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INTRODUCTION
Soil health is the capacity of soil to maintain important ecosystem functions, such as sustaining living organisms (i.e., plants, animals, and humans). In general, free-living nematodes contribute for soil health as important key species in nutrient cycles and in the multitrophic interactions with other soil microbes. However, the presence of plant-parasitic nematodes (PPN), may cause detrimental effects on plant hosts and other soil microorganisms, disrupting the equilibrium of soil ecosystem services (Brussaard, 1997).
The root-lesion nematodes (RLN) from genus Pratylenchus Filipjev, 1936 (Nematoda: Pratylenchidae) are migratory PPN that attack a wide variety of important food and fed crops (e.g., potato, carrot, soybean, maize, alfafa) causing severe economic constraints in the affected cultures (Jones et al., 2013). Plants infected by the RLNs present a stunted and necrotic root system (as result of RLN feeding habits) with poor development in aboveground plant parts. Pratylenchus penetrans is one of the most devastating RLN being reported in more than 400 plant hosts worldwide (Vicente et al., 2021). In Europe, P. penetrans has been detected in potato cultures in Portugal (Esteves et al., 2015) and in association with olive trees in Spain, Italy, and Turkey (Ali et al., 2014).
With the continuous withdrawal of chemical nematicides, such as Aldicarb, Carbofuran and 1,3-Dichloropropene, the current PPN control measures are far from being effective and sustainable. Plant-derived compounds, mostly plant secondary metabolites (e.g., alkaloids, essential oils components, phenolic compounds) are safer to humans and environment and have already been applied against important PPN pests (Barbosa et al., 2012; Faria et al., 2021). Within the framework of the national project PratyOmics - Plant metabolomics for the control of the root-lesion nematode Pratylenchus penetrans (PTDC/ASP-PLA/0197/2020), the present work aims to evaluate the nematicidal activity of seven plant-derived compounds in direct contact bioassays against P. penetrans.
MATERIAL AND METHODS
Nematode Culture
Pratylenchus penetrans A44L4 was obtained in portuguese potato fields in 2010 by NematoLab team (Centre for Functional Ecology, University of Coimbra) (Esteves, et al., 2015). Nematodes are routinely multiplied in carrot disks according to Boisseau & Sarah (2008) and, when needed, extracted for 24 h in distilled water containing 50 μg/mL carbenecillin and kanamycin. Following, a nematode suspension was prepared with approximately 50-75 mixed-stage RLN per 100 μL distilled water.
Nematicidal Activity
Tested compounds (Table 1) were diluted in acetone (purity 99.8%, Carl Roth GmbH + Co. KG) to 2 mg/mL and maintained at -20ºC until use. Bioassays were conduted in a 96-well microtiter plate. For each compound, 1 μL of the solution was added to a mixed-stage nematode suspension (99 μL). Plates were covered with their lid and maintained at 25°C in the dark. After 24 h, dead and live nematodes were counted under a binocular microscope Olympus SZX-12 (Olympus Corporation, Tokyo, Japan). Nematodes were considered dead if they failed to respond to the gentle physical prodding with a needle. Two independent biological trials were performed with five replicates at similar conditions. Negative control was performed with acetone (purity 99.8%, Carl Roth GmbH + Co. KG.).
Data Analysis
Corrected mortality values (Mc) were obtained following the Schneider-Orelli formula (Puntener, 1981): MC = MT - M0 / 100 - M0, in which M0 is the mortality in the control, and MT is the mortality in treatments.
RESULTS AND DISCUSSION
Mean corrected mortality values obtained for control treatments, using acetone, was 2.2%. For the seven phenolic compounds tested((+)-catechin, caffeic acid, coumaric acid, ferulic acid, gallic acid, gentisic acid and quercetin), P. penetrans mortality was relatively low, after 24 h of direct contact (Table 1). The highest Mc value was 5.8% recorded for gentisic acid, while the lowest Mc (1.0%) was obtained for gallic acid. The monoterpene carvacrol, reached full mortality (Mc=100%).
Table 1 Nematicidal activity of commercial pure compounds (2 mg/mL in acetone) against Pratylenchus penetrans exposed for 24 h
| Compound | Grade Supplier | Class | Corrected mortality (Mc) |
|---|---|---|---|
| (+)-Catechin | 98% - Aldrich | I | 1.98 ± 1.31 |
| Caffeic acid | 98% HPLC - Fluka | I | 4.64 ± 1.54 |
| Coumaric acid | 98% HPLC - Fluka | I | 1.88 ± 0.81 |
| Ferulic acid | for research only - Extrasynthese | I | 1.99 ± 0.84 |
| Gallic acid | for research only - Extrasynthese | I | 1.00 ± 1.02 |
| Gentisic acid | for research only - Extrasynthese | I | 5.83 ± 2.53 |
| Quercetin | 95% - Sigma-Aldrich | I | 1.04 ± 0.87 |
| Carvacrol | 98% - Sigma-Aldrich | II | 100.00 ± 0.00 |
Class: I, Phenolic compounds; II, Monoterpene
The phenolic compounds showed little or no nematicidal effect in P. penetrans, and only caffeic acid and gentisic acid could induce a higher Mc than the negative control (acetone). Only the monoterpene cravacol showed a high activity against this RLN.
Previous studies (Nguyen et al., 2013, d'Errico et al., 2018) showed that the efficiency of gallic acid, extracted from Terminalia nigrovenulosa, and SaviotaN (extracted from chestnut) against Meloidogyne incognita. However, in P. penetrans, its activity is reduced. Wuyts et al. (2006) showed that coumaric acid, ferulic acid, caffeic acid and quercetin displayed no activity (i.e., motility) in this nematode. According to our results, the seven phenolic compounds had little or no effect to P. penetrans, when tested individually, supporting the results observed by Wuyts et al. (2006). So far, this is the first work where the nematicidal activity of catechin, caffeic acid, gallic acid and gentisic acid is assessed against P. penetrans. Several monoterpenes have been already tested in P. penetrans. Rong and Qing (2000) showed that citronellol, carvacrol and (-)-perillaldehyde are potential nematicides against this RLN.
Plant secondary metabolites such as terpenoids and phenolics, have different mode of actions in nematodes (e.g., attract or repel, stimulate or inhibit egg-hatching, or exhibit nematicidal properties) (Ntalli et al., 2020). Beside these direct effects, and in the context of soil health and ecological equilibrium, these compounds can interact with multiple target sites, and therefore reduce the likehood of developing pest resistance (Chitwood et al., 2002).
CONCLUSIONS
Plant-based compounds with nematicidal effect against PPN might be good alternatives to synthetic nematicides and are less harmful for soil health. However, it is important to conduct previous rigorous tests, since not all compounds are effective. In PratyOmics, we will employ plant metabolomics on resistant/susceptible potato cultivars of P. penetrans to discover new bioactive compounds with antagonistic effect against this RLN.
