This work is licensed under the Creative Commons Attribution 4.0 International License.
Weeds compete with crop plants and soil organisms for resourses through the production of allelochemicals like phenolic acids, terpenes, terpenoids, glycosides, alkaloids, and flavonoids (Whittaker and Feeny, 1971; Blum, 1996; Keating, 1999). A major phyto-nematode control research issue is the study of herbal preparations rich in allelochemicals with nematicidal activity, of no adverse effects to non-target organisms and easy biodegradability. The use of green manures as soil bioamendments may be a suitable nematode control tool for many crop systems, especially if the botanical species to be incorporated are readily available in situ, like weeds. Solanum nigrum Linn. and Datura stramonium Linn., commonly known as black nightshade and jimsonweed, are two Solanaceous, highly invasive and globally distributed weeds that exhibit a range of biological properties (Zhou et al., 2012; Abbasi et al., 2015; Sher et al., 2015).
Although S. nigrum can be infected by Meloidogyne incognita (Robab et al., 2012) it also exhibits nematicidal activity. Specifically, its dried ground seed powder incorporated in soil at the rate of 5 g kg−1 lessens root galling and increases host shoot length (Radwan et al., 2012). Moreover, the water extract of S. nigrum at a concentration of 10 mg ml−1 induces morphological changes in the body structure of the root-lesion nematode Pratylenchus goodeyi, greatly affects movement and causes mortality (Gouveia et al., 2014). Interestingly, root extracts of S. nigrum are traditionally used in the treatment of animal worms and abdominal pain (Jagtap et al., 2013).
Solanum nigrum is a major source of various chemical groups of nematicidal compounds like alkaloids (Jagtap et al., 2013; Sammani et al., 2013), glycoalkaloids (Li et al., 2007; Ding et al., 2013), saponins (Jagtap et al., 2013), phenols (Gharbi et al., 2017), fatty acids (Dhellot et al., 2006; Mohy-ud-din et al., 2010) and tannins (Jagtap et al., 2013). An alkaloid named drupacine was found to exhibit an EC50 value of 76.3 µg ml−1 on M. incognita second stage juveniles (J2) and to reduce egg hatch by 36% after immersion in 1.0 mg ml−1 (Wen et al., 2013). Similarly, 4-quinolone waltherione and waltherione A, have been reported to have larvicidal activity against M. incognita (EC50 values of 0.09 and 0.27 μg ml−1 at 48 h) and egg hatch inhibition activity (91.9 and 87.4% after 7 days of exposure to 1.25 μg ml−1) (Jang et al., 2015). Saponins like solanigroside A and solanigroside B (Zhou et al., 2007) as well as oleanane-type triterpenoid saponins exhibit LC50 values against M. incognita ranging from 70.1 to 94.7 μg ml−1 after 48 h (Li et al., 2013); while a saponin based commercial nematicide from Quillaja saponaria has been registered for nematode control in Europe (Giannakou, 2011). 4-methylphenol is of significant in vitro activity against M. javanica (Yang et al., 2015) while Zhang and co-workers have demonstrated that fatty acids like caproic, caprylic, capric, lauric, myristic, and palmitic cause significantly high mortality to M. incognita J2 (Zhang et al., 2012). Tannic acid has been proven nematicidal as well (Hewlett et al., 1997). We also, in our previous studies, have demonstrated that acetic and hexanoic acid, as components of Melia azedarach, are effective against root-knot nematodes in terms of J2 paralysis activity (Ntalli et al., 2010).
Similar to S. nigrum, D. stramonium is a host for root-knot nematodes and even increases populations of Meloidogyne species if not controlled effectively (Ntidi et al., 2012). Nonetheless, hot water and ethanol extracts of D. stramonium seeds tested at 25 to 100 mg ml−1 caused 75% to 100% mortality of M. incognita J2 (Chaudhary et al., 2013). Similarly, leaf and stem extracts of D. stramonium tested at 500 mg L−1 against J2 resulted in relatively high mortality rates of 68 and 70% after 72 h of exposure (Elbadri et al., 2008). When tested in pot experiments, dried ground leaves of D. stramonium mixed with soil at the rate of 1 to 10 g kg−1 soil significantly suppressed M. incognita populations and root galling as they decomposed, but high rates proved to be phytotoxic (Radwan et al., 2006). Pre-plant treatments with D. stramonium leaf extracts at 0.5% to 1% significantly reduced gall numbers (Mateeva and Ivanova, 2000). Furthermore, aqueous leaf extracts of D. stramonium inhibited egg hatch and killed M. incognita larvae (Rao et al., 1986).
Chemical composition studies on D. stramonium seeds revealed N-trans-feruloyl tryptamine, hyoscyamilactol, scopoletin, umckalin, daturaolone, daturadiol, N-trans-ferulicacyl-tyramine, cleomiscosin A, fraxetin, scopolamine, 1-acetyl-7-hydrox-beta-carbol-ine, 7-hydroxy-beta-carboline1-propionic acid (Li et al., 2012). Scopolamine is a muscarinic antagonist (Lee et al., 2000) and one of the most important alkaloids present in D. stramonium (Ma et al., 2015).
The scope of this study was to (i) evaluate the nematicidal activity of S. nigrum and D. stramonium in terms of (a) J2 paralysis, (b) egg hatch inhibition, and (c) inhibition of nematode development in host roots and (ii) to delineate the chemical composition of active extracts after derivatization by GC–MS.
Materials and Methods
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: ((J2Dx - J2D0)/total) × 100 where Dx = day after the start of the assay. Cumulative percent undifferentiated egg hatch was calculated using the formula: ((EggsD0 - EggsDx)/total) × 100 where Dx = 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 27oC, 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(EC5O))]} 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.
Results
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 EC50/96h 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.
Efficacy of Datura stramonium and Solanum nigrum methanol extracts against Meloidogyne incognita and Meloidogyne javanica.1
D. stramonium
S. nigrum
Immersion period
M. incognita
M. javanica
M. incognita
M. javanica
1d
968 ± 98
>8,000
409 ± 56
686 ± 98
2d
553 ± 85
581 ± 73
507 ± 72
792 ± 95
3d
427 ± 75
427 ± 23
418 ± 78
954 ± 96
1Half maximal effective concentration EC50 ± SD (μg ml−1) calculated after 1, 2, and 3 days of nematode immersion in test solutions.
Egg hatch inhibition in free eggs treated with the test compounds
The cumulative undifferentiated egg hatch was decreased significantly by both D. stramonium and S. nigrum extracts at 100 μg mL−1 at day 6, while in successive assessments the activity increased for D. stramonium and decreased for S. nigrum (Tables 2, 3). Concerning the percent of J2 released from eggs immersed in the two methanol extracts, again D. stramonium was more active since it differed from control at 10 μg ml−1 at day 2 while S. nigrum differed from the control only at concentrations equal or higher than 100 μg ml−1 (day 2). In the next assessment date at day 6, activity increased for D. stramonium and decreased for S. nigrum differing from control at 1 and 100 μg ml−1, respectively. At day 10 the percent J2 release in control decreased naturally and thus J2 release differences among treatments were not evident thereafter (Tables 4, 5).
Effect of Datura stramonium methanol extract on cumulative percent hatch of Meloidogyne incognita undifferentiated eggs.1
D. stramonium cumulative undifferentiated egg hatch
μg ml−1
Day 2
Day 6
Day 10
Day 14
1,000
9 ± 1.0a
9 ± 1.0a
9 ± 1.5a
9 ± 1.5a
100
10 ± 5.0a
10 ± 5.0a
10 ± 5.0a
10 ± 5.0a
10
17 ± 6.5a
23 ± 6.5ab
23 ± 6.5a
23 ± 6.5a
1
14 ± 6.0a
24 ± 4.5ab
23 ± 6.5a
23 ± 6.5a
0
13 ± 8.0a
35 ± 9.0b
47 ± 9.0b
47 ± 9.0b
1The cumulative percent hatch of undifferentiated eggs ± SD was calculated using the formula: ((eggsD0 - eggsDx)/total) × 100. Eggs (20–30 per well) were collected and distributed in 24-well plates and were incubated at 27°C for 14 days in test solutions or plane water. Eggs were counted at 2, 6, 10, and 14 days post experiment establishment. Each percent data represent the mean ± SD from two experiments performed in time, with five replicates per treatment each. Values within each day were compared using Tuckey’s test and those followed by different letters are significantly different at (P ≤ 0.05).
Effect of Solanum nigrum methanol extract on cumulative percent hatch of Meloidogyne incognita undifferentiated eggs.1
S. nigrum cumulative undifferentiated egg hatch
μg ml−1
Day 2
Day 6
Day 10
Day 14
1,000
9 ± 0.5a
8 ± 0.0a
9 ± 6.0a
9 ± 6.0a
100
15 ± 2.0a
16 ± 4.0a
16 ± 4.0a
16 ± 4.0a
10
15 ± 2.0a
22 ± 1.0ab
27 ± 1.5ab
27 ± 1.0ab
1
17 ± 2.0ab
24 ± 3.5ab
27 ± 5.0ab
27 ± 5.0ab
0
25 ± 4.0b
35 ± 9.0b
47 ± 9.0b
47 ± 9.0b
1The cumulative percent hatch of undifferentiated eggs ± SD was calculated using the formula: ((eggsD0 - eggsDx)/total) × 100. Eggs (20–30 per well) were collected and distributed in 24-well plates and were incubated at 27oC for 14 days in test solutions or plane water. Eggs were counted at 2, 6, 10, and 14 days post experiment establishment. Each percent data represent the mean ± SD from two experiments performed in time, with five replicates per treatment each. Values within each day were compared using Tuckey’s test and those followed by different letters are significantly different at (P ≤ 0.05).
Effect of Datura stramonium methanol extract on cumulative percent release of Meloidogyne incognita J2.1
D. stramonium percent J2 release
μg ml−1
Day 2
Day 6
Day 10
Day 14
1,000
3 ± 1.5a
1 ± 1.0a
1 ± 1.0a
1 ± 1.0a
100
6 ± 4.0a
5 ± 3.5a
4 ± 3.0a
4 ± 3.0a
10
20 ± 5.5ab
5 ± 5.0a
4 ±1.5a
4 ± 1.5a
1
31 ± 6.5bc
5 ± 4.0a
5 ± 3.5a
5 ± 3.5a
0
40 ± 5.5c
20 ± 5.5b
8 ± 4.5a
5 ± 3.5a
1The cumulative percent release of J2 ± SD was calculated using the formula: ((J2Dx - J2D0)/total) × 100. Eggs (20–30 per well) were collected and distributed in 24-well plates and were incubated at 27°C for 14 days in test solutions or plane water. Released J2 were counted at 2, 6, 10, and 14 days post experiment establishment. Each percent data represent the mean ± SD from two experiments performed in time, with five replicates per treatment each. Values within each day were compared using Tuckey’s test and those followed by different letters are significantly different at (P ≤ 0.05).
Effect of Solanum nigrum methanol extract on cumulative percent release of Meloidogyne incognita J2.1
S. nigrum percent J2 release
μg ml−1
Day 2
Day 6
Day 10
Day 14
1,000
2 ± 2.0a
2 ± 2.0a
2 ± 2.0a
2 ± 2.0a
100
11 ± 6.0ab
5 ± 4.5ab
2 ± 1.5a
2 ± 1.5a
10
23 ± 2.0bc
6 ± 2.0ab
2 ± 1.0a
2 ± 1.0a
1
33 ± 6.5c
10 ± 5.0ab
11 ± 6.5a
6 ± 3.5a
0
40 ± 5.5c
20 ± 5.5b
8 ± 4.5a
±3.5a
1The cumulative percent release of Meloidogyne incognita J2 ± SD was calculated using the formula: ((J2Dx - J2D0)/total) × 100. Eggs (20–30 per well) were collected and distributed in 24-well plates and were incubated at 27oC for 14 days in test solutions or plane water. Released J2 were counted at 2, 6, 10, and 14 days post experiment establishment. Each percent data represent the mean ± SD from two experiments performed in time, with five replicates per treatment each. Values within each day were compared using Tuckey’s test and those followed by different letters are significantly different at (P ≤ 0.05).
Inhibition of nematode development
Meloidogyne incognita densities in tomato roots and gall formation were significantly supressed when D. stramonium and S. nigrum powders were incorporated in the nematode infested soil. Meloidogyne incognita development in artificially inoculated tomato plants treated with the weed powders was reduced with EC50 values for female per gram root counts calculated for S. nigrum and D. stramonium of 1.13 and 11.40 mg g−1, respectively. Galls/g root were similar (Table 6), with no phytotoxicity evident at the dose range of the treatments used for the bioassay.
Efficacy of weed paste (decomposing tissues) on Meloidogyne incognita as calculated in pot experiments.
Females/g root
Galls/g root
EC50 (mg g−1)
SE
95% CI
EC50(mg g−1)
SE
95% CI
Datura stramonium
11.40
0.92
9.48–13.32
12.85
1.19
10.39–15.33
Solanum nigrum
1.13
0.17
0.78–1.48
1.15
0.17
0.79–1.51
SE, Standard error; CI, Confidence interval.
GC–MS analysis
The low-molecular weight polar compounds extracted from both plants S. nigrum and D. stramonium were submitted to derivatization and were chemically analyzed using GC–MS. We were able to detect amino acids, carbohydrates, carboxylic acids and some compounds with non-elucidated structures termed unknowns, from U1 to U16 (Table 7). The metabolites with the highest concentrations present in both plants were fructose, sucrose and U1 with concentrations ranging from 20 to 90 mg L−1. Although sugars like glucose and galactose were abundant in D. stramonium aqueous extracts, they were not detectable in S. nigrum extracts. Likewise, S. nigrum was richer in palmitic acid and glycerol. On the other hand, the chloroform extracts of both plants showed high levels of alkaloids such as dehydrohyoscinamine, atropine, and scopolamine (Table 8). Fatty acids and monoglycerides were also present at high concentrations; for example, palmitic acid was present at 1,694 mg L−1 only in S. nigrum and monoheptadecanoate glycerol at 466 mg L−1 in D. stramonium. When the alkaloid rich extract was tested against J2, no paralysis was evidenced at the concentration range of 100 to 1,000 μg ml−1 (data not shown).
Small polar metabolites extracted from Datura stramonium and Solanum nigrum.
Concentration (mg L−1)
n°
RTa
LRIb
m/z
Quantitative masse
Cmpd
D. stramonium
S. nigrum
1
18.303
1,099
147–233–133
147
Propanedioic acid (2TMS)
ND
48.52
2
19.498
1,166
147–205–299
147
Glycerol (3TMS)
3.61
43.36
3
19.982
1,193
147–247–129
147
U1
78.45
89.87
4
20.331
1,215
147–189–292
147
Lactic acid (2TMS)
28.29
11.70
5
20.699
1,240
147–175–117
147
U2
1.68
1.13
6
21.238
1,276
138–168–227
138
U3
7.60
ND
7
21.723
1,309
147–189–233
147
U4
6.47
2.22
8
22.473
1,361
147–233–245
147
Malic acid (2TMS)
4.90
1.70
9
22.706
1,377
147–189–219
147
U5
34.81
13.35
10
22.793
1,383
217–205–147
217
Erythrose (1MEOX) (3TMS)
ND
2.81
11
23.006
1,398
254–269–180
254
U6
–
–
12
23.161
1,410
271–169–147
147
5-hydroxymethyl 2-Furoic acid (2TMS)
14.15
ND
13
23.206
1,413
117–147–217
117
U7
–
–
14
23.243
1,416
205–292–147
147
Threonic acid (4TMS)
1.52
ND
15
23.616
1,445
147–334–245
334
U8
1.09
ND
16
24.63
1,525
103–147–217
217
Xylitol (5TMS)
1.32
1.13
17
25.339
1,583
117–147–147
117
U9
–
–
18
25.518
1,598
217–319–147
217
Altrose (5TMS)
3.96
ND
19
25.7
1,614
231–147–133
231
U10
–
–
20
25.872
1,629
246–147–129
147
U11
–
–
21
26.126
1,651
217–257–379
217
U12
–
–
22
26.462
1,679
204–379–147
204
Lyxose (1MEOX) (4TMS)
t
t
23
26.878
1,716
217–307–103
217
Arabinitol (5TMS)
2.72
2.22
24
26.889
1,717
345–255–147
345
U13
–
–
25
27.08
1,735
147–217–307
217
Fructose oxime (6TMS)
99.41
60.26
26
27.363
1,761
205–319–147
319
Glucose oxime (6TMS)
161.16
ND
27
27.562
1,779
319–205–160
319
Galactose oxime (6TMS)
43.89
ND
28
27.72
1,794
319–205–147
319
Glucitol tms
ND
18.40
29
28.313
1,851
95–83–195
95
U14
–
–
30
28.427
1,862
313–129–117
313
Palmitic acid (TMS)
ND
21.06
31
29.189
1,937
217–305–318
305
Myo-inositol (6TMS)
10.10
1.43
32
29.683
1,986
319–205–72
319
U15
–
–
33
30.64
2,087
124–361–140
124
Atropine TMS
6.04
1.23
34
31.03
2,130
98–217–330
330
Methyl-5,8-epoxyretinoate
1.87
2.34
35
31.33
2,164
59–72–126
59
Oleamide
11.28
–
36
33.11
2,387
371–147–203
371
Monopalmitin (2TMS)
4.20
2.60
37
33.73
2,436
217–289–361
217
U16
–
–
38
33.89
2,447
361–217–147
361
Sucrose (8TMS)
46.74
20.16
39
34.01
2,456
217–230–147
217
Alpha.-DL-arabinofuranoside, methyl (3TMS)
6,8658
–
40
34.54
2,491
399–217–147
399
Monostearin (2TMS)
3.21
2.06
aRetention time; bLinear retention index;
ND, Not detected; TMS, trimethylsilyl; U, unknown.
Chemical composition of chloroform extraction of Datura stramonium and Solanum nigrum.
ND, not detected; TMS, trimethylsilyl; U, unknown.
Discussion
A number of weeds have been studied as alternatives to synthetic nematicides. For instance, water and ethanol leaf extracts of Euphorbia hirta, Phyllanthus amarus, Cassia obtusifolia, Sida acuta, and Andropogon gayanus have been found to provoke 100% mortality on M. incognita juveniles at 15% to 20% (w/v) (Olabiyi et al., 2008). Chaudhary and co-workers have reported on 75% to 100% mortality of juveniles of M. incognita after treatment with hot water and ethanol extracts of D. stramonium seed at 25 to 100 mg ml−1 (Chaudhary et al., 2013), while Elbadri and co-workers have reported high nematicidal activity levels for D. stramonium seed extracts on M. incognita J2 at 500 ppm (Elbadri et al., 2008). Herein we report on a higher Meloidogyne sp. paralysis activity exhibited by S. nigrum and D. stramonium methanol extracts, since the EC50/3d values were calculated at around 420 μg ml−1. Interestingly the chloroform extracts of D. stramonium and S. nigrum were not active against the phytonematodes, thus suggesting the absence of activity for the alkaloids fraction. As previously demonstrated for other nematicidal plant extracts (Ntalli et al., 2013) M. incognita was found more susceptible than M. javanica when exposed to D. stramonium and S. nigrum extracts. When the weed extracts were tested for egg hatch inhibition, D. stramonium was more effective than S. nigrum in suppressing both cumulating undifferentiated egg hatch and J2 release from eggs. Similarly, water extracts of Luffa cylindrica and Momordica charantia significantly inhibited the hatching of Meloidogyne spp. eggs (Ononuju and Nzenwa, 2011). To the best of our knowledge this is the first report on the paralysis activity and egg hatch inhibition activity of S. nigrum and D. stramonium methanol extracts against Meloidogyne spp.
Interestingly when the S. nigrum seeds paste was used to treat nematode infested soil the EC50 value for reducing females per gram of tomato roots was the lowest ever reported for similar treatments by our group, namely 1.13 mg g−1 (Ntalli et al., 2010; Caboni et al., 2012; Caboni et al., 2013; Aissani et al., 2015; Caboni et al., 2015). Also Radwan and co-workers have reported on S. nigrum powder activity on M. incognita but at higher concentration levels, namely 5 g kg−1 (Radwan et al., 2012). It seems S. nigrum paste incorporated in the nematode infested soil was more active than the extract and more effective than D. stramonium.
Our results, on the chemical composition of the weeds under study agreed with former broad chemical screening studies (Jimoh et al., 2010). Steenkamp et al. (2004) also detected atropine and scopolamine by high performance liquid chromatography in D. stramonium. Additionally, linoleic acid, present at 3,268 mg L−1 in S.nigrum was reported for its nematicidal activity on Caenorhabditis elegans with EC50 value as low as 5 mg L−1 (Stadler et al., 1993). This work reports for the first time, ten metabolites in D. stramonium and S. nigrum using GC–MS after methoxylation and sylilation.
It appears that the complexity of the biological interactions among chemical constituents adds to the overall efficacy of the material. We previously found that soil incorporation of powdered plant materials had lower EC50 values for nematicidal activity than the respective extracts (Ntalli et al., 2010, Aissani et al., 2015, Caboni et al., 2015). Farmers do in fact utilize complex materials like waste resources, oil seed cake, and gutter oil to help manage M. incognita (Zhang et al., 2012) and efficacy is the sum of activities of active(s) against various nematode growth stages (egg, J2, and female laying eggs). The efficacy of the botanical nematicidals along with their side effects on non-target organisms, easiness of preparation, and cost effectiveness contribute to their overall significancy (Ntalli and Caboni, 2017). Biofumigation has been advocated as an eco-friendly tactic to manage plant-parasitic nematodes amongst which the number-one target has been Meloidogyne sp. (Jones et al., 2013) and Brassicas is the oldest green manure amendment for their control globally (Fourie et al., 2016). Here we prove that the production/release of nematicidal allelochemicals by S. nigrum when its seeds are crashed and incorporated into the soil is among the best reported (EC50 = 1.13 mg of S. nigrum powder per gram of soil) by our group. Since both S. nigrum and D. stramonium are widespread weed species, their soil incorporation could be an interesting alternative nematode control tool.