Chemical Characterization of Plant Extracts and Evaluation of their Nematicidal and Phytotoxic Potential

Nacobbus aberrans ranks among the “top ten” plant-parasitic nematodes of phytosanitary importance. It causes significant losses in commercial interest crops in America and is a potential risk in the European Union. The nematicidal and phytotoxic activities of seven plant extracts against N. aberrans and Solanum lycopersicum were evaluated in vitro, respectively. The chemical nature of three nematicidal extracts (EC50,48h ≤ 113 µg mL−1) was studied through NMR analysis. Plant extracts showed nematicidal activity on second-stage juveniles (J2): (≥87%) at 1000 µg mL−1 after 72 h, and their EC50 values were 71.4–468.1 and 31.5–299.8 µg mL−1 after 24 and 48 h, respectively. Extracts with the best nematicidal potential (EC50,48h < 113 µg mL−1) were those from Adenophyllum aurantium, Alloispermum integrifolium, and Tournefortia densiflora, which inhibited L. esculentum seed growth by 100% at 20 µg mL−1. Stigmasterol (1), β-sitosterol (2), and α-terthienyl (3) were identified from A. aurantium, while 1, 2, lutein (4), centaurin (5), patuletin-7-β-O-glucoside (6), pendulin (7), and penduletin (8) were identified from A. integrifolium. From T. densiflora extract, allantoin (9), 9-O-angeloyl-retronecine (10), and its N-oxide (11) were identified. The present research is the first to report the effect of T. densiflora, A. integrifolium, and A. aurantium against N. aberrans and chemically characterized nematicidal extracts that may provide alternative sources of botanical nematicides.


Phytotoxicity Test
Most nematicidal extracts against N. aberrans extracts showed 100% inhibition (−100%) of L. esculentum radicle growth at 20 µg mL −1 , except extracts from H. terenbinthinaceus (−37%) (Figure 4). T. densiflora R extracts were the most phytotoxic with 40 and 38% inhibition at 0.02 µg mL −1 (Figure 4). Potentially, these extracts could act as soil disinfection agents. Also, A. cuspidata, A. subviscida, and T. densiflora A extracts showed hormetic effects: a 20 µg mL −1 solution inhibited radicle growth inhibition while a 0.02 µg mL −1 solution promoted it. Nematostatic and nematicidal effects observed by treatments with EC50,48h < 113 µg mL −1 relate to secondary metabolites like sterols, flavonoids, thiophenes, or alkaloids (PAs and allantoin), possibly biosynthesized in plants as a stress response. In general, sterols are involved in plants' growth and fertility as hormonal precursors and cell membranes' functional components. Nematodes also need sterols for their survival, but they cannot biosynthesize them de novo, so the nematodes readily absorb sterols. For example, Meloidogyne arenaria, M. incognita, and Pratilenchus agilis incorporate and transform sterols into necessary derivatives in their growth and reproduction [56]. Thus, nematodes should elicit a biological response to some sterols. Also, plant-nematode interactions require flavonoids and might be required for nematode reproduction. However, some flavonoids with specific structural arrangements have shown toxic effects on specific targets such as enzymes. Finally, thiophenes could inhibit enzymes like superoxide dismutase [57] and damage DNA [58]. The transformation of secondary metabolites to more toxic compounds also happened with PAs, as mentioned before.

Chemicals
All reagents and solvents (ACS grade), LiChroprep RP-18, and SiO2 supports for column and plate chromatography were obtained from Merck (MA, USA). Amberlite XAD16, α-terthienyl, β-sitosterol, stigmasterol, deuterated solvents, and dimethyl sulfoxide (DMSO-Hybri-Max) were obtained from Sigma Chemical (St. Louis, MO, USA). Nematostatic and nematicidal effects observed by treatments with EC 50,48h < 113 µg mL −1 relate to secondary metabolites like sterols, flavonoids, thiophenes, or alkaloids (PAs and allantoin), possibly biosynthesized in plants as a stress response. In general, sterols are involved in plants' growth and fertility as hormonal precursors and cell membranes' functional components. Nematodes also need sterols for their survival, but they cannot biosynthesize them de novo, so the nematodes readily absorb sterols. For example, Meloidogyne arenaria, M. incognita, and Pratilenchus agilis incorporate and transform sterols into necessary derivatives in their growth and reproduction [56]. Thus, nematodes should elicit a biological response to some sterols. Also, plant-nematode interactions require flavonoids and might be required for nematode reproduction. However, some flavonoids with specific structural arrangements have shown toxic effects on specific targets such as enzymes. Finally, thiophenes could inhibit enzymes like superoxide dismutase [57] and damage DNA [58]. The transformation of secondary metabolites to more toxic compounds also happened with PAs, as mentioned before.

Chemicals
All reagents and solvents (ACS grade), LiChroprep RP-18, and SiO 2 supports for column and plate chromatography were obtained from Merck (MA, USA). Amberlite

Plant Species
The plant species were collected in Oaxaca, Mexico (See Table 6), and voucher specimens were deposited in the Herbarium of Forest Sciences, Universidad Autonoma de Chapingo, Texcoco (Estado de México, México). The scientific name, collection site, voucher number, plant part used, and extraction solvent are listed in Table 6.

Preparation of Extracts
Extracts were prepared according to procedures previously described [52]. All extracts were kept at 4 • C and protected from light and moisture until further use.

Identification of Compounds from A. integrifolium
Methanol extract of A. integrifolium (23.1 g) was partitioned with ethyl acetate (3 times) to obtain 10.1 g of ethyl acetate soluble fraction (ESF) and 13 g of methanol soluble fraction (MSF). ESF was subjected to column chromatography (SiO 2 ) and eluted with mixtures of AcOEt: n-hexanes to obtain a mixture of chlorophylls "a" and "b" (80 mg), and a dark solid (155.8 mg). The solid was re-chromatographed (SiO 2 ) with the same eluents to obtain lutein (4, 9.3 mg) ( Figure 1) and a mixture (27.1 mg) of stigmasterol (1) and β-sitosterol (2). MFS (13 g) was solubilized in water and supported on a column of Amberlite XAD16; after two washes with water, the compounds retained were eluted with methanol to obtain a residue (1.5 g) free from simple carbohydrates. The residue (1.0 g) was eluted in a chromatography column (C 18 ) using methanol:water mixtures as eluent. Chromatographic separation yielded 72 mg of four quercetagetin derivatives in binaries mixtures; its approximate composition was calculated by integrating 1 H NMR areas of their characteristic signals. These compounds were identified as centaurin (5, 28.1 mg), patuletin-7-β-O-glucoside (6, 1.7 mg), pendulin (7, 6.4 mg), and penduletin (8, 1.0 mg) (Figure 1) from the analysis of their NMR data (Supplementary Table S2).

Nematodes
Mature egg masses of N. aberrans were extracted from infected roots of tomato plants (Lycopersicum esculentum Mill., 1768 or Solanum lycopersicum), propagated at Colegio de Postgraduados, Montecillo, Texcoco, Mexico. Egg masses were gently washed with water to remove adhered soil and a NaOCl 0.53% solution until the gelatinous matrix dissolved. Then they were washed with distilled water on a mesh sieve (#400) and incubated in distilled water at 25 • C for 5 days. Emerging J2 individuals were used in all experiments.

Assay
Test solutions were prepared in DMSO with 0.5% Tween 20 at 10, 100, 1000 µg mL −1 for extracts, while concentrations at 100 µg mL −1 were used for compounds and fractions. Fluopyram 50% (Verango, Bayer) and abamectin 5.41% (Oregon 60C-FMC) at 5, 10, 15, 25, 30 and 50 µg mL −1 (dissolved in distilled water) were tested as positive control. Treatments (5 µL) and between 100 and 150 J 2 individuals in 95 µL of water were added to 96-well plates (Falcon, USA) and incubated at 25 • C. DMSO with 0.5% Tween 20 (5 µL) in 95 µL of water was used as blank. Previously, non-effect on J2 mobility was shown at 24, 36, 48, 60, and 72 h with the solvents used (Supplementary Table S3). Percentages of J2 immobility were recorded after 12, 24, 36, 48, 60, and 72 h by counting mobile and immobile J 2 individuals under a stereomicroscope at 240X. A nematode was considered immobile if the nematode failed to respond to stimulation with a needle. After that, the J 2 individuals at 1000 µg mL −1 (extracts) and 100 µg mL −1 (commercial stigmasterol, α-terthienyl, and β-sitosterol) were washed on a 400-mesh filter with distilled water to remove the excess test substance (extracts and commercial compounds). The treatments were replaced with distilled water to allow a possible recovery of the J2 individuals after 24 h. If they remained immobile, they were assumed to be dead, and the effect was considered nematicide. If any J 2 individual regained mobility, the effect was considered nematostatic (paralysis). All treatments (extracts, isolated and commercial compounds) and control were replicated five times, and the experiments were performed two times. The immobility percentage was calculated using the equation: i = 100 × (1 − n t /n c ); where i = immobility percentage, n t = active J 2 in the treatment, and n c = active J 2 in the blank [60].

Phytotoxicity Assay
Experiments were conducted with L. esculentum F1 seeds var. Sheva according to the methodology described [61]. Prior to evaluation, all extracts were dissolved in a 0.5% DMSO/H 2 O solution at 20, 2.0, 0.2, 0.02, and 0.002 µg mL −1 concentrations to obtain solids-free solutions. Commercial herbicide (Glyphosate) was used as a positive control at the same concentrations, while 0.5% DMSO/H 2 O was used as blank (100% growth).

Statistical Analysis
All experimental data were subjected to an analysis of variance (ANOVA) using Statistica Pro (Stat Soft, Japan). Treatment means were tested with Tukey's HSD multiple comparison test at 0.05% or 0.01% probability levels.

Conclusions
To our knowledge, our results show for the first time the nematicidal activity against N. aberrans from T. densiflora, A. integrifolium, and A. aurantium extracts. In this research, we identified several compounds present in the nematicidal extracts against J2 individuals of N. aberrans containing: (a) flavonoids (A. integrifolium); (b) triterpene-type compounds (A. aurantium, A. integrifolium), (c) thiophene-type compounds (A. aurantium) and (d) alkaloids (T. densiflora). We identify 5-8 and 9-10 from A. integrifolium and T. densiflora, respectively. Moreover, we described the phytotoxic effect of all extracts on tomato radicle growth. Further research of these plant extracts will allow us to identify more compounds responsible for the nematicidal activity and provide alternative nontoxic crop protection chemicals.
Supplementary Materials: The following are available online. Table S1: Effect of plant extracts at 10 µg mL −1 on the immobility of N. aberrans J 2 s individuals after different exposure times; Table S2: 1 H and 13 C data for compounds 5-8. 400 MHz, 100 MHz CD 3 OD; Table S3: Effect of DMSO with 0.5% Tween on immobility of N. aberrans J2s after different exposure times.