Unlike the free-living form in soil, plant-parasitic nematodes (PPNs) are scourge for agriculture worldwide. These small roundworms are able to infect thousands of plant species and cause a disastrous global crops yield losses (Blok et al., 2008). Although PPNs have different lifestyles and feeding strategies, all species use a hollow, protrusible syringe-like stylet to penetrate the wall of plant cells, produce and inject secretions to facilitate infection, and then withdraw nutrients from the plant.
The principal source of nematode secretions are three enlarged oesophageal or “salivary” glands – two subventral glands (SvG) and one dorsal gland (DG) (Figure 4), adapted to enhance secretory activity for plant infection. Besides the oesophageal glands, the cuticle and the amphids, the principal chemosensory organs of nematodes made up of 12 sensory neurons, have been also demonstrated to secrete proteins during plant infection (Perry, 1996; Semblat et al., 2001; Curtis, 2007).
Figure 4: Secretory organs of a typical plant-parasitic nematode (adapted from Haegeman et al. 2012).
(A)-(E) In situ hybridisation images of nematode genes specifically expressed in secretory organs. (A) SPRYSEC in G. pallida (Jones et al., 2009) (B) SXP-RAL2 protein in G. rostochiensis (Jones et al., 2000) (C) SXP-RAL2 protein in G. rostochiensis (Jones et al., 2000) (D) chorismate mutase in G. pallida (Jones et al., 2003) (E) CL1191Contig1_1 protein in M. incognita (Bellafiore et al., 2008) Plant parasitic nematodes are classified according to their lifestyle and feeding habits. Those that feed externally on the root are called ectoparasites, whereas the nematodes that feed internally from different inner cell types are classified as endoparasites. They are further sub-classified into sedentary, fixed at a feeding site, or migratory, moving and feeding inside the root or the shoot (Figure 5) (Decraemer & Hunt, 2013).
Ectoparasite nematodes tend to gather in the soil rhizosphere (soil on and around root) to browse along root and then to feed. They can have a long stylet that helps them to penetrate the plant root, goes deeply inside for rich nutrients in plant cells. This feeding strategy makes them easier to switch hosts but also be harmed by environment or predators. The sedentary ectoparasite Tylenchulus semipenetrans are responsible for losses in citrus, olive and grapevine trees, whereas the migratory ectoparasite Xiphinema spp. (Figure 6) can transmit important plant viruses to grapes.
Figure 5: Phylogeny and lifestyle of Tylenchida (adapted from Bert et al. 2011; Sijmons et al. 1994)
(A) Phylogeny of Tylenchida. (B) Schematic representation of feeding sites and feeding structure of some selected root parasitic nematodes. 1= Migratory ectoparasites: 1A, Tylenchorhynchus dubius; 1B, Trichodorus spp.; 1C, Xiphinema index; 1D, Longidorus elongates; 2= Sedentary ectoparasites: Criconemalla xenoplax; 3: Migratory ecto-endoparasites: Helicotylenchus spp.; 4: Migratory endoparasites: Pratylenchus spp.; 5: Sedentary endoparasites: 5A, Trophotylenchulus obscurus; 5B, Tylenchulus semipenetrans; 5C, Verutus volvingentis; 5D, Cryphodera utahensis; 5E, Rotylencholus reniformis; 5F, Heterodera spp.; 5G, Meloidogyne spp.
Migratory endoparasitic nematodes cause massive plant tissue necrosis because of their migration and feeding. As they have no permanent feeding site, they simply withdraw the nutrients using their stylet, killing the plant cell and moving ahead of the lesion. Some examples of migratory endoparasites are Pratylenchus (lesion nematode-Figure 6), Radopholus (burrowing nematode), Hirschmaniella (rice root nematode). Furthermore, these nematodes could cause extensive wounds in plant roots, that leads to a potential secondary infection by bacteria and fungi (Zunke, 1990).
(A) Migratory ectoparasite Xiphinema spp. (B) Symptom of viruses transmitted by Xiphinema spp. on grapevine leaf (C) Migratory endoparasite Pratylenchus sp. (D) Symptom of Pratylenchus sp. on wheat include lower leaf yellowing, decreased tillers and wilting (Photo credit by: (A) NC State University (B) http://plpnemweb.ucdavis.edu/ (C) Courtesy D. Wixted (D) Kirsty Owen, DAFF)
Sedentary endoparasites are, among PPNs, the most economical and dangerous ones. These pests enter host roots, establish a specialized feeding site within the root tissue and feed internally. They are represented by two major threats: root-knot nematodes (RKNs, Meloidogyne spp.) and cyst nematodes (CNs, Globodera spp. and Heterodera spp.). Both nematodes group preferentially infect plant root from the elongation zone and induced the formation of multinucleate and hypertrophied feeding cells (Figure 7). However, their ways to achieve the feeding site are different. The CN J2s migrate intracellular by cutting cortical cell walls and migrating through cells until they reach the differentiating vascular. By contrast, RKNs migrate intercellular. RKN J2s move towards the root tip until they reach the root apex, and then migrate back up until they reach a site near the vascular cylinder (Figure 7) (Perry & Moens, 2011). While CNs are mainly found in a few plant species, RKNs show a capacity to infect almost cultivated plants throughout the world. In the next part of the introduction, I will focus on the description of RKNs.
Figure 7: Parasitic strategies of cyst nematodes and root-knot nematodes during migration.
(A) CNs migrate intracellular and wound the plant cells while (B) RKNs migrate intercellular without impact on plant cells; (C) Feeding sites induced by CNs; (D) Feeding sites induced by RKNs. (Photo credit: (A), (B), (D) INRA Sophia-Antipolis; (C) www.apsnet.org)
Root-knot nematodes (RKNs, Meloidogyne species) are ones of the most economically devastating plant pathogens in the world (Trudgill & Blok, 2001), that causes a global crop losses of about 10 billion euros per year. RKNs could be found in the temperate and tropical regions all over the world (Blok et al., 2008; Abad & Williamson, 2010) and are able to infect thousands of plant species (Figure 8). These microscopic worms induce typical root deformations, known as galls, which result a weak and poor-yielding plants. Until 2009, 97 RKN species have been described (Hunt & Handoo, 2009), in which those with asexual reproduction are the most damaging pests, e.g. M. incognita, M. javanica, M. arenaria and M. enterolobii (Figure 9). Climate change could promote nematodes to produce more generations per year, thus increase the risk of nematode infections (Ghini et al., 2008). Therefore, novel and efficacy strategies need to be developed to against these pests and secure global food production.
(A) RKN symptom on cucumber root (B) M. graminicola infestation on rice field (C) M. javanica infestation on tomato (D) RKN damage in carrot field in New York state (Photo credit to: (A) INRA Sophia-Antipolis (B) Roger Lopez-Chaves, Universidad de Costa Rica (C) http://www.nagref-her.gr/ (D) Courtesy G.S. Abawi).
There are three reproduction modes in RKNs: mitotic parthenogenesis, meitotic parthenogenesis and amphimixis (sexual reproduction) (Figure 9). There are a few RKNs that reproduce sexually (M. carolinensis, M. megatyla, M. microtyla, M. pini). They have a restricted distribution, a poor host-range and less impact in agriculture. Some species (M. hapla, M. chitwoodi, M. fallax) reproduce by cross-fertilization when males are present; or by meiotic (automixis) parthenogenesis when males are absent. Mitotic parthenogenesis, is the mode of reproduction of the most important RKN species in term of host-range and agronomic impact (M. incognita, M. javanica, M. arenaria, M. enterolobii) (Castagnone-Sereno et al., 2013). This lack of sexual reproduction prevents the use of classical genetic approach to study these nematode species. Although information on nematode reproduction is still missing and lacking for several species, it is admitted that mitotic parthenogenesis species have wider host-range than the meiotic or amphimixis ones.
Figure 9: Mode of reproduction and reproductive pathway of root-knot nematodes (genus Meloidogyne) (Castagnone-Sereno et al., 2013).
(A) Consensus tree of phylogenetic relationships in root-knot nematodes (genus eloidogyne) and modes of reproduction (The tree is based on the analysis of SSU rDNA sequence); (B) Schematic representation of reproductive pathways in root-knot nematodes. 2n represents the somatic chromosome number independently of the ploidy level. Abbreviations: pb1, first polar body; pb2, second polar body.
Root-knot nematode control
There are several methods commonly used to control RKNs. These methods can be divided in to three main types: chemical control, biological control and resistant plants. The main goal of these methods is to limit the nematode population under an economically viable threshold. In reality, crop rotation is not efficient to control RKN due to large host range of these pests.
For long times, nematodes have been controlled using chemical nematicides. There are two types of nematicides, soil fumigants (gas) and non-fumigants (liquid or solid). Soil fumigants became popular because they limited practical methods; they drastically reduced nematode populations in the soil, and were cost effective for most crops. Non-fumigant nematicides such as fenamiphos (Nemacur) and aldicarb (Temik) were based upon the same kinds of active ingredients as many insecticides (i.e. nerve poisons) and could be applied in liquid or granular formulations (Lambert & Bekal, 2002). While non-fumigant nematicides reduce nematode populations, their effectiveness is not as consistent as that of fumigant nematicides. However, nematicides induced severe impacts to environment and human health. Therefore, in Europe, most of nematicides were banned by the application of Council Directive 91/414/EEC, except four active molecules: ethoprophos, fenamiphos (aka fenamiphos), fosthiazate and oxamyl. However, they are expensive, not so efficient, and the authorization to use these products will soon be expired in 2017.
An alternative nematode control method is to use natural predators or pathogens of nematodes. Most researches focused on isolating soil microorganisms, mainly fungi and bacteria as potential microbial control agents (Davies & Spiegel, 2011). The fungi Arthrobotrys irregularis was reported to be a predator for Meloidogyne spp. thanks to the formation of hyphae in lasso shaped (patent INRA n°7817624, (Cayrol, 1978)). The bacteria Pasteuria penetrans is also documented as control agent for Meloidogyne spp. The spore of this specie is able to parasite on the nematode and blocks its multiplication (Djian-Caporalino & Panchaud-Mattei, 1998). However, the efficiency and use of these biological agents were limited to some specific soil conditions and their commercialization was also limited by the difficulties and costs of the production of these agents.
Plant resistance constitutes an effective control method. Plant breeders cross natural nematode resistance genes (R-genes) into cultivated plant species to improve their resistance to nematodes. So far, there are about 30 R-genes to RKNs have been identified, mostly in the Solanaceae family, such as Mi-1.2 in tomato and Me genes in pepper, and Ma in Myrobalan plum (Castagnone-Sereno, 2006; Williamson & Kumar, 2006; Claverie et al., 2011). Only two genes, Mi-1.2 and Ma, were cloned (Figure 10). The Mi-1.2 gene encodes a typical nucleotide binding leucine rich repeat (NB/LRR) type resistance gene, which confers resistance to M. incognita, M. javanica, M. arenaria (Rossi et al., 1998). Mi-1.2 is remarkable in that it also confers resistance to the potato aphid (Macrosiphum euphorbiae) and whitefly (Bemisia tabaci). The Ma gene is a Toll/Interleukin1 Receptor (TIR)-NBS/LRR (TNL) gene that confers a high and wide-spectrum RKN resistance comprising, besides the mitotic parthenogenetic RKNs controlled by Mi-1.2 gene, the uncontrolled M. enterolobii (Claverie et al., 2011). The wide-spectrum and resistance induced by Ma gene may be best explained by an indirect interaction – guard hypothesis between the resistance gene product and putative nematode avirulence factors (Claverie et al., 2011). Whereas efficiency of several R-genes against RKNs is temperature-dependent, e.g. the tomato Mi-1.2 gene or the pepper N gene, Ma, as pepper Me3 and Me1 are stable at high temperature (Djian-Caporalino et al., 1999).
Table of contents :
1. Generalities about Nematodes
1.1. Nematode structure
1.2. Nematode lifestyles
1.3. Nematode systematics
2. Plant-parasitic nematodes
3. Root-knot nematodes
3.1. Reproduction mode
3.2. Root-knot nematode control
3.3. RKN life cycle
4. Giant cells: formation and main characteristics
4.1. Cell cycle and cytoskeleton reorganization during giant cell formation.
4.2. Importance of the metabolism in GC
5. RKN effectors
5.1. Identification of RKN effectors.
5.2. Functional analysis of effectors
6. Transcriptomic approach to identify nematode effectors
CHAPTER 1: Identification of Parasitism Effectors Expressed During Plant Infection from the Transcriptome of Meloidogyne incognita
Materials and Methods
Supplementary data chapter 1
CHAPTER 2: Transcriptome Profiling of the Root-Knot Nematode Meloidogyne enterolobii During Parasitism and Identification of Novel Effector Proteins
Material and methods
GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES
1. Identification of parasitism effectors expressed during plant infection from the transcriptome of Meloidogyne incognita.
2. Transcriptome profiling of the root-knot nematode Meloidogyne enterolobii during parasitism and identification of novel effector proteins
3. Toward new resistance strategies against RKN.
Annex 1: In situ Hybridisation protocol for localisation of gene expression in nematode
Annex 2: siRNA soaking protocol and resistance test
Annex 3 : Curriculum vitae