Nematicidal Screening of Aqueous Extracts from Plants of the Yucatan Peninsula and Ecotoxicity

Active metabolites from plants are considered safer than synthetic chemicals for the control of plant-parasitic nematodes of the genus Meloidogyne. In the present work, 75 aqueous extracts (AEs) from different vegetative parts of 34 native plant species of the Yucatan Peninsula were evaluated against second-stage juveniles (J2s) of Meloidogyne incognita and M. javanica in microdilution assays. The highest mortality (M) against both Meloidogyne species was produced by the foliar AE from Alseis yucatanensis (M ≥ 94%) and Helicteres baruensis (M ≥ 77%) at 3% w/v after 72 h. Other active AEs at 3% were from the leaves of Croton itzaeus and stems of H. baruensis (M: 87–90%) on M. javanica and the stems of Annona primigenia and the leaves of Morella cerifera on M. incognita (M: 92–97%). The AEs from A. yucatanensis had the lowest LD50 against M. incognita (0.36% w/v), and against M. javanica (3.80% w/v). In an acute ecotoxicity assay of the most promising AEs using non-target earthworms (Eisenia fetida), the AE of A. yucatanensis had slight acute toxicity (LD50: 2.80% w/v), and the rest of the most active AEs were not ecotoxic. These tropical plants are potential candidates for further studies as biorational agents for controlling Meloidogyne species.


Introduction
Plant-parasitic nematodes cause 8.8-14% of the annual crop losses worldwide, at an estimated cost of approximately USD 173 billion [1,2]. Root-knot nematodes (RKNs) are one of the groups (Meloidogyne sp.) with the highest pathogenic capacity and the greatest number of hosts. Of the 105 described species in the genus Meloidogyne, M. arenaria, M. hapla, M. incognita, and M. javanica in particular parasitize many important crops [2][3][4]. These RKN species attack the root vascular system, causing water and nutrient transport deficiencies, wilting and chlorosis, retarding plant growth, and reducing yields [5,6]. In addition, they suppress the host defense system, making plants more susceptible to other plant pathogens such as bacteria, fungi, and viruses [7,8].
Meloidogyne has been controlled using agronomic practices such as fallow, crop rotation, resistant varieties, and mainly by synthetic chemical nematicides [9]. However, the use of synthetic nematicides such as methyl bromide, 1,3-dichloropropene, carbamates (oxamyl), and organophosphates (fenamiphos), among others, is being restricted because of their high toxicity [10,11]. Requirements for low or no residual synthetic pesticides in the food chain have led to an increased demand to use alternatives with low or no toxicity [9,12]. Plant extracts or their metabolites have been shown to have nematicidal activity [13,14]. For example, the aqueous extracts (AEs) of Azadirachta indica [15], Taxus baccata [16], and Xanthium strumarium [17] cause high mortality to M. incognita, and the
In general, the AEs from the leaves of A. yucatanensis and H. baruensis were the most active against both nematodes. The AE of A. yucatanensis had the lowest LD 50 against both species, 0.36% (w/v) on M. incognita and 3.80% w/v on M. javanica, and the lowest LD 90 (1.83% w/v) against M. incognita. Interestingly, the LD 90 was very similar for the AEs of A. yucatanensis and H. baruensis against M. javanica, 5.64 and 5.71% (w/v), respectively. In addition, there was a significant difference in the LD 50 and LD 90 between the two plant species against M. incognita but not for M. javanica (Table 3, Figures 1 and 2).

Ecotoxicity of the AE of Alseis yucatanensis on Eisenia fetida
The AE from the leaves of A. yucatanensis had an ecotoxicological effect within 72 h on adults of E. fetida at 3% (w/v) and was lethal at 6% (w/v). Treatments with serial dilutions of this AE showed that the LD 50 was 2.80% (w/v) and the LD 90 was 4.72% (w/v) ( Table 4). By contrast, the AE of H. baruensis, and other most active AEs from leaves of A. primigenia, C. itzaeus and M. cerifera, were not ecotoxic to adults of E. fetida in this acute assay ( Figure 3).  Helicteres baruensis Croton arboreus (S), C. itzaeus (S), Diospyros sp. (L), H. baruensis (S), M. glabra (L), and Tabernaemontana donnell-smithii (L). When these nine highly toxic AEs from eight different plant species were tested at 3% (w/v), five caused high mortality (>75%): AEs from the leaves of A. yucatanensis, C. itzaeus, C. latifolia, and H. baruensis and from the stems of H. baruensis (Table 2). In general, the AEs from the leaves of A. yucatanensis and H. baruensis were the most active against both nematodes. The AE of A. yucatanensis had the lowest LD50 against both species, 0.36% (w/v) on M. incognita and 3.80% w/v on M. javanica, and the lowest LD90 (1.83% w/v) against M. incognita. Interestingly, the LD90 was very similar for the AEs of A. yucatanensis and H. baruensis against M. javanica, 5.64 and 5.71% (w/v), respectively. In addition, there was a significant difference in the LD50 and LD90 between the two plant species against M. incognita but not for M. javanica (Table 3, Figures 1 and 2).

Ecotoxicity of the AE of Alseis yucatanensis on Eisenia fetida
The AE from the leaves of A. yucatanensis had an ecotoxicological effect within 72 h on adults of E. fetida at 3% (w/v) and was lethal at 6% (w/v). Treatments with serial dilutions of this AE showed that the LD50 was 2.80% (w/v) and the LD90 was 4.72% (w/v) ( Table 4). By contrast, the AE of H. baruensis, and other most active AEs from leaves of A. primigenia, C. itzaeus and M. cerifera, were not ecotoxic to adults of E. fetida in this acute assay (Figure 3).

Discussion
The present contribution is part of the first bioprospecting research on native plant extracts against M. javanica and the second against M. incognita. Further, it enriches our knowledge about the natural nematicidal properties of some plant species of the flora of the Yucatan Peninsula. Previous studies on 55 plant extracts from 20 native plants showed that the AE from C. urticifolia (2.6%, w/v) and ethanol extracts from C. urticifolia, E. winzerlingii, and Tephrosia cinerea (500 ppm) were effective against M. incognita [26][27][28]. We found no reports of nematicidal activity for the plant species studied here after an exhaustive literature search. Bioprospecting of the 75 AEs from native plant species for activity against the J2s of M. javanica and M. incognita led to the detection of 33 AEs (44%) from 20 plant species (59% of the total) with high effectiveness (M = 90-100%) at 6 w/v. At 3% (w/v), 22 AEs (20% of the total) caused >75% mortality against at least one nematode species.
The J2s of M. incognita were more sensitive to plant extracts; 19 AEs (25% of the total

Discussion
The present contribution is part of the first bioprospecting research on native plant extracts against M. javanica and the second against M. incognita. Further, it enriches our knowledge about the natural nematicidal properties of some plant species of the flora of the Yucatan Peninsula. Previous studies on 55 plant extracts from 20 native plants showed that the AE from C. urticifolia (2.6%, w/v) and ethanol extracts from C. urticifolia, E. winzerlingii, and Tephrosia cinerea (500 ppm) were effective against M. incognita [26][27][28]. We found no reports of nematicidal activity for the plant species studied here after an exhaustive literature search. Bioprospecting of the 75 AEs from native plant species for activity against the J2s of M. javanica and M. incognita led to the detection of 33 AEs (44%) from 20 plant species (59% of the total) with high effectiveness (M = 90-100%) at 6 w/v. At 3% (w/v), 22 AEs (20% of the total) caused >75% mortality against at least one nematode species.
The J2s of M. incognita were more sensitive to plant extracts; 19 AEs (25% of the total tested) caused >75% mortality at 3%. Other authors reported similar mortality to what we found, but the AE concentrations were higher. For example, AEs from Achyranthes aspera, Ageratum conyzoides, and Solanum xanthocarpum leaves at 33% (w/v) caused 84-98% mortality after 48 h of exposure to J2 larvae of M. incognita [29,30]. AEs from C. longa, N. sativa, and P. nigrum caused 36% mortality against the J2s of M. javanica at 40% (w/v) after 72 h [18]. Likewise, AEs from the seeds and shoots of A. indica and leaves of Nerium oleander and Olea europea caused 53-65% mortality at 10% (w/v) after 48 h [31]. The AEs tested here induced higher nematicidal activity against M. incognita J2s at lower concentrations. Furthermore, the LD 50 and LD 90 of the AE from the leaves of A. yucatanensis against the J2s of M. javanica after 72 h were lower than those documented for the AEs from the leaves of Cinnamomum zeylanicum (LD 50 of 15.38 mg/mL and LD 90 of 24.73 mg/mL) and Eugenia caryophyllata (LD 50 = 17.91 mg/mL and LD 90 = 23.45 mg/mL) after 24 h [32]. AEs obtained with water extraction at 100 • C, for 10 h and 14 h at 25 ºC, from C. abyssinica at 1:10 (w/v) were reported to cause 72.2% mortality in J2s of M. javanica after 24 h [19]. In particular, more AEs have been tested against M. incognita than against M. javanica based on our search of the literature. Reports of plant extracts tested against M. javanica more commonly focused on organic extracts and essential oils [14]. On the other hand, the nematicidal potential of hydrolate distilled waters that remain after hydro-or steam distillation, and separation of the essential oil, have been amply demonstrated [33]. Specifically, the nematicidal effects in vitro and in vivo of hydrolates from Spanish aromatic plant species Alium sativum, Artemisia absintium, Lavandula × intermedia var. Super, L. luisieri, Thymus vulgaris, T. zygis, and purple garlic have been tested against M. javanica [34][35][36][37]. Furthermore, hydrolates from Greek aromatic species Origanum vulgare, Mentha piperita, and Melissa officinalis, Satureja hellenica, and C. cyminum showed in vitro high activity towards M. incognita and M. javanica [38][39][40].
In the present work, 25% of the AEs had greater lethality against J2s at 3% w/v than 6% w/v, but the difference in lethality was significant for only eight of these AEs (>12% difference): AEs from Alvaradoa amorphoides leaves and roots, Bakeridesia notolophium leaves, Byrsonima bucidifolia leaves, Licaria sp. leaves, M. cerifera stem, P. cubana stem bark, and Paullinia sp. leaves. This phenomenon of lower concentrations causing greater mortality than higher ones has been reported previously. For example, leaf extracts from C. urticifolia at 500 ppm caused 90% mortality, but 250 ppm caused 97% mortality against J2s of M. incognita [26]. Similarly, the extracts of Achillea wilhelmsii at 6% (w/v) caused 10.6% mortality, and 15.6% at 3% (w/v) [41]. These data could be due to pipetting errors, temperature variations, or variations in the extract dosage and exposure duration [42]. Moreover, during incubation, precipitates can be formed in the 6% concentration and adhere to the microplate walls, lowering the concentration of exposure.
The present contribution adds to the few works on the in vitro activity of plant extracts against M. javanica and M. incognita. For example, organic extracts and fractions of Eugenia winzerlingii leaves caused 100% mortality at 1 µg/µL against J2s of M. javanica and M. incognita within 72 h; such activity was conferred by decanoic, undecanoic, and dodecanoic acids [28]. The essential oils of S. hellenica caused 100% paralysis of the J2s of M. javanica and M. incognita at 200 µL/L after 96 h due to the activity of p-cymene and carvacrol [39].
Our nematicidal bioassays indicate that the AEs from the leaves of A. yucatanensis and H. baruensis are the most promising against M. incognita and M. javanica. Among the 22 plant species belonging to the genus Alseis (Rubiaceae), only two are described for Mexico, A. yucatanensis and A. hondurensis Standl. Both tree species are endemic to Southern Mexico and part of Central America [24,43]. Our report is the first to show the nematicidal activity of an extract from A. yucatanensis against J2s of M. incognita and M. javanica. Studies by the working group have documented that the AE of the leaves of A. yucatanensis had no antifungal activity at 2000 µg/mL against the phytopathogens Fusarium equiseti and F. oxysporum [44]. The only reported activity of this species is the vasorelaxation (VR) of aortic tissue in rats with a median effective dose of 0.12 mg/mL of the AE from the bark [45]. For other species of the genus, ethanol and acetone-water (7:3) extracts of the leaves of Alseis blackiana were reported to have moderate in vitro antiviral activity (VR of 0.5 to 1.0) against HSV-1 and HSV-2 (herpes simplex virus) at 100 µg/mL [46]. Nematicidal activity of some Rubiaceae species has been reported: soil amendments at 1.0% (w/w) of plant debris from Coffea arabiga reduced the incidence of root galls on Cucurbita pepo caused by M. arenaria, and an aqueous extract of Moringa pterygosperma leaves at 1:5 caused 100% mortality of the J2s of M. incognita after 24 h [47]. However, nothing is known about the phytochemistry of A. yucatanensis. In the genus Alseis, only alkaloid components from Alseis blackiana were detected by a positive Dragendorff's test [48]. Indole alkaloids have been widely reported from members of the families Rubiaceae, Apocynaceae, and Loganiaceae, which have prominent activity against diverse biological targets [49]. The application of powdered leaves (1, 3, and 5 g) of Catharanthus roseus is highly effective (M = 71.8, 71.6, and 72.6%) in controlling M. incognita infection in potted pumpkins (Cucumis sativum) [50]. Waltheriones E and A from Triumfetta grandidens are effective against M. incognita [51], and harmine from Peganum harmala is active against M. javanica [52].
The genus Helicteres (Malvaceae) consists of 60 species distributed mainly in America, with four species described from Mexico and three of them, H. baruensis, H. guazumifolia, and H. mexicana, from the Yucatan Peninsula [53,54]. This genus is found in tropical and subtropical regions, mainly in deciduous forests. The plant H. baruensis (Mayan name of sutup) is first reported with nematicidal activity for the species and genus. Petroleum ether and chloroform extracts from H. baruensis have action against Salmonella enteritidis and Bacillus cereus; this activity is attributed to sterols and alkaloids [55], but their ethanol and AE did not have antifungal activity against F. equiseti or F. oxysporum [44]. Further, their methanol and dichloromethane extracts had no anti-neoplastic activity against the LNCaP prostate cancer cell line [56]. In addition to sterols and alkaloids, polyphenols are also present in its leaves [57].
Earthworms have been used for in vitro ecotoxicity assays of pesticides to evaluate environmental risk. The filter paper contact test is used to determine the initial acute toxicity of soil contaminants [22]. In the present study, the ecotoxicity of A. yucatanensis caused slight acute toxicity towards E. fetida. Other studies on the AE, such as artificial soil tests [22] and chronic ecotoxicity tests, should be conducted next. These toxicity tests have not been commonly reported for natural extracts or metabolites from plants or other organisms [21]. Among these, an essential oil from Piper betle at 1000 µg/cm 2 was innocuous to E. fetida; mortality was only 5.4% after 48 h in the filter paper contact test [58]. Cucurbitacin E from Citrus colocynthis had good efficacy after 48 h against Spodoptera litura with LD 50 of 15.84 ppm and LD 90 of 67.70 ppm, and at 100 ppm, it induced 11% mortality of E. fetida [59], corroborating its lack of effect against this non-target soil organism.
After application, the plant extracts readily decompose when exposed to environmental factors such as light and temperature [60]. Therefore, the nematicidal extracts from A. yucatanensis and H. baruensis and other promising AEs should be further tested in the greenhouse and field for toxic or beneficial effects on plants. The active compounds also need to be identified, purified, and tested for toxicity against additional non-target organisms.  33 43.2 W). The leaves, stem, stem bark, roots, and root bark were collected from most plant species, and complete plants were collected for some species. One specimen of each collected species was deposited in the Herbarium-Fibroteca U Najil Tikin Xiw"of the Centro de Investigación Científica de Yucatán (Tables 1 and 2).

Materials and Methods
The plant material was dried in a lamp chamber (50-60 • C) for 3 days and crushed in a grinder (Model 1520, Pagani, Azcapotzalco, Mexico) with blades (5 mm screen) and kept in the dark in a cold room until use. A total of 75 samples from the 34 plant species were obtained.

Preparation of Aqueous Extracts (AEs)
The powdered plant material (1.5 g) was added to 20 mL of boiling distilled water for 5 min, then cooled and filtered through cheesecloth cotton and filter paper (Whatman No. 1). The water volume was brought to 25 mL to obtain an AE at an initial concentration of 6% (w/v). All AEs were frozen (−17.5 ± 0.5 • C) and lyophilized (Labconco FreeZone 2.5, model 7670520, Houston, TX, USA) for 15 h to reduce the volume to 50% and obtain a concentration of 12% (w/v) of the original plant material for each extract. The concentrated AEs were then sterilized using a 0.22 µm Millipore filter (Merck-Millipore, Burlington, MA, USA) and frozen until use.

Nematicide Bioassay
The nematode populations of M. javanica were obtained from the Instituto de Ciencias Agrarias (25 ± 1 • C/70% relative humidity (RH)), and M. incognita from the Tecnológico Nacional de México-Campus Conkal (30 ± 2 • C/90% RH). The populations of both species were maintained on Solanum lycopersicum (var. Marmande) plants in plastic pots in a greenhouse. For bioassays, egg masses were manually recovered from galled roots and incubated for 72 h in sterile water at 30 ± 2 • C for M. incognita and 25 ± 1 • C for M. javanica. Hatched second-instar juveniles (J2s) were adjusted to a final concentration of 100 J2s/100 µL of distilled water [14,26].
Each well of a 96-well U-bottom plate (NEST Biotechnology, Wuxi, Jiangsu, China) received 100 µL of the AE (12%, w/v) and 100 µL of the nematode suspension. As a positive control, the synthetic nematicide Vydate ® (oxamyl 235 g/L) was used at 1 µL/mL, and the suspension of nematodes in distilled water (100 J2s/100 µL) was used as a negative control. For each treatment, four replicate wells were evaluated. The experimental plates were sealed and incubated in the dark using the same conditions used for the egg masses (vide supra) described by Moo-Koh et al. [14,61].
Any immobile J2 larvae that were insensitive to a needle touch were counted as dead at 72 h and expressed as percentage mortality (M%). The data obtained from the nematicidal activity are presented as corrected percentages of J2s mortality (Schneide-Orelli formula: % efficiency = (b − k/100 − k) × 100, where b = % of dead individuals in the treatment and k = % of dead individuals in the control [62]. The extracts that caused the greatest mortality were then evaluated at serial dilutions (6, 3, 1.5% w/v or less). The LD 50 and LD 90 values were determined using a probit analysis with SAS ver. 9.4 for Windows (SAS Institute, Cary, NC, USA).

Eisenia Fetida Assay
For the E. fetida assay (a contact test on filter paper, OECD 207 1984), 1 mL of the test AE at 6 and 3% (w/v) was dropped onto a Whatman No. 1 filter paper (6.4 cm long × 7.8 cm wide) at a final concentration of 1200 and 600 µg/cm 2 and kept in a hood extractor until completely dry. The filter paper was then placed in a glass vial (8 cm long × 3 cm in diameter). The earthworms were purchased (PlanetaMexico, Mexico City, Mexico) and maintained in organic horse manure and soil at 28 ± 2 • C until they were adults (2 months old) and weighing 300-600 mg each. Then, they were washed with water and kept on moist filter paper in the dark for 3 h to ensure that all intestinal contents had been released. Water was used as a negative control and a synthetic nematicide (Vydate ® (oxamyl 42%), Dupont) as a positive control at 1 µg/mL (0.02 µg/cm 2 ). Each vial received 1 mL of distilled water to keep the filter paper moist and one earthworm deposited; then, vials were covered with a gauze-type cloth that was secured with a rubber band to ensure ventilation and retain the earthworms in the vial. For each treatment, 20 replicate vials were used. The vials were placed in a humid chamber at 26 ± 2 • C in the dark. Every 24 h for 72 h, the worms were checked and considered dead if they did not respond to a small stimulus in the anterior region. Any pathological behaviors or symptoms were also recorded [22].
Serial dilutions (6, 3, 1.5% w/v or less) of the AE with ecotoxicity were tested and worms checked every 24 h for 72 h as described above. The results were processed in SAS software (SAS Institute, Cary, NC, USA) to obtain the LD 50 and LD 90 .

Conclusions
Our knowledge of Mexican flora as potential nematicides has been enriched. In this approach to surveying plant species in the Yucatan Peninsula for bioactivity against phytopathogenic nematodes, we confirmed that native species caused high mortality against the J2s of the phytonematodes M. incognita and M. javanica. The AEs from A. yucatanensis and H. baruensis leaves were the most effective; the first had low ecotoxicity, and the second none. With high efficacy in vitro against both nematodes, the aqueous extracts of A. yucatanensis and H. baruensis are highly promising candidates for developing products to control Meloidogyne nematodes.