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BACKGROUND: Double‐stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as 'Candidatus Liberibacter', which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV‐LASER) to promote dsRNA penetration into leaves and vasculature. RESULTS: UV‐LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100‐, 300‐ and 600‐bp dsRNAs that, in general, were correlated with size. The 100‐bp dsRNA required no pretreatment, whereas 300‐ and 600‐bp dsRNAs entered the vasculature after UV‐LASER treatment only and UV‐LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant‐derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta‐delivered dsRNA (200–250 bp) was determined by feeding third‐instar psyllids on tomato leaves post UV‐LASER adjuvant/surfactant treatment, with or without psyllid cdc42‐ and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real‐time polymerase chain reaction with reverse transcription (RT–qPCR) amplification. At 10 days post the ingestion‐access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. CONCLUSION: Results indicated that UV‐LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Simple Summary: The Asian citrus psyllid is an insect pest of citrus trees that transmits the causal agent of huanglongbing disease. Double-stranded RNA molecules trigger RNA interference (RNAi), a naturally occurring process in eukaryotes that modulates the expression of genes involved in host innate immunity. Use of dsRNA biopesticides offers an alternative to traditional insecticides. In this study, the concentration of dsRNA required to achieve optimal knockdown and mortality for screening exogenous dsRNA by oral delivery was 200 ng/μL. Implementing this pre-established dsRNA regime for laboratory screening of ACP RNAi will enable high-throughput discovery of dsRNA targets and aid in evaluating RNAi biopesticide formulations. Establishing a working threshold can also prevent the use of excessive amounts of dsRNA, which is expensive, can negatively influence RNAi efficiency, and potentially trigger off-target effects. The Asian citrus psyllid (ACP) is a citrus pest and insect vector of "Candidatus Liberibacter asiaticus", the causal agent of citrus greening disease. Double-stranded RNA (dsRNA) biopesticides that trigger RNA interference (RNAi) offer an alternative to traditional insecticides. Standardized laboratory screening of dsRNA requires establishing the minimal effective concentration(s) that result in effective RNAi "penetrance" and trigger RNAi, resulting in one or more measurable phenotypes, herein, significant gene knockdown and the potential for mortality. In this study, knockdown was evaluated for a range of dsRNA concentrations of three ACP candidate genes, clathrin heavy chain (CHC), vacuolar ATPase subunit A (vATPase-A), and sucrose non-fermenting protein 7 (Snf7). Gene knockdown was quantified for ACP teneral adults and 3rd instar nymphs allowed a 48 h ingestion-access period (IAP) on 10, 50,100, 200, and 500 ng/µL dsRNA dissolved in 20% sucrose followed by a 5-day post-IAP on orange jasmine shoots. Significant gene knockdown (p < 0.05) in ACP third instar nymphs and adults ranged from 12–34% and 18–39%, 5 days post-IAP on dsRNA at 10–500 and 100–500 ng/µL, respectively. The threshold concentration beyond which no significant gene knockdown and adult mortality was observed post-48 h IAP and 10-day IAP, respectively, was determined as 200 ng/µL, a concentration indicative of optimal RNAi penetrance. [ABSTRACT FROM AUTHOR]

BACKGROUND: RNA‐mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence‐specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double‐stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold‐change in gene expression using a quantitative polymerase chain reaction. RESULTS: Knockdown of the D24 target, at 39%–45%, resulted in 51% PoP mortality by 10 days post‐ingestion (dpi) of dsRNA. Knockdown of C7, at 38%–61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co‐delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%–12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP. CONCLUSIONS: Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]