Acaricidal and Repellent Activity of Zanthoxylum myriacanthum (Rutaceae) Fruit Extracts Against Tetranychus urticae and Tetranychus truncatus (Acari: Tetranychidae)

The family Tetranychidae is comprised of approximately 1,250 species of spider mites. Over 100 species are pests damaging a variety of plants in field crops, orchards, greenhouses, and nurseries (Le Goff et al. 2009, Migeon et al. 2010). The two-spotted spider mite, Tetranychus urticae Koch, is an important mite pest worldwide (Flamini 2006). It is a serious pest of strawberry, shrubs, and ornamental trees in the northern part of Thailand (Charanasri et al. 1988). The cassava red mite, Tetranychus truncatus Ehara, is a major mite pest throughout Asia and the Pacific Islands (Bolland et al. 1998). In Thailand, they are known to be serious pests in cassava-growing areas in the eastern region. Both mite species are often found on the lower leaf surface and use their stylets to suck fluid from leaf cells resulting in foliar chlorosis, leaf desiccation and drop, and reductions in yield (Kumral et al. 2010).

Alternatives to conventional chemical acaricides are needed. Overuse of synthetic pesticides has negative effects on nontarget organisms, human health, and the environment. To address these problems, plant extracts are applied to control mite pests because these natural products have a short environmental persistence and low mammalian toxicity compared with various chemical pesticides (Attia et al. 2012). Zanthoxylum myriacanthum Wall. ex Hook. f., locally known as makwhaen in northern Thailand, belongs to the Rutaceae family (Suksathan et al. 2009) and can be found in tropical and temperate areas (Bubpawan et al. 2015). In northern Thailand, its fruit is normally used as a condiment and/or spice (Sriwichai et al. 2019). Limonene (67.1%), α-pinene (6.4%), β-myrcene (3.8%), and linalool (3.0%) are the major components of makwhaen essential oil (Li et al. 2014). The essential oil from the fruit of a related species, Zanthoxylum limonella (Dennst.) Alston, has insecticidal effects on Aedes aegypti (L.) fourth instars with a median lethal concentration (LC₅₀) of 24.61 ppm (LC₉₅, 55.81 ppm) in 24 h (Pitasawat et al. 2007). However, little is known about the acaricidal activity of Z. myriacanthum extracts on polyphagous mite pests. Therefore, this research was focused on the adulticidal, ovicidal, and repellent properties of makwhaen (Z. myriacanthum) crude extracts against two-spotted spider mite and cassava red mite under laboratory conditions.

Mite rearing. Two-spotted spider mites and red cassava mites were obtained from the Acarology Laboratory in Department of Entomology, Kasetsart University (Bangkok, Thailand). Mites were reared in plastic boxes (17.5-cm width [W] × 25-cm length [L] × 4-cm height [H]) containing a mulberry, Morus alba L., leaf placed on tissue paper on a moistened sponge (13-cm W × 22.5-cm L × 2.5-cm H). A mite-infested mulberry leaf was cut into small pieces and placed on the fresh leaf to allow mites to move to the fresh leaves as previously described by Auamcharoen and Chandrapatya (2015). Each spider mite species was maintained in separate boxes in the laboratory at ambient room conditions (27 ± 2°C, 10-h light [L]: 14-h dark [D]). All experiments were conducted under conditions similar to those for mite rearing.

Crude extract preparation. Makwhaen dried fruits were collected from the local orchard in Mueang District, Lampang Province, Thailand, in September 2018. The extraction method was that of Janlaor and Auamcharoen (2021). Briefly, 1 kg of ground dried makwhaen fruit was immersed separately with 2 L of each organic solvent (hexane, methylene chloride, or methanol) in a glass bottle (5-L capacity) at room temperature for 3 d. During this time, the extract glass bottle was shaken every day. The residue was extracted two more times. The combined solvent solution from each extraction was filtered through a cheesecloth and refiltered again with Whatman no. 1 filter paper (GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, UK). The hexane, methylene chloride, and methanol solutions were evaporated separately using a rotary evaporator under reduced pressure to receive the hexane extract, methylene chloride extract, and methanol extract. The crude extracts were kept separately in amber bottles in the refrigerator (10 ± 2°C) until used in bioassays.

Adulticidal bioassays. A cork borer was used to cut 2-cm diameter discs from fresh mulberry leaves. Three discs each were placed abaxial surface up on moistened cotton in a 9-cm-diameter glass petri dish. Twenty adult females of the same age of each mite species were introduced to each disc by using a fine paint brush. Mites on the 3 leaf discs were sprayed with 500 µL of 6, 8, 10, and 12% (w/v) concentrations of makwhaen extracts using a plastic atomizer. Mites on the mulberry leaf discs in the control were treated with 1% (v/v) Tween-20 (BDH Laboratory Supplies, Poole, UK) plus water. Each treatment was replicated four times with three leaf discs per replicate. The number of dead mites on each leaf disc adult was counted at 24 h after exposure.

Ovicidal bioassays. The bioassay arenas with the three leaf discs were established as in the previous bioassay. Twenty adult females of the same age of each mite species were transferred to each disc by using a fine brush, where they remained for 24 h to lay eggs. The adults were then removed from each disc after 24 h. Leaf discs and eggs were treated with 500 µL of the appropriate extract solution at concentrations of 6, 8, 10, and 12% (w/v) using a plastic atomizer. Controls were treated with 1% Tween-20 in water as done in the previous bioassay. Each treatment was replicated four times with three leaf discs per replicate. The number of hatched eggs on each leaf disc was recorded 5 d after treatment.

Repellent bioassays. The repellency of the extracts was assessed using the methods of Da Camara et al. (2015) and Sararit and Auamcharoen (2020) (Fig. 1). Each assay arena was a 9-cm glass petri dish with a moistened rectangular pad of cotton on the bottom. Two 2-cm-diameter mulberry leaf discs and a 1-cm-diameter filter paper disc (Whatman no. 1) were arranged linearly on the cotton filter paper that was cut into 1-cm lengths, coated on the bottom with laminating film, and placed between two mulberry leaf discs. Two rectangles of filter paper (Whatman no. 1) measuring 0.5-cm W × 1-cm L were positioned so that each served as a bridge from the center-positioned filter paper disc to an outer leaf disc. One of the bridges was treated with 15 µL of an appropriate extract solution (6, 8, 10, and 12% [w/v] concentrations), and the other was treated with 1% Tween-20 plus water to serve as a control. Solutions on both bridges were allowed to evaporate for 10 min, after which 20 adult female mites were placed on the middle filter paper disc in the arena. The number of mites on each mulberry leaf disc was counted at 1, 2, 3, 4, 5, 24, 48, and 72 h after treatment. The percent repellence was calculated using the following formula: percent repellency = [(C – T)/(C + T)] × 100. In the formula, C is the number of mites on the leaf disc on the control side and T is the number of mites on the leaf disc on the treated side (Akhtar et al. 2012). Each treatment was replicated four times.

Extract analysis. Gas chromatography-mass spectrometry (GC-MS) analysis was performed with a 5973 Agilent GC 6890N Series autosampler (Agilent, Santa Clara, CA). Each sample contained 2 µL of extract. The initial temperature was programmed at 70°C, then increased to 160°C at the rate of 2°C/min, then increased to 220°C at a rate of 2°C/min, and held for 10 min for total run time of 85 min. Other operating parameters were high purity helium as the gas carrier and a flow rate of 1 mL/min, and the injector and ion source temperatures were 230 and 280°C, respectively. A mass spectra (MS) 40-550 Atomic Mass Unit was used. The MS and retention indices of extract constituents were identified by comparison to a computer library (National Institute of standard and Technology [NIST], Mass Spectral Search Program and Chemstation Wiley Spectral Library, Gaithersburg, MD).

Statistical analyses. Each of the bioassays was performed under a completely randomized design, and data were subjected to analysis of variance. Data reported as a percentage were transformed using the arcsine square root transformation before analysis. Tukey's honestly significant difference test was used for the separation of the treatment means (R Development Core Team 2016). The LC₅₀s were calculated by probit analysis (Finney 1971) using SPSS software version 19.0 (Statistical Package for the Social Sciences, Version 19.0, Armonk, NY).

Adulticidal bioassays. The mean ± SE mortality of T. urticae adults at 24 h after exposure ranged from 52.1 ± 4.3 to 73.3 ± 5.1% with the hexane extract and 45.4 ± 5.8 to 85.8 ± 4.5% with the methylene chloride extract; however, mortality with the methanol extract was only 14.2 ± 2.1 to 42.5 ± 8.4% (Table 1). Similar levels of mortality were observed with T. truncatus treated with the hexane (36.3 ± 6.0 to 73.3 ± 6.6%) and methylene chloride (51.3 ± 6.6 to 85.0 ± 3.5%) extracts. Over the four concentrations (6, 8, 10, and 12%), mortality following exposure to the methanol extract was markedly higher in T. truncatus adults (73.8 ± 8.8 to 95.8 ± 1.5%) than that in T. urticae adults (14.2 ± 2.1 to 42.5 ± 8.4%). Statistically significant differences were detected among treatment means within extract treatments and mite species; however, statistical separation among the multiple means was affected by the variation in the data. Regardless, the mortality response of the two mite species was, in general, positively related to the concentration of the extract.

The LC₅₀ for the hexane extract against T. truncatus was 7.94% (95% fiducial limits [FL] = 5.15–10.30; slope ± SE, 0.22 ± 0.02). For the methylene chloride extract, the LC₅₀s were 7.55% (95% FL = 5.71–9.00; slope ± SE, 0.25 ± 0.02) with T. urticae and 6.88% (95% FL = 3.67–8.89; slope ± SE, 0.24 ± 0.02) with T. truncatus, which were not statistically different based on overlapping 95% FL of these values. To the contrary, the LC₅₀s of the methanol extract against T. urticae and T. truncatus were 12.34% (95% FL = 11.41–13.76; slope ± SE, 0.19 ± 0.03) and 5.36% (95% FL = –3.53–8.02; slope ± SE, 0.28 ± 0.02), respectively, which were statistically different based on nonoverlapping 95% FL values.

Ovicidal activity. The only extract that exhibited appreciable ovicidal activity was the hexane extract against T. truncatus that reduced egg hatch by 55.4%, 62.4%, 59.8%, and 68.7% at the 6%, 8%, 10%, and 12% concentrations, respectively, at 5 d after exposure (Table 2). These means did not differ statistically from each other, but they differed significantly (F = 61.74; df = 5, 66; P < 0.001) from the controls. All other treatments limited egg hatch by only 45% or less.

Repellent bioassays. The repellency of the concentrations of the three extracts was determined by mite response to filter paper strips treated with the chemical. The filter paper strips acted as bridges to an untreated leaf disc from an untreated filter paper disc. Mites were allowed paired choices between untreated leaf discs via the treated bridge and an untreated bridge. Numbers of mites were counted on each leaf disc and each bridge at times following treatment for a period of 72 h. Based on a percentage of repellency derived from those counts, we found no statistical differences in the concentrations or types of extracts for both T. urticae and T. truncatus adult females at 1, 5, 24, 48, and 72 h after treatment (Table 3).

Extract analysis. Fifteen and 18 constituents were identified from the hexane and methylene chloride extracts of Z. myriacanthum dried fruits, respectively, with different elution time and amount (Tables 4, 5). The main components in the hexane extract were DL-limonene (29.75%), sabinene (9.76%), caryophyllene oxide (4.70%), 1,3-cyclooctane (3.85%), and α-terpineol (2.71%) (Table 4). Limonene (40.70%), sabinene (16.60%), 1,3-cyclooctane (3.71%), trans-sabinene hydrate (3.11%), and (-)-caryophyllene oxide (2.79%) were the main constituents of the methylene chloride extract (Table 5).

Our results revealed that the 10% and 12% concentrations of the methylene chloride and hexane extracts of Z. myriacanthum fruit caused >70 % mortality of T. urticae and T. truncatus adult mites 24 h after exposure. Based on the LC₅₀s, the methylene chloride extract exhibited a stronger adulticidal effect on T. urticae than the methanol extract. Against T. truncatus, three extracts presented similar adulticidal activity. Our results were similar to those of other studies. Tewary et al. (2005) reported that 5,000 and 10,000 ppm of petroleum ether extract of Zanthoxylum armatum L. leaves caused 35 and 38% T. urticae adult mortality, respectively, at 48 h after treatment. Attia et al. (2012) reported the contact toxicity property of extracts in three plant species belonging to Rutaceae family as well as Z. myriacanthum. The distillate of Haplophyllum tuberculatum Forssk. demonstrated high activity against larvae and adult females of T. urticae (94% and 93% mortality, respectively) at 72 h posttreatment, followed by Ruta chalepensis L. (66% and 61% mortality, respectively), and Citrus aurantium L. (63% and 55% mortality, respectively).

Moreover, an essential oil in other Zanthoxylum species also possesses biological activities on the red flour beetle (Tribolium castaneum [Herbst]) and mosquito species. Wanna and Satongrod (2020) reported that a 5% concentration of essential oil from dried seeds of Z. limonella Alston showed the highest activity on T. castaneum at 120 and 48 h for adults and larvae, respectively, whereas 10% concentration caused 100% mortality of T. castaneum eggs at 14 d. The Z. limonella essential oil hydrolate presented strong larvicidal activity on Aedes albopictus (Skuse) and Culex quinquefasciatus Say after 24 h. Against Ae. albopictus, the LC₅₀ and LC₉₀ values were 11 and 19.4% (v/v), whereas those values were 15.5 and 25.8% (v/v) against Cx. quinquefasciatus (Rabha et al. 2012). Soonwera and Phasomkusolsil (2017) reported that essential oil from Z. limonella dried fruit yielded an LC₅₀ of 6.0% and 5.7% after 24 h for Ae. aegypti and Cx. quinquefasciatus adults, respectively. The concentration of 10% caused 100% mortality after 12 and 24 h of Ae. aegypti and Cx. quinquefasciatus larvae, respectively.

Among three tested extracts of Z. myriacanthum, the hexane extract at all tested concentrations (6–12%) exhibited ovicidal activity against T. truncatus, with >50% of eggs remaining unhatched eggs at the end of experiment. This finding suggests that the hexane extract is more suitable as an ovicide for T. truncatus than that for T. urticae. Unfortunately, the methylene chloride and methanol extracts had less ovicidal activity against the eggs of both species. Further work should be directed to the oviposition activity of the three extracts of Z. myriacanthum against T. urticae and T. truncatus, especially in light of the results of Soonwera and Phasomkusolsil (2017) who reported that Z. limonella essential oil deterred the ovipositional activity of Ae. aegypti and Cx. quinquefasciatus gravid females.

Hexane, methylene chloride, and methanol extracts of Z. myriacanthum repelled T. urticae and T. truncatus adults at levels >60% from 5 to 72 h postexposure. Da Camara et al. (2015) demonstrated that essential oils from Citrus sinensis Osbeck var. pera and C. aurantium fruit skins were repellent at levels of 43.4 and 90.6%, respectively, against T. urticae adults at 1 h after exposure, which is a similar repellency effect to those obtained in our study. Furthermore, the essential oils from Z. limonella act as a repellent against mosquitoes. The fruit oil at a 30% concentration in mustard oil mixture repelled Ae. albopictus with the longest protection time (296–304 min) (Das et al. 2003). The fruit essential oil protected experiment participants from Ae. aegypti, Cx. quinquefasciatus, and Anopheles dirus Peyton & Harrison for 2 h using an arm-in-cage test (Trongtokit et al. 2005). The Z. limonella essential oil at a 10% concentration demonstrated 100% and 99.5% repellency against Ae. aegypti and Cx. quinquefasciatus, respectively (Soonwera and Phasomkusolsil 2017).

Dried Z. myriacanthum fruit extracted with different solvents in this study showed variations in the chemical compositions. Limonene was the major chemical compound in the hexane extract (29.75%) and the methylene chloride extract (40.70%), corroborating the findings of a number of previous studies in which limonene was identified as the main chemical constituent of Zanthoxylum species (Li et al. 2014, 2016, Sriwichai et al. 2019). Limonene and sabinene were the major components from essential oils of fresh and dried Z. myriacanthum fruits (Sriwichai et al. 2019), whereas Li et al. (2014) found that limonene was the main compound in the essential oil of fresh Z. myriacanthum fruits. Also, Li et al. (2014, 2016) demonstrated that the main constituent was limonene in Zanthoxylum schinifolium Sieb. et Zucc. and Zanthoxylum bungeanum Maxim. Moreover, d-limonene also was the major chemical compound in C. sinensis and C. aurantium essential oils (Da Camara et al. 2015). Kim et al. (2013) found limonene (22.83%) in essential oil of Anethum graveolens L. This plant essential oil had high repellency and fumigant activities against T. urticae and T. truncatus (Sararit and Auamcharoen 2020). Consequently, limonene in hexane and methylene chloride extracts of Z. myriacanthum in this study may be the causative factors for the observed adulticidal, ovicidal, and repellency activities against T. urticae and T. truncatus. Limonene (255.44 mg/L) caused moderate toxicity on T. urticae by using direct contact application methods, whereas limonene at 125 mg/L displayed high egg mortality (Badawy et al. 2010). d-Limonene showed 71.3% repellency activity against T. urticae at 1 h of exposure (Da Camara et al. 2015).

Based on our results, the hexane, methylene chloride, and methanol extracts of Z. myriacanthum demonstrated potential for use in managing adults and eggs of T. urticae and T. truncatus. These extracts can be used as acaricides for controlling the population of both mites or can be applied as repellent products to protect plants from spider mite damage. Furthermore, Charoenying et al. (2010) reported that the crude ethyl acetate extract of Z. limonella Aston fruits at a 1,000-ppm concentration showed efficacy in inhibiting Chinese amaranth (Amaranthus tricolor L.). This result indicates that Z. limonella fruit extract may contain allelopathic toxins and may have potential as an allelopathic product. Xanthoxyline, a phenolic compound isolated from Z. limonella fruits at 2,500 µM also completely inhibited seed germination and growth of Chinese amaranth, and it inhibited barnyard grass (Echinochloa crus-galli [L.] Beauv.) seed germination, shoot length, and root length by 43.6%, 71.6%, and 87.5%, respectively. From their results, we might postulate the allelopathic effects of Z. myriacanthum extracts. Further studies should investigate the allelopathic and phytotoxic effects of Z. myriacanthum extracts on cultivated plants before developing them as botanical acaricides against spider mites on infested plants. The results will guide producers in the decision to use appropriate plant extracts for controlling target pests.

This research is supported in part by the Graduate Program Scholarship from The Graduate School, Kasetsart University.