Direct nematicidal effects of methyl jasmonate and acibenzolar-S-methyl against Meloidogyne incognita

Abstract The aim of this study was to examine the nematicidal properties of two defence inducers against the root-knot nematode Meloidogyne incognita. A direct-contact bioassay was applied to evaluate the nematicidal effects of acibenzolar-S-methyl (ASM) and methyl jasmonate (MEJA) on second-stage juveniles (J2). Nematodes were incubated in different concentrations of these compounds, and the numbers of immobile nematodes were counted after 24 and 48 h post incubation. Tap water was then added to verify whether the nematodes recovered or remained dead at 72 h. The percentage of dead nematodes was used as indicator for the toxicity of the different solutions. Our results show that ASM, in the formulation of Bion®, and MEJA have nematicidal properties.


Introduction
Root-knot nematodes (RKN; Meloidogyne spp.) are pathogenic sedentary endoparasites of the plant root system. They occur worldwide and their host range includes many important agricultural crops on which they can cause major damage and yield losses ; Bartlem et al. 2014). Induction of the host plant's resistance is an option that is being investigated as an alternative strategy to the use of nematicides for the management of plant-parasitic nematodes. Various agents can induce plant's resistance to subsequent pathogen attack upon treatment (Walters et al. 2013). One such resistance inducer is the synthetic molecule acibenzolar-S-methyl (ASM), a derivative of salicylic acid. ASM is marketed under the trade name Bion® in Europe (Syngenta Ltd, Basel, Switzerland) and has been shown to be a successful activator of the plant's defence system, enabling a reduction in RKN infection (Molinari 2016). The plant hormone methyl jasmonate (MEJA) can also elicit the plant's defence system against RKN (Fujimoto et al. 2011). Defence elicitors can be applied by foliar sprays, but also soil drenching, root dipping and seed treatment (Pankaj et al. 2013;Molinari 2016) and thus might be able to exert a direct nematicidal effect against plant-parasitic nematodes (Ntalli & Caboni 2012;Mukhtar et al. 2013;Suryawanshi et al. 2014). The objective of this study was therefore to investigate the direct nematicidal effect of these two elicitors on Meloidogyne incognita which is considered one of the most important plant-parasitic nematodes (Moens et al. 2009).

Results and discussion
Compared to tap water, incubation in demineralised water did not have a significant effect on the mortality of J2 (data not shown). For both tap and demineralised water, mortality percentages ranged between 2 and 18%. The mortality percentages of J2 in solutions of 1-16 mM of acetic acid (positive control; Wuyts et al. 2006) were significantly (p ≤ 0.05) higher compared to tap water controls (100% at all time intervals; data not shown).
Addition of ASM (in the formulation of Bion®) had a significant (p ≤ 0.05) nematicidal effect on the J2 in all three experiments compared to control treatments: nematicidal concentrations ranged from 0.625 mM (80% at 24 h and 100% at 48 h) up to 2.5 mM, the highest tested concentration (80-94% at 24 h and 91-100% at 48 h; Figure 1(A)). LC50 was determined at 0.78 mM. Molinari (2016) also observed a nematicidal effect at 0.5 mM ASM (30% mortality after 24 h incubation). On the other hand, Owen et al. (2002) found no effect of incubation in ASM on the mortality percentage of a mixture of M. incognita, Meloidogyne javanica and Meloidogyne arenaria. However, the authors used only a maximum concentration of 50 μg/mL ASM (0.24 mM), which also did not have an effect in our experiment. Chinnasri et al. (2003) found that incubation with 200 mg/L ASM, corresponding to 0.9 mM, did not affect either Rotylenchus reniformis or J2 of M. javanica, not even after 120 h of incubation. This result is not in accordance with our results where the dilution closest to 0.9 mM (0.625 mM) had nematicidal effects on the J2 of M. incognita.
Exposure to MEJA had a significant (p ≤ 0.05) effect on the nematodes. In the experiments, 1 and 2 mM showed a significant (p ≤ 0.05) effect on the mortality percentage at 48 h but not at 24 h. Mortality percentages at 48 h ranged from 13 to 18% for 1 mM and 31-34% for 2 mM (Figure 1(B)). LC50 was estimated to be 3 mM, which was a higher concentration then we tested. This LC50 is somewhat higher than observed by Gavin et al. (2013). These authors studied the nematicidal effect of MEJA on Pratylenchus zeae and Helicotylenchus spp. Concentrations of 3.125-50 ppm (0.1375-2.2 mM) MEJA were used. Maximum P. zeae mortality percentages observed were 48% (25 ppm; 1.1 mM) and 60% (50 ppm; 2.2 mM) after 12 and 24 h incubation, respectively. The maximum Helicotylenchus spp. mortality percentage was 66% after 12 h incubation and 71% after 24 h incubation in 12.5 ppm MEJA (0.55 mM). In all cases, non-motile J2 did not recover their motility for at least 12 h by rinsing in water after exposure to MEJA (Gavin et al. 2013). Similar to our results, no recovery was observed either.

Conclusion
The objective of this study was to investigate the direct nematicidal effect of two defence elicitors on M. incognita. So far to our knowledge, this is the first report of nematicidal effects of MEJA on M. incognita. Our results of ASM confirm earlier work and can be used further to investigate a new way to a new nematode control practice. Interestingly, both ASM and MEJA have been previously observed to be able to induce plant resistance against nematodes at the nematicidal concentrations (Owen et al. 2002;Fujimoto et al. 2011;Molinari 2016). In future experiments, nematicidal effects of resistance inducers must be taken into account when developing experiments with the objective to study induced (systemic) resistance against plant-parasitic nematodes.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) to N. Schouteden. Figure 1. effect of several dilutions of Bion® (asM; a) and methyl jasmonate (MeJa; B) on the mortality percentages of second-stage juveniles (J2) of Meloidogyne incognita at 24 h (light grey), 48 h (medium grey) after incubation and on recovery (dark grey) compared to the control treatments with tap water and acetic acid (4 mM). error bars represent standard deviation. an aNoVa was carried out on arcsin (x/100) transformed data. different letters indicate a significant difference (p ≤ 0.05) of treatment according to the tukey t-test.