Crop protection potential of the leaves of Juglans mandshurica Maxim.: The phenoloxidase promoting related insecticidal effect of its ethanolic extract

Abstract

Juglans mandshurica Maxim. (JM) is cultivated for timber production, food, and the pharmaceutical industry. Although its leaves (JML) are usually discarded as agricultural waste, they have attracted researchers’ attention for reutilization. The present research was therefore carried out to investigate the crop protection potential of JML. First, phenoloxidase-related insecticidal effects were predicted according to in vitro tyrosinase-potentiating property of JML extracts using its 70% and 90% ethanolic extracts from different collection dates (June, July, and August). This was subsequently verified by using diverse bioassay experiments to evaluate its in vivo bioactivity against Plutella xylostella Linnaeus, Aphis gossypii Glover, and Pieris rapae Linne. The results showed that, 90% ethanolic JML extract (20 mg/mL) had the strongest selective anti-feeding activity, with 87.74% and 79.35% deterrence against A. gossypii and P. rapae, respectively. After four days of oral administration, the 70% ethanolic JML extract (50 mg/mL) showed ∼100% corrected mortality in all test insects. Furthermore, regarding contact toxicities, the JML extracts (25 mg/mL) induced ∼71%, 56%, and 82% corrected mortalities against P. xylostella, P. rapae, and A. gossypii, respectively. Among them, JML extracts showed the strongest growth inhibition against P. xylostella (34.12%) and P. rapae (41.23%) after 24 h treatment. Second, the insecticidal mechanism of JML was investigated by targeting the phenoloxidase (PO) of P. xylostella. The insect’s PO activity was promoted in vitro with the maximum efficacy of 35.09% from 9 mg/mL of JML extract, and this increasing potential could also be observed in the in vivo evaluation. For instance, after the administration of LC70 of JML extract till the 4th instar of the insect, stronger PO activity was detected compared to the control group (77.3% vs. 58.0%). The level of L-Dopa, a key intermediate of PO, significantly increased (31.11 μg/mL) in the hemolymph of insects when treated with the LC70 of JML extract compared with the control group (4.29 μg/mL). The qRT-PCR-mediated mRNA expression analysis confirmed increased PO expression in P. xylostella compared to the control group after JML extract administration. Finally, UPLC-Q-TOF-MS coupled with partial least squared discriminant analysis (PLS-DA) was used to rapidly profile and characterize the 18 compounds as the index ingredients that were related to PO enzymatic activity potentiation or insecticidal effects in the JML extracts. Therefore, our research highlights the potential of J. mandshurica for insecticide development and demonstrates that JML’s abnormal promotion of insect PO activity contributes to its toxicity against pests.