Plant-Parasitic Nematodes and Their Management: A Focus on New Nematicides

Abstract

Plant parasitic nematodes are microscopic organisms that inhabit soil and plant tissues. Among such organisms, those of the genera Meloidogyne, Heterodera, Globodera, and Pratylenchus spp., are the most harmful, as they affect more than 2500 different species of plants, generating worldwide economic losses of over US$100 billion per year. These nematodes constitute a notable threat to the country’s progress and food security. Almost half of the global market for nematicides, which corresponds to US$ 1 billion per year, is used to control these nematodes. Non-fumigant nematicides are the most widely used in their control; however, many of them, such as carbamates and organophosphorus, are banned by environmental protection agencies because of their undesirable effects on non-target organisms. In the last 10 years, a new series of nematicides have emerged with different mechanisms of action than the old non-fumigant nematicides. Tioxazafen and fluazaindolizine are some of the latest new-generation nematicides that have come on the market. The rational design of new nematicides through in silico approaches combined with studies of the genetics and biochemistry of these microorganisms will help to better understand their management and control, aiming to reduce the environmental impact caused by the irrational use of nematicides.

1. Introduction

According to the United Nations (UN), the global population will reach 8.5 billion by 2030 [1]. As a result, the World Bank projects that food consumption will rise by 60–80% [2]. Although the Food and Agriculture Organization of the United Nations (FAO) has as one of its goals to end world poverty, the fact is that food security has been declining rapidly since 2015 [3]. Agricultural pests are clearly one of the many elements leading to a country’s or region’s food security being undermined [4]. Pests and plant diseases, according to the FAO, account for 40% of global food production losses, amounting to more than US$250 billion each year [5]. As a result, dealing with plant pests and diseases is critical to attaining higher agricultural sustainability and security, particularly in the post-pandemic age.

Although microorganisms play a vital role in agriculture, many of them act as phytopathogens, reducing agricultural yield seriously [6, 7]. Plant parasitic nematodes (PPNs) are microorganisms that have a deleterious impact on the harvested of numerous food crops such as soybean (Glicine max), corn (Zea mays), potato (Solanum tuberosum), tomato (Solanum lycopersicum), rice (Oryza sativa) and carrot (Daucus carota) [8, 9]. There are about 4000 PPNs species, but only a small number are linked to economic losses owing to decreased agricultural yields, which are estimated to be worth more than US$100 billion each year [10].

Because of their complex interaction with host plants, extensive host range, and the degree of damage caused by infection, root-knot nematodes (RKNs), cyst nematodes (CNs), and root-lesion nematodes (RLNs) were at the top of the list of the most economically important species [11]. Thus, Heterodera glycines, for example, has a significant influence on the agricultural economies of the United States of America (USA) and Brazil (Figure 1), since it affects one of the most important crops for global protein and carbohydrate production: soybeans [11]. Currently, Brazil leads the world in soybean production, with more than 114 million tons produced per year, followed by the USA with 97 million tons per year [12, 13]. In the USA alone, economic losses caused by H. glycines exceed US$1.0 billion per year [14].

Figure 1.

Geographical distribution map Heterodera glycines. The yellow areas and circles correspond to regions of the world where the nematode is currently present. Source: EPPO global database (2023).

Some of the approaches used to manage and control PPNs include resistant cultivars, crop rotation, biological management, and chemical compounds [8]. Chemical methods are the most efficient and therefore most widely used in the management of PPNs; however, many of the chemicals used are highly hazardous to human and environmental health, including carbamates (CMs), organophosphorus (OPs), organic halides, and pyridinylmethylbenzamides [15].

The arsenal of nematicides accessible for the management of PPNs has been substantially restricted by current environmental protection laws. This has increased demand for novel nematicides that are more selective, less toxic, and compatible with sustainable agricultural guidelines. As a result, various nematicides have been introduced to the market over the last decades. This is the case of tioxazafen, Fluopyram, and fluazaindolizine, which are considered new-generation nematicides as they are less toxic to mammals and the environment, suggesting that they have mechanisms of action distinct from those observed for CMs and OPs types.

2. PPNs: overview

PPNs are microscopic and ubiquitous organisms that live in soil and plant tissues. Despite their inability to travel great distances, anthropogenic actions can lead them to be transmitted via contaminated plant material, soil, and agricultural machinery [11]. In addition to preventing plant roots from absorbing nutrients, PPNs may make plants more vulnerable to secondary phytopathogens such as fungi, bacteria, and viruses [16].

Many agriculturally important PPNs are members of the Tylenchida order, which includes endoparasites from the Heteroderidae and Pratylenchidae families. The Heteroderidae family includes the most important agricultural genera: RKNs (Meloidogyne spp.) and CNs (Heterodera and Globodera spp.). The family Pratylenchidae, on the other hand, comprises migratory endoparasite RLNs (Pratylenchus spp.), with over 60 species documented. All of the species in those genera are capable of infecting over 3000 distinct plant species, making them a global threat [17, 18].

PPNs, unlike free-living nematodes, have a hollow and protractile stylet that allows them to pass through plant cell walls, but not the plasma membrane, enabling them to feed on cell contents via the feeding tube that is solely connected with the stylet [19]. PPNs release a cocktail of polymeric or non-polymeric effector proteins that degrade and modify cytoplasmic components, causing the host cell’s physiology and morphology to change [20]. The force for the nematode to feed on the cellular contents is provided by a muscular pump in the basal nodes (metacorpus). In this process, a complex plant-parasite interaction occurs, involving a sequence of recognition and response events that vary depending on the kind of parasite and host plant [21]. Although many aspects of the pathogenesis process are unknown, it is evident that acquiring this information is necessary for the creation of new and more effective techniques of treatment and control.

3. Biology and parasitism of the main PPNs of agricultural importance

3.1 Root-knot nematodes (Meloidogyne spp.)

RKN are obligate biotrophic endoparasites that require a host plant to complete their life cycle [20]. There are more than 90 species, but only a few have worldwide agricultural value. The most damaging species include: Meloidogyne incognita, M. javanica, M. hapla, M. enterolobii and M. arenaria [22]. They generally feed and reproduce inside the roots of plants, where they promote the establishment of permanent feeding sites, which subsequently give birth to galls or root-knots. Because these root-knots can be mistaken with those formed by other PPNs such as Nacobbus aberrans and Subanguina radicicola, their identification and diagnosis are essential, as Meloidogyne is generally more aggressive than the latter [22].

The RKNs life cycle starts when an adult female deposits their eggs (~500) in gelatinous-textured sacs (egg masses) generated by six anal glands (Figure 2). This gelatinous sac is made up of glycoproteins, which, in addition to protecting the egg, serve as a regulator of embryonic development by detecting changes in the temperature and humidity of the environment. With the advance in feeding, the female becomes more voluminous and the posterior section of her body is extended in such a way that allows the egg mass to be exposed outside the root, thus forming the nodes or galls [23]. The galls make it easier for microorganisms in the rhizosphere to come in contact with the eggs; however, their gelatinous structure has been shown to have antimicrobial properties that protect them from attack by microorganisms [24].

Figure 2.

Schematic representation of the root-knot nematode life cycle. 1: Egg in development, 2: J2 larvae entering root, 3 and 4: Swollen nematodes feeding in root, 5: Free male on the ground, 6: Mature female breaking through root surface with egg sac.

After embryogenesis, J1 undergoes the first molt (1° ecdysis), which will give birth to the infective form known as the second instar juvenile (J2). When temperature and humidity conditions are favorable, hatching of the eggs occurs, releasing the J2 nematodes. Unlike other PPNs, which require stimuli from plant roots for hatching, Meloidogyne spp. hatching can occur with or without plant stimuli [25]. In the soil, J2 carries out a pre-parasitic motile phase. During this period, it is susceptible to biotic and abiotic stresses, surviving, before infecting a root of a susceptible host, at the expense of its lipid reserves [26]. Meloidogyne spp. stages that can be discovered in the soil include J2 and mature males. Although the mechanisms by which J2 finds a susceptible plant are not fully understood, it is known that plant root exudates contain substances such as carbon dioxide, tannic acids, flavonoids, and volatile organic acids that may regulate J2 arrival or leave events [27, 28]. In addition, PPNs can be attracted by chemicals released during the attack by herbivores or insects. Thus, for example, it has been shown in Tylenchulus spp. that they are more easily attracted to plants infested by insect larvae than to non-infested plants [29].

J2 enters and migrates via the vascular cylinder of a vulnerable plant after penetrating very close to the root tip where there is a lack of completely developed endodermis (elongation zone) [30]. During this action, the intermediate lamella softens mechanically and enzymatically [31]. After traveling a short distance into the plant, J2 becomes sessile due to the atrophy of its somatic muscle (except for the head). The head is buried in the periphery of the vascular tissue cells (zone of cell differentiation), where it feeds (through a feeding tube) on the protoxylem and protophloem contents before the cells differentiate into specialized nurse cells known as giant cells (GCs) [32]. After the establishment of a permanent feeding site, the nematode becomes sedentary, growing significantly in size until it reaches maturity.

Although the mechanisms by which GCs are formed are not fully understood in detail, it is known that, during feeding, parasites secrete effector proteins that modify cell division events through reorganization of the cytoskeleton and signaling pathways [33]. This cocktail of effector molecules, which facilitate the establishment of the feeding site, is mainly produced by the dorsal gland. Peptides, virulence factors, β-1,4 endogluconases, polygalacturonases, xylanases, chorismate mutase, among others, are some of the molecules that compose the biochemical arsenal of the RKNs secretome [34].

The cell wall between the daughter nuclei is created in telophase during normal plant cell division; however, in the creation of GCs, the cell splits without cytokinesis and hence without cell wall development. This causes multinuclear GCs to form, as well as the proliferation of other organelles such as endoplasmic reticulum, mitochondria, and ribosomes [35]. These metabolic and physiological changes give GCs the characteristics of metabolically active cells, with the inner growth of the wall forming an extensive labyrinth network that increases the surface area of exchange with the associated membrane of xylem vessels [36]. Although less visible, phloem sieve components develop around large cells to ensure phloem continuity [37].

Compared with uninfected plants, tissue from RKNs-infected plants shows a significant increase in the phytohormone auxin [38]. Auxins, like ethylene, have a role in the formation of cell wall ingrowths by promoting the production of cell wall modifying proteins as well as proteins implicated in acidic growth (proton pumps) [39, 40]. These and others evidence imply that the parasite’s modulation of this phytohormone is a key factor in the creation of GCs. In addition to auxins, cytokinins, which are involved in cell division, are elevated in infected tissue, suggesting that they may actively contribute to the parasite-feeding site formation [41].

The plant defense system is also affected by the effector proteins controlling the phytohormone pathway: salicylic acid (SA) and jasmonic acid (JA). In general, the SA pathway protects against biotrophic and hemibiotrophic pathogens such as endoparasitic sedentary [42]. The JA pathway, on the other hand, is involved in plant defenses against necrotrophic pathogens and leaf-chewing insects [43].

The capacity of biotrophic and hemibiotrophic pathogens to modify plant defenses is important to host tissue colonization success. The first line of activated protection against RKNs is the pathogen-associated molecular pattern (PAMPs) triggered immunity (PTI) [44]. In response to the attack (PTI response), the plant triggers the synthesis of reactive oxygen species (ROS), as well as the production of protein kinases (MAPKs) and the activation of the JA and SA pathways [45]. SA regulates the expression of several genes involved in the synthesis of proteins mediating the pathogenic response, while the JA pathway regulates the expression of genes encoding for proteins involved in the synthesis of thionin, defensin, and phytoalexin, among others [45, 46]. The level of response and plant-parasite interaction seems to be influenced by the type of plant, as well as by the type of parasite and its infective state [20].

After the establishment of the feeding site, J2 undergoes significant morphological changes, including two successive molts (J3 and J4). Unlike J2, these last two stages do not feed, as they lack a functional stylet [47]. Under appropriate environmental conditions and with sufficient food availability, J4 undergoes a final ecdysis, the female acquires the classical pear shape (500 times the volume of J2) and the male is left free in the environment. Before reaching full maturity, the cells of the genital primordium of the female divide to give rise to the rectal glands, which will later secrete the gelatinous material with which she will protect her eggs. On the other hand, the male characteristics (testis and vas deferens) differentiate from the posterior end, where they connect with the rectum [25].

It has been recorded that, within the genus Meloidogyne, there are three different forms of reproduction. On the one side, there is the fertilization of oocytes by the male (amphimixis), while, on the other hand, there is parthenogenesis, which can be meiotic or mitotic. In the first case, there is a fusion of the pronucleus of an ovule, generated by meiosis, with a polar body (automixis); while, in the second case, the oocyte divides by mitosis, where one of the nuclei deteriorates and the other remains to give rise to the embryo [48, 49].

As in CNs, sex chromosomes are absent, so sex is strongly influenced, at least in parthenogenetic species, by environmental conditions as well as by host plant conditions. Lack of food, overcrowding, extreme temperatures, and soil dryness are some of the conditions that favor the development of males that reproduce by meiotic parthenogenesis [50]. These males rarely fertilize females, and when they do, mitotic parthenogenesis takes place without any fusion with the sperm nucleus.

3.2 Cyst nematodes (Heterodera and Globodera spp.)

After RKNs, the CNs constitute the second group of sedentary endoparasites with the greatest impact on world agriculture. Among the most important species are Heterodera glycines, also known as the soybean cyst nematode (SNC); Globodera pallida and G. rostochiensis, both known as potato cyst nematodes (PCNs); and, finally, nematodes that attack cereals such as Heterodera avenae and H. filipjevi [22].

Like RKNs, the life cycle comprises an egg stage, four juveniles (J1,2,3,4), and adults (male and female). However, there are important differences that will be discussed below.

Unlike Meloidogyne, in CNs, the eggs may be retained in the female’s body, or, depending on the species, deposited in a gelatinous sac. After their deaths, the female body transforms into the cyst, which protects and holds the eggs inside. Depending on its maturation stages, it can acquire different shades ranging from black-brown to yellow. These color changes are known to be due to the activity of the enzyme polyphenol oxidases, which, in addition to catalyzing the hydroxylation of phenolic compounds, also catalyze the polymerization of o-quinones on the cyst cuticle [51].

In the cyst, the embryo can remain for many years in a state of metabolic suspension (dormancy stage) when environmental conditions are unfavorable. This dormant state is essential for the survival and pathogenesis of the CNs. In general, normal embryo development requires the action of internal signaling molecules, which are regulated by biotic and abiotic conditions [52]. Not all species have this dormancy stage in their life cycle; however, for those that do, it can be of two types depending on the dormancy strategy used: quiescence and diapause. Quiescence can be an obligate or facultative response to poor environmental conditions, which is reversed when external conditions are ideal. On the other hand, diapause, which can also be obligate or facultative, is a state in which development of the embryo (J1) is completely arrested until the metabolic requirements are not satisfied, so that good environmental conditions are not sufficient to reverse the process [53].

Although the egg may hatch spontaneously, there are, however, factors that stimulate hatching. These may be environmental (temperature, soil moisture, oxygen availability, etc.) or derived from root exudates [54]. The effects of such factors on egg hatching vary between species of CNs [55]. The hatching of H. glycines and G. rostochiensis eggs is partly dependent on factors derived from root exudates. Terpenoids (glycinoeclepin A and solanoeclepin A, B), glycoalkaloids (α-solanine and α-chaconine), metavanadate, picrolonic acid, and phenanthroline derivates, are some of the factors identified in root exudates that stimulate egg hatching [56, 57, 58]. These factors can be used as agrochemicals in the control of PPNs, since they can be applied in infested fields, stimulating the release of J2 before planting the crop.

Although the exit of J2 from the cyst constitutes the last stage of the hatching process, it is known that for this to occur, changes in the permeability of the membranes that cover the egg must take place. These changes are essential since they trigger the activation of second messengers that promote the following stages of development, such as the activation of metabolic pathways and J1 ecdysis [59]. Three membranes are known to surround the egg. The inner one is formed mainly by lipids and is semi-permeable to water and small ions; the intermediate, constituted by chitin microfilaments that give resistance and flexibility to the egg; and, finally, an outer one, is formed by lipoproteins (vitelline layer), which are believed to be essential for egg fertilization [60].

Unlike RKNs, where J2 activation can occur before changes in eggshell structure, in CNs, changes in eggshell membrane permeability are required for J2 activation. Within the egg, the movement of the juveniles is reduced by the low turgor pressure of the perivitelline fluid. It is mainly composed of the disaccharide trehalose (α-D-glucopyranosyl 1–1, α-D-glucopyranoside), which not only serves as an energy source for the nematode but also exerts a high osmotic pressure that reduces the water content of the perivitelline fluid [61]. Reduction of the trehalose content, and thus hydration (increased turgor pressure), is a prerequisite for egg hatching [60]. Although this is true for many species, there are others (i.e., Heterodera schachtii) that, on the contrary, have a low osmotic pressure of the perivitelline fluid allowing them to hatch under osmotic stress conditions [62]. Changes in the permeability of eggshells to Ca²⁺ ions, modifications in their lipoproteins, as well as the presence of zinc-dependent enzymes that mediate hatching, are other factors known to be involved in such phenomena [61, 63]. After the first ecdysis, J2 becomes metabolically active and uses its stylet to exit the egg. Once out of the egg, it is believed that it leaves the cyst through the hole generated by the detachment of the head from the female’s body once it has reached full cyst maturation [64].

Free in the soil, J2 is vulnerable to environmental conditions, so it must find a suitable root host plant before its lipid reserves are depleted. Under optimal conditions, localization time for G. pallida and G. rostochiensis has been estimated between 6 and 11 days [65]. In soil, J2 must orient in a three-dimensional matrix, responding to changes in the gradient of a wide variety of physical and chemical stimuli (i.e., CO₂, temperature, pH, redox potential, ethylene, phytohormones, etc.). Some are primarily involved in orientation, while others are in root attraction. It is generally accepted that these signals are perceived through amphids [66].

Once J2 reaches the root of a susceptible plant, it enters through the elongation areas of the root tip, as described for Meloidogyne spp. As in RKNs, penetration enzymes such as cellulases β-1,4 endogluconases are produced; however, there is a difference in their entry into plant tissues, as in CNs the movement is intracellular, while in RKNs it is intercellularly [67]. In CNs, the permanent feeding site is known as a syncytium (composed of about 200 cells). As in RKNs, its formation is a complex process that requires the presence of effector proteins modulating the plant-parasite response. At first, the parasite introduces its stylet into the initial syncytial cell (ISC) in order to evaluate the type of response. When the response is unsatisfactory (covering the stylet by callose), the nematode withdraws its stylet to continue trying with another cell close to the one initially selected. Once the ISC is selected, the maturation process of the syncytium begins, which will become its feeding site for life [68].

In general, the process of syncytium formation is a highly complex process, which, as in RKNs, involves changes in gene expression in both the plant and the nematode. After 7 hours of ISC preparation, the sub ventral glands atrophy while the dorsal gland becomes more active [69]. The stylet is then removed from the ISC and reintroduced. It is at this point that the effector proteins contained in the dorsal gland are released into the cytoplasm of the ISCs [68]. As mentioned above, the secreted proteins have the function of establishing syncytium, which is facilitated by the dissolution of the cell wall separating adjacent cells from the ISC [70]. In Arabidopsis thaliana, it has been observed that J2 of H. schachtii can select procambial or cambial cells for ISC formation. In the first case, the syncytium extends to the xylem and phloem vessels, whereas in the latter case, it maintains contact with the phloem through companion cells [71, 72].

Syncytial cells, like GC in RKNs, undergo morphological changes. The nucleus enlarges by endoreduplication, and the central vacuole breaks down and gives rise to a large number of small vacuoles; on the other hand, the cytoplasm expands to accommodate a large number of ribosomes, mitochondria, endoplasmic reticulum, etc. [68]. In addition to these morphological changes, cells also experience metabolic changes that involve the overexpression of genes that are part of primary metabolism. These changes are influenced by effector proteins that modulate the transport and function of phytohormones. Thus, for example, chorismate mutase, an enzyme isolated from several PPNs and phytopathogenic fungi, is implicated in the initial pathogenesis of infection since it is known to alter the shikimic acid pathway, which is essential for plant-nematode interaction, since through it, precursors for auxins, salicylic acid, and a wide variety of phenolic compounds are generated [72, 73].

After the establishment of the feeding site, the nematode develops to the adult stage. The determination of males and females is influenced by environmental conditions and the supply of nutrients from the host plant. Females, unlike males, have a higher demand for nutrients, so their syncytia are larger than those formed by males. As adults, males, unlike females, are mobile and leave the root to find and fertilize a female. On the other hand, the female enlarges considerably to maintain the eggs (~600 inside the cyst and ~ 200 outside it) and thus restart the parasite’s life cycle, which has been estimated at 4 weeks for H. glycines under favorable conditions [60].

3.3 Root-lesion nematodes (Pratylenchus spp.)

The RLNs are obligate biotrophic migratory endoparasites nematodes, without a permanent feeding site. There are more than 90 species registered with a cosmopolitan distribution. After RKNs and CNs, it is the third most important genus in agriculture. The species with the greatest agricultural economic impact are Pratylenchus penetrans; P. thornei; P. neglectus; P. zeae; P. vulnus and P. coffeae [22].

As migratory endoparasites, they move intra- and inter-cellularly through the root tissues, generating superficial cracking of the root with internal rotting of the tubers that predispose to secondary infections by fungi and bacteria. Unlike the infections generated by RKNs and CNs, where the galls and cysts are observed with the naked eye on infested plants, in RLNs it is not easy to identify signs or symptoms that warn about their presence [74].

Its life cycle is relatively simple. This can last between 3 and 6 weeks, depending on the environmental conditions and the host. The eggs are deposited by the female either in the soil, the roots, or the tubers. Like RKNs and CNs, J2 hatches from the egg, which later develops through J3, J4, and the adult stages: male and female. All are vermiform (worm-like), infective, and motile. This ability to remain mobile during all phases of their cycle allows them to freely enter and exit the host plant [75]. Males commonly reproduce by parthenogenesis, and in some species, they are frequent, while in others they are not [76].

Like other PPNs, they are attracted to root elongation zones by different chemotactic molecules released by the host plant. They have a short, robust, hollow-mouthed stylet, which they use to break the plant cell wall by repeatedly pushing it. J2 and J3 tend to feed on the root hairs; however, they can also do from the epidermal, cortical, or stellar cells [77]. Prior to ingestion of the plant cell’s cytoplasmic contents, the dorsal glands are activated to secrete effector proteins that accumulate in the ampulla behind the buccal stylet. Feeding time can be short or last a few hours. In the latter case the cell may die, however, during the brief feeding, the stylet is removed, and the puncture site is sealed with no contents leaking out. Also, their intracellular displacement is another cause of cell death even when feeding occurs for short periods of time [78, 79].

Initial invasion is followed by the penetration of other nematodes, attracted by compounds released from damaged cells. Migration together with the feeding points generates brown lesions (necrotic areas,), which vary according to the type of host plant and parasite species. This roam feeding behavior leads to loss of plant growth and the appearance of leaf chlorosis and swelling and atrophy of the roots [80].

Although not all aspects of the biology of parasitism in RLNs are known, it is known that many of the effector proteins are different from those produced by RKNs and CNs. It is to be expected that, due to their migratory behavior and their intermittent feeding, the arsenal of effector proteins to manipulate the response of the host plant is less extensive than that of the sedentary endoparasites. Thus, for example, no orthologous sequences have been found for effector proteins essential for the establishment of infection in sedentary endoparasites such as 19C07 (auxin influx in syncytium], chorismate mutase, 10A06 (antioxidant genes in syncytium), CLE peptide, auxin, peptide hormones (CEPs), cytokinin, among others [74]. In silico studies of alignment between conserved domains of effector protein sequences have shown that these are different among sedentary nematode species. Thus, for example, it has been shown that among RLNs and RKNs, there are only a few effector proteins in common, among which cysteine proteases, pepsin inhibitor-3, astacin, and a domain of unknown function called DUF148 stand out. Although the function of this domain is not clear, it is believed that it may be part of the SXP/RAL-2 (ANIS5) family of proteins that bind cations such as calcium and magnesium [81].

Transcriptomic studies in P. thornei and P. coffeae have provided evidence of the secretion of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and peroxiredoxin, which block the action of reactive oxygen species (ROS) produced by the host plant. On the other hand, the resistance of some cultivars to infection is due to the presence and accumulation of secondary metabolites; however, due to their ability to move freely through plant tissues, many of the RLNs escape the defense activities of the host plant [74, 82].

Like other nematode species, Pratylenchus spp. have acquired the ability to remain in the anhydrobiotic state for long periods of time; however, their success rate is moderate compared to other nematodes such as Dytilenchus dipsaci or G. rostochiensis [83]. The rate of loss of soil moisture (dehydration), the stage of development of the parasite, the presence of plant residues (root and stem), as well as genetic characteristics, are some of the factors that affect the development of anhydrobiosis of P. penetrans in dry soil [84]. Unlike J4, which is resilient, the other forms of the parasite are sensitive to changes in soil moisture. Eggs, on the other hand, survive changes better than motile forms of the parasite [85]. In addition to withstanding dry soil, these can also survive freezing as anhydrobionts. The loss of water from their bodies reduces the formation of ice needles that can pierce internal organ cells [86]. Although the reproductive capacity is not affected by anhydrobiosis, the reserve lipid content is managed differently during this stage, since it has been shown that lipid consumption is higher in females than in males [85, 87].

4. Management: a focus on new nematicides

4.1 Overview

The management and control of PPNs are of crucial importance in agriculture since many of them can endanger the food security of a country due to the loss of productive yield of the crops. After the USA and China, Brazil is the third country with the highest grain production in the world. According to the FAO [88], Brazil leads world soybean production, with 134 million tons produced in 2021, 10% more than that produced by the USA: ~120 million tons. It is estimated that 20% of soybean production in Brazil is affected by H. glycines, which constitutes a phytosanitary problem that generates economic losses of more than R$16 billion per year [89].

Although crop rotation, genetic improvement, and biological control are some of the alternatives currently available for the management and control of PPNs (Figure 3), chemical control is the most widely used method worldwide [90, 91]. In developing countries, where access to agricultural technology is limited, and where the cost-benefit factor is of paramount importance for the economy, the use of agrochemicals is still the method of choice, due to their ease of implementation and availability [92, 93].

Figure 3.

Flow chart summarizing the different approaches that currently exist for the management and control of PPNs.

Almost half of the global nematicides market, worth US$ 1.3 billion, is used to control species of the genera Meloidogyne and Heterodera [94, 95]. However, with increasing requirements for food safety and environmental protection, several nematicides are no longer considered suitable for modern agriculture, as there are much data showing their high toxicity to the environment as a whole. For example, the use of methyl bromide and 1,3 dichloropropene (fumigant nematicides) are currently banned for the control of PPNs [96, 97]. On the other hand, non-fumigant nematicides such as organophosphorus (OPs) and carbamates (CMs), are also prohibited in many parts of the world, due to their high toxicity to humans and the environment [98].

OPs and CMs owe their high toxicity to their low selectivity in their mechanism of action, since they are reversible inhibitors of the cholinesterase enzyme (AChE). This is because many are used as broad-spectrum pesticides (insecticides, acaricides, nematicides, and rodenticides) and as chemical weapons [99]. Aldicard, carbofuran, fenamiphos, and oxamyl are some of the OPs and CMs in which cholinesterase inhibition of PPNs has been demonstrated. Since the concentration of nematicide in agricultural soils is not high enough to kill nematodes, many of these compounds exert a nematostatic function with temporary paralysis that interferes with host feeding and egg hatching [100].

Although resistance has been reported under controlled laboratory conditions in PPNs, there are no proven examples in the scientific literature documenting dramatic changes in tolerance to nematicides that lead to suspicions about the development of resistance under field application conditions [100]. On the other hand, field resistance to agrochemicals has been widely demonstrated in different species of agricultural pests; however, it is believed that in PPNs the risk of developing resistance under field conditions is theoretically unlikely [101]. The arguments supporting this hypothesis are based on the application methods of nematicides and the ecological behaviors of PPNs. These characteristics reduce the effects of selective pressure to develop resistance, which can be overcome by the indiscriminate use of nematicides with different mechanisms of action [102].

Despite the fact that the global market for nematicides is much smaller than that of other agrochemicals such as insecticides (US$16.4 billion in 2019), fungicides (US$13.4 billion in 2019), and herbicides (US$32.6 billion in 2019), the fact remains that the market continues to grow despite strong regulatory rules limiting their use. Thus, for example, in 2011 dividends were approximately US$1 billion; however, by 2022 it was US$1.78 billion, an increase of approximately 43% [95, 100]. Post-modern control of PPNs requires cost-effective and selective targeted control strategies that meet the environmental safety demands of both growers and consumers. Thus, in the last 10 years, new nematicides have emerged (Figure 4) with low persistence in the environment and with selective mechanisms of action different from those of OPs and CMs. Of these new nematicides, only fluopyram and cyclobutrifluram have a currently known mechanism of action on PPNs [103, 104].

Figure 4.

Chemical structures of new nematicides.

4.2 Fluopyram and cyclobutrifluram

Marketed as an active ingredient in Verango®, Velum® and Indemnify®, fluopyram (N-[2-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]ethyl]-2-(trifluoromethyl)benzamide) is a new generation nematicide-fungicide developed by Bayer Crop Science in 2012. Like fluopyram, cyclobutrifluram is a trifluoromethylpyridine developed and pantented by Syngenta in 2013 and that is marketed under the names Vaniva®, Tymirium®, and Victrato® [105]. Both interfere with the electron transport chain of the mitochondrial system (complex II) through inhibition of the enzyme succinate dehydrogenase (SDH). This mechanism has been reported for both PPNs and plant pathogenic fungi [106].

In eukaryotic organisms, SDH catalyzes the oxidation of succinate to fumarate as part of a mechanism to transfer electrons through the quinone pool. On the other hand, in nematodes and anaerobic helminths, complex II is associated with another enzyme that performs the reverse reaction (oxidation of reduced quinones) known as fumarate reductase (FRD). This is an adaptation to low oxygen pressures that prevent completely aerobic respiration. Such conditions are typical in intestinal nematodes; however, in free-living nematodes such as Caenorhabditis elegans, FDR is present as well as SDH, suggesting that FDR is an inducible enzyme under anaerobic conditions [107].

SDH consists of four subunits (A-D) that differ in structure and function. Thus, for example, the SDHA sub-unit is the largest and is where the active site of the enzyme is located. On the other hand, the SDHB subunit contains iron-sulfur groups that mediate electron transfer to ubiquinone, which is located at the interface between the SDHB, SDHC, and SDHD subunits. It is there that many SDHIs bind to interfere with the catalytic activity of complex II [107]. Sequence alignments between sub-units of C. elegans and Meloidogyne spp. show that there is a high degree of similarity [108, 109]. It has been shown in in vitro studies that fluopyram binds to complex II of C. elegans with an inhibition constant (Ki) of 2.0 nM [110]. Additionally, it has been possible to generate in vitro, mutants resistant to fluopyram and other SDHI [100]. Although more expensive than other SDHIs such as flutolanil and solatenol (US$650/ha approx.), fluopyram is the only nematicide that is effective for both C. elegans and PPNs [103, 111]. Studies on mammalian SDHs show that fluopyram has little effect on the activity of these enzymes, suggesting that it is a compound that is highly selective for nematode SDHs. These findings explain the toxicological profile of fluopyram, which is considered, at moderate doses, safe for other life forms [110].

Compared to fluensulfone, fluopyram works fast-acting. At a concentration of 2.0 mg/L, 100% of the J2 of Meloidogyne incognita are immobilized after 24 h of exposure in-vitro. At the same concentration, 48 hours are required for fluensulfone to immobilize 100% of the nematodes [112]. The half maximal effective concentration (EC₅₀) required to immobilize J2s of M. incognita is 0.7 mg/L, which is comparatively lower than that achieved by fluensulfone (50 mg/L) [100, 94]. Despite its strong nematicidal activity against M. incognita, fluopyram is nematostatic against Heterodera schachtii at a concentration of 20 mg/L [113]. It has also shown good in-vitro activity against R. reniformis and Ditylenchus dipsaci with visible effects after 2 hours of exposure at a concentration of 5.0 mg/L and 3.0 mg/L, respectively [103, 114].

Unlike fluensulfone, abmectin, and aldicard, fluopyram has a poor ovicidal effect. At a concentration of 2.5 mg/L, they have a slight effect on the inhibition of the hatching of M. incognita eggs [104]. On the other hand, an 81% reduction in the inhibition of G. pallida egg hatching has been observed in vitro at a concentration of 5 mg/L. However, they are easily recovered after washing. At concentrations greater than 50 mg/L hatching inhibition is achieved without recovery [115]. In greenhouse and field trials on tomatoes, carrots, cotton (Gossypium hirsutum), potato, and soybean, fluopyram has been effective at managing PPNs [116, 117, 118].

4.3 Fluensulfone

Fluensulfone (5-chloro-2-(3,4,4-trifluorobut-3-enylsulfonyl)-1,3-thiazole) is the active ingredient of Nemitz®, a nematicide belonging to the group of fluroalkenyl sulfones, used for the control of PPNs in a wide variety of agricultural crops [119]. First registered in the USA in 2014 [120], it was developed by ADAMA and Control Solutions for Quali-Pro brand, in the past Makhteshim Agan Industries Ltd. It exists in three commercial formulations, one for turfgrass (Nimitz® Pro G) and two for horticulture: granules (Nimitz® 2% GR) and emulsion (Nimitz® and Nimitz® 480 EC).

Unlike OPs and CMs, fluensulfone is less toxic to humans and non-target organisms. It is moderately toxic after oral exposure, and has low acute toxicity following dermal application or inhalation in rabbits and rats [121]. The lethal dose via oral administration (LD₅₀) in rats is 671 mg/kg body weight, which is five hundred times safer than the old nematicides such as aldicard (1 mg/kg of body weight) [122]. Despite the above, it has been reported to be toxic to aquatic organisms. Thus, for example, the EC₅₀ for Daphnia magna is 0.35 mg/L after 48 h of exposure and 0.04 mg/L for Pseudokirchneriella subcapitata after 72 h of exposure [123]. In addition, fluensulfone is phytotoxic, thus it should be applied 7 days before planting [124]. Unlike soil fumigants, there is no need for a limiting buffer zone, as the re-entry time is essentially only 12 hours after application.

Although there are few studies that demonstrate the impact of fluensulfone on free-living nematodes, it is known to be less toxic to C. elegans than to M. incognita [125]. Unlike fostiozate, fluensulfone has been reported not to affect the biodiversity of free-living nematodes [126]. In tests on turfgrass soil, fluensulfone has been observed to have a lower impact on the density and biodiversity of free-living nematodes when compared to fluopyram and abamectin; however, it reduced the green grass cover [127].

The immobilization in the form of a straight rod generated by fluensulfone when applied to J2 differs from that produced by OPs, suggesting a different mechanism of action than AChE inhibitors [128]. However, the exact mechanism of action on PPNs remains unknown. Some hypotheses suggest that fluensulfone possibly inhibits the enzyme acyl-CoA dehydrogenase, which is key in mobilizing lipids to obtain energy [129]; however, others suggest that its mechanism of action may be related to that of fluopyram [100, 103]. In insects, the difluoroalkenyl derivatives were found to inhibit β-oxidation of fatty acids in the mitochondria [130].

Compared to OPs and fluopyran, fluensulfone produces a slower but irreversible paralysis. In vitro tests show that after removal with water of fluensulfone from J2 Meloidogyne hapla exposed to 1.0 mg/L for 24 h, 90% remained immobile [131]. In addition, J2 of M. incognita exposed for 17 h to 4.0 mg/L fluensulfone lost the ability to infect lettuce seedlings after rinsing with water [112]. This loss of infective capacity has also been reflected in the number of galls that form when plants are infected with nematodes that have had fluensulfone removed with water.

Like other nematicides, the susceptibility of PPNs to fluensulfone depends on the genus and species, even registering variations between the same species. In migrating nematodes such as Ditylenchus dipsaci, Bursaphelenchus xylophilus, and Aphelenchoides spp., it has been shown that they are more tolerant to fluensulfone than M. javanica [132]. While J2 of M. javinica is immobilized at a concentration of 0.25 mg/L for 48 h, in B. xylophilus and D. dipsaci a concentration higher than 16 mg/L is required for complete immobilization of these nematodes after 48 h of exposure [131, 132]. Among nematodes of the genus Meloidogyne, it has been seen that M. javanica is more tolerant than M. incognita and M. hapla. Thus, for example, the EC₅₀ for M. javanica after 48 h of exposure is 0.83 mg/L; while for M. incognita and M. hapla it is 0.12 and 0.41 mg/L, respectively [100, 128]. Although its ovicidal effect is better than that of fluopyram, it is limited, requiring concentrations greater than 50 mg/L for a 50% reduction in hatching of M. incognita eggs; however, its effect is irreversible [115].

Currently, fluensulfone is approved for the control of different PPNs species (Meloidogyne, Pratylenchus, Hoplolaimus, Globodera, etc.) in a wide variety of crops such as tomato, potato, pepper, cabbage, squash, and strawberry, among others [100, 133]. Depending on the number of nematodes (population density per gram of soil), the type of crop rotation, and the presence of resistant plants, the average application rate is between 1.9 and 3.3 kg/ha [100, 129]. There are different forms of application; however, the most recommended are drip injection or broadcast by mechanical incorporation with a single application 7 days before planting [129]. It has been estimated that its half-life in soil is 36 days, with a dissipation rate (DT₅₀) of between 20 and 50 days, which can vary depending on the type of soil and environmental conditions [134, 135].

4.4 Tioxazafen: seed nematicide

Registered in 2017 as a seed nematicide (NemaStrike™ ST), tioxazafen is a phenyloxadiazole (formally 3-phenyl-5-thiophen-2-yl-1,2,4-oxadiazole) developed by Monsanto Company, now Bayer CropScience, for the management and control of nematodes in cotton, corn, and soybean crops [136]. Unlike other nematicides, the development of tioxazafen involved the use of in silico approaches such as computational screening, scaffold hopping, and ligand-based model. After evaluating the nematicidal activity of different models of a series of stilbene, chalcone, and azobenzene compounds (SCA series), tioxazafen emerges as the best candidate for combining intrinsic efficacy, longevity in soil, and synthetic accessibility [137].

Although its mechanism of action is not known with certainty, it is hypothesized that its nematicidal activity is due to ribosomal disruption. Polymorphism studies on C. elegans mutants that are tolerant to high doses of tioxazafen have revealed that resistance is caused by a single nucleotide change in the gene coding for the L3 subunit of the mitochondrial ribosome [138]. On the other hand, in silico studies of the prediction of the target of small molecules through virtual screening, modeling, and molecular docking indicate that another possible mechanism of action of tioxazafen is through the inhibition of chaperone proteins [Arley, unpublished].

It is known through in vitro bioassays and X-ray crystallographic studies that benzisoxazoles and oxadiazoles are potent selective inhibitors of Hsp90 chaperone proteins [139, 140]. The Hsp90 protein is an essential chaperone in the folding of several proteins known as clients such as protein kinases, transcription factors, and hormone receptors, among others [141]. In nematodes such as Brugia pahangi, Brugia malayi, Schistosoma mansoni, and Ancylostoma caninum, Hsp90 is essential for survival inside the host [142, 143]. In C. elegans, Hsp90 is essential for larval development (L2-L4) and nematode longevity, since its chaperone activity stabilizes the nuclear transcription factor DAF-16/FOX, responsible for activating genes involved in longevity, lipogenesis, oxidative stress, glycolysis, innate immunity, and reproduction [144].

Incorporated as an active ingredient in fungicide-insecticide formulations (Acceleron®), tioxazafen has low water solubility (0.0125 mg/mL) and reduced mobility in soil with an octanol/water partition coefficient (Log P) of 3.26. These characteristics make tioxazafen a moderately lipophilic compound that, according to estimates, has an ambient half-life of more than 100 days under aerobic conditions [138]. According to in vitro and in vivo studies, tioxazafen and its derivatives (thiophenic acid, benzamidine, etc.) do not pose any risk to terrestrial invertebrates, amphibians, reptiles, pollinating insects, and aquatic plants. However, it has been shown that consumption of tioxazafen-treated seeds can be harmful to birds and some terrestrial vertebrates [145]. In tests using radiolabeled tioxazafen on soybeans, the chemical was shown to be dispersed mostly in the root area without penetrating the vascular tissues [137].

In studies in growth chambers and in the field, it has been possible to evaluate the larvicidal and ovicidal power of tioxazafen, as well as its ability to inhibit reproduction and colonization of the host plant. The results suggest that tioxazafen exerts a direct nematicidal action on PPNs at doses below those required for OPs and CMs (50 mg/L) [136, 138]. The EC₅₀ for M. incognita and R. reniformes has been estimated to be approximately 60 mg/L after 24 hours of exposure. No recovery of motility was observed after the removal of tioxazafen with water. In bioassays, its ovicidal power is reached at concentrations above 3.0 mg/L [136].

Trials in growth chambers and greenhouses show that tioxazafen is an excellent controller of several key nematodes when applied to seeds of soybean, corn, and cotton. In field trials with corn and soybean cultivars conducted by the USA EUP (Experimental Use Permit) in 40 locations that had nematode populations three times greater than those needed to cause economically significant damage, it was observed that tioxazafen increased the productive yield more efficiently than those observed for commercial use nematicides [137].

In Brazil, the nematicidal power of tioxazafen on Heterodera glycines, M. incognita, M. javanica, P. brachyurus, and P. zae has been reported in soybean, corn, and cotton crops [146]. According to the results, to achieve a control equal to or greater than that of the positive control (imidacloprid + thiodicarb), 0.250 mg per seed is required to control M. javanica and P. brachyurus; while for the control of H. glycines and M. incognita, 0.500 and 0.750 mg per seed is required, respectively. When compared to untreated plants, phytotoxicity experiments on house vegetation demonstrate that tioxazafen has no effect on plant growth or biomass [146]. In tomato plants, however, it has been discovered that its larvicidal effectiveness is dependent on the concentration and species of PPNs, with Pratylenchus and Rotylenchus spp. requiring a larger dose of the product [146]. Although the efficiency of tioxazafen has been widely demonstrated both in vitro and in field studies, since 2020 tioxazafen has been voluntarily withdrawn by the manufacturer; however, tioxazafen is easily accessible synthetically, which is why it continues to be used in research on the rational design of bioactive compounds [136, 147].

4.5 Fluazaindolizine: selective nematicide

Corteva Agriscience™, an agricultural division of the DowDuPont company (formerly DuPont Crop Protection), discovered and developed Salibro™, based on the new active substance fluazaindolizine (Reklemel™ Active). It is the first member of the new chemical class of N-phenylsulfonylimidazopyridine-2-carboxamide nematicides [148]. Fluazaindolizine, which was first registered with the EPA in July 2021, is a pesticide used to control PPNs on crops such as carrots, squash, tomatoes, eggplant, potatoes, and taro, as well as some fruits such as oranges, peaches, almonds, and grapes [149]. Their discovery began with high-throughput screening of an internal compound library against RKNs. After evaluating different 2,5-disubstituted arylsulfonamides, fluazaindolizine was selected due to its ecotoxicological and nematicidal profile [148]. It is suitable for a broad range of application techniques, including drip irrigation, bed spraying, and soil incorporation, among others [150].

In vitro studies on targets associated with old nematicides such as AChE, mitochondrial electron transporters, glutamate-regulated chloride channels (GluCl), and nicotinic receptors (nAChRs); show that fluazaindolizine has a mechanism of action different from that of other nematicides [148]. However, its exact mechanism on PPNs remains unknown. After five days of exposure at a concentration of 30 μM, no significant impact was observed on the motility and mortality of non-target organisms such as C. elegands, Drosophila melanogaster, and Diabrotica undecimpunctata. On the other hand, in silico studies of structure-activity relationship show that a possible mechanism of action of fluazaindolizine is through the inhibition of the enzyme pantoate synthase [Arley, unpublished]. The enzyme is responsible for the synthesis of pantothenate (vitamin B₅), which is the precursor of coenzyme A (CoA), which in turn is an essential cofactor for the synthesis and degradation of fatty acids, as well as for the TCA cycle [151]. In PPNs there is a strict requirement for this vitamin, thus, for example, it has been observed that there is a horizontal transfer of genes for the synthesis of B complex vitamins (B₁, B₅, B_6, and B₇) between H. glycines and bacteria and fungi [152, 153].

Under in vitro conditions, it has been observed that the sublethal concentration of fluazaindolizine is between 5 and 250 mg/L. At concentrations greater than 500 mg/L, an acute poisoning of Meloidogyne is observed, without recovery after treatment with water [100, 148]. This loss of motility and infective capacity after treatment with water is affected even at concentrations of 5 mg/L [154]. The LC₅₀ for RKNs is 177 mg/L at 72 h. The nematicidal effect of fluazaindolizine is both concentration and time-dependent. Additionally, it has been observed that there is a different susceptibility to fluazaindolizine between species and populations of the Meloidogyne spp., [155]. It does not present ovicidal activity at concentrations lower than 400 mg/L on Meloidogyne spp.; however, at concentrations greater than 500 mg/L, an 82% reduction in hatching of M. incognita eggs was observed after 3 days of exposure [156].

Greenhouse and field trials on tomato, strawberry, carrot, etc. plants show that fluazaindolizine is an effective nematicide for the treatment of M. incognita [157, 158]. Depending on the type of crop, application method, and soil type, the recommended application rate is between 0.25 and 2.0 kg/ha. In tomato fields infested by M. javanica, the application of 1.12 kg/ha of fluazaindolizine significantly reduced the number of galls for up to 2 months after planting. However, there was no change in yield or nematode population when compared to the control [159]. Similar results were obtained at concentrations lower than 1.0 kg/ha [100, 160]. Other studies indicate that at concentrations between 1.0 and 2.0 kg/ha fluazaindolizine does not reduce, after 30 days of application, the populations of Belonolaimus longicaudatus, P. penetrans, and M. hapla. On the other hand, the nematicidal power of flueazaindolizin e on other PPN species, as well as the influence of environmental factors on its activity, remains to be investigated.

Although the selective effect of fluazaindolizine on non-target organisms is known, there is still toxicological information on the safety of this new nematicide on human and environmental health. The LD₅₀ in rats via oral administration is 1187 mg/kg [100]. The main target organs in mammals are the urinary tract, hematopoietic tissue, and liver or gallbladder [161]. It is carcinogenic in high concentrations and has little impact on the reproductive system of rats and rabbits. Despite the possible toxicological effects of fluazaindolizine for mammals, it is known to be an easily photodegradable compound. The pH does not affect its degradation in soil; however, humic acid, iron (Fe-III), and nitrate (NO₃⁻) ions negatively affect the photodegradation of fluazaindolizine. It has been proposed that the possible mechanism of photodegradation of fluazaindolizine involves imidazole-ring, dechlorination, hydroxyl substitution, ring-opening, cleavage, oxidation, and decarboxylation [162, 163].

5. Conclusion

Because of their cosmopolitan nature and high reproductive success, PPNs are a problem for medium- and large-scale agriculture. Although the leading countries in high-precision agriculture have the technical and economic resources to deal with this problem, this is not the case in developing countries, where traditional pest control methods are insufficient to adequately manage PPNs. Understanding the molecular basis of the plant-nematode interaction is essential for proper control, as this will allow the development of more rational and specific methods for PPN control. Omics and in silico approaches based on the integrated analysis of genes and proteins involved in pathogenesis activities (invasion, feeding, colonization, etc) are key factors for the development of new, more selective, and cost-effective nematicides. Finally, organic and ecologically friendly agriculture requires the integration of new technologies with traditional control and management methods in order to reduce the impact on non-target organisms.

Acknowledgments

The author Acknowledgments Chemaxon Ltd. for the license granted for the use of the MarvinSketch software (v.22.18) with which the chemical structures of new nematicides were designed.

Conflict of interest

The author declares no conflict of interest.