Diagnosis and Molecular Variability of an Argentinean Population of Nacobbus aberrans with Some Observations on Histopathology in Tomato

Abstract

Diagnosis of an Argentinean population of Nacobbus sp. infecting sweet pepper (lamuyo) was carried out including morphology, scanning electron microscopy, and molecular studies. In light of our morphometric, molecular and host-range results, we consider the studied population to belong to N. aberrans s. l., and by host range tests the population is assigned to the "sugar beet group." ITS-PCR analysis on individual male and immature female specimens of this population yielded amplification products of approximately 922 bp. RFLP profiles and sequencing of the ITS region revealed that, in addition to the host group, the present population can be assigned to the "Argentina 2" group. Disease development and histopathology were investigated with glasshouse observations using tomato, pepper, sugar beet and potato seedlings exposed to nematode infection for 45 days at 28 +/- 2 degrees C. Histopathology of tomato roots confirmed that all immature stages and young females and males are migratory, whereas mature females are obligate sedentary endoparasites. Rather than syncytia, large regions of cortical necrosis and cavities were detected in tomato swellings infected by juveniles. However, syncytia were associated only with adult females. Large root galls, hyperplasia, abnormal proliferation of lateral roots and asymmetry of root structure were common anatomical changes induced by the nematode feeding in tomato roots.

Fig. 1.

LM and SEM micrographs of different Nacobbus aberrans life stages. A–C) juvenile stage: entire body (A); anterior body portion and tail (B and C). D–G) immature female: entire body (D); vulva-tail region (E); lateral field (F); anterior and posterior body end (G,H). I,J) Male anterior and posterior body portion. K–N) SEM micrographs of adult females: entire body (K); anterior end in profile (L) and face view in (M); vulva terminus area (N) (Scale bars in K = 400 μm; all others = 25 μm).

Fig. 2

Tomato root systems (A–C of tomato cv. Platense), (D–E of lamuyo pepper cv. Zafiro), with several so-called "rosary-like beads" induced by Nacobbus aberrans after 45 d nematode infection. Note at the insert (C) the large egg-mass, on the surface of large gall.

Fig. 3

Anatomical changes induced by Nacobbus aberrans in tomato roots. Necrosis and cavities induced by juvenile stages in (A); large multicellular syncytia in B, C and D. Posterior female body and male into the galled root tissues in (E); Syncytial mononucleate cells showing hypertrophied nuclei and nucleoli. Abbreviations used: ne = necrosis; S = syncytium; N = nematode; f = Female; Hn = hypertrophied nucleus; m = male. (Scale bar = 50 μm)

Fig. 4

Restriction patterns of the amplified ITS region of individual male specimen of Nacobbus aberrans from Argentina. A: Alu I; D: Dde I; Hae: Hae III; H: Hinf I; Hp: Hpa II; R: Rsa I; and M: 100 bp DNA ladder.

Fig. 5

Maximum parsimony tree resulting from analysis of alignments of internal transcribed spacer ITS sequences of Nacobbus aberrans populations from different geographic areas and Meloidogyne arenaria as outgroup. * = the consensus sequence of A3 population was identified from several ITS sequences of A3 population. Bootstrap supports more than 50% are given for appropriate clade.

Fig. 6

Portion of the multi-alignment of different isolates of Nacobbus aberrans. The alignment corresponds to a region located at the 5’ end of the ITS1. The outlined horizontal lines mark the three hypothetical groups; the upper sequence group is the first one, the intermediate the second one and last sequence group the third one. The accession numbers of each isolate are: Al: AY254359; A2: AY254360; Bl: AY254361; B2: AY254362; B3: AY254363; B4: AY254364; E2: AY254365; Ml: AY254366; M2: AY254367; M3: AY254368; M4: AY254369; Nl: AY827831; N2: AY8278832; N3: AY827833; N4: AY827834; N5: AY827835; N6: AY827836; N13L: DQ017478; N19S: DQ017479; N211L: DQ017476; and N23S: DQ017473.