The cassava red mite, Tetranychus truncatus Ehara (Acari: Tetranychidae), is a serious spider mite pest of cassava (Manihot esculenta Crantz) and mulberry (Morus alba L.). These mites have a high reproductive rate and also a short life cycle (Sakunwarin et al. 2003), which contributes to the pest status. Their damage to plant leaves appears as chlorotic spots or areas which affect photosynthetic processes (Tomczyk and Kropczynska 1985).

Spider mites are routinely managed by applying conventional synthetic pesticides that are efficacious and easily available (Nauen et al. 2001). Nevertheless, chemistries used repeatedly and/or for extended times may result in the development of resistance in target populations, negative impacts on natural enemies, and deposition of harmful residues in the environment (Leeuwen et al. 2010; Ullah and Gotoh 2013). The use of natural products from plants (e.g., crude extracts, essential oils) is an alternative to conventional acaricides and their negative impacts.

Acorus calamus L. (Acoraceae), commonly known as sweet flag or calamus, is a perennial wetlands monocot that is native to India (Amit and Vandana 2013). Previous studies have shown that it has antibiotic effects against bacteria and fungi and insecticidal properties against some insects (Eswara Reddy et al. 2016; Koul 1987; Lee et al. 2004; Phongpaichit et al. 2005; Singh et al. 2010; Thaenthanee et al. 2014). The essential oil of A. calamus contains various chemical constituents depending on the plant parts (leaves or rhizomes) extracted, with β-asarone as one of the major chemicals identified (Liu et al. 2013; Lohani et al. 2012). This study was conducted to determine residual and direct contact toxicities of crude extracts and essential oils obtained from A. calamus rhizomes in laboratory bioassays against eggs and adult females of T. truncatus.

Materials and Methods

Mite colony. The stock colony of T. truncatus was initiated with from mites collected from infested cassava, Ma. esculenta, leaves collected at Kasetsart University (Bangkok, Thailand). Mites were fed on mulberry, Mo. alba, leaves placed on tissue paper on a moistened sponge in a plastic box (15 × 21 × 4 cm; width × length × height) and maintained at room temperature (27 ± 2°C and 10L: 14D). Leaves were replenished periodically by placing the infested leaf on a fresh leaf to allow mites to transfer to the fresh leaf as per methods of Auamcharoen and Chandrapatya (2015). All bioassays were conducted and maintained at the same conditions as for mite rearing.

Plant materials. Sweet flag, A. calamus, rhizomes were collected from Nonthaburi province, Thailand, in August 2016. The plant material was identified by Mr. Sukid Rueangruea (Department of National Parks, Wildlife and Plant Conservation, Bangkok, Thailand), where the voucher specimen (BKF No. 194351) was deposited in the Forest Herbarium of that facility. Extracts were taken from fresh rhizomes immediately after collection and transported to the laboratory. Extracts from dried rhizomes were from those that had been air-dried for 1 wk after collection.

Crude extract preparation. Crude extracts utilized in the bioassays were obtained by two extraction methods—the polar sequential extraction method and the individual extraction method. The process of the polar sequential extraction method was that of Aryani and Auamcharoen (2016) and Pancharoen et al. (2014). Briefly, 1 kg of ground fresh or dried rhizomes was placed in a glass bottle (5-L capacity) with 2 L of hexane for 3 d. The resulting residue was subjected to the same extraction process two additional times. The combined hexane solutions from these extractions were filtered using a Whatman No. 1 filter paper (GE Healthcare U.K. Limited, Amersham Place, Little Chalfont, Buckinghamshire, U.K.) and dried using a rotary evaporator under reduced pressure to receive the hexane extract. The residue from hexane extraction was re-extracted with methylene chloride and, finally, the methylene chloride extraction residue was re-extracted with methanol. The methylene chloride and methanol solutions were separately evaporated to yield methylene chloride and methanol extracts, respectively. The individual extraction method did not involve sequential extractions. Fresh or dried rhizomes were extracted separately using only hexane, methylene chloride, or methanol at the same conditions previously described. Each of the crude extracts was placed in refrigeration (10 ± 2°C) until used in the bioassays.

Essential oils distillation. The essential oil distillation process is described by Wongtong and Nawanich (2001) and Torres et al. (2014). Briefly, fresh (1 kg) or dried (300 g) rhizomes were distilled by water (2.5 L) for 8 h using a Clevenger-type apparatus. The essential oils layer was separated from the water layer using a glass pipette dropper and stored in glass vials at 10 ± 2°C until used in the bioassays.

Preliminary screening of extracts and oils. Crude extracts and essential oils were screened for residual and direct contact toxicities against T. truncatus mites at concentrations of 2.5 and 5% (v/v). The materials showing efficacy against the mites were further evaluated for biological activity against T. truncatus eggs and adult females.

Residual contact toxicity bioassays. Mulberry leaf discs (2-cm diam) were placed lower-surface up on moistened cotton in 9-cm diam glass Petri dishes with 3 discs per dish. Droplets (50 µl) of the essential oil solution at concentrations of 0.625, 1.25, 2.5, 5 and 10% (v/v) were deposited on appropriate leaf discs. Mulberry leaf discs treated with Tween-20 (BDH Laboratory Supplies, Poole, BH15 1TD, U.K.) (1%, v/v) in water were used as the untreated controls. The solvent was allowed to evaporate, after which 20 T. truncatus adult females from the colony were placed on each disc using a fine paint brush. All treatments were replicated three times with three leaf discs per replication (Petri dish). The number of dead mites on each leaf disc was recorded every 24 h for three consecutive days.

Direct contact toxicity bioassays. For egg bioassays, mulberry leaf discs (2-cm diam) were placed on moistened cotton in 9-cm diam glass Petri dishes. Twenty T. truncatus adult females were placed on each leaf disc allowing them to oviposit on the discs. Adults were removed from the discs after 24 h. Three leaf discs with eggs were sprayed with 500 µl of 0.625, 1.25, 2.5, 5, and 10% (v/v) concentrations of the crude extract or the essential oil solution using a plastic atomizer. Eggs treated with Tween-20 (1%, v/v) in water served as controls. The number of unhatched eggs was counted 7 d later.

For adult mite bioassays, 20 adult females from the colony were placed on each 2-cm diam leaf disc in glass Petri dishes. These were then sprayed with 500 µl of crude extract solution at concentrations of either 0.625, 1.25, 2.5, 5, or 10% (v/v) using a plastic atomizer. Tween-20 (1%, v/v) in water was sprayed on leaf discs for the control. The number of dead mites on each leaf disc was counted at 24, 48, and 72 h after treatment.

Statistical analyses. Data were subjected to analysis of variance (ANOVA) following correction for control mortality (Abbott 1925). A Tukey's honestly significant difference (HSD) test was applied to compare the treatment means (R Development Core Team 2016). Median lethal concentrations (LC₅₀s) were estimated by probit analysis (Finney 1971) using SPSS (Statistical Package for the Social Sciences, Version 19.0, Armonk, NY).

Results

Residual toxicity. Mortality of T. truncatus adult females placed on leaf discs previously treated with essential oils extracted from A. calamus rhizomes varied with concentration and source (dried versus fresh) of the oil (Table 1). Cumulative percentage mortality 3 d after placement of female mites on the treated surfaces ranged from a mean (±SE) of 4.8 ± 13.5% at the 0.625% concentration to 73.8 ± 4.1% at the 10% concentration using oil from fresh rhizomes. Mean cumulative mortality following exposure to oils extracted from dried rhizomes was 17.1 ± 7.3% at the 0.625% concentration and 91.8 ± 2.3% at the 10% concentration. Mortality in the 5 and 10% concentrations did not differ statistically with the oil extracted from fresh rhizomes, but mortality with the 10% concentration (91.8 ± 2.3%) of the oil from dried rhizomes differed significantly (F = 18.4; df = 4, 40; P < 0.001) from that with the 5% concentration (47.4 ± 6.1%). The LC₅₀s of the respective preparations were 5.75% (95% fiducial limits [FL] = 3.39–12.01; Slope ± SE = 0.23 ± 0.02) for oils from fresh rhizomes and 4.97% (95% FL = 3.76–6.81; Slope ± SE = 0.28 ± 0.02) for oils from dried rhizomes, which are not statistically significant based on overlapping 95% FL of these values.

Table 1 Cumulative mean (±SE) percent mortality of T. truncatus adult females following exposure to leaf surfaces treated with essential oils extracted from fresh and dried rhizomes of A. calamus.

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Ovicidal activity. Contact toxicity of essential oils and crude methanol extracts against T. truncatus eggs was determined by applying oils or extracts directly on the eggs. No eggs hatched following treatment with a 10% concentration of essential oil extracted from fresh rhizomes, while only 3.7 ± 1.7% of eggs hatched following treatment with 5% concentration of the oil (Table 2). These treatments did not differ statistically, but the higher concentrations differed significantly (F = 88.02; df = 4, 40; P < 2e-16) from the remaining concentrations tested. Oil extracted from dried rhizomes appeared be less effective as an ovicide with percentage hatch ranging from 94.3 ± 2.3% to 71.2 ± 3.9% following treatment with oil concentrations ≤5% (Table 2). Ovicidal activity was noted only with the 10% concentration of essential oil extracted from dried rhizomes with percentage hatch of 8.2 ± 2.6% (Table 2). The LC₅₀s of essential oil from fresh and dried rhizomes were 2.18% (95% FL = 1.74–2.78; Slope ± SE = 0.79 ± 0.06) and 5.91% (95% FL = 4.69–7.78; Slope ± SE = 0.31 ± 0.02), respectively, which are statistically significant based on nonoverlapping 95% FL of these values.

Table 2 Mean (±SE) percent hatched eggs of T. truncatus treated with essential oils extracted from fresh and dried rhizomes of A. calamus.

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The crude methanol extract derived by the individual extraction method with dried rhizomes reduced egg hatch by 73.7 ± 5.1% at the 10% concentration (Table 3). Other concentrations of this extract were less effective in reducing egg hatch (82.6–42.2%). Extracts using polar sequential extraction methods with hexane and methylene chloride demonstrated low ovicidal activity (<51% reduction in hatch) on eggs of T. truncatus (Table 3).

Table 3 Mean (±SE) percent hatched eggs of T. truncatus treated with extracts from dried rhizomes of A. calamus.

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Direct contact toxicity against adult females. Mortality of adult female mites was determined following exposure to various concentrations of compounds extracted from dried or fresh rhizomes by different extraction methods and solvents (Table 4). Mortality exceeded 90% with treatments of 2.5 and 5% concentrations of methylene chloride (individual method) extracts from fresh rhizomes, 5% concentration of methylene chloride (individual method) extract from dried rhizomes, and 5 and 10% concentrations of methanol (individual method) extracts from dried rhizomes. Mortality following treatment with 1.25, 2.5, 5.0, and 10.0% of the methylene chloride (polar sequential method) extracts from fresh rhizomes was >91% (Table 4). The LC₅₀s for the extracts ranged from 1.31% (95% FL = 1.03–1.65; Slope ± SE = 1.37 ± 0.11) for the methylene chloride extracts from fresh rhizomes obtained by the individual extraction method to 2.52% (95% FL = 1.51–4.47; Slope ± SE = 0.55 ± 0.04) for the methylene chloride extracts from dried rhizomes obtained by the individual extraction method (Table 4). The LC₅₀ with adult females are not statistically significantly based on overlapping 95% FL of these values.

Table 4 Cumulative mean (±SE) percent mortality of T. truncatus adult females following exposure to various concentrations of extracts from fresh and dried rhizomes of A. calamus.

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Table 4 Continued.

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Table 4 Continued.

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Discussion

Many plant extracts have biological activity against tetranychid spider mites in laboratory conditions. Residues of hexane extracts from Annona vepretorum Mart. (Annonaceae) yielded an LC₅₀ value of 50.61 mg/ml against the two-spotted spider mite, Tetranychus urticae (Koch), while methanol extracts exhibited LC₅₀s of 10.96 mg/ml for topical + residue exposure and 22.07 mg/ml for residual toxicity only (Fernandes et al. 2017). Residues of ethanol extracts from Annona muricata L. seeds killed 93.3% of T. urticae mites, with the residual effects extended 120 h after treatment when >80% mortality was recorded (Maciel et al. 2015). Our results showed similar responses with the essential oils from fresh and dried rhizomes of A. calamus having residual activity resulting in >70% T. truncatus mite mortality 24 h after application of a 10% concentration of the extract. Based on LC₅₀s, essential oils from fresh and dried rhizomes of A. calamus appear to possess a potential for development as a residual miticides.

We also demonstrated that essential oil from rhizomes was an effective ovicide with >91% reduction in egg hatch following exposure to 5 and 10% concentrations of extracts from fresh rhizomes and 10% concentration of extracts from dried rhizomes. Similarly, Choi et al. (2004) also found that essential oils from caraway seed (Carum carvi L., Apiaceae), citronella java (Cymbopogon nardus (L.) Rendle, Gramineae), lemon eucalyptus (Eucalyptus citriodora Hook., Myrtaceae), penny-royal (Mentha pulegium L., Lamiaceae), peppermint (Mentha piperita L., Lamiaceae), and spearmint (Mentha spicata L., Lamiaceae) had ovicidal effects against T. urticae. Erdogan et al. (2012), however, determined that oils from Helichrysum arenarium L. (Asteraceae), Rhododendron luteum Sweet (Ericaceae), Veratrum album L. (Liliaceae), Tanacetum parthenium L. (Asteraceae), and Allium sativum L. (Amaryllidaceae) had no ovicidal effects on T. urticae. In this study, the 10% concentration of methanol (individual method) extract from dried rhizomes of A. calamus showed a moderate ovicidal effect with 73.7% reduction in egg hatch. Similarly, Sarmah et al. (2009) reported relatively high ovicidal effects of aqueous extracts from either Xanthium strumarium (L.) or A. calamus against the spider mite Oligonychus coffeae (Nietner); however, extracts from Polygonum hydropiper (L.) and Clerodendron infortunatum (L.) proved less efficacious.

We observed that direct contact toxicity against adult T. truncatus is linked to the polarity of the organic solvents used for extraction. Methylene chloride extracts from fresh and dried rhizomes of A. calamus demonstrated higher acaricidal activity than did methanol extracts. This result corresponds to that of Roy et al. (2011) who tested the acaricidal activity of petroleum ether, acetone, and methanol extracts of P. hydropiper obtained by cold percolation and sequential extraction methods against Oligonychus coffeae with acetone fractions showing the greatest acaricidal activity, followed by petroleum ether and methanol fractions. Activity also showed a positive concentration-dependent response. Roy et al. (2018) subsequently reported acaricidal properties of karanja oil, mustard oil, olive oil, sesame oil, castor oil, groundnut oil, and rose oil against O. coffeae. Water extracts from juazeiro, Ziziphus joazeiro von Martius, leaves applied to cotton foliage infested with Tetranychus ludeni Zacher caused mortality levels ranging from 17 to 81% (Ferraz et al. 2017).

Based on our results, using methylene chloride as the organic solvent to extract oil from fresh rhizomes of A. calamus by the polar sequential extraction method yielded a product that has greater potential for further development as an acaricide than those derived by the individual extraction method. However, the polar sequential extraction method is a longer process than the individual extraction method. With the polar sequential extraction method, methylene chloride will be used to extract the residues of plant materials after extraction with hexane; yet, methylene chloride can be used immediately to extract plant materials using the individual extraction method. On the other hand, the polar sequential extraction method is more cost effective in that residues of plant materials can be extracted several times with organic solvents before discarding. Under the individual extraction method, the residue of plant material extracted with only one solvent was discarded after extraction. Mite mortality in the control treatments interfered with our ability to discern cumulative T. truncatus mortality over longer periods of exposure.

Based on the experimental results, the essential oils from fresh and dried rhizomes of A. calamus had residual contact toxicity on T. truncatus adult females and direct contact toxicity on T. truncatus eggs. The essential oil from fresh rhizomes was more toxic than the essential oil from dried rhizomes against the egg stage of mites. On the contrary, the methylene chloride extracts from fresh A. calamus rhizomes extracted by individual extraction method and polar sequential extraction method displayed direct contact toxicity to T. truncatus adult females. Future studies should be directed to investigating the activities of the different active constituents in the essential oil and extracts of A. calamus rhizomes. This plant product is appropriate to develop as bio-acaricides against mites in the field.