Multigene phylogeny of root-knot nematodes and molecular characterization of Meloidogyne nataliei Golden, Rose & Bird, 1981 (Nematoda: Tylenchida)

Abstract

The root-knot nematodes of the genus Meloidogyne are highly adapted, obligate plant parasites, consisting of nearly one hundred valid species, and are considered the most economically important group of plant-parasitic nematodes. Six Meloidogyne species: M. arenaria, M. hapla, M. incognita, M. microtyla, M. naasi and M. nataliei were previously reported in Michigan, USA. For this study, Meloidogyne nataliei was isolated from the grapevine Vitis labrusca from the type locality in Michigan, USA, and was characterized using isozyme analysis and ribosomal and mitochondrial gene sequences. No malate dehydrogenase activity was detected using macerate of one, five, six, seven or ten females of M. nataliei per well. However, one strong band (EST = S1; Rm: 27.4) of esterase activity was detected when using homogenates of ten egg-laying females per well. Phylogenetic analyses of sequences of the partial 18S ribosomal RNA, D2-D3 of 28S rRNA, internal transcribed spacer of rRNA, mitochondrial cytochrome oxidase subunit I genes and the cytochrome oxidase subunit II-16S rRNA intergeneric fragment from fifty-five valid Meloidogyne species and M. nataliei were conducted using Bayesian inference and maximum likelihood methods. From these results, we infer 11 distinct clades among studied species, with M. nataliei and M. indica composing a basal lineage. Seventy five percent of these species belong to seven clades within the Meloidogyne superclade. Characterization of these clades is provided and evolutionary trends within the root-knot nematodes are discussed.

Figure 1

(A,B) Infected roots of grape with females protruding (arrows), some cover by a massive eggs mass. (Scale bars: 0.5 mm).

Figure 2

General morphology of Meloidogyne nataliei (LM). (A–G) Second-stage juveniles; (H–J,L–N,P): Male; (K,O): Female. (A,O) Entire body. (B,C,H) Anterior region in median lateral view. (D,I) Anterior region in surface lateral view. (E,J) Pharyngeal region. (K) Perineal pattern. (L) Mid-body lateral field. (F,G,M,N,P) Caudal region. (Scale bars: A,O: 100 µm; B–J, K–N, P = 10 µm).

Figure 3

Esterase (EST) and malate dehydrogenase (MDH) phenotypes from egg-laying females of Meloidogyne nataliei, and M. javanica and M. hapla used as controls. C1- extract from one female of M. javanica (J3; N1) per well; C2 - extract of five females of M. hapla (H1; H1) per well; Lanes 1, 2, and 3 - extract from five females of M. nataliei (S1) per well.

Figure 4

Bayesian 50% majority rule consensus tree as inferred from 18S rRNA, ITS1 rRNA, D2-D3 expansion segments of 28S rRNA, COI gene and COII-16S rRNA sequence alignment under the GTR + I + G model. Branch support of over 70% is given for appropriate clades and it is indicated as: posterior probabilities value in Bayesian inference analysis/bootstrap value from maximum-likelihood analysis. (n = ? – chromosome number information unknown).