To quantify the efficiency of pesticide delivery at the root level, I solved the model for the Cy5 dispersion constant Dp and the rate constant of Cy5 absorption to soil kPS . I then used the average values of DNP, kNPS, Dp and kPS to optimally estimate kPF, . Again, the model output matched the empirical data closely. The rate of Cy5 release kPF can be ranked from highest to lowest as follows: PhMV >>> CPMV > TMGMV >> MSNP. Interestingly, these results do not match the release profile of Cy5 in the dialysis assay , suggesting that the interaction between nanoparticles and soil has a major influence on the release rate.Testing experimentally whether a new nanoparticle formulation is a suitable candidate for pesticide delivery to the rhizosphere is time consuming and expensive; each nanoparticle described above required 7 soil column experiments and I used an average of 100 SDS denaturing gels to solve the soil transport profile of the nanocarriers through 2 to 30 cm of soil depth . The model described above minimizes the time and cost required to evaluate a novel nanopesticide. In conjunction with the model, the only experimental work required is to run the new nanocarrier candidate through a 4 cm deep soil column, a mobility test well recognized and established by the Organization for Economic Cooperation and Development.This experimental data is necessary to establish the value of DNP and kNPS, to predict the nanopesticides behaviour at any other soil depth . The VLP bacteriophage Qβ,package of blueberries which has been investigated as a drug carrier for medical application but not for pesticide delivery,was tested for its ability to transport through soil .

Qβ was predicted to transport through soil similarly to CPMV and TMGMV, further confirming the superior soil mobility of VNPs/VLPs over synthetic materials. The data obtained from the six different nanoparticle tested indicate that kNPS is linearly related to the surface area of MSNP, TMGMV, Qβ, and CPMV; however, PLGA and PhMV behaviors are different. . PhMV nanoparticles have been shown to have known positive zeta potential at a pH of 7.4. On the other hand, both clay and organic matter have a net negative surface charge, which may explain the enhanced soil binding of PhMV. I suspect the polymer nature of PLGA and its strong electronegativity also promote its enhanced binding to organic matter present in the soil. The linear relationship of kNPS of other nanoparticles with surface area suggests that the binding for the nanoparticles follows a mechanism that depends on the surface area such as a mechanism based on van der Waals forces. Even though such analysis is limited due to limited number of particles tested, it does suggest that the model parameters have physical basis that may be elucidated. Nematode endoparasites infect 3,000 different plant species including many crops,and are most abundant ~24 cm beneath the soil surface.Based on our empirical and modelling results, I selected TMGMV to deliver the nematicide abamectin.Abamectin is insoluble in water and binds strongly to organic matter in the top layer of soil, so its effect in the rhizosphere is limited and it is an ideal candidate for nanopesticide delivery using TMGMV. I used our nanopesticide model to determine how much TMGMV formulation must be applied to maintain the IC50 concentration of abamectin 24 cm beneath the surface for at least 24 h. A conjugated formulation would be better than encapsulation to avoid premature release, and the linkage should be stable enough to allow the carrier to reach the target depth before the cargo is dispersed, such as a labile ester with a half-life release rate of 4 days.

The IC50 value of abamectin is 1.309 x 10-4 mg cm- 3 , and at least this concentration must therefore be achieved in the rhizosphere.I modelled various flow rates representing the typical range of crop irrigation systems, and used a common irrigation regimen of 1 h three times a week, the first irrigation taking place immediately after nanopesticide application. The values of DNP and kNPS for TMGMV were determined as above, and in place of abamectin I used the values for the chemically similar Cy5. I assumed complete release at the root level due to the hydrolysis of the labile ester linkage over the course of a few days. The simulation output revealed that the mass of nanopesticide needed to maintain the target abamectin concentration for 24 h was dependent on the flow rate. Without no irrigation, neither free nor conjugated abamectin would achieve that concentration due to the extremely slow rate of diffusion. At a flow rate of 0.5 cm3 min-1 , the lowest dose of TMGMV abamectin required to maintain the target abamectin concentration 24 cm below the surface was 0.1056 mg cm-2 . The model therefore offers a powerful tool to optimize the dose regimen that must be use to maximize the efficacy of pesticides in the rhizosphere. Biological pests, including pathogens, arthropods, nematodes, and weeds are responsible for major losses in crop yields.262 In modern agriculture, pest management often relies on the use of synthetic chemicals that are sparingly soluble and absorb to soil particles with high affinity. Consequently, contemporary pesticides generally have poor bio-availability, and therefore require applications in large quantities to achieve an effective dose.The accumulation of these chemicals in the environment contaminates both land and water sources, which leads to off-target toxicity to other species, including domestic animals and humans .As a result, an increasing number of pesticides have been withdrawn from the market due to the tightening of regulatory guidelines.

The persistence of pesticide traces in the environment in concentrations below their effective dose has also resulted in the build-up of target resistance, ultimately rendering some pesticide formulations obsolete.Because these compounds are not being efficiently replaced, there is currently a gap in the market which threatens our food safety and security. Advances in nanotechnology have led to the development of agrochemical nanomaterials to protect crops from various pests.The encapsulation or conjugation of pesticides in/to nanocarriers improves their stability and solubility, preventing their premature degradation by photolysis or biodegradation. Compared to free pesticides, nanocarriers can have enhanced soil mobility and increase the pesticide’s potential for interaction with target pests at lower doses.While nanocarriers can bring significant benefits to the agricultural industry, some health and environmental risks remain to be solved. The majority of nanopesticides in the development pipeline are based on metallic compounds, synthetic or natural polymers, which tend to persist in the environment, and in some cases can cause acidification of soil, impairing its fertility. Thus, there is a need to design eco-friendly nanocarriers with low-toxicity and favorable bio-degradation. To this end, we and others have proposed to repurpose the capsids of plant viruses for pesticide delivery applications. For example,nft growing system the delivery of anthelmintic drugs to endoparasitic nematodes using the icosahedral red clover necrotic mosaic virus and the rod-shaped tobacco mild green mosaic virus has been reported. Plant viruses are already part of the natural soil ecosystem and are not known to cause adverse effects in humans or animals. We have focused on the high aspect ratio nanoparticles derived from the nucleoprotein assembly of TMGMV because the high aspect ratio offers a larger surface area to be modified with pesticide payload compared to spherical nanoparticles. More importantly, we reported enhanced soil mobility of TMGMV and accumulation at the crop root level, where nematodes reside.To pave the way for environmental and field applications of TMGMV, we set out to develop non-infectious formulations thereof. Infectious TMGMV has been approved by the EPA for use as a bioherbicide; its use is restricted to its application in the state of Florida for the treatment of the invasive tropical soda apple weed.

While TMGMV has a rather narrow host range, it does infect solanaceous plants, including tomato, chili peppers, and eggplants. Therefore, to enable broad applicability it is desired to prepare non-infectious nanoparticle formulations. The inactivation of plant viruses was first explored in 1936 using various chemical treatments such as formalin, hydrogen peroxide, or even sodium nitrite.264 Generally these chemical treatments either crosslink or oxidize the nucleic acids and/or proteins. Since then, extensive work has been reported on the use of ultraviolet radiation as an effective methodto inactivate tobamovirus using tobacco mosaic virus and TMGMV as models.UV irradiation causes RNA-protein crosslinks as well as dimerization of adjacent uracils, both of which inhibit RNA replication and translation.TMGMV, the U2 strain of TMV, was found to be 5.5x more sensitive to UV inactivation.Since isolated RNA from TMV and TMGMV were equally sensitive to UV inactivation, the difference was attributed in differences in packaging of the RNA. While TMV and TMGMV share the same structure, both are nucleoprotein assemblies measuring 300×18 nm with a 4 nm-wide central channel, it appears that packaging of the TMV coat protein protects its RNA more efficiently from UV damage. Nonetheless, this previous body of data relied on visual local lesion quantification to record the level of infectivity post viral inactivation; this method has now been outperformed by the far more sensitive polymerase chain reaction, which quantifies the presence of viral RNA within the inoculated leaves. Therefore, follow-up studies revisiting the UV inactivation of TMGMV were warranted. In addition to testing UV treatment, we also considered two commonly used chemical treatments, namely βPL and formalin. βPL and formalin are more commonly used in the medical field to produce inactivated vaccines; for example, these reagents are used to produce non-virulent enterovirus , hepatitis A, polio, and influenza virus vaccines. βPL induces the acylation or alkylation of nucleotides and amino acids. On the other hand, formalin induces chemical RNA and protein crosslinking, including RNA-protein crosslinks.Here, we compared the inactivation of TMGMV particles by UV light against that of chemical treatment using βPL or formalin. To test whether the inactivated formulation remained infective, Nicotiana tabacum Tennessee 86 , N. tabacum Samsun-NN, and tropical soda apple  were inoculated and challenged with the various TMGMV particle preparations. Visual inspection of plants and reverse-transcription polymerase chain reaction was conducted on individual leaves to quantify infectivity with a high degree of sensitivity. Nicotiana tabacum Tennessee 86 , N. tabacum Samsun-NN, and tropical soda apple plants were seeded in 30 x 20 x 3.5 cm aluminum baking trays using Sungrow® Mix #3 Professional Mix and maintained on a greenhouse bench at the USDAARS-U.S. Horticulatural Research Laboratory, Fort Piece, FL 34945. Seedlings were transplanted individually in 3.8 liter plastic pot and allowed to grow in the greenhouse. When the plants were about 30 days old, fully developed new leaves were mechanically inoculated by gently abrading with a Q-tips® swab dipped in native or inactivated TMGMV or buffer . Five plants were inoculated for each treatment conditions in addition to a negative control . Leaves were imaged and harvested individually 22 days or 16 days post-inoculation and stored at -80°C until further processing.Inoculated leaves were submerged in liquid nitrogen for 1 min in a mortar and pulverized using a pestle into a thin powder. The pulverized leaves were suspended into UltraPure DNase/RNase free distilled water using 1 mL per gram of leaves and vortexed for 1 min prior to centrifugation at 13,000 g for 10 min to pellet down the leaf material. 500 μL of the supernatant was denatured by adding 1/4 vol. of 10 % SDS under heating for 10 min at 60°C. Samples were then treated with 2 volumes of UltraPure phenol:chloroform:isoamylalcohol , mixed by vortexing for 1 min, and centrifuged at 13,000 g for 10 min. The upper phase containing the RNA was transferred into a fresh tube and the extraction was repeated an additional two times. The RNA extract was added to 2 volumes of 100% ethanol and further purified and concentrated using the Quick-RNA™ Miniprep kit . The final purified RNA was suspended in 30 μL of UltraPure DNase/RNase free distilled water and stored at -80°C until further analysis. The concentration was determined by UV-visible spectroscopy at 260 nm using the extinction coefficient for single-stranded RNA: 25 ng mL−1 cm−1 .We set out to study the effect of UV light exposure as well as βPL and formalin treatment on TMGMV structural integrity and its genome stability. Dose escalation studies were performed, and resulting inactivated TMGMV particles were characterized by size exclusion chromatography , dynamic light scattering , and transmission electron microscopy to assess their physical state . Independent of the treatment modality or concentration, SEC indicated intact TMGMV particles; free or broken coat proteins were not detected .