Identification of Nematicidal Constituents of Notopterygium incisum Rhizomes against Bursaphelenchus xylophilus and Meloidogyne incognita

During a screening program for new agrochemicals from Chinese medicinal herbs, the ethanol extract of Notopterygium incisum rhizomes was found to possess strong nematicidal activity against the two species of nematodes, Bursaphelenchus xylophilus and Meloidogyne incognita. Based on bioactivity-guided fractionation, the four constituents were isolated from the ethanol extract and identified as columbianetin, falcarindiol, falcarinol, and isoimperatorin. Among the four isolated constituents, two acetylenic compounds, falcarindiol and falcarinol (2.20–12.60 μg/mL and 1.06–4.96 μg/mL, respectively) exhibited stronger nematicidal activity than two furanocoumarins, columbianetin, and isoimperatorin (21.83–103.44 μg/mL and 17.21–30.91 μg/mL, respectively) against the two species of nematodes, B. xylophilus and M. incognita. The four isolated constituents also displayed phototoxic activity against the nematodes. The results indicate that the ethanol extract of N. incisum and its four isolated constituents have potential for development into natural nematicides for control of plant-parasitic nematodes.

During the screening program for new agrochemicals from Chinese medicinal herbs and wild plants, the ethanol extract of the rhizomes of Notopterygium incisum Ting ex H. T. Chang (Family: Umbelliferae) (Figure S1) was found to possess nematicidal toxicity against the two species of nematodes, B. xylophilus and M. incognita.Notopterygium incisum is perennial herb native to China (Gansu, Qinghai, Shaanxi, Sichuan province, and Tibet Autonomous Region).It grows at a range of 1600 to 5000 m above sea level and lives among forest edges and scrubs that border the grasslands of these elevated slopes [20].The rhizomes of N. incisum known as "Qianghuo" in Chinese, is a well-known traditional Chinese medicinal herbs.They are used in herbal preparations for a series of symptoms from the common cold, chills, fever, rheumatoid arthritis, and general limb or body aches and pains [14].Previous phytochemical investigations revealed that the rhizomes of N. incisum contained phenolic compounds, alkaloids, polyacetylenes, coumarins, sesquiterpenoids, and glycosides [21][22][23][24][25][26][27][28][29][30][31][32][33][34].Pharmacological studies have revealed that its extracts or constituents possess anti-inflammatory, antioxidative, analgesic, and anti-influenza activity as well as antiproliferative activity against several cancer cell lines [21,22].However, a literature survey shows that there is no report on nematicidal activity of the ethanol extract of N. incisum rhizomes.Thus, the objective of this study was to investigate the nematicidal activity of the ethanol extract of N. incisum rhizomes against the two species of nematodes and isolation of active constituents from the ethanol extract.

Isolated Bioactive Compounds
Four bioactive compounds were isolated and based on bioassay-guided fractionation and identified based on their spectroscopic data and comparison with literature vales.Their chemical structures are given in Figure 1.
During the screening program for new agrochemicals from Chinese medicinal herbs and wild plants, the ethanol extract of the rhizomes of Notopterygium incisum Ting ex H. T. Chang (Family: Umbelliferae) (Figure S1) was found to possess nematicidal toxicity against the two species of nematodes, B. xylophilus and M. incognita.Notopterygium incisum is perennial herb native to China (Gansu, Qinghai, Shaanxi, Sichuan province, and Tibet Autonomous Region).It grows at a range of 1600 to 5000 m above sea level and lives among forest edges and scrubs that border the grasslands of these elevated slopes [20].The rhizomes of N. incisum known as "Qianghuo" in Chinese, is a wellknown traditional Chinese medicinal herbs.They are used in herbal preparations for a series of symptoms from the common cold, chills, fever, rheumatoid arthritis, and general limb or body aches and pains [14].Previous phytochemical investigations revealed that the rhizomes of N. incisum contained phenolic compounds, alkaloids, polyacetylenes, coumarins, sesquiterpenoids, and glycosides [21][22][23][24][25][26][27][28][29][30][31][32][33][34].Pharmacological studies have revealed that its extracts or constituents possess anti-inflammatory, antioxidative, analgesic, and anti-influenza activity as well as antiproliferative activity against several cancer cell lines [21,22].However, a literature survey shows that there is no report on nematicidal activity of the ethanol extract of N. incisum rhizomes.Thus, the objective of this study was to investigate the nematicidal activity of the ethanol extract of N. incisum rhizomes against the two species of nematodes and isolation of active constituents from the ethanol extract.

Isolated Bioactive Compounds
Four bioactive compounds were isolated and based on bioassay-guided fractionation and identified based on their spectroscopic data and comparison with literature vales.Their chemical structures are given in Figure 1.

Nematicidal Activity
Among the four isolated constituents, falcarindiol and falcarinol exhibited strong nematicidal activity against the pine wood nematode (B.xylophilus) in dark with LC 50 values of 2.20 µg/mL and 12.62 µg/mL, respectively.Isoimperatorin and columbianetin also had LC 50 values of 21.83 µg/mL and 103.44 µg/mL against B. xylophilus, respectively (Table 1).Moreover, based on LC 50 values, the two polyacetylene compounds demonstrated stronger nematicidal activity (no overlap of the 95% FLs) than a positive control, 2% avermectin (dark, LC 50 = 3.57 µg/mL) and two coumarins (isoimperatorin and columbianetin) against the pine wood nematodes (Table 1).When using 12 h light and 15 min UV light treatment, the nematicidal activity of the four isolated constituents against B. xylophilus increased.For example, falcarindiol had LC 50 values of 0.95 µg/mL (light) and 0.73 µg/mL (UV) against B. xylophilus, two-to-three times more toxic than in dark (Table 1) while falcarinol showed almost nine times more toxic in UV light treatment than in dark against B. xylophilus.Moreover, after 15 min UV light treatment, the two coumarins also possessed two-to-three times more toxic to the pine wood nematodes than the dark treatment.Based on LC 50 values, the two polyacetylene compounds (falcarindiol and falcarinol, in dark LC 50 = 1.08 µg/mL and 4.96 µg/mL, respectively) exhibited stronger nematicidal activity (no overlap of the 95% FLs) than two coumarins (isoimperatorin and columbianetin, dark treatment LC 50 = 7.57 µg/mL and 30.91 µg/mL, respectively) against the southern root-knot nematodes (M.incognita) (Table 2).Falcarindiol also possessed the same level of nematicidal activity against the southern root-knot nematodes as the positive control, 2% avermectin (in dark, LC 50 = 1.25 µg/mL) and the three other constituents showed less toxic than the positive control (Table 2).When using 15 min UV light treatment, falcarindiol, falcarinol, and isoimperatorin demonstrated almost five times more toxic to the southern root-knot nematodes than in dark treatment while columbianetin showed only two times more toxic (Table 2).
In the previous reports, several naturally occurring polyacetylenes have been demonstrated to possess toxic or phototoxic activity against insects, especially larval mosquitoes [35][36][37][38][39][40].Falcarinol and falcarindiol were also demonstrated to possess larvicidal activity against the common house mosquito (Culex pipiens) with 24 h LC 50 values of 3.49 ppm and 6.51 ppm, respectively [41].There were several reports on nematicidal activity of natural occurring polyacetylenes (derived from Carthamus tinctorius) against plant parasitic nematodes such as, rice white tip nematode (Aphelencoides besseyi) with more than 80% mortality after five days at a concentration of 10 ppm treatment [42,43].Moreover, 14 polyacetylenes or their thiophene derivates, isolated from species of the Asteraceae were found to possess toxicity against adult nematodes (Caenorhabditis elegans) and toxicity of the compounds was found to be enhanced greatly by irradiation with near-UV radiation or natural sunlight [44].However, their investigation with falcarindiol showed no photoactivation with UVA radiation [41].Moreover, falcarinol and falcarindiol have been identified as antifungal compounds in many Apiaceae plant species inhibiting spore germination of different fungi in concentrations ranging from 20 to 200 µg/mL [45].Falcarinol and falcarindiol also exhibited significant anti-Candida, antibacterial, and antimycobacterial activity, with an ability to kill Mycobacterium tuberculosis and isoniazid-resistant Mycobacterium avium at 10 µg/disk in a disk diffusion assay [46].However, this is the first report of nematicidal activity and photoactivation with UV treatment of falcarinol and falcarindiol against the two species of nematodes.The mode of action of falcarinol and falcarindiol on nematodes was not investigated; however, the mechanism for antifungal activity of falcarinol and falcarindiol is believed to involve disruption of cell membranes [47].Thus, falcarinol, the more lipophilic compound, was more toxic than falcarindiol against the two species of nematodes, the more polar acetylene (Table 1).However, it was also suggested that the potent insecticidal action of falcarinol might be related to the GABAergic block associated with the higher intake expected in herbivorous insects because GABA A receptors are important targets of neuroactive pesticides [48,49].Columbianetin and isoimperatorin had been demonstrated to possess insecticidal activity against several insects, such as the cabbage aphid (Brevicoryne brassicae), Egyptian cottonworm (Spodoptera littoralis), larvae of Aedes albopictus, A. aegypti, Culex pipiens pallens [50][51][52].Columbianetin also exhibited antibacterial, antifungal, and cytotoxic activity [53][54][55].However, this is first report of nematicidal activity against the two species of plant-parasitic nematodes.
The above findings suggest that nematicidal activity of the ethanol extract of N. incisum rhizomes and its four isolated constituents-especially the two polyacetylene compounds-against the two species of plant-parasitic nematodes is quite promising.As they are currently commonly used, nematicides are synthetic pesticides and these synthetic pesticides are also highly toxic to humans and other non-target organisms, the ethanol extract and its four isolated constituents show potential to be developed as possible natural nematicides for the control of B. xylophilus and M. incognita.
In traditional Chinese medicine, N. incisum rhizomes are used in herbal preparations for a series of symptoms from the common cold, chills, fever, rheumatoid arthritis, and general limb or body aches and pains [20].It thus seems that this medicinal herb is quite safe for human consumption because it has been used as a medicinal herb for hundreds of years.Moreover, the four isolated constituents are naturally present in many vegetables and fruits, e.g., carrot, celery, parsley, coriander, ginseng, etc.Thus it seems that the four isolated constituents are quite safe for human consumption.However, no experimental data about the safety of this herb is available so far, so to develop a practical application for the ethanol extract and the isolated constituents as novel nematicides, further research on the safety of the extract/compounds to humans is needed.

Plant Material and Extraction
The dried rhizomes of N. incisum (5 kg) were purchased from Anguo Chinese Medicinal Herbs Market (Anguo, Hebei, China), which harvested from Gansu Province, China.The species was identified by Dr QR Liu, College of Life Sciences, Beijing Normal University, Beijing, China.A voucher specimen of N. incisum (No.CMH-Qianghuo-Gansu-2015-08) was deposited at the museum of the Department of Entomology, China Agricultural University, Beijing, China.
The dried rhizomes were cut into pieces and then successively extracted with 40 L different concentrations of ethanol/ tap water mixtures (95%, 75%, 50%, by volume) and tap water at room temperature for three days.The extracts were then filtered, mixed and concentrated under vacuum to afford crude extract (800 g).After that, the crude extract was diluted with distilled water (2 L) and then successively partitioned with the same volume of n-hexane, chloroform, ethyl acetate and n-butyl alcohol.Each partition was concentrated separately to obtain four solvent fractions (27 g, 86 g, 31 g, 32 g) and stored in brown glass bottles at 4 • C for further experiments.

Bioassay-Directed Fractionation
The crude ethanol extract and its four solvent fractions were screened for their nematicidal potential against juveniles of the two species of nematodes as described below.On the basis of 72 h mortalities at a concentration of 1000 µg/mL, the most active (chloroform fraction) was subjected to further fractionation.

Nematodes
Eggs of M. incognita were extracted from infected roots of tomato (Solanum lycopersicum L.).All the tomatoes were reared in a growth chamber (16:8 h L:D, 25-28 • C, 75%-80% RH).When reaching the five-leaf stage, tomato plants were used for inoculations; and 43 days later, infected tomato plants were uprooted and the roots were washed free of soil with tap water.Egg masses were hand-picked using sterilized tweezers from infected roots and rinsed three times with sterilized distilled water.To obtain second-stage juveniles (J2) of M. incognita, egg masses were placed on a mesh nylon filter (openings 30 µm in diameter) [56].J2s that passed through the filter were collected daily and used for bioassays immediately.
B. xylophilus was collected from sawdusts of infected pine woods in Fuling District, Chongqing City, China (29.70 • N and 107.39 • E) in September 2015, and obtained through the modified Baermann funnel technique [57].Colonies of B. xylophilus were maintained on Botrytis cinerea cultures.The fungus B. cinerea was cultured on potato dextrose agar (PDA) in a growth chamber (25-28 • C in dark).When B. cinerea was fully grown on PDA, the plates were inoculated with B. xylophilus and cultured in the growth chamber (25-28 • C in dark) until the fungal mycelium were completely consumed by B. xylophilus.Then, B. xylophilus were collected using the modified Baermann funnel technique [48], washed with a mixture of 0.1% streptomycin sulfate and 0.002% actinone three times to remove any surface bacterial or fungal contaminants and immediately used for bioassays.

Nematicidal Activity Bioassays
Nematicidal activity bioassays of M. incognita were taken under laboratory conditions at 25-28 • C. The standard nematode suspensions of M. incognita were prepared by appropriate dilution with sterilized distilled water to get approximately 100 J2s/mL.All the tested extracts or compounds were dissolved in ethanol and diluted with distilled water to obtain stock solutions of double the treatment concentrations, which were determined by a series of range-finding tests.However, the final concentration of ethanol in each treatment never exceeded 1% (by volume).Then, each well of 24-well tissue culture plates were added with 500 µL standard J2 suspension.Numbers of active J2 in every well were counted under a stereoscope at 10× and 5× before 500 µL stock solution was added to the corresponding well.Plates were then covered with rice paper to avoid evaporation.Each test was composed of five concentrations with three replicates.Commercial avermectin (purchased from Aladdin-Reagent Company, Shanghai, China)-serving as a positive control and distilled water containing ethanol (1%, by volume)-was used as a negative control.Both treated and control J2 nematodes were placed in a growth chamber at 25-28 • C in dark or in light.To measure photoactive effect, 15 min UV radiation was provided and the treated sets were placed to the growth chamber at 25-28 • C in dark.Mortality recordings were taken 72 h after treatment.J2 nematodes that showed no movements when stimulated with a fine needle were considered to be dead.
Nematicidal activity bioassays of B. xylophilus were taken as almost the same as M. incognita bioassays, but only juveniles were used.

Data Analysis
Data were corrected for control mortality using Abbott's formula [61].LC 50 and LC 90 values, along with 95% confidence limits (CLs), and chi-square values were calculated using SPSS 14.0 software.LC 50 values of the tested materials were considered to be significantly different when their 95% confidence limits (CLs) failed to overlap.Chi-square values were significant at the p < 0.05 level.

Conclusions
The present work indicated that the ethanol extract of N. incisum rhizomes and its four isolated constituents demonstrated strong nematicidal activity against B. xylophilus and Meloidogyne incognita.Our results suggested that the extract of N. incisum rhizomes and its four isolated constituents, especially falcarinol and falcarindiol, have potential for development into natural nematicides for control of plant-parasitic nematodes.

Figure 1 .
Figure 1.Constituent compounds isolated from the ethanol extract of Notopterygium incisum rhizomes.Figure 1. Constituent compounds isolated from the ethanol extract of Notopterygium incisum rhizomes.

Figure 1 .
Figure 1.Constituent compounds isolated from the ethanol extract of Notopterygium incisum rhizomes.Figure 1. Constituent compounds isolated from the ethanol extract of Notopterygium incisum rhizomes.