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Pyriproxyfen was purchased from Sigma-Aldrich (St. Louis, MO, USA). Plant diterpenes (methyl linderone) were isolated from Lindera erythrocarpa, as described in a previous study (Lee et al., 2015). Each reagent was prepared as a stock solution in dimethyl sulfoxide (DMSO) for the Yeast two-hybrid β-galactosidase assays (Y2H assays). Plant extracts (i.e., the methanol extracts of 1628 plant species) were received from the Korean Plant Extracts Bank (Daejeon, Korea).

The yeast two-hybrid binding test using quantitative β-galactosidase assay was carried out with the Y187 strain transformed with the JH receptor of M. persicae and its partner, Met-SRC, as described by Shin et al. (2018). The transformed Y187 strain was grown according to the protocol established by Shin et al. (2018). The optical density value of yeast (growth and treatment yeast cell) were read on a microplate reader (Spectramax 190, Molecular Devices Corporation, San Jose, CA) at a wavelength of 420 or 600 nm.

To screen the JHD activity of plants, 100 μL of grown yeast cells (OD₆₀₀ = 0.2–0.3) were treated with both 0.1 ppm of pyriproxyfen and 10 ppm of each plant extract in 96-well plates. A positive control of 0.1 ppm of pyriproxyfen with 10 ppm of methyl linderone and a negative control of 0.1 ppm of pyriproxyfen with control solvent (DMSO) were included in each plate. The cells were incubated for 3 h and then subjected to an OD₄₂₀ measurement, which yielded quantitative estimates of β-galactosidase activity. Subsequently, the OD₄₂₀ values were normalized to an arbitrary unit of JHD. Methyl linderone was used as a positive control, owing to its strong interference with pyriproxyfen-mediated Met-SRC binding in tested M. persicae β-galactosidase assay systems (Shin et al., 2018). The binding interference by 10 ppm methyl linderone was calculated as a single arbitrary unit of JHD activity. The averages of duplicate experiments (in arbitrary units) were then used to calculate the specific JHD activity of each plant extract, using the following formula:

where A represents the JHD activity (if A < 0, then A = 0), OD₄₂₀ Control is the absorbance of yeast cells treated with JH, OD₄₂₀ PE is the absorbance of yeast cells treated with 0.1 ppm of pyriproxyfen and 10 ppm of plant extract, and OD₄₂₀ ML10 is the absorbance of cells treated with 0.1 ppm of pyriproxyfen and 10 ppm of methyl linderone.

We tested 3648 plant extracts (from 167 families, including 1628 species), directly adding each extract to the yeast culture to investigate whether the pyriproxyfen-mediated Met-SRC binding could be disrupted by plant extracts in the two-hybrid yeast assay (Table 1). We selected the threshold for high JHD activity based on the results of a previous study (Oh et al., 2017). We set the high JHD value (JHD > 0.7) based on the JHD value (JHD > 0.5) indicated in the previous study (Oh et al., 2017). Among the plant extracts evaluated, in our study, 320 plant extracts were found to disrupt the pyriproxyfen-mediated binding of M. persicae to Met-SRC. We summarized the high JHD value (JHD > 0.7) based on the JHD value (JHD > 0.5) indicated in the previous study (Oh et al., 2017). Based on this, among 320 plant extracts, 272 (angiosperms) and 46 (gymnosperms) extracts were identified (Supplementary Table 1). The 20 most active extracts were identified and selected, and the extract from Camellia (Theaceae) showed the highest JHD activity (Fig. 1). Furthermore, most of the plants whose extracts showed superior JHD activity corresponded to gymnosperms and angiosperms (Fig. 2).

Fig. 1. 
Plant species with high juvenile hormone disruptor (JHD) activity among plant extracts evaluated as treatments against M. persicae. Values and error bars indicate means ± SD, respectively.

Fig. 2. 
Average juvenile hormone disruptor (JHD) activity values per plant groups. Values and error bars indicate means ± SD, respectively (Numbers in parentheses indicate plant extract).

Extracts from the four major vascular plant groups, namely the Pteridophyta, Lycopodiophyta, gymnosperms, and angiosperms, had significant effects on M. persicae two-hybrid yeast assay. We found that the JHD activity was particularly elevated in gymnosperms, with a mean value significantly higher than that of the Pteridophyta, Lycopodiophyta, and angiosperms, respectively (Fig. 2). Furthermore, after determining the average JHD activity for Pteridophyta, Lycopodiophyta, and gymnosperm extracts, excluding less than 5 extracts, the top 10 plants were selected and identified from more than five extracts of angiosperms (Fig. 3). High JHD activity was evaluated from the extracts of two gymnosperm families, namely Cupressaceae, which contained the species Platycladus orientalis, and Pinaceae, which included pines (Fig. 3A), as well as for the Lycopodiophyta family Selaginellaceae (Fig. 3D), and Dryopteridaceae (Pteridophyta; Fig. 3C). In addition, JHD activity was also measured in angiosperm, namely Magnoliaceae and Alismataceae (Fig. 3B). The JHD activity of both Cupressaceae and Pinaceae was higher than that of other plant taxa and higher than the average value for all plant extracts. Also, this study confirmed that the effect of JHD on the M. persicae-specific JHD assay system differs according to plant group. We found that the JHD activity was particularly concentrated in the gymnosperms (Oh et al., 2017); the mean JHD activity of the gymnosperm extracts was significantly higher than that of the Lycopodiophyta, Pteridophyta, and angiosperms, respectively.

Fig. 3. 
Average juvenile hormone disruptor (JHD) activity values per plant family, subdivided in plant group. (A) gymnosperms; (B) angiosperms; (C) Pteridophyta; (D) Lycopodiophyta. Values and error bars indicate means ± SD, respectively. (Numbers in parentheses indicate plant extract)

In addition, camellidin II from the camellia flower, which was shown to have high JHD activity in this study, is known to be an antifeedant for Eurema hecabe larvae (Numata et al., 1987). Secondary metabolites which are produced by plants, such as camellidin II, are known to react against insects that harm plants and to elicit the production of substances similar to the hormones of those insects (Toong et al., 1988; Dinan, 2001). Accordingly, plant extracts containing secondary metabolites can potentially be developed as novel insecticides that are safe and environment-friendly. Notably, the present screening method with plant extracts can rapidly identify candidates for new insecticide substances. Therefore, our results indicate that the discovered JHD compounds could be used to develop novel insecticides.