Volatiles affect the physiological processes of insect, and are also the main basis for selecting target plants for barrier crops protection. Previous studies have found that cucumbers intercropping with celery (A. graveolens) could reduce the number and oviposition of B. tabaci on cucumbers (Tu and Qin 2017). Also, B. tabaci populations were reduced 84% in intercrops of tomato and C. sativum compared with tomato alone. In our study, we found that A. graveolens, A. rugosa, and C. sativum had a strong repellent effect on B. tabaci (Fig. 1a). Our results provided a new barrier plant (A. rugosa) for “push-pull” strategy and possible new target plant for controlling B. tabaci. Besides, we found that d-limonene also had a strong repellent effect on B. tabaci when its concentration was 10− 1-10− 5 g/ml, so we infered that it might be the main reason for the repellent of plants. RNAi results showed that the preference of B. tabaci to d-limonene was changed, and its repellence to the three plants were also disappeared (Fig. 3, 4a, 4b). Our findings confirmed the original hypothesis that d-limonene played an important role in the avoidance properties of plants.
OBPs are important components of the olfactory system and play a key role in the recognition of signals in B. tabaci, and its relationship with volatiles was also revealed. For example, BtabOBP1 played key role in recognition of R-curcumene, caused differences in B. tabaci perference on wild and cultivated tomatoes (Zhang et al. 2016; Hua et al. 2013; Zhan et al. 2021). BtabOBP1 and BtabOBP4 could bind β-ionone and affected the localization of B. tabaci to egg-laying sites (Li et al. 2019). Results from our study showed that d-limonene can bind BtabOBP3, which was consistent with the qPCR results, indicating that BtabOBP3 played a role in the olfactory response of B. tabaci MED to d-limonene (Fig. 2, 6). BtabOBP3 has been found highly expressed in the head and binded with odorant molecules, such as β-ionone, trans-cinnamaldehyde, trans-2-hexenal, linalool, naphthalene, cedrol, 1,8-cineole, β- citronellol (Wang et al. 2020). Our results confirmed that the recognition of d-limonene by B. tabaci was also related to BtabOBP3. BtabOBP3 played a key role in the odor recognition process of B. tabaci. In addition, the transmission rate of ToCV was reduced by 83.3% after feeding dsBtabOBP3, so BtabOBP3 was likely to become an essential target for controlling B. tabaci (Shi et al. 2019).
D-limonene has a high volatility and toxicity to insects but lower to humans, so it is used as a green pesticide (Wu et al. 2019). For example, d-limonene acted as an insect repellant to control the population of B. tabaci and reduced their feeding on host plants (Johnston et al. 2022). Also, the main components of Zanthoxylum bungeanum essential oil were d-limonene and linalool, which had good toxicity effect on Tribolium castaneum (Liang et al. 2022). Most of the reports have been biased towards measuring the fumigation toxicity of plant essential oils, while our experiment focused on the synergistic effect between d-limonene and different pesticides. D-limonene could control B. tabaci and the LC50 was 81.623 ppm. Compared with a single pesticide, the LC50 of mixtures were decreased by 25.2% (L + B) and 38.7% (L + F), respectively. Our results showed that d-limonene was a good agent to improve the efficacy and reduce the application amount of other insecticides, and it also could control B. tabaci.
Reducing the expression of the Bta11975 gene could reduce the transmission rate of B. tabaci to tomato chlorosis virus (Lu et al. 2021). Silencing the key gene BtTPS in the trehalose synthesis pathway could lead to 90% mortality in B. tabaci nymphs (Gong et al. 2022). This indicated that pest control could be achieved by inhibiting the expression of specific genes in relevant pathways. We discovered for the first time that BtabOBP3 is the target gene of d-limonene that could increase the control effect of B. tabaci. The LC50 of d-limonene decreased by 32.6%, the LC50 of mixtures decreased by 38.4% (L + B) and 33.1% (L + F) after feeding dsBtabOBP3 compared to feeding dsGFP (Table 4). These results suggested that interfering with the expression of target genes could increase the control efficacy of B. tabaci. For example, the detoxification ability of B. tabaci studies could be inhibited by down-regulating the expression of P450 CYP6CM1 gene (Li et al. 2015). In addition, Citrus sinensis essential oil (95.1% d-limonene) could against Musca domestica by fumigation and increased when mixed with P450 inhibitors (Rossi and Palacios 2013). The above research on control effect has focused on the detoxification gene, while we found that OBP could also be used as a target gene for pest control. The control effect of d-limonene on B. tabaci could be enhanced by dsBtabOBP3.
Based on the above results, we conclude that d-limonene has a certain promoting effect on the control of B. tabaci, and found the target gene through molecular mechanism. In summary, this study found for the first time that BtabOBP3 is the target gene of d-limonene and dsBtabOBP3 enhanced the control effect of B. tabaci, which provided a certain means and basis for the comprehensive control of B. tabaci. However, future research is still required to determine the specific molecular mechanism and associated pathways of B. tabaci response to d-limonene.