WO2018046312A2 - Perfectionnements apportés ou liés au silençage génique - Google Patents

Perfectionnements apportés ou liés au silençage génique Download PDF

Info

Publication number
WO2018046312A2
WO2018046312A2 PCT/EP2017/071388 EP2017071388W WO2018046312A2 WO 2018046312 A2 WO2018046312 A2 WO 2018046312A2 EP 2017071388 W EP2017071388 W EP 2017071388W WO 2018046312 A2 WO2018046312 A2 WO 2018046312A2
Authority
WO
WIPO (PCT)
Prior art keywords
dsrna
plant
stink bug
carbohydrate
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2017/071388
Other languages
English (en)
Other versions
WO2018046312A3 (fr
Inventor
Myriam Beghyn
Yann Naudet
Lies Degrave
Lien DE SCHRIJVER
Kevin V DONOHUE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Devgen NV
Syngenta Participations AG
Original Assignee
Devgen NV
Syngenta Participations AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Devgen NV, Syngenta Participations AG filed Critical Devgen NV
Publication of WO2018046312A2 publication Critical patent/WO2018046312A2/fr
Publication of WO2018046312A3 publication Critical patent/WO2018046312A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
    • A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell

Definitions

  • the present invention relates to the control of gene expression using double stranded RNA (dsRNA).
  • dsRNA double stranded RNA
  • the invention relates to compositions which enhance the uptake of dsRNA administered external to a target organism to enable effective gene silencing in that target organism.
  • RNA interference is a well-established technique used to down regulate gene expression by using dsRNA or small interfering RNA (siRNA) to trigger degradation of mRNA of a particular gene of interest, thus preventing translation.
  • RNAi has not only provided a means of functionally analysing genes, but has been used for the control of pest organisms.
  • a problem with the application of dsRNA for the control of pest organisms concerns the poor uptake of dsRNA by a pest organism when the dsRNA is applied exogenously at a locus at which they exist.
  • Exogenous dsRNA refers to dsRNA which is applied to a target organism such that it is incorporated into a target organism and results in the effective silencing of an essential target gene.
  • dsRNA may be exogenously applied in a composition in its native form, or applied whilst incorporated in an organism different to that of the target organism.
  • compositions comprising dsRNA onto a food source of the target organism, wherein the dsRNA is ingested by the organism, resulting in effective silencing of an essential gene target.
  • dsRNA control Whilst this technique has provided an efficient means of dsRNA control in a number of target organisms, such as coleopteran pest species, many other pest species are refractory to this method of dsRNA delivery. Therefore the application of dsRNA for use as a pest control agent is significantly limited.
  • the present invention provides at least to some extent a solution to the problem of exogenous dsRNA delivery in those pest species which are refractory to those methods presently used in the art.
  • a pesticidal composition comprising a dsRNA and a carbohydrate, wherein said dsRNA is capable of RNAi gene silencing and comprises a strand that is complementary to at least part of a nucleotide sequence of a gene from a plant pest organism.
  • the composition of the invention is substantially devoid of salts.
  • the composition may comprise additional components, suitable components having a function of a binding agent, and/or an anti-desiccant and/or a humectant a rheology modifier and/or a bulking agent.
  • Suitable binding agents include emulsifiable mineral oil (Actipron), carrageenan, molasses, starch, dextrine, ethyl cellulose, methacrylate polymers, gelatine, glucose, arabic gum or other vegetable gums, tragacanth gum (dried sap), honey, whole or skimmed milk,
  • Suitable anti-descants include calcium alginate, water absorbing polymers, cellulose, glycerol and molasses
  • Suitable humectants include glycerol, polyacrylamide gel, adipic acid, alginic acid, acetamide-B, mercaptoethylamide, boric acid, calcium stearate, cellulose acetate, stearic acid or calcium stearate, gellan, alginate, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and polyvinylpyrrolidone/vinyl acetate copolymer.
  • Suitable rheology modifiers include water absorbing polyacrylamide (Alcosrob AB3), calcium, alginate, dextran, pre gelatinised corn flour, carob gum, sodium carboxymethylcellulose, fumed silicon dioxide calcium gluconate, acacia gum or xanthan gum.
  • Suitable bulking agents include attapulgite clay, bentonite clay, kaolinite, calcium
  • montmorillonite calcium carbonate, celatom, celite, ground clay, kaoline, diatomaceous earth and ground limestone.
  • the pesticidal composition of the invention comprises a dsRNA which is synthetically transcribed in vitro.
  • the pesticidal composition of the invention comprises a dsRNA which is transcribed by a microorganism.
  • the microorganism is inactivated.
  • microorganism By 'inactivated' it is meant that the microorganism is treated by heat or with a chemical such that it is no longer capable of propagation.
  • Suitable microorganisms include those selected from the group of bacteria, fungi, algae and yeast.
  • the dsRNA is present in the composition as a component of a cell lysate.
  • the cell lysate may be a complete lysate, characterized in that all cells have undergone lysis, or the lysate may contain a proportion of intact cells. Therefore, dsRNA of the invention may be present in a cell lysate, intact cells or a proportion present in both.
  • the carbohydrate present in the pesticidal composition is provided at a concentration that is sufficient to promote the plant pest organism to take up dsRNA to enable effective RNAi gene silencing.
  • the carbohydrate is present in the composition at an amount from 0.01 % to 20% of total volume of the composition.
  • the carbohydrate comprises at least one glucose residue and/or at least one fructose residue.
  • the carbohydrate is sucrose, maltose, glucose, fructose or raffinose.
  • the carbohydrate is a sugar alcohol. Suitable sugar alcohols include sorbitol, mannitol and myo-inositol.
  • the plant pest organism is of the order Hemiptera.
  • the Hemiptera plant pest may be selected from the group consisting of green stink bug Acrosternum hilare (Say), Brochymena quadripustulata (Fabricius) rough stink bug, Chlorochroa sayi (Stal) Say stink bug, Coptosoma xanthogramma (White) black stink bug, Euschistus servus (Say) brown stink bug, Euschistus tristigmus (Say) dusky stink bug, Euschistus variolarius (Palisot de Beauvois) onespotted stink bug, Halyomorpha halys (Stal) brown marmorated stink bug, Nezara viridula (Linnaeus) southern green stink bug, Oebalus pugnax (Fabricius) rice stink bug, Perillus bioculatus (Fabricius) twospotted stink bug, Piezodorus guildinii (Westwood) redbanded stink bug, Plautia stali Scott oriental stink bug, Thyanta
  • the pesticidal composition of the invention comprises at least one other pesticidally active ingredient in combination with the dsRNA of the invention.
  • the pesticidally active ingredient may target the same plant pest organism or a different plant pest organism. Suitable pesticides are those commonly known to the skilled artisan.
  • the present invention further provides a method for controlling a plant pest organism comprising applying to the plant, to the seed of a plant, or locus of a plant or seed the composition of the invention.
  • a plant pest organism Preferably the composition is ingested by a plant pest organism.
  • Another aspect of the invention relates to a method for controlling a plant pest organism comprising applying to a plant, sequentially and in any order, a dsRNA capable of RNAi gene silencing comprising a strand that is complementary to at least part of a nucleotide sequence of a gene from a plant pest organism, a carbohydrate and optionally another pesticidally active ingredient.
  • the present invention also relates to the use of the composition of the invention for controlling plant pest growth and/or infestation.
  • the present invention also relates to the use of a carbohydrate, in a composition comprising pesticidal dsRNA, to satiate the appetite of a pest so that the production of salivary nucleases by the pest s substantially inhibited.
  • the carbohydrate is sucrose
  • the pest is a plant pest
  • the plant is a soybean plant
  • the pest is a stink bug
  • the carbohydrate is present in the composition at an amount of 0.01 % to 20% of the total volume of the composition.
  • the present invention also relates to a method of substantially inhibiting the production of salivary nucleases in a pest fed a composition comprising a pesticidal dsRNA by including a carbohydrate in the composition.
  • the carbohydrate is sucrose
  • the pest is a plant pest
  • the plant is a soybean plant
  • the pest is a stink bug
  • the carbohydrate is present in the composition at an amount of 0.01 % to 20% of the total volume of the composition.
  • Stink Bugs were reared at 26°C, 65% relative humidity, with 16:8 hours light: dark cycle.
  • Adults were kept in insect cages (50 cm x 50 cm x 80 cm) and fed with fresh runner beans -Phaseolus coccineus- and crushed peanuts- Arachis hypogaea L.
  • a soybean plant- Glycine max- served as an extra food source and oviposition substrate. Every 2 weeks, fresh eggs were collected from 3 cages.
  • Egg masses were cut out of the plant leaves, and used to seed a container (15 cm x 25 cm x 1 1 cm) lined with paper tissue, closed with a mesh and stored in the rearing chamber (26°C, 65% RH, with 16:8 hours I: d).
  • Runner beans - Phaseolus coccineus - were added when first instars appeared. From the third instar onwards, a tray containing a cotton boll soaked in tap water and a tray containing crushed peanuts was added. When the nymphs molted to fourth instar, they were transferred to larger boxes (27 cm x 27 cm x 13 cm), with double food/liquid supply than the younger stages. Every 3 days, tissue, beans, peanuts and water were refreshed.
  • Second instars were transferred to boxes containing runner beans on a paper tissue and boxes were closed with a mesh. From the third instar onwards, a tray containing a cotton boll soaked in water and a tray containing sunflower seeds was added until adulthood. Every 3 days, tissue, beans, seeds and water were refreshed.
  • Feeding sachets were made by placing a parafilm strip (15.5cm/1 1 cm) on a 96-well filter plate (Millipore) which is placed on a vacuum manifold (Millipore) and connected to vacuum pump (Laboport). The vacuum pump was switched on and when wells were slightly depressed, extra pressure was given by rolling a 96-well rubber mat on top of the parafilm to form 96 U- shaped wells. When the vacuum manifold was switched off, wells were filled with 25 ⁇ of feeding solution (carbohydrate + dsRNA), sealed with an Ampliseal (Greiner bio-one) and removed from the manifold.
  • feeding solution Carbohydrate + dsRNA
  • Complex diet was prepared according to manufacturer's description and used within 7 days of preparation.
  • the nymphal survival rate was recorded from 3 to 10 days post exposure to dsRNA.
  • a lethal RNAi effect was expected from day 6 onward.
  • the assay system was validated using dsRNA corresponding to a known target as the positive control and a
  • fluorescent protein dsRNA (GFP) as the negative control.
  • the positive control for testing the different carbohydates was dsRNA made up in 15% carbohydrate (in particular sucrose) solution, and the negative control for the carbohydrate testing was dsRNA made up in ultrapure water (MilliQ).
  • carbohydrate in particular sucrose
  • dsRNA made up in ultrapure water (MilliQ).
  • dsRNA template material was generated by PCR using two target specific primers containing the T7 RNA polymerase promoter sequence at their 5'-end.
  • the PCR reaction was analyzed on agarose gel and purified using the QIAquick PCR purification kit (Cat.n°28006, Qiagen).
  • the purified template was quantified using the DropSense96 (Trinean).
  • the template was used in an in vitro transcription reaction (RiboMAXTM Large Scale RNA Production System-T7 P1300, Promega). To generate at least 1 mg purified dsRNA an equivalent of 20 reactions was set-up and incubated overnight at 37°C.
  • RNAs produced by transcription from the templates were allowed to anneal, were then treated with DNase and RNase, and were purified by precipitation.
  • An extra purification step using a VIVAPSIN20 column with MWCO 30kDA (VS2022, Sartorius) was included for the removal of residual salt and unincorporated nucleotides.
  • the samples were analyzed on agarose gel.
  • Monosaccharides tested were glucose and fructose. Disaccharides tested were sucrose (control), and maltose. The tested oligosaccharide was raffinose and carbohydrate alcohols were mannitol, myo-inositol and sorbitol.
  • Two feeding mixtures per carbohydrate were prepared by mixing 390 ⁇ 25% carbohydrate solution with 260 ⁇ 2.5 ⁇ 9/ ⁇ solution of dsRNA. All solutions were prepared in Ultrapure water (MilliQ). The control solution contained GFP synthetic dsRNA and the target solution contained positive control synthetic dsRNA, which were both diluted in MilliQ. Feeding assays were set up and nymphal survival was recorded as described above.
  • dsRNA solutions and carbohydrate stocks were combined- as described in the first round of testing i.e. to a final concentration of synthetic dsRNA of 1 ⁇ g/ ⁇ l and carbohydrate concentrations of 15%, 10%, 5% and 1 %. Feeding assays were set up with second instar nymphs and nymphal survival was recorded as described above.
  • RNAi response was observed when nymphs were exposed to synthetic dsRNA in the presence of sucrose, glucose, fructose and maltose at all concentrations tested.
  • dsRNA was provided in raffinose
  • the RNAi effect could only be achieved when using a final concentration of 10% and 5 % (m/v %) (Fig.6).
  • other carbohydrates were toxic (high mortality during the first days of feeding) or their presence was unable to induce a clear RNAi response in the nymphs (Fig.7, Fig.8).
  • An RNAi response may be observed if lower concentrations were used.
  • the sequence of interest was cloned as a hairpin (inverted repeats separated by a spacer sequence) behind an IPTG-inducible promoter.
  • the resulting dsRNA expression plasmid was then transformed into a suitable bacterial host.
  • the production of dsRNA was done by fermentation.
  • the bacteria containing the dsRNA expression plasmid were grown in defined medium to a specific optical density and induced by addition of IPTG for a specific time period. After induction, the bacteria were harvested by centrifugation, resuspended in water to a defined optical density and inactivated by heat treatment.
  • Carbohydrates sucrose, maltose and lactose where tested in combination with bacterial lysate expressing a fluorescent protein dsRNA (GFP) and target dsRNA fragment.
  • Carbohydrates were tested at 15% (m/v %) and 5% (m/v%) final concentrations.
  • Bacterial lysate was tested at concentrations of 0.5U, 0.25, 0.125 and 0.0625U per nymph 20U/ml, 10U/ml, 5U/ml and 2.5U/ml respectively.
  • 1 unit (U) corresponds to the amount of bacteria present in 1 ml culture with an OD600 nm value of 1 prior to (heat) inactivation.
  • the tested carbohydrates were made up in solutions of 30% and 10% (m/v) concentrations.
  • Bacteria expressing GFP and target dsRNA were diluted to solutions of 40U/ml, 20U/ml, 10U/ml and 5U/ml. All dilutions were made in ultrapure water (MilliQ).
  • Carbohydrate solutions and bacterial lysate suspensions were mixed in equal proportions i.e. 1 : 1. Feeding assays were set up on second instar nymphs and nymphal mortality was recorded as described above.
  • RNAi response is visible when nymphs were exposed to all concentrations of bacterial lysate containing the target dsRNA mixed with both dilutions of sucrose and maltose (5% and 15%). No RNAi response was seen when nymphs were exposed to the bacterial lysate in combination with 15% and 5% lactose (Fig.13-Fig.14).
  • Both FITC inulin solutions were mixed with bacterial lysate expressing non targeting GFP, resulting in a solution with a final concentration of 75U/ml bacterial lysate, 2 mg/ml FITC inulin and 15% sucrose and a solution with a final concentration of bacterial lysate of 75U/ml and 2mg/ml FITC inulin in the absence of a carbohydrate.
  • the 8 nymphs from each plant were pooled in a 1.5 ml vial and washed twice with MilliQ.
  • the nymphs were placed on a tissue to dry and individually transferred to a single well, containing a metal bead, of a deep-well plate containing 1 ml tubes- strips (Machery -Nagel).
  • the plates were flash-frozen in liquid nitrogen and placed in the Tissue-Lyzer (Qiagen) for 1 min at 30 Hz. After maceration of the nymphs, the plates were centrifuged for 10 minutes at 4000 g, 200 ⁇ MilliQ was added to each well and mixed. Plates were centrifuged a second time for 10 minutes at 4000 g.
  • Jack soy bean seeds were grown until reaching V2-V3 development stage as previously described.
  • the plants were sprayed with a 15% carbohydrate solution eg. sucrose, containing 75U/ml bacterial lysate expressing non target GFP dsRNA and a solution containing 75U/ml bacterial lysate expressing non target GFP dsRNA in the absence of a carbohydrate.
  • carbohydrate solution eg. sucrose
  • 75U/ml bacterial lysate expressing non target GFP dsRNA a solution containing 75U/ml bacterial lysate expressing non target GFP dsRNA in the absence of a carbohydrate.
  • a total volume of 2 ml was sprayed on the plant and replicated 3 times. Thirty second instar nymphs, were placed on each sprayed plant. Spraying of the plants, rearing of insects, caging and storage of the plants was performed as previously described.
  • the nymphal survival rate was recorded from 3 to 10 days post exposure to the bacterial lysate. Clear differences in survival rate of the nymphs were demonstrated in this experiment. A very low survival rate of nymphs was observed on plants sprayed with bacterial lysate expressing non target GFP dsRNA in the absence of sucrose (Fig.16). In contrast, the nymphs on plants sprayed with the bacterial lysate in the presence of sucrose could survive throughout the assay.
  • RNAi effects with bacterial lysate expressing lethal target dsRNA were performed in the presence of a carbohydrate, which was selected as a positive control for subsequent assays.
  • Jack soy bean seeds were grown until reaching V2-V3 stage as previously described.
  • the plants were sprayed with a 15 % sucrose solution containing 75U/ml bacterial lysate expressing non targeting GFP or target dsRNA.
  • a total volume of 2 ml was sprayed on the plant and replicated 3 times. Thirty second instar nymphs, were placed on each sprayed plant. Spraying of the plants, rearing of insects, caging and storage of the plants was performed as previously described.
  • nymphs from each plant were transferred to a 20-cm petridish (Greiner) containing a filter paper (Greiner) and a runner bean (Phaseolus coccineus). The nymphal mortality was recorded from 3 to 10 days post exposure to the Al.
  • RNAi on planta (Continuous exposure) - second instar nymph As shown in Fig.17 and Fig.18, spraying plants with a 15% sucrose suspension of bacterial lysate expressing target dsRNA led to a dramatic increase in insect mortality when compared to GFP control. RNAi on planta (Continuous exposure) - second instar nymph
  • Jack soy bean seeds were grown until reaching V2-V3 development stage as previously described.
  • the plants were sprayed with a 15 % sucrose solution containing 75U/ml bacterial lysate expressing non targeting GFP or target dsRNA.
  • a total volume of 2 ml was sprayed on the plant and replicated 3 times.
  • Twenty five second instar nymphs were placed on each sprayed plant and placed in a 2 compartment box (Fig.19) coated with Fluon PTFE (Blades Biological Ltd) to prevent nymphal escape. Spraying of the plants, rearing of insects and storage of the plants was performed as previously described. Pictures were taken daily to capture plant protection and nymphal mortality was recorded on day 3, 10 and 14 days post exposure to the Al.
  • RNAi on planta Continuous exposure
  • Jack soy bean seeds were grown until reaching V3 development stage as previously described.
  • the plants were sprayed with a 15 % sucrose solution containing 75U/ml bacterial
  • lysate expressing non targeting GFP dsRNA or target dsRNA For each condition a total volume of 2 ml was sprayed on one plant and replicated 3 times.
  • Spraying of the plants, rearing of insects and storage of the plants was performed as previously described.
  • RNAi on planta Continuous exposure
  • Jack soy bean seeds were grown until reaching V3 development stage as previously described. Plants were sprayed with a 15 % sucrose solution containing 75U/ml bacterial lysate expressing non targeting GFP dsRNA or target dsRNA. For each condition a total volume of 2 ml was sprayed on one plant. Each condition was replicated 12 times. The 12 plants were placed in 1 insect cage and second, third, fourth and fifth instar nymphs (8 each) were placed in the insect cage. Pictures were taken on a daily basis to capture plant protection and mortality was recorded on day 3, 6, 10, 14 and 16 days post exposure to the Al.
  • Fig.1 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the absence of a carbohydrate and in the presence of 15% sucrose.
  • the RNAi response of nymphs feeding on the target dsRNA in the presence of sucrose is visible in the second week post feeding. No RNAi response is observed when feeding on target dsRNA in the absence of a carbohydrate.
  • Fig.2 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the presence of 1 % and 15% sucrose. The RNAi response of nymphs when feeding on the target dsRNA in the presence of sucrose is visible in the second week post feeding.
  • Fig.3 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the presence of 5% and 10% glucose.
  • the RNAi response of nymphs when feeding on the target dsRNA in the presence of glucose is visible in the second week post feeding.
  • Fig.4 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the presence of 5% and 10% fructose.
  • the RNAi response of nymphs when feeding on target dsRNA in the presence of fructose is visible in the second week post feeding.
  • Fig.5 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the presence of 1 % and 15% maltose.
  • the RNAi response of nymphs when feeding on target dsRNA in the presence of maltose is visible in the second week post feeding.
  • Fig.6 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the presence of 5% and 10% raffinose.
  • the RNAi response of nymphs when feeding on target dsRNA in the presence of raffinose is visible in the second week post feeding.
  • Fig.7 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the presence of 1 % and 5% mannitol.
  • Fig.8 shows the mortality of second instar nymphs after feeding on non-target (GFP) and lethal target naked dsRNA in an in vitro assay in the presence of 15% and 1 % sorbitol.
  • the decrease in mortality of nymphs during the first week of feeding on non-target dsRNA indicates that at the given concentrations the sorbitol is toxic.
  • Fig. 9 shows the mortality of second instar nymphs after feeding on bacterial lysate expressing non-target (GFP) and lethal target dsRNA in the presence of 15% sucrose.
  • GFP non-target
  • the RNAi response of nymphs when feeding on target dsRNA in the presence of sucrose is visible in the second week post feeding.
  • Fig. 10 shows the mortality of second instar nymphs after feeding on bacterial lysate expressing non-target (GFP) and lethal target dsRNA in the presence of 5% sucrose.
  • GFP non-target
  • the RNAi response of nymphs when feeding on target dsRNA in the presence of sucrose is visible in the second week post feeding.
  • Fig. 11 shows the mortality of second instar nymphs after feeding on bacterial lysate expressing non-target (GFP) and lethal target dsRNA in the presence of 15% maltose.
  • GFP non-target
  • the RNAi response of nymphs when feeding on target dsRNA in the presence of sucrose is visible in the second week post feeding.
  • Fig. 12 shows the mortality of second instar nymphs after feeding on bacterial lysate expressing non-target (GFP) and lethal target dsRNA in the presence of 5% maltose.
  • the RNAi response of nymphs when feeding on target dsRNA in the presence of sucrose is visible in the second week post feeding.
  • Fig. 13 shows the mortality of second instar nymphs after feeding on bacterial lysate expressing non-target (GFP) and lethal target dsRNA in the presence of 15% lactose. No RNAi response of nymphs when feeding on target dsRNA in the presence of lactose is visible in the second week post feeding.
  • Fig. 14 shows the mortality of second instar nymphs after feeding on bacterial lysate expressing non-target (GFP) and lethal target dsRNA in the presence of 5% lactose. No RNAi response of nymphs when feeding on target dsRNA in the presence of lactose is visible in the second week post feeding.
  • Fig. 15 shows high uptake of FITC fluorescent dye nymphs on V1 -V2 plants sprayed with a suspension of 75U/ml of bacterially expressed GFP dsRNA in 15% sucrose and very low uptake of FITC fluorescent dye in absence of 15% sucrose.
  • Fig. 16 shows low survival rate of nymphs on V2-V3 soybean plants sprayed with a suspension of 75U/ml bacterial lysate expressing GFP dsRNA and high survival rate on plants sprayed with a suspension of 75U/ml bacterial lysate expressing GFP dsRNA in the presence of sucrose.
  • Fig. 17 shows low survival rate of a first species of Stink Bug nymphs when exposed for 3d to plants sprayed with a 15% sucrose suspension of 75U/ml bacterial lysate expressing target dsRNA compared to the negative control (GFP).
  • Fig.18 shows low survival rate of a second species of Stink Bug nymphs when exposed for 3 days to plants sprayed with a 15% sucrose suspension of 75U/ml bacterial lysate expressing lethal target dsRNA compared to the negative control (GFP).
  • Fig. 19 shows plant protection of the soy bean plant when the plants were sprayed with a solution of 15% sucrose containing bacterial lysate expressing dsRNA of a lethal target dsRNA compared to the damage incurred on the control plants (sprayed with GFP dsRNA).
  • Fig. 20 shows high mortality of nymphs continuously exposed to V2-V3soy bean plants sprayed with a suspension of 75U/ml bacterial lysate expressing lethal target dsRNA compared to the mortality of nymphs exposed to control (GFP) bacterial lysate.
  • Fig. 21 shows high mortality rate of adults on V3 soybean plants sprayed with a 15% sucrose solution containing 75U/ml bacterial lysate expressing target dsRNA compared to low mortality of adults on V3 soybean plants sprayed with a suspension of 75U/ml bacterial lysate expressing GFP dsRNA
  • Fig. 22 shows high mortality of all Stink bug nymphal stages continuously exposed to 12 V3 soy bean plants sprayed with a solution of 15% sucrose containing 75U/ml bacterial lysate expressing lethal target dsRNA compared to the mortality of nymphs exposed to control (GFP) bacterial lysate.
  • Fig. 23 shows plant protection 16 days post infestation with nymphs (all stages), when the plants were sprayed with a solution of 15% sucrose containing bacterial lysate expressing dsRNA of a lethal target dsRNA (Target dsRNA) compared to the damage incurred on the control plants (Non target dsRNA).
  • Tiget dsRNA lethal target dsRNA

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Plant Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

La présente invention concerne une composition pesticide comprenant un acide ribonucléique double brin (ARNdb) et un glucide, ledit ARNdb permettant le silençage génique d'ARNi et comprenant un brin qui est complémentaire d'au moins une partie d'une séquence nucléotidique d'un gène provenant d'un organisme nuisible végétal.
PCT/EP2017/071388 2016-09-06 2017-08-24 Perfectionnements apportés ou liés au silençage génique Ceased WO2018046312A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662383666P 2016-09-06 2016-09-06
US62383666 2016-09-06

Publications (2)

Publication Number Publication Date
WO2018046312A2 true WO2018046312A2 (fr) 2018-03-15
WO2018046312A3 WO2018046312A3 (fr) 2018-04-19

Family

ID=59738332

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/071388 Ceased WO2018046312A2 (fr) 2016-09-06 2017-08-24 Perfectionnements apportés ou liés au silençage génique

Country Status (2)

Country Link
AR (1) AR109465A1 (fr)
WO (1) WO2018046312A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115161306A (zh) * 2022-06-29 2022-10-11 江苏省农业科学院 绿盲蝽rna降解酶、其编码基因、载体、菌株及其应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPR621501A0 (en) * 2001-07-06 2001-08-02 Commonwealth Scientific And Industrial Research Organisation Delivery of ds rna
PL1687435T3 (pl) * 2003-11-17 2012-02-29 Bayer Cropscience Nv Odporność na owady z użyciem hamowania ekspresji genów
CA2622671C (fr) * 2005-09-16 2020-12-22 Devgen Nv Procede de protection phytosanitaire a base d'interference arn
EP2633048B1 (fr) * 2010-10-27 2019-07-31 Devgen NV Régulation à la baisse de l'expression génique chez des insectes nuisibles
US8575328B2 (en) * 2010-12-14 2013-11-05 The United States Of America, As Represented By The Secretary Of Agriculture Formicidae (ant) control using double-stranded RNA constructs
CN103562394B (zh) * 2011-04-20 2019-09-03 德福根有限公司 在昆虫害虫中下调基因表达
BR112016022711A2 (pt) * 2014-04-01 2017-10-31 Monsanto Technology Llc composições e métodos para controle de pragas de inseto
US9580709B2 (en) * 2014-08-22 2017-02-28 The United States Of America, As Represented By The Secretary Of Agriculture Double stranded RNA constructs for aphid control

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115161306A (zh) * 2022-06-29 2022-10-11 江苏省农业科学院 绿盲蝽rna降解酶、其编码基因、载体、菌株及其应用
CN115161306B (zh) * 2022-06-29 2023-09-15 江苏省农业科学院 绿盲蝽rna降解酶、其编码基因、载体、菌株及其应用

Also Published As

Publication number Publication date
WO2018046312A3 (fr) 2018-04-19
AR109465A1 (es) 2018-12-12

Similar Documents

Publication Publication Date Title
Valetti et al. Growth promotion of rapeseed (Brassica napus) associated with the inoculation of phosphate solubilizing bacteria
Nabti et al. Restoration of growth of durum wheat (Triticum durum var. waha) under saline conditions due to inoculation with the rhizosphere bacterium Azospirillum brasilense NH and extracts of the marine alga Ulva lactuca
Glassner et al. Bacterial niches inside seeds of Cucumis melo L.
Song et al. Seed defense biopriming with bacterial cyclodipeptides triggers immunity in cucumber and pepper
Bruno et al. Insights into the immune response of the black soldier fly larvae to bacteria
CN106061243B (zh) 用于提高作物产量和防虫害的真菌内生菌
Diaz et al. 'Bacillus' spp. as plant growth-promoting bacteria in cotton under greenhouse conditions
Xu et al. Revealing the underlying mechanisms mediated by endophytic actinobacteria to enhance the rhizobia-chickpea (Cicer arietinum L.) symbiosis
Becerra et al. Wolbachia-free heteropterans do not produce defensive chemicals or alarm pheromones
Senthilraja et al. Chitin-based bioformulation of Beauveria bassiana and Pseudomonas fluorescens for improved control of leafminer and collar rot in groundnut
Feng et al. OfGNBP silencing enhances the toxicity of Serratia marcescens Bizio (SM1) to Odontotermes formosanus (Shiraki)
EP2765185A2 (fr) Souche de Brevibacillus parabrevis et composition à libération contrôlée à base de celle-ci
Ncube et al. Fusarium ear rot and fumonisins in maize kernels when comparing a Bt hybrid with its non-Bt isohybrid and under conventional insecticide control of Busseola fusca infestations
Ahmed et al. Biofilm formation and indole-3-acetic acid production by two rhizospheric unicellular cyanobacteria
CN107603984A (zh) 用于控制昆虫侵袭的核苷酸序列及其方法
Franco et al. Colletotrichum falcatum modulates the olfactory behavior of the sugarcane borer, favoring pathogen infection
WO2018046312A2 (fr) Perfectionnements apportés ou liés au silençage génique
Sun et al. No adverse effects of transgenic maize on population dynamics of endophytic Bacillus subtilis strain B916‐gfp
Reyad et al. Biocontrol of root rot in Geranium with antimycotic rhizobateria
Alallaf et al. Desertifilum tharense methanol extract inhibits vascular wilt caused by Fusarium oxysporum f. sp. lycopersici and promotes tomato growth
Tsavkelova Bacteria associated with orchid roots
Vatanparast et al. Development and efficacy of dsRNA pesticides targeting the Colorado potato beetle with enhanced stability via chitosan formulations
Darmawan et al. Utilizing organic waste for biopesticide: efficacy and mortality test against Spodoptera litura with entomopathogenic microorganisms
Sorokan et al. Endophytic strain Bacillus subtilis 26D promotes Solanum tuberosum L. defense against Leptinotarsa decemlineata say
Parker Reducing Cereal Aphids On Barley By Exploiting Beneficial Species Interactions With Rhizobacteria And Natural Enemies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17758505

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17758505

Country of ref document: EP

Kind code of ref document: A2