Abstract: Natural products are an important resource for finding new pesticides. Additionally, preparation of elemental nano-particles of natural products was proven to be promising as anti-microbial agents. Present study aimed to examine the petroleum ether, ethyl acetate and ethanol extracts of Urtica urens and their silver-nano formulations as nematicides against root-knot nematode Meloidogyne incognita. Leaf extracts of U. urens were prepared through successive extraction and the Ag-nanoparticles of extracts and the reference nematicide (rugby®) were synthesized. Results revealed that Ag-nano formulations of extracts were effective in the management of M. incognita. There was an 11-fold of activity between Ag-rugby and the least toxic extract (ethyl acetate) against eggs. The LC50 values showed significant effects of Ag-rugby, rugby® and Ag-petroleum ether against the larvae compared with petroleum ether, ethanol and ethyl acetate extracts. Petroleum ether extract and its Ag nano-particles might be considered as environmental-safe and effective nematicides alternatives against the invasive M. incognita.
INTRODUCTION
Nematode infestation causes significant economic loss worldwide that was estimated by about $80 billion (Handoo, 1998). About 4,100 species of plant-parasitic nematodes have been identified and most of these species are restricted to specific geographical areas but Meloidogyne spp. is distributed globally (Nicol et al., 2011). Meloidogyne incognita infects a wide range of host plants causing the formation of root galls. The nematode initiates development of multinucleate giant cells, which serve as permanent feeding sites within the root and provide nutrition for the developing worm to complete its life cycle. Root-knot nematodes are considered one of the most serious soil-borne pests of many crops in Egypt (Balbaa, 2010) and worldwide (Anderson and Mulvey, 1979; Roberts et al., 2005; Sikora et al., 2007).
Management of root-knot nematodes are difficult because of its wide host range and ability to survive in various environmental conditions. Control strategies include the use of biological control predators and parasites, cover crops (Tagetes spp., sorghum-sudangrass and sunn hemp plants) through allelochemicals, fertilization, crop rotation, organic amendments, resistant cultivars, sanitation, solarization and nematicides (McSorley, 2002; Wang et al., 2007). Nematicides are the most commonly used management method, but they are costly and contaminate the environment (Adegbite et al., 2005) so alternatives are required.
Researchers investigated the use of plant natural products such as essential oils and other plant metabolic derivatives as nematicides. For example, saponins from Asparagus adscendens, Albizzia chinensis and Acacia concinna were active against Meloidogyne incognita (Meher et al., 1988). Thiarubrine A (a naturally occurring disulphide polyine) of Aspilia mossambicensis was very effective to control Meloidogyne javanica (Rodriguez, 1988). The combinations of thymol and benzaldehyde exhibited broad-spectrum nematicidal effect against root-knot and cyst nematode (Soler-Serratosa et al., 1996). The alkaloid 1,2-dehydropyrrolizidine was found in many plants (Asteraceae, Boraginaceae, Fabaceae and Apocynaceae) and had effective nematicidal activity against the Northern root-knot nematode Meloidogyne hapla (Thoden et al., 2009). Burning (dwarf) and stinging nettle (Urtica spp.) are rich source of secondary metabolites including 5-hydroxytryptamine, (-)-3,4-divanillyltetrahydrofuran lignin, acetylcholine, acetic acid, butyric acid, formic acid, histamine, leukotrienes and the steroids; stigmasterol, stimast-4-en-3-one and campesterol (Emmelin and Feldberg, 1949; Hirano et al., 1994; Wagner et al., 1994; Hryb et al., 1995; Schottner et al., 1997; Jimoh et al., 2010).
Leaf extracts of U. urens showed nematicidal properties against the citrus nematode Tylenchulus semipenetrans (Mohammad et al., 1981) and it was among several weeds that were effectively controlled M. incognita (Ornat and Sorribas, 2008). Stinging nettle (Urtica dioica) significantly reduced numbers of several species of nematodes (Nasiri et al., 2014). Increasing the biological activity and bio-compatibility of plant extracts through the preparation of new formulation might be useful. Synthesis of nanoparticles using plants extracts was reported to increase the anti-pathogenic activity.
Nanoparticles (up to 100 nm) offer improved properties compared to the larger particles because of small particle size, which help in distribution and coverage of plants (Wiesman and Chapagain, 2006). Biosynthetic of metal (Ag, Au, Cu and Cd) nano-formulation of plant extracts has received an increasing attention because of their potential application in pest control. Of particular interest, Ag nano-particles revealed antibacterial (Sathishkumar et al., 2009), antivirals (Rogers et al., 2008) and antifungal activities (Panacek et al., 2009). Therefore, present study investigated the effectiveness of Ag-nano formulations of polar and non-polar extracts of Urtica urens against the root-knot nematode (M. incognita).
MATERIALS AND METHODS
Rugby® 20% SC (Cadusafos: organophosphorodithioate; an insecticide and a nematicide) was used in present study as a reference nematicide and was purchased from local suppliers. Solvents, silver nitrate (ACS reagent, >99%, 209139, Sigma-Aldrich) and other chemicals were of analytical grade and purchased from reputed local suppliers.
Plant collection and sample preparation: Plants of burning nettle (U. urens) were collected from tomato and eggplant fields at Al-Nubaryia area, Albeheira Governorate, Egypt. Plants were identified and washed under running tap water and dried using tissue paper. Leaves were freeze-dried at -60 to -70°C for 2 days (Christ Freeze-Dryer, Gamma 1-16 LSC, Osterode, Germany). Freeze-dried samples were ground to a fine powder and stored in at -20°C freezer until extraction.
Plant extracts preparation: Approximately 25 g of freeze-dried leave samples were extracted successively in soxhlet apparatus with 200 mL of each of petroleum ether (PE), ethyl acetate (EA) and absolute ethanol (E) at 60-80°C for 6 h. Before, each successive extraction, the powder was carefully spread on a sheet of paper to dry at room temperature. Extracts were dried over anhydrous sodium sulfate and the solvent was completely evaporated under reduced pressure using a rotary evaporator (Unipan vacuum rotary evaporator type 350 P, Poland). Dried crude extracts were kept in tightly closed brown bottles at -20°C until use.
Synthesis of silver nanoparticles: About 10 mL silver nitrate (5 mM) were added slowly to 10 mL of the plant crude extract (2500 mg L–1) and 5 mL of 10 mM ascorbic acid (as reducing agent) in brown glass bottles (ca. 90 mL). Then the mixture was incubated at room temperature in dark place for 24 h. Mixture color was changed into brown indicating the formation of silver nanoparticles (Fig. 1) (Cruz et al., 2010; Annamalai et al., 2011). The Ag-nanoparticles of rugby® was prepared following a modified method of Tien et al. (2008). The reduction reaction of AgNO3 to the nematicide was started by adding 10 mL of AgNO3 (5 mM) slowly to 10 mL of rugby® in amber screw cap tubes at room temperature. Then tubes were incubated at 40°C for 12 h. The reddish-brown color solution was synthesized and was used as the Ag-rugby® formulation.
UV/vis spectra and scanning electron microscopy analysis: The synthesized Ag-nanoparticles were verified using the distinctive absorption pattern from 300-700 nm (Jenway UV/vis spectrophotometer, Model 6305, Bibby Scientific Limited, Staffordshire, UK). The morphology and size of the nano-particles were measured and photographed by the Scanning Electron Microscope (SEM) (Model JSM-5300, JEOL, USA) after coating the sample using the ion sputtering device (Fine Coat, Model JFC-1100E, JEOL, USA). In the SEM, an electron beam was focused into affine probe and raster scanned over a small rectangular slide. As the beam interacts with the sample, it generates signals (i.e., secondary electrons, internal currents and/or photon emission), which can be appropriately detected.
| Fig. 1(a-d): | Scanning electron microscopy images of (a) Ag-nanoparticles of rugby, extracts of U. urens, (b) Ethyl acetate, (c) Ethanol and (d) petroleum ether |
Root-knot nematode (M. incognita) culture: Root-knot nematode (M. incognita) was collected from eggplants at Al-Nubaryia area and used to establish a nematode culture in the greenhouse on eggplant plants. Eggplants were planted in plastic pots (30 cm diameter, one plant per pot) that filled with autoclave-sterilized peat moss. Plants were grown under greenhouse conditions (24±2°C and 70±5% RH), watered once every 3-4 days. Fertilization was done every 6-8 days with commercial fertilizer that has N (19%), P (19%), K (19%), S (5.94 %), Fe (1000 mg L–1), Zn (500 mg L–1) and Mn (500 mg L–1) (Farmers VASCON, Moubarak Industries City, Quesna, Menoufia, Egypt) at 1 kg/1000 L of water. The M. incognita egg and larval suspensions were prepared from infected roots with obvious galls (about 60 days of inoculation).
Nematicidal activity against eggs and larvae of M. incognita: The efficiency of the U. urens extracts and their nano-formulations to eggs and larvae of M. incognita were tested by applying 1 mL of series of concentrations 125, 250, 500 and 1000 mg L–1 of each treatment to 1 mL of the eggs or larvae suspension. Egg suspension (100 eggs mL–1) and 50 larvae (2nd stage) mL–1 of M. incognita were prepared using a special microscopic slide for counting the nematodes and used for each replicate. Glass tubes were incubated at 27±2°C for 2 and 7 days for larvae and eggs, respectively. Eggs hatchability and larval mortality were evaluated. Assays were repeated twice and each treatment was replicated 5 times. Average results of the two assay times were compared with the recommended nematicide and water as control. The median lethal concentration (LC50) and toxicity index values of each treatment were calculated using Log dose Probit (LdP) line software (Ehabsoft, Cairo, Egypt).
RESULTS
Synthesis of Ag-nano formulation: Data presented in Fig. 1 showed that the reduction reaction of rugby® and U. urens extracts with silver nitrate was successful in the synthesis of circular nano-sized particles. The ESM photographs showed that the size of Ag nano-particles of rugby® (Ag-rugby, Fig. 1a) was homogeneous. The U. urens extracts were reduced by silver nitrate; petroleum ether extract (Ag-PE, Fig. 1d) showed more homogeneous and smaller size compared to ethyl acetate (60-112 nm) (Ag-EA, Fig. 1c) and ethanol extract (80-111 nm) (Ag-E, Fig. 1b). Nano-formulation of Ag and petroleum ether extract was the most homogenous particle size compared with rugby®, ethyl and extracts of ethyl acetate and ethanol of U. urens.
Biological activity against eggs stage of M. incognita: Efficacy of natural extracts of U. urens in the management of M. incognita compared to rugby® (recommended nematicide) and their Ag-nano formulations (Ag-rugby, Ag-PE, Ag-EA and Ag-E) were assayed under laboratory condition (Table 1). Ag-rugby suppressed the hatchability of eggs with an LC50 value of 5.62 mg L–1 compared to 7.21 mg L–1 for rugby® itself. The Ag-PE had the most suppressive effect to eggs hatchability compared to other extracts but was less than Ag-rugby and rugby®. Petroleum ether extract was more toxic (LC50; 28.77 mg L–1) than ethanol, Ag-E, Ag-EA and ethyl acetate extracts with LC50 values of 40.92, 42.39, 45.71, 47.92 and 61.96 mg L–1, respectively.
Biological activity against 2nd larval stage of M. incognita: Results in Table 2 showed the nematicidal activity of natural extracts of U. urens and their Ag-nano formulations compared with rugby® and its Ag-nano-sized formulation. The toxicity order was Ag-rugby, rugby®, Ag-PE, Ag-E, Ag-EA, petroleum ether extract, ethyl acetate extract and ethanol extract with LC50 values of 6.72, 7.81, 40.92, 42.39, 61.96, 169.23, 175.36 and 188.81 mg L–1, respectively. The Ag-rugby, rugby® and Ag-PE were more toxic to 2nd larval stage compared to petroleum ether, ethanol and ethyl acetate extracts.
| Table 1: | Median lethal concentration (LC50) and toxicity index values of the ethanol, ethyl acetate and petroleum ether extracts of U. urens and their nano-formulations compared to the nematicide rugby® and its Ag-nano particles against eggs of M. incognita |
| 1Comparison among all tested materials according to the most toxic one (Sun, 1950) | |
| Table 2: | Median lethal concentration (LC50) and toxicity index values of the ethanol, ethyl acetate and petroleum ether extracts of U. urens and their nano-formulations compared to the nematicide rugby® and its Ag-nano formulation against 2nd larval stage of M. incognita |
| 1Comparison among all tested materials according to the most toxic one (Sun, 1950) | |
Limits of confidence of LC50 values showed significant effects between Ag-rugby, rugby® and Ag-PE and petroleum ether, ethanol and ethyl acetate extracts.
DISCUSSION
Nanoparticles (up to 100 nm) offer improved properties and activities compared to the original material (Wiesman and Chapagain, 2006). To the best of our knowledge, few researchers tested the nematicidal activity of extracts of burning nettle against nematode; Mohammad et al. (1981) reported that leave extracts of U. urens effectively control the Tylenchulus semipenetrans nematode, Decker (1982) listed U. urens (stinging nettle) among plants that control root-knot nematode and Nasiri et al. (2014) found that exudates of U. dioica effectively controlled the Pratylenchus, Aphelenchoides and Helicotylenchus nematodes.
Present study was the first study to report about testing the efficacy of Ag-nano formulations of extracts of U. urens against the worldwide nematode M. incognita. Results reported herein revealed an 11-fold of activity between Ag-rugby and the least toxic extract (ethyl acetate) against eggs. However, limits of confidence of the median lethal concentrations showed overlap among tested extracts, nano-extracts, rugby® and Ag-rugby, which highlights similar suppressive effects of eggs hatchability. There was small difference in the activity between extracts of U. urens and their corresponding nano-formulations, where only a 1.3-fold of activity was reported. No difference was noticed between the ethanol extract and its Ag-formulation.
Examination of the slope of the LDP-lines reported similarity in the nematicidal action among rugby® and the petroleum ether extract, which were different from that of the ethanol and ethyl ether extracts. Ag-petroleum ether extract was the most effective against both eggs and larval stages this might be due to the fact that Ag-PE had the most homogenous particle size. Moreover, present study showed similar effects of the petroleum ether extracts and their Ag nano-particles and rugby®, which highlights the potential use of such components in controlling such invasive pest with environmentally-friendly and less toxic plant extracts.
ACKNOWLEDGMENT
The authors would like to acknowledge the Scanning Electron Microscope (SEM) Unit at Faculty of Science, Alexandria University for renting the equipment.