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Variations in ribosomal DNA sequences and phylogeny of Globodera parasitising solanaceous plants
Abstract
The D3 expansion region of the 28S gene and the ITS1-5.8S-ITS2 region of rDNA sequences from Globodera rostochiensis, G. pallida, G. tabacum tabacum, G. tabacum virginiae and G. tabacum solanacearum have been aligned and compared, There are no nucleotide differences in the D3 region sequences between G. rostochiensis and G, pallida. Sequence analysis and RFLPs of ITS-PCR products showed that several haplotypes are present in the genomes of G. rostochiensis and G. pallida populations. Restriction patterns of PCR products for eight enzymes for differentiation of these two species are given. Phylogenetic analysis of 41 ITS region sequences obtained from populations and species of the subfamily Punctoderinae revealed four distinct main clades within Globodera parasitising solanaceous plants: G. rostochiensis, G. tabacum, G. pallida and an undescribed Globodera sp. from South America. The utility of RFLP profiles and sequences of the rDNA are discussed for diagnostics and phylogeny of Globodera.
... The genus Nothotylenchus Thorne, 1941, a member of the family Anguinidae Nicoll, 1935, is mainly delimited by non-valvate and non-muscular fusiform to slightly swollen metacorpus (Siddiqi, 2000;Andrássy, 2007). It is further characterized by fungal feeding behavior and pharyngeal glands not forming a long overlap over the intestine with glands nuclei located anterior to pharyngo-intestinal junction (Subbotin and Riley, 2012). ...
... Primers for LSU rDNA D2-D3 amplification were forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGT-3´) and reverse primer D3B (5´-TCGGAAGGAACCAGCT ACTA-3´) (Nunn, 1992). The primers rDNA1 ( 5´-T T G A T T A C G T C C C T G C C C T T T -3´) (Subbotin et al., 2000) and TW81 (5´-GTTTCCGTAGGTGAACCTGC-3´) (Joyce et al., 1994) were used to amplify ITS rDNA. Forward 1096F (5´-GGTAATTCTGGAGCTAATAC-3´) and reverse 1912R ...
... Validity of the genus Nothotylenchus is now well established (Siddiqi, 2000;Andrássy, 2007, Subbotin andRiley, 2012;Hashemi and Karegar, 2020). In general morphology, it looks similar to Ditylenchus Filipjev, 1936, but could be separated from it using a combination of characters viz. a non-valvate and non-muscular metacorpus and pharyngeal glands nuclei located anterior to pharyngo-intestinal junction (Siddiqi, 2000;Andrássy, 2007, Subbotin andRiley, 2012). ...
Nothotylenchus savadkoohensis n. sp. was recovered from rotten wood samples of an unidentified forest tree in the Mazandaran province and described herein. It is mainly characterized by an elongated conoid tail ending in a sharply pointed tip and four lines in the lateral field. Females of the new species have 379–662 μm long bodies with 5.8–6.9 μm long stylets ending in fine posteriorly sloping knobs, the metacorpus not valvate, the pharyngeal bulb slightly overlapping the intestine, and the vulva at 76.5–84.0% of body length. Males are also common and have 13.0–14.5 μm long spicules and bursa cloacal. By having an elongated conoid tail and four lines in the lateral field, the new species comes close to four known species, namely N. acris, N. acutus, N. antricolus , and N. truncatus . The morphological differences between the new species and the abovementioned species are discussed. The new species was sequenced for its D2–D3 segment of LSU and ITS rDNA regions. In the LSU phylogenetic tree, the currently available LSU sequences of the genus Nothotylenchus occupied distant placements from each other and the LSU sequence of the new species formed clade with a sequence assigned to Neotylenchus sp. In ITS phylogeny, the newly generated sequence of the new species formed a clade with a clade that includes sequences of Ditylenchus sp. and Neomisticius platypi and N. variabilis .
... The cosmopolitan genus Tylenchorhynchus (Cobb, 1913), is one of the biggest groups of plant-parasitic nematodes, which are migratory ectoparasites of the various plants (Siddiqi, 2000). The genus Bitylenchus (Filipjev, 1934) is very similar to the genus Tylen chorhynchus. ...
... Some nematologists place Bitylenchus as a junior synonym of Tylenchorhynchus (Fortuner and Luc, 1987;Handoo, 2000;Geraert, 2011), but other nematologists recognized both as valid genera (Gómez Barcina et al., 1992;Siddiqi, 2000;Andrássy, 2007). In the study by Handoo et al. (2014) about integrative taxonomy of the genera Bitylenchus and Tylenchorhynchus, these two genera were clearly separated from each other and the monophyly of the genus Bitylenchus was accepted only after the exclusion of B. ventrosignatus (Tobar-Jiménez, 1969) Siddiqi, 1986. ...
... Some nematologists place Bitylenchus as a junior synonym of Tylenchorhynchus (Fortuner and Luc, 1987;Handoo, 2000;Geraert, 2011), but other nematologists recognized both as valid genera (Gómez Barcina et al., 1992;Siddiqi, 2000;Andrássy, 2007). In the study by Handoo et al. (2014) about integrative taxonomy of the genera Bitylenchus and Tylenchorhynchus, these two genera were clearly separated from each other and the monophyly of the genus Bitylenchus was accepted only after the exclusion of B. ventrosignatus (Tobar-Jiménez, 1969) Siddiqi, 1986. Hosseinvand et al. (2020, from their phylogenetic analyses in order to accept the hypothesis of Bitylenchus and Tylenchorhynchus as valid genera. ...
During a survey on the biodiversity of plant-parasitic nematodes in Khuzestan province (southwest Iran), Bitylenchus hispaniensis was discovered around the rhizosphere of the euphrates poplar tree. The morphological and morphometric data were provided for the recovered species. To the best of our knowledge, this is the first report of B. hispaniensis from Iran and for the first time in association with euphrates poplar worldwide. Molecular phylogenetic analyses of the Iranian population of B. hispaniensis using the D2-D3 expansion segments of 28S rDNA and internal transcribed spacer (ITS rDNA) sequences using Bayesian inference (BI), showed a maximally supported clade with other sequences of the species.
... The availability of molecular data for Globodera had a significant impact on the systematics of this group, reshaping concepts of its species relationships and origins. Distinct genetic differences among G. pallida populations spanning Europe and other regions and those found in South America were revealed by sequence and phylogenetic analyses of the ITS-rRNA (Blok et al., 1998;Subbotin et al., 2000Subbotin et al., , 2011Madani et al., 2010;Skantar et al., 2011;Hoolahan et al., 2012a), cytb (Picard et al., 2007Plantard et al., 2008;Pylypenko et al., 2008;Madani et al., 2010;Geric Stare et al., 2013) and COI (Chitambo et al., 2019) genes. The analysis of partial cytb gene sequences and microsatellites of G. pallida collected in different regions allowed the identification of the origin of western European populations with a high degree of certainty (Picard et al., 2007Plantard et al., 2008). ...
... Alignments with the ITS rRNA, COI and cytb gene sequences were created using ClustalX 1.83 (Chenna et al., 2003) with default parameters. New sequences were aligned with corresponding published gene sequences (Ferris et al., 1995(Ferris et al., , 1999Blok et al., 1998;Subbotin et al., 2000Subbotin et al., , 2001Subbotin et al., , 2011Sabo et al., 2002;Manduric & Andersson, 2004;Širca & Urek, 2004;Picard et al., 2007Picard et al., , 2008Plantard et al., 2008;Pylypenko et al., 2008;Grenier et al., 2010;Madani et al., 2010;Skantar et al., 2011;Geric Stare et al., 2013;Lax et al., 2014;Chitambo et al., 2019 and others). Several alignments were created: i) ITS rRNA gene alignment containing only reference sequences of each Globodera species; ii) ITS rRNA gene alignment containing all available sequences of Globodera; iii) COI gene alignment containing sequences of all studied samples; iv) COI gene sequence alignment containing reference haplotype sequences for all Globodera species; v) cytb gene alignment containing sequences of all studied samples; vi) cytb gene sequence alignment containing reference haplotype sequences for all Globodera species; and vii) several COI and cytb gene alignments containing sequences of certain species. ...
... Phylogenetic relationships within Globodera were studied and discussed in detail using ITS rRNA gene sequences by Subbotin et al. (2000), Skantar et al. (2011, Knoetze et al. (2013Knoetze et al. ( , 2017a, Lax et al. (2014) and other authors. The phylogeny of this genus inferred from the COI gene in the present study is mainly congruent with that from the ITS rRNA gene, although it does not provide a distinct resolution between some species. ...
Globodera presently contains 13 valid and three as yet undescribed species. Three species, G. rostochiensis, G. pallida and G. ellingtonae, the potato cyst nematodes (PCN), cause significant economic losses on potatoes around the world. In our study we provide comprehensive phylogenetic analyses of 455 ITS rRNA, 219 COI and 164 cytb gene sequences of 11 valid and two undescribed species of Globodera using Bayesian inference, maximum likelihood and statistical parsimony. New 205 COI, 116 cytb and 21 ITS rRNA gene sequences were obtained from 148 populations of these species collected from 23 countries. The phylogenetic analysis revealed that Globodera displayed two main clades in the trees: i) Globodera from South and North America parasitising plants from Solanaceae; and ii) Globodera from Africa, Europe, Asia and New Zealand parasitising plants from Asteraceae and other families. Based on the results of phylogeographical analysis and age estimation of clades with a molecular clock approach, it is hypothesised that Globodera species originated and diversified from several centres of speciation located in mountain regions and then dispersed across the world from these regions during the Pleistocene. High genetic diversity of Bolivian populations of G. rostochiensis was observed for both mtDNA genes. Analysis of phylogenetic relationships of G. pallida and G. rostochiensis populations revealed incongruence in topology between networks inferred from mtDNA genes, which might be an indication of possible recombination and selective introgression events through gene flow between previously isolated populations. This puts some limitations on the use of the mtDNA marker as universal DNA barcoding identifier for PCN. Globodera bravoae syn. n. is proposed as a junior synonym of G. mexicana.
... In contrast, the differentiation of two races of devastating plant parasitic nematode Globodera rostochiensis (Roj and Ro2/3) was carried out using RAPD with similar conditions [39]. RAPD markers are suitable candidates for differentiating Meloidogyne species using isolated genomic DNA from nematodes, acting as a template [40,41]. RAPD characterization of the single female of the Meloidogyne species led to an observation that amplification patterns from a single female root-knot nematode are stable over three successive generations, leading to the assumption of the mitotic parthenogenetic reproductive mode of this nematode [42]. ...
... For instance, Höglund and co-workers employed this technology to uncover genetic differences in parasitic nematodes, including lungworms [43]. The principle of selective and accurate amplification underpins the AFLP approach, which was developed in response to challenges with endonuclease digestion of genomic DNA and adaptor ligation [40]. This method investigates gene expression to discover possible parasitic diseases, such as the potato cyst nematode (Globodera rostochiensis) [44]. ...
Citation: Bhat, K.A.; Mir, R.A.; Farooq, A.; Manzoor, M.; Hami, A.; Allie, K.A.; Wani, S.M.; Khan, M.N.; Sayyed, R.Z.; Poczai, P.; et al.
... In contrast, the differentiation of two races of devastating plant parasitic nematode Globodera rostochiensis (Roj and Ro2/3) was carried out using RAPD with similar conditions [39]. RAPD markers are suitable candidates for differentiating Meloidogyne species using isolated genomic DNA from nematodes, acting as a template [40,41]. RAPD characterization of the single female of the Meloidogyne species led to an observation that amplification patterns from a single female root-knot nematode are stable over three successive generations, leading to the assumption of the mitotic parthenogenetic reproductive mode of this nematode [42]. ...
... For instance, Höglund and co-workers employed this technology to uncover genetic differences in parasitic nematodes, including lungworms [43]. The principle of selective and accurate amplification underpins the AFLP approach, which was developed in response to challenges with endonuclease digestion of genomic DNA and adaptor ligation [40]. This method investigates gene expression to discover possible parasitic diseases, such as the potato cyst nematode (Globodera rostochiensis) [44]. ...
Abstract
Nematodes are non-segmented roundworms evenly distributed with various habitats ranging to approximately every ecological extremity. These are the least studied organisms despite being the most diversified group. Nematodes are the most critical equilibrium-maintaining factors, having implications on the yield and health of plants as well as well-being of animals. However, taxonomic knowledge about nematodes is scarce. As a result of the lack of precise taxonomic features, nematode taxonomy remains uncertain. Morphology-based identification has proved inefficacious in identifying and exploring the diversity of nematodes, as there are insufficient morphological variations. Different molecular and new evolving methodologies have been employed to augment morphology-based approaches and bypass these difficulties with varying effectiveness. These identification techniques vary from molecular-based targeting DNA or protein-based targeting amino acid sequences to methods for image processing. High-throughput approaches such as next-generation sequencing have also been added to this league. These alternative approaches have helped to classify nematodes and enhanced the base for increased diversity and phylogeny of nematodes, thus helping to formulate increasingly more nematode bases for use as model organisms to study different hot topics about human well-being. Here, we discuss all the methods of nematode identification as an essential shift from classical morphometric studies to the most important modern-day and molecular approaches for their identification. Classification varies from DNA/protein-based methods to the use of new emerging methods. However, the priority of the method relies on the quality, quantity, and availability of nematode resources and down-streaming applications. This paper reviews all currently offered methods for the detection of nematodes and known/unknown and cryptic or sibling species, emphasizing modern-day methods and budding molecular techniques.
Keywords: emerging methods; identification; meta-barcoding; morphology; PCR; nematodes
Citation: Bhat, K.A.; Mir, R.A.; Farooq, A.; Manzoor, M.; Hami, A.; Allie, K.A.; Wani, S.M.; Khan, M.N.; Sayyed, R.Z.; Poczai, P.; et al.
... They found one population of G. pallida from South America more distinct then the others, in different molecular method: RFLP and sequence analysis (Blok et al., 1998) and earlier in SSR and RAPD method (Blok et al., 1997) in comparison of ITS2 region of all tested populations. Identical approaches carry out on 16 Ukrainian population of G. pallida and G. rostochiensis by Pypylenko et al. (2008) shows similarity of Globodera pallida population in 97,8% based on sequences 938bp in comparison to published sequences of European population of PCN described by Blok et al. (1998) and Subbotin et al. (2000). Polymorphism of sequence of the first inter-nal transcribed spacer (ITS1) has been widely used to identifying and assessing genetic variability of three Russian population of Globodera rostochiensis comes from different geographical localities (Chrisanfova et al., 2008). ...
... RFPLs analysis of ITS-PCR products were carry out by Subbotin in 2000 on group of Russian populations of Globodera rostochiensis and the other Globodera species (Subbotin et al., 2000). Researchers used RFLP catalogue and sequence information from different populations or species and set of digestion enzymes to compare PCR products of nematode populations. ...
The cyst nematodes belonging to the genus Globodera are big worldwide problem in countries were Sola-naceaous plants growing. Knowledge of species-composition in populations of Globodera rostochiensis and Globodera pallida is very important for selection of appropriate measure of nematode regulations occurrence. Inter- and intraspecific variability among species of Globodera rostochiensis and Globodera pallida were studied intensively with the use of molecular analyses of DNA methods. This review summarize and compare of methods chosen to distinguishing between Globodera, both pathotypes and species.
... Ribosomal genes exhibit enough conserved inter-specific neutral genetic variation as to inform species delimitation without being prone to marker saturation [15][16][17][18]. For cyst nematodes identification, although several methods have been used, DNA-based approaches have shown to be more accurate to separate G. pallida from G. rostochiensis and other Globodera species and, ribosomal regions have also shown to be useful markers to distinguish species within the genus [12,17,[19][20][21]. For new occurrences of Globodera spp., sequencing of DNA fragments is also recommended, especially for regions where genetic data has not been reported before and for PCN species that may not follow a typical profile [17,22]. ...
... Resulting sequences were assembled in Sequencher 1 software version 5.1 (Gene Codes Corporation, Ann Arbor, MI USA) and manually reviewed for base calling errors. Partial 28S rRNA and ITS1-2 + 5.8S rRNA gene sequences from G. pallida, G. mexicana, G. rostochiensis, G. tabacum, G. ellingtonae, and G. artemisiae, were retrieved from GenBank nucleotide database and included in the alignment (Table 3) [8,19,21,[35][36][37][38][39][40][41][42][43]. Sequences of Punctodera punctata and P. chalcoensis also obtained from GenBank (AF274416.1, ...
Potato cyst nematodes (PCN) from the genus Globodera spp. cause major losses in the potato (Solanum tuberosum) industry worldwide. Despite their importance, at present little is known about the status of this plant pathogen in cultivated potatoes in Colombia. In this study, a total of 589 samples collected from 75 geographic localities in nine potato producing regions of Colombia (Cundinamarca, Boyacá, Antioquia, Nariño, Santander, Norte de Santander, Tolima, Caldas and Cauca) were assayed for the presence of potato cyst nematodes. Fifty-seven percent of samples tested positive for PCN. Based on phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene, all populations but one were identified as Globodera pallida. Sequences of G. pallida from Colombia formed a monophyletic group closely related to Peruvian populations, with the lowest average number of nucleotide substitutions per site (Dxy = 0.002) and net nucleotide substitutions per site (Da = 0.001), when compared to G. pallida populations from Europe, South and North America. A single sample formed a well-supported subclade along with G. rostochiensis and G. tabacum from Japan, USA and Argentina. To our knowledge this is the first comprehensive survey of Globodera populations from Colombia that includes genetic data. Our findings on species diversity and phylogenetic relationships of Globodera populations from Colombia may help elucidate the status and distribution of Globodera species, and lead to the development of accurate management strategies for the potato cyst nematodes.
... The species Heterodera schachtii belongs to the Schachtii group, the latter group also includes H. betae Wouts, Rumpenhorst & Sturhan, 2001, H. ciceri Vovlas, Greco & di Vito, 1985, H. daverti Wouts & Sturhan, 1979, H. galeopsidis Goffart, 1936, H. glycines Ichinohe, 1952, H. lespedezae Golden & Cobb, 1963, H. medicaginis Kirjanova & Krall, 1971, H. rosii Duggan & Brennan, 1966and H. trifolii Goffart, 1932(Subbotin et al., 2000a. The Humuli group however, includes the species H. humuli Filipjev, 1934 also includes Heterodera fici Kirjanova, 1954, H. litoralis Wouts & Sturhan, 1996, H. ripae Subbotin, Sturhan, Rumpenhorst & Moens, 2003, H. turcomanica Kirjanova & Shagalina, 1965and H. vallicola Eroshenko, Subbotin & Kazachenko, 2001(Subbotin et al., 2000b. The reported species of the genus Heterodera (23 species in total) in Iran prior to 2011 are given in Ghaderi et al. (2012). ...
... A final extension was performed at 72°C for 10 min (Alvani et al., 2016;Pedram 2017). The used primers for amplifying the ITS rDNA were forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (Joyce et al., 1994), and rDNA1 (5´-TTGATTACGTCCCTGCCCTTT-3´) and rDNA 1.58S (5´-ACGAGCCGAGTGATCCACCG-3´) as used by Subbotin et al. (2000b). Primers for 28S rDNA D2/D3 amplification were forward primer D2A (5'-ACAAGTACCGTGAGGGAAAGT-3') and reverse primer D3B (5'-TGCGAAGGAACCAGCTACTA-3') (Nunn 1992). ...
... The D2-D3 expansion segments of large-subunit (LSU) rDNA were amplified using primers forward D2A (5 0 -ACAAG-TACCGTGAGGGAAAGTTG-3 0 ) and reverse D3B (5 0 -TCGGAAG-GAACCAGCTACTA-3 0 ) (Nunn 1992). The internal transcribed spacer 1 region (ITS1) was amplified using two primer pairs: forward rDNA1 (5 0 -TTGATTACGTCCCTGCCCTTT-3 0 ) and reverse rDNA2 (5 0 -TTTCACTCGCCGTTACTAAGG-3 0 ) (Subbotin et al. 2000); forward TW81 (5 0 -GTTTCCGTAGGTGAACCTGC-3 0 ) and reverse AB28 (5 0 -ATATGCTTAAGTTCAGCGGGT-3 0 ) (Joyce et al. 1994). The PCR mixture (30 ml) contained the following: 15 μl Taq DNA polymerase 2× Master Mix RED, 2mM MgCl2 (Ampliqon, Odenese, Denmark), 8 μl distilled water, 1 μl of each primer (10 pmlo/μL), and 5 μl of DNA template. ...
Four species of the genus Longidorus were recovered from southern (Bushehr province) and southeastern (Southern Khorasan province) Iran. The first species, L. paratabrizicus n. sp. represents a new member to the genus and is characterised by 4.8–5.6 mm long females with anteriorly flattened lip region separated from the rest of the body by depression, amphidial fovea pocket-shaped without lobes, tail conical, dorsally convex, ventrally almost straight with bluntly rounded tip and males in population. By having similar lip region and tail shape, the new species most closely resembles five species viz. L. artemisiae, L. globulicauda, L. patuxentensis, L. sturhani, and L. tabrizicus. It represents the cryptic form of the last species. The second species belongs to L. mirus, recovered in both southern and southeastern Iran, representing the first record of the species after its original description. As an update to the characteristics of this species, it’s all juvenile developmental stages were recovered and described. The criteria to separate L. mirus from two closely related species, L. auratus and L. africanus, are discussed. The third species belongs to L. persicus, a new record in southern Iran. The fourth species, L. orientalis was recovered in high population density in association with date palm trees in Bushehr province. The phylogenetic relationships of the new species and recovered populations of L. mirus and L. persicus were reconstructed using two ribosomal markers and the resulted topologies were discussed.
... Primers for amplification of ITS rDNA were: forward rDNA1 (5'-TTG ATT ACG TCC CTG CCC TTT-3') and reverse rDNA1.58S (5'-ACG AGC CGA GTG ATC CAC CG-3') (Subbotin et al., 2000). Primers for amplification of 18S rDNA were: forward SSUF22 (5'-TCC AAG GAA GGC AGC AGG C-3') and reverse SSUR13 (5'-GGG CAT CAC AGA CCT GTT A-3') (Dorris et al., 2002). ...
The Columbia lance nematode, Hoplolaimus columbus, is an important pest that can cause severe damage to a wide host range of agricultural crops. During a survey on the biodiversity of plant-parasitic nematodes in the Misan province, southeast Iraq, H. columbus was discovered around the rhizosphere of oleander. The morphological and morphometric data were provided for the recovered species. The phylogenetic relationships of the Iraqi population of H. columbus with representatives of the family Hoplolaimidae were reconstructed using partial sequences of the small subunit, D2-D3 expansion segments of the large subunit, and internal transcribed spacer regions of ribosomal DNA based on Bayesian inference. In three inferred SSU, LSU and ITS phylogenies, Iraqi H. columbus belonged to the H. columbus / H. seinhorsti clade. To our knowledge, this is the first report of the species from Iraq.
... Primers for amplification of ITS rDNA were: forward rDNA1 (5′-TTG ATT ACG TCC CTG CCC TTT -3′), and reverse rDNA1.58S (5′-ACG AGC CGA GTG ATC CAC CG -3′) (Subbotin et al., 2000). To amplify the abovementioned loci, the polymerase chain reactions (PCRs) were performed as described by Azimi & Abdolkhani (2023). ...
During a survey on the biodiversity of plant-parasitic nematodes in the Misan province, southeast Iraq, a population of Psilenchus hilarulus was discovered around the rhizosphere of okra. The study included the analysis of the morphological and morphometric characteristics of the species that were recovered. These characteristics were then compared to those of other populations that have been reported from other locations. The phylogenetic relationships of the Iraqi population of P. hilarulus with representatives of tylenchid taxa were reconstructed using the partial sequences of the small subunit (SSU), D2-D3 expansion segments of large subunit (LSU), and internal transcribed spacer (ITS) regions of ribosomal DNA, based on Bayesian inference. In the phylogenetic trees inferred from SSU and LSU sequences, the sequences of genus Psilenchus formed a clade separate from the representatives of Tylenchidae and Merliniidae. In the SSU tree, the Iraqi population occupied a placement inside a major clade that includes the sequences assigned to P. hilarulus, P. cucrumerus and Psilenchus sp. In LSU tree, new LSU sequences formed a clade with a major clade that includes sequences assigned to P. hilarulus, P. cucrumerus and P. vinciguerrae. The first ITS sequence of the genus, the ITS rDNA of the Iraqi population of P. hilarulus, was utilized to reconstruct and analyze the corresponding phylogenetic tree. This appears to be the initial documentation of P. hilarulus emerging from Iraq.
... (5 -ACGAGCCGAGTGATCCACCG-3 ) (Subbotin et al., 2000). PCR was performed in a total vol. of 30 μl (10.6 μl distilled water, 15 μl Taq DNA Polymerase Master Mix RED 2.0x (Ampliqon), 1.2 μl of each primer (10 pmol μl −1 ), and 2 μl of DNA template). ...
A population of Xiphinema artemisiae was recovered from Iran, representing the first report after its original description from a meadow pasture in northern Caucasus, Russia. The Iranian population was recovered from the rhizosphere of grasses in the Damavand region, Tehran province. The females of the recovered populations are characterised by a lip region separated from the rest of body by a depression, a 5.1-5.6 mm long body, a 129-138 μ m long odontostyle, 76-85 μ m long odontophore, a uterus having a pseudo-Z-organ and crystalloid bodies observed in some specimens, and a short, rounded, dorsally more convex tail with a subcentral mucron, four juvenile developmental stages (the first stage was not recovered) and functional males. The Iranian population is morphologically and morphometrically similar to the type population and minor morphological differences can be attributed to geographical interpopulation differences. Molecular phylogenetic relationships of the Iranian population of X. artemisiae with other species were reconstructed using sequences of three genomic ribosomal markers, viz . small, large subunit D2-D3 and internal transcribed spacer (SSU, LSU D2-D3 and ITS) rDNA.
... Subsequently, the product was loaded on a 1.5% agarose gel. The primer set used for the amplification was the ITS primers: 5′-TTG ATT ACG TCC CTG CCC TTT-3′ (forward) and 5′-TTT CAC TCG CCG TTA CTA AGG-3′ (reverse) (Subbotin et al. 2000). The PCR Page 3 of 14 Shamseldean et al. ...
Background
Isolation of novel species of entomopathogenic nematodes (EPNs) with biocontrol potential against important insect pests is very important for the sustainable management of economic pests damaging food crops and providing protection to the agricultural environment. This study was aimed to new indigenous EPN isolates from Egyptian agricultural soils and studies its biocontrol potential for further use in the biological control programs. Five out of 15 soil samples obtained from a farm located at the Cairo–Alexandria desert highway was positive for the presence of EPN, using the greater wax moth baiting method.
Results
Sequencing of the internal transcribed spacer (ITS) region of 4 of the nematode isolates suggested that they belong to the species Heterorhabditis indica . However, one isolate does not show a high similarity to any of the H. indica previously recorded in the database of the Gen Bank and hence was identified as a new Heterorhabditis species and was deposited at the National Center for Biotechnology Information (NCBI) and registered under accession no. (OP555450) under the name of Heterorhabditis alii . This new species was also registered in the ZooBank under the registration link of: LSID urn: lsid: zoobank.org: act: 306F9D57-CC30-4B8E-8B19-4F0E42B08F34. No males were found in this species. Morphological characterization using the light microscope (LM) and scanning electron microscope (SEM) confirmed the identification of this nematode as a new species of the genus Heterorhabditis . Moreover, virulence of this new species against the fall armyworm (FAW), Spodoptera frugiperda (Smith 1797) (Lepidoptera: Noctuidae) was tested in comparison with the foreign EPN species, Heterorhabditis bacteriophora (HP88) and the local Heterorhabditis indica (Mango 2 isolate) and proved to be more effective against this devastative insect pest than the two compared species.
Conclusions
The present study found out a new species of the EPN genus, Heterorhabditis in Egypt. Our results were confirmed by both morphological and molecular analyses. The efficacy of this new species against the FAW proved to be a potent and safe biocontrol agent that can be used in biological control programs against this invasive insect pest of corn in Egypt and other global countries.
... For the molecular phylogenetic studies, four live nematode specimens (two females and two juveniles) were selected. Each specimen was transferred to an Eppendorf tube containing 10 μl ddH2O, 8 μl lysis buffer (125 mM KCl, 25 mM Tris-Cl pH 8.3, 3.75 mM MgCl2, 2.5 mM DTT, 1.125% Tween 20, 0.025% gelatine), and 2 μl proteinase K (600 μg/ml), and crushed for 2 min with a micro-homogeniser (Subbotin et al. 2000). The tubes were frozen at -80°C (15 min), then incubated at 65°C (1 h) and at 95°C (10 min), consecutively. ...
During a survey of soil nematodes in 2022, a new species of the genus Longidorus , described here as Longidorus zanjanensis sp. nov., was discovered in the rhizosphere of Astragalus sp. in Zanjan Province, Iran. The new needle nematode is described and illustrated based on morphological, morphometric, and molecular traits. Further, its females are characterized by having a long body ranging 5.6–7.7 mm long, lip region anteriorly flattened and almost continuous or slightly offset by a depression with body contour, ca 16.5–18.5 μm wide, amphidial fovea pouch-like without basal lobes, guiding ring at 35–41 μm distance from the anterior end, and an odontostyle and odontophore ranging 102–115 and 47–75 μm long, respectively. The pharyngeal bulb is 123–153 μm long, female reproductive system didelphic–amphidelphic containing sperm, vulva almost equatorial, located at 46.7–51.4% of body length, tail short, rounded to bluntly conoid, bearing two pairs of caudal pores and terminus widely rounded with distinct radial lines in hyaline region (39–50 μm long, c = 122.4–189.4, c’ = 0.6–0.8). Males are common, making up to 60% of the adults, and are functional, with spicules 68.0–80.0 μm long, as well as having 8–14 ventromedian copulatory supplements. All four juvenile life developmental stages were present, with the tail of first-stage juvenile conoid shape, dorso-ventrally curved with rounded terminus. The polytomous codes delimiting the new species are: A4-B3-C3-D3-E1-F34-G12-H1-I2-J1-K6. Morphologically, the new species comes close to eight known species of the genus, namely L. apulus , L. armeniacae , L. crassus , L. kheirii , L. soosanae , L. proximus , L. pauli, and L. ferrisi. The morphological differences between the new species and the aforementioned species are discussed. Molecular phylogenetic analyses based on D2-D3 of large subunit (LSU) and internal transcribed spacer 1 (ITS1) rRNA sequences indicate that Longidorus zanjanensis sp. nov. is closely related to L. hyrcanus , L. soosanae , and L. elongatus.
... Primers for amplification of ITS rDNA were forward primer rDNA1 (5′-TTGATTACGTCCCTGCCCTTT-3′) and reverse primer rDNA1.58S (5′-ACGAGCCGAGTGATCCACCG-3′) (Subbotin et al. 2000). To amplify the above-mentioned segments of DNA, the polymerase chain reactions (PCRs) were performed as described previously (Jumaah & Azimi 2022 ...
During an investigation on the biodiversity of plant-parasitic nematodes in Misan province (southeast of Iraq), two populations of Pratylenchus thornei were isolated from the rhizosphere of faba bean. The morphological and morphometric data were provided for the recovered populations. Both populations were similar to each other, but they were somewhat different in terms of the tail shape and body length. The tail in the Alkahla population was subcylindrical, tail terminus truncated and sometimes with a small projection. The tail in the Ali-Algharbi population was conical with an almost round to broadly rounded terminus. The morphological and morphometric characters of both populations agree with the type population of the species and some other populations reported from different areas. Molecular phylogenetic analysis of the Iraqi populations of P. thornei using the large subunit ribosomal RNA gene (LSU rDNA D2-D3) and internal transcribed spacer (ITS rDNA) sequences using Bayesian inference (BI), showed that they form maximally supported clades with other sequences of the species. The present study is the first report of P. thornei from Iraq based on morphological and molecular data.
... The primers used for the ITS region amplification were the forward primer rDNA1 (5 -TTGATTACGTCCCTGC CCTTT-3 ) and reverse primer rDNA1.58S (5 -ACGAGC CGAGTGATCCACCG-3 ) (Subbotin et al., 2000). The primers for amplifying COI were forward primer COIF (5 -GATTTTTTGGKCATCCWGARG-3 ) (He et al., 2005) and reverse primer XIPHR2 (5 -GTACATAATGAA AATGTGCCAC-3 ) (Lazarova et al., 2006). ...
A new species of the dagger nematode, genus Xiphinema , was recovered from the rhizospheric soil samples collected from rangelands in Semnan province, north-central Iran, and described based upon both morphological and molecular data. Xiphinema sangesarense n. sp. is characterised by 3.7-4.8 mm long females having 136-154 μ m long odontostyle, 74-87 μ m long odontophore, two equally developed genital tracts with crystalloid bodies in the tubular part of the uterus, short dorsally convex, ventrally slightly convex tail with a mucron at the end, four juvenile developmental stages and males present, functional. The new species most closely resembles X. azarbaijanense and is regarded as its tentative cryptic form, but was separated from it by reproductive mode, and morphological and molecular differences. Compared to X. afratakhtehense , the second similar species, it could be separated based on morphological differences, especially characteristics of juveniles and molecular data. Furthermore, the new species has close morphology with five know species, namely: X. aequum , X. horvatovicae , X. illyricum , X. macedonicum and X. vuittenezi , in terms of morphological characteristics (mainly structure of uterus and similar female tail), and their differences are discussed. In phylogenetic analyses of the new species using several sequences of two partial large subunit (LSU) D2-D3, and internal transcribed spacer (ITS) sequences, the new species formed a clade with Xiphinema sp. in the LSU tree, and in the ITS tree it formed a clade with X. afratakhtehense . In the cytochrome c oxidase I ( COI ) gene tree, sequences of the new species occupied a placement inside the clade of several sequences of X. afratakhtehense .
... For molecular phylogenetic analyses, two females, after morphological examination on temporary slides, were selected for DNA preparation in a small drop of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0, 100 QIAGEN Inc., Valencia CA) on separate clean slides and each was squished separately with a clean cover slip and collected using a pipette by adding 60 μl TE buffer. To amplify each segment, the following primers were used: for the SSU segment of rDNA, forward 988F (5′-CTC AAA GAT TAA GCC ATG C-3′), reverse 1912R (5′-TTT ACG GTC AGA ACT AGG G-3′) and forward 1813F (5′-CTG CGT GAG AGG TGA AAT -3′) and reverse 2646R (5′-GCT ACC TTG TTA CGA CTT TT-3′) (Holterman et al., 2006), for the LSU segment, forward D2A (5′-ACA AGT ACC GTG AGG GAA AGT-3′), reverse D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) (Nunn, 1992) and KK28S-1 (5′-AAG GAT TCC CTT AGT AAC GGC GAG TG-3′) (Kiontke et al., 2004), and for the ITS segment, forward rDNA1 (5′-TTG ATT ACG TCC CTG CCC TTT-3′) and reverse rDNA 1.58S (5′-ACG AGC CGA GTG ATC CAC CG-3′) (Subbotin et al., 2000). To amplify the above-mentioned segments of DNA, the polymerase chain reactions (PCRs) were performed as described previously (Pour Ehtesham et al., 2021). ...
Longidorus soosanae n. sp. is described and depicted based on morphological and molecular criteria. It was recovered from the rhizosphere of Fagus sp. in Golestan province, northern Iran. The new species is primarily characterized by its anterior region of the body distinctly tapered from the guiding ring towards the anterior end, forming a bottle-shaped appearance. Further, it is characterized by medium-sized females ranging 5.2–7.5 mm long, the lip region not set-off from the rest of the body, flat at the apex and 15–17 μm wide, pocket-shaped amphidal fovea lacking distinct basal lobes, guiding ring at 31–39 μm distance from the anterior end, and an odontostyle and odontophore ranging 92–103 and 59–72 μm long, respectively. The pharyngeal bulb is 92–130 μm long, vulva at 45.8–52.6% of body length and tail short, dorsally more convex-rounded. Males make up to 44% of the adults and are functional, having 50–64 μm long spicules and 9–13 ventromedian supplements. Only the fourth juvenile developmental stage (J4) was recovered. In reliance on morphological perspectives (odontostyle length, lip region shape, body length, guiding ring distance from anterior end, shape of amphidial fovea, shape of tail, index a and male presence/absence), the new species was compared with seven species of the genus including: L. alaskaensis, L. elongatus, L. goodeyi, L. iuglandis, L. juglandicola, L. panderaltum and L. proximus. From molecular perspectives, it is closely related to L. elongatus. Phylogenetic relationships of the new species with other selected relevant genera and species were reconstructed based on small and large subunits (SSU and LSU) and internal transcribed spacer (ITS) sequences and the obtained topologies were discussed.
... Most real-time PCR methods for the detection of plant pathogenic nematodes rely on the ribosomal ITS gene region (reviewed in Braun- Kiewnick and Kiewnick [2018]). However, several studies have found intraspecific and intra-genomic variation in ITS, indicating that multiple divergent copies of the ITS gene region exist in nematode genomes (Thiéry and Mugniéry 1996;Blok et al., 1998;Subbotin et al., 2000Subbotin et al., , 2011, thus limiting its use for real-time PCR diagnostics. ...
The stem and bulb nematode Ditylenchus dipsaci is a destructive nematode pest on many crops and is internationally quarantined in many countries, whereas Ditylenchus weischeri, only known to infect a weed plant (Cirsium arvense), is an unregulated nematode species with no known economic importance. In this study, we used comparative genomics to identify multiple gene regions and developed novel real-time PCR assays for the detection of D. dipsaci and D. weischeri. We sequenced the genomes of two mixed-stage nematode populations of D. dipsaci and two mixed-stage nematode populations of D. weischeri. The assembled genomes of D. dipsaci were 228.2 Mb and 239.5 Mb, and the genomes of D. weischeri were 177.0 Mb and 196.3 Mb. Depending on the species, 21,403–27,365 gene models were predicted. Using orthologous group analysis, single-copy and species-specific genes were identified. Primers and probes were designed targeting two species-specific genes in each species. The assays detected as low as 12 pg of DNA from the target species, or as few as five nematodes, with a Cq of 31 cycles or less. Our study provides genome data for two additional D. dipsaci isolates and two D. weischeri isolates, and four new and validated molecular assays to be used for rapid detection and identification of the two species.
... Primers for amplification of ITS rDNA were forward primer rDNA1 (5 -TTGATTACGTCCCTG CCCTTT-3 ) and reverse primer rDNA1.58S (5 -AC GAGCCGAGTGATCCACCG-3 ) (Subbotin et al., 2000). Primers for amplification of 18S rDNA were forward primer SSUF22 (5 -TCCAAGGAAGGCAGCAGGC-3 ) and reverse primer SSUR13 (5 -GGGCATCACAGACCT GTTA-3 ) (Dorris et al, 2002). ...
Ditylenchus pedrami n. sp., recovered from the rhizospheric soil of date palm in Khuzestan province, southwest Iran, is described and illustrated based upon morphological and molecular data. The new species is characterised by having six lines in the lateral field, lip region smooth and continuous with body contour, stylet 9-11 μm long, median pharyngeal bulb oval with small valve, pharyngeal bulb offset from the intestine, V = 83.9 (80.2-88.1), conoid tail with a pointed, dull or rounded tip and males with 20.1 (17-24) μm long spicules. Morphologically, the new species comes close to D. africanus, D. anchilisposomus, D. australiae, D. clarus, D. clavicaudatus and D. parcevivens, mainly by having shared features like six lines in the lateral fields, stylet length and somewhat similar tail tip. The new species was also compared with D. stenurus and D. sarvarae, two species with close phylogenetic affinities to it. The phylogenetic relationships of the new species with representatives of the family Anguinidae were reconstructed and discussed using partial sequences of the small subunit, D2-D3 expansion segments of the large subunit, and internal transcribed spacer regions of ribosomal DNA (SSU, LSU D2-D3 and ITS rDNA) based on Bayesian inference (BI).
... Sequences of these 17 isolates were different when compared with the reference sequences. Similarly, Subbotin et al. (2000) have also found several haplotypes within the genome of G. rostochiensis. This could be either due to the nature of genome in different isolates or random effect of genetic drift (Picard et al. 2006). ...
Quarantine species of potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, were reported from Nilgiris during 1961. The studies were carried out at ICAR-CPRS, Muthorai, Ooty and ICAR-CPRI, Shimla during 2015–17. To investigate the distribution of PCN, soil samples were collected from potato growing areas of Nilgiris and were identified based on morphological criteria and ITS-1 region. Molecular characterization using ITS-1 region specific primers revealed the presence of pure population of G. rostochiensis in 50% of the samples, G. pallida in 10.7% of the samples, mixed population in 28.6% of the samples and absence of both the species in 10.7% of the samples. The phylogenetic analysis inferred by the sequence of the ITS-1 region confirmed 92–100% genetic similarities in Globodera spp. Seventeen isolates of G. rostochiensis showed 92–99% genetic similarity and rest four 92–100% similarities. Whereas, genetic similarity among the ten isolates of G. pallida was 96.1–99.4%. In the morphometric characters J2s of G. rostochiensis exhibited shorter body length (459.8 μm) than G. pallida (493.7 μm). G. rostochiensis and G. pallida had difference in mean stylet length (21.1 μm and 23.4 μm respectively), hyaline tail terminal length (28.3 μm and 24.2 μm respectively) and shape of stylet knob. Highest mean value of vulval basin-anus distance (65.3 μm), number of cuticular ridges between vulval basin-anus (18.4) and Granek’s ratio (4.0 μm) was recorded in G. rostochiensis than G. pallida. Therefore, the present study will help to take appropriate and region specific PCN management decisions according to species dominance in that area.
... (5'-ACGAGCCGAGTGATCCACCG-3') (Subbotin et al., 2000). The 30 µl PCR mixture contained 10 µl of distilled water, 15 µl of Master Mix 2X (Ampliqon, Odenese, Denmark), 1 µl of each primer (10 pmol/µl), and 3 µl of DNA template. ...
During a survey on the biodiversity of plant-parasitic nematodes in Misan province (southeast Iraq), Tylenchorhynchus clarus and T. zeae were discovered around the rhizosphere of sugarcane and pumpkin, respectively. The morphological and morphometric data were provided for the recovered species. The morphological characters of both populations are in agreement with the type populations and other populations of them. To the best of our knowledge, this is the first report of these two species from Iraq, and a first report of the association of T. zeae with pumpkin. Molecular phylogenetic analyses of the Iraqi populations of T. clarus and T. zeae using the D2–D3 expansion segments of 28S rDNA and internal transcribed spacer (ITS) rDNA sequences using Bayesian inference (BI), showed they form maximally supported clades with other sequences of both species.
... Sequences from the samples were compared with GenBank accessions from other nematode species using the BLASTn homology search program (Madani et al. 2010;Subbotin et al. 2000Subbotin et al. , 2001. The published sequences of ITS, 28S, and COI from G. rostochiensis and other species were selected and downloaded. ...
On a global basis, potato cyst nematodes Globodera spp. Skarbilovich, 1959 (Behrens, 1975) are one of the most serious soil-borne pathogens in potato (Solanum tuberosum L.) production. In 2019-2020, 188 soil samples were taken from rhizosphere soil associated with the roots of stunted and chlorotic potato plants in the main potato-growing areas of Yunnan and Sichuan provinces of China. G. rostochiensis Wollenweber, 1923 (Skarbilovich, 1959) was recovered from 112 of the samples. Nematode identification was as confirmed by morphometric, light microscopy, electron microscopy and molecular methodologies. Population densities of G. rostochiensis ranged from 47.0 to 69.0 eggs/g soil. A BLASTn homology search program was used to compare the sequences of populations of G. rostrochienses from Yunnan and Sichuan provinces with populations of other Heteroderinae spp. and populations of G. rostochiensis from other nations. While potatoes have been grown in China for at least 400 years and the nation produces more potatoes than any other country, potato cyst nematodes were not reported in China until 2022.
... For amplifying the ITS rDNA, the forward primer rDNA1 (5 -TTGATTACGTCCCTGCCCTTT-3 ) and reverse primer rDNA1.58S (5 -ACGAGCCGAGTGATCCACCG-3 ), as used by Subbotin et al. (2000), were used. The 30 μl PCR mixture contained: 15 μl Taq DNA polymerase 2x Master Mix RED, 2 mM MgCl 2 (Ampliqon), 8 μl distilled water, 1 μl of each primer, and 5 μl of DNA template. ...
... The SSU rDNA was amplified using the forward primer F22 (5'-TCC AAG GAA GGC AGC AGG C-3') (Dorris et al., 2002) and the reverse primer 2646R (5'-GCT ACC TTG TTA CGA CTT TT-3') (Holterman et al., 2006). The primers used for amplifying the ITS rDNA were the forward primer rDNA1 (5'-TTG ATT ACG TCC CTG CCC TTT-3') and the reverse primer rDNA2 (5'-TTT CAC TCG CCG TTA CTA AGG-3') (Subbotin et al., 2000). PCR was carried out in a final volume of 35 μl with the addition of 12.1 μl distilled water, 17.5 μl Taq 2X PCR Master Mix (AMPLIQON, Denmark), 1.2 μl of each primers and 3 μl DNA template. ...
The entomopathogenic nematode, Steinernema borjomiense, was isolated from north Iran, and identified herein based upon morphological and molecular data. The Iranian isolate of this species is characterised by its 645-910 µm long infective juveniles, having brown-golden spicules 52-67 µm long in first, and 30-51 µm long in second generation males. The pathogenicity of the recovered population of S. borjomiense was evaluated on the larvae of two exotic invasive forest pests in north Iran, the box tree moth (BTM), Cydalima perspectalis, and the fall webworm (FWW), Hyphantria cunea, under laboratory conditions. The results indicated that the lethal concentration 50 (LC 50) values of nematode were 60.2 and 180.3 IJ larva-1 on fifth and fourth instar larvae of BTM; and 96.7 and 213.8 IJ larva-1 on fifth and fourth instar larvae of FWW, respectively, after 48 h at 25°C and 60% relative humidity (RH). Together, the present results corroborated the efficacy of this isolate for biocontrolling BTM and FWW in laboratory conditions.
... The internal transcribed spacer 1 (ITS1) fragment was amplified using the forward primer rDNA1 (5´-TTG ATT ACG TCC CTG CCC TTT-3´) and the reverse primer rDNA1.58S (5´-ACG AGC CGA GTG ATC CAC CG-3´) (Subbotin et al., 2000). PCR was carried out (for both the aforementioned fragments) in a total volume of 30 μl (11 μl distilled water, 15 μl 2x Master mix (Ampliqon, Denmark), 1 μl of each primer (10 pMol/μl), and 2 μl of DNA template). ...
Longidorus armeniacae n. sp. is described and illustrated using morphological and molecular data. It was recovered from the rhizosphere of apricot trees in Semnan province, northeast Iran. The new species is characterized by having females with plump (body width at vulva 80–123 μm, a = 44.5–68.2), medium sized body 5.0–7.4 mm long, lip region 16–20 μm wide, continuous with body contour, amphidial fovea small, pouch-like, guiding ring at 29–35 μm distance from the anterior end, odontostyle and odontophore 101–121 and 68–89 μm long, respectively, pharyngeal bulb 125–154 μm long, vulva at 48.8–59.3%, tail short (c′ = 0.5–0.7), rounded, sometimes varying to short conical; males common with 80–107 μm long spicules, 8–13 ventromedian supplements; four juvenile developmental stages present and tail in J1 with a digitate mucro. The morphological differences of the new species with 15 species having comparable morphology and/or close phylogenetic affinities namely L. arthensis, L. athesinus, L. caespiticola, L. carniolensis, L. cholevae, L. helveticus, L. iuglandis, L. lignosus, L. magnus, L. picenus, L. pius, L. poessneckensis, L. profundorum, L. raskii and L. uroshis are discussed. The polytomous identification codes of the new species are as follows: A45-B34-C23-D3-E1-F234-G1-H1-I2-J1-K7. The phylogenetic relationships of the new species and other selected species of the genus were reconstructed using LSU D2-D3 and the ITS rDNA sequences.
... com/NEBcutter2/). The sequences obtained in the present study were submitted to the GenBank database and compared with known sequences using the BLASTn homology search program (Subbotin et al., 2000Madani et al., 2010). The download sequence files were changed to FASTA format using Clustalx 1.83. ...
A new species of cyst-forming nematode, Heterodera amaranthusiae n. sp., is described and illustrated from the weed, Amaranthus retroflexus , in a potato field in Yunnan Province, China. It is characterised by having canary to russet-brown and asymmetric lemon-shaped cyst, distinct neck, bifenestrate vulval cone, relatively short vulval slit of 29 (28-32) μ m, bullae absent and underbridge absent or weak if present. Second-stage juveniles are characterised by a well-developed stylet of 23 (22-25) μ m with robust shaft and basal knobs concave anteriorly, tail conoid, 51 (48-58) μ m long and hyaline region comprising 48 (41-53)% of its length. Morphologically and morphometrically it most resembles H. vallicola in the Humuli group. The ITS, 28S and COI gene sequences of H. amaranthusiae n. sp. clearly differentiate it from other Heterodera species. For diagnostic purposes, restriction enzyme analysis of the ITS region and three restriction enzymes, Alu I, Bsu RI ( Hae III) and Cfo I ( Hha I), were selected, clearly distinguishing H. amaranthusiae n. sp. from representative species in the Humuli group. Phylogenetic relationships with other species of the genus, inferred from two ribosomal regions and the cytochrome oxidase c subunit 1 region, based on Bayesian analysis, consistently showed that H. amaranthusiae n. sp. clustered with high support with other Humuli group species but with separate species status.
... The intraspecific polymorphism revealed in European populations of G. pallida by various molecular markers provided new information to solve this issue. However, those studies did not include any Peruvian populations (Phillips et al., 1992;Folkertsma et al., 1994;Folkertsma et al., 1996a;Folkertsma et al., 1996b;Folkertsma et al.,2001;Thiery and Mugniery, 1996;Burrows et al., 1996;Manduric and Andersson, 2003) or did not allow precise identification of the origin of European populations due to the low representation of Peruvian samples (Blok and Phillips 1995;Blok et al. 1997;1998;Subbotin 2000, Grenier et al. 2001. Moreover, the molecular markers used in those s tudies were not always adequate enough for phylogeographical purpose (cf. ...
... Primers for amplification of ITS rDNA were forward primer rDNA1 (5'-TTG ATT ACG TCC CTG CCC TTT-3') and reverse primer rDNA1.58S (5'-ACG AGC CGA GTG ATC CAC CG-3') (Subbotin et al., 2000). The 25 μl PCR mixture contained 14.5 μl of distilled water, 3 μl of 10× PCR buffer, 0.5 μl of 10 mM dNTP mixture, 1.5 μl of 50 mM MgCl 2 , 1 μl of each primer (10 pmol μl -1 ), 0.5 μl of Taq DNA polymerase (Cinna Gen, Tehran, Iran; 5 U μl -1 ), and 3 μl of DNA template. ...
During a survey on the biodiversity of plant-parasitic nematodes in okra fields of Khuzestan province (southwest Iran), two species Helicotylenchus abunaamai and H. dihystera were recovered and identified. The morphological and morphometric data were provided for the recovered species and their differences with the type and some other populations were discussed. To the best of our knowledge, this is the first report of these two species in okra fields worldwide. In molecular phylogenetic analyses using the D2-D3 expansion segments of large subunit (LSU D2-D3) and internal transcribed spacer (ITS rDNA) sequences using Bayesian inference (BI) and maximal number of species of the genus, Iranian population of H. abunaamai formed a maximally supported clade with H. caudatus and H. digonicus; and the Iranian population of H. dihystera fell into a clade including several isolates of the species. In ITS phylogeny, the Iranian population of H. abunaamai formed a maximally supported clade with H. crenacauda and an unidentified species of the genus; and the Iranian population of H. dihystera fell into a clade including other isolates of the species. The variations of D2-D3 and ITS sequences of Helicotylenchus dihystera in comparisons with other available sequences of the species were discussed. H. abunaamai was molecularly characterised for the first time and this is the first molecular study of an Iranian population of H. dihystera.
... In some cases, the forward primer KK28S-1 (5′-AAGGATTCCCTTAGTAACGG CGAGTG-3′) and reverse primer KK28S-4 (5′-GCGG TATTTGCTACTACCAYYAMGATCTGC-3′) (Kiontke et al. 2004) were also used. The internal transcribed spacer 1 (ITS1) was amplified using the forward primer rDNA1 (5′-TTGATTACGTCCCTGCCCTTT-3′) and reverse primer rDNA 1.58S (5′-ACGA GCCGAGTGATCCACCG-3′) (Subbotin et al. 2000). The PCR carried out in total 30 μl volume contained: 15 μl Taq DNA Polymerase 2x Master Mix RED, 2 mM MgCl 2 (Ampliqon-Denmark), 10 μl distilled water, 1 μl of each primer, and 3 μl of DNA template. ...
Five populations of a new dagger nematode species were recovered from natural grasslands and forests of north and northwest Iran, and described based upon morphological and molecular data in present paper. Xiphinema hyrcaniense n. sp. is characterized by 3.9–5.5 mm long females, having 102–142 μm long odontostyle, 64–88 μm long odontophore, guiding ring located at 115–147 μm distance from anterior end, two equally developed genital branches having crystalloids in tubular part of uteri and pseudo-Z-organ at their junction with pars dilatata uteri, short, rounded to dorsally more convex tail with a mucro or in few specimens, without it. Common males with 72–95 μm long spicules and four juvenile developmental stages. The new species is similar to six known species belonging to artificial morphospecies group 5, especially looking closest to X. montenegrinum in its general morphology, but could be separated by ontogenesis of tail shape and morphometric indices. In molecular phylogenetic analyses using partial large subunit, and internal transcribe spacer 1 ribosomal DNA (LSU D2-D3 and ITS1 rDNA) sequences, the new species formed a clade with X. cretense and Xiphinema sp. in LSU; and in ITS1 tree, with X. dentatum and X. paradentatum.
... There have been many phylogenetic analyses of species within the genus Globodera (e.g., [5,73,[76][77][78][79][80][81][82][83][84][85][86]). A recent study, based on a phylogenetic analysis of gene sequences of three molecular markers (455 ITS rRNA, 219 COI, and 164 cytb) of 11 valid and 2 undescribed species of Globodera [87], found that Globodera displayed two main clades in their phylogenetic trees: (i) Globodera from South and North America parasitizing plants from Solanaceae; and (ii) Globodera from Africa, Europe, Asia, and New Zealand parasitizing plants from Asteraceae and other families. ...
The scope of this paper is limited to the taxonomy, detection, and reliable morphological and molecular identification of the potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis. It describes the nomenclature, hosts, life cycle, pathotypes, and symptoms of the two species. It also provides detailed instructions for soil sampling and extraction of cysts from soil. The primary focus of the paper is the presentation of accurate and effective methods to identify the two principal PCN species.
... Sequences of these 17 isolates were different when compared with the reference sequences. Similarly, Subbotin et al. (2000) have also found several haplotypes within the genome of G. rostochiensis. This could be either due to the nature of genome in different isolates or random effect of genetic drift (Picard et al. 2006). ...
Quarantine species of potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, were reported from Nilgiris during 1961. The studies were carried out at ICAR-CPRS, Muthorai, Ooty and ICAR-CPRI, Shimla during 2015-17. To investigate the distribution of PCN, soil samples were collected from potato growing areas of Nilgiris and were identified based on morphological criteria and ITS-1 region. Molecular characterization using ITS-1 region specific primers revealed the presence of pure population of G. rostochiensis in 50% of the samples, G. pallida in 10.7% of the samples, mixed population in 28.6% of the samples and absence of both the species in 10.7% of the samples. The phylogenetic analysis inferred by the sequence of the ITS-1 region confirmed 92-100% genetic similarities in Globodera spp. Seventeen isolates of G. rostochiensis showed 92-99% genetic similarity and rest four 92-100% similarities. Whereas, genetic similarity among the ten isolates of G. pallida was 96.1-99.4%. In the morphometric characters J 2 s of G. rostochiensis exhibited shorter body length (459.8 μm) than G. pallida (493.7 μm). G. rostochiensis and G. pallida had difference in mean stylet length (21.1 μm and 23.4 μm respectively), hyaline tail terminal length (28.3 μm and 24.2 μm respectively) and shape of stylet knob. Highest mean value of vulval basin-anus distance (65.3 μm), number of cuticular ridges between vulval basin-anus (18.4) and Granek's ratio (4.0 μm) was recorded in G. rostochiensis than G. pallida. Therefore, the present study will help to take appropriate and region specific PCN management decisions according to species dominance in that area.
... The ITS1 region was amplified using the forward primer rDNA1 (5´-TTGATTACGTCCCTGCCCTTT-3´) and reverse primer rDNA1.58S (5´-ACGAGCCGAGTGATCCAC CG-3´) (Subbotin et al., 2000). The polymerase chain reaction (PCR) cycles and sequen cing of amplified fragments were according to Heydari et al. (2020). ...
Ektaphelenchoides pini, the type species of the genus Ektaphelenchoides, was recovered from wood and bark samples of a dead broadleaf forest tree collected from the forests of Golestan province in north of Iran. The recovered population is mainly characterized by massive wide spicules of males with well-developed condylus marked by indentations at the apex and simple distal tip. It was further characterized by 756 to 947 μm long females having a cephalic region slightly separated from the rest body by a shallow depression, 20 to 23 μm stylet with wide lumen lacking conophore and knobs, excretory pore (E pore) at about one metacorpus length behind it, or 92 to 106 μm from anterior end and hemizonid just posterior to it, vagina anteriorly inclined, post uterine sac (PUS) ca 1.2 times vulval body width long, posterior body region elongate conoid, ending to a filiform tip, no functional rectum, a vestigial anus and common males with dorsally convex tail ending to an elongate spike and two pairs of precloacal (P2) and caudal (P3) large papillae at 5 to 6 μm distance anterior to cloacal opening and 30 to 41% of tail, respectively and lacking the single precloacal papilla (P1). In molecular phylogenetic analyses using small and large subunit ribosomal DNA (SSU, LSU rDNA) sequences, the Iranian population of E. pini fell in a clade including species of three genera Ektaphelenchus, Ektaphelenchoides, and Devibursaphelenchus in SSU, and a clade including species of Ektaphelenchus and Ektaphelenchoides in LSU tree, in close association with an isolate identified as E. pini in the latter phylogeny with high (0.99) Bayesian posterior probability (BPP). The comparisons with the type and French populations, as well as phylogenetic affinities of the species using ribosomal data, are discussed. This is the first report of E. pini from Iran, and its first simultaneous morphological and molecular phylogenetic study. New observations on some species of the genus were also presented and discussed.
Ektaphelenchoides pini, the type species of the genus Ektaphelenchoides, was recovered from wood and bark samples of a dead broadleaf forest tree collected from the forests of Golestan province in north of Iran. The recovered population is mainly characterized by massive wide spicules of males with well-developed condylus marked by indentations at the apex and simple distal tip. It was further characterized by 756 to 947 μm long females having a cephalic region slightly separated from the rest body by a shallow depression, 20 to 23 μm stylet with wide lumen lacking conophore and knobs, excretory pore (E pore) at about one metacorpus length behind it, or 92 to 106 μm from anterior end and hemizonid just posterior to it, vagina anteriorly inclined, post uterine sac (PUS) ca 1.2 times vulval body width long, posterior body region elongate conoid, ending to a filiform tip, no functional rectum, a vestigial anus and common males with dorsally convex tail ending to an elongate spike and two pairs of precloacal (P2) and caudal (P3) large papillae at 5 to 6 μm distance anterior to cloacal opening and 30 to 41% of tail, respectively and lacking the single precloacal papilla (P1). In molecular phylogenetic analyses using small and large subunit ribosomal DNA (SSU, LSU rDNA) sequences, the Iranian population of E. pini fell in a clade including species of three genera Ektaphelenchus, Ektaphelenchoides, and Devibursaphelenchus in SSU, and a clade including species of Ektaphelenchus and Ektaphelenchoides in LSU tree, in close association with an isolate identified as E. pini in the latter phylogeny with high (0.99) Bayesian posterior probability (BPP). The comparisons with the type and French populations, as well as phylogenetic affinities of the species using ribosomal data, are discussed. This is the first report of E. pini from Iran, and its first simultaneous morphological and molecular phylogenetic study. New observations on some species of the genus were also presented and discussed.
... Sequences of these 17 isolates were different when compared with the reference sequences. Similarly, Subbotin et al. (2000) have also found several haplotypes within the genome of G. rostochiensis. This could be either due to the nature of genome in different isolates or random effect of genetic drift (Picard et al. 2006). ...
Quarantine species of potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, were reported from Nilgiris during 1961. The studies were carried out at ICAR-CPRS, Muthorai, Ooty and ICAR-CPRI, Shimla during 2015-17. To investigate the distribution of PCN, soil samples were collected from potato growing areas of Nilgiris and were identified based on morphological criteria and ITS-1 region. Molecular characterization using ITS-1 region specific primers revealed the presence of pure population of G. rostochiensis in 50% of the samples, G. pallida in 10.7% of the samples, mixed population in 28.6% of the samples and absence of both the species in 10.7% of the samples. The phylogenetic analysis inferred by the sequence of the ITS-1 region confirmed 92-100% genetic similarities in Globodera spp. Seventeen isolates of G. rostochiensis showed 92-99% genetic similarity and rest four 92-100% similarities. Whereas, genetic similarity among the ten isolates of G. pallida was 96.1-99.4%. In the morphometric characters J 2 s of G. rostochiensis exhibited shorter body length (459.8 μm) than G. pallida (493.7 μm). G. rostochiensis and G. pallida had difference in mean stylet length (21.1 μm and 23.4 μm respectively), hyaline tail terminal length (28.3 μm and 24.2 μm respectively) and shape of stylet knob. Highest mean value of vulval basin-anus distance (65.3 μm), number of cuticular ridges between vulval basin-anus (18.4) and Granek's ratio (4.0 μm) was recorded in G. rostochiensis than G. pallida. Therefore, the present study will help to take appropriate and region specific PCN management decisions according to species dominance in that area.
... The internal transcribed spacer 1 (ITS1) fragment was amplified using the forward primer rDNA1 (5′-TTG ATT ACG TCC CTG CCC TTT-3′) and the reverse primer rDNA1.58 s (5′-ACG AGC CGA GTG ATC CAC CG-3′) (Subbotin et al. 2000). PCR was carried out (for both the aforementioned fragments) in a total volume of 40 μl (12 μl distilled water, 20 μl 2x Master mix (Ampliqon, Denmark), 2 μl of each primer (10 pMol/μl), and 4 μl of DNA template). ...
Longidorus behshahrensis n. sp. is described and illustrated using morphological and molecular data. It was recovered from the rhizosphere of a wild cherry tree in Mazandaran province, northeastern Iran. The new species is characterized by having 6.8–8.4 mm long females, lip region separated from the rest of the body by a shallow depression, amphidial fovea pocket shaped not bilobed, guiding ring at 25–31 μm from the anterior end, 81–91 and 51–63 μm long odontostyle and odontophore, respectively, 109–130 μm long pharyngeal bulb, didelphic-amphidelphic reproductive system with long tubular bipartite uteri with sperm, vulva at 44–54%, tail bluntly conical, dorsally convex, ventrally concave with widely rounded terminus, hyaline region with discernible radial lines; males common with 50–60 μm long spicules and 9–11 ventromedian single supplements. Only the two latter juvenile developmental stages were recovered for the new species. Codes for identifying the new species are A3-B1-C23-D2-E1-F34-G23-H2-I2. Morphologically, the new species comes close to eight known species of the genus namely L. artemisiae, L. dunensis, L. elongatus, L. euonymus, L. goodeyi, L. iranicus, L. protae and L. tabrizicus. The morphological differences of the new species and the aforementioned species are discussed. In molecular phylogenetic analyses using LSU rDNA D2-D3 sequences, the position of the new species among closely related species was not resolved due to polytomy. In ITS1 phylogeny, the new species formed a clade with L. distinctus.
... Globodera (Subbotin, Perry, Warry, & Halford, 2000), Nematodirus (Heise et al., 1999;Nadler, Hoberg, Hudspech, & Rickard, 2000) and ...
Ribosomal RNA genes have long been a favored locus in phylogenetic and metabarcoding studies. Within a genome, rRNA loci are organized as tandem repeated arrays and the copies are homogenized through the process of concerted evolution. However, some level of rRNA variation (intragenomic polymorphism) is known to persist and be maintained in the genomes of many species. In nematode worms, the extent of rRNA polymorphism (RP) across species and the evolutionary and life history factors that contribute to the maintenance of intragenomic RP is largely unknown. Here we present an extensive analysis across 30 terrestrial nematode species representing a range of free‐living and parasitic taxa isolated worldwide. Our results indicate that RP is common and widespread, ribosome function appears to be maintained despite mutational changes, and intragenomic variants are stable in the genome and neutrally evolving. However, levels of variation were varied widely across rRNA locus and species, with some taxa observed to lack RP entirely. Higher levels of RP were significantly correlated with shorter generation time and high reproductive rates, and population‐level factors may play a role in the geographic and phylogenetic structuring of rRNA variants observed in genera such as Rotylenchulus and Pratylenchus. Although RP did not dramatically impact the clustering and recovery of taxa in mock metabarcoding analyses, the present study has significant implications for global biodiversity estimates of nematode species derived from environmental rRNA amplicon studies, as well as our understanding of the evolutionary and ecological factors shaping genetic diversity across the nematode Tree of Life.
... The internal transcribed spacer 1 (ITS1) fragment was amplified using the forward primer rDNA1 (5′-TTGAT-TACGTCCCTGCCCTTT-3′) and the reverse primer rD-NA1.58s (5′-ACGAGCCGAGTGATCCACCG-3′) (Subbotin et al., 2000). PCR was carried out for both the aforementioned fragments in a total volume of 40 μ l (12 μ l distilled water, 20 μ l 2x Master mix (Ampliqon, Denmark), 2 μ l of each primer (10 pMol/μ l), and 4 μ l of DNA template). ...
The morphology and morphometric characteristics of a Longidorus population recovered from a wheat-potato field in Hamadan province, western Iran, fit well with those given for two species, L. proximus and L. israelensis. The Iranian population was characterized by 5.6 to 8.6-mm-long females having a 17 to 21-µm-wide lip region separated from the rest of the body by a shallow depression, pocket-shaped amphidial fo-vea with a simple base and a ventral enlargement, a guiding ring at 31 to 40 µm distance from the anterior end, 108 to 127-µm-long odontostyle, 58 to 64-µm-long odontophore, 101 to 129-µm-long pharyngeal bulb with remarkably larger dorsal gland nucleus (at 49 to 53% of the bulb length) and two smaller ventrosublateral nuclei (at 66 to 76% of the phar-yngeal bulb length), four juvenile developmental stages, and a rare male. The morphological and molecular data corroborated its assignment to the species L. proximus. In molecular phylogenetic analyses using partial LSU rDNA D2-D3 sequences, the presently studied Iranian population and previously sequenced isolates of L. proximus formed a clade with L. cretensis, L. iranicus, L. pseudoelongatus, and L. closelongatus, all except L. pseudoelongatus with no available data, having the similar phar-yngeal gland nuclei size and arrangement. In internal transcribed spacer 1 (ITS1) phylogeny, it formed a clade with L. sturhani and four aforemen-tioned species. The characters delimiting the two species L. proximus and L. israelensis were discussed and a new synonymy was proposed.
Paratylenchus vandenbrandei , has been recovered from the rhizospheric soil of Euphrates poplar ( Populus euphratica ) in the Karkheh protected area of Khuzestan province, southwestern Iran. The species was identified as P. vandenbrandei by the presence of three lines in the lateral fields; conoid rounded lip region; presence of submedian lobes, a stylet 24.0–28.8 μm long; an excretory pore at the level of the anterior part of the pharyngeal bulb; a round-to-oval spermatheca; presence of vulval flaps; and a conoid tail, with a terminus that is rounded or slightly pointed in some specimens. Males have a conoid tail, with a rounded-to-slightly-pointed terminus. The phylogenetic relationships of the species were reconstructed and investigated using partial sequencing of the D2-D3 expansion segments of large subunits, as well as internal transcribed spacer regions (LSU D2-D3 and ITS rDNA) based on Bayesian inference (BI). P. vandenbrandei has formed a clade with P. neonanus , P. minor , P. nainianus , P. chongqinjensis , P. pedrami , P. baldaccii , P. leptos and P. rostrocaudatus with maximal support (BPP = 1.00). To the best of our knowledge, this is the first report of P. vandenbrandei in Iran and the first molecular characterization of the species worldwide.
Nanotechnology plays a vital role in agriculture development. This role exists in many agricultural aspects such as fertilizer production, pesticide production, and pest management. Using nanotechnology, it could reduce the cost of food production and environmental pollution and at the same time increase the yield of crops. Nano-sensors are considered one of the most important components in nanotechnology branches. The nano-sensors used in diagnosis or detection of diseases may be chemical, biological, or mechanical components. Sometimes biological sensors are used so are called nanobiosensor. Recently, nano-sensors were used for monitoring of plant diseases. It was known that the sooner the disease is detected, the sooner the treatment is possible. Some nano-sensors have been synthesized for detection of plant parasitic nematode infection and also gave a fast solution to the management of this pest. So, many scientists considered that the nano-sensors are the main tolls in disease mentoring.
During a survey on the biodiversity of plant-parasitic nematodes in Khuzestan province (southwest Iran), Ditylenchus filimus was discovered in the rhizosphere of date palm. The morphological and morphometric data were provided for the recovered species. The morphological characters of Iranian population are in agreement with the type population and other populations of this species. Molecular phylogenetic analyses of the species with representatives of the family Anguinidae were discussed using partial sequences of the small subunit, D2-D3 expansion segments of the large subunit, and internal transcribed spacer regions of ribosomal DNA (SSU, LSU D2-D3 and ITS rDNA) based on Bayesian inference (BI). To the best of our knowledge, this is the first report of molecular characterization of this species. Key Words: 18S rDNA, D2-D3 LSU rDNA, ITS rDNA, Ditylenchus, morphometric data, phylogeny
Plant-parasitic nematodes are among the most influential soil organisms worldwide and have a detrimental impact on wheat productivity; they also play a major role in maintaining soil diversity. However, limited information is available on the biodiversity of nematodes associated with wheat cropping systems in Turkey. To address this knowledge gap, 45 wheat fields in the Afyonkarahisar Province of Turkey were sampled in 2021 in order to extract and identify nematodes. A total of 14 genera and 44 species of these nematodes were identified based on their physical traits and ITS DNA sequences examination. Shannon diversity index was used to determine the prevalence and biodiversity features of these nematodes. This study represents the first comprehensive research on plant-parasitic nematodes from wheat-growing regions in the Afyonkarahisar Province. The genera Helicotylenchus, Heterodera, Merlinius, Pratylenchoides and Pratylenchus dominated with a high relative abundance percentage >60%, and were extremely common (>90% for each). The average Shannon index of nematode species in the wheat fields was 2.20, with an evenness value of 0.81, indicating moderate diversification and good nematode evenness. This study also revealed a significant correlation between nematode genus biodiversity and edaphic, climatic and geographical factors. Finally, this research demonstrated a remarkable diversity of soil nematodes associated with wheat crops and they have the potential to be useful tools for extensive soil bio-study.
This is a compilation of articles published in the Special Issue Systematics, Morphological, and Molecular Characterization of Economically Important Plant–Parasitic Nematodes: A Themed Issue in Honor of Dr. Gary Bauchan in Plants. It includes a series of original research (seven) and review articles (four) focused on plant-parasitic nematodes including two new species description, Pratylenchus dakotaensis n.sp. and Xiphinema malaka n. sp. Nematodes are one of the most
important pests globally and can cause up to 14% loss of food crops. In total, nematodes cause over $100 billion in global crop damage annually. To date, only a few thousand PPN species have been described. Nematode identification has traditionally relied on morphological and anatomical characters using light microscopy and, in some cases, scanning electron microscopy (SCN). Lately, integrative studies combining molecular diagnosis with morphology and taxonomy are used to
accurately identify and describe nematode species. Detailed analyses of morphological and molecular data have both significantly contributed to our overall understanding of the dynamic and complex nature of plant–nematode interactions. We are grateful to all the authors who submitted their work to be included in this special issue.
A new species of the genus Longidorus, L. hyrcanus n. sp., is described and illustrated based upon two populations, recovered in two distant points in northern Iran. Morphologically, it is characterized by having 5.0–5.8 mm long females with rounded lip region continuous with body contour, amphidial fovea pocket-shaped, asymmetrically bilobed at base, odontostyle 110–127 μm long, tail short, rounded or bluntly conoid, with widely rounded terminus, four juvenile developmental stages and common males. The polytomous codes delimiting the new species are: A45-B12-C34-D1-E2-F23-G12-H12-I2-J1-K1. The new species resembles twelve known species of the genus mainly by having similarities in rounded lip region continuous with body contour and tail short, rounded or bluntly conoid, with widely rounded terminus and/or having close phylogenetic affinities (e.g. L. elongatus) namely: Longidorus baeticus, L. crataegi, L. elongatus, L. fasciatus, L. hangzhouensis, L. igoris, L. iranicus, L. iuglandis, L. orientalis, L. pacensis, L. profundorum and L. raskii. The morphological differences of the new species with aforementioned species are discussed. The phylogenetic relationships of the new species with other relevant species of the genus were reconstructed using D2-D3 expansion segments of large ribosomal subunit and internal transcribed spacer (LSU D2-D3 and ITS) rDNA sequences, and the resolved topologies were discussed.
Summary – A new species of Scutylenchus was recovered from soil collected in the rhizosphere of forest trees in natural Asalem forests,
Gilan province, north Iran. The new species is described and illustrated based upon morphological and molecular data. Scutylenchus
quercinus n. sp. is mainly characterised by having four lines in the lateral fields, thus delimiting it from currently known species of
the genus, which have six lines. It is further characterised by 605-709 μm long females having a slightly offset cephalic region with
five or six annuli, stylet 19-21 μm long with conus ca 50% of its total length and three slightly posteriorly sloping knobs, vulva in a
cavity, with epiptygma, vagina vera squarish in lateral view, female tail subcylindrical with smooth end, and males with 26-30 μm long
spicules. The new species was morphologically compared with eight similar species. The molecular phylogenetic relationships of the
new species with other relevant species and genera were reconstructed using partial small, large subunit and internal transcribed spacer
ribosomal DNA (SSU, LSU D2-D3 and ITS1 rDNA) sequences and the resulting topologies were discussed.
Background and Objectives:
The Karkheh protected area is considered as one of the protected areas of Iran, which harbors a remarkable diversity of vegetations. A population belonging to the genus Pratylenchus was recovered and identified as P. mediterraneus during a survey on the biodiversity of the plant-parasitic nematodes in this region of Khuzestan province. This study aims to characterize the population based on the morphological and morphometric characteristics and evaluate its phylogenetic affinities using LSU D2-D3 and ITS rDNA sequences. Moreover, its phylogenetic relationships was investigated with other relevant genera and species.
Materials and Methods:
Several soil samples were collected from the rhizosphere of tamarisk trees in Karkheh protected area. The centrifugal-floatation and tray methods were used for extracting the nematodes from the soil samples. The collected specimens were fixed and transferred to the pure glycerin after extracting the nematodes by using the modified De Grisse method. Then, permanent microscopic slides were prepared from the processed nematodes. The species was identified by using a light microscope equipped with a drawing tube based on the morphological and morphometric characteristics, and valid keys. The molecular phylogenetic analyses were performed by using partial sequences of the D2-D3 expansion segments of the large subunit, and internal transcribed spacer (LSU D2-D3 and ITS rDNA) regions based on the Bayesian inference under the GTR + G + I model.
Results:
A population of the genus Pratylenchus was recovered in the present study. The morphological, morphometric and molecular studies based on the D2-D3 domains of the 28S and ITS rRNA gene indicated that the recovered population belongs to P. mediterraneus. The morphological, morphometric, and molecular data of the Iranian population of the species were presented for the first time in this study. In the phylogenetic tree inferred using the 28S rRNA gene sequences, the newly generated sequence of the Iranian population of P. mediterraneus formed a clade with other sequences of the species, and some sequences assigned to P. thornei. All other sequences of P. thornei occupied a clade, in close phylogenetic relationship with the aforementioned clade. The newly generated sequence of the Iranian population formed a maximally supported clade with other sequences of the species in the phylogenetic tree inferred using the ITS rRNA gene.
Discussion:
The two species P. thornei and P. mediterraneus are morphologically very similar and can mainly be separated from each other by presence/absence of male and a functional spermatheca. Their separation is further corroborated by using the partial sequences of 28S and ITS rDNA. The sequences of the two species form the separate clades, and the identification of some sequences occupying the same clade with the sequences of P. mediterraneus need further validations in lack of the morphological data.
A survey was carried out to estimate the frequency and occurrence of potato cyst nematode (PCN) in potato cultivating fields in different districts of Himachal Pradesh. A total of 109 composite soil samples were collected from 5 districts of Himachal Pradesh, India. Nematode cysts were extracted from the soil using standard procedure. The population density and percentage of occurrence of cyst nematode were calculated. According to the results district, Mandi had the highest PCN cyst population density, and the percentage of occurrence is 100%. District Kangra had the least PCN cyst population density and the percentage of occurrence is 66.6%. This survey showed the occurrence Globodera rostochiensis district-wise. The collected cysts were identified by morphological and molecular techniques, up to species level based on nucleotide homology and phylogenetic analysis. According to morphometrics distance between the vulva and anus is very less (65.69 ±22.2) µm. The outcome sequences are showed 95-99.05% similarity with sequence I.D KJ636272.1 from the Netherlands. This survey indicates that PCN Globodera rostochiensis were widely distributed in the potato cultivating fields of Himachal Pradesh. The present study helped determine the distribution of PCN in the soil and species dominance in particular areas. Hence, it helps design different strategies against PCN management.
Les nématodes à kyste du genre Globodera sont parmi les phytoparasites les plus étudiés de par l’impact économique qu’ils peuvent engendrer sur les productions agricoles. Originaire des hauts plateaux de la cordillère des Andes ce genre aurait été importé en Europe à la fin du XIX siècle. L’existence d’un complexe d’espèce cryptique chez G. pallida, nématode phytoparasites d’importance au sein du genre, est questionnée. La révision taxonomique de cette espèce peut avoir des conséquences fortes en termes d’épidémiologie, d’évaluation et de gestion des risques. Durant ce travail une approche de taxonomie intégrative impliquant trois champs disciplinaires, génétique, morphométrique et biologique, a été développée sur un panel de populations sud-américaines pour délimiter de nouvelles frontières au sein de cette espèce. L’exploration de la diversité génétique a permis de révéler l’exitence de deux groupes fortement distants de G. pallidaCette étude questionne aussi les processus impliqués dans cette divergence qui semblent distincts pour ces deux groupes. L’existence d’une différenciation morphologique a été observée grâce à un outil d’analyse d’image automatisé créé spécifiquement pour ce travail. Le développement de l’outil constitue une approche novatrice pour l’étude de la morphométrie dans ce genre et plus généralement chez les nématodes. Cette différenciation s’appuie sur l’intégration combinée de trois métriques adaptées à la morphométrie automatisée. Enfin, l’étude de la capacité de ces entités à se développer aux dépens de certaines plantes a elle aussi rapporté des
Two populations of Xiphinema persicum n. sp. belonging to the X. americanum-group, were recovered from Semnan province, and described and illustrated based upon morphological, morphometric and molecular data. The type population of the new species is characterized by 2233–2736 μm long females, offset lip region, anteriorly flat to slightly rounded and separated from the rest body by constriction, 82–87 μm long odontostyle, two equally developed genital branches with visible endosymbiont bacteria in ovaries under light microscope, vulva at 52.8–55.5%, 27–32 μm long dorsally convex and ventrally straight to slightly convex tail with a rounded tip, or having a wide mucro-like differentiation, rare male with six ventromedian supplements and four juvenile developmental stages. The new species was morphologically compared with six similar species, X. bricolense, X. californicum, X. incertum, X. pachtaicum, X. santos and X. simile. The latter species has closest morphology and phylogenetic affinities to the new species in both large subunit and internal transcribed spacer 1 (LSU and ITS1 rDNA) trees; but X. persicum n. sp. has four juvenile developmental stages (vs. three), longer odontostyle, and posteriorly located guiding ring compared to it. The phylogenetic relationships of the endosymbiont bacterium of the new species with other isolates was reconstructed using the partial sequences of 16S rDNA.
Potato cyst nematodes (PCN) from the genus Globodera spp. cause major losses in potato ( Solanum tuberosum ) industry worldwide. Despite their importance, at present little is known about the status of this plant pathogen in cultivated potatoes in Colombia. In this study, a total of 589 samples collected from 75 geographic localities from nine potato producing departments of Colombia were assayed for the presence of potato cyst nematodes. Fifty-seven percent of samples tested positive for PCN. All populations but one were identified as Globodera pallida , with conspicuous morphometric variation found among populations. Based on phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene, G. pallida from Colombia formed a monophyletic group closely related to Peruvian populations, with the lowest average number of nucleotide substitutions per site ( Dxy = 0.002) and net nucleotide substitutions per site ( Da = 0.001), when compared to G. pallida populations from South, North America and Europe. A single sample formed a well-supported subclade along with G. rostochiensis and G. tabacum from Japan, USA and Argentina. To our knowledge this is the first comprehensive survey of Globodera populations from Colombia that includes morphological and genetic data. Our findings on species diversity and phylogenetic relationships of Globodera populations from Colombia may help elucidate the status and distribution of Globodera species, and lead to the development of accurate management strategies for the potato cyst nematodes.
Three populations of Xiphinema primum n. sp. and two populations of X. pachtaicum were recovered from natural forests and cultural regions of northern Iran. Both species belong to the X. americanum-group and were characterized by their morphological, morphometric and molecular data. The new species, which was recovered in three locations, belongs to the X. brevicolle-complex and is characterized by 2124-2981 μm long females with a widely rounded lip region separated from the rest of the body by a depression, 103-125 μm long odontostyle, two equally developed genital branches with endosymbiont bacteria inside the ovary, which are visible under light microscope (LM), vulva located at 51.8-58.0%, the tail is 26-37 μm long with a bluntly rounded end and four juvenile developmental stages. It was morphologically compared with nine similar species viz. X. brevicolle, X. diffusum, X. incognitum, X. himalayense, X. luci, X. parabrevicolle, X. paramonovi, X. parataylori and X. taylori. The second species, X. pachtaicum, was recovered in two geographically distant points close to city of Amol. Molecular phylogenetic studies of the new species were performed using partial sequences of the D2-D3 expansion segments of the large subunit ribosomal RNA gene (LSU rDNA D2-D3), the internal-transcribed spacer rDNA (ITS = ITS1+5.8S+ITS2), and the mitochondrial cytochrome c oxidase I gene (COI mtDNA) regions. The Iranian population of X. pachtaicum was also phylogenetically studied based upon its LSU rDNA D2-D3 sequences. Both species were also inspected for their putative endosymbiont bacteria. Candidatus Xiphinematobacter sp. was detected from two examined populations of the new species, whereas the second endosymbiont bacterium, detected from three examined isolates of X. pachtaicum, was related to the plant and fungal endosymbionts of the family Burkholderiaceae. The phylogenetic analyses of the two endosymbiont bacteria were performed using partial sequences of 16S rDNA. In cophylogenetic analyses, significant levels of cophylogenetic signal were observed using both LSU rDNA D2-D3 and COI mtDNA markers of the host nematodes and 16S rDNA marker of the endosymbiont bacteria.
Twenty nine potato cyst nematode populations from Russia, five populations from England and one population each from the Ukraine, New Zealand, Germany and The Netherlands were compared using protein electrophoresis, restriction fragment length polymorphism of ribosomal DNA (rDNA-RFLP) and random amplified polymorphic DNA (RAPD) techniques. All populations from Russia were identified as Globodera rostochiensis and RAPD analysis revealed substantial genomic diversity.
De nombreuses techniques biochimiques et moléculaires ont été utilisées pour l'étude de la variabilité génétique des nématodes à kystes de la pomme de terre en vue de trouver des marqueurs liés aux pathotypes ou de définir des groupements à caractéristiques biologiques. Au cours de la présente étude ont été utilisées des amorces en séquence répétitive simple (SSR) appliquées à des populations de #Globodera rostochiensis$ et #G. pallida$. Huit de ces SSR ont été testées dont trois donnent des résultats reproductibles. Les résultats obtenus permettent une séparation nette des deux espèces de même qu'une différenciation entre populations d'une même espèce. Certains des groupements obtenus paraissent plus liés à l'origine géographique qu'aux caractéristiques de virulence. (Résumé d'auteur)
We report a new transformation, the LogDet, that is consistent for sequences with differing nucleotide composition and that have arisen under simple but asymmetric stochastic models of evolution. This transformation is required because existing methods tend to group sequences on the basis of their nucleotide composition, irrespective of their evolutionary history. This effect of differing nucleotide frequencies is illustrated by using a tree-selection criterion on a simple distance measure defined solely on the basis of base composition, independent of the actual sequences. The new LogDet transformation uses determinants of the observed divergence matrices and works because multiplication of determinants (real numbers) is commutative, whereas multiplication of matrices is not,except in special symmetric cases. The use of determinants thus allows more general models of evolution with a symmetric rates of nucleotide change. The transformation is illustrated on a theoretical data set (where existing methods select the wrong tree) and with three biological data sets: chloroplasts, birds/mammals (nuclear), and honeybees ( mitochondrial ) . The LogDet transformation reinforces the logical distinction between transformations on the data and tree-selection criteria. The overall conclusions from this study are that irregular A,C,G,T compositions are an important and possible general cause of patterns that can mislead tree-reconstruction methods, even when high bootstrap values are obtained. Consequently, many published studies may need to be reexamined.
A morphometric evaluation of second-stage juveniles (J2), males, females, cysts, and eggs of several isolates of the tobacco cyst nematode (TCN) complex, Globodera tabacum tabacum (GTT), G. t. virginiae (GTV), and G. t. solanacearum (GTS) is presented. Morphometrics of eggs, J2, and males are considerably less variable than of females and cysts. No measurements of eggs and J2 are useful for identification of the three subspecies. Distance from the median bulb and excretory pore to the head end in J2 and males is quite stable. Stylet knob width of males is useful for identifying GTV isolates and tail length in separating males of GTT isolates from GTV and GTS. Body length/width (L/W) ratio of females and cysts discriminates GTT from GTV and GTS; stylet knob width is an auxiliary character for identifying GTV. This subspecies complex has a continuum of values for the other characters. Data suggest a close relationship between GTV and GTS, which also occur in close proximity in Virginia.
Morphological comparisons with light microscopy and scanning electron microscopy were made among second-stage juveniles (J2) and males of several isolates of the three subspecies of the tobacco cyst nematode complex, Globodera tabacum sspp. tabacum, virginiae, and solanacearum. Observations focused on the anterior region, (including head shape, lip pattern, and stylet morphology) and the tail region (including tail shape in J2 and spicules in males). The three subspecies could not be separated on the basis of any of these characters.
The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida.
The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).
To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses.
Belonolaimus isolates from six U.S. states were compared by restriction endonuclease digestion of amplified first internal transcribed spacer region (ITS1) of the nuclear ribosomal genes. Seven restriction enzymes were selected for evaluation based on restriction sites inferred from the nucleotide sequence of a South Carolina Belonolaimus isolate. Amplified product size from individuals of each isolate was approximately 700 bp. All Midwestern isolates gave distinct restriction digestion patterns. Isolates identified morphologically as Belonolaimus longicaudatus from Florida, South Carolina, and Palm Springs, California, were identical for ITS1 restriction patterns. The correlation between ITS1 restriction patterns and the distribution of B. longicaudatus isolates suggest that the California isolate is a relatively recent introduction into the state.
DNA sequences and other molecular data compared among organisms may contain phylogenetic signal, or they may be randomized with respect to phylogenetic history. Some method is needed to distinguish phylogenetic signal from random noise to avoid analysis of data that have been randomized with respect to the historical relationships of the taxa being compared. We analyzed 8,000 random data matrices consisting of 10-500 binary or four-state characters and 5-25 taxa to study several options for detecting signal in systematic data bases. Analysis of random data often yields a single most-parsimonious tree, especially if the number of characters examined is large and the number of taxa examined is small (both often true in molecular studies). The most-parsimonious tree inferred from random data may also be considerably shorter than the second-best alternative. The distribution of tree lengths of all tree topologies (or a random sample thereof) provides a sensitive measure of phylogenetic signal: data matrices with phylogenetic signal produce tree-length distributions that are strongly skewed to the left, whereas those composed of random noise are closer to symmetrical. In simulations of phylogeny with varying rates of mutation (up to levels that produce random variation among taxa), the skewness of tree-length distributions is closely related to the success of parsimony in finding the true phylogeny. Tables of critical values of a skewness test statistic, g1, are provided for binary and four-state characters for 10-500 characters and 5-25 taxa. These tables can be used in a rapid and efficient test for significant structure in data matrices for phylogenetic analysis.
Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
Morphological and genome variation between 32 nematode isolates identified originally asPratylenchus coffeae, P. gutierrezi, P. loosi, and P. pseudocoffeae were characterized to estimate phylogenetic relationships among them. All isolates have numerous males, and two lip annuli. Viewed en face with scanning electron microscopy, the first lip annulus is divided into lateral and median sectors in several isolates from coffee (Central America and Indonesia), and in one From aster (Florida). The first lip annulus is smooth in all other isolates, including six from coffee (Brazil and Indonesia). Principal component analysis (PCA) of one morphological (smooth face vs divided face) and three weakly-allometric morphometric variables (V, a, length of stylet) revealed seven assemblages of isolates. The PCA-derived assemblages conform closely to phylogenetic relationships inferred from analysis of 28S rDNA sequences. Nevertheless, identity within the D2/D3 expansion segment was not absolute for all isolates within the morphological assemblages, indicating the possibility that several assemblages are species complexes. Based on face morphology, five isolates (smooth faces) from coffee near the type locality for P. coffeaein eastern Java, Indonesia are different species than preserved museum specimens (divided faces) collected in the same area. Moreover, the D2/D3 sequence of a Java isolate suggests that it may be conspecific with isolates of P. coffeae sensu lato (all with identical sequences and smooth faces) from citrus, banana, yam, aglaonema and cocoyam, but not with isolates from citrus in Oman, nor banana in Ghana. Morphology and D2/D3 sequence of a P. gutierrezi topotype isolate revealed the likelihood that it is not conspecific with two other isolates with divided faces from coffee in Central America, Morphology and genetic sequence data for isolates from coffee and citrus in Sao Paulo State in Brazil, indicate that they are one or more undescribed species. When compared to the morphology and D2/D3 sequence of P. loosi from tea in Sri Lanka, isolates recently described as P. loosi from Paspalum notatum and Panicum hemitomon in Florida are apparently two undescribed species.
Molecular examination of the ribosomal internal transcribed spacer (ITS) region in potato cyst nematodes (PCN) is described. The ITS was amplified and sequenced from a number of PCN collections. A low level of sequence variation was found between Globodera rostochiensis, G. pallida, and a Peruvian PCN collection, but no variation within Australasian collections of species was noted. Polymerase chain reaction (PCR) primers based upon the G. rostochiensis–G. pallida sequence differences were designed and successfully used to identify mixed PCN species in a single PCR reaction.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Forty populations of Globodera pallida originating from either South America or field sites in Europe were tested for their reproductive ability on a susceptible potato cultivar and five genotypes with quantitative resistance from either Solanum vernei or S. tuberosum ssp. andigena CPC 2802 in a glasshouse pot experiment. The results showed that there was a wide and continuous range of virulence (85% to 4% reproduction) to all the resistant genotypes. This range was as broad within the European populations as within those from S. America. There was a significant host clone x nematode population interaction, largely accounted for by grouping the clones according to the source of resistance and the populations by their continent of origin. Populations from Europe were relatively more virulent on genotypes derived from S. vernei. In contrast, populations from S. America were relatively more virulent on genotypes derived from S. tuberosum ssp. andigena CPC 2802. Examination of the data from the European populations also showed a significant host x population interaction tending to separate British from mainland European populations.
Bursaphelenchus species can cause serious economic damage to pine forestry, and are widely distributed across the globe. The genetic structure of the B. mucronatus-B. xylophilus species group was investigated using sequences of the internal transcribed spacers of the ribosomal RNA gene repeat as a marker. Analysis of the ITS from 11 Bursaphelenchus isolates, using Aphelenchoides rhytium as an outgroup, supports the perception that the two species are distinct genetic entities. Significant divergence was however found within each species. ITS sequence analysis does not support a separation of European and Japanese B. mucronatus into distinct species-level taxa. Phylogenie des especes de Bursaphelenchus issue d'une analyse des sequences du DNA ribosomique de l'espaceur interne transcrit - Les especes de Bursaphelenchus peuvent provoquer de serieux degats aux cultures de pins et sont largement reparties dans le monde. La structure genetique des especes du groupe B.mucronatus-B. xylophilus a ete etudiee en utilisant comme marqueur des sequences repetees de l'espaceur interne transcrit du gene de l'ARN ribosomique. L'analyse des ITS de 11 isolats de Bursaphelenchus, en utilisant Aphelenchoides rhytium comme un extra-groupe, conforte l'idee que les deux especes sont des entites genetiques distinctes. Une divergence significative a cependant ete trouvee a l'interieur de chaque especes. L'analyse des sequences des ITS n'accredite pas la separation des B. mucronatus europeen et japonais en deux especes distinctes.
Restriction enzyme analysis of ribosomal DNA (rDNA) sequences was used to distinguish species and isolates of root-knot nematodes. DNA fragments containing the internal transcribed spacer (ITS) rDNA were amplified from total DNA of 27 geographic isolates each of Meloidogyne hapla and M. chitwoodi and from one isolate each of M. incognita and M. javanica by polymerase chain reaction (PCR). The amount of DNA present in a single juvenile was sufficient to amplify these PCR products. The amplified ITS fragments were relatively short compared to those that have been found for the genera Aphelenchoides, Caenorhabditis, Ditylenchus, Heterodera, and Xiphinema. Digestion of the ITS regions with AluI, DraI, and HinfI distinguished M. hapla and M. chitwoodi from each other as well as from M. incognita and M. javanica. Results indicated that different ITS sequences are present within a single individual of M. hapla. Three isolates of M. chitwoodi that produce isozyme patterns distinct from other M. chitwoodi isolates also could be distinguished by ITS restriction fragment length polymorphisms. The possibility that they do not belong to M. chitwoodi proper is discussed.
La variabilité inter et intraspécifique de #Globodera$ parasites des Solanacées est évaluée par leurs gammes d'hôtes et leurs profils RAPD. Divers espèces et cultivars de tabac sont testés vis-à-vis de #Globodera tabacum$ sensu lato et de #G. "mexicana"$. les profils RAPD sont étudiés sur 26 populations appartenant aux six espèces (publiées ou non) de #Globodera$. De fortes différences dans la gamme d'hôtes permettent de classer les populations en groupes de virulence. Huit amorces Operon de dix bases (OPG-1, OPG-2, OPG-3, OPG-4, OPG-5, OPL-3, OPL-4, OPL-6) permettent d'identifier 222 marqueurs RAPD. Divers programmes d'analyses en grappes et en composante principale sont réalisés en utilisant ces marqueurs, avec de très faibles différences de classification entre programmes. Les dendrogrammes obtenus montrent l'existence de quatres unités taxinomiques, à savoir #G. rostochiensis$, #G. pallida$, #G. tabacum$ et #G. "mexicana"$. Ils sont semblables à ceux obtenus précédemmenet avec l'analyse de restriction des ITS. Ils révèlent des apparentements congruents avec ceux déterminés par la gamme d'hôtes et non contradictoires avec les barrières génétiques précédemment mises en évidence par les hybridations in vitro. (Résumé d'auteur)
This study examined the ribosomal cistron of Ditylenchus destructor, D. myceliophagus and seven host races of D. dipsaci from different geographic locations. The three species showed restriction fragment length polymorphisms (RFLPs) in the ribosomal cistron, the 18S rDNA gene, and the ribosomal internal transcribed spacer (ITS). Southern blot analysis with a 7.5-kb ribosomal cistron probe differentiated the five host races of D. dipsaci examined. Polymerase chain reaction (PCR) amplification of the ITS, followed by digestion with some restriction endonucleases (but not others), produced restriction fragments diagnostic of the giant race. Because the PCR product from D. myceliophagus and the host races of D. dipsaci was about 900 base pairs and the ITS size in D. destructor populations was 1,200 base pairs, mixtures of populations could be detected by PCR amplification. ITS fragments differentiated between D. dipsaci and Aphelenchoides rhyntium in mixed populations. This study establishes the feasibility of differentiation of the host races of D. dipsaci by probing Southern blots with the whole ribosomal cistron.
Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3' end of the 18S gene and the 5' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.
We have sequenced one complete rDNA tandem repeat from the nematode C. elegans. By comparative analysis we derive secondary
structures for the 18s, 5.8s, and 26s rRNA molecules, and comment on other important features of the sequence. We also present
the sequence of a Junction between the rDNA and non-ribosomal DNA. Finally, we use our data to quantify the evolutionary relationships
among several organisms currently studied in developmental biology.
We used nucleotide sequences of the large subunit ribosomal genes (26S rDNA) to examine evolutionary relationships among species of the genus Pratylenchus (Order: Tylenchida, Family: Pratylenchidae), commonly known as root-lesion nematodes. Ten species of Pratylenchus were studied including, P. penetrans, P. crenatus, P. minyus, P. vulnus, P. thornei, P. musicola, P. coffeae, P. hexincisus, P. scribneri, and P. brachyurus. The species Hirschmanniella belli, Meloidogyne javanica, Heterorhabditis bacteriophora, Nacobbus aberrans, Radopholus similis, and Xiphinema index were used as outgroups. Based on parsimony analyses of approximately 307 aligned nucleotides of the D3 expansion region of the 26S rDNA, it is clear that species of Pratylenchus are a paraphyletic assemblage. The outgroup taxon H. belli shares a common ancestor with the clade that includes P. vulnus and P. crenatus while N. aberrans and R. similis share a common ancestor with 5 other species included in this study.
We report variation in the rDNA internal transcribed spacers (ITSs) of aphid species, the first for these insects. Variation at 6 sites within ITS1 sequences of the green peach aphid, Myzus persicae, identified two haplotypes coexisting within the same individuals, indicating that molecular drive has not homogenised different copies of rDNA. During this study, we found that PCR can cause a precise 58-bp loss in the amplified copies of an ITS haplotype (type 1). This occurs in all detectable copies under routine PCR conditions, at different annealing temperatures and with Pfu and Taq polymerases. In addition, "hot-start" PCR exclusively copied a different, rare haplotype (type 2). These observations have important considerations for using PCR, as large deletions in PCR products may not reflect real deletions in the genome, and changes in PCR conditions may be needed to copy cryptic haplotypes.
Among root knot nematodes of the genus Meloidogyne, the polyploid obligate mitotic parthenogens M. arenaria, M. javanica, and M. incognita are widespread and common agricultural pests. Although these named forms are distinguishable by closely related mitochondrial DNA (mtDNA) haplotypes, detailed sequence analyses of internal transcribed spacers (ITSs) of nuclear ribosomal genes reveal extremely high diversity, even within individual nematodes. This ITS diversity is broadly structured into two very different groups that are 12%-18% divergent: one with low diversity (< 1.0%) and one with high diversity (6%-7%). In both of these groups, identical sequences can be found within individual nematodes of different mtDNA haplotypes (i.e., among species). Analysis of genetic variance indicates that more than 90% of ITS diversity can be found within an individual nematode, with small but statistically significant (5%-10%; P < 0.05) variance distributed among mtDNA lineages. The evolutionarily distinct parthenogen M. hapla shows a similar pattern of ITS diversity, with two divergent groups of ITSs within each individual. In contrast, two diploid amphimictic species have only one lineage of ITSs with low diversity (< 0.2%). The presence of divergent lineages of rDNA in the apomictic taxa is unlikely to be due to differences among pseudogenes. Instead, we suggest that the diversity of ITSs in M. arenaria, M. javanica, and M. incognita is due to hybrid origins from closely related females (as inferred from mtDNA) and combinations of more diverse paternal lineages.
Genetic variation between populations of Globodera pallida, primarily from Britain but including populations from continental Europe and South America and two Globodera rostochiensis populations, was examined using random amplified polymorphic DNA (RAPD). Fourteen primers were used and 250 amplification products observed. A comparison was made of the similarities between the species and, within G. pallida, between populations from Britain, The Netherlands, Germany, and Switzerland, of the pathotypes Pa2 and Pa3. In addition, one Pa1 population and two others from South America were included. On the basis of the RAPD analysis, all the Pa2-Pa3 populations, except one from Scotland (Luffness), constituted a single group with no clear distinction based on pathotype designation. The Luffness population is known to be distinct in its virulence. The data indicated that the main Pa2-Pa3 group could be subdivided based on geographic origin, but this is not well supported by bootstrap analysis. The Pa1 population and the two populations from South America all formed distinct groups.
Potato cyst nema-tode diagnostics; morphology, different hosts and biochem-ical technique Potato cystnematode
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DifferencesbetweenITSregionsofiso-latesof root-knotnematodes Meloidogynehapla and M. chit-woodi
- C Zijlstra Lever
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Eff ciency of ribosomal DNA sequence comparison among relative Globodera rostochiensis populations
- A Uehara
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KUSHIDA, A., UEHARA, T. & MOMOTA, Y. (1998). Eff ciency
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Potato cyst nematode diagnostics using the polymerase chain reaction Proceedings of plant health and the European single market symposium
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FLEMING, C.C. & MOWAT, D.J. (1993). Potato cyst nematode
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D. (Ed.) Proceedings of plant health and the European single
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GeneDoc: analysis and visualization of genetic variation TREEVIEW: An application to view phylogenetic trees on personal computer
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The retention index and the rescaled consistency index
FARRIS, J.S. (1989). The retention index and the rescaled
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Heteroderidae , cyst and non cyst-forming nematodes Manual of agricultural nematology
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(Ed.). Manual of agricultural nematology. New York, NY,
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Nematode parasites of potatoes Plant parasitic nematodes in temperate agriculture
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parasites of Solanaceous plants, revealed by Random Ampli ed Polymorphic DNA (RAPD) and correlation with biological features
- Intra-And Interspeci C Variability In Globodera
Intra-and interspeci c variability in Globodera, parasites of
Solanaceous plants, revealed by Random Ampli ed Polymorphic DNA (RAPD) and correlation with biological features.
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