Nematicidal Weeds, Solanum nigrum and Datura stramonium

Nematode rearing and collection

Populations of M. incognita and M. javanica both of Greek origin were reared on tomato (Solanum lycopersicum Mill.) cv. Belladonna. Freshly hatched (24 h) J2 as well as eggs of different growth stages were extracted from egg masses according to Hussey and Barker (1973) from 60 day-old (d) infested roots, to be used for the bioassays. The egg masses were handpicked from the tomato roots under a stereoscope.

Chemicals

Methanol, chloroform, and hexane were of high-performance liquid chromatography grade. All chemical standards were obtained from Sigma-Aldrich (Milano, Italy).

Plant material extraction procedure

Dry plant material, 5 g of S. nigrum (seeds) and D. stramonium (shoots), were extracted in 50 ml methanol for 30 min in a sonicator apparatus. After exhaustive evaporation of the solvent the yields in dry material were measured at 12.3 ± 0.07 and 12.6 ± 0.01% (w/w) for D. stramonium and S. nigrum, respectively. The extracts were used directly for bioassays with nematodes and chemical composition analysis without evaporation.

J2 paralysis bioassays

J2s were extracted as described previously after hatch in modified Baermann funnels. Hatched juveniles were discarded after the first 2 days. Thereafter, hatched J2 less than or equal to 2 days-old were used for the paralysis experiments. The D. stramonium and S. nigrum extracts were diluted in DMSO, brought to volume with water and tested for paralysis activity in Cellstar 96-well cell culture plates (Greiner Bio-One) at a ratio of 1:1 (v/v) with nematodes’ suspension. The final concentration of DMSO in test wells did not exceed 1% (v/v). Distilled water served as a control together with the carrier control (DMSO). Each well contained 15 J2s and the test concentrations of both extracts ranged from 100 to 1,000 μg ml⁻¹. Border wells with J2s immersed in distilled water alone served as controls for fumigant activity test (Ntalli et al., 2011). Multiwell plates were covered to avoid evaporation and were maintained in the dark at 20°C. Juveniles were ranked into two distinct categories, moving and paralysed, with the aid of an inverted microscope (Euromex, The Netherlands) at ×40 after 1d, 2d, and 3d. After evaluation, J2 were washed through a 20 μm sieve, to remove the test compounds, and were immersed in water alone to determine if motility was regained. Numbers of motile and paralysed J2s were assessed by pricking the juvenile body with a needle, and they were counted. Nematodes that did not move at this point were considered dead. J2 paralysis bioassays were performed three times, and every treatment was replicated six times.

Egg hatch inhibition in free eggs treated with the test compounds

The egg hatch inhibition tests were performed in microwell assays (Ntalli et al., 2016). Briefly, nematodes were pipetted into 24-well cell culture plates (Greiner Bio-One), with 0.5 mL treatment at double the test concentration and 0.5 mL nematode inoculum (20 eggs) in sterile distilled water per well. Datura stramonium and S. nigrum methanol extracts were dissolved in DMSO and then brought to volume with water to reach desired concentrations. The final concentration of DMSO in test solutions did not exceed 1% as this concentration did not harm nematodes. The bioassay treatments were: 0.0 μg ml⁻¹ (water control), 0.0 μg ml⁻¹ (carrier control), and 1, 10, 100, and 1,000 μg ml⁻¹, extract in the carrier. Five wells were used per treatment, and the plates were covered by plastic adhesive sheets to prevent volatiles escaping to adjacent wells. Hatch quantification was done by directly counting undifferentiated eggs and J2 in each well at day 0 using an inverted microscope at ×40. Thereafter, assessments were performed after 2, 6, 10, and 14 days. Cumulative percent J2 release was calculated using the formula: ((J2_Dx - J2_D0)/total) × 100 where D_x = day after the start of the assay. Cumulative percent undifferentiated egg hatch was calculated using the formula: ((Eggs_D0 - Eggs_Dx)/total) × 100 where D_x = day after the start of the assay.

Inhibition of nematode development

Procedures were according to Ntalli et al. (2010). Briefly artificially inoculated with M. incognita tomato plants were then treated with powders of S. nigrum seeds and D. stramonium shoots, in a dose response from to 0.1 to 100 mg g⁻¹. After the completion of a biological cycle at 27^oC, 60% RH at 16 h photoperiod, plants were uprooted and roots were stained with acid fuchsin (Byrd et al., 1983). The following variables were assessed: fresh root weight, fresh shoot weight, and total number of female nematodes and galls per gram of root at ×10 magnification under uniform illumination by transparent light. The experiment was performed twice, and the treatments were arranged in a completely randomised design with five replicates.

Sample extraction for GC–MS analysis

For small polar metabolite analysis, the following procedure was used. Powdered plant material (100 mg) was extracted with 2 mL solvent mix chloroform/methanol (2/1, v/v), and three replications were made. After dispersion, the whole mixture was agitated for 15 to 20 min in an orbital shaker at room temperature. The mixture was centrifuged to recover the liquid phase. The supernatant was washed with 400 µL of 0.9% KCl solution in water and vortexed for 1 min. After centrifugation at 2,000 rpm, the water phase was evaporated to dryness under a nitrogen stream. Afterwards, the residue was suspended in 50 µL of methoxyamine hydrochloride (10 mg ml⁻¹) in pyridine. After 17 h, 50 mL of N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) were added and kept for 1 hr at room temperature before 600 µL of a solution of 2-dodecanone in hexane (20 mg L⁻¹) were added and samples were GC/MS analyzed. Derivatized atropine, linoleic acid and monostearin were used for GC/MS calibration.

For the alkaloidal compounds analysis, extraction with chloroform was performed as follows. Powdered plant material (100 mg) was extracted with a mix of 5 mL of a 0.1 N sodium hydroxide in water and 5 mL of CHCl₃. A solution of caffeine in methanol (1 mg ml⁻¹) was added as internal standard (I.S.). After 5 min centrifugation, the chloroform phase was separated and evaporated to dryness under a nitrogen stream. The residue was suspended in 100 µL of BSTFA and kept for 1 h at 70°C for silylation. After a 10-fold dilution with hexane, samples were GC/MS analyzed.

GC–MS conditions

One microliter of derivatized plant extract was injected in splitless mode into a 6,850 gas chromatograph coupled with a mass spectrometer 5,973 Network (Agilent Technologies, Santa Clara, CA, USA) equipped with a 30 m × 0.25 mm ID silica capillary column, which was chemically bonded with 0.25 μm DB-5MS stationary phase (J&W scientific, Folsom, CA, USA). The injector temperature was kept at 200°C and the mobile phase flow was 1 mL min⁻¹. The column temperature gradient was as follows: 50°C for 10 min, then increased from 50 to 300 at a rate of 10°C min⁻¹ and finally held at 300°C for 4 min. The transfer line and the ion source temperatures were respectively 280°C and 180°C. Ions were generated at 70 eV with electron ionization and were recorded at 1.6 scan sec⁻¹ over the mass range m/z 50 to 550. GC–MS data analysis was conducted by integrating each resolved chromatogram peak and normalizing the area for the corrected total area of the chromatogram. These peaks were examined for their mass spectra and identification of the peaks was attempted using the NIST 08 library after deconvolution with AMDIS.

Statistical analysis

Treatments of motility experiments were replicated six times, and each experiment was performed twice. The percentages of paralyzed J2 observed in the microwell assays after 1 h were corrected by eliminating the natural death/paralysis in the water control according to the Schneider Orelli’s formula: Corrected % = {(Mortality percent in treatment - Mortality percent in control)/(100 - Mortality percent in control)} × 100 and they were analysed (ANOVA) after being combined over time. Since the ANOVA indicated no significant treatment by time interaction, means were averaged over experiments. Corrected percentages of paralyzed J2 treated with the weed extracts were subjected to nonlinear regression analysis using the log–logistic equation proposed by Seefeldt et al.: Y = C + (D − C)/{1 + exp[b (log(x) − log(EC_5O))]} where C = the lower limit, D = the upper limit, b = the slope at the EC₅₀, and EC₅₀ = the test solution concentration required for 50% death/paralysis of nematodes after normalizing with the control (natural death/paralysis). In the regression equation, the test concentration was the independent variable (x) and the paralyzed J2 (percentage increase over water control) was the dependent variable (y). The mean value of the six replicates per each test concentration and immersion period was used to calculate the EC₅₀ value.

Egg hatch inhibition treatments were replicated five times, and each bioassay was performed twice. Because the ANOVA indicated no significant treatment by time interaction, means were averaged over experiments. In egg hatch inhibition bioassays, treatment means were compared using Tukey’s test at P ≤ 0.05. Statistical analysis was performed using SPSS 20.

Pot bioassays were organised in a complete randomized design with five replications and were performed twice. Since ANOVAs indicated no significant treatment by time interaction (between runs of experiment), means were averaged over experiments. The data from the pot bioassays were expressed as a percentage decrease in the number of females or galls per gram of root corrected according to the control, using the Abbott’s formula: corrected percent = 100 × {1 − [females number in treated plot/females number in control plot]}. Data were fit to the log-logistic model (Seefeldt et al., 1995) to estimate the concentration that caused a 50% decrease in females and galls per gram of root (EC₅₀ value). In this regression equation, the test compounds (% w/w) were the independent variables (x) and the female nematodes, or galls, (as the percentage decrease over the water control) was the dependent variable (y). Because ANOVAs indicated no significant treatment by time interaction (between runs of experiments), means were averaged over experiments. Treatments means were compared using Tukey’s test at P ≤ 0.05.

J2 paralysis bioassays

Paralysis activity of M. incognita was more affected by D. stramonium and S. nigrum extracts than M. javanica (Table 1). Clear time and dose response relationships were established for D. stramonium and the EC_50/96h values were 427 μg ml⁻¹ for both M. incognita and M. javanica (Table 1). Solanum nigrum demonstrated a nematostatic effect and the mortality was stabilized 3 days post J2 immersion in test solutions.