Rosane H. C. Curtis

Cuticle Surface Coat of Plant-Parasitic Nematodes

The surface coat (SC) of the plant-parasitic nematode cuticle is an understudied area of current research, even though it likely plays key roles in both nematode-plant and nematode-microbe interactions. Although in several ways Caenorhabditis elegans is a poor model for plant-parasitic nematodes, it is a useful starting point for investigations of the cuticle and its SC, especially in the light of recent work using this species as a model for innate immunity and the generic biology underpinning much host-parasite biology. We review the research focused on the involvement of the SC of plant-parasitic nematodes. Using the insights gained from animal-parasitic nematodes and other sequenced nematodes, we discuss the key roles that the SC may play.

Chemotaxis can take plant-parasitic nematodes to the source of a chemo-attractant via the shortest possible routes

It has long been recognized that chemotaxis is the primary means by which nematodes locate host plants. Nonetheless, chemotaxis has received scant attention. We show that chemotaxis is predicted to take nematodes to a source of a chemo-attractant via the shortest possible routes through the labyrinth of air-filled or water-filled channels within a soil through which the attractant diffuses. There are just two provisos: (i) all of the channels through which the attractant diffuses are accessible to the nematodes and (ii) nematodes can resolve all chemical gradients no matter how small. Previously, this remarkable consequence of chemotaxis had gone unnoticed. The predictions are supported by experimental studies of the movement patterns of the root-knot nematodes Meloidogyne incognita and Meloidogyne graminicola in modified Y-chamber olfactometers filled with Pluronic gel. By providing two routes to a source of the attractant, one long and one short, our experiments, the first to demonstrate the routes taken by nematodes to plant roots, serve to test our predictions. Our data show that nematodes take the most direct route to their preferred hosts (as predicted) but often take the longest route towards poor hosts. We hypothesize that a complex of repellent and attractant chemicals influences the interaction between nematodes and their hosts.

Comparison of host recognition, invasion, development and reproduction of Meloidogyne graminicola and M. incognita on rice and tomato

The rice root-knot nematode Meloidogyne graminicola normally infects rice, wheat and several other graminaceous plants. Meloidogyne incognita is a serious pest of dicotyledonous crops, although it can infect and reproduce on some cereals. This paper demonstrates and compares host recognition, development and reproduction of these two species of root-knot nematodes on rice and tomato plants. Attraction bioassays in pluronic gel clearly showed that M. incognita preferred tomato roots to rice or mustard roots, whilst M. graminicola was more attracted towards rice compared with tomato or mustard roots. Based on the attraction data from this study, it can be hypothesised that either: i ) the blend of attractants and repellents are different in good and poor hosts; or ii ) relatively long-range attractants, together with shorter-range repellents, might affect nematode movement patterns. Some host specific attractants might also be involved. Meloidogyne incognita was able to invade and develop to adult female but did not produce eggs in rice roots. By contrast, M. graminicola developed and reproduced faster on both rice and tomato plants compared with M. incognita. Nevertheless, second-stage juveniles of both these root-knot nematodes showed a similar pattern of distribution inside the roots, preferring to accumulate at the root tips of rice or in the vascular cylinder and cortical region of tomato.

Host status of cultivated plants to Meloidogyne hispanica

The reproduction of a Meloidogyne hispanica isolate from Portugal was evaluated in 63 plant species/cultivars, in pot assays at 25 ± 2.0°C, on the basis of root gall index (GI) and reproduction factor (Rf = final/initial egg density) at 60 days after inoculation. Cultivars of aubergine, bean, beetroot, broccoli, carnation, corn, cucumber, French garlic, lettuce, melon, onion, parsley, pea, potato, spinach, and tobacco and two of cabbage were susceptible (3 ≤ GI ≤ 5; 1.15 ≤ Rf ≤ 262.86). Cabbage cv. Bacalan, cauliflower cv. Temporão and pepper cv. Zafiro R2 were hypersusceptible or poor hosts (Rf < 1; GI > 2) and pepper cvs. Aurelio and Solero were resistant (0.0 ≤ GI ≤ 0.4; 0.00 ≤ Rf ≤ 0.03). The response of the pepper cultivars and the Mi-1 resistant tomato cv. Rossol was also conducted in pots using two inoculum levels and four temperatures, three growth chamber (25 ± 2.7°C, 29.3 ± 1.8°C and 33.6 ± 1.2°C) and one outdoors (24.4 ± 8.2°C). At 24.4 ± 8.2°C and 25 ± 2.7°C, the reproduction on the resistant tomato was significantly lower compared to the susceptible cv. Easypeel. At all temperatures, resistance was evident for the pepper cultivars, despite the fact they were not found to contain any of the Me1, Me3, Me7 and N genes. The eggs obtained on cv. Aurelio at 33.6 ± 1.2°C were used to get a selected resistance breaking isolate of M. hispanica that was able to reproduce on the three pepper cultivars. Our results suggest that the initial M. hispanica isolate is a mixture of virulent and avirulent individuals. The pepper cultivars tested, have potential to reduce M. hispanica populations in agro-ecosystems under certain conditions, but they should be used as a part of an integrated management strategy in order to prevent the development of virulent populations.

Transcriptional changes of the root-knot nematode Meloidogyne incognita in response to Arabidopsis thaliana root signals.

Root-knot nematodes are obligate parasites that invade roots and induce the formation of specialized feeding structures. Although physiological and molecular changes inside the root leading to feeding site formation have been studied, very little is known about the molecular events preceding root penetration by nematodes. In order to investigate the influence of root exudates on nematode gene expression before plant invasion and to identify new genes potentially involved in parasitism, sterile root exudates from the model plant Arabidopsis thaliana were produced and used to treat Meloidogyne incognita pre-parasitic second-stage juveniles. After confirming the activity of A. thaliana root exudates (ARE) on M. incognita stylet thrusting, six new candidate genes identified by cDNA-AFLP were confirmed by qRT-PCR as being differentially expressed after incubation for one hour with ARE. Using an in vitro inoculation method that focuses on the events preceding the root penetration, we show that five of these genes are differentially expressed within hours of nematode exposure to A. thaliana roots. We also show that these genes are up-regulated post nematode penetration during migration and feeding site initiation. This study demonstrates that preceding root invasion plant-parasitic nematodes are able to perceive root signals and to respond by changing their behaviour and gene expression.

Do phytohormones influence nematode invasion and feeding site establishment

Phytohormones mediate important aspects of developmental processes in plants and may participate in various aspects of plant nematode interactions. Several reports demonstrate that nematode infection interferes with plant auxin flow and accumulation, and that auxin is necessary for feeding site formation. Other plant hormones, such as ethylene, might be modulating several cellular processes during feeding site formation. So far, the nematode molecules involved in activating these processes in planta have not been identified. Auxin can act as a signalling molecule by inducing changes in the surface cuticle and behaviour of Meloidogyne spp. and these changes might be essential for infection. Auxin binds to the chemosensory organs, amphids and phasmids and also to several tail neurons of Meloidogyne spp. This interaction might allow these nematodes to sense and follow an auxin gradient through amphidial or phasmidial receptors during penetration and migration inside roots.

Biometrical, Biochemical, and Molecular Diagnosis of Portuguese Meloidogyne hispanica Isolates

Meloidogyne hispanica infects many economically important crops worldwide. The accurate identification of this pathogen is essential for the establishment of efficient and sustainable integrated pest management programs. Portuguese M. hispanica isolates were studied by biometrical, biochemical, and molecular characteristics. Biometrical characteristics of M. hispanica females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern (Hi4) for M. hispanica esterases that allowed for species differentiation. Molecular analysis of the mtDNA region from COII and 16S rRNA genes resulted in amplification products (1,800 bp) similar to M. hispanica, M. ethiopica, and M. javanica, and the described HinfI was unable to discriminate M. hispanica from the other two species. Analysis of the mtDNA sequences revealed altered nucleotides among the isolates that created new restriction sites for AluI and DraIII. The resulting restriction patterns successfully discriminated between the three species, providing a new tool for Meloidogyne identification. Finally, the phylogenetic relationship between M. hispanica and several Meloidogyne spp. sequences was analyzed using mtDNA, confirming the divergence between meiotic and mitotic species and revealing the proximity of M. hispanica to closely related species. Based on the studies conducted, the application of isozyme or polymerase chain reaction restriction fragment length polymorphism analysis would be a useful and efficient methodology for M. hispanica identification.

Parallels between plant and animal parasitic nematodes

The simplicity of the nematode body plan, associated morphology and low cell number belies their molecular complexity which, in combination, have provided for unrivalled success amongst the metazoa, with nematodes dominating biomass statistics. Their structural simplicity means that most nematodes, free-living or parasitic, have much in common with parallels in almost every facet of their biology. One major difference between the parasitic nematodes and their free-living cousins is their propensity for host interaction, a fact that is believed to have driven more rapid molecular evolutionary change as they strive to compete with their host for resources and survival in the face of host defence strategies. The fact that both animal and plant parasitism by nematodes arose independently on multiple occasions points to a diversity of starting points for their parasitic way of life. Despite this, animal and plant parasitic nematodes show many of the same traits that are believed to contribute to their success, including: their ability to arrest development at key stages in their life cycle; their ability to locate and infect their host; their ability to manipulate their interface with the host so that they can survive for extended periods and derive appropriate nutrients. Commonalities in the adoption of these strategies in both animal and plant parasites mean that there are many parallels between them, not just in their basic biology which is common across nematode life strategies, but in their parasite-specific adaptations and behaviours that enable them to succeed as infectious organisms living within and upon host species.

Characterization of the venom allergen—like protein (vap-1) and the fatty acid and retinol binding protein (far-1) genes in Meloidogyne hispanica

The root-knot nematode (RKN) Meloidogyne hispanica has been found in all continents associated with a wide host range, including economically, important plants and can be considered a species of emerging importance. Considerable progress has been made to identify nematode effector genes as they are important targets for the development of novel control strategies. The effector genes, venom allergen-like protein (vap-1) and fatty acid and retinol binding protein (far-1), were identified, isolated and sequenced in M. hispanica (Mhi-vap-1 and Mhi-far-1) using the genome information available for the RKNs M. incognita and M. hapla. These genes are differentially expressed during M. hispanica development and their amplification products were observed from cDNA of the eggs, second-stage juveniles (J2) and adult females. However, Mhi-vap-1 showed the highest level of expression in J2. In situ hybridization analysis revealed that the Mhi-vap-1 and Mhi-far-1 transcripts are accumulated within the J2 subventral oesophageal glands. The specific expression in the subventral oesophagel glands and presence of the secretion signal peptide for both genes suggests that these proteins are secreted by the J2 and may play a role in the early parasitic stage of the infection process. These genes were also isolated and sequenced in M. arenaria, M. incognita and M. javanica; and phylogenetic analysis revealed that the predicted protein sequences belonging to M. hispanica and several other species of plant-parasitic nematodes have a high degree of conservation.

Immunolocalization of a putative cuticular collagen protein in several developmental stages of Meloidogyne arenaria, Globodera pallida and G. rostochiensis.

The monoclonal antibody IACR-CCNj.3d has previously been used to isolate a gene (gp-col-8) with strong similarity to cuticular collagen from a mixed stage Globodera pallida cDNA expression library. The antibody has also been shown to label specifically the amphidial canal of pre-parasitic second stage juveniles (J2) of several plant nematode species without any reactivity on the cuticular surface, indicating that this protein is either not present or is inaccessible on the cuticular surface. This paper investigates the cross-reactivity of Mab IACR-CCNj.3d with Meloidogyne arenaria and the localization of the putative collagen protein on the cuticular surface of parasitic stages in planta and on the cuticular surface of juveniles inside eggs. The antigen was shown to be present in all developmental stages of the two species of potato cyst nematodes and M. arenaria. The antibody bound strongly to the amphidial canal and hypodermis of pre-parasitic J2 and adult females. The antigen was present on the cuticular surface of the sausage-shaped J2 in planta and of first stage juveniles (J1) inside the eggs. The presence of collagen on the surface of the cuticle of moulting stages of plant parasitic nematodes has been observed for the first time. It is clear that this protein has a role in the construction of the cuticle of the first stage juveniles and parasitic second stage juveniles, during moulting inside the eggs and in the root tissue, respectively.