Morphological and molecular characterisation of one new and several known species of the reniform nematode, Rotylenchulus Linford & Oliveira, 1940 (Hoplolaimidae: Rotylenchulinae), and a phylogeny of the genus

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The reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant roots and are mainly distributed in tropical and subtropical regions. In this study, we provide morphological and molecular characterisation of six out of ten presently known valid species of Rotylenchulus: R. clavicaudatus, R. leptus, R. macrodoratus, R. macrosoma, R. reniformis and R. sacchari from South Africa, USA, Italy and Spain. Rotylenchulus parvus was only studied morphologically. A new species, R. macrosomoides sp. n., isolated from soil and roots of sugarcane in South Africa, is described. The phylogeny of Rotylenchulus, as inferred from the analyses of D2-D3 of 28S rRNA, ITS rRNA, coxI mtDNA and hsp90 gene sequences, is presented. The study revealed that R. reniformis and R. macrosoma have a sister relationship, but that relationships between other Rotylenchulus species remain unresolved. The phylogenetic analysis also confirmed the hypothesis that this genus originated from the Afrotropical zoogeographical region. Our study revealed that R. reniformis and R. macrosomoides sp. n. have two distinct rRNA gene types and R. macrosoma have three rRNA gene types in their genomes. PCR with species-specific primers was developed for rapid diagnostics of R. reniformis.

Nematology

International Journal of Fundamental and Applied Nematological Research

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Figures

  • Rotylenchulus macrosomoides sp. n. A-E, immature female. A: Anterior part of body; B: Vulval region with reflexed ovaries; C: Lateral field at mid-body; D, E: Tails. F-H, male. F: Anterior region; G: Lateral field at mid-body; H: Tail. I, J, juvenile. I: Anterior part of body; J: Tail. K, L, mature female. K, L: Habitus. (Scale bar: A-J = 30 μm; K, L = 100 μm.)

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  • Rotylenchulus macrosomoides sp. n. A-D, immature female. A, B: Lateral view of lip region; C: En face view; D: Lateral field at mid-body. E-G, female. E: Vulval area; F: Entire; G: Tail region.

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  • Rotylenchulus macrosomoides sp. n. A-E, male. A: Lateral view of lip region; B: En face view of lip region; C: Lateral field at mid-body; D: Entire body; E: Tail region. F, G, immature female. F: Lateral view of lip region; G: En face view.

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  • Rotylenchulus clavicaudatus. A-C, F, immature female. A: Anterior part of body; B: Vulval area with reflexed ovaries; C: Lateral field at mid-body; F: Tail. D, H, J, male. D: Anterior part of body; H: Tail; J: Lateral field at mid-body. E, G, I, juvenile. E: Anterior part of body; G: Tail; I: Lateral field at mid-body. (Scale bar = 30 μm.)

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  • Rotylenchulus clavicaudatus. A-G, immature female. A: En face view; B, C: Lip region, lateral view; D: Lateral field at mid-body; E: Vulval region; F, G: Tails. H, I, male. H: Tail region; I: Lateral field at mid-body.

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  • Rotylenchulus leptus. A-D, mature Female. A: En face view; B: Lateral field near tail; C: Tail; D: Entire. E-J, immature female. E, F: En face view; G: Lateral view of lip region; H: Vulval area; I, J: Tail. (Scale bar: A-C = 10 μm; D = 100 μm; E-J = 5 μm.)

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  • Olive root parasitised by a mature female of Rotylenchulus macrodoratus. Note the gelatinous matrix covering the female body and a male (marked by an arrow). (Scale bar = 100 μm.)

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  • Rotylenchulus macrosoma. A: Olive root parasitised by a swollen female; B, C: Entire immature female and male, respectively; D, E: Immature female anterior region; F: Immature female tail region; G: Male tail region; H: Mature female tail region, inset = tail tip. Abbreviations: a = anus; dgo = dorsal gland orifice; V = vulva. (Scale bars: A-D = 50 μm; E, F = 20 μm; G = 10 μm; H = 100 μm.)

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  • Rotylenchulus parvus. Mature female. A-E: Various body postures. (Scale bar: A = 5 μm; B-E = 30 μm.)

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  • Rotylenchulus parvus. Mature female. A, B: Lip region lateral and en face; C: Vulval bulge; D: Tail region; E, F: Different body postures. (Scale bar: A, B = 5 μm; C, D = 10 μm; E, F = 50 μm.)

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  • Rotylenchulus sacchari. A, B, H-K, immature female. A: Anterior part of body; B: Vulval area with reflexed ovaries; H-K: Tail regions. C-F, male. C: Anterior part of body; D: Lateral field at mid-body; E, F: Tail region. G, L, M, juvenile J4. G: Anterior region; L, M: Tail region. (Scale bar = 30 μm.)

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  • Rotylenchulus sacchari. A-D, juvenile (?J2). A, C: Ovarial primordial area; B: Anterior part of body; D: Tail. E-G, juvenile J2. E: Ovarial primordial area; F: Anterior part of body; G: Tail. Rotylenchulus leptus. H-L, immature female. H: Anterior part of body; I: Vulval region; J: Lateral field at mid-body; K, L: Tails. M-O, juvenile. M: Lateral field at mid-body; N: Tail; O: Anterior region. (Scale bar = 30 μm.)

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  • Rotylenchulus sacchari. Immature female. A: lateral view of lip region; B, C: Two en face views; D: Vulval area; E: Lateral field at mid-body; F: Tail.

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  • Phylogenetic relationships between Rotylenchulus species. Bayesian 50% majority rule consensus tree as inferred from the analysis of the D2-D3 of 28S rRNA gene dataset under the GTR + I + G model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold font.

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  • Putative consensus secondary structures of the D2 expansion segment of 28S rRNA for R. reniformis. A: Type A; B: Type B.

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  • Phylogenetic relationships between Rotylenchulus species. Bayesian 50% majority rule consensus tree as inferred from the analysis of the ITS1 region dataset under the GTR + I + G model. The large clade of R. reniformis with unresolved relationships between sequences is indicated as a triangle. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold font.

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  • Phylogenetic relationships between Rotylenchulus species. Bayesian 50% majority rule consensus tree as inferred from the analysis of the 5.8S + ITS2 region dataset under the GTR + I + G model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold font.

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  • Phylogenetic relationships between Rotylenchulus species. Bayesian 50% majority rule consensus tree as inferred from the analysis of the partial hsp90 gene dataset under the GTR + G model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold font.

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  • Phylogenetic relationships between Rotylenchulus species. Bayesian 50% majority rule consensus tree as inferred from the analysis of the partial coxI gene dataset under the GTR + G model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences are in bold font.

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  • PCR with the Rotylenchulus reniformis specific primers. A: D2A and R_renif_R1A primer combination; B: D2A and R_renif_R2B primer combination. Lanes: M = 100 bp DNA marker (Promega); 1 = R. sacchari; 2 = R. macrosomoides sp. n.; 3 = R. reniformis (CD997); 4 = R. reniformis (CD1153); 5, 6 = R. reniformis (CD1395); 7 = R. reniformis (CD747); 8 = control without DNA.

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  • PCR with the Rotylenchulus reniformis specific primers. Multiplex PCR with D2A and R_renif_R1B and R_renif_R2A primer combination. Lanes: M = 100 bp DNA marker (Promega); 1 = R. reniformis (CD747); 2 = R. reniformis (CD997); 3 = R. reniformis (CD1153); 4 = control without DNA.

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