GC-MS Analysis of the Essential Oil from Seseli mairei H. Wolff (Apiaceae) Roots and Their Nematicidal Activity

The essential oil (EO) was extracted from aerial parts with insecticidal and fungicidal activity. Herein, the hydro-distilled essential oils of Seseli mairei H. Wolff roots were determined by GC-MS. A total of 37 components were identified, (E)-beta-caryophyllene (10.49%), β-geranylgeranyl (6.64%), (E)-2-decenal (6.17%) and germacrene-D (4.28%). The essential oil of Seseli mairei H. Wolff had nematicidal toxicity against Bursaphelenchus xylophilus with a LC50 value of 53.45 μg/mL. The subsequent bioassay-guided investigation led to the isolation of three active constituents: falcarinol, (E)-2-decenal, and octanoic acid. The falcarinol demonstrated the strongest toxicity against B. Xylophilus (LC50 = 8.52 μg/mL). The octanoic acid and (E)-2-decenal also exhibited moderate toxicity against B. xylophilus (LC50 = 65.56 and 176.34 μg/mL, respectively). The LC50 of falcarinol for the toxicity of B. xylophilus was 7.7 and 21 times than that of octanoic acid and (E)-2-decenal, respectively. Our findings demonstrate that the essential oil from Seseli mairei H. Wolff roots and their isolates may be developed as a promising natural nematicide.


Introduction
Plant-parasitic nematodes (e.g., Meloidogyne spp., Bursaphelenchus spp., Heterodera and Globodera spp.) may cause severe damage to plants and crops. It is estimated that the annual loss in global crop production due to plant-parasitic nematodes is around 8.8-14.6% of crop yields [1]. The pine wood nematode (PWN), Bursaphelenchus xylophilus, can lead to pine wilt disease, and has resulted in $ 1 billion economic losses annually [1,2]. Since its discovery in Jiangsu province, China in 1982, PWN has been found in 14 provinces of China [3], which causes extensive damage in pine forests. In the last decades, these nematodes were controlled mainly by the overuse of chemically synthesized nematicides and soil fumigants. Heavily repeated application of these chemicals has resulted in many problems such as pest resurgence, resistance to nematicides, pesticide residues in plants, groundwater contamination, and fatality in non-target organisms [4]. Therefore, it is necessary to develop the less toxic activity pesticides. Phytochemicals found in a variety of plants show a great potential in controlling nematodes [5]. Essential oil (EO) is a natural volatile substance, with complex mixture of mainly terpenoids, was found to stand out for nematicidal toxicity [4][5][6][7][8][9][10][11]. According to previous literature, some EO extracted from diverse plants such as plants in Boswellia carterii, Cymbopogon citrates, Eugenia caryophyllata, etc., exhibited nematicidal activities against PWN [4,12].
Seseli mairei H. Wolff, a perennial herbaceous plant with erect stems growing 15 to 80 cm tall, known as "Zhu Ye Fang Feng", is mainly distributed in Yunnan, Sichuan, Guizhou, and Guangxi provinces of China, as well as northern Thailand [13]. Its roots are brown cylinder with sweet taste, as herbal remedies for the treatment of inflammation, swelling, rheumatism, pain, and common cold in traditional Chinese medicine [14]. Previously phytochemical Molecules 2023, 28, 2205 2 of 7 investigation on Seseli mairei H. Wolff has identified a number of coumarins, phenylpropanoids, triterpenoids and polyacetylenes [13][14][15][16][17][18]. At present, there are no reports on the volatile compounds and nematicidal activities of Seseli mairei H. Wolff roots. Therefore, we aimed to evaluate the chemical components and nematicidal activity of EO from Seseli mairei H. Wolff roots against PWN.
In summary, the nematicidal toxicity of the EO of Seseli mairei H. Wolff and the isolated compounds, especially falcarinol, is lesser compared with the chemical nematicides, which may be developed as potential natural nematicides for the control of plant-parasitic nematodes. Therefore, plant EO can play a vital role in the management of plant-parasitic nematodes and reduce the risks possessed by synthetic chemicals. Nevertheless, further studies on the practical application of EO as novel nematicides are needed to enhance the stability and efficacy as well as to improve cost effectiveness.

Plant Material and EO Extraction
The air-dried roots ( Voucher specimen (no. 001-tssf-01657) was deposited at the museum of School of Chemistry, Biology and Environment, Yuxi Normal University, China. The plant specimens were chopped into small pieces. Each small piece (500 g) was added into 2000 mL of tap water and boiled in a Clevenger apparatus, followed by steam distillation for 6 h. Steam condensate (including EO) from distillation was harvested and placed in a flask. The EO was extracted with the same volume of n-hexane (a non-polar solvent with moderate boiling point) using a separation funnel. Evaporation of the solvent was conducted using a vacuum rotary evaporator. The samples were dried over anhydrous Na 2 SO 4 , and n-hexane was evaporated to obtain EO [10,27]. The EO was kept at 4 • C until further analysis.

Nematodes
The PWN (B. xylophilus) was extracted from chips of the infected pine wood harvested in Shuifu city, Yunnan province, China (28.63 • N latitude and 104.40 • E longitude, altitude 500 m) in September 2020, and extracted with the modified Baermann funnel method [17]. After rinsing 3 times with sterile distilled water (H 2 O), the PWN isolate was reared on Botrytis cinerea cultures. The gray mold fungus (B. cinerea) was cultured on potato dextrose agar (PDA) in a growth chamber at 27-29 • C in darkness. Subsequently, the plate was inoculated with B. xylophilus and maintained in the growth chamber at 27-29 • C in darkness until the fungal mycelium was fully digested by the PWN. After that, the PWN were harvested using the modified Baermann funnel method [31], rinsed 3 times with a mixture of 0.002% actinone and 0.1% streptomycin sulfate to eliminate any surface fungal or bacterial contaminants, and then employed for bioassays immediately.

Nematicidal Activity
Range-finding tests were performed to select the appropriate testing concentrations of EO and its isolates. Standard nematode suspension was prepared by dilution with sterilized H 2 O to obtain 100 juveniles/mL. Subsequently, 500 µL standard juvenile suspensions was introduced into a 24-well tissue culture plate. The number of active juveniles in each well was counted using a stereoscope at 5× and 10× before the addition of 500 µL stock solution. The final concentration of ethanol was less than 1% [25]. To avoid evaporation, filter paper was used to cover the plates [10]. Each test was consisted of 5 concentrations with four replicates. Rotenone (Aladdin, Shanghai, China) and H 2 O containing ethanol (1%) were employed as positive and negative controls, respectively. Both control and treated juveniles were incubated in the growth chamber at 27-29 • C in darkness. After treatment for 72 h, the mortality rate was determined. Juveniles were considered to be dead if they had no movement after stimulation with a fine needle.

GC-MS Analysis
GC-MS analysis was conducted using an Agilent system containing an Agilent Chem Station, a model 5973N mass selective detector, and a model 6890N gas chromatograph (Agilent Technologies Inc, City of Santa Clara, USA). The GC column was HP-5ms Capillary GC column [poly-(5%-diphenyl/95%-dimethylsiloxane), 30 m × 0.25 mm, 0.25 μm film thickness]. The oven temperature was initiated at 60 °C for 60 s, increased at 10 °C/min to 180 °C for 60 s, and further increased at 20 °C/min to 280 °C for 15 min. The injector temperature was kept at 270 °C. The sample (1 μL,) was diluted with acetone (1:100), and then injected at a ratio of 1:10. Helium was employed as the carrier gas, and the flow rate was 1.0 mL/min. Spectral scanning was conducted within the range of 20-550 m/z at 2 scans/s. The EO components were identified by comparing their mass spectra and retention index according to previous literature [32,33] and presented in NIST 98 as well as those of authentic compounds prepared in our laboratory. A homologues series of n-alkanes (C8-C24) were employed as reference points for calculating retention indices. Component relative percentage was determined according to GC peak areas without using the correction factors.

GC-MS Analysis
GC-MS analysis was conducted using an Agilent system containing an Agilent Chem Station, a model 5973N mass selective detector, and a model 6890N gas chromatograph (Agilent Technologies Inc, City of Santa Clara, USA). The GC column was HP-5ms Capillary GC column [poly-(5%-diphenyl/95%-dimethylsiloxane), 30 m × 0.25 mm, 0.25 µm film thickness]. The oven temperature was initiated at 60 • C for 60 s, increased at 10 • C/min to 180 • C for 60 s, and further increased at 20 • C/min to 280 • C for 15 min. The injector temperature was kept at 270 • C. The sample (1 µL,) was diluted with acetone (1:100), and then injected at a ratio of 1:10. Helium was employed as the carrier gas, and the flow rate was 1.0 mL/min. Spectral scanning was conducted within the range of 20-550 m/z at 2 scans/s. The EO components were identified by comparing their mass spectra and retention index according to previous literature [32,33] and presented in NIST 98 as well as those of authentic compounds prepared in our laboratory. A homologues series of n-alkanes (C 8 -C 24 ) were employed as reference points for calculating retention indices. Component relative percentage was determined according to GC peak areas without using the correction factors.

Data Analysis
Abbott's formula was used to correct the mortality data. To calculate LC 50 values and their 95% confidence intervals, Probit analysis was conducted on the obtained results using the PriProbit Program version 1.6.3 (Kyoto University, Kyoto, Japan) [34].