Spray-Induced Gene Silencing and dsRNA Biopesticides: Delivery Technologies, Regulatory Landscape, and Resistance Management
Introduction
Spray-Induced Gene
Silencing (SIGS) represents a novel and environmentally friendly approach to
crop protection, leveraging the natural mechanism of RNA interference (RNAi) to
combat pests and pathogens without genetic modification of the crop plant. In
SIGS, externally applied double-stranded RNAs (dsRNA) or small interfering RNAs
(siRNAs) are sprayed onto plant surfaces, where they are taken up either by the
plant or the target pest/pathogen. These RNAs trigger sequence-specific gene
silencing in the pest or pathogen, thereby suppressing its virulence or
survival. This approach is an alternative to Host-Induced Gene Silencing
(HIGS), which requires transgenic plants expressing dsRNA. SIGS does not alter
the plant genome, making it broadly acceptable and compliant with non-GMO
practices. Following the recent milestone of Ledprona – the first
sprayable dsRNA biopesticide approved by the U.S. EPA in late 2023 – interest
in SIGS has surged in academia and industry. This review provides an in-depth
examination of SIGS and dsRNA biopesticides, with a focus on delivery
technologies, global regulatory frameworks, biosafety/off-target
considerations, and strategies for resistance management.
At its core, RNAi is a
natural gene regulation and immune mechanism in eukaryotes, whereby dsRNA is
processed into siRNAs that guide the degradation of complementary mRNA,
effectively “silencing” target genes. This precise, sequence-specific mode of
action gives SIGS a key advantage over traditional chemical pesticides: it can
target pests and pathogens with minimal impact on non-target organisms and the
environment. Early studies demonstrated that spraying dsRNA targeting essential
genes of fungal pathogens could dramatically reduce disease severity, for
example achieving over 80% reduction in Botrytis (grey mold) lesions on
grapevines. Similar success has been seen against insect pests and viruses,
establishing proof-of-concept that exogenous RNA can induce “cross-kingdom”
gene silencing (also called environmental or spray-induced RNAi) to protect
plants.
Despite these
promising results, SIGS faces practical challenges before it can be widely
adopted in the field. Naked dsRNA is inherently unstable on plant surfaces,
susceptible to degradation by UV light and RNases, and often has difficulty
penetrating leaf cuticles or insect gut barriers. Moreover, achieving
consistent efficacy under variable environmental conditions (rainfall,
sunlight, etc.) and against diverse pests requires advanced delivery methods.
Researchers are actively developing innovative carriers (nanoparticles,
liposomes, clay nanosheets, etc.) to protect dsRNA and improve its uptake by
target organisms. At the same time, regulatory agencies around the world are
formulating guidelines to ensure the safe use of dsRNA biopesticides, given
their novel mode of action. Important considerations include off-target effects
on non-target species, environmental fate of applied RNA, and potential for
pests to develop RNAi resistance. The sections below delve into (1) the broad
spectrum of SIGS applications across insects, fungi, viruses (and even weeds),
(2) the cutting-edge delivery technologies enabling effective dsRNA sprays, (3)
the evolving regulatory landscape and real-world approvals, (4) biosafety and
off-target impact assessment, and (5) resistance management strategies to
sustain the long-term efficacy of dsRNA biopesticides.
Figure 1: Schematic
of Spray-Induced Gene Silencing (SIGS) in crop protection. Exogenously applied
dsRNA (e.g., via foliar drone spray) adheres to plant surfaces and is either
taken up by pest/pathogen cells directly or by plant tissue and then transferred
to the pest. Once inside the target organism, the dsRNA is processed by Dicer
into siRNAs, which are loaded into the RNA-induced silencing complex (RISC).
The RISC–siRNA complex then binds and cleaves complementary mRNA in the
pest/pathogen, silencing the expression of vital genes. This targeted gene
knockdown impairs the pest or pathogen (e.g., causing death or loss of
virulence), thereby enhancing disease resistance in the plant. Importantly, the
dsRNA is designed to not match plant genes, so it does not affect the crop’s
own cells.
SIGS Applications
in Crop Protection
SIGS for Insect
Pest Control: Foliar
application of dsRNA has been explored as a way to control a variety of insect
pests. In practice, dsRNA is sprayed onto plant leaves and stems, where it
either adsorbs to the surface or is taken up into plant tissues. In
plant-feeding insects (especially chewing or sucking insects), ingestion of
dsRNA-coated tissues can trigger an RNAi response that knocks down essential
insect genes. For instance, Ledprona (EPA-approved in 2023) targets the psmb5
gene of the Colorado potato beetle (Leptinotarsa decemlineata), which
encodes a proteasome subunit critical for cell survival. By silencing this
gene, the dsRNA causes larval mortality and protects potato crops. Field and
lab studies have shown that Ledprona (trade name “Calantha”) indeed
causes high mortality in potato beetle populations and reduces feeding damage
on treated plants. Many other insect targets have been investigated: genes
involved in metabolism, digestion, or immunity of pests like cotton bollworm,
fall armyworm, aphids, and beetles have been silenced via dsRNA feeding,
resulting in impaired development or death (up to ~80–100% mortality in some
cases). Notably, the susceptibility to SIGS can vary among insect orders –
coleopteran pests (beetles) tend to be highly susceptible, whereas some
lepidopterans (moths/butterflies) are less so due to factors like
dsRNA-degrading gut nucleases. Nonetheless, advances in formulation (e.g.,
nanoparticle carriers) are improving RNAi efficiency even in less susceptible
insects. A major appeal of dsRNA insecticides is their specificity: the dsRNA
can be designed to target a gene unique to the pest, avoiding impacts on
beneficial insects, wildlife, or humans. This makes SIGS an attractive tool for
Integrated Pest Management (IPM), enabling the suppression of pest populations
while conserving natural enemies and pollinators.
SIGS for Fungal
Disease Control: Many fungal
pathogens of plants have been shown to naturally take up external RNAs from
their environment, providing an opportunity to target them with dsRNA sprays.
Pioneering work by Koch et al. (2016) demonstrated that spraying dsRNA
targeting Fusarium genes (e.g., ergosterol biosynthesis enzymes) on
barley could significantly reduce fungal infection – transcripts of the
targeted genes dropped ~50%, and pathogen growth was curtailed. Similarly,
spraying dsRNAs against Botrytis cinerea Dicer-like genes (key to the
fungus’s pathogenicity) reduced grey mold disease severity on fruits and
vegetables by over 80%. These successes arise because fungi such as B.
cinerea, Sclerotinia sclerotiorum, Verticillium dahliae, etc., can take up
dsRNA from plant surfaces (often via clathrin-mediated endocytosis) and trigger
gene silencing internally. However, uptake efficiency varies: some fungi (e.g.,
Colletotrichum species) show poor dsRNA uptake, which can limit SIGS
effectiveness. For fungi where uptake is efficient, the outcomes can be
powerful – targeting vital fungal genes can inhibit spore germination, hyphal
growth, or virulence factor production, essentially “vaccinating” the plant
against the pathogen. A recent study showed that dsRNA sprays against Austropuccinia
psidii (myrtle rust) had both preventive and curative effects on infected
plants, reducing disease severity when applied either before or after
infection. One important consideration is that fungi, being eukaryotes like
plants, may share some gene sequences with their host; thus dsRNA design must
avoid sequences that could match plant transcripts (to prevent any off-target
silencing in the crop). Overall, SIGS offers a promising alternative to
chemical fungicides, especially as fungicide resistance grows in many pathogen
populations. By targeting pathogen-specific genes (for example, those unique to
fungal metabolism or virulence), dsRNA sprays can suppress diseases like
powdery mildew, rusts, and wilts without the broad toxicity associated with
conventional fungicides.
SIGS for Viral
Disease Protection: Plants are
vulnerable to numerous viruses, against which traditional control options are
very limited. RNA interference is a natural antiviral defense in plants –
infected plants produce virus-derived small RNAs that help silence viral
genomes. SIGS builds on this by externally providing dsRNA matching the virus,
thereby amplifying the silencing signal against the virus. Spraying dsRNA
targeting essential viral genes can protect plants from infection or reduce the
severity of symptoms. For example, spraying dsRNA corresponding to Tomato
spotted wilt virus (TSWV) genes significantly delayed symptom development
in tomato plants. Other studies have shown dsRNA or hairpin RNA sprays can curb
viruses like Tobacco mosaic virus, Potato virus Y, Zucchini yellow mosaic
virus, and Alfalfa mosaic virus, resulting in lower viral loads and
milder disease symptoms in the host plants. An important innovation in this
arena was the use of layered double hydroxide clay nanosheets (known as
“BioClay”) to deliver dsRNA for virus control. Mitter et al.
demonstrated that dsRNA against Pepper mild mottle virus loaded onto
clay particles and sprayed on leaves conferred protection for 20+ days,
whereas naked dsRNA washed off quickly. The clay gradually releases dsRNA,
allowing newly grown leaves to also gain protection as the dsRNA moves
systemically to some extent. This highlights the synergy between RNAi and
delivery technology, which is further discussed in the next section. Overall,
dsRNA sprays can be a game-changer for managing plant viruses by providing a
highly specific, residue-free means of immunity. Unlike conventional antivirals
or cross-protection strategies, SIGS can be quickly retargeted to new virus
strains (important given viruses’ high mutation rates) and leaves no persistent
chemicals on the crop.
SIGS for Weed
Management (Emerging): While
most SIGS research has targeted insect and disease pests, there is interest in
using dsRNA to control weeds. Weeds compete with crops for resources and often
evolve resistance to herbicides, so RNAi could offer a novel mode of action. In
principle, a dsRNA could be designed against a weed-specific gene such that
spraying it on unwanted plants causes gene knockdown and growth inhibition. One
study provided a proof-of-concept by spraying dsRNA targeting a chlorophyll-binding
protein gene in the noxious vine Mikania micrantha, which caused the
weed’s leaves to yellow and the plants to wilt. Another patent proposal
described using SIGS to silence genes that confer herbicide resistance in weeds
(e.g., to restore glyphosate sensitivity in resistant Amaranthus
pigweeds). However, weed applications of SIGS are still in their infancy and
face unique challenges. Unlike pest insects or pathogens, weeds are plants –
their genetic makeup often overlaps with crops. Designing a dsRNA that kills a
weed without affecting a related crop species or other flora requires exquisite
specificity. For example, many grasses or brassica weeds share genes with crop
varieties, raising risk of off-target crop injury if those sequences aren’t
carefully excluded. Delivery is another hurdle: ensuring the dsRNA penetrates
into the weed plant’s tissues (perhaps through stomata or cut wounds) in
sufficient quantity to trigger RNAi. Stability of the dsRNA in the field (sunlight,
rain) is critical, as weeds may require prolonged suppression. Given these
obstacles, and the fact that weeds can often regrow from untouched meristems or
seeds, SIGS for weed control is not yet practical on a large scale. To
progress, research is needed to expand genomic resources for weeds (to find
unique target genes), and to improve delivery methods that could, for instance,
translocate siRNAs throughout the weed plant. Despite the current limitations,
this remains a fascinating frontier – in the future, “RNAi herbicides” might
complement traditional herbicides, especially to manage herbicide-resistant
weeds in an ecologically benign way.
In summary, SIGS
technology has demonstrated broad applicability across multiple pest domains
– from chewing insects and sap-suckers, to fungal and viral pathogens, and even
to parasitic or invasive plants. In each case, the appeal lies in specificity
and adaptability. Unlike conventional pesticides that are chemically
indiscriminate, dsRNA will only affect organisms that have a matching gene
sequence, greatly reducing impacts on non-target species. Moreover, if a new
pest threat emerges, RNAi solutions can be developed rapidly by identifying a
suitable gene target and synthesizing the corresponding dsRNA (a process much
faster and cheaper now than developing a new chemical pesticide). However,
translating these promising lab and greenhouse results to consistent field
success requires overcoming several biological and technological challenges –
most critically, efficient delivery of dsRNA and maintaining its stability in
the agro-ecosystem. In the next section, we discuss the state-of-the-art
delivery technologies that enable sprayable dsRNA biopesticides to perform
under field conditions.
Delivery
Technologies for dsRNA Biopesticides
One of the central
challenges in SIGS is how to deliver dsRNA molecules effectively to
their intended targets in the field. When simply sprayed in naked form, dsRNA
faces multiple hurdles: it can be washed off by rain, degraded by UV radiation
or nucleases, and may struggle to cross the waxy plant cuticle or insect gut
lining. To address these issues, researchers have developed a variety of
delivery and formulation technologies. Broadly, the goals of these technologies
are to protect the dsRNA from degradation, facilitate its uptake
into the plant or pest, and extend the duration of its effect so that
fewer applications are needed. Below we review major delivery strategies,
including nanoparticle carriers, chemical additives, and innovative formulations
that have emerged in recent years.
Naked dsRNA Sprays
and Formulation Aids
Early SIGS experiments
often used naked (unformulated) dsRNA sprayed onto plants, sometimes with a
simple surfactant or buffer. This can work under controlled conditions – for
example, when a plant is kept out of rain and the pest/pathogen is introduced soon
after dsRNA application. In such scenarios, the dsRNA can adhere to leaf
surfaces and be taken up by fungi or ingested by insects before it degrades.
However, under realistic field conditions, unprotected dsRNA has a very short
window of efficacy. Studies show that unformulated dsRNA may persist only hours
to a couple of days on foliage due to environmental exposure, and it generally
does not survive a rainfall event (washing off the leaves). For instance,
experiments with naked dsRNA against plant viruses found that the RNA was
undetectable on leaves after just 5–7 days and provided only transient
protection unless frequently re-applied.
To improve
performance, simple formulation additives can be used. Surfactants or
wetting agents help dsRNA spread and stick to leaf surfaces uniformly,
potentially aiding its absorption into tissues. Sticker or film-forming
agents can create a thin layer that shields dsRNA from being immediately
washed off (e.g., materials like xanthan gum or certain polymers that leave a
residue). Another approach is pegylation or end-capping of dsRNA –
chemically modifying the RNA ends or backbone to make it less prone to nuclease
digestion (some studies have explored 2’ fluoro or 2’ O-methyl nucleotide
modifications for stability, although one must ensure these don’t impair
gene-silencing efficiency). Yet, even with these measures, naked dsRNA is
considered the “baseline” – often insufficient alone for long-lasting field
protection. Thus, attention has shifted to more advanced carriers, especially
nanoparticle-based systems, which are described below.
Nanoparticle-Based
Carriers for dsRNA Delivery
Encapsulating or
binding dsRNA to nanoparticles (NPs) has emerged as a powerful strategy to
enhance stability and delivery efficiency. The use of nanocarriers offers
multiple benefits:
- Protection from Degradation: NPs can shield dsRNA from environmental
RNases and UV light. For example, encapsulation in a nanoparticle matrix
or within vesicles prevents direct exposure of dsRNA to degrading enzymes
and solar radiation. This prolongs the dsRNA’s life on the plant surface
and in the pest’s gut. Studies have shown that certain nanoparticle
formulations prevent dsRNA breakdown even in the alkaline, nuclease-rich
gut environment of insects, which otherwise rapidly degrade naked RNA.
- Improved Uptake: Nano-formulations can facilitate the
entry of dsRNA into target organisms. In insects, NPs help dsRNA cross gut
barriers by protecting it through the peritrophic membrane and aiding
endocytic uptake by gut cells. In fungi, positively charged nanoparticles
can enhance dsRNA attachment to fungal cell walls and promote
internalization via endocytosis. Some NPs even exploit cellular uptake
pathways – for instance, liposomes fuse with cell membranes to deliver their
RNA cargo inside.
- Controlled Release: Many nanocarriers provide a slow-release
effect. Rather than dumping all dsRNA at once (where it could be quickly
degraded), the carrier can release dsRNA gradually over time. This extends
the duration of gene silencing activity. A dramatic example is the BioClay
system (detailed below) where dsRNA persisted on leaves for 30 days and
continued to be effective against viruses for at least 20 days after one
spray.
- Targeting and Penetration: Some advanced nanoparticles are being
designed to target specific pests (e.g. by using ligands or antibodies on
the NP that bind to a pest’s cuticle or gut receptors). While still mostly
in experimental stages, this could increase specificity and reduce the
amount of dsRNA needed. Additionally, extremely small particles (like
carbon dots or quantum dots) can penetrate leaf cuticles or insect
exoskeletons to a degree, potentially allowing novel delivery routes
(e.g., topical absorption without ingestion).
Various types of
nanocarriers have been tested for dsRNA, including polymeric nanoparticles,
liposomes, dendrimers, viral-like particles, inorganic clay nanosheets, and
others. We will highlight a few notable examples:
Polymer-Based
Nanoparticles (Chitosan and Others)
Chitosan nanoparticles
(CNPs) are one of the most
studied carriers for dsRNA. Chitosan is a natural, biodegradable polymer
derived from chitin (found in crustacean shells) and has a positive charge in
acidic conditions. This allows chitosan to electrostatically bind the
negatively charged dsRNA, forming nano-sized complexes (polyplexes). Research
has shown that chitosan-complexed dsRNA is protected from nuclease digestion
and remains intact longer inside insect bodies. For example, dsRNA against Helicoverpa
(cotton bollworm) genes, when loaded onto chitosan NPs, induced much higher
mortality of the pest compared to naked dsRNA, both in lab and field tests.
Chitosan nanoparticles improved RNAi in pests like Spodoptera frugiperda
and Ostrinia nubilalis by preventing gut degradation and facilitating
dsRNA entry into the hemolymph. Additionally, chitosan is attractive for
agriculture because it is cheap, non-toxic, and even has innate antimicrobial
properties that could benefit plant health. Modified chitosans (like chitosan cross-linked
with tripolyphosphate) have achieved >70% kill rates of mosquito larvae via
RNAi, demonstrating the broad utility of this carrier. One must note, however,
that chitosan can bind indiscriminately to other negatively charged substances
(like plant cell walls or proteins), so formulation optimization is needed to
maximize delivery to the target pest.
Apart from chitosan,
researchers have developed dendrimer and star polymer systems for dsRNA.
Dendrimers are highly branched polymers that can carry multiple dsRNA molecules
and potentially protect them by steric hindrance. “Star” polycations (which
have multiple arms that bind RNA) have been used to deliver dsRNA into aphids
and other pests, significantly improving gene knockdown efficacy. For instance,
a fluorescent core-shell nanoparticle functionalized with cationic polymers
delivered dsRNA into Aphis gossypii (cotton aphid) and achieved gene
silencing in the aphid that was otherwise not possible with naked dsRNA. Some
star polymer formulations have shown the ability to increase dsRNA stability on
plant leaves by more than 3-fold and also enhance uptake by pests compared to
dsRNA alone. The downside of these specialized polymers is often their cost of
production and synthesis complexity, but they illustrate the potential for
highly efficient delivery vectors.
Lipid-Based
Carriers and Artificial Vesicles
Lipid nanoparticles
and liposomes are widely used in medical RNA delivery (including mRNA vaccines)
and are now being applied to plant protection RNAi. Liposomes are
essentially tiny bilayer spheres (like artificial cells) that can encapsulate
dsRNA inside. They merge with cell membranes, releasing dsRNA into the cell,
which is ideal for getting RNA into plant or fungal cells. In the context of
SIGS, liposome-encapsulated dsRNAs have been tested for insect delivery: one
study encapsulated dsRNA targeting a moth gene into lipid vesicles and observed
improved knockdown and mortality compared to free dsRNA. Lipid-based
formulations also help dsRNA cross the plant cuticle; some experiments showed
increased uptake of siRNA into sprayed leaves when delivered in cationic lipid
nanoemulsions versus aqueous buffer. Another approach is using virus-like
particles (VLPs) – essentially empty viral capsids (proteins that form a
shell) loaded with dsRNA. VLPs can protect dsRNA from degradation and naturally
enter cells (since viruses evolved to deliver RNA/DNA). Sarkar & Roy-Barman
(2021) demonstrated that VLPs could be used to package dsRNA and deliver it to
pest insects effectively, taking advantage of the capsid’s ability to evade the
host’s extracellular defenses. While still in developmental stages, lipid and
VLP carriers are attractive because they are bio-derived and biodegradable, and
some (like certain lipids) are already approved as safe food or pharma
additives, potentially easing regulatory approval for agricultural use.
Layered Double
Hydroxide Clay (BioClay)
One of the
breakthrough delivery systems for SIGS is the use of layered double
hydroxide (LDH) clay nanosheets, popularly termed BioClay. These are
nanoscopic sheets of inorganic clay (for example, magnesium-aluminum hydroxide)
with a high binding affinity for nucleic acids. Researchers in Australia
(Mitter et al., 2017) found that by simply mixing dsRNA with LDH in
solution, the dsRNA would adsorb onto the clay sheets, forming a stable complex
that can be sprayed onto plants. The implications are impressive: once bound,
the dsRNA does not wash off with water and is shielded from UV and nucleases.
Mitter’s team showed that after a single spray of BioClay carrying
virus-targeting dsRNA, the dsRNA could still be detected on the leaves 30
days later, and plants were protected from the virus for at least 20 days.
Essentially, the clay acts as a slow-release reservoir. As the LDH particles
gradually break down (a process accelerated by slightly acidic conditions on
leaf surfaces), they release dsRNA over time. BioClay has several advantages:
(1) The materials are non-toxic and already present in soils (being similar to
natural clays), so they add no toxic residue. (2) The formulation is relatively
simple and shelf-stable; dry powder of dsRNA-clay can be stored and mixed into
water for spraying. (3) It greatly reduces the frequency of application
– a farmer could spray before a forecasted pest outbreak and the dsRNA remains
effective for weeks. Field trials of BioClay formulations have been reported
against viruses and fungal pathogens with promising results, and this
technology is on the cusp of commercial deployment. In fact, the first
RNA-based product submitted for approval in Brazil (by GreenLight Biosciences
in 2025) uses a form of this technology for controlling grapevine powdery
mildew, highlighting that no cold-chain is needed and it can be mixed with
other chemicals easily. One consideration with clay carriers is that, being
inorganic particles, they could potentially accumulate in soil if overused.
However, LDH clays are generally considered environmentally benign, and the
quantities used per hectare are low. Ongoing research is examining how long
clay-bound dsRNA persists in various soil types (initial studies indicate they
do eventually degrade and may even be consumed by soil microbes along with the
dsRNA).
Other Novel
Carriers
Emerging
nanotechnologies are continually being integrated with SIGS. Carbon-based
nanocarriers like graphene oxide and carbon quantum dots have been shown to
bind and deliver dsRNA efficiently. A recent study used graphene quantum dots
(GQDs) to deliver dsRNA targeting Fusarium fungus, achieving suppression
of Fusarium head blight in wheat. The GQDs presumably helped the dsRNA permeate
into wheat heads and protected it from rapid degradation. Another area is biopolymer
composites – for example, synthetic polymers that respond to pH or enzymes
to release dsRNA at the right site (like in an insect midgut). Some researchers
are also exploring silica nanoparticles or DNA-based nanostructures
(e.g., DNA origami loaded with dsRNA) as carriers. Additionally, artificial
extracellular vesicles (nano-sized vesicles made from plant or bacterial
lipids) have been tested; these mimic natural vesicles that plants use for
signaling, thus might be readily taken up by fungal pathogens.
Finally, beyond
carriers, application methods are part of delivery technology. While
foliar spray is the main method (and the focus of this review), it's worth
noting alternatives such as trunk injection for trees, root drench or seed
coating with dsRNA. Trunk injection has been used experimentally to introduce
dsRNA into the xylem, resulting in systemic distribution throughout a tree or
grapevine. This method was able to protect new foliage and even fruits from
pathogens in some cases, but it is labor-intensive and not practical for
broad-acre crops. Seed or grain coatings with dsRNA are being explored so that
seedlings have built-in protection against soil-borne pests (though dsRNA
stability on seeds is a challenge unless a protective matrix is used).
In summary, delivery
innovations are transforming dsRNA biopesticides from a neat idea into a
field-ready reality. The combination of SIGS with nanotechnology is
especially promising: encapsulating dsRNA in various nanocarriers has
repeatedly been shown to boost stability in harsh conditions and improve
delivery to target sites. Encapsulation can also reduce the cost by lowering
the needed dosage and frequency of application. As these technologies mature,
we are seeing products that can be applied just like conventional sprays – for
example, GreenLight’s dsRNA fungicide can be tank-mixed with other pesticides
and does not require special handling, according to developers. This ease of
use will be critical for farmer adoption. The next section examines how
regulatory bodies are treating these novel biopesticides worldwide, especially
now that the first products are moving from labs and trial plots into
commercial fields.
Regulatory
Landscape and Global Approvals
The regulatory
framework for dsRNA biopesticides is actively evolving, as authorities balance
the promise of this new technology with the need to ensure safety for health
and environment. Unlike transgenic RNAi crops (which fall under GMO
regulations), sprayable dsRNA products occupy something of a new category. They
are biochemical pesticides by nature (essentially an active ingredient
that is a nucleic acid sequence), but their specificity and mode of action
differ markedly from conventional chemicals. Below, we outline the regulatory
approaches in key regions and any notable approvals or field trial clearances.
- United States (EPA): In the U.S., dsRNA products are regulated
by the Environmental Protection Agency (EPA) under the biopesticide
framework. The EPA has a Biopesticides and Pollution Prevention Division
that evaluates these products similarly to other biochemical or microbial
pesticides. A landmark decision came in December 2023 when EPA
granted the first commercial registration for a sprayable dsRNA pesticide,
Ledprona, for use on potato crops. This registration is
time-limited for three years, a cautious approach often used for novel
actives to gather additional data. Ledprona targets the Colorado potato
beetle and was approved after EPA’s scientific review concluded it “has
no unreasonable adverse effects” and “no risks of concern to human
health or the environment”. Notably, the EPA worked with international
experts via the OECD during this assessment, reflecting the novelty of
dsRNA risk assessment. The EPA also established an exemption from
tolerance for residues of Ledprona dsRNA on potato, meaning no maximum
residue limit is needed due to its safety profile. This exemption,
published in the Federal Register in October 2023, signifies that EPA
toxicologists found negligible risk if humans consume trace amounts
of the dsRNA on food (RNA is a normal part of our diet and is digested
into nucleotides). The U.S. approach thus far treats dsRNA biopesticides
as biochemical pesticides, which typically have a streamlined
registration compared to synthetic chemicals, provided that data show low
toxicity and environmental impact. It’s worth noting that prior to
Ledprona’s full registration, EPA had granted Experimental Use Permits
(EUPs) for field trials of dsRNA sprays, indicating regulatory openness to
facilitating development. The United States is expected to see more
submissions soon (e.g., products for corn rootworm or soybean pests), and
EPA’s handling of Ledprona sets a precedent for evaluating factors like
off-target genome matches, environmental persistence, and
species-specificity.
- European Union (EFSA and EU Regulations): The EU currently has no fully approved
dsRNA spray products, but research trials have been conducted in Europe
(for instance, field tests in Belgium and Slovenia on Colorado potato
beetle dsRNA were reported). In the EU regulatory system, exogenously
applied dsRNAs are generally treated as conventional plant protection
products (PPPs) under Regulation EC 1107/2009, which means they must
be approved as active substances and then formulated products authorized,
similar to any new pesticide. However, there has been some debate: dsRNA
molecules themselves are not “organisms” or living GMOs, so they are not
regulated under the GMO Directive 2001/18 unless a living GMO is
involved in the product (e.g., a microbial dsRNA factory present in the
formulation). The consensus emerging is that dsRNA sprays are
chemical/biochemical agents. According to a review by Dalakouras et
al. 2024, in the EU “dsRNAs are considered chemical pesticides” for
regulatory purposes. This means a full risk assessment is required,
covering human health (toxicology, residues) and environmental effects on
non-target species, just as for any pesticide, but with appropriate
adaptations for RNAi mode of action. EFSA (European Food Safety Authority)
has published guidance documents on data requirements for RNAi-based GM
plants and is working on guidance for sprayable RNAi products. An
interesting twist is that some EU member states classify certain
dsRNA uses differently: for example, if a dsRNA is produced by a GM
bacterium but the bacterium is removed, the product might be seen as
non-GMO (only containing the RNA). A case-by-case interpretation can occur
until formal guidance is harmonized. The EU’s stance is also influenced by
the European Green Deal and Farm to Fork strategy which aim to reduce synthetic
pesticide use – RNA biopesticides could be welcomed under these
sustainability goals. Nonetheless, the EU public and regulatory scrutiny
on anything biotech is high, so demonstrating that dsRNA products pose no
threat to beneficial insects (like bees, butterflies) and won’t disrupt
ecosystems is critical for approval. It is expected that the first dsRNA
active substance approvals in the EU may happen in the next few years,
potentially with Colorado potato beetle dsRNA (since that pest is a major
problem in Europe and an EU research network has been evaluating RNAi for
it).
- Brazil and Latin America: Brazil is moving quickly in this area. In
October 2025, GreenLight Biosciences announced it had submitted for
registration the country’s first RNA-based fungicide, targeting
grapevine powdery mildew. Brazilian regulatory authorities (Ministry of
Agriculture – MAPA, together with Anvisa for health and Ibama for
environment) are reviewing this. Notably, Brazil’s biosafety commission
CTNBio has classified sprayable dsRNA products as non-GMO, since
they contain no living modified organisms and do not alter genomes. This
classification greatly simplifies the approval process – the product is
evaluated like a biochemical pesticide, without the layers of GMO regulation.
GreenLight’s Brazilian submission highlights some favorable points: the
dsRNA leaves no residues (and thus no MRL needed), it can be applied close
to harvest with no export barriers, and it doesn’t require cold storage in
distribution. Brazil’s existing pesticide law doesn’t explicitly mention
dsRNA, but the country has historically been progressive in adopting
biotech crops and biological controls, so approvals could be swift if data
show safety and efficacy. Indeed, GreenLight’s reps noted their RNA product
meets stringent EU and North American residue standards, implying
confidence in regulatory acceptance. We also see interest in RNAi products
in other Latin American countries (Argentina, Chile) often in
collaboration with companies and academic groups, as they participated in
global trials for the powdery mildew product.
- Australia: Australia has been at the forefront of
SIGS research (e.g., the BioClay technology was developed at the
University of Queensland). The regulatory body there (APVMA) has to date
not announced a specific approval, but they did allow field trials of
BioClay on crops like cowpea to protect against viruses around 2017–2018.
Given Australia’s push for agtech innovation, it’s likely that
BioClay-based products or others will be registered in the near future,
especially for high-value crops and to address pesticide resistance
issues. Australian regulators will likely examine these as agricultural
chemicals, but an interesting aspect is that Australia has a category for
“biological” or low-risk products that might expedite things.
- Canada: Health Canada’s PMRA (Pest Management Regulatory Agency) has not
yet had a public case of a dsRNA spray, but as a close regulatory partner
to the US EPA, they may follow EPA’s scientific approach. If a product is
registered in the US, Canadian approval often follows with some delay,
provided Canadian-specific data (like pollinator safety, if needed) are
addressed.
- International Guidelines: To assist all regulatory agencies, the
Organisation for Economic Co-operation and Development (OECD) has
been coordinating expert groups to develop harmonized risk assessment
guidance for dsRNA pesticides. In 2020, the OECD published
“Considerations for the Environmental Risk Assessment of dsRNA-based
Pesticides” and in 2023 a complementary document on human health risk assessment.
These documents cover how to evaluate issues like: the likelihood of dsRNA
affecting non-target organisms (based on sequence similarity and the
presence of RNAi machinery in those organisms), the degradation and
persistence of dsRNA in various environmental compartments (soil, water,
plant surfaces), and any potential for dsRNA to trigger immune responses
(for instance, long dsRNA can trigger interferon responses in mammals at
high doses, but typical environmental exposures are far below those levels).
Regulators worldwide are using these guidelines to shape their evaluation
protocols. Generally, the consensus so far is that dsRNA pesticides can
be considered “low-risk” when designed properly, because: (1) they are
not toxic chemicals, but rather naturally occurring molecules that degrade
into basic nutrients; (2) they can be made very specific to the target
organism’s genes; (3) they do not amplify or replicate (unlike a
biocontrol organism, dsRNA has no life of its own); and (4) they usually
do not persist long in the environment.
In summary, the
regulatory landscape is cautiously optimistic. The first approvals (like EPA’s
registration of Ledprona) set important precedents, demonstrating that
regulators are willing to embrace novel RNAi technology when evidence shows
safety and need. Global harmonization efforts via OECD mean that data packages
prepared for one jurisdiction may be useful in others, potentially accelerating
approvals across countries. Still, regulators emphasize the importance of case-by-case
evaluation, because each dsRNA is different in sequence and each use case
may have different non-target exposure profiles. For instance, a dsRNA sprayed
in an orchard might drift to wild plants or expose pollinators to trace
amounts, so those scenarios must be considered. Risk mitigation measures (such
as buffer zones, timing of sprays to avoid pollinator activity, etc.) could be
imposed if needed, but so far the assessments indicate very low risk profiles.
In fact, EPA noted that Ledprona provided an additional tool for resistance
management and could replace more toxic insecticides, aligning with the goal of
safer pest control. A challenge for regulators is also public perception –
ensuring that the farming community and public understand that an “RNA
pesticide” is not a transgenic crop and does not alter genomes. Clear
communication, as done in Brazil where the product is explicitly labeled
non-GMO by CTNBio, will help acceptance.
As of 2025, field
approvals exist in the U.S. (Ledprona on potatoes) and are pending or in
trial phase in Brazil (RNA fungicide in grapes) and possibly the EU
(experimental trials under exemptions). The next few years will likely see a
rapid expansion of submissions – covering pests like corn rootworm, soybean
aphid, varroa mite (in beehives), and pathogens like wheat rust or banana
viruses. Each will test the regulatory frameworks and provide learning. It’s an
exciting era where regulations are catching up with technology to enable the
deployment of dsRNA biopesticides as part of sustainable crop protection.
Biosafety and
Off-Target Considerations
A critical component
of bringing dsRNA biopesticides to market is ensuring their biosafety –
that they do not cause unintended harm to non-target organisms or the
environment. Fortunately, many features of SIGS inherently promote safety, but
careful assessment is still required. Here we discuss off-target effects,
environmental fate, and overall biosafety considerations for dsRNA sprays:
- Specificity and Off-Target Effects: The primary safety advantage of dsRNA
pesticides is their high specificity. A dsRNA is designed to have at least
20 consecutive nucleotides complementary to a target gene in the
pest/pathogen. The probability of the exact same 20nt sequence occurring
in a non-target organism’s genome (and being in an expressed gene) is
extremely low, especially if that non-target is not closely related to the
pest. In the design phase, developers routinely use bioinformatics to
compare the dsRNA sequence against genomes/transcriptomes of beneficial
insects, mammals, birds, etc. Any sequence with significant matches
(usually ≥21 nt contiguous) to off-target genes would be discarded. As a
result, the final product should ideally only silence the intended gene in
the intended pest. Empirical studies support that off-target effects are
minimal: for example, Zotti & Smagghe (2015) reviewed RNAi in pest
control and found it to be “relatively specific with few off-target
effects”. In another case, dsRNA designed for western corn rootworm
had no observable impact on ladybird beetles or lacewing predators in
laboratory feeding tests, indicating that even if they ingested some
dsRNA, it did not match their genes to cause harm. However, caution is
warranted: there are rare scenarios where partial sequence overlap
might cause a mild off-target knockdown in a non-target organism. Some
genes are conserved across species (e.g., actin, tubulins), but a
responsible dsRNA design will avoid targeting such broadly conserved
genes. Additionally, plants and animals have differing codon usage and
sequence motifs, further reducing unintended interactions. Modern dsRNA
products like the RNA fungicide in Brazil explicitly state that the chosen
gene has no overlap with any non-target organism’s genetic code. This
“safe by design” approach is a cornerstone of dsRNA biosafety.
- Impact on Beneficial Insects and Soil
Organisms: A key
stakeholder concern is usually, “Will this RNA spray harm bees,
butterflies, earthworms, or other beneficials?” Based on current evidence,
the answer is: highly unlikely, if properly designed. Bees, for instance,
do have an RNAi pathway (they use it for antiviral defense), but a dsRNA
pesticide would need to have sequence similarity to a bee gene to affect
them. Developers ensure no such similarity. Experiments where bees were
exposed to plant-incorporated dsRNA (in GM plants) showed no adverse
effects on bee health or development. Similarly, tests on parasitoid wasps
(which might consume host fluids containing dsRNA) found no evidence of
harm, suggesting that any dsRNA they encounter either doesn’t match their
genes or is at too low an amount to matter. Earthworms and soil microbes
would likely encounter dsRNA after it washes off or the plant decays. RNA
is a nutrient source for microbes, so soil microbiota may even benefit
slightly from an input of RNA – importantly, since the dsRNA does not
replicate, it will just be another bit of organic matter. A study on the
environmental fate of a Monsanto-developed dsRNA (DvSnf7) showed that it degraded
in various soils with half-lives on the order of days, and no accumulation
was observed. The degraded RNA is simply recycled in the soil food web.
The EPA, in its review of Ledprona, concluded there were “no effects to
listed species under the Endangered Species Act”, indicating that they
did not find any credible risk to any non-target species from that dsRNA.
However, regulators often require testing on a representative set of
non-targets (e.g., a pollinator, an aquatic invertebrate, a bird, fish,
etc.) to confirm there’s no unexpected toxicity. So far, those tests have
not revealed issues – which is not surprising, given RNA is present in all
the food we and wildlife eat (every plant and microbial cell is full of
RNA). The difference here is the dsRNA is designed to be effective in the
pest that has matching mRNA; in other organisms it is just another piece
of dietary RNA.
- Human Safety: Humans are also exposed only to small
amounts of these dsRNAs, mostly by eating produce that had been treated. As
mentioned, the EPA granted a tolerance exemption for Ledprona dsRNA on
food, meaning they judged any residues to be safe. Our digestive system
rapidly breaks down RNA – there is no evidence that dietary RNAi from
plants transfers gene-silencing activity to people. (Humans routinely
consume siRNAs and dsRNAs from food plants without effect; any plant you
eat has microRNAs that do not survive the gut in active form.) The only
theoretical human health concern could be if someone has an allergic
reaction to the formulation components, but the dsRNA itself is not an
allergen or toxin. Inhalation of spray mist by applicators is another
point to examine – RNA is not known to cause respiratory issues (it’s not
like a chemical that can irritate mucous membranes), but formulations will
be tested for any adjuvants that might. So far, dsRNA products are
expected to be labeled with the lowest toxicity category (IV) for EPA,
requiring minimal protective equipment.
- Environmental Persistence: One favorable aspect of dsRNA is that it degrades
naturally and relatively quickly in the environment, which prevents
long-term contamination. Ribonucleases (RNases) are enzymes ubiquitously
present in soils, water, on plant surfaces, and produced by microbes.
These enzymes break RNA into harmless nucleotides. Studies have shown that
dsRNA on plant leaves often degrades within a few days to a couple of
weeks depending on weather. In soil, raw dsRNA typically persists only
briefly (hours to days) because soil microbes consume it; one study noted
that when dsRNA was added to various soils, its half-life was under 48
hours in active microbiologically rich soil. Even in water (e.g., if
runoff carries some dsRNA to a pond), RNases in water and sunlight would
degrade it. Thus, unlike many chemical pesticides which can persist for
months or years and bioaccumulate, dsRNA is a non-polluting,
non-bioaccumulative agent. This contributes to its alignment with
sustainable agriculture goals. Of course, if dsRNA is formulated in a
protective carrier (like clay or polymer), its degradation is delayed as
intended on the crop. But eventually, once released, it still faces the
same natural breakdown. The GreenLight fungicide example explicitly notes
that their RNA degrades quickly with no toxic metabolites, and even frames
it as “RNA serves as food for microorganisms” during degradation.
- Potential Ecological Effects: While direct toxicity is unlikely, one
might ask if there are any subtle ecological ripple effects. For instance,
could knocking down a pest also affect its predators due to reduced food
supply? This is a desired effect actually (pest suppression), and
typically IPM considers that if a pest is controlled, its predators might
temporarily have less prey, but those predators often have alternative
prey or will rebound when needed. Another hypothetical concern: could
environmental RNAi occur in a non-target? For example, if a bird eats many
insects that died from RNAi, could the dsRNA move into the bird’s system?
Research indicates that while dsRNA can transit in food chains (e.g., a
spider eating an insect that had fed on dsRNA-laced plant sap can end up
with trace dsRNA in its gut), it does not usually persist or cause effects
unless the sequence by wild coincidence matched the spider’s genes. The
cross-kingdom RNAi typically requires some level of sequence match and
uptake mechanism. Most vertebrates have robust barriers and innate immune
responses to exogenous long dsRNA (triggering interferon responses and
then degrading the RNA), so it is improbable for a dsRNA pesticide to
silence a gene in a vertebrate. Nonetheless, regulatory risk assessments
consider worst-case exposure and still found no cause for concern.
- Public Perception and GMO Concerns: An indirect “biosafety” issue is public misunderstanding. Because RNAi technology arose from genetic engineering contexts, some people might think spraying dsRNA could somehow make a crop GMO or alter DNA. It’s important to communicate that dsRNA spray is non-transgenic and non-heritable. The sprayed RNA does not integrate into genomes; it simply triggers a temporary suppression of target genes in pests. Public outreach by developers (like the statements that CTNBio in Brazil classified it non-GMO and that it leaves no residues) help alleviate these concerns. Indeed, one of the reasons SIGS was developed was to avoid the controversies of transgenic plants – you get the benefit of RNAi without modifying the plant’s DNA. Regulators have echoed this distinction; for example, EPA’s messaging on Ledprona emphasized it is “not a genetically modified organism” and only uses a natural mechanism to silence pest genes.
Figure 2: Summary
of biosafety considerations for SIGS-dsRNA technology. The Advantages
(green) include highly targeted action, rapid adaptability to new pests,
reduced reliance on synthetic chemical pesticides, eco-friendliness and
compatibility with sustainable agriculture, and potential for improved crop
yields via better pest control. Potential Risks (red) and concerns
involve off-target gene effects in non-target species (minimized by careful
design), ecological impacts (e.g., on food webs) which are considered low, the
possibility of pests/pathogens developing resistance to dsRNA, stability
challenges (RNA can degrade quickly), and public perception issues (e.g.,
confusion with GMOs). Drawbacks or hurdles (blue) include still-limited
field knowledge, variable efficacy under real-world conditions, delivery and
environmental factors affecting performance, regulatory challenges in different
regions, and currently higher production costs (though decreasing). Overall,
the consensus is that the benefits can be harnessed while mitigating risks
through design and management strategies.
In conclusion, the
biosafety profile of dsRNA biopesticides is remarkably favorable when products
are designed and used correctly. Their specificity reduces collateral damage to
ecosystems, and their biodegradability means they don’t persist as pollutants.
Of course, ongoing vigilance is needed: as more products enter the market,
continued monitoring for any unexpected effects (e.g., perhaps some species we
didn’t think of shows sensitivity) is prudent. But to date, both experimental
evidence and regulatory evaluations indicate that dsRNA sprays can achieve pest
and pathogen control with significantly less risk to the environment and
human health than conventional broad-spectrum pesticides. This bodes well for
public acceptance and for integrating SIGS into environmentally conscious
farming systems. The final piece of the puzzle, which we address next, is how
to manage the efficacy of these tools long-term – specifically,
preventing or delaying target pests from developing resistance to RNAi, and
integrating dsRNA sprays into holistic pest management strategies.
Resistance
Management Strategies for RNAi-Based Biopesticides
Like any pest control
method, the sustainability of SIGS will depend on how well we can manage and
mitigate resistance development in target populations. Insects, fungi,
and other pests have historically evolved resistance to chemical pesticides
when exposed repeatedly over time – RNA-based pesticides will be no exception
if not used wisely. However, RNAi offers some unique angles for resistance
management that traditional chemicals do not. In this section, we discuss
potential resistance mechanisms to dsRNA, evidence from lab selection
experiments, and strategies (both in deployment and design) to maintain
effectiveness of dsRNA biopesticides as part of Integrated Pest Management
(IPM).
Mechanisms of
Resistance to dsRNA: Pests
could theoretically evade RNAi in a few ways:
- Target Site Mutations: The simplest mechanism is a mutation in
the target gene’s sequence that reduces the dsRNA’s ability to bind and
silence it. Since RNAi requires sequence complementarity, even a single
nucleotide change in the pest’s mRNA at a crucial spot could hinder
binding of the siRNA. Over time, pest populations under dsRNA pressure
might accumulate such mutations (especially if the dsRNA targets a small
region). This is analogous to how pests develop resistance to Bt toxins
via mutations in gut receptors, for example. There is evidence this can
happen: laboratory selection of western corn rootworm with sublethal dsRNA
led to colonies with single nucleotide polymorphisms in the target gene
that made them less susceptible. Similarly, if a fungus population has
high genetic variability, some individuals might naturally carry sequence
variants less recognized by the dsRNA.
- Reduced Uptake: Pests may become resistant by losing or
down-regulating the mechanisms that allow dsRNA uptake into their cells.
In insects, dsRNA uptake in the gut can involve endocytosis and specific
transport proteins (e.g., SID-1-like channels in some species). If a
subpopulation has a mutation that impairs these uptake pathways, those
individuals might survive dsRNA treatment and pass on that trait. For
instance, studies on fruit flies (Bactrocera dorsalis) found that
strains refractory to RNAi had lower expression of key endocytosis genes;
when those genes were experimentally up-regulated, the RNAi sensitivity
was restored. In western corn rootworm, a lab-selected resistant strain
showed greatly reduced dsRNA uptake in midgut cells compared to susceptible
strain. This suggests the insect evolved a mechanism to prevent dsRNA
entry (possibly changes in gut membrane composition or transporters). In
fungi, theoretically, a mutation that reduces clathrin-mediated
endocytosis would lower dsRNA uptake and confer resistance to SIGS,
although this is harder for a fungus to “choose” since endocytosis is
vital for its normal function.
- Increased Degradation: Pests could ramp up expression of
nucleases that degrade dsRNA. Many insects have dsRNases in their gut lumen.
Continuous exposure to dsRNA might select for individuals producing more
robust dsRNase activity, thus destroying the dsRNA before it can act. Some
studies have noted that lepidopteran pests, which are naturally less
RNAi-susceptible, have very high nuclease activity – an innate form of
resistance. Whether pests can adaptively increase nuclease production in
response to dsRNA exposure isn’t fully documented yet, but it’s a
plausible route.
- Altered RNAi Machinery: Another subtler mechanism would be changes
in the RNAi pathway inside cells – for example, a pest might down-regulate
Dicer or Argonaute proteins in certain tissues, reducing the RNAi effect.
However, since those proteins often have other roles and RNAi is also part
of the pest’s normal gene regulation/antiviral defense, heavily modifying
them might come at a fitness cost to the pest.
It’s worth noting that
not all species are equally prone to RNAi resistance. Some pests already have
variants that make them naturally less sensitive (for example, certain strains
of Lygus plant bugs simply don’t take up dsRNA well, so they are
“refractory” from the start). In contrast, Colorado potato beetle is very
RNAi-susceptible, but even there, lab experiments found that continuous dsRNA
exposure could select for less susceptible populations over generations.
Interestingly, one study on Colorado potato beetle cells in vitro found that
resistant cells had lower expression of a dsRNA-binding protein (StaufenC)
necessary for RNAi, indicating the cells effectively dampened their RNAi
machinery to survive.
Integration into
IPM and Mitigation Tactics:
The good news is that RNAi resistance can be managed – and potentially
more easily managed than conventional pesticide resistance – through several
approaches:
- Gene Rotation or Mixtures: Unlike a chemical with a fixed mode of
action, dsRNA agents can be re-designed to target different genes. If a
pest shows signs of resistance to one dsRNA (say via a target mutation),
the simplest fix might be to switch to a dsRNA targeting another essential
gene in that pest. This is analogous to rotating insecticide modes of
action, but here the modes of action are all RNAi, just different gene
targets. Because producing a new dsRNA is relatively fast (especially with
current cell-free production platforms), a pipeline of alternate targets
can be maintained. In fact, a unique advantage of RNAi noted by
researchers is that if a target gene mutates, we can easily mitigate
that resistance by using a dsRNA targeting a different region or a
different gene. For practical deployment, companies might formulate
products with multiple dsRNAs combined, each against a different target
gene in the pest – a strategy to make it very difficult for the pest to
adapt since it would need simultaneous mutations in multiple genes.
- Use in Rotation with Other Controls: Classic IPM would suggest not relying
solely on one tool. DsRNA sprays could be alternated or combined in a
season with other control measures, such as Bt bioinsecticides,
conventional chemicals (preferably softer ones), or cultural controls. For
the Colorado potato beetle, for example, an IPM program might include crop
rotation (to disrupt pest life cycle), a dsRNA spray during peak larval
emergence, and perhaps a different bioinsecticide later if needed. This
reduces selection pressure specifically for RNAi resistance because the
pest population is periodically hit with other stresses. In EPA’s approval
of Ledprona, they explicitly mention it as a tool in resistance management
– CPB is notorious for becoming resistant to chemicals, so adding RNAi to
the arsenal helps, but conversely we should protect RNAi from resistance
by not overusing it exclusively.
- High Dose and Refuge Strategy: Borrowing from transgenic Bt crop
management, one could employ a “high dose/refuge” approach. The idea is to
apply a sufficiently high dose of dsRNA so that nearly all pests ingest a
lethal amount (minimizing survivors that could breed resistance), and to
provide unsprayed refuge areas where some pests not exposed to dsRNA
survive to dilute any resistant genes. In practice, for sprays, a refuge
might be an unsprayed strip or untreated plants. High dose is achievable
if production is cheap enough to apply a robust concentration of dsRNA
that knocks down even partially resistant individuals. Of course, unlike a
continuously expressed Bt in a plant, spray residues diminish, so timing
and coverage matter for achieving an effective dose.
- Monitor and Respond: Resistance management also involves
monitoring pest populations for early signs of resistance. This could be
done via bioassays (collecting field populations and testing if they still
respond to the dsRNA) or molecular surveillance (checking if target gene
sequences in the population are shifting away from the dsRNA’s sequence).
The paper by Pallis et al. (2023) on baseline susceptibility of US
potato beetle populations to Ledprona is a good example of a
pre-deployment study. They found some geographic variation, with one
population (New York) being naturally less susceptible, possibly due to
general fitness or slightly different genetic background. Knowing this
helps set strategy – e.g., in areas with lower susceptibility, one might
use a higher rate or mix with another control. If field failures of dsRNA
sprays are observed, companies can pivot quickly to alternative dsRNA
targets as mentioned.
- Preserving RNAi Pathway Health: Interestingly, one could think about
measures to avoid pests "shutting down" their RNAi pathways. In
theory, sublethal exposure that allows pests to survive could encourage
them to adapt by diminishing RNAi response. Therefore, ensuring the
initial products are used in a way that gives a lethal knockdown (where
intended) is important. Also, there’s research into synergists that
could inhibit pest nucleases or otherwise boost RNAi efficacy (like
knocking down a pest’s own nuclease gene along with the target gene). This
is a bit high-tech but could be envisioned in the future: a combined dsRNA
targeting both an essential gene and a negative regulator of RNAi in the
pest to double-whammy them.
- Resistance in Pathogens: In fungi or viruses, resistance might
manifest as the pathogen population evolving so that the target gene
sequence diverges or the pathogen stops taking up the dsRNA. For viruses, high
mutation rates mean a dsRNA targeting a very specific sequence could
become obsolete if the virus mutates there. The counter is to target
highly conserved viral regions or multiple regions at once. For example,
target a gene that the virus cannot easily mutate without losing
fitness (perhaps an RNA polymerase gene segment). For fungi, if one strain
becomes less uptake-competent, it might also be less virulent (since these
mechanisms are often tied to normal growth). Nonetheless, rotating targets
and mixing control methods (like combining SIGS with biocontrol fungi or
low-dose fungicides) can prevent any one fungal genotype from dominating.
Integration with
Other IPM Tactics: One of the
most compelling aspects of SIGS is how well it can integrate into IPM programs
geared toward sustainable pest management. Because dsRNA is species-specific
and non-toxic to others, it can be used alongside biological controls
(predators, parasitoids, biopesticidal microbes) without harming them. For
instance, one could release ladybugs for aphid control while spraying a dsRNA
to take out a virus in the crop – the ladybugs are unaffected by the dsRNA
(since it’s designed for the virus) and can continue their beneficial role.
Even more synergistic, recent research suggests some biocontrol agents might enhance
RNAi: certain plant-colonizing beneficial bacteria and fungi induce plant
defenses or produce compounds that could stress pests, making them more
susceptible to dsRNA’s effects. Combining such approaches (volatile organic
compounds from biocontrols plus dsRNA sprays) might yield greater pest
suppression than either alone. Precision agriculture tools (like drones for
targeted spraying and digital monitoring of pest levels) also dovetail nicely
with SIGS – one can imagine using pest population data to decide when/where to
apply dsRNA, thereby using it only when necessary, which inherently slows
resistance development.
In summary, while the
risk of resistance to dsRNA biopesticides is real, we have a robust toolkit to
manage it. Many of the principles from traditional resistance management apply
(rotation, mixtures, refuges, monitoring), but with RNAi we also have the
flexibility of redesigning our active ingredient in ways not possible with
conventional chemicals. This adaptability – making a new sequence – is akin to
having an ever-refreshing arsenal, so long as we can identify new essential
genes in the pest (which - thanks to genomics - is easier than ever). The
integration of SIGS into IPM, rather than using it as a standalone silver
bullet, will ensure its longevity. By using dsRNA sprays judiciously and in
concert with other control measures, we can suppress pest populations, delay
the onset of resistance, and prolong the effective life of these biopesticides.
Given the rapid advances in production and formulation, if a particular dsRNA
loses efficacy, it might only be a matter of a season before the
next-generation solution is available – a very different scenario from chemical
pesticides where development is slow and costly.
Conclusion and
Future Perspectives
Spray-Induced Gene
Silencing has moved from conceptual promise to practical reality within the
span of a decade. The approval of the first dsRNA biopesticide products and the
on-going field trials worldwide mark the dawn of a new era in crop protection –
one where information molecules (nucleic acids) can be used as
eco-friendly weapons against pests and pathogens. This technology aligns
strongly with the modern agricultural trajectory: increasing precision,
sustainability, and safety of pest management.
In this review, we
highlighted how SIGS can target a broad spectrum of crop threats with surgical
precision, leveraging natural biological pathways to disarm enemies while
sparing allies. We explored the cutting-edge delivery systems, from
nanocarriers to BioClay, that address the key hurdles of stability and uptake,
thereby transforming dsRNA applications from ephemeral to long-lasting in the
field. We surveyed the regulatory landscape, noting a generally positive reception
by agencies once data are provided, and a recognition that dsRNA products can
help achieve pesticide reduction goals without sacrificing crop yields.
Biosafety considerations overwhelmingly indicate that these tools can be used
without harming non-target organisms or the environment, especially when guided
by thoughtful design and risk assessment. Finally, we delved into resistance
management, concluding that while vigilance is needed, the inherent
adaptability of RNAi (and IPM-compatible use patterns) equip us better than
ever to sustain efficacy.
Looking forward,
several trends and needs can be anticipated:
- Expansion of Targets: We will likely see SIGS being developed
for more complex pest issues, including those that have been hard to
control by other means. For instance, research may yield dsRNA sprays for
sap-sucking insects like aphids/whiteflies (perhaps using novel delivery
aids to get RNA into phloem), for nematodes (via soil drenches or root
uptake of dsRNA), and even for managing plant parasitic weeds like Orobanche
or Striga (spraying the crop or soil with dsRNA that those
parasitic seedlings absorb). As more genomes are sequenced, identifying
unique target genes in such pests becomes easier. The generalist scope
(all crops, all pests) mentioned by stakeholders is ambitious but
conceivable – ultimately a library of RNA sprays could exist so
that for any new outbreak or emerging pest, a matching RNA can be quickly
formulated.
- Cost Reduction and Production Scaling: While the cost of dsRNA production has
dropped dramatically (from ~$100,000 per gram in early 2000s down to a few
dollars per gram or less with new techniques), continued improvements will
make large-scale use economically feasible for even low-value crops.
Advances such as cell-free enzymatic synthesis (as GreenLight’s platform
uses, costing <$0.50 per gram), or fermentation in bioreactors using
engineered microbes or yeasts, will bring costs down and volumes up. This
is crucial because high volume staple crops (wheat, corn, rice) require
cost per hectare comparable to existing solutions. The good news is,
unlike many biotechnologies, RNA production enjoys economies of scale and
does not rely on scarce materials – it’s basically nucleotides and
enzymes. We may even envision on-site RNA production units on farms or
local cooperatives for just-in-time dsRNA synthesis tailored to local pest
sequences.
- Formulation Innovation: The next generation of formulations might
incorporate smart delivery – e.g., dsRNA that is released only when
a pest is present (perhaps triggered by pH or pest digestive enzymes), or
formulations that can be applied in advance (like a seed treatment or a
slow-release granule in soil) to protect plants over an extended period.
Nanotech will continue to play a role: virus-like particles, biodegradable
polymer films, and even plant-made nanoparticles (e.g., exosome-like
vesicles from plants) are on the horizon. These could improve targeting
(maybe foliar sprays that only activate on insect feeding) and reduce
amounts needed. Furthermore, integration with other agrichemicals in
“all-in-one” products could happen – for instance, a spray that contains a
dsRNA plus a nutrient or plus a microbe, to give plants a one-shot
treatment that boosts growth and guards against pests.
- Regulatory and Public Acceptance: As more products prove themselves safe
and effective, we anticipate regulatory harmonization to simplify bringing
dsRNA products to market globally. Perhaps an international consensus on
data requirements will emerge, and countries with smaller regulatory
capacity can rely on approvals from places like US/EU/Australia as
reference. Public acceptance will grow as success stories emerge, but
outreach remains important. Emphasizing that these are non-toxic, non-GMO,
and leave no residues will position dsRNA biopesticides as “green”
solutions. If marketed correctly, consumers might even prefer produce
protected by RNAi (similar to how many welcomed the reduction of synthetic
pesticides). That said, clear communication is key to avoid
misconceptions. The term “biopesticide” is apt, but even better might be
framing it as a nature-derived, eco-friendly pest solution.
- Understanding the RNAi Ecology: There are still scientific questions to
explore. For example, what is the minimum dose and spray coverage needed
for systemic effects in certain plants? How do plant traits (cuticle
thickness, leaf morphology) affect dsRNA uptake? Could plants be
bred/seleceted for better dsRNA absorption to enhance SIGS (a fascinating
synergy of breeding and biotech)? Additionally, more knowledge on how
different pests process dsRNA will help tailor approaches (for instance,
overcoming the “RNAi refractory” species by maybe adding a viral
suppressor of RNAi – a bit ironically – to temporarily disable pest defenses
so dsRNA can work). Monitoring for off-target effects in the field (like
metagenomic studies on soil or insect communities where dsRNA is used
extensively) can validate assumptions of safety in real agricultural
ecosystems. So far, expectations are being met: dsRNA’s specificity and
fast degradation indeed result in minimal disturbance to non-targets.
- Resistance Monitoring and
Counter-Strategies:
If/when resistance alleles to RNAi are documented in field populations, a
rapid response pipeline will be needed. This might involve switching
products or stacking multiple dsRNAs. The adaptability of SIGS is a huge
asset here – one can even envision a software that, given a detected pest
genome variant, designs a new dsRNA on the fly. Nonetheless, prudent usage
up front (IPM, not over-relying on single mode) remains the cornerstone to
delay such issues.
In closing, sprayable
dsRNA biopesticides represent a paradigm shift in crop protection. They embody
the concept of “precision agriculture” at the molecular level – using
sequence-specific agents to achieve control with minimal collateral damage.
This aligns with the pressing need to reduce chemical pesticide loads in our
environment and to develop pest management solutions that can keep pace with
the ever-evolving challenges of agriculture (be it climate-driven pest
outbreaks or emerging resistance to older chemistries). SIGS is not a silver
bullet or a standalone solution for every problem, but as part of an integrated
approach, it offers a powerful, versatile tool. The path from concept to field
has been rapid; within just a few years we went from lab tests to EPA approval
and large-scale trials. As research continues and more products gain traction,
we can expect SIGS to move from a “niche” or novel approach to a mainstream
pillar of crop protection – potentially as common in the farmer’s toolkit
as spraying insecticides or deploying GM traits has been. The difference is
that this pillar stands on a foundation of biotechnology that is more
harmonized with ecological principles: harnessing natural processes (RNAi) to
defend plants in a way that is both effective and sustainable. The continued
convergence of plant science, entomology, pathology, nanotechnology, and
regulatory science will ensure that spray-on RNAi technologies fulfill their
potential in feeding the world with fewer compromises to environmental and
human health.
Acknowledgments: The author thanks the numerous researchers
whose pioneering work in RNAi and plant protection has been cited in this review.
Special appreciation to the teams developing delivery innovations and to
regulatory scientists for their careful evaluation of this new technology. This
interdisciplinary progress paves the way for a greener and safer agricultural
future.
Dalakouras, A., Koidou, V., & Papadopoulou, K. (2024). DsRNA-based pesticides: Considerations for efficiency
and risk assessment. Chemosphere, 352, 141530.
Dietz-Pfeilstetter, A.,
Mendelsohn, M., Gathmann, A., & Klinkenbuß, D. (2021). Considerations and regulatory approaches in the USA and
in the EU for dsRNA-based externally applied pesticides for plant protection. Frontiers in Plant Science,
12, 682387.
EFSA Panel on Genetically
Modified Organisms. (2025). Risk
assessment considerations for RNAi‐based genetically modified plants. EFSA Journal, 23(3), e9321.
Fischer, J. R., et al. (2019). Dissipation of DvSnf7 double‐stranded RNA in Brazilian
soils. Agricultural & Environmental
Letters, 4(1), 190016.
He,
L., Huang, Y., & Tang, X. (2022).
RNAi-based pest control: Production, application and the fate of dsRNA. Frontiers in Bioengineering and
Biotechnology, 10, 1080576.
Khan,
F., et al. (2024). Spraying dsRNA with
chitosan formulation improves control of the western flower thrips,
Frankliniella occidentalis, in a greenhouse. Insect Molecular Biology,
34(4), 552–569.
Mitter, N., Worrall, E. A.,
Robinson, K. E., Li, P., Jain, R. G., Taochy, C., Fletcher, S. J., Carroll, B.
J., Lu, G. Q., & Xu, Z. P. (2017).
Clay nanosheets for topical delivery of RNAi for sustained protection against
plant viruses. Nature Plants, 3(2), 16207.
OECD. (2023). Considerations for the human health risk
assessment of externally applied dsRNA-based pesticides (Series on
Pesticides and Biocides, No. 110). OECD Publishing.
Office of the Federal Register.
(2023, October 5). Ledprona
double-stranded RNA; exemption from the requirement of a tolerance. Federal Register, 88(192),
69039–69043.
Pallis, S., Alyokhin, A.,
Manley, B., Rodrigues, T. B., Barnes, E. R., & Narva, K. (2023). Baseline susceptibility to a novel dsRNA-based
insecticide across US populations of Colorado potato beetle. Agriculture, 13(12), 2283.
Santos, G. (2025, October 27). GreenLight files for registration of Brazil's first
RNA-based fungicide. Cultivar Magazine.
Stokstad,
E. (2024, June 27). The perfect
pesticide? RNA kills crop-destroying beetles with unprecedented accuracy. Science.
Sun,
C., Imran, M., Feng, X., Shen, X., & Sun, Z. (2025). Spray-induced gene silencing for crop protection: recent
advances and emerging trends. Frontiers in Plant Science, 16, 1527944.
U.S. Environmental Protection
Agency. (2023, December 22). EPA registers novel pesticide
technology for potato crops.
Volpato, A., et al. (2023). Artificial nanovesicles for dsRNA delivery in spray
induced gene silencing for crop protection. bioRxiv.
Yang, W., Wang, B., Lei, G.,
Chen, G., & Liu, D. (2022). Advances
in nanocarriers to improve the stability of dsRNA in the environment. Frontiers in Bioengineering and
Biotechnology, 10, 974646.
