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New Frontiers in Pest Control: Chitosan Nanoparticles-Shielded dsRNA as an Effective Topical RNAi Spray for Gram Podborer Biocontrol

  • Henry Kolge
    Henry Kolge
    Nanobioscience Group, Agharkar Research Institute, Pune 411004, India
    Savitribai Phule Pune University, Pune 411007, India
    More by Henry Kolge
  • Kartiki Kadam
    Kartiki Kadam
    Nanobioscience Group, Agharkar Research Institute, Pune 411004, India
  • Sharad Galande
    Sharad Galande
    Agricultural Entomology Section, College of Agriculture, Mahatma Phule Krishi Vidyapeeth, Pune 411005, India
  • Vikram Lanjekar
    Vikram Lanjekar
    Biodiversity and Bioenergy, Agharkar Research Institute, Pune 411004, India
  • , and 
  • Vandana Ghormade*
    Vandana Ghormade
    Nanobioscience Group, Agharkar Research Institute, Pune 411004, India
    Savitribai Phule Pune University, Pune 411007, India
    *Email: [email protected]
Cite this: ACS Appl. Bio Mater. 2021, 4, 6, 5145–5157
Publication Date (Web):June 4, 2021
Copyright © 2021 American Chemical Society

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    Abstract Image

    Chickpea pod borer, Helicoverpa armigera, displays resistance to chemical insecticides and transgenics. The potential nontransformative RNAi approach of specific gene silencing by mRNA breakdown through exogenous double-stranded (dsRNA) delivery to Helicoverpa faces problems of degradation by nucleases and insect gut pH. We demonstrate that chitosan nanoparticles (CNPs) effectively mediate specific dsRNA delivery against Helicoverpa armigerajuvenile hormone methyltransferase (JHAMT) and acetylcholine esterase (ACHE) target genes. Ionotropically synthesized cationic CNPs (100 nm size, +32 mV charge) loaded dsRNA efficiently and protected it effectively from degradation by nucleases and insect gut pH. Tagging CNPs with Calcofluor fluorescence illustrated its efficient uptake in columnar insect gut cells. The potential of CNPs-mediated dsRNA delivery was elucidated with effective silencing of green fluorescent protein transformed Sf9 cells. Furthermore, CNPs–dsRNA complexes were stable for 5 d on leaf surfaces, and their ingestion with leaf effectively silenced H. armigeraJHAMT and ACHE genes to suppress related enzyme activities and caused 100% insect mortality. Further, in planta bioassay with CNPs–dsRNA spray confirmed the RNAi induced insect mortality. Moreover, CNPs–dsRNA fed nontarget insects Spodoptera litura and Drosophila melanogaster were unaffected, and no toxicity was observed for CNPs in cell line studies. Remarkably, only two low dose (0.028 g/ha) topical CNPs-ache-dsRNA sprays on chickpea displayed reduced pod damage with high yields on par with chemical control in the field, which was followed by CNPs-jhamt-dsRNA nanoformulation. These studies can pave the way for the development of topical application of CNPs–dsRNA spray as a safe, specific, innovative insecticide for sustainable crop protection.

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    Supporting Information

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    • Efficiency of designed dsRNA; ninhydrin assay for N/P ratio; uptake of CNPs in insect gut; protocols for GFP gene silencing in Sf9 cells and analysis by confocal microscopy, FACS, and fluorimetry; screening of CNPs–dsRNA complex with artificial feed bioassay; real-time qPCR for gene expression analysis; enzyme assays of acetylcholine esterase and juvenile hormone methyltransferase; field evaluation of CNPs–dsRNA on chickpea for efficacy against H. armigera, phytotoxicity, and grain yield; synthesis and characterization of the CNPs–dsRNA complex; real-time qPCR analyses of JHAMT and ACHE silencing in leaf feed bioassay; and nontarget effect and specificity of the H. armigera dsRNAs (PDF)

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