New reduced-risk agricultural nematicides - rationale and review

Table 1.

The lack of nematicide research by industry from the 1960s to the last decade is in part due to the cryptic nature of nematodes, and the difficulty of recognizing and assessing impacts on crop yield, which often leads to an underestimation of the damage that they can cause. In addition, and probably the main reason for the research gap, is that the nematicide market is very small when compared with the herbicide, fungicide, and insecticide markets (Kang et al., 2016, Fig. 1). The entire history of nematicides in fact is one of accidental discoveries, as all of them were initially discovered not as nematicides, but rather as sterilants or fumigants (methyl bromide, 1,3-dichloropropene, metam), insecticides (oxamyl, ethoprop, and other organophosphates and carbamates), fungicides (fluopyram), or animal health drugs (abamectin).

Figure 1:

From discovery to product – what does it take?

Once the initial nematicidal activity has been found, the focus is to improve this activity. At the same time, other activities need to be done, such as studies on toxicology and environmental impact, mode-of-action, soil behavior, formulations, patent situation, cost of manufacturing, and use rates and potential uses. The early discovery research is always confidential, and it is typically not until a few years before registration, that the research is made public. Figure 2 outlines a nematicide discovery process in industry, starting from idea generation, to the discovery process, and up to the commercial development phase. The entire process is highly integrated and requires a wide range of experts working together. It is estimated that only one in 140,000 active ingredients discovered today will pass the rigorous testing requirements to become a registered pest management product (Sparks, 2013).

Figure 2:

Majority of research done on new nematicides is focused on their risk assessment on the environment and human health, that include potential impacts on wildlife, fish, plants, and other non-target organisms. Safety to non-target organisms is becoming increasingly important, and safety studies include organisms such as collembola, soil and predatory mites, honeybees, spiders, and water fleas. For soil-applied nematicides, the product’s impact on the soil environment is an important source of concern, and many regulatory requirements are put in place to address this. Pesticide registration nowadays is a very complex, highly regulated, and involved process from start to finish.

The cost of bringing a new chemical active ingredient to market is increasing every year, and is now estimated to be more than US$250 million, about 10-fold what it was in the 1960s (Sparks, 2013). Similarly, the average time from product discovery to market launch has increased and is now >10 years. This trend will probably continue, making it increasingly harder for smaller firms to bring new products to the market, as they simply cannot afford to invest the time and money, much less deal with the substantial amount of regulatory documentation.

Chemical name	Chemical structure	Water solubility	Soil 1/2 life	Mode-of-action	Signal words
Fumigant (1,3-D)		Gas	Short < 14 d	unknown	Danger
Carbamate (oxamyl)		240,000 ppm	Short 7 d	AChEa	Danger
Fluensulfone		545 ppm	Short 7-17 d	Beta oxidation inhibitor	Caution
Fluopyram		10 ppm	Long > 200 d	SDHIb	Caution
Fluazaindolizine		2000 ppm	Medium 30 d	unknown	TBD
Spirotetramat		30 ppm	Short (< 1 d)	ACCc inhibitor	Warning
Tioxazafen		1.24 PPM	Long (48-303 d)	Disrupts ribosomal activity	Caution

Tioxazafen (Nemastrike^®, Monsanto/Bayer) was originally discovered by Divergence, which was acquired by Monsanto in 2011, which in turn was acquired by Bayer in 2018 (Table 3). The compound is a new systemic nematicide belonging to the chemical class oxadiazoles (Slomczynska et al., 2015). Its mode-of-action may be linked to disruption of ribosomal activity of nematodes. Few data have been made available on this compound, which is registered as a seed treatment for corn, cotton or soybean only. At the time of writing, tioxazafen was no longer offered for use in 2020, citing safety concerns.

Spirotetramat (Movento^®, Monsanto/Bayer) is a spirocyclic tetramic acid derivative and was first marketed as a fully systemic insecticide (Nauen et al., 2008; Table 2). Spirotetramat has unique translocation properties in that it is translocated within the entire vascular system (upwards and downwards through the xylem and phloem, respectively). To be active against nematodes, the compound needs to be hydrolyzed in the plant to the active spirotetramat-enol form (Vang et al., 2016). In C. elegans, spirotetramat-enol leads to arrested larval development and disruption of the life cycle (Vang et al., 2016; Gutbrod et al., 2018). The nematicide also inhibits acetyl-CoA carboxylase activity, storage of lipids, fatty acid composition, and disruption of surface coat synthesis in C. elegans (Gutbrod et al., 2018). Silencing of acetyl-CoA carboxylase in Heterodera schachtii by RNAi mimicked the effects of spitrotetramat-enol, indicating that the mode-of-action is inhibition of acetyl-CoA carboxylase (Gutbrod et al., 2018). Concentrations of spirotetramat-enol necessary to result in 95% of a C. elegans population having arrested development were 44 to 48 ppm (Vang et al., 2016). There is some evidence that spirotetramat-enol needs to be ingested by plant-parasitic nematodes to elicit an effect. Meloidogyne incognita and Mesocriconema xenoplax were exposed to 0.017 and 0.026 kg/ha spirotetramat in vitro, which reduced mobility of M. xenoplax but not of M. incognita (Shirley et al., 2019). In another study, egg hatch of Rotylenchuls reniformis was not inhibited after exposure to spirotetramat (Waisen et al., 2019).

Fluensulfone, fluopyram, and fluazindolizine are new nematicides that all have a trifluoro (3-F) group in their molecular structures and are hereby referred to as 3-F nematicides. These nematicides have a much safer toxicity profile than the older nematicides (fumigants, organophosphates, carbamates) (Table 2). However, they are quite different in terms of their chemical and physical properties, and their modes-of-action.

Fluensulfone (Nimitz^®, Adama) was the first of the new chemical nematicides and was first registered in the US in 2014 (EPA, 2014). Fluensulfone is a member of the class of 1,3-thiazoles and was originally discovered by Nihon Bayer in 2001. This compound is a specific nematicide and was the first of its kind to become registered. Fluensulfone has a soil half-life up to 36 days (Ludlow, 2015b, Table 2). Under field conditions, fluensulfone had a 50% dissipation rate (DT₅₀) between 23.7 and 24.3 days depending on soil type, similar to that of fosthiazate (Norshie et al., 2017). When treated with similar doses of fluensulfone, Caenorhabditis elegans and M. incognita respond with immobility and eventual death (Kearn et al., 2014). This included C. elegans mutants resistant to organophosphates and carbamates, indicating that cholinesterase inhibition is not the target for this compound. The unpublished mode-of-action of fluensulfone is that it is a fatty acid beta oxidation inhibitor. More recently, Kearn et al. (2017) have found that in G. pallida second-stage juveniles (J2) exposed to fluensulfone exposure led to increased lipid content, loss of cell viability, and tissue degeneration symptoms not seen in adult and dauer C. elegans (Kearn et al., 2017). Additionally, exposed Globodera pallida J2 had reduced stylet thrusting and reduced mobility along with coiling posture; the rate of these symptoms correlated strongly with the concentration of fluensulfone. The higher the dosage the faster symptoms appeared; at 146 ppm of fluensulfone G. pallida J2 experienced the coiling posture after just 30 min.

Several in vitro studies have been conducted to determine the effects of fluensulfone on Meloidogyne species. Doses as low as 25 ppm impacted M. incognita J2 activity after 24 hr exposure, and egg hatch was reduced at 95 ppm (Moreira and Desaeger, 2019; Wram and Zasada, 2019). Meloidogyne species and populations within a species varied in their response to fluensulfone (Oka and Saroya, 2019). Meloidogyne incognita was more sensitive to fluensulfone than M. javanica. The fluensulfone 17 hr LC₅₀ was ten-fold (0.48 vs 0.12 mg/L) different between two M. incognita populations. Exposing M. javanica J2 to fluensulfone at sublethal concentrations for 17 hr was able to reduce the number of J2 attracted to lettuce root tips in pluronic agar and those that invaded produced smaller galls (Oka and Saroya, 2019). Migratory plant-parasitic nematodes showed different responses in vitro (Oka, 2014). Bursaphelenchus xylophilus and Ditylenchus dipsaci were not impacted by exposure to fluensulfone at high concentrations (16 mg/L) after 48 hr, unlike Aphelenchoides besseyi and Aphelenchoides fragariae which had > 50% immobility after 48 hr of exposure. More than 60% of Pratylenchus penetrans and P. thornei were immobilized after exposure to fluensulfone of 4 mg/L and stayed immobilized even after removal of the compound (Oka, 2014). Xiphinema index was also impacted irreversibly by exposure to fluensulfone at doses as low as 1 mg/L with 60% immobility after 48 hr of exposure and a 24 hr rinse (Oka, 2014).

Fluopyram (Verango^®, Velum^®, Indemnify®, Bayer CropScience) was initially discovered and patented by Bayer as a fungicide (WIPO, 2005), and was first registered as a fungicide in 2012 under the trade name Luna (EPA, 2012). The nematicidal activity of fluopyram was not known or disclosed until 2008, when Nihon Nohyaku CO filed a nematicide use patent for fluopyram. The first nematicide registration was in Honduras (Agrow, 2013) on banana under the name Verango, and later in South Africa (AgroNews, 2015) and US (EPA, 2015) under the name Velum. Fluopyram is a member of the class of benzamides, and a Fungicide Resistance Action Committee code 7 fungicide. It is an inhibitor of the enzyme succinate dehydrogenase in fungi (Veloukas and Karaoglanidis, 2012). Heiken (2017) confirmed this to be the likely mode-of-action in nematodes, demonstrating that succinate dehydrogenase knockdown mutants of C. elegans had a roughly 2.6-fold increase in sensitivity to fluopyram. Unlike the other 3-F nematicides, fluopyram has a very long soil half-life, up to 746 days depending on soil type (Ludlow 2015a, Table 2).

Fluopyram is a very fast-acting and potent nematicide, with M. incognita J2 exposed to fluopyram at 5.58 ppm for 2 hr showing reduced mobility by 80%, and the 24 hr ED₅₀ for M. incogntia being as low as 1 ppm (Faske and Hurd, 2015; Wram and Zasada, 2019). Fluopyram also showed good activity against Rotylenchulus reniformis, with the 2-hr EC₅₀ roughly 2x that for M. incognita (Faske and Hurd, 2015). However, fluopyram also showed to be nematistatic in in vitro assays, with a 58% recovery of mobility of M. incognita J2 after removal of the nematicide. Although nematistatic, in that same study, exposure to fluopyram for 1 hr to 1.3 and 3.3 ug/ml for M. incognita and R. reniformis significantly reduced reproduction of nematodes on tomato. Wram and Zasada (2019) also demonstrated that fluopyram is a poor ovicide, with only a slight reduction in M. incognita egg hatch at 2.5 ppm.

Fluazaindolizine (Salibro^®, DuPont/Corteva) is the latest of the new chemical nematicides and is expected to be registered in 2021 (Lahm et al., 2017). Like fluensulfone, fluazaindolizine specifically targets nematodes, and no other (fungicidal or insecticidal) activity has been reported. Fluazaindolizine is a member of the class of carboxamides. Its mode-of-action is unknown, however it is distinct from that of carbamates/organophosphates, or any other known nematicides (Lahm et al., 2017). Fluazaindolizine was unable to inhibit motility and mortality of C. elegans adults and Drosophila melanogaster egg and adult stages at concentrations of 200 and 300 ppm, respectively (Lahm et al., 2017). However, M. incognita J2 exposed to 5 to 50 ppm of fluazaindolizine were increasingly immobile and eventually dead 24 to 96 hr after exposure (Lahm et al., 2017).

Fluazaindolizine has irreversible effects on M. incognita even after 24 hr of exposure (Thoden and Wiles, 2019; Wram and Zasada, 2019). However, based on nematode motility, a 24-hr EC₅₀ for M. incognita J2 for this compound is over 2× that of fluensulfone and over 200× that of fluopyram (Wram and Zasada, 2019), making this a slower-acting nematicide. In a soil environment, exposure to fluazaindolizine reduced the ability of M. hapla and M. incognita to move through sand at concentrations of 50 ppm (Thoden and Wiles, 2019). Fluazaindolizine was also a poor ovicide, with reductions in M. incognita egg hatch only at concentrations over 250 ppm for 7 days of exposure (Thoden and Wiles, 2019; Wram and Zasada, 2019). When effects of this compound on free-living nematodes were considered, there was no reduction in Acrobeloides buetschlii egg hatch over 5 days with exposure to fluazaindolizine at concentrations as high as 250 ppm (Thoden and Wiles, 2019). There was also no effect on A. buetschlii motility at concentrations as high as 250 ppm after 144 hr of exposure.

Similar to fluensulfone, there can be some variation in how populations of the same plant-parasitic nematode species respond to fluazaindolizine. Five populations of M. incognita and five populations of M. javanica were examined after exposure to fluazaindolizine for their effects on J2 mobility and motility (Thoden et al., 2019). After 24 hr of exposure to fluazaindolizine at 50 ppm the percentage of affected Meloidogyne J2 in all populations tested ranged from 42 to 86%. When Meloidogyne J2 were pre-exposed for 24 hr at 50 ppm, the ability to move through a sand layer varied from 53 to 100% across populations. In general, M. javanica mobility and motility was slightly less impacted by exposure to fluazaindolizine compared with M. incognita.

The 3-F nematicides (fluensulfone, fluopyram, fluazaindolizine) have been evaluated on a variety of crops for their ability to suppress a diversity of plant-parasitic nematodes (Table 4). However, most have focused on their efficacy against Meloidogyne species in vegetable crops. Several greenhouse studies evaluated the efficacy of these compounds on controlling M. incognita on susceptible tomato cultivars at concentrations ranging from labeled rates to 24-hr sublethal pre-exposure doses. At sublethal doses fluazaindolizine and fluensulfone suppressed M. incognita reproduction more than fluopyram, although actual sublethal doses were much lower for the latter (Thoden and Wiles, 2019; Wram and Zasada, 2019). In another study, half and full labeled rates of any of these compounds did not suppress reproduction of M. incognita (Silva et al., 2019).

As stated above, it appears that fluopyram may be a poor ovicide. Fluopyram applied at the labeled rate (249 g a.i./ha) to the soil surface of tomato plants two days post inoculation with M. incognita eggs had no impact on nematode reproduction when compared to the untreated control (Heiken, 2017). However, fluopyram applied at the labeled rate two weeks after tomatoes were inoculated with M. incognita eggs, reduced the number of eggs per gram of root by 91% compared with the untreated control, demonstrating that application timing is important.

Both fluopyram and fluensulfone were effective nematicides against M. incognita in lima bean in both greenhouse and microplot studies (Jones et al., 2017). With similar control to oxamyl, fluopyram at a rate of 0.22 L a.i./ha reduced galling 55 and 64%. Fluensulfone was the most effective against M. incognita in this study, with > 81% reduction in galling on lima beans when applied at 2.34 L a.i./ha.

The impact of initial nematode density on efficacy of the 3-F compounds has also been explored (Hajihassani et al., 2019). In this microplot study, initial population densities of M. incognita ranged from 1,000 to 20,000 J2/microplot. Fluensulfone and fluazaindolizine had the lowest final soil population levels compared with oxamyl and fluopyram. At low inoculation densities there was no difference between the nematicide in reducing root galls, but at high nematode densities fluopyram, fluazaindolizine, and fluensulfone had greater gall reduction compared to oxamyl.

Becker et al. (2019) found contradictory results when examining the effects of these compounds on carrot production over multiple years. Fluensulfone and fluopyram were unable to consistently lower final population densities of M. incognita, unlike fluazaindolizine. Fluopyram was also less consistent in reducing harvest galling and only protected the top 1/3 of the carrot tap root, unlike fluensulfone and fluazaindolizine. In cucumber, drip applied fluensulfone at a rate of 3.0 kg a.i./ha was able to reduce M. incognita J2 population densities by 73% along with a reduction in root galling (Morris et al., 2016).

Desaeger and Watson (2019) also found that the use of these compounds could help prevent rapid re-infestation of roots. When field grown tomatoes were treated with drip applied non-fumigant nematicides, fluensulfone had the most consistent suppression of M. javanica compared to treatments of fluazaindolizine, fluopyram, and treatment combinations of oxamyl and fluopyram, oxamyl and fluazaindolizine, and fluensulfone and oxamyl. In chloropicrin treated soils there was an increase in nematode population at the end of the growing season, unlike in non-fumigant nematicide treated soils that had consistently lower levels of M. javanica.

When evaluating these compounds for control of nematodes outside of Meloidogyne species, there have been mixed results. Watson and Desaeger (2019) conducted a field study with Belonolaimus longicaudatus, Meloidogyne hapla, Pratylenchus penetrans and found that of the non-fumigant nematicides tested (fluopyram, fluensulfone, fluazaindolizine) fluopyram was the only nematicide to reduce B. longicaudatus and improve strawberry yield. Fluopyram was able to somewhat reduce P. penetrans in soil and roots compared with the control and metam-potassium, but this effect was not significant. Neither fluensulfone or fluazaindolizine were able to suppress B. longicaudatus, M. hapla, P. penetrans or increase vigor or yield of strawberry.

In a greenhouse study, Oka (2019) also evaluated the effects of fluensulfone exposure pre- and post-plant on Xiphinema index and Longidorus vineacola in fig and pepper. When soil infested with X. index was treated with 5 mg/L of fluensulfone 1 week prior to introducing a fig transplant, it reduced the number of X. index recovered with no difference observed with rates of fluensulfone up to 20 mg/L. Longidorus vineacola was more sensitive to fluensulfone, with 2 mg/L fluensulfone applied one-week prior to transplant showing to suppress this nematode (Oka, 2019). Treatments that were applied one-week post-plant were less effective for both nematodes with >2× pre-plant effective doses needed to reduce the number of X. index and L. vineacola recovered (Oka, 2019). In a greenhouse assay, fluensulfone applied at 2 mg/L reduced population densities of P. penetrans and P. thornei growing on lettuce and chickpea, respectively (Oka, 2014).

Several studies have evaluated the effects of fluensulfone on plant-parasitic nematodes infecting tuber crops like potato and sweet potato (Table 4). A full rate of fluensulfone in liquid and granular form resulted in comparable suppression of G. pallida to that by fosthiazate, but was not as effective as oxamyl (Norshie et al., 2016). Fluensulfone suppressed B. longicaudatus, Pratylenchus sp. and Paratrichodorus sp. on potato over three years (Grabau et al., 2019). Fluensuflone at all rates tested (2.92, 4.11, 5.87, 8.20 L/ha) consistently reduced densities of B. longicaudatus at harvest and was comparable to treatment with Telone II (1,3-dichloropropene at 61 L/ha). However, fluensulfone did not consistently suppress Pratylenchus sp. or Paratrichodorus sp. across years. In the same study (Grabau et al., 2019), potato yield was consistently higher in fluensulfone treated plots, especially with lower rates of fluensulfone. However, both Telone II and fluensulfone resulted in end of harvest final nematode population densities that were greater than initial densities of nematodes. Ploeg et al. (2019) explored the effects of fluensulfone and application timing of fluensulfone on suppression of M. incognita on sweet potato. Fluensulfone was applied at 3.36 kg/ha 2 to 7 days pre-plant, followed by 2 post-plant applications of 1.68 kg/ha at 26 and 58 days. Marketable yield was increased for both treatments of fluensulfone, but similarly to results from potato (Grabau et al., 2019), final soil population densities of M. incognita were 8 to 13 times higher than initial population densities. However, Ploeg et al. (2019) found that both fluensulfone treatments were able to reduce M. incognita eggs/g of sweet potato root by 80%, which could contribute to the increased marketable yield.

As indicated, the bulk of efficacy data of 3-F nematicides is on Meloidogyne spp., and all products seem to be quite effective against these nematodes (Oka et al., 2012; Morris et al., 2016; Heiken, 2017; Jones et al., 2017; Desaeger and Watson, 2019) (Table 4). Much less data is available on other plant-parasitic nematodes, but as stated above, indications are that several nematodes such as Pratylenchus spp. and Belonolaimus longicaudatus may be more tolerant to fluazaindolizine, and a lesser extent fluensulfone (Grabau et al., 2019; Watson and Desaeger, 2019). Although fluopyram overall shows more broad-spectrum activity (Moreira and Desaeger, 2019), it does not seem to affect lance nematodes (Hoplolaimus spp.) in turf (Crow, 2017). It is not clear why certain plant-parasitic nematodes might be less sensitive, but possibly differences in cuticle permeability, or detoxification mechanisms inside the nematode are involved. This may also explain their reported relative inactivity against non-plant-parasitic nematodes, including entomopathogenic nematodes (Moreira and Desaeger, 2019; Thoden and Wiles, 2019). Fluopyram, as stated earlier, seems to have more broad-spectrum activity, including against non-plant-parasitic nematodes (Moreira and Desaeger, 2019; Waldo et al., 2019). More long-term field studies are needed to verify this, but the relative inactivity towards beneficial nematodes would certainly be a good thing.

Differences in nematicide efficacy across trials may be due to the physical properties of the new products, especially their solubility in water and half-life in soil (Table 3). Water solubility is important in that it will determine how well the molecule moves through the soil profile. Higher water solubility will give better soil coverage and distribution of the active but will also increase the risk of leaching. Soil half-life determines how long the molecule stays active in the soil. Longer soil half-life will give longer soil residual activity and more extended nematode control, but also increases the risk of soil accumulation. Fluensulfone and fluazaindolizine are relatively similar in terms of solubility and soil half-life, but fluopyram is quite different, having low water solubility, and a much longer soil half-life (Table 3). Overall, all the 3-F nematicides have much lower water solubility, but longer soil half-lives than oxamyl. A lack of uniform soil distribution, combined with the typically patchy field distribution of nematodes, could explain some of the variability observed in the above field trials. Also, the lack of standardization in terms of sampling procedure, sampling time, and extraction method is another source of variability. Like in any other discipline, a keen understanding of the pest, i.e. nematode biology and plant-nematode interactions, is critical to understand, interpret, and validate the inherent variability of nematode field trial data. It should come as no surprise that a background specific to applied nematology is now a discipline in high demand, but also in short supply. Fortunately, both industry and universities have started to notice this gap in expertise, and many have started hiring applied nematologists again.