ToBRFV and Its Effects
Tomatoes represent one of the most cultivated horticultural crops worldwide, with over 5 million hectares of cultivated area and more than 182 million tons of tomatoes produced globally.
The tomato brown rugose fruit virus (ToBRFV) is a destructive virus with a significant economic impact. ToBRFV can infect up to 100% of a crop and yield losses of between 25-70% have been reported, with fruits being unmarketable. ToBRFV, like other members of the Tobamovirus genus, is highly stable and can simply be transmitted mechanically through contact, such as by workers’ hands, clothing, contaminated tools, direct plant-to-plant contact, and propagation materials.
Foliar (leaf) symptoms include mosaic patterning which can range in severity from obvious to subtle. Leaf deformation may also occur such as puckering and narrowing, blistering, or reduction in leaflet size. Plants may also exhibit wilted leaves. Necrosis may develop on pedicels (stems), calyces, petioles, and flowers.
There is no cure for ToBRFV and there are no treatments available, therefore the only way to remove the virus from a crop is by destroying the plants and quarantining cultivation areas. The best means of prevention is to use virus-free planting material.
Proof of Concept for RNA Based Treatment
A semi-commercial net house experiment was conducted in the Agricultural Research Organization (ARO) – Volcani Center, Israel. The objectives were to determine the efficacy of the RNAway based treatment against ToBRFV and to compare it to naked RNA and scrambled (non-target) RNA controls.
Semi-Commercial trial design
Included 5 treatment groups: healthy plants, infected plants untreated, infected plants treated with naked RNA, scrambled RNA, and RNAway based treatment.
The plants were applied with a single foliar spray and subsequently inoculated with the virus after 3 days. Next, plants were additionally sprayed 4 times, once every 2 weeks throughout the growing cycle.
Semi-commercial trial setup
Disease Assessment
Disease assessment was determined 4 and 7 weeks post inoculation. Disease severity was qualitatively determined using a six–grade scale 0-5 as follows:
0 – Symptomless, 1 – Very low severity, 2 – Low severity, 3 – Medium severity, 4 – High severity, 5 – Very high severity
Images describe that at 4 and 7 weeks after inoculation, only a few plants that were sprayed with the RNAway based treatment were assessed with low symptoms of ToBRFV compared to the naked RNA, scrambled RNA, and infected groups in which all plants had observable symptoms.
Visual disease assessment 4 and 7 weeks post inoculation
Disease severity 4 weeks post inoculation
Disease severity 7 weeks post inoculation
Fruit Quantity
7 weeks after inoculation, the number of fruits was counted on each plant.
Fruit quantity in the RNAway based treatment group was higher and similar to the quantity of fruits in healthy plants, compared to the reduced quantity of fruits in naked RNA, scrambled RNA, and infected groups.
Fruit quantification 7 weeks post inoculation
ELISA
3 weeks after inoculation with the virus, leaf samples were taken from the top of each plant for quantitative ELISA testing.
As indicated by the green and red arrows in the ELISA graph below, to the left of the dotted line is considered healthy, and to the right is considered infected. Half of the treated plants were negative for ToBRFV presence even after almost one month post inoculation. Although some plants treated with the RNAway based treatment were positive, overall fruit quantity and disease severity were not significantly different than the healthy group. This shows that the RNAway based treatment can fight the effects of the virus and mitigate disease symptoms even if the plant becomes infected.
ELISA results 3 weeks post inoculation
