Exogenously applied double-stranded RNA (dsRNA) molecules onto tomato leaves, moved rapidly from local to systemic leaves and were uptaken by agricultural pests namely aphids, whiteflies and mites. Four small interfering RNAs, deriving from the applied dsRNA, were molecularly detected in plants, aphids and mites but not in whiteflies. Double-stranded RNA (dsRNA) acts as the elicitor molecule of the RNA silencing (RNA interference, RNAi), the endogenous and evolutionary conserved surveillance system present in all eukaryotes. DsRNAs and their subsequent degradation products, namely the small interfering RNAs (siRNAs), act in a sequence-specific manner to control gene expression. Exogenous application of dsRNAs onto plants elicits resistance against plant viruses. In the present work, exogenously applied dsRNA molecules, derived from Zucchini yellow mosaic virus (ZYMV) HC-Pro region, onto tomato plants were detected in aphids (Myzus persicae), whiteflies (Trialeurodes vaporariorum) and mites (Tetranychus urticae) that were fed on treated as well as systemic tomato leaves. Furthermore, four siRNAs, deriving from the dsRNA applied, were detected in tomato and the agricultural pests fed on treated tomato plants. More specifically, dsRNA was detected in agricultural pests at 3 and 10 dpt (days post treatment) in dsRNA-treated leaves and at 14 dpt in systemic leaves. In addition, using stem-loop RT-PCR, siRNAs were detected in agricultural pests at 3 and 10 dpt in aphids and mites. Surprisingly, in whiteflies carrying the applied dsRNA, siRNAs were not molecularly detected. Our results showed that, upon exogenous application of dsRNAs molecules, these moved rapidly within tomato and were uptaken by agricultural pests fed on treated tomato. As a result, this non-transgenic method has the potential to control important crop pests via RNA silencing of vital genes of the respective pests.

RNAi-mediated insect pest management has recently shown promising results against the most serious pest of tomato, the tomato leafminer, Tuta absoluta. This study aimed to investigate whether dsRNA (dsTa-αCOP) designed to target the T. absoluta-αCOP gene could cause adverse effects to its biocontrol agent, the mirid predator, Nesidiocoris tenuis. Oral exposure of N. tenuis to dsRNA (dsNt-αCOP) designed to target N. tenuis-αCOP resulted in a 61%, 67% and 55% reduction in its transcript level in comparison to the sucrose, dsGFP and dsTa-αCOP treatments, respectively. In addition, significantly higher mortality of 57% was recorded in dsNt-αCOP-treated N. tenuis when compared to the sucrose (7%), dsGFP (10%) and dsTa-αCOP (10%) treatments. Moreover, the predation rate of ~33–39 Ephestia kuehniella eggs per N. tenuis adult dramatically reduced to almost half in the surviving dsNt-αCOP-treated N. tenuis. This worst-case exposure scenario confirmed for the first time that the RNAi machinery is functional in this species and that the risk of exposure through the oral route is possible. In contrast, dsTa-αCOP did not cause any sub-lethal effects to N. tenuis upon oral exposure. Oral exposure of T. absoluta to dsTa-αCOP resulted in 50% mortality. In the context of a biosafety risk assessment of RNAi-mediated insect management, investigating the effects on non-target organisms is essential in order to include this method as part of an integrated pest management strategy. Based on our laboratory assays, RNAi-mediated control is compatible with the biological control of T. absoluta by its natural enemy N. tenuis, adding the RNAi approach in the armoire of integrated pest management of T. absoluta.

The phytophagy of the predator Nesidiocoris tenuis (Hemiptera: Miridae) can trigger defense responses in tomato plants against pests, such as two spotted spider mite Tetranychus urticae (Acari: Tetranychidae) and South American leaf miner Tuta absoluta (Lepidoptera: Gelechiidae). The expression of genes governing Jasmonic Acid (JA) biosynthesis pathway and fluctuations in the levels of underlying metabolites have been rarely studied in mirid-infested plants. In the present study, fifteen 3rd instar nymphs of N.tenuis were caged on each top and lower leaf of tomato plants for 4 d to induce plant defense; after this period the predators were removed. With regard to T. absoluta, oviposition preference; larval period; and pupal weight were significantly reduced in N. tenuis-punctured plants. T. urticae adults exhibited a significantly higher escape tendency and reduced survival on punctured plants. Metabolomics confirmed such observations revealing substantial differences between N. tenuis-punctured and unpunctured (control) plants. Metabolites directly associated with the activation of the JA defense pathway, such as the precursor α-linolenic acid, had increased concentrations. The expression of the defense-related genes PI-II, MYC2, VSP2, and HEL was increased in the top leaves and only VSP2 and MBP2 in the lower leaves; interestingly, in the middle (unpunctured) leaves VSP2, HEL, and MBP2 were also upregulated, indicating systemic signaling. Collectively, phytophagy of N. tenuis caused adverse effects on T. absoluta and T. urticae, whereas the multi-omics approach (phenomics, metabolomics, and genomics) offered valuable insights into the nature of the plant defense responses and provided useful evidence for future applications in integrated pest management, plausibly resulting in the reduction in the required pesticide volumes.

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