & Sturhan, 1995). S AMPLE PREPARATION FOR MOLECULAR STUDIES A single cyst was placed in 10 ? l of double distilled water on a glass slide and crushed under a dissecting microscope. J2s and eggs were transferred into a sterile Eppendorf tube containing 8 ? l lysis buffer (500 mM KCl, 100 mM Tris-Cl pH 8.3, 15 mM MgCl2 , 10 mM DTT, 4.5% Tween 20, 0.1% gelatin) and homogenised. Two ? l of proteinase K (600 ? g/ml) were then added. After freezing (? 80° C, at least 10 min) the tubes were incubated at 65° C for 1 h and then at 95° C for 10 min. PCR REACTION After centrifugation(1 min; 16 000 g) 10 ? l of the DNA suspension was added to the PCR reaction mixture containing 10 ? l 10? Taq incubation buffer with 25 mM MgCl2 (Appligene, B&L Systems, Boechout, Belgium);
4 ? l dNTP-mixture 5 mM each (Eurogentec, Seraing, Belgium), 1 ? l (1.5 M) of each primer (synthesised by Eurogentec), 0.8U Taq Polymerase (Appligene, B&L Systems) and double distilled water to a nal volume of 100 ? l. Primers AB 28 (5? ATATGCTTAAGTTCAGCGGGT 3? ) and TW 81 (5? GTTTCCGTAGGTGAACCTGC 3? ) as described by Joyce et al. (1994) were used in the PCR reaction. The DNA-ampli cation pro le carried out in a GeneE (New Brunswick Scienti c, Wezembeek-Oppem, Belgium). DNA thermal cycler consisted of 4 min 94° C; 35 cycles of 1 min 94° C, 1.5 min 62° C, and 2 min 72° C; and 5 min 72° C. After DNA ampli cation, 5 ? l product was run on a 1% agarose gel. The remainder was stored at ? 20° C. RFLP Seven ? l of each PCR-product was digested with one of the following twelve restriction enzymes: AluI, BsuRI, Bsh1236I, Bsp143I, HindIII, HinfI, Hin6I, MspI, MvaI, PstI, RsaI and TaqI, in the buffer stipulated by the manufacturer. The digested DNA was loaded on a 1.5% agarose gel, separated by electrophoresis, stained with ethidium bromide and visualised and photographedunder UV light. Digested PCR products loaded on a polyacrylamide gel were separated by electrophoresis on a Multiphor II Electrophoresis Unit (Pharmacia Biotech, Roosendaal, The Netherlands), silver stained (Pharmacia Biotech) and photographed. Procedures for obtaining PCR ampli ed products and endonuclease digestion of these products were repeated at least three times to verify the results. S TATISTICAL ANALYSES Morphometrical data were statistically analysed with the STATISTICA (version 5.0) computer package. Canonical discriminant analysis (CDA) was used to assess the relative similarity of 24 populations based on four morphometrical characters of the juveniles. We used the unweighted pair group cluster analysis, as recommended by Wishart (1978) and Orloci (1978), to compile a dendrogram clustering the populations at different levels on a scale of similarity. The similarity values were calculated as S = (1 ? D / 10) * 100 (Brown & Topham, 1985), where D is the Mahalanobis distance calculated in the CDA. The number of clusters was estimated and the cophenetic correlation calculated by methods described by Aldenderfer and Blash eld (1989). All digested DNA bands separated by electrophoresis on a 1.5% agarose gel were recorded as a binary matrix
Vol. 1(2), 1999 Country Isolate number Source Studies England New Zealand Germany Germany France France Belgium France Spain India France Russia Russia Russia Ukraine Russia Sweden Tadzhikistan Russia Spain Scotland Sweden Scotland Russia Russia Russia Belgium New Zealand Belgium Har Hac Hav1 Hav2 Hav3 Hav4 Hav5 Hav6 Hav7 Hav8 Hav9 Hf1 Hf2 Hf3 Hf4 Hf5 Hf6 Hf7 Hf8 Hf9 Hh1 Hh2 Hir Hlat Hsp1.1 Hsp1.2 Hsp2 Hlit Hsh J. Rowe, IACR-Rothamsted, Harpenden, UK W. Wouts, Auckland, New Zealand D. Sturhan, BBA, Münster, Germany D. Sturhan, BBA, Münster, Germany R. Rivoal, INRA, France R. Rivoal, INRA, France S.A. Subbotin, Institute of Parasitology, Moscow, Russia R. Rivoal, INRA, France D. Romero, Centro de Ciencias Medioambientales, Madrid, Spain J. Rowe, IACR-Rothamsted, Harpenden, UK R. Rivoal, INRA, France V.P. Balakhnina, Institute of Helminthology, Moscow, Russia L. Nasonova, Nizhnii Novgorod, Russia S.A. Subbotin, Institute of Parasitology, Moscow, Russia V. Termino, Kiev, Ukraine E. Osipova, Institute of Helminthology, Moscow, Russia A. Ireholm, Swedish University of Agriculture Sciences, Sweden A.R. Madzhidov, Dushanbe, Tadzhikistan L. Nasonova, Nizhnii Novgorod, Russia D. Romero, Centro de Ciencias Medioambientales, Madrid, Spain S.A. Subbotin, Institute of Parasitology, Moscow, Russia J. Rowe, IACR-Rothamsted, Harpenden, UK S.A. Subbotin, Institute of Parasitology, Moscow, Russia S.A. Subbotin, Institute of Parasitology, Moscow, Russia S.A. Subbotin, Institute of Parasitology, Moscow, Russia S.A. Subbotin, Institute of Parasitology, Moscow, Russia S.A. Subbotin, Institute of Parasitology, Moscow, Russia W. Wouts, Auckland, New Zealand M. Moens, Agricultural Research Centre, Merelbeke, Belgium CDA, RFLP RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP CDA, RFLP RFLP CDA, RFLP CDA RFLP CDA, RFLP CDA, RFLP CDA CDA, RFLP CDA, RFLP CDA, RFLP CDA CDA, RFLP RFLP RFLP Identi cation of Heterodera avenae group species
Table 1. Nematode populations of the genus Heterodera used in this study.
H. arenaria H. aucklandica H. avenae
H. iri H. latipons H. spp.
H. litoralis H. schachtii
Lincolnshire One Tree Hill, Auckland Taaken, Lower Saxony Rinkam, Bavaria Argentan, Fr3 strain St. Georges-du-Bois, Fr2 strain Knokke, West Vlaanderen Nuisement-sur-Coole, Fr4 strain Santa Olalla Desert region Villasavary, Fr1 strain Baimak, Bashkiria Gorodets, Nizhnii Novgorod region Pushkin, Leningrad region Chabany, Kiev region Saratov Etelhem Dushanbe Vad, Nizhnii Novgorod region Torralba de Calatrava Montrose Sk. Fagerhult (paratypes) Forfar Rostov region Putilovo, Leningrad region Kurilovo, Moscow region Zarren, West Vlaanderen Glen Innes, Auckland Unknown
S.A. Subbotin et al.
Table 2. Morphometrics of cysts and juveniles (means in ? m ± S.E.) of studied populations of seven species from Heterodera avenae group. Species (Population) Vulval areas of cyst n Fenestra Semi-fenestral Vulval bridge Vulval slit length width width length H. lipjevi (Baimak, Russia)*, Hf1 H. lipjevi (Gorodets, Russia)*, Hf2 H. lipjevi (Pushkin, Russia)*, Hf3 H. lipjevi (Chabany, Ukraine)*, Hf4 H. lipjevi (Etelhem, Sweden), Hf6 H. lipjevi (Dushanbe)*, Hf7 H. lipjevi (Spain), Hf9 H. avenae (Taaken, Germany)*, Hav1 H. avenae (Rinkam, Germany)*, Hav2 H. avenae (Argentan, France), Hav3 H. avenae (St. Georges, France), Hav4 H. avenae (Nuisement, France), Hav6 H. avenae (Knokke, Belgium), Hav5 H. avenae (Villasavary, France), Hav9 H. avenae (desert reg. India), Hav8 H. avenae (Spain), Hav7 H. arenaria (England), Har H. iri (Scotland), Hir Heterodera sp1.1 (Putilovo, Russia)* Heterodera sp1.2 (Kurilovo, Russia) Heterodera sp2 (Zarren, Belgium) H. latipons (Rostov, Russia), Hlat H. hordecalis (Sweden), Hh1 H. hordecalis (Scotland), Hh2 18 53± 0.9 (48-60) 15 51± 0.9 (45-58) 6 52± 1.8 (45-58) 21 55± 0.9 (48-63) 8 52± 1.4 (45-58) 16 54± 1.1 (50-61) 6 50± 1.1 (48-55) 20 48± 0.7 (43-53) 20 48± 1.1 (40-55) 11 50± 1.1 (45-58) 8 47± 1.0 (43-50) 3 46± 1.9 (44-50) 10 45± 1.4 (38-53) 5 46± 0.6 (45-48) 5 48± 1.1 (45-50) 5 45± 3.8 (38-50) 4 53± 1.8 (50-58) 5 48± 1.0 (45-50) 13 43± 0.7 (38-48) 11 43± 1.3 (40-50) 9 49± 1.8 (38-55) 12 60± 1.6 (50-70) 14 57± 0.9 (52-66) 10 63± 1.8 (58-75) 30± 0.8 (28-38) 28± 0.6 (25-33) 27± 1.6 (20-30) 29± 0.6 (25-33) 28± 1.1 (21-33) 28± 0.8 (24-32) 27± 0.5 (25-29) 25± 0.6 (20-30) 23± 0.5 (18-27) 25± 0.6 (23-30) 24± 0.7 (20-25) 21± 0.8 (20-23) 22± 1.1 (18-28) 24± 0.6 (23-26) 21± 0.6 (20-23) 23± 0.6 (23-24) 26± 1.6 (23-30) 27± 0.8 (25-30) 23± 0.8 (18-28) 25± 0.4 (23-28) 24± 1.0 (20-28) 23± 0.5 (20-25) 22± 0.5 (20-26) 25± 0.9 (20-30) n Body length Juveniles Stylet length Tail Hyaline part length of tail length 39± 0.6 (36-45) 33± 0.7 (28-38) 37± 0.7 (34-41) 35± 0.5 (31-39) 33± 0.6 (30-37) 31± 0.6 (29-36) 36± 0.6 (31-41) 45± 0.6 (40-49) 44± 0.7 (39-50) 44± 0.8 (39-51) 48± 1.0 (41-56) 45± 0.9 (36-50) 47± 1.1 (38-57) 44± 0.5 (40-48) 38± 1.3 (31-46) 41± 0.5 (37-44) 51± 0.7 (46-56) 56± 0.7 (47-61) 42± 0.7 (39-45) 45± 0.8 (39-53) 46± 1.1 (38-52) 32± 0.7 (26-38) 33± 0.6 (30-36) 35± 0.8 (30-42)
8.1± 0.4 10.9± 0.4 20 552± 3.7 25.4± 0.2 60± 0.9 (5.0-10.0) (7.5-15.0) (514-573) (23.5-26.5) (55-67) 9.4± 0.4 10.3± 0.3 20 526± 4.9 24.5± 0.1 55± 0.9 (7.5-11.3) (9.3-12.5) (485-570) (23.9-25.5) (50-62) 7.9 ± 0.8 14.0± 0.6 15 539± 4.5 25.2± 0.2 60± 0.6 (5.0-10.0) (12.5-15.0) (504-568) (24.5-26.0) (56-62) 10.5± 0.3 11.9± 0.3 20 520± 7.1 24.9± 0.2 54± 0.9 (7.5-12.5) (9.5-14.3) (478-577) (23.5-26.0) (50-60) 9.9± 0.5 10.1± 0.5 14 509± 5.2 24.3± 0.1 56± 1.1 (7.5-12.5) (7.5-12.5) (477-552) (24.5-24.8) (46-60) 8.8± 0.4 10.9± 0.6 16 519± 4.7 24.8± 0.2 55± 0.6 (6.9-10.9) (7.9-15.8) (494-537) (23.2-25.6) (52-59) 11.8± 0.7 11.0± 0.6 20 526± 6.0 26.4± 0.2 57± 0.7 (9.3-13.3) (9.3-12.5) (484-572) (25.5-27.5) (52-63) 7.3± 0.2 10.1± 0.3 20 566± 4.8 26.3± 0.3 69± 1.0 (6.3-7.5) (8.0-12.5) (522-598) (23.3-29.3) (59-80) 7.1± 0.4 8.9± 0.3 20 557± 4.8 26.5± 0.3 69± 0.9 (5.0-10.8) (6.3-10.8) (491-595) (23.5-29.1) (61-76) 8.6± 0.3 9.6± 0.2 20 568± 4.5 26.2± 0.1 66± 0.8 (7.5-10.0) (8.8-10.2) (538-602) (25.5-27.5) (57-71) 9.4± 0.4 9.8± 0.3 24 519± 3.0 26.6± 0.1 70± 0.7 (7.5-10.8) (7.5-10.5) (488-541) (25.5-27.5) (66-77) 10.8± 0.8 9.8± 0.2 20 516± 8.8 26.4± 0.2 66± 0.9 (10.0-12.5) (9.5-10) (449-570) (24.5-27.5) (56-75) 7.4± 0.2 10.2± 0.3 20 571± 13 27.5± 0.3 69± 1.0 (6.3-8.8) (9.3-12.5) (423-644) (25.5-29.6) (59-77) 10± 0.4 9.7± 0.6 20 563± 5.5 26.9± 0.2 69± 0.8 (8.8-11.3) (7.5-10.8) (505-560) (25.5-28.6) (62-76) 10.6± 0.6 8.7± 0.5 15 505± 7.4 26.1± 0.2 61± 0.8 (9.3-12.5) (7.5-10.0) (453-559) (24.5-27.5) (56-65) 10.4± 1.5 10± 0.1 20 553± 6.0 26.4± 0.2 67± 0.6 (7.5-12.5) (9.8-10.2) (478-597) (24.5-28.6) (61-74) 8.4± 0.8 11.4± 0.6 20 633± 8.0 29.4± 0.2 77± 1.3 (6.8-10.0) (10-13) (536-671) (27.5-30.6) (63-84) 6.6± 0.7 12.6± 0.8 18 593± 4.1 26.9± 0.1 85± 1.3 (5.0-8.8) (10-15) (562-639) (25.8-27.5) (77-97) 7.0± 0.5 10.6± 0.4 20 504± 4.5 24.9± 0.2 65± 0.9 (5.0-10.0) (8.8-12.5) (471-532) (22.4-26.0) (59-69) 9.8± 0.3 8.6± 0.3 20 522± 5.2 24.4± 0.2 69± 1.1 (8.8-11.8) (7.5-10.8) (464-563) (22.9-25.5) (53-77) 6.4± 0.4 9.4± 0.3 20 494± 5.8 24.9± 0.1 69± 1.2 (5.0-7.5) (7.5-10.0) (434-545) (24.0-26.5) (62-78) 28± 1.2 7.3± 0.3 20 485± 6.4 23.4± 0.1 53± 0.7 (23-38) (5.9-9.3) (421-552) (22.4-24.5) (43-59) 28± 0.6 20± 0.4 11 442± 4.1 24.4± 0.1 51± 0.4 (24-32) (18-22) (417-462) (24.0-24.8) (50-54) 26± 1.1 22± 1.1 20 470± 5.5 25.6± 0.2 54± 0.9 (18-30) (18-28) (407-507) (24.5-27.5) (48-62)
*Measurements made by Subbotin et al. (1996). 198 Nematology
Identi cation of Heterodera avenae group species
Table 3. Factor structure for canonical variables of Heterodera species juveniles from 24 populations. Axis 1 Body length Stylet length Hyaline part of tail length Tail length Canonical correlation Cumulative 0 .457 0 .597 0 .818 0 .845 Axis 2 0 .612 0 .672 –0 .193 –0 .134 Axis 3 0 .634 –0 .436 0 .183 0 .433 Axis 4 0 .141 –0 .030 0 .520 –0 .283
0 .921747 0 .812422 0 .694518 0 .405525 0 .647918 0 .870565 0 .977429 1 .00000
of 0 and 1 corresponding to the absence or presence of individualbands. The matrix was given as input data to the Phylogeny Inference Package (PHYLIP, version 3.572). Gendist was the Phylip program used to compute the genetic Nei’s distance. Cluster analysis by the unweighted pair group method with arithmetic mean (UPGMA) was performed by the Neighbor program. Bootstrap analysis, using 1000 bootstrapped data sets, was performed to determine statistical constancy of the classi cation.
M ORPHOMETRICAL AND MORPHOLOGICAL
CHARACT ERISTICS OF SPECIES
The J2 and cyst morphometrical data are presented in Table 2. Measurements are means followed by their standard error; the range is placed in parenthesis. The CDA of the 24 populations calculated four canonical variables. Cumulated, the rst two variables accounted for 87% of the variance and the rst three for almost 98% of the variance. The tail length and the length of the hyaline part of the tail had the highest correlation with the rst variable; the stylet length was best correlated with the second variable and body length with the third one (Table 3). Two-dimensional scatterplots of population means of canonical variables were generated (Fig. 1A, B). They allow the population grouping to be compared with species identi cation and the similarities among species to be estimated. The results of the unweighted pair group cluster analysis are presented as a dendrogram (Fig. 2). The populations were clustered in nine distinct groups at a similarity level of 80% (cophenetic correlation = 0.87). H. lipjevi populations were grouped in two clusters. Six populations(Hf1, Hf2, Hf3, Hf4, Hf6 and Hf7) formed a cluster at 83% similarity; Hf9 (Torralba de Calatrava,
Vol. 1(2), 1999
Spain) was clustered with the Indian populationof H. avenae (Hav8). J2 of H. lipjevi were easily distinguished from the closely related European H. avenae populations by a shorter tail (average 54-60 vs 66-70 ? m) and a shorter hyaline part of tail (31-39 vs 44-48 ? m). They were also distinguished from H. avenae by the shape of the stylet knobs (distinctly concave anteriorly) and the rounded tail terminus. The Spanish population of H. lipjevi differed from other populationsby a longer stylet length (Table 2). Fenestra length and mean semifenestral width of vulval areas of cysts of H. lipjevi were larger than of H. avenae cysts (average 50-55 vs 45-50 ? m, 27-30 vs 22-25 ? m, respectively). Vulval cyst cones of H. lipjevi had an underbridge. H. latipons clustered with six H. lipjevi populations, the two H. hordecalis (Hh1 and Hh2) populations clustered with to Hf9 and Hav8. H. latipons and H. hordecalis were easily distinguished from other species and from each other by differences in vulval slit length, the vulval bridge width, the strong underbridge and the lack of distinct bullae in the vulval cone of the cyst. H. avenae populations were grouped in three clusters. The two French populations (Hav4 and Hav6) were separated from the other European populations (Hav1, Hav2, Hav3, Hav5, Hav7, and Hav9); the Indian population (Hav8) was grouped with H. lipjevi from Spain (Hf9). A shorter J2 body length, tail length and hyaline part of tail length distinguished the Indian population from the other H. avenae populations. The French populations (Hav4 and Hav6) differed from the other European populations by a shorter J2 body length (Table 2). A weak underbridge was present in some specimens of these two populations. Stylet knobs of J2s from all H. avenae populations were at or sometimes slightly concave anteriorly and the tail terminus was usually narrowly rounded. The Heterodera spp. populations all from grasslands formed a distinct cluster at almost 68% similarity with the large H. avenae group. The body length, the fenestral length and the vulval bridge width differed slightly between the populations (Table 2). Shapes of stylet knobs and tail terminus, however, were similar. Stylet knobs were at or sometimes slightly concave anteriorly and the tail terminus was narrowly rounded. J2s of these species were distinguished from the similar European H. avenae populations by a shorter stylet length (24.4-24.9 vs 26.227.5 ? m) and a shorter hyaline part of tail length (42-46 vs 44-48 ? m).
S.A. Subbotin et al.
Fig. 1. Canonical discriminant analysis. Scatterplots of means of 24 populations of the Heterodera avenae group on (A) the rst and the second axes and on (B) the rst and third canonical axes (for species codes, see Table 1).
Fig. 2. Similarity dendrogram of 24 populations from the Heterodera avenae group as computed by canonical discriminant analysis of four morphometrical characters (for isolate numbers, see Table 1).
H. arenaria and H. iri constituted a very distinct group with only 36% similarity with the other populations. H. arenaria signi cantly differed from all other species by a longer body and stylet length of the J2s and a larger fenestra length of the cysts. The stylet knobs of the J2s were distinctly concave anteriorly and the tail terminus rounded. H. arenaria had only 52% similarity to H. iri. This latter species was distinguished from the
other species by a longer tail and hyaline part of the tail. The stylet knobs of the J2s were at or sometimes slightly concave anteriorly; the tail terminus was narrowly rounded. PCR AMPLIFICATION AND RFLP ANALYSIS The ampli cation of the rDNA ITS regions of each population yielded one fragment of approximately 1060
Identi cation of Heterodera avenae group species
bp. No PCR products were obtained in the control lacking the DNA template. HindIII was the only enzyme that did not restrict any of the ITS. Most enzymes separated all the species of the group. The restriction pattern obtained with BsuRI (data not shown), PstI (Fig. 3A) and TaqI (Fig. 3B) clearly distinguished H. lipjevi. There was no intraspeci c polymorphism within H. lipjevi. AluI (Fig. 3C) separated European H. avenae populations and H. arenaria from the other species of the group; no tested enzyme allowed the differentiation of these two species from each other. AluI (Fig. 3C), Hin6I (Fig. 3D), Bsh1236I (data not shown), BsuRI (data not shown) and RsaI (Fig. 3E) yielded RFLP distinguishing H. latipons. BsuRI, Bsh1236I and MvaI separated H. hordecalis. MspI distinguished H. latipons and H. hordecalis from others. Digestion with Hin6I (Fig. 3D) distinguished Heterodera sp2. (Hsp2) and H. aucklandica from the other species; TaqI (Fig. 3B) and HinfI (Fig. 3F) distinguished these species from Heterodera sp1. (Hsp1.1). No enzyme enabled the differentiation of H. aucklandica from Heterodera sp.2 (Hsp2). H. iri was separated by RFLP generated by Bsh1236I (data not shown), Hin6I (Fig. 3D) and MspI (data not shown). For the distinction of Heterodera sp1. (Hsp1.1) at least two enzymes were necessary. Table 4 groups the data obtained from all RFLPs and shows the enzymes that can be used to separate species. Intraspeci c polymorphismwas revealed within H. avenae. AluI (Fig. 3C) and RsaI (Fig. 3E) did not digest PCR ampli ed products of European populations, but did digest PCR products of the Indian population (Hav8). Digestion by both enzymes also showed heterogeneity in ITS regions of the three French populations (Hav4, Hav6 and Hav9). For these populations two additional bands were obtained. The sum of the three fragments was approximately 2120 bp, i.e., about twice the size of the undigested ampli ed product. These additional bands were of the same length as those in the restriction patterns of the Indian population. Different intensity of these bands was observed between cysts from the St. Georges-duBois population (Hav4). AluI digestion of PCR products of each of three cysts showed clear additional bands for all three cysts; RsaI digestion of these products, however, produced distinct additional bands for only two cysts (Fig. 4). Very weak additional bands were repeatedly obtained in some H. avenae populations after digestion by Bsh1236I. A total of 90 scored fragments were obtained with eleven enzymes and used for analysis. The dendrogram constructed from Nei’s distances with UPGMA analysis
Vol. 1(2), 1999
revealed seven main clusters (Fig. 5). Cluster I contained all populations of H. lipjevi; cluster II contained populations of H. avenae, H. arenaria and the Heterodera spp. from grasses. The remaining ve clusters were each composed of single species: H. iri, H. latipons, H. hordecalis, H. schachtii and H. litoralis. R ESTRICTION ENDONUCLEASE CLEAVAGE MAPS Restriction patterns obtained after digestion of the PCR product of H. avenae populations Hav1, Hav2, Hav3, Hav5 and Hav7 and of the H. lipjevi populations corresponded to ITS sequences of the Swedish strict H. avenae isolate and Swedish East Gotland strain isolate, respectively, as published by Ferris et al. (1994). Based on our ITS-RFLP data and the sequence data published by Ferris et al. (1994) restriction endonuclease cleavage maps of ITS region of H. aucklandica, H. avenae, H. iri, H. lipjevi and Heterodera spp. were constructed for ve enzymes (Fig. 6).
Our comparative study showed that morphological and morphometrical divergences between species and populations correspond generally with genetic differences. H. arenaria, however, has to be considered as an exception. J2s of this species were easily distinguished from juveniles of other species by their larger size. The H. arenaria restriction patterns, however, did not differ from those of H. avenae. Different authors found good separations of H. arenaria from other species of the H. avenae group. Stone and Hill (1982), when using principal coordinate analysis of six J2s’ numerical characters found H. arenaria well separated from H. avenae and H. mani. Ibrahim and Rowe (1995) showed that H. arenaria could be separated from closely related species by non-speci c esterases. The dendrogramconstructed with molecular data showed two distinct clusters within the H. avenae group, which correspond with the two morphological groups of Wouts and Sturhan (1995): the H. avenae group sensu stricto (H. avenae, H. mani, H. lipjevi, H. iri, H. aucklandica) and the H. latipons group (H. latipons and H. hordecalis) supporting their view and adding evidence to the observations of Krall and Krall (1978), Shagalina and Krall (1981) and Sturhan and Wouts (1995) that there is an extensive variation within the H. avenae group and con rms the view that this group is not monophyletic.
S.A. Subbotin et al.
Fig. 3. Restriction fragments of ampli ed ITS regions of species belonging to the Heterodera avenae group. A: PstI; B: TaqI; C: AluI; D: Hin6I; E: RsaI; F: HinfI (for species codes, see Table 1, lane M: 100 bp DNA ladder).
Identi cation of Heterodera avenae group species
Table 4. Number of different RFLP pro les yielded by a single enzyme of the ITS regions of cyst nematodes. Species AluI H. avenae (type A) H. arenaria H. avenae (type B) H. avenae (types A+B) H. lipjevi Heterodera sp.1 Heterodera sp.2 H. aucklandica H. iri H. latipons H. hordecalis H. schachtii H. litoralis 1(*) 1 2 3 2 2 2 2 2 4 5 6 5 BsuRI 1 1 1 1 2 1 1 1 1 3 4 5 6 Bsh1236I 1 1 1 1 1 1 1 1 2 3 4 5 6 Bsp143I 1 1 1 1 1 1 1 1 2 2 1 1 1 Restriction enzymes HinfI 1 1 1 1 2 2 1 1 2 2 2 3 4 Hin6I 1 1 1 1 1 1 2 2 3 4 1 5 6 MspI 1 1 1 1 1 1 1 1 2 3 3 4 5 MvaI 1 1 1 1 1 1 1 1 2 2 3 4 5 PstI 1 1 1 1 2 1 1 1 1 1 1 1 3 RsaI 1 1 2 3 2 2 2 2 2 4 1 5 6 TaqI 1 1 1 1 2 1 3 3 3 3 4 3 4
(*) a same number indicates species with identical patterns.
Fig. 4. Restriction fragments of ampli ed ITS regions of individuals from two populations of Heterodera avenae. Lane 1: 100 bp DNA ladder; lane 2: unrestricted PCR product; lanes 3-8: digestion with RsaI; lanes 9-14 digestion with AluI; lanes 3 and 9: cyst 1 from St. Georges-du-Bois, 4 and 10: cyst 2 from St. Georges-du-Bois, 5 and 11: cyst 3 from St. Georgesdu-Bois; lanes 6 and 12: cyst 1 from Nuisement-sur-Coole, 7 and 13: cyst 2 from Nuisement-sur-Coole, 8 and 14: cyst 3 from Nuisement-sur-Coole.
H. litoralis — with a bifenestral vulval cone but not belonging to the H. avenae group because of its vulval slit of about 40 ? m — has a greater genetic distance from the H. avenae group than from H. schachtii (Wouts & Sturhan, 1996). This fact supports Krall and Krall (1978) who stated that the bifenestrate con guration of the vulVol. 1(2), 1999
val area evolutionary developed in different cyst nematode groups independently.Our dendrogram of genetic diversity of the H. avenae group (Fig. 5) constructed with RFLP data corresponds to a previously published dendrogram by Bekal et al. (1997), but shows additional relationships with other species of this group. Our results support those obtained by Sturhan and Rumpenhorst (1996) and Bekal et al. (1997) that the ‘Gotland strain’ from Sweden is identical to populations of H. lipjevi. Similarities of ITS regions sequences of Pushkin (Hf3) and Vad (Hf8) populations of H. lipjevi were found to be over 99% similar to the East and West Gotland strain isolates from Sweden (V. Ferris, pers. comm.). Differences in protein patterns and in rDNA between different populations of H. avenae and the ‘Gotland strain’ or H. lipjevi have been reported by several authors (Ferris et al., 1989, 1994; Bossis & Rivoal, 1996; Rumpenhorst et al., 1996; Sturhan, 1996; Sturhan & Rumpenhorst, 1996; Subbotin et al., 1996; Bekal et al., 1997). At present, H. lipjevi is found in Tadzhikistan, Uzbekistan, Iran, Turkey, Bulgaria, Russia, Ukraine, Estonia, Poland, Germany, England and Sweden. The main centre of its distribution is considered to be the East European-orient region (Rumpenhorst et al., 1996; Sturhan & Rumpenhorst, 1996; Subbotin et al., 1996). Using twelve restriction enzymes we did not nd any intraspeci c polymorphism within the ITS region of H. lipjevi populations.The Spanish populationfrom Torralba de Calatrava (Hf9) did not differ in our rDNA restriction analysis; however, it was clearly distinguished by CDA.
S.A. Subbotin et al.
Fig. 5. Dendrogram constructed from Nei’s distance and showing the clustering of 26 populations of species from the Heterodera avenae group based on rDNA-RFLP data. Bootstrap values (%) based on 1000 resamplings are given on appropriate clusters.
Our study and data published by Valdeolivas and Romero (1990) show that the J2s of this population have a longer stylet than those of typical H. lipjevi populations (average 26.4, 26.9 vs 24.3-25.4, respectively). Protein electrophoresis also revealed differences between the Spanish population and other H. lipjevi populations (Bossis & Rivoal, 1996; Sturhan & Rumpenhorst, 1996). Molecular intraspeci c polymorphism was observed within H. avenae populations. Extended digestion period, mixing experiments with control DNA, repeated ampli cations, and ampli cation with primers described by Vrain et al. (1992) suggest that this heterogeneity is not the result of partial digestion. At least two types of ITS regions were identi ed: type A for most European populations (Hav1, Hav2, Hav3, Hav5 and Hav7), type B for the Indian population (Hav8) and their combination (type A+B) for three French populations(Hav4, Hav6 and Hav9). These three genetic types generally corresponded to morphological types analogous to the same populations. The Indian population (Hav8) found in a desert region clearly differs from all others. AluI and RsaI digested the ITS region and permitted differentiation of this population from the other H. avenae populations. Ferris et al. (1994) published the ITS-sequence of the Australian Rainbow isolate of H. avenae. From this sequence, it can
be concluded that AluI and RsaI digest this population at the same sites. ITS restriction patterns produced by TaqI, however, may distinguish the Australian from the Indian population. Protein electrophoretic studies also showed a great similarity between the Australian populationand the population from Delhi (Sturhan & Rumpenhorst, 1996). Further investigationsare needed to clarify the taxonomic status of Indian populations. The polymorphism observed by Bekal et al. (1997) between H. avenae populations was different from ours. The number of genetic population types, therefore, may be higher than as yet has been identi ed. Three French populations(Hav4, Hav6 and Hav9) showed heterogeneity in the ITS1 region. The additional bands generated by AluI and RsaI were of the same length as those obtained for the Indian population. Therefore, we think that the French specimens we examined were composed of a mixture of ITS types A and B. These two additional bands produced an additional cluster for the French populations. Two of these populations (Hav4 and Hav6) belong to the second group of H. avenae pathotypes or Ha12 (Andersen & Andersen, 1982) and were separated by differences in morphometrics and CDA. ITS heterogeneity was revealed in many nematode species (Zijlstra et al., 1995; Thiéry & Mugniéry, 1996; Cherry et al.,
Identi cation of Heterodera avenae group species
1997) and, perhaps, re ects the result of evolutionary interactions between populationsand between species. The graminaceous cyst nematode populations used in our study (Hsp1.1, Hsp1.2 and Hsp2) formed a distinct separate cluster. They were morphologicallyand morphometrically easily distinguished from other species from the H. avenae group. Population Hsp1.1 from Putilovo is similar in morphology and morphometrics to the socalled ‘German grassland Heterodera species’ populations, which will be described as a new species (Sturhan, pers. comm.). This similarity is also supported by protein electrophoresis (Sturhan & Rumpenhorst, 1996; Subbotin et al., 1996). Discriminant analysis of four J2 numerical characters showed conspeci ty of populations from Putilovo and Kurilovo (Hsp1.1 and Hsp1.2). The morphology of J2s and cysts of the Belgian grassland population (Hsp2), is very similar to that of the Russian grassland populations (Hsp1.1 and Hsp1.2) and the morphology detailed in the original description of H. aucklandica, but differs from the latter by a shorter tail (average 65-66 vs 76) (Wouts & Sturhan, 1995). The ITS regions of Hsp2 were similar to those of H. aucklandica but differed from Hsp1.1 by the RFLPs obtained with four enzymes: HinfI, Hin6I, TaqI and Tru9I (data not published). The level of morphometric similarity between these European ‘grassland populations’ is rather high, suggesting that these populationsshould not be considered as belonging to a separate species, but only as belonging to a subspecies. Our study showed that rDNA-RFLPs and multivariation analysis of morphometric characters can distinctly separate species and populations within the H. avenae group. The H. avenae group sensu stricto is a complex of more or less distinct populations differentiated at species or subspecies level. Further DNA observations, and more detailed morphological, morphometrical, biological, ecological and biogeographical studies are needed to identify at which taxonomic level populationsof cyst-forming nematodes can be separated. The creation of a catalogue of RFLPs of the ITS region of cyst forming nematode species would facilitate the identi cation of species and population.
Fig. 6. Restriction endonucleasecleavage maps of ITS regions of Heterodera avenae group species, constructed on the base of our RFLP data and sequence data published by Ferris et al. (1994). ( — restriction site). Vol. 1(2), 1999
This work was partly nancially supported by the Russian Foundation of Fundamental Researches (Grant No 96-04-48470). The authors thank Drs V.P. Balakhnina, D.J.F. Brown, A. Ireholm, A. Madzhidov, L. Nasonova,
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E. Osipova, J.A. Rowe, R. Rivoal, D. Sturhan, V. Termeno and W.M. Wouts for supplying nematode populations.We also gratefully acknowledge Dr W.M. Wouts for critical reading of the manuscript.
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