Activity of the Ethanolic Extract Obtained from Citrus microcarpa Pericarps against Meloidogyne enterolobii, and Chemical Composition Analysis

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Xiaoli Dou College of Tropical Agriculture and Forestry/Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, Hainan University, Haikou 570228, China

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Yani Wu College of Tropical Agriculture and Forestry/Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, Hainan University, Haikou 570228, China

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Jiguang Luo Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Xiaopeng Yin Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Meiying Fu Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Xiangping Zeng Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Huifang Wang Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Abstract

Citrus microcarpa is a popular nutritious fruit that is widely cultivated in China. In recent years, many compounds with significant pharmacological activities have been isolated successfully from the pericarp of C. microcarpa. However, to date, there are no reports on the activity of C. microcarpa pericarp against root-knot nematodes. This study used the ethanolic extract from the pericarp of Hainan C. microcarpa and the impregnation method to determine its activity on J2 Meloidogyne enterolobii specimens and on single-egg hatching. The results showed that when J2 individuals were treated with 50 mg⋅mL–1 of the extract, the lethal concentration 50 values after 24 and 48 hours were 17.124 and 8.858 mg⋅mL–1, respectively. The mortality rate of nematodes after 48 hours of treatment was 100%, and the inhibition rate of single-egg hatching after 24 hours was 89.29%. The ethanolic extract of C. microcarpa peels showed high inhibitory and lethal activity against the M. enterolobii. The analysis of the chemical composition of the extract revealed 28 substances with insecticidal and antibacterial effects, including lignans, flavonoids, fatty acids, organic acids, terpenoids, and imidazole. The formulas of the chemical structures and pharmacological effects of these potential insecticidal and antibacterial substances were elucidated to provide a scientific basis and a theoretical reference for the use of C. microcarpa pericarps as a raw material for the development of new, natural plant nematicides.

Meloidogyne enterolobii is one of the most harmful root-knot nematodes (Kiewnick et al. 2009). It was first reported on elephant ear bean trees in Danzhou City, Hainan Province, China, in 1983, and is currently found in various regions of the world, including the Americas, Africa, and Europe (Lian et al. 2015). This nematode has a wide host range that includes most monocotyledonous crop species, dicotyledonous grasses, and woody plants (Liu et al. 2024). Even crops such as tomatoes (Mi-1 gene), chili peppers (N gene), and cowpeas (Rk gene) carrying disease resistance genes can be parasitized, resulting in heavy yield losses that can reach more than 65% (Long et al. 2015). Recent investigations have reported that, except for Wuzhishan City, all crops in 18 cities and counties in Hainan Province, China, were affected by the root-knot nematode of elephant ear bean, Meloidogyne enterolobii, which has thus become a dominant pathogenic nematode population in crops grown in Hainan Province, where prevention and control of M. enterolobii has become of paramount importance for the rescue of agricultural production (Li et al. 2020; Yang and Eisenback 1983).

Citrus peels are rich in essential volatile oils and have a special aroma. The multicomponent, natural flavoring-oil extracted from these peels is highly valued for its numerous applications in the field of insecticidal and bactericidal compound development (Zhang et al. 2024). Thus, for example, the Citrus ethanolic extract shows inhibitory activity on Lepidoptera (Spodoptera frugiperda) and Diptera (Bactrocera oleae, Ceratitis capitata adults, and Aedes aegypti) (Sarma et al. 2019; Siskos et al. 2009; Villafañe et al. 2011). Similarly, lemon peel extract shows inhibitory activity against gram-positive bacteria (Ekwenye and Edeha 2010; Harfouch et al. 2019). In turn, Citrus extract shows excellent antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Salmonella (Lin et al. 2021; Meng 2013; Roanisca and Mahardika 2020). Citrus microcarpa is an evergreen shrub of the Citrus L. genus in the Rutaceae family. A native to China, it is mainly distributed in Hainan Province (Duan et al. 2015). Unlike other Citrus species, C. microcarpa has very high nutritional and medicinal values, such that it is known as the “king of plants.” In ancient times, the unique medicinal value of C. microcarpa was recorded in the Compendium of Materia Medica, which states that it has strong effects, including generating fluids, quenching thirst, strengthening the stomach, and reducing digestion. The C. microcarpa pericarp contains abundant bioactive substances such as flavonoids, coumarins, alkaloids, and volatile oils with various effects, including antibacterial, insecticidal, and antiviral effects (Cao and Pan 2022). Indeed, in recent years, the variety of pharmacological activities of C. microcarpa have been reported often. Thus, for example, using gas chromatography (GC)–mass spectrometry (MS) technology, Su et al. (2020) identified the essential oil component limonene, the greatest content of which was observed in the pericarp of C. macrocarpa. Limonene shows biological activity against crop diseases and pests, as well as against weeds (Liu 2019). Furthermore, its lethal effects on rhizobial nematodes have been confirmed in Indian soils (Saxena et al. 1987). Thus, C. microcarpa pericarps seemingly contain substances with an inhibitory effect on M. enterolobii.

To date, chemical control remains the prevailing tool for root-knot nematode control, with the concomitant threat of the environmentally dangerous residual effects of chemical control agents. Alternatively, plant-derived pesticides are characterized by low toxicity, easy degradation in natural environments, no residual effects, and no accumulation in the environment. Given all these advantages, these plant-derived compounds are safe to use and protect the environment. Therefore, developing and using plant-based pesticides to reduce chemical control of crop diseases, pests, and weeds is in line with current needs of human society and nature (Liang 2019). Hence the efforts involved in the search for plant-based pesticides have become a research hot spot (Dan et al. 2011; Liu 2019). However, to date, neither the activity associated with root-knot nematodes, the chemical structural formulas, nor the pharmacological effects of these compounds have been reported. We study explored the inhibitory activity of the ethanolic extract from C. microcarpa pericarps on M. enterolobii and screened potential active substances preliminarily. The chemical structural formulas and pharmacological effects of the active substances were elucidated, thus providing a scientific basis and a theoretical reference for the development of C. microcarpa pericarps as a new, natural plant-derived raw material for the development of environmentally friendly effective nematicides.

Materials and Methods

Test plants

The test plant material, C. macrocarpa fruit, was purchased in Jun 2023 at a fruit market in the Qiongshan District, Haikou City, Hainan Province, China. Fruit with lime-green, fresh, and smooth skin were selected and confirmed to be C. microcarpa by Chen Xuyu (Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences & Peking Union Medical College, Haikou City, Hainan Province, China).

Test nematode

Second-stage juvenile (J2) specimens of the root-knot nematode M. enterolobii were collected from pepper crop plants in Honghua Village, Jiyang District, Sanya City, Hainan Province, China (lat. 18.339471°N, long. 109.569134°E) and were reared in the laboratory of the Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Hainan Province, Haikou, China).

Instrumentation and reagents

Reagents.

The reagents used in the experiments described included pure ethanol, analytical grade (Hengshun Chemical Co., Ltd., Wenzhou, China); dimethyl sulfoxide (DMSO; Shanghai McLean Biochemical Technology Co., Ltd., China), 90% abamectin original powder (Aino Pharmaceutical Co., Ltd., North China); 2-octanol, methoxamine salt (Tokyo Chemical Industry Co., Ltd., Japan); chloroform and pyridine (Shanghai Adamas Reagent Co., Ltd., China); ribol (Shima Co., Ltd., Japan); BSTFA (with 1% trimethylchlorosilane, v/v; REGIS Technologies, IL, Morton Grove, USA); FAMEs (Dr. Ehrenstorfer GBH, Augsburg, Germany), and double distilled (ddH2O).

Instrumentation.

The instruments used in the experiments included a rotating evaporator (Heidelberg, Schwarzbach, Germany); a 7890A gas chromatograph, a 7890B gas chromatograph, a 5977B mass spectrometer, a DB-5MS (30 m × 250 μM × 0.25 μm) and a DB Wax (30 m × 250 μM × 0.25 μm) chromatographic column (Agilent, Santa Clara, CA, USA); a PEGASUS HT mass spectrometer (Lico, San Jose, USA); a Heraeus Fresco17 centrifuge and a Forma 900 series ultra-low temperature refrigerator (Thermo Fisher Scientific, Waltham, MA, USA); a BSA124S-CW analytical balance (Sedolis Scientific Instruments Co., Ltd., Beijing, China); a JXFSTPRP-24 grinder (Jingxin Technology Co., Ltd., Shanghai, China); a YM-080S ultrasonic instrument (Fangao Microelectronics Co., Ltd., Shenzhen, China); a DHG-9023A oven (Yiheng Scientific Instrument Co., Ltd., Shanghai, China), and a LNG-T98 vacuum dryer (Huamei Biochemical Instrument Factory, Taicang City, China).

Root-knot nematode species validation

Nematode DNA was extracted as described by Wang et al. (2011). The molecular identification of root-knot nematode referred to the method of Long et al. (2006), using sequence characterized amplified region (SCAR)-labeled primers Me-F/Me-R (5′-AACTTTTGTGAAAGTGCCGCTG-3′/5′-TCAGTTCAGGCAGGATCAACC-3′) of the root-knot nematode to amplify specific bands. The primers used in this experiment were synthesized by Sangong Bioengineering Technology Service Co., Shanghai, China.

Preparation of plant extracts

Using an ultrasonic extraction method, C. microcarpa pericarps were peeled off and cleaned of impurities on the surface with sterile water. Pericarps were then placed in a constant-temperature oven at 60 °C and dried to a constant mass. A wall crusher was used to crush fruit peels, which were then passed through a 50-μm mesh sieve. Dried pericarp powder samples (10 g each) were placed in 500-mL triangular flasks, to which was added 100 mL of 95% ethanol solution in a 1:10 ratio. Ultrasound extraction was performed at 35 °C at 80 W of power for 2 h before filtration. The obtained filtrate was evaporated at 40 °C using a rotary evaporator to form a paste, which was then stored at 4 °C until use.

Determination of nematicidal activity

Using the maceration method, a nematode suspension with 100 nematodes in it was added to each well on a 24-well cell culture plate with 500 μL of the sample solution, which was brought to 1 mL with sterile water to yield sample concentrations of 50, 25, 12.5, 6.25, and 3.125 mg⋅mL–1. Because the ethanolic extract from C. microcarpa pericarp turned into a paste after evaporation and required the use of organic solvents for solubilization, we tested using acetone and DMSO for such solubilization of the extract. We found that acetone, but not DMSO, caused J2 juveniles to die; thus, DMSO was ultimately selected as the cosolvent. To eliminate the influence of the cosolvent, DMSO was set as a blank control, 90% abamectin original powder as a pharmaceutical control, and sterile water as a negative control. Each concentration was tested in triplicate. After placing the processed cell culture plate at 25 °C for 24 and 48 h, treated nematodes were transferred to a centrifuge tube for centrifugation, after which the supernatants were aspirated carefully. Nematodes were washed twice with sterile water and placed into a 3-cm culture dish, and sterile water was added to resuscitate them. After 48 h of resuscitation, nematodes were observed using a stereomicroscope. Living nematodes show a curved body shape and move by peristaltic coiling and extending, whereas dead nematodes appear stiff. Using acupuncture, if nematodes remain stiff and do not move, it is judged they are dead (Wang et al. 2013). The mortality rate and the corrected mortality rate of nematodes were calculated per Eqs. [1] and [2], respectively:
Mortality rate =(No. of dead nematodesNo. of nematodes in the treatment)× 100%
and
Corrected mortality rate =(Treatment nematode mortality rate  Control nematode mortality rate1Control nematode mortality rate)× 100%.

Effects of plant extracts on single-egg hatching of nematodes

Fresh, plump yellow-brown egg sacs were selected for incubation experiments. Egg sacs were placed in a 1.0% sodium hypochlorite solution and shaken for 1 min before centrifugation. The supernatant was discarded and the bottom solution was washed three times with sterilized water. Subsequently, water was added and the mixture was shaken to collect the supernatant to obtain a single egg suspension (Haseeb et al. 2005; Lu et al. 2006). A single-egg suspension (100 particles/well) was added to each well in a 12-well cell culture plate; then, 500 μL of sample solution was added to each well, and sterile water was used to bring the volume in each well to 1 mL for a final sample concentration of 50, 25, 12.5, 6.25, or 3.125 mg⋅mL–1. Sterile water and DMSO were used as controls, and each treatment was repeated three times. After treatment at 25 °C for 4, 8, 16, 24, and 48 h, the extraction solution adhered to the surface of the egg was centrifuged and washed with sterile water. The egg was then placed in sterile water for further incubation at 25 °C in an incubator. After 4 d, egg hatching was observed using a stereomicroscope. The hatching and incubation inhibition rates were calculated according to Qi et al. (2011), using Eqs. [3] and [4], respectively:
Hatching rate =(Total no. of incubationsTotal no. of eggs)×100%
and
Incubation inhibition rate=(Control hatching rateTreatment hatching rateControl hatching rate)×100%.

Analysis of the chemical composition of C. microcarpa peel extracts

To identify the bioactive substances in C. microcarpa pericarps, ultrahigh-performance liquid chromatography (UHPLC)–MS was used to separate compounds that are difficult to volatilize and are thermally unstable. GC-MS was used to separate volatile and thermally stable compounds, and GC-MS-volatile organic compounds (VOCs) was used to separate flavor-volatiles and characterize the active compounds comprehensively.

UHPLC-MS analysis.

A total of 100 μL of the supernatant of the ethanolic pericarp extract was placed in a feed vial and analyzed by UHPLC-MS.

GC–time-of-flight-MS analysis.

Analysis by GC–time-of-flight (TOF)–MS was performed using an Agilent 7890 gas chromatograph coupled with a TOF mass spectrometer. The system used a DB-5MS capillary column. A 1-μL aliquot of sample was injected in splitless mode. Helium was used as the carrier gas. The front inlet purge flow was 3 mL⋅min–1, and the gas flow rate through the column was 1 mL⋅min–1. The initial temperature was kept at 50 °C for 1 min, raised to 310 °C at a rate of 10 °C⋅min–1, and kept at that point for 8 min. The injection, transfer line, and ion source temperatures were 280, 280, and 250 °C, respectively. The energy used was –70 eV in the electron impact mode. MS data were acquired in full-scan mode with an m/z range of 50 to 500 at a rate of 12.5 spectra per second after a solvent delay of 6.4 min.

Analysis of GC-MS-VOCs.

The solid-phase microextraction (SPME) cycle of the PAL rail system (a liquid automatic sampler system) was as follows: the incubation temperature was 60 °C, the preheat time was 15 min, the incubation time was 30 min, and the desorption time was 4 min. GC-MS analysis was performed using an Agilent 7890 gas chromatograph system coupled with a 5977B mass spectrometer. The system used DB-Wax, and the injection was in the splitless mode. Helium was used as the carrier gas, the front inlet purge flow was 3 mL⋅min–1, and the gas flow rate through the column was 1 mL⋅min–1. The initial temperature was kept at 40 °C for 4 min, then was raised to 245 °C at a rate of 5 °C⋅min–1, and was kept at that point for 5 min. The injection, transfer line, ion source, and quad temperatures were 250, 250, 230, and 150 °C, respectively. The energy was –70 eV in electron impact mode. MS data were acquired in scan mode with an m/z range of 20 to 400 and a solvent delay of 2.13 min.

Data processing and analysis

Data processing for root-knot nematode bioactivity assay.

The data were analyzed using Excel (WPS Office 12.1.0.16729) and IBM SPSS Statistics 27 statistical analysis software to calculate the mortality rate in each treatment and to determine the lethal concentration 50 (LC50) value of the C. microcarpa peel extract.

Qualitative analysis of the chemical composition of the pericarp.

The peak extraction, baseline correction, deconvolution, peak integration, peak alignment, and mass spectral matching were performed to analyze the mass spectral data using ChromaTOF software v. 4.3x (LECO) (Dunn et al. 2011; Kind et al. 2009). Using the LECO-Fiehn Rtx5 database, mass spectral and retention time index matching were performed, combined, and compared with the compounds in the relevant databases (KEGG and HMDB, as well as the self-constructed database of Shanghai Baiyi Biomedical Technology Co. Ltd.) to characterize the substances present in the ethanolic extract of the pericarps.

Results

Confirmation of root-knot nematode species identity

Using the SCAR–polymerase chain reaction identification method, a single band of 236 bp was identified using Me-F/Me-R–specific primers for the root-knot nematode M. enterolobii. No other bands were observed (Fig. 1).

Fig. 1.
Fig. 1.

Amplification results of sequence characterized amplified region marker-specific primers for root-knot nematodes. M = DNA marker D2000; 1–5 = root-knot nematode Meloidogyne enterolobii; 6 = blank control.

Citation: HortScience 59, 6; 10.21273/HORTSCI17817-24

Nematocidal effect of C. microcarpa pericarp extracts

The bioactivity assay results showed that the ethanolic extract of the pericarps exhibited strong bioactivity against J2 M. enterolobii, which was significantly greater than those of the blank control (DMSO) and the negative control (sterile water). Furthermore, the inhibitory activity tended to increase with time. The LC50 value was 17.124 and 8.858 mg⋅mL–1 after 24 and 48 h of treatment, respectively, and the nematode mortality rate reached 100% after 48 h at 50 mg⋅mL–1 of pericarp extract (Tables 1 and 2).

Table 1.

Toxicity of different treatments against J2 Meloidogyne enterolobii.

Table 1.
Table 2.

Toxicity of Citrus microcarpa pericarp extract on Meloidogyne enterolobii.

Table 2.

Effects of C. microcarpa pericarp extract on the hatching of nematode eggs

The ethanol extract of C. microcarpa pericarps at different concentrations has inhibitory effects on the hatching of nematode eggs in M. enterolobii. With the extension of treatment time and the increase of concentration, the inhibition rate of egg hatching also increases. The concentration of 50 mg⋅mL–1 has the best effect on nematode egg hatching, and the inhibition rate reaches 89.29% after 24 h of treatment (Fig. 2).

Fig. 2.
Fig. 2.

Effects of Citrus microcarpa pericarp extract on the hatching of nematode eggs. The data in the figure are mean ± the standard error. Different letters within the same column group show significant differences at P < 0.05.

Citation: HortScience 59, 6; 10.21273/HORTSCI17817-24

Chemical composition analysis of C. microcarpa pericarps

UHPLC-MS analysis.

UHPLC-MS analysis yielded 611 peaks that were analyzed by reviewing the literature. Eight lignans and flavonoids with insecticidal and bacteriostatic effects were screened from the extract preliminarily (Table 3; Fig. 3).

Table 3.

Ultrahigh performance liquid chromatography–mass spectrometry analysis of some portions of the ethanolic extract of C. microcarpa pericarps.

Table 3.
Fig. 3.
Fig. 3.

Total ion flow diagram of ultrahigh performance liquid chromatography–mass spectrometry of the ethanolic extract of Citrus microcarpa pericarps. (A) Positive ion mode. (B) Negative ion mode.

Citation: HortScience 59, 6; 10.21273/HORTSCI17817-24

GC-TOF-MS analysis.

The GC-TOF-MS analysis revealed 671 peaks that were analyzed through a review of the literature. Preliminary screening of the extract revealed 10 organic acids and fatty acids with insecticidal and bacteriostatic effects (Table 4; Fig. 4).

Table 4.

Gas chromatography–time-of-flight mass spectrometry analysis of some chemical constituents of the ethanolic extract of C. microcarpa pericarps.

Table 4.
Fig. 4.
Fig. 4.

Total ion flow diagram of gas chromatography–time-of-flight mass spectrometry of the ethanolic extract of Citrus microcarpa pericarps. TIC = total ion current.

Citation: HortScience 59, 6; 10.21273/HORTSCI17817-24

GC-MS-VOC analysis.

The GC-MS-VOC analysis yielded 1427 peaks, and preliminary screening of 10 terpenoids and imidazoles with insecticidal and bacteriostatic effects from the extract was achieved by review of the literature (Table 5; Fig. 5).

Table 5.

Gas chromatography–mass spectrometry–volatile organic compound analysis of some chemical constituents of the ethanolic extract of Citrus microcarpa pericarps.

Table 5.
Fig. 5.
Fig. 5.

Citrus microcarpa pericarp headspace volatility gas chromatography–mass spectrometry–volatile organic compounds total ion flow map. TIC = total ion current.

Citation: HortScience 59, 6; 10.21273/HORTSCI17817-24

Discussion

There are many types of citrus plants that have both edible and pharmacological value. Furthermore, to date, numerous studies have reported on the insecticidal and antibacterial activities of extracts from citrus medicinal plants. Our study found that the ethanolic extract of the pericarp of the local plant resource C. macrocarpa, grown in Hainan, China, has strong biological activity against J2 M. enterolobii. and hatching of its eggs. Specifically, using UHPLC-MS, GC-TOF-MS, and GC-MS-VOCs, we detected 2979 active substances through chemical composition analysis of ethanolic extracts. In addition, we organized and analyzed 28 of these substances with insecticidal and antibacterial effects, including lignans, flavonoids, fatty acids, organic acids, terpenes, and imidazoles (Frota et al. 2023; Liu et al. 2002; Ma and Gu 2003). In recent years, researchers have isolated more than 100 monomeric components from medicinal plants, including flavonoids, coumarins, alkaloids, organic acids, and terpenoids that exhibit strong nematicidal activity. Furthermore, there are studies reporting that flavonoids such as luteolin and quercetin can inhibit plant parasitic nematodes (Bano et al. 2020; Chin et al. 2018); terpene compounds, such as carvacrol and the aromatic compound eugenol, which can effectively kill Caenorhabditis elegans (Tsao and Yu 2000); monoterpenoid compounds, such as geraniol, aromatic alcohol, citral, and so on (Abdel-Rahman et al. 2013), which have strong lethal effects on threads; organic acid compounds, such as oxalic and citric acids, which have high inhibitory activity on southern root-knot nematodes (Liu et al. 2011); and the aldehyde cinnamaldehyde, which has high inhibitory activity against southern root-knot nematodes, Poaceae root-knot nematodes, and pine wood nematodes (D’Addabbo et al. 2020). The results of our study further indicated that the ethanolic extract of C. microcarpa pericarp contains substances with strong inhibitory and lethal effects on M. enterolobii, thus providing a sound basis for the development of plant-based nematicides from C. macrocarpa extracts. However, we investigated only the nematicidal activity of ethanolic extracts from C. macrocarpa. Further research is needed to establish unequivocally the identity of the components that play a major role, and the underlying mechanism of action.

Conclusion

In our study ethanol was used as a solvent to analyze the chemical components in the ethanolic extracts from C. microcarpa pericarps. Twenty-eight potential active substances were identified along with their corresponding chemical structural formulas. The compounds that have been reported to show strong nematode inhibitory activity in plants were isolated from the ethanolic extract obtained from C. microcarpa pericarps, indicating that such extract contains more nematicidal bioactive substances, and that 95% ethanol is effective for the extraction of bioactive substances from the pericarp. The chemical composition and pharmacological effects of plant extracts are the foundation and core of the development of plant-based pesticides. The composition and content of substances extracted by different solvents may vary, and the biological activities exhibited may also vary. Thus, for example, Peng (2014) pointed out that different extracts have the following effects on larvae upon contact: killing, growth inhibition, and delayed molting time. Solvent efficacy with respect to these effects ranked in the following order: ethanol extract > chloroform extract > acetone extract, which confirmed that ethanol has the greatest biological activity as an extraction solvent. Therefore, in future research, other solvents can be used for the extraction of bioactive compounds from plant tissues to compare their biological activities, such as to obtain other effective compounds and provide a basis for the development of plant-derived nematicides and other insecticidal and antibacterial bioactive substances.

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  • Kind T, Wohlgemuth G, Lee DY, Lu Y, Palazoglu M, Shahbaz S, Fiehn O. 2009. FiehnLib: Mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Anal Chem. 81(24):1003810048. https://doi.org/10.1021/ac9019522.

    • Search Google Scholar
    • Export Citation
  • Li Z, Long H, Sun Y, et al. 2020. Occurrence and distribution of root-knot nematodes in vegetables in Hainan Province. Plant Protect. 46(6):213–216, 245. https://doi.org/10.16688/j.zwbh.2019440.

    • Search Google Scholar
    • Export Citation
  • Lian DM, Wang HF, Zhao ZX, et al. 2015. Cloning and prokaryotic expression of the Hsp70 gene in Meloidogyae erterolohii. J Plant Pathol. 45(1):713. https://doi.org/10.13926/j.cnki.apps.2015.01.002.

    • Search Google Scholar
    • Export Citation
  • Liang C. 2019. Screening and component identification of extracts from 10 tropical plants against plant pathogenic fungi and root knot nematodes (PhD Diss). Hainan University, Haikou, China. https://doi.org/10.27073/d.cnki.ghadu.2019.000853.

  • Lin X, Cao S, Sun J, Lu D, Zhong B, Chun J. 2021. The chemical compositions, and antibacterial and antioxidant activities of four types of citrus essential oils. Molecules. 26(11):3412. https://doi.org/10.3390/molecules26113412.

    • Search Google Scholar
    • Export Citation
  • Liu P. 2019. Main types and application prospects of plant-based insecticides. Qinghai Agric For Technol. (4):57–60, 68.

  • Liu DD, Duan YX, Chen LJ, et al. 2011. Toxicity and greenhouse control effects of six plant derived compounds on southern root knot nematodes. Henan Agric Sci. 40(5):111113. https://doi.org/10.15933/j.cnki.1004-3268.2011.05.018.

    • Search Google Scholar
    • Export Citation
  • Liu GQ, Gao JM, Wu WJ. 2002. New progress in research on plant derived insecticidal components. Northwest Botanical J. 22(3):703713. https://doi.org/10.3321/j.issn:1000-4025.2002.03.037.

    • Search Google Scholar
    • Export Citation
  • Liu Y, Li CY, Yao ZH, et al. 2024. Research progress on chemical control of crop root knot nematode disease. J Pest Sci. https://doi.org/10.16801/j.issn.1008-7303.2024.0004.

    • Search Google Scholar
    • Export Citation
  • Liu YQ, Liu ZX, Gao B, et al. 2019. Progress in the application of limonene in the field of pesticides. China Plant Protect J. 39(8).

  • Long H, Liu H, Xu JH. 2006. PCR identification and detection method for M. enterolobii. J Plant Pathol. 36(2):109115. https://doi.org/10.13926/j.cnki.apps.2006.02.003.

    • Search Google Scholar
    • Export Citation
  • Long H, Sun Y, Bai C, et al. 2015. Identification study of Meloidogyae erterolohii in Hainan Province. J Trop Crops. 36(2):371376.

  • Ma CZ, Gu ZR. 2003. Fatty acid compounds have lethal effects on insects and field control experiments on cabbage insects. Shanghai Agric J. 19(3):101104. https://doi.org/10.3969/j.issn.1000-3924.2003.03.029.

    • Search Google Scholar
    • Export Citation
  • Meng P. 2013. Extraction, purification, structural identification, and biological activity study of limonoids from golden citrus (PhD Diss). Fujian Agriculture and Forestry University, Fuzhou, China.

  • Nasiou E, Giannakou OI. 2018. Effect of geraniol, a plant-based alcohol monoterpene oil, against Meloidogyne javanica. Eur J Plant Pathol. 152(3):701710. https://doi.org/10.1007/s10658-018-1512-x.

    • Search Google Scholar
    • Export Citation
  • Peng H. 2014. Extraction and bioassay of insecticidal active substances from water hyacinth (PhD Diss). Hainan Normal University, Haikou, China. https://doi.org/10.7666/d.D345001.

  • Qi YH, Cao SF, Lv HP, Du H. 2011. The effects of different pesticides on the hatching of southern root knot nematode eggs and the activity of 2nd instar larvae. Northwest Agric J. 20(9):184189. https://doi.org/10.3969/j.issn.1004-1389.2011.09.037.

    • Search Google Scholar
    • Export Citation
  • Roanisca O, Mahardika RG. 2020. Citrus × microcarpa bunge fruit extract as antibacterial against Staphylococcus aureus. IOP Conf Ser Earth Environ Sci. 599:012043. https://doi.org/10.1088/1755-1315/599/1/012043.

    • Search Google Scholar
    • Export Citation
  • Sarma R, Adhikari K, Mahanta S, Khanikor B. 2019. Insecticidal activities of Citrus aurantifolia essential oil against Aedes aegypti (Diptera: Culicidae). Toxicol Rep. 6:10911096. https://doi.org/10.1016/j.toxrep.2019.10.009.

    • Search Google Scholar
    • Export Citation
  • Saxena DB, Goswami BK, Tomar SJ. 1987. Nematicidal activity of some essential oils against Meloidogyne incognita. Nematologia Mediterranea. 8(2):195201.

    • Search Google Scholar
    • Export Citation
  • Siskos EP, Konstantopoulou MA, Mazomenos BE. 2009. Insecticidal activity of Citrus aurantium peel extract against Bactrocera oleae and Ceratitis capitata adults (Diptera: Tephritidae). J Appl Entomol. 133(2):108116. https://doi.org/10.1111/j.1439-0418.2008.01312.x.

    • Search Google Scholar
    • Export Citation
  • Su M, Yang J, Zhou X, et al. 2020. GC-MS analysis of the essential oil composition of kumquat rind. Guangzhou Chemical Indus. 48(10):8993.

    • Search Google Scholar
    • Export Citation
  • Tsao R, Yu Q. 2000. Nematicidal activity of monoterpenoid compounds against economically important nematodes in agriculture. J Essent Oil Res. 12(3):350354. https://doi.org/10.1080/10412905.2000.9699533.

    • Search Google Scholar
    • Export Citation
  • Villafañe E, Tolosa D, Bardón A, Neske A. 2011. Toxic effects of Citrus aurantium and C. limon essential oils on Spodoptera frugiperda (Lepidoptera: Noctuidae). Nat Prod Commun. 6(9):13891392. https://doi.org/10.1002/med.20253.

    • Search Google Scholar
    • Export Citation
  • Wang LP, Wan XC, Hou RY, et al. 2013. The inhibitory effect of plant extracts and preparations such as Camellia oleifera. Anhui Nongye Daxue Xuebao. 40(4):642648. https://doi.org/10.13610/j.cnki.1672-352x.2013.04.011.

    • Search Google Scholar
    • Export Citation
  • Wang JL, Zhang JC, Gu JF. 2011. DNA extraction method for single nematode. Plant Quarantine. 25(2):3235.

  • Yang B, Eisenback JD. 1983. Meloidogyne enterolobii n. sp. (Meloidogynidae), a root-knot nematode parasitizing pacara earpod tree in China. J Nematol. 15(3):381391. https://doi.org/10.2307/1380543.

    • Search Google Scholar
    • Export Citation
  • Zhang FY, Ye WF, Wang XC, et al. 2024. Fingerprint analysis of essential oils from citrus plants. Guangdong Chemical. 51(4):139142.

  • Fig. 1.

    Amplification results of sequence characterized amplified region marker-specific primers for root-knot nematodes. M = DNA marker D2000; 1–5 = root-knot nematode Meloidogyne enterolobii; 6 = blank control.

  • Fig. 2.

    Effects of Citrus microcarpa pericarp extract on the hatching of nematode eggs. The data in the figure are mean ± the standard error. Different letters within the same column group show significant differences at P < 0.05.

  • Fig. 3.

    Total ion flow diagram of ultrahigh performance liquid chromatography–mass spectrometry of the ethanolic extract of Citrus microcarpa pericarps. (A) Positive ion mode. (B) Negative ion mode.

  • Fig. 4.

    Total ion flow diagram of gas chromatography–time-of-flight mass spectrometry of the ethanolic extract of Citrus microcarpa pericarps. TIC = total ion current.

  • Fig. 5.

    Citrus microcarpa pericarp headspace volatility gas chromatography–mass spectrometry–volatile organic compounds total ion flow map. TIC = total ion current.

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  • Kind T, Wohlgemuth G, Lee DY, Lu Y, Palazoglu M, Shahbaz S, Fiehn O. 2009. FiehnLib: Mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Anal Chem. 81(24):1003810048. https://doi.org/10.1021/ac9019522.

    • Search Google Scholar
    • Export Citation
  • Li Z, Long H, Sun Y, et al. 2020. Occurrence and distribution of root-knot nematodes in vegetables in Hainan Province. Plant Protect. 46(6):213–216, 245. https://doi.org/10.16688/j.zwbh.2019440.

    • Search Google Scholar
    • Export Citation
  • Lian DM, Wang HF, Zhao ZX, et al. 2015. Cloning and prokaryotic expression of the Hsp70 gene in Meloidogyae erterolohii. J Plant Pathol. 45(1):713. https://doi.org/10.13926/j.cnki.apps.2015.01.002.

    • Search Google Scholar
    • Export Citation
  • Liang C. 2019. Screening and component identification of extracts from 10 tropical plants against plant pathogenic fungi and root knot nematodes (PhD Diss). Hainan University, Haikou, China. https://doi.org/10.27073/d.cnki.ghadu.2019.000853.

  • Lin X, Cao S, Sun J, Lu D, Zhong B, Chun J. 2021. The chemical compositions, and antibacterial and antioxidant activities of four types of citrus essential oils. Molecules. 26(11):3412. https://doi.org/10.3390/molecules26113412.

    • Search Google Scholar
    • Export Citation
  • Liu P. 2019. Main types and application prospects of plant-based insecticides. Qinghai Agric For Technol. (4):57–60, 68.

  • Liu DD, Duan YX, Chen LJ, et al. 2011. Toxicity and greenhouse control effects of six plant derived compounds on southern root knot nematodes. Henan Agric Sci. 40(5):111113. https://doi.org/10.15933/j.cnki.1004-3268.2011.05.018.

    • Search Google Scholar
    • Export Citation
  • Liu GQ, Gao JM, Wu WJ. 2002. New progress in research on plant derived insecticidal components. Northwest Botanical J. 22(3):703713. https://doi.org/10.3321/j.issn:1000-4025.2002.03.037.

    • Search Google Scholar
    • Export Citation
  • Liu Y, Li CY, Yao ZH, et al. 2024. Research progress on chemical control of crop root knot nematode disease. J Pest Sci. https://doi.org/10.16801/j.issn.1008-7303.2024.0004.

    • Search Google Scholar
    • Export Citation
  • Liu YQ, Liu ZX, Gao B, et al. 2019. Progress in the application of limonene in the field of pesticides. China Plant Protect J. 39(8).

  • Long H, Liu H, Xu JH. 2006. PCR identification and detection method for M. enterolobii. J Plant Pathol. 36(2):109115. https://doi.org/10.13926/j.cnki.apps.2006.02.003.

    • Search Google Scholar
    • Export Citation
  • Long H, Sun Y, Bai C, et al. 2015. Identification study of Meloidogyae erterolohii in Hainan Province. J Trop Crops. 36(2):371376.

  • Ma CZ, Gu ZR. 2003. Fatty acid compounds have lethal effects on insects and field control experiments on cabbage insects. Shanghai Agric J. 19(3):101104. https://doi.org/10.3969/j.issn.1000-3924.2003.03.029.

    • Search Google Scholar
    • Export Citation
  • Meng P. 2013. Extraction, purification, structural identification, and biological activity study of limonoids from golden citrus (PhD Diss). Fujian Agriculture and Forestry University, Fuzhou, China.

  • Nasiou E, Giannakou OI. 2018. Effect of geraniol, a plant-based alcohol monoterpene oil, against Meloidogyne javanica. Eur J Plant Pathol. 152(3):701710. https://doi.org/10.1007/s10658-018-1512-x.

    • Search Google Scholar
    • Export Citation
  • Peng H. 2014. Extraction and bioassay of insecticidal active substances from water hyacinth (PhD Diss). Hainan Normal University, Haikou, China. https://doi.org/10.7666/d.D345001.

  • Qi YH, Cao SF, Lv HP, Du H. 2011. The effects of different pesticides on the hatching of southern root knot nematode eggs and the activity of 2nd instar larvae. Northwest Agric J. 20(9):184189. https://doi.org/10.3969/j.issn.1004-1389.2011.09.037.

    • Search Google Scholar
    • Export Citation
  • Roanisca O, Mahardika RG. 2020. Citrus × microcarpa bunge fruit extract as antibacterial against Staphylococcus aureus. IOP Conf Ser Earth Environ Sci. 599:012043. https://doi.org/10.1088/1755-1315/599/1/012043.

    • Search Google Scholar
    • Export Citation
  • Sarma R, Adhikari K, Mahanta S, Khanikor B. 2019. Insecticidal activities of Citrus aurantifolia essential oil against Aedes aegypti (Diptera: Culicidae). Toxicol Rep. 6:10911096. https://doi.org/10.1016/j.toxrep.2019.10.009.

    • Search Google Scholar
    • Export Citation
  • Saxena DB, Goswami BK, Tomar SJ. 1987. Nematicidal activity of some essential oils against Meloidogyne incognita. Nematologia Mediterranea. 8(2):195201.

    • Search Google Scholar
    • Export Citation
  • Siskos EP, Konstantopoulou MA, Mazomenos BE. 2009. Insecticidal activity of Citrus aurantium peel extract against Bactrocera oleae and Ceratitis capitata adults (Diptera: Tephritidae). J Appl Entomol. 133(2):108116. https://doi.org/10.1111/j.1439-0418.2008.01312.x.

    • Search Google Scholar
    • Export Citation
  • Su M, Yang J, Zhou X, et al. 2020. GC-MS analysis of the essential oil composition of kumquat rind. Guangzhou Chemical Indus. 48(10):8993.

    • Search Google Scholar
    • Export Citation
  • Tsao R, Yu Q. 2000. Nematicidal activity of monoterpenoid compounds against economically important nematodes in agriculture. J Essent Oil Res. 12(3):350354. https://doi.org/10.1080/10412905.2000.9699533.

    • Search Google Scholar
    • Export Citation
  • Villafañe E, Tolosa D, Bardón A, Neske A. 2011. Toxic effects of Citrus aurantium and C. limon essential oils on Spodoptera frugiperda (Lepidoptera: Noctuidae). Nat Prod Commun. 6(9):13891392. https://doi.org/10.1002/med.20253.

    • Search Google Scholar
    • Export Citation
  • Wang LP, Wan XC, Hou RY, et al. 2013. The inhibitory effect of plant extracts and preparations such as Camellia oleifera. Anhui Nongye Daxue Xuebao. 40(4):642648. https://doi.org/10.13610/j.cnki.1672-352x.2013.04.011.

    • Search Google Scholar
    • Export Citation
  • Wang JL, Zhang JC, Gu JF. 2011. DNA extraction method for single nematode. Plant Quarantine. 25(2):3235.

  • Yang B, Eisenback JD. 1983. Meloidogyne enterolobii n. sp. (Meloidogynidae), a root-knot nematode parasitizing pacara earpod tree in China. J Nematol. 15(3):381391. https://doi.org/10.2307/1380543.

    • Search Google Scholar
    • Export Citation
  • Zhang FY, Ye WF, Wang XC, et al. 2024. Fingerprint analysis of essential oils from citrus plants. Guangdong Chemical. 51(4):139142.

Supplementary Materials

Xiaoli Dou College of Tropical Agriculture and Forestry/Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, Hainan University, Haikou 570228, China

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Yani Wu College of Tropical Agriculture and Forestry/Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, Hainan University, Haikou 570228, China

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Jiguang Luo Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Xiaopeng Yin Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Meiying Fu Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Xiangping Zeng Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agro-Products, Hainan Academy of Agricultural Sciences)/Scientific Observation and Experiment Station of Crop Pests in Haikou, Ministry of Agriculture and Rural Affairs/Key Laboratory of Plant Diseases and Pests of Hainan Province, Haikou 571100, China

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Contributor Notes

This work was supported by the China Agricultural Research System of Ministry of Finance (MOF) and Ministry of Agriculture and Rural Affairs (MARA) (CARS-16-E18), the Hainan Province Science and Technology Special Fund (ZDKJ202002), and the Natural Science Foundation of Hainan Province (321QN363).

Data from this study are presented in the figures and tables in this article, and in the supplemental material.

W.H. conceptualized the study and designed the research proposal. D.X. implemented the research process, collected and organized the data, and wrote the article. Y.W. and X.Y. searched for relevant literature and purchased the experimental materials. L.L. obtained the research funding and designed the framework of the article. M.F. and X.Z. revised the article. All authors read and agreed to the published version of the manuscript.

H.W. is the corresponding author. E-mail: wanghuifang@hnaas.org.cn.

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  • Fig. 1.

    Amplification results of sequence characterized amplified region marker-specific primers for root-knot nematodes. M = DNA marker D2000; 1–5 = root-knot nematode Meloidogyne enterolobii; 6 = blank control.

  • Fig. 2.

    Effects of Citrus microcarpa pericarp extract on the hatching of nematode eggs. The data in the figure are mean ± the standard error. Different letters within the same column group show significant differences at P < 0.05.

  • Fig. 3.

    Total ion flow diagram of ultrahigh performance liquid chromatography–mass spectrometry of the ethanolic extract of Citrus microcarpa pericarps. (A) Positive ion mode. (B) Negative ion mode.

  • Fig. 4.

    Total ion flow diagram of gas chromatography–time-of-flight mass spectrometry of the ethanolic extract of Citrus microcarpa pericarps. TIC = total ion current.

  • Fig. 5.

    Citrus microcarpa pericarp headspace volatility gas chromatography–mass spectrometry–volatile organic compounds total ion flow map. TIC = total ion current.

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