Impacting of Root-Knot Nematodes on Tomato: Current Status and Potential Horizons for Its Managing

Abstract

Root-Knot Nematodes (Meloidogyne spp.) are very serious pathogen on tomato plants among the worldwide. They are widely distributed in soil and causes a highly economical losses for more than 5000 plant species. Therefore, many managements’ strategies are applicable to decrease their effectiveness such as resistant genotypes, soil solarisation and chemical control. Until now, chemical control is the most applied strategy for nematode management. Although nematicides are highly impacted for nematode suppression but environmentally not safety and very toxic. Consequently, several promising studies revealed that root-knot nematode (RKN) can inhibit nematode reproduction based on the susceptibility of their plant host. The plant effectors play a vital role during nematode infection and effect on plant response to nematode requirements. To understand well the relationship between nematode and their host, the molecular and immunolocalization methods illustrated some proteins which are expressed by plant genes involved in plant–nematode interaction. This chapter will focus on the latest status and future perspectives for nematode management.

1. Introduction

Phyto-parasitic nematode; Meloidogyne spp. which are known root-knot nematodes (RKNs) and the most widespread soil-borne obligate plant parasites that are sedentary endoparasites in plant roots [1]. More than identified 100 species belonged under Meloidogyne spp. capable infect almost all vascular plant species and distributed in the tropical and subtropical regions [2]. The most common species in the tropical regions are Meloidogyne incognita, M. javanica and M. arenaria while M. hapla, M. fallax and M. chitwoodi are distributed in the cooler regions [3]. Since the humid climate is the most favorable conditions for survival and reproduction of RKNs, the most damage and crop losses pronounced in tropical regions [4]. Tomato plants are grown round the worldwide over all the year and are considered one of the most important hosts for RKNs. Accordingly, these nematodes cause billions of dollars in losses of the tomato crop annually. The success of parasitism and life cycle of these nematodes depends on their induction for nematode-feeding sites (NFS) within the root tissues of plant host [5]. Therefore, root-knot nematode infection formed many tumeres on infected roots called galls that contain NFS within it. Many applicable processes can reduce and manage RNK in filed. Agriculture practices, physical, and biological methods together significantly more effective than pesticides. Unfortunately, nematicides application still used until now since it is the most management strategy to reduce the damage of RKNs. Although, the nematicides highly effect on nematode and infection parameters but the apprehensions on environmental safety and human health risks have led to restriction of the nematicides application, which is commonly expensive and toxic, particularly in sustenance agriculture system. Another approach to inhibit RKNs and their population in soil is the cultivation of resistant plants that depends on natural resistance inherited by resistance genes. However, this management process requires long-term experiments and to date, there are relatively few resistance genes identified. Currently, a new promising approach for suppress RKNs depends on some information became available for about genes that are involved in the relationship between RKNs and their host during infection. Thus, a good understanding of the interaction between plant and nematode enables us to develop a new strategy to reduce the risk of RNKs.

2. Historical brief

Thru the nineteenth century in 1855, Miles Josef Berkeley was the first to record and attributed galls on cucumber roots to nematodes. Later in 1872, Greef titled the pathogen of root galls, as Heterodera radicicola. In 1879, Cornu also detected root galls on sainfoin plants (Onobrychis sativus Lam.) in the Loire valley, France and described it as root-knot caused by nematode and named it as Anguillula marioni. While, in 1884, Müller turn this name to Heterodera again. Afterward, Treub in Java, Indonesia named the root gall producing nematode as Heterodera javanica in 1885. During 1887, Göeldi described and illustrated a RKN from coffee plants in Brazil briefly and named it Meloidogyne exigua. In the meantime, Neal called it as Anguillula arenaria in 1889 in the United States of America, while Cobb in 1890 named it as Tylenchus arenaria in New Zealand. At the beginning of twentieth century in 1901, Prayer published the first research article relating to RKN that was describing a nematode disease as root galling on banana in Egypt. Then, Kofoid and White have named the root gall inducing nematode as Oxyuris incognita in 1919. Generally, the name Heterodera marioni was widely used for RKN until 1949, when Chitwood re-established the genus Meloidogyne suggested by Göeldi in 1887, and retained four species M. javanica, M. arenaria, M. exigua and M. incognita and described M. hapla and a variety of M. incognita he termed M. incognita var. acrita [1].

3. Economic impact

Economically, RKN cause several billion dollars of losses annually that is estimated to be totally between US$80 to US$110 billion per year for agriculture crops around the world [6, 7]. The severity of damage and losses caused by Meloidogyne depending on the nematode specie, susceptibility of host, crop rotation, season, and soil type [8]. Beside the directly effect of RKN as a plant pathogen, Meloidogyne species also able to interact with other soil-borne pathogens especially vascular wilt pathogens and root-rot pathogens. Consequently, the synergistic effect of RKN and other plant pathogens increase plant damages and crop losses as well. According to the mechanical force of RKN that cause wounds, and their physiological effect on the plant, this interactions effects on the susceptibility and response of host plants to root-rot infection, and lead to breakdown plant resistance particularly for wilt diseases [9, 10]. Tomato (Solanum lycopersicum L.) is one of the most popular vegetable crops worldwide and grown on more than 5 million hectares. Annually, tomato plants producing nearly 243 million tons of tomato fruits around the worldwide estimated about US$ 1.6 billion [11]. Commonly, RKN caused more than 85% of the damage to agricultural crops [12], and 68% of tomato yield lost per year [13, 14]. The damage thresholds of Meloidogyne depends on their specie, race, and plant type. The average of thresholds has been determined for several crops is approximately 0.5–2 J2/g of soil [15]. Regarding to symptoms caused by RKN infection on their host, many galls formed on plant root system (Figure 1a) and counting the nematode feeding sites in the vascular tissues that shelter adult females (Figure 1b). Thus, these galls effect on the uptake of nutrients and water by the plant [16]. In addition, plants revealed foliar symptoms such as preharvest wilting, yellowing of leaves, general reduction of plant growth, floral abortions and decrease of both fruits number and quality, as well as death of the plant in severe infections (Figure 1c) [17]. Besides the direct losses of Meloidogyne spp., the global cost of nematicides marketing is annually developing. The world’s increasing focus on controlling the incidences of plant-parasitic nematodes and improving crop yields to ensure food security led to spend US$1.8 billion in 2022. According to increasing the environmental concerns worldwide and improved crop yields, the governments funding is focusing for increase the integrated pest management strategies and to produce new chemical and bio-nematicides as alternatives to traditional synthetic pesticides. Therefore, the cost of creating a new chemical active ingredient is increasing every year and is now estimated to be more than US$250 million [18].

Figure 1.

Symptoms of root-knot nematodes. Galled root system of infected tomato plants (a), histological symptoms of root galls illustrated the developed nematode-feeding site containing adult female (b). Dead, stunted, chlorotic, and preharvest wilting plants caused by root-knot nematode in the field of tomato (c). (*) giant cells, (n) nematode and em = egg-mass. Bars = 200 μm (C) (a, reprinted from DOI: 10.1007/s41348-022-00642-3), (b, reprinted from DOI: 10.24425/jppr.2019.126040) and (c, reprinted from https://www.growingproduce.com/vegetables/field-scouting-guide-root-knot-nematode/#slide=148494-148490-2).

4. Biological life cycle

Stereotypically, nematode’s life cycle including six stages; an egg, four juvenile stages and the adult stage. A molting phase occurs between each juvenile and adult stage. Concerning RKN, the parasitic cycle (Figure 2) commences when the J2 penetrates a root in the zone of elongation (Figure 2a) [19]. Afterwards, J2 successes to move intercellularly through the cortex toward the root tip without causing damage to the root cells (Figure 2b). The reason of simplicity penetration and roaming within the root is due to the mechanical force of nematode’s stylet and their secretions that including cell-wall-degrading enzymes produced from specialized glands [20, 21, 22]. After that, J2 turns around and moves back up into the differentiating vascular cylinder until it reaches the region where the protoxylem is just beginning to form (Figure 2c), where it establishes a long-term feeding site. The J2 induces the redifferentiation of five to seven parenchyma root cells for the development of the nematode feeding site structure (Figure 2d) [23]. These feeding cells form to multinucleate giant cells inducted by the injection of secretions produced from the dorsal esophageal gland of J2 [24]. When J2 starting feeding, becomes sedentary and directly exchange its shape from a vermiform to fusiform shape after the second and the third molts that differentiates non-feeding phases (J3 and J4) [25]. Then, J4 undergoes the fourth molt to differentiates to adult stage. In optimal conditions, almost all J4 differentiates to young females that feeding resumes again through giant cells. Consequently, the developing females becomes mature and swollen, pear-shaped, and lays approximately 500–2000 eggs embedded and clustered in a gelatinous matrix called egg-mass attached on the root surface (Figure 2e). Within the egg (Figure 2f), the first stage juvenile (J1) forming (Figure 2g) and molting to differentiate to second-stage juvenile (J2) which hatches in soil and looking for another plant roots to repeat the disease cycle. Normally this cycle between initial infection and laying egg-masses takes 21 days at 25°C. According to the environmental conditions, plant response and nutrient availability, sometimes during droughty condition or in resistant host, males differentiate and directly leaving the root without feeding [26]. Typically, RKN reproduction by parthenogenesis, although males are frequently found and seem to have no role in sexual reproduction [1].

Figure 2.

Root-knot nematode parasitic cycle. The J2 is the only stage that can penetrate the roots closely behind the root tip (a). Intercellularly J2 migrate toward the root tip (b). Behind the root cap, the infective stage (J2) turns-up to the vascular tissue (c). The J2 stops moving and induce approximately 7 feeding cells by its secretions to form nematode feeding site (d) which are rapidly differentiated into multinucleated giant cells (*). Adult female still partially inside the gall and lays its eggs in a gelatinous matrix (egg-mass) (e) attached with root surface. Inside the egg (f) the nematode embryo rapidly develops into J1 (first stage juvenile) (g) that hatch from egg to looking for another plant root to penetrate again.

5. The parasitic approach

According to understanding the plant-nematode interaction relationship, root-knot nematodes have evolved strategies to suppress host immune responses for the development of its feeding sites. The activation of plant immune responses depends on specific molecules that recognize nematode signaling. Recently, genetic sequencing analyses led to identification of molecular components that secreted from RKN during parasitism. These analyses have contributed to our overall understanding of the dynamic and complex nature of plant-nematode interactions. These molecules called efforts and produced in three esophageal salivary glands classified to two subventral glands (SvG) and one dorsal gland (DG). The effectors secreted by SvG allowing J2 penetration and migration in the root while proteins secreted during parasitism are produced by SvG and particularly by DG [27]. Also, some effectors produced in other secretory organs, such as chemosensory amphids [22]. Proteomic analysis has identified around 500 proteins secreted by preparasitic J2s or feeding females of M. incognita [28]. Furthermore, during this molecular dialog there is other secreted proteinaceous effectors such as phytohormones, have been shown to favor the plant-nematode interaction [29]. Among proteins secreted by preparasitic J2s, cell wall-degrading effectors have been detected to support its penetration and migration within the root (Figure 2a, b), and effectors suppressing plant defenses have been described [30, 31]. Root-knot nematodes are sedentary obligate biotrophic pathogens establish a relationship with their host plants, inducing the redifferentiation of root cells into specialized feeding cells for a long-term. Vitally, the successful establishment of nematode feeding cells is critical for nematode development and its reproduction. Nematode feeding cells (NFC) called giant cells (GCs) forming when J2 settle down to start feeding and stimulated by other secreted effectors from J2 that injected via its syringe-like stylet at the beginning of feeding process. Fully differentiated GCs are enlarged cells that estimated around more than 300 times larger than normal cells and are converted into multinucleate cells through synchronous nuclear divisions without cell division (Figure 3a) [23]. Therefore, GCs may contain more than a hundred polyploid nuclei that may have undergone extensive endoreduplication (Figure 3b) [16, 32]. All these dramatical changes lead to the formation of nematode feeding sites that containing GCs surrounded by active mitotic cells called neighboring cells which lead to display a typical root gall [32, 33, 34]. Moreover, the plant cytoskeleton in GCs is totally random as a response to RKN induction during nematode feeding. Both actin filaments and microtubules have observed during RKN infection in Arabidopsis thaliana and shown a dense network of cortical microtubules and thick microtubule bundles were formed at giant cell cortex (Figure 4), also shown a dense actin network and actin cables illustrated in GC and neighboring cells (Figure 5) [35, 36]. Genetically, the formation of GC requires extensive changes to gene expression for several gene involved in the plant-nematode interaction [37]. Currently, some studies focused on several genes involved in plant response for RKN infection and the development of their NFS that can be promise to a novel possibility horizon for management of Meloidogyne spp. in the future.

Figure 3.

Giant cell formation. The white arrows indicate to mitotic activity illustrated the nuclear divisions without cytokinesis (a), red arrows indicate to multinuclear in the fully forming giant cells. Bars = 5 μm. DOI: 10.1371/journal.ppat.1002343.

Figure 4.

In vivo observation of cortical microtubules (CMTs) cytoskeleton arrays during giant cell development in roots of Arabidopsis thaliana (microtubule-binding domain MBD-GFP). (A), (B) and (C) overlays of differential interference contrast transmission. (a’), (B′) and (C′) images of confocal laser scanning microscopy showing that a dense network of CMTs and thick microtubule bundles were presented at giant cell cortex. n, nematode; NC, neighboring cell. bars = 5 μm (a - B′), 10 μm (C and C′). DOI: 10.1371/journal.ppat.1002343.

Figure 5.

Actin cytoskeleton organization in galls inducted by RKN on the root of in roots of Arabidopsis thaliana. Dense actin network in giant cells and neighboring cells forming 10 days after infection (a, a’) and 21 days after infection (b, b’). G, gall and *, giant cell. the actin cytoskeleton is depicted in gray levels on a yellow background. Bars = 50 mm. DOI:10.1105/tpc.109.069104.

6. Management strategies

Many applicable processes are impacting on RKN and their damage on plant hosts. Many years ago, until now, the traditional methods including almost all practise related with agronomic methods and chemical control procedures using nematicides are the most familiar techniques for nematode control. Various efforts have focused on this problem in worldwide particularly in developing countries. One of the most widely project for control of Meloidogyne spp. was The International Meloidogyne Project (IMP) coordinated by North Carolina State University has been performed extensive research for several years to help the developing nations to reduce crop loss caused by RKN. This project has been lunched in 1975 with a teamwork was including more than 100 nematologists participated from more than 70 developing countries. The classical methods for nematode control involve the use of agricultural practices, chemical control using nematicides and resistant plants. Significantly, nematicides are successfully reduce nematode’s populations in soil and increase both quantity and quality of agricultural crops. However, they are unsafe highly toxic chemicals consequently, the employed nematicides are highly pollutants and dangerous to our environment. Hence, many alternative chemicals which are eco-friendly can use to increase the immunity of plant host for example some plant extracts. In addition, biological control by using numerous bioagents against nematode can inhibit nematode activity and their production. Commercially, there is some bio-nematicides are common applicable as a substitute for nematicides.

6.1 Traditional practices

6.1.1 Chemical practices

The safety of agroecosystem is facing a major challenge that require protect it from the toxicity of pesticides and increase the outcomes of crops simultaneously. Commonly, chemical controls of RKN using a various nematicides is the most impacting for all Meloidogyne spp. and positively decrease crop losses; however, numerous nematicides are being phased out due to environmental and health concerns [7]. Therefore, the alternative chemicals that have both the nematocidal effect and to be eco-friendly is highly required. Recently, several botanical-based nematocidal are being commercially marketed. The extracts of Neem plant (Azadirachta indica) have the most famous nematicidal formulations such as Neemrich, Neemix, Neemazal, Neemgold, and Neemax [38]. Also, allicin (diallyl thiosulfinate) extracted from garlic plant (Allium sativum) is a nematicidal compound that highly effective against M. incognita and improve tomato yield [39]. In parallel, various plant extracts have been synthesized to ensure more safety for ecosystem than the commonly known synthetic chemical nematicides.

6.1.2 Agronomic practices

Agricultural practices are non-chemical management tactics such as crop rotation with non-host crops or resistant cultivars, and these policies are an economical method for nematode management. Although RKN distributed on more than 5000 plant species [40], there are some plants have shown to be poor host such as wheat, corn, sorghum and garlic that can cultivate during crop rotation [7, 41, 42]. The cultivation of non-host crops or resistant cultivars during crop rotation able to suppress RKN populations by decreasing their eggs and infective juveniles (J2) in soil [43]. Concerning tomato crops, the rotation to non-hosts should be for a minimum of 3 years [44]. Also, the exclusion of weed plants is important avoidance strategy for other alternative hosts to reduce nematode population in soil because many weed species may serve as hosts to RKN [45, 46]. Irrigation water is an important facility for nematode transfer. Thus, sanitation of farm equipment, and plant seedling can avoid transferring the pathogen to non-infested fields [47]. Additional procedures such as fallowing soil, soil solarization, steaming, and flooding can decrease the survival rate of nematode’s eggs and infective stage (J2) [44]. Furthermore, physical techniques such as soil solarization before planting can be combined with cultural processes for effective control of RKN. The application of soil solarization technique usually during summer season for 8 weeks using transparent polyethylene sheets for wet soil mulching [48]. Under the influence of the sun’s heat, water evaporates from the soil to condense on the inner surface of polyethylene sheet in the form of droplets as a lens that collect the sun’s rays, which leads to a rise in the soil temperature in the upper 40 cm and reducing both eggs and juveniles survivals [49]. Additional traditional method among agricultural practice is soil amendments with organic manure for improving soil structure, physical and chemical soil properties, temperature, and humidity conditions as well as the nutrient content necessary for plant growth and their immunity for pathogens. The previous studies shown that soil amendment using farm manure and extracts from marigold (Tagetes spp.) let to release toxic compounds that can harm plant parasitic nematodes [50] and have also been activated the biocontrol agents in soil [47]. The high rate of soil amendments using organic materials has a significant effect on nematode populations in soil [51]. Normally, the indirectly outcomes of soil amendments using organic manures are increasing the activity of many benefit bioagents in soil that can suppress the population levels of many plant pathogens including RKN and may be able to induce systemic resistance of plant species as well [52].

6.1.3 Biological control agents

Biological control methods using living soil-habitat microorganisms (bacteria and fungi) that effect on the nematode’s population unites in soil (eggs and/or J2) by secreted the natural bioactive substances [53]. Many studies and experiments focused mainly on bacteria and fungi that are revealed antagonistic effect against RKN. The results of these research achieved to produce some commercial biological products against certain Meloidogyne spp. These products are usually developed from bioagents which can attach with nematode cuticle or to parasitize eggs-masses subsequently decreasing nematodes population in soil. Some bacterial isolates shown a highly activity against RKNs infected tomato such as Pseudomonas jessenii, P. protegens, Bacillus thuringiensis and Serratia plymuthica. Additionally, some fungal isolates for example Purpureocillium lilacinus, Trichoderma harzianum, Arthrobotrys oligospora, Lecanicillium muscarium Gliocladium spp., Pochonia chlamydosporia and Paecillomyces lilacinus [54, 55, 56]. Furthermore, some endophytic agents such as Fusarium oxysporum (FO162) can induce systemic resistance against Meloidogyne spp. in tomato [57] and plant growth-promoting rhizobacteria (PGPR) as well [58]. One of the most important PGPR is strain LMG27872 of Paenibacillus polymyxa that increasing the percentage of J2 mortality and reducing number of galls and egg hatching of M. incognita in tomato [59]. Similarly, the same effect detected by two bacterial isolates ZHA296 and ZHA178 of Paenibacillus castaneae [60]. Also, both bacterial strains; BZR 86 and BZR 277 of Bacillus velezensis inhibited M. incognita and improved plant health, and crop productivity under greenhouse condition [61]. Moreover, Bacills amyloquefaciens, B. megaterium, Pseudomonas fluorescens, and P. putida have a potential effect against RKN in the laboratory as well as in field conditions [62]. Among endophytic fungi, Arbuscular mycorrhizal fungi (AMF) that are soil fungi and symbiosis with the plant roots. These fungi extremely benefit for plant healthy by acting enhanced plant tolerance for RKN due to induce plant systemic resistance and provide plant nutrients [63, 64].

6.1.4 Resistant genotypes

Planting resistant cultivars is one of the environmentally friendly methods to reduce RKN in tomato. The plant resistance for RKN depends on the genotypes that are restrict or prevent nematode reproduction in their plant host. At least 10 plant resistance genes (R-genes; Mi-1, Mi-2, Mi-3, Mi-4, Mi-5, Mi-6, Mi-7, Mi-8, Mi-9, and Mi-HT) for Meloidogyne spp. have been identified in tomato plant [65] Among these genes, only five genes (Mi-1, Mi-3, Mi-5, Mi-9, and Mi-HT) have been mapped. Concerning tomato-resistant genotypes, Mi-1 gene is the most common that was originally identified in Solanum peruvianum and transferred into S. lycopersicum [66]. This gene confers resistance to M. incognita, M. javanica, and M. arenaria [44, 66]. The Gene map of Mi-1 gene localized it to the short arm of tomato chromosome 6 [67]. There are two homologs of Mi-1 gene coded by Mi-1.1 and Mi-1.2 that were identified at the Mi locus. The Mi-1.2 gene conferred resistance to 15 populations of Meloidogyne spp. [68]. In contrast, the tomato genotypes that are possess Mi-1.1 gene but lack the Mi1.2 gene demonstrated highly compatible with M. javanica [69, 70]. While the resistant genotypes that possess Mi1.2 gene can delay or suppress the development and reproduction of nematodes [70]. In addition, 83 WRKY genes have identified in tomato plants [71]. One or more members of this gene family such as SlWRKY72, SlWRKY73, or SlWRKY74 have been examined as contributing positively to both PAMP-triggered immunity (PTI) and Mi-1-mediated effector-triggered immunity (ETI) against M. javanica [72, 73]. Also, the SlWRKY80 gene was required for Mi-1-mediated resistance against RKN [74]. Thus, these genes could play an important role during nematode infection in investigated resistant tomato genotypes as Mi-1-mediated effector-triggered immunity. Subsequently, all these sources of resistance can become valuable additions to nematode management strategies in the future considering that the natural resistance require long-term experiments. Also, genetic resistance in the host plant could be breakdown when there is a high population density of the nematode [75].

6.2 Innovative methods

The chemical activation of the plant’s natural defense mechanisms should be involved as alternative safety strategy for management of RKN. Some chemicals are inducers challenging localized hypersensitive reactive which involves recognition proceedings between plant and pathogen. In systemic manner, plants have other mechanisms that boundary pathogen access and their reproduction. For instance, several defense genes in plant up regulated by salicylic acid and Benzothiadiazole [76, 77, 78]. The defensive proteins that called pathogenesis-related proteins expressed by these genes in resistance or tolerance plant. Chemical induction of “systemic acquired resistance” is detected by using Benzothiadiazole in tomato and grapevines plants to suppress infection of M. incognita [79] and by using hydroxyurea in tomato to inhibit progress infection of M. javanica [80]. Also, chitosan stimulated production of defense-related chemicals in tomato plant and was associated through improvement process of resistance to root-knot nematode as well [81]. In addition, some botanical extracts and synthetic compounds able to be a resistance inducer to plant pathogen [82, 83]. Many chemicals able to encourage systemic resistance in various plant species to different pathogens such as Oomycetes, fungi, bacteria, viruses, and nematodes [84, 85]. During plant defense mechanisms, Jasmonic acid (JA) and salicylic acid (SA) play an important role as plant growth regulator [86] and shown different reactions in plant resistance responses against root-knot nematodes [87, 88, 89, 90, 91]. As a reaction to biotic and abiotic agent, γ-Aminobutyric Acid (GABA) is a non-protein amino acid rapidly accumulated within tomato plant tissues and has an important role in plants during plant-pathogen interaction particularly RKN [92]. Also, GABA has an isomer named β-Aminobutyric Acid (BABA) that is known as an inducer for plant disease resistance (ISR) when applied to various plants host during RKN infection [93, 94]. Also, BABA plays a role as an inducer for Systemic Acquired Resistance (SAR) against M. javanica [95]. Consequently, treatment of tomato plants by BABA reduced damage of root knot nematode. Also, suppress M. javanica on pineapple, and exposed induce resistance against M. javanica in cucumber [95]. Among commercial products, both Agrispon and Sincocin are liquid concentrate derived from plant extracts. Commercially, Agrispon sold as a plant fertilizer that led to improve root building, plant growth and their yield without a negative environmental impact. Also, Sincocin used in plant fertility programs and improves plant’s ability to resist a variety of pathogens and environmental stresses.

6.3 Novel approaches

Among advanced techniques, microarray analysis technique that can add more detail for about plant response to Meloidogyne spp. infection through identification of some genes involved in the pathogenicity relationship between RKN and their plant host. Specifically, genes implicated in cell wall formation, transport processes and plant defense responses during NFS and GC formation. As well, the development of RNA interference (RNAi) and complementary DNA (cDNA) technology led to characterized nematode secretions as parasitism effectors and should explain the molecular events and regulatory mechanisms during RKN infection. Consequently, it is easy to be following the proteins that are expressed by these target genes in NFS via immunolocalization analysis either In vitro by using In situ hybridization or immunofluorescence technique, or In vivo using green fluorescence protein (GFP) fused with target gene. Among manipulation occurred in GCs at the cytoskeletal level, the previous studies observed that the disruption of the cytoskeleton is possibly a requirement to allow RKN to complete their life cycle [96]. Thus, the cytoskeleton is involved in the process of RKN infection. The genetical studies for Arabidopsis thaliana genome identified seven actin-depolymerizing factor (ADF) genes that are upregulated in GC during infection of by RKN. Particularly the expression of ADF2 gene increased between 14 and 21 d after RKN inoculation resulting in accumulation of actin filaments in GC. The knockdown of ADF2 gene using RNAi reveals that ADF2 protein expressed by this gene is required for normal cell development and plant growth. Thus, during nematode infection, decreasing level of ADF2 protein led to reducing F-actin turnover and inhibition the expansion of GCs in NFS and gall formation as well. Accordingly, these effects, the development of RKN is delay and their reproduction significantly decreasing [35]. Recently, several cytoskeleton-associated proteins facilitating cytoskeletal remodeling and defense signaling findings have discovered. Furthermore, the reorganization of the actin cytoskeleton is revealed to further feedback-regulate reactive oxygen species (ROS) production and trigger salicylic acid (SA) signaling [97]. Beside actine filaments, microtubules are a member in the cytoskeleton network and have also an important role in mitotic activity during cell division. Regarding A. thaliana, there are two genes; TUBG1 and TUBG2 which are nucleate the cortical cytoplasm microtubules and regulate their dynamic. Furthermore, GCP3 and GCP4 genes which are nucleate the mitotic microtubules during cell division. During RKN infection, these four genes are upregulating in the cytoplasm and at cell wall of GC causing increasing of microtubule nucleation in cytoplasm and forming a complex network of cortical microtubules at the cell wall of GC and become randomly organized. Without affecting of un-infected plant growth, the knockout of either or both TUBG1 and TUBG2 genes let to delaying of nematode life cycle and gall formation, and decreasing their population [36, 98]. Based on these studies that focused on cytoskeleton and its coordinating genes during RKN infection, could be cytoskeleton has an extremely important role of the plant immunity and plant susceptibility for nematode infection as well [99]. Concerning nematode secretions that containing effectors destroy the cytoskeleton, Meloidogyne incognita secrete an effector called MiPFN3 (Meloidogyne incognita Profilin 3). This protein effector can bind to actin monomers, disrupt actin polymerization, and reduce the filamentous actin network [100]. That is means that the manipulation of the cytoskeleton by RKN may be a tactic to promote its parasitism. Additionally, Arabidopsis genome comprising two pectate lyase-like genes (PLL), PLL18 (At3g27400) and PLL19 (At4g24780). Upregulation of these genes detected in the developed NFS during infection of M. incognita. While the mutant lines that loss one of these genes negatively influences the development of GC [101]. Similarly, Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 revealed less susceptible to RKN suggesting a requirement of cytokinin signaling for GC and feeding site formation [102]. According to the endoreduplication of DNA is required for the mitotic activity during GC formation. Therefore, potential DNA damage in the genome of gall cells is evident. WEE1 gene is particularly involved in DNA damage checkpoint control and encoding for a protein kinase that controls cell cycle [103]. Nematode feeding site demonstrate transcriptional activation of the DNA damage checkpoint kinase WEE1. The interrupted nuclei phenotype in GC indicated to the accumulation of mitotic defects. The WEE1-knockout line in Arabidopsis and WEE1-knockdowen line downregulation in tomato repressed RKN infection and their reproduction [104].

7. Conclusion

The highly impacting of root-knot nematode (Meloidogyne spp.) in tomato and other plant host requires an urgent integration management for this plant pathogen. Since the toxicity of nematicides is the critical point for our environmental safety. The alternative eco-friendly chemicals that have a nematocidal effect must be applied together with the application of agronomic and biological control methods, and cultivation of resistance genotypes in strategy of integrated pest management. Genetic analysis of Meloidogyne spp. identified genes that encoding specific effectors promoting parasitism process modulating the plant’s defense system to successfully forming and establishment of a nematode feeding site (NFS) [105]. Consequently, the functional analyses of these effector could be led to the identification of susceptibility genes with potential for use in resistance breeding [106, 107]. However, these susceptibility genes mostly have a vital role for plant physiology and development. Interfering with host protein recognition by nematode effectors may be an interesting way of preserving important plant functions whilst breaking the susceptibility of the plant to nematode. The breeding of new line harboring mutations that are less susceptible to nematode infection may be achieved with new technologies, such as the TILLING and CRISPR/Cas9 technologies [108, 109]. According to the knowledge for about the functions of effector/target which are required to improve the compatibility between nematode and their plant host, it can guide this strategy to stop this interaction and engineer durable disease resistance as a novel process to root-knot nematode management.