The Hawaiian beet webworm (Spoladea recurvalis) is widely distributed worldwide, including countries in Africa and Asia, such as Korea (Bae and Paek, 2006;Kim et al., 2014;Jeyasankar and Gokilamani, 2016;Ehsine and Aoun, 2020;Husain et al., 2020;Kwon et al., 2021). Although S. recurvalis is considered to belong to the Spoladea genus in most countries, including Korea (Bae and Paek, 2006), it is considered to belong to the Hymenia genus in some countries. The larvae attack Chenopodiaceae, Amaranthaceae, and Liliaceae, and they fold leaves using thread and feed the interior soft tissue of the stem(Jeyasankar and Gokilamani, 2016;Kim et al., 2014). Depending on the environment, S. recurvalis passes either through the 4^th-instar larval stage (Jeyasankar and Gokilamani, 2016) or 5^th-instar (Lee et al., 2013). Egg hatching is possible at 15°C, but development into the 2^nd-instar does not occur, and the total developmental period is 14.6-51.0 days at 17.5-35.0°C (Lee et al., 2013). In South Korea, representative host crops, such as spinach, beet, beetroot, and amaranth, that are damaged by S. recurvalis show a decrease in the quantity (Bae and Paek, 2006;Kim et al., 2014;Kwon et al., 2021).

The major insect pests of spinach that have been reported are aphids, mites, spinach leaf miner (Pegomya Hyoscyami), tomato leaf miner (Liriomyza bryoniae), and tortoise beetle (Cassida nebulosa). However, studies on the occurrence and optimal control time of pests through precise diagnosis are still lacking in South Korea (RDA, 2011). When S. recurvalis occurs on spinach, young larvae start to attack the tender leaves first, followed by the rear side of leaves. As mature leaves are folded, the commercial value and quantity are significantly damaged when left without control (Fig. 1-A, B).

Several studies have been conducted to establish control strategies against S. recurvalis in South Korea, including a study on the occurrence patterns in several vegetable greenhouses (Lim et al., 2012;Kwon et al., 2021), temperature-dependent development models (Lee et al., 2013), and determination of the economic injury level of S. recurvalis on beet (Kim et al., 2014). For controlling S. recurvalis, several studies have been conducted globally using insecticides, microorganisms, and plant extracts (Jeyasankar and Gokilamani, 2016;Muralikrishna et al., 2019). However, in South Korea, as of 2019, 13 active ingredients for the chemical control of S. recurvalis are currently registered for three minor crops, such as leaf beet and pigweed, under the government pesticide registration project in accordance with the positive list system (PLS) (RDA, 2022). As no insecticide is registered for spinach in South Korea, there is an urgent need of an insecticide to control S. recurvalis occurrence.

Therefore, this study was conducted to establish an effective control strategy against S. recurvalis and to register five insecticides for spinach by inspecting insecticidal susceptibility, residual activity, and field control tests.

Materials and methods

Insect rearing

The S. recurvalis used in the insecticidal activity testing was mass-reared using a population that occurred on a spinach field in the Hwaseong region of Gyeonggi Province was collected in 2019. After sowing in a square acrylic container (30 × 30 × 30 cm), 30-day-old spinach seedlings were supplied as food to breed at least three generations in the laboratory. The breeding conditions were 25±1°C, 60%–70% relative humidity, and 16L:8D as the photoperiod.

Test Chemical Agent

A total of five chemical agents were used in the insecticidal susceptibility testing of S. recurvalis larvae (Table 1). At the time of conducting these experiments, these insecticides were not registered for the control of S. recurvalis on spinach in South Korea.

Insecticidal activity in laboratory

The insecticidal effect on 3^rd-instar S. recurvalis larvae was investigated. Each insecticide was diluted to seven concentrations below the recommended concentration. Spinach leaves were added as a food source to the insect breeding dish (Φ9.0 × 4.0 cm, SPL, Korea) with filter paper on top of moistened cotton wool. After inoculating the S. recurvalis larvae, the diluted insecticide was sprayed on the larvae body 5 times (approximately 10 ml) using a small sprayer (4 × 14 cm, 100 ml). Mortality was evaluated after 24, 48, and 72 h after spraying, and dead individuals were enumerated in the untreated group. The corrected mortality (%) was calculated using the Abbott formula (Abbott, 1925).

Dead individuals were determined when they did not stand upright or move at least the length of their body when nudged with a brush. The test was conducted under laboratory conditions (25±1°C, 50%–60%, 16L:8D) in three repetitions of 20 larvae per repetition.

Residual effect

The residual effects of the five insecticides on the 3^rd-instar S. recurvalis larvae were tested. The recommended diluted concentration of each insecticide was sprayed sufficiently on the leaves of spinach seedlings placed in a greenhouse exposed to natural light. After 3, 5, and 7 days from chemical spraying, they were moved to the laboratory and used for the test. The leaves sprayed in advance were fed to the larvae, and the mortality was evaluated after 24, 48, and 72 h under laboratory conditions (25±2°C, 50%–60%, 16L:8D). The test was repeated thrice, with 20 larvae per repetition.

Control Effect in Field

The field control effect was conducted for the five insecticides at two spinach (cultivar Ultra) greenhouses in Hwaseong and Yeoncheon in Gyeonggi Province. The spinach was sown directly in the greenhouse, and a drip irrigation system was used to provide water. To unify the density and larval developmental stage of S. recurvalis before treatment, 50 larvae were selected and inoculated per replicate at Yeoncheon (September 15) and Hwaseong (September 24). The plot was arranged in repetitions of three randomized block designs, and a net cage (BugDorm-3 rearing cage, Seoul, Korea) covered the inoculation point to minimize loss due to external factors such as birds and larval self-defeating (Fig. 1-C). Each insecticide was diluted to the recommended concentration, and the number of living larvae was determined after 3, 5, and 7 days from insecticide spraying. The control value (%) was calculated as [(percent living in untreated–percent living in treated)/percent living in untreated] × 100. In addition, these five insecticides were not registered for spinach, and the presence of external phytotoxicity was examined in the shoots and leaves after 3, 5, and 7 days from spraying at the recommended and double concentrations.

Statistical Analysis

The indoor insecticidal activity and field control effect for each insecticide were compared using the SAS statistics program (SAS Institute, 2011) by testing the difference between means using the Duncan’s multiple range test (DMRT).

Results

Insecticidal activity in laboratory

Table 2 shows the insecticidal activity of the five insecticides that have not been registered for spinach against S. recurvalis. Pyridalyl EW, with a yet unreported mode of action, showed 100% insecticidal activity after 24 h of spraying at the recommended concentration. Chlorantraniliprole WP, a compound of the diamides group, showed a 98.3% insecticidal activity after 48 h of spraying. Lufenuron EC, chromafenozide EC, and tebufenozide WP, which belong to the insect growth regulator group, exhibited low insecticidal activities of 45.0%, 26.7%, and 50.0% after 24 h of spraying, which then increased to 96.7%, 96.7%, and 100% after 72 h, respectively. After 72 h of insecticide spraying, lufenuron EC, chromafenozide EC, chlorantraniliprole WP, tebufenozide WP, and pyridalyl EW showed insecticidal activities of 90% or more, even at 2 (12.5 ppm), 4 (12.5 ppm), 8 (2.5 ppm), 4 (20.0 ppm), and 8 (12.5 ppm) times diluted concentrations.

Residual effects testing

Fig. 2 shows the residual effect of the five insecticides on 3^rd-instar S. recurvalis larvae. When the larvae were inoculated on the spinach leaves after 3, 5, and 7 days from insecticide spraying, high insecticidal activities were observed, similar to the insecticidal activity observed in the laboratory (day 3: df = 5; F = 239.43; p < 0.01; day 5: df = 5; F = 89.95; p < 0.01; and day 7: df = 5; F = 216.64; p < 0.01). Chromafenozide EC, chlorantraniliprole WP, tebufenozide WP, and pyridalyl EW showed 98.3%, 100%, 95.0%, and 100% mortality when the S. recurvalis larvae were exposed 7 days after spraying, respectively. In particular, pyridalyl EW showed a high residual effect and showed the most rapid insecticidal activity (86.7%) after 24 h of exposure to S. recurvalis larvae 7 days after treatment. In contrast, lufenuron EC showed 96.8% insecticidal activity after 72 h of exposure to S. recurvalis larvae 3 days after treatment, but the insecticidal activity decreased to less than 75% 5 days after treatment.

Control effect field testing

Field control tests were conducted for the five insecticides at the two spinach greenhouses located in Hwaseong and Yeoncheon in the Gyeonggi Province. The control value showed an increasing trend as the number of days passed since insecticide spraying in both locations. Lufenuron EC, chromafenozide EC, chlorantraniliprole WP, tebufenozide WP, and pyridalyl EW showed high control effects of 98.5%, 100%, 97.1%, 100%, and 100% after 7 days of treatment at Hwaseong region, respectively (Tables 3, 4), and the control values were 99.3%, 100%, 97.1%, 100%, and 100% at Yeoncheon region, respectively (Tables 5, 6). In addition, these insecticides did not cause any phytotoxicity to the leaves and stems of spinach, even at double concentrations (Table 7).

Discussion

This study provides effective five insecticides against S. recurvalis through insecticidal susceptibility, residual activity, and field control tests. In this study, the five insecticides used also showed similarly high insecticidal activity at the recommended concentrations, and it was observed that S. recurvalis showed very high susceptibility even when exposed to leaves 7 days after insecticide spraying, even at concentrations below the recommended values. Therefore, it was determined that these insecticides will result in an effective control at the early stage of S. recurvalis occurrence. Five insecticides showed a time difference in insecticidal activity depending on the mode of action, but all insecticides showed high insecticidal activity against the S. recurvalis larvae (3^rd-instar). Studies on the insecticidal activity of insecticides against S. recurvalis are insufficient. Moreover, there have been no reports on insecticide resistance. In the bioassay of 11 insecticides of 10 modes of action against the S. recurvalis larvae (2^nd-instar), Muralikrishna et al. (2019) reported that all 11 insecticides, including chlorantraniliprole, showed 100% insecticidal activity after 36 h of spraying. This result was similar to our results that chlorantraniliprole, a diamide insecticide, showed a 97.1% control on S. recurvalis at the recommended concentration even in the field conditions. However, the resistance of diamide insecticides to moth pests, including Spodoptera exigua, is rapidly progressing in Korea and China (Nauen and Steinbach, 2016;Cho et al., 2018;Wang et al., 2021). Therefore, to prevent the emergence of insecticide-resistant populations in the future, an alternating insecticide spraying program with different modes of action should be developed.

In the residual effect test of five insecticides, it can be deduced that lufenuron EC should be sprayed at least every 3 days, and chromafenozide EC, chlorantraniliprole WP, tebufenozide WP, and pyridalyl EW sprayed at 7 day intervals to control S. recurvalis larvae on spinach. However, in this experiment, spinach, which was pre-treated with the insecticides, was exposed to sunlight but was not in contact with water through the drip irrigation system. Therefore, the efficacy duration may decrease depending on various irrigation methods, such as sprinklers.

In the field test in spinach greenhouse at two regions, lufenuron EC, chromafenozide EC, chlorantraniliprole WP, tebufenozide WP, and pyridalyl EW showed high control effect of 97.1% or more and did not cause any phytotoxicity to the leaves and stems of spinach, even at double concentrations. In the absence of pesticides registered in spinach for the control of S. recurvalis, the results of this study will be basic data for establishing an integrated control strategy for S. recurvalis. Besides, all tests were conducted according to the pesticide registration test method of South Korea, so this result means these five insecticides are very likely to be registered in spinach.

Although most of the spinach from Gyeonggi area is cultivated in greenhouses, it is sometimes grown in open fields. This difference can result in different insecticidal activities due to photosynthesis from natural light and rain (Hulbert et al., 2011;Hossain et al., 2013). However, when the control of the open field was conducted by selecting minimal rainfall periods, it was expected that control effects would be similar to those under greenhouse conditions.

These results provide basic data for establishing chemical control strategies for S. recurvalis in various crops including spinach. Furthermore, in order to satisfy consumers who want eco-friendly agricultural products and minimize adverse effects on environment, biological pesticides such as neem extracts (Muralikrishna et al., 2019), B. bassiana (Opisa et al., 2018), natural enemies, and even resistant cultivars need to be considered (Othim et al., 2018). In the future, we will do further study to make integrated pest management (IPM) program by mixing the five pesticides selected in this study with various insecticidal plant extracts to minimize the use of pesticides.