. 2019 Oct 25;10:1319. doi: 10.3389/fpls.2019.01319

Table 3.

Non-transformative delivery strategies for insects, pathogens, and virus management.

Target pest	Crop	Delivery strategy	Target gene		Molecule	Size	Molecule concentration	Results	Reference
	Insects
Plutella xylostella	Kale	Foliar spray	AChE2		siRNA	18–27 bp	200 µg/ml	Approximately 60% mortality.	(Gong et al., 2013)
Leptinotarsa decemlineata	Potato	Foliar spray	Actin		dsRNA	50 – 297 bp	5 μg leaf ⁻¹	Significant mortality in dsRNA length-depend pattern.	(San Miguel and Scott, 2016)
Diaprepes abbreviates	Citrus	Foliar spray	Not informed		dsRNA	Not informed	Not informed	Control started 4-5 days after dsRNA application.	(de Andrade and Hunter, 2016)
Diaphorina citri;	Citrus approximately 2.5 m tall and Grapevines	Trunk injection; root drench	Arginine kinase		dsRNA	Not informed	2 g in 15 liters of water	Insects successfully uptake dsRNA from the treated plants; dsRNA was detected in plants for at least 57 days.	(Hunter et al., 2012)
Bactericera cockerelli;
Homalodisca vitripennis
Nilaparvata lugens	Rice	Roots soaking	Ces		dsRNA	Not informed	1 mL (1.0 mg mL⁻¹ of water)	Gene knocked down; nymph mortality.	(Li et al., 2015)
Nilaparvata lugens	Rice	Roots soaking	CYP18A1		dsRNA		1 mL (1.0 mg mL⁻¹ of water)	Gene knocked down; nymph mortality.
Ostrinia furnacalis	Maize	Irrigation	KTI		dsRNA		10 mL (0.5 mg mL⁻¹ water)	Gene knocked down; larval mortality.
Myzus persicae	Tomato	Foliar application	ZYMV HC-Pro		dsRNA	588 bp	10.5 µg dsRNA in 10 µL water	Insect successfully uptake dsRNA; the dsRNA was processed into siRNA by the insect RNAi machinery.	(Gogoi et al., 2017)
Tetranychus urticae
Trialeurodes vaporariorum								Low dsRNA uptake; No siRNA in insects.
Halyomorpha halys	Green beans	Soaking	JHAMT		dsRNA	200-500 bp	300 µl (0.017 μg μL^-1 of water)	Significant reduction in gene expression.	(Ghosh et al., 2017)
Halyomorpha halys	Green beans	Soaking	Vg		dsRNA	200-500 bp	300 µl (0.067 μg μL^-1 of water)	Significant reduction in gene expression.	(Ghosh et al., 2017)
Planococcus citri	Tobacco	VIGS using recombinant TMV	Actin		siRNA	Not informed	–	Crawlers feed on recombinant TMV-infected plants showed lower fecundity and pronounced death.	(Khan et al., 2013)
			CHS1
			V-ATPase
Bactericera cockerelli	Tomato	VIGS using recombinant TMV	Actin		siRNA	21 nt	–	Gene knocked down in insects feed on these plants; Insects fed on infected tomatillo plants showed a decreased progeny production.	(Wuriyanghan and Falk, 2013)
	Tomatillo
	Tobacco
Diaphorina citri	Citrus	VIGS using recombinant CTV	Awd		siRNA	20-22 nt	–	Adults showed malformed-wing phenotype and increased mortality.	(Hajeri et al., 2014)
Phenacoccus solenopsis	Tobacco	VIGS using recombinant PVX	Bur		siRNA	–	–	Insects fed on treated plants showed physical deformities or died.	(Khan et al., 2018)
Phenacoccus solenopsis	Tobacco	VIGS using recombinant PVX	V-ATPase		siRNA	–	–		(Khan et al., 2018)
Drosophila melanogaster	–	VIGS using recombinant FHV; microinjection	RPS13		siRNA	–	–	Significantly higher mortality in insects.	(Taning et al., 2018)
			Vha26
			Alpha COP
Helicoverpa armigera	–	dsRNA expressed in bacteria, using recombinant E. coli strain HT115; artificial diet coated with engineered bacteria	AK		dsRNA	379-426 bp	30 µL (10⁹ cells)	Knocked down the target gene caused drastic reductions in body weight, body length, and pupation rate, resulting in high mortality.	(Ai et al., 2018)
Spodoptera exigua	Chinese cabbage	dsRNA expressed in bacteria, using recombinant E. coli strain HT115	INT		dsRNA	410 bp	10⁷ cells per larva	Significant reduction of the SeINT expression resulting in insect mortality; Pretreatment with an ultra-sonication increased the insecticidal activity of the recombinant bacteria, and treated larvae became s susceptible to Cry toxin.	(Kim et al., 2015)
Spodoptera exigua	–	dsRNA expressed in bacteria, using recombinant E. coli strain HT115; artificial diet containing engineered bacteria	CHSA		dsRNA	635 bp	High dose (250X), medium dose (50X), and low dose (10X) based on the dilution factors.	Significant reduction in survival rates. Levels of target gene expression, tissue structure, and survival rates were dose-dependent.	(Tian et al., 2009)
Lymantria dispar	–	dsRNA expressed in bacteria, using recombinant E. coli strain HT115; diet with engineered bacteria	Locus 365		dsRNA	–	300 μl of bacteria culture	Target-gene knocked down, reduction in body mass and egg masses.	(Ghosh and Gundersen-Rindal, 2017)
Lymantria dispar	–		Locus 28365		dsRNA	–	300 μl of bacteria culture		(Ghosh and Gundersen-Rindal, 2017)
Mythimna separata	–	dsRNA expressed in bacteria, using recombinant E. coli strain HT115; artificial diet containing engineered bacteria	Chi		dsRNA	700 bp	–	Target gene knocked down after oral delivery of engineered bacteria, resulting in resulted in increased mortality and reduction in body weight of the feeding larvae.w	(Ganbaatar et al., 2017)
Bactrocera dorsalis	–	dsRNA expressed in bacteria, using recombinant E. coli strain HT115; artificial diet containing engineered bacteria	Rpl19		dsRNA	–	200 ml 250X of bacteria culture expressing dsRNA.	Successful gene silencing of the target genes after insects were fed on a diet containing engineered bacteria. An over-expression of the target genes after continuously supply of engineered bacteria was also observed.	(Li et al., 2011)
			V-ATPase
			Rab11
			Noa
Bemisia tabaci	Hibiscus	dsRNA expressed in fungus, using engineered Isaria fumosorosea	TLR7		dsRNA	548 bp	2x10⁷,1x10⁷,5×10⁶, 2.5x10⁶ spores mL^-1	The engineered IfB01-TRL7 strain increased the mortality of whitefly nymphs compared to the IfB01 strain. The IfB01-TRL7 strain also show higher virulence, with decreased and shortened values of LC50 and LT50.	(Chen et al., 2015)
Manduca sexta	Tobacco	VIGS using recombinant TRV	DCL1	In tobacco plants	dsRNA	≥ 300 bp	–	Knocked down of the DCL target genes in engineered tobacco plants to express a 312 bp fragment of MsCYP6B46 gene increased the gene silencing results.	(Kumar et al., 2012)
			DCL2
			DCL3
			DCL4
			CYP6	In tobacco hornworm
Diseases
Fusarium graminearum	Barley	Foliar spray	CYP3		dsRNA	791 bp	500 μL (20 ng μL^-1 of water)	Inhibition of fungal growth.	(Koch et al., 2016)
SCMV	Maize	Bacterial crude extract foliar spraying (E. coli strain HT115)	CP		dsRNA	147-247 bp	One-half diluted extraction crude	Inhibition of SCMV infection.	(Gan et al., 2010)
Botrytis cinerea	Tomato, Strawberry, Grape, Lettuce, Onion, Rose	Foliar application	DCL1		sRNA	21-24 nt	400 µl (20 ng µL^–1)	Both sRNA and dsRNA were uptake by the fungus resulting in fungal growth inhibition.	(Wang et al., 2016b)
			DCL1		dsRNA	252 bp
			DCL2		sRNA	21-14 nt
			DCL2		dsRNA	238 bp
Sclerotinia sclerotiorum	Canola	Foliar spray	59 target genes		dsRNA	200-450 bp	10–25 µL of 200–500 ng dsRNA plus 0.02–0.03% Silwet L-77.	From the 59 dsRNAs tested, 20 showed antifungal activity with a reduction in lesion size ranging from 26–85%.	(McLoughlin et al., 2018)
Botrytis cinerea	Canola	Foliar spray	59 target genes		dsRNA	200-450 bp	10–25 µL of 200–500 ng dsRNA plus 0.02–0.03% Silwet L-77.		(McLoughlin et al., 2018)
BCMV	Tobacco; cowpea	Foliar spray	Nib		dsRNA naked or loaded onto LDH	480 bp	100 μg of in a 1 mL or 250 ng of dsRNA.	Plants were protected from aphid-mediated virus transmission.	(Worrall et al., 2019)
BCMV	Tobacco; cowpea	Foliar spray	CP		dsRNA naked or loaded onto LDH	461 bp	100 μg of in a 1 mL or 250 ng of dsRNA.		(Worrall et al., 2019)
Fusarium asiaticum	Wheat	Foliar spray	Myosin 5		dsRNA	496 bp	0.1 pM	Reduced pathogen sensitivity to phenamacril with a reduction in infection.	(Song et al., 2018)
PPV	Tobacco	Bacterial crude extract foliar spraying (E. coli strain HT115)	IR 54		hpRNA	977 bp	Dilution series (1/2 to 1/20) using 3 µg of total nucleic acid/µl.	Dilutions of 1/10 or less did not display disease symptoms upon completion of their life cycles	(Tenllado et al., 2003)
PMMoV	Tobacco		HC; CP		dsRNA	1492 bp; 1081 bp	One-half diluted French Press preparations derived from engineered bacteria.	Plants treated with dsRNA-expressing preparations showed no virus symptoms (HC: 82% or CP: 73%).	(Tenllado et al., 2003)
TMV	Tobacco	Bacterial crude extract foliar spraying (Different E. coli strain tested)	CP		dsRNA	480 bp	One-half diluted French Press preparations derived from engineered bacteria.	M-JM109 or M-JM109lacY strains and the pGEM-CP480 vector exhibited the best results producing great quantities of dsRNA. Tobacco plants sprayed with dsRNA crude bacterial extract showed inhibition in TMV infection.	(Yin et al., 2009)
PMMoV	Tobacco	Foliar spray	RP		dsRNA naked or loaded onto LDH	977 bp	125 µL per cm² (1.25 µg of dsRNA and/or 3.75 µg of LDH).	Virus protection for at least 20 days.	(Mitter et al., 2017a)
CMV	Cowpea	Foliar spray	2b supressor		dsRNA naked or loaded onto LDH	330 bp	125 µL per cm² (1.25 µg of dsRNA and/or 3.75 µg of LDH).	Virus protection for at least 20 days.	(Mitter et al., 2017a)
Fusarium asiaticum.	Wheat	Foliar spray after leaves were wounded using quartz sand	β2-tubulinX		dsRNA	480 bp	40 ng μL⁻¹ of water	Antifungal activity against these fungi with a reduction in the dosage of carbendazim fungicides necessary to control the pathogens.	(Gu et al., 2019)
Botrytis cinerea	Cucumber
Magnaporthe oryzae	Barley
Colletotrichum truncatum	Soybean

AChE2, acetylcholine esterase; CP, Coat Protein; Ces, carboxylesterase; ZYMV, Zucchini yellow mosaic virus; JHAMT, Juvenile hormone acid O-methyltransferase; Vg, Vitellogenin; CYP: cytochrome P450; KT, Kunitz-type trypsin inhibitor; DCL, Dicer-like; BCMV: Bean common mosaic virus; PMMoV, Pepper mild mottle virus; CMV, Cucumber mosaic virus; LDH, double-layered hydroxide; RP, Replicase; CTV, Citrus tristeza virus; Awd, abnormal wing disc; BUR, Bursicon; FHV, Flock house virus; RPS13, Ribosomal protein S13; Vha26, Vacuolar H[+]-ATPase 26kD E subunit; Alpha COP, Alpha-coatomer protein; AK, Arginine kinase; INT, β1 integrin gene; CHSA, Chitin synthase gene A; Chi, chitinase; Rpl19, ribosomal protein Rpl19; Sec23, Protein transport protein sec23; vATPaseE, Vacuolar ATP synthase subunit E; vATPaseB, Vacuolar ATP synthase subunit B; COPβ, Coatomer subunit beta; SCMV, Sugarcane Mosaic Virus; HC, Helper component; IR, replicase; TLR7, Toll-like receptor 7; LC50, Lethal Concentration 50; LT50, Lethal Time 50; VIGS, Virus-induced gene silencing.