Recent advances in biodegradable matrices for active ingredient release in crop protection: Towards attaining sustainability in agriculture.

Climate changes, emerging species of plant pests, and deficits of clean water and arable land have made availability of food to the ever-increasing global population a challenge. Excessive use of synthetic pesticides to meet ever-increasing production needs has resulted in development of resistance in pest populations, as well as significant ecotoxicity, which has directly and indirectly impacted all life-forms on earth. To meet the goal of providing safe, sufficient, and high-quality food globally with minimal environmental impact, one strategy is to focus on targeted delivery of pesticides using eco-friendly and biodegradable carriers that are derived from naturally available materials. Herein, we discuss some of the recent approaches to use biodegradable matrices in crop protection, while exploring their design and efficiency. We summarize by discussing associated challenges with the existing approaches and future trends that can lead the world to more sustainable agricultural practices.

Loading and release mechanism of red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin.

Loading and release mechanisms of Red clover necrotic mosaicvirus (RCNMV) derived plant viral nanoparticle (PVN) are shown for controlled delivery of the anticancer drug, doxorubicin (Dox). Previous studies demonstrate that RCNMV's structure and unique response to divalent cation depletion and re-addition enables Dox infusion to the viral capsid through a pore formation mechanism. However, by controlling the net charge of RCNMV outer surface and accessibility of RCNMV interior cavity, tunable release of PVN is possible via manipulation of the Dox loading capacity and binding locations (external surface-binding or internal capsid-encapsulation) with the RCNMV capsid. Bimodal release kinetics is achieved via a rapid release of surface-Dox followed by a slow release of encapsulated Dox. Moreover, the rate of Dox release and the amount of released Dox increases with an increase in environmental pH or a decrease in concentration of divalent cations. This pH-responsive Dox release from PVN is controlled by Fickian diffusion kinetics where the release rate is dependent on the location of the bound or loaded active molecule. In summary, controllable release of Dox-loaded PVNs is imparted by 1) formulation conditions and 2) driven by the capsid's pH- and ion- responsive functions in a given environment.

New insight into the structure of RNA in red clover necrotic mosaic virus and the role of divalent cations revealed by small-angle neutron scattering.

Red clover necrotic mosaic virus (RCNMV) is a 36-nm-diameter, T = 3 icosahedral plant virus with a genome that is split between two single-stranded RNA molecules of approximately 3.9 kb and 1.5 kb, as well as a 400-nucleotide degradation product. The structure of the virus capsid and its response to removing Ca(2+) and Mg(2+) was previously studied by cryo-electron microscopy (cryo-EM) (Sherman et al. J Virol 80:10395-10406, 2006) but the structure of the RNA was only partially resolved in that study. To better understand the organization of the RNA and conformational changes resulting from the removal of divalent cations, small-angle neutron scattering with contrast variation experiments were performed. The results expand upon the cryo-EM results by clearly showing that virtually all of the RNA is contained in a thin shell that is in contact with the interior domains of the viral capsid protein, and they provide new insight into changes in the RNA packing that result from removal of divalent cations.

Plant-biomass-based hybrid seed wraps mitigate yield and post-harvest losses among smallholder farmers in sub-Saharan Africa.

Sustainable practices that reduce food loss are essential for enhancing global food security. We report a 'wrap and plant' seed treatment platform to protect crops from soil-borne pathogens. Developed from the abundantly available wastes of banana harvest and recycled old, corrugated cardboard boxes via chemical-free pulping, these paper-like biodegradable seed wraps exhibit tunable integrity and bioavailability of loaded moieties. These wraps were used for nematode control on yam (Dioscorea cayenensis-rotundata) seed pieces in Benin, a major producer of this staple crop in the sub-Saharan African 'yam belt'. Our seed wraps loaded with ultra-low-volume abamectin (1/100 ≤ commercial formulation) consistently controlled yam nematode (Scutellonema bradys) populations while considerably increasing the yield at various locations over 2015-2018. Substantial reduction in post-harvest tuber weight loss and cracking was observed after 3 and 5 months of storage, contributing to increased value, nutrition and stakeholders' preference for the wrap and plant treatment.

Crystallization and preliminary X-ray diffraction analysis of red clover necrotic mosaic virus.

Red clover necrotic mosaic virus (RCNMV) is a species that belongs to the Tombusviridae family of plant viruses with a T = 3 icosahedral capsid. RCNMV virions were purified and were crystallized for X-ray analysis using the hanging-drop vapor-diffusion method. Self-rotation functions and systematic absences identified the space group as I23, with two virions in the unit cell. The crystals diffracted to better than 4 Šresolution but were very radiation-sensitive, causing rapid decay of the high-resolution reflections. The data were processed to 6 Šin the analysis presented here.

Tailored Lignocellulose-Based Biodegradable Matrices with Effective Cargo Delivery for Crop Protection.

Controlled release and targeted delivery of agrochemicals are crucial for achieving effective crop protection with minimal damage to the environment. This work presents an innovative and cost-effective approach to fabricate lignocellulose-based biodegradable porous matrices capable of slow and sustained release of the loaded molecules for effective crop protection. The matrix exhibits tunable physicochemical properties which, when coupled with our unique "wrap-and-plant" concept, help to utilize it as a defense against soil-borne pests while providing controlled release of crop protection moieties. The tailored matrix is produced by mechanical treatment of the lignocellulosic fibers obtained from banana plants. The effect of different extents of mechanical treatments of the lignocellulosic fibers on the protective properties of the developed matrices is systematically investigated. While variation in mechanical treatment affects the morphology, strength, and porosity of the matrices, the specific composition and structure of the fibers are also capable of influencing their release profile. To corroborate this hypothesis, the effect of morphology and lignin content changes on the release of rhodamine B and abamectin as model cargos is investigated. These results, compared with those of the matrices developed from non-banana fibrous sources, reveal a unique release profile of the matrices developed from banana fibers, thereby making them strong candidates for crop protection applications.

Infusion of dye molecules into Red clover necrotic mosaic virus.

The Red clover necrotic mosaic virus capsid is utilized to package and release molecules through reversible depletion and re-addition of divalent cations.

Plant Virus-Based Nanoparticles for the Delivery of Agronomic Compounds as a Suspension Concentrate.

Nanoparticle formulations of agrichemicals may enhance their performance while simultaneously mitigating any adverse environmental effects. Red clover necrotic mosaic virus (RCNMV) is a soil-transmitted plant virus with many inherent attributes that allow it to function as a plant virus-based nanoparticle (PVN) when loaded with biologically active ingredients. Here we describe how to formulate a PVN loaded with the nematicide abamectin (Abm) beginning with the propagation of the virus through the formulation, deactivation, and characterization of the finished product.

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding.

Transfer RNA structure involves complex folding interactions of the TPsiC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TPsiC domain, rT54, Psi55 and m5C49, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA(Phe) T-half molecules (nucleosides 40-72) with the corresponding unmodified D-half molecule (nucleosides 1-30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg2+ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K(d)s (rT54 = 6 +/- 2, m5C49 = 11 +/- 2, Psi55 = 14 +/- 5, and rT54,Psi55 = 11 +/- 3 microM) significantly lower than that of the unmodified T-half (40 +/- 10 microM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA(Phe) and native yeast tRNA(Phe), as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA's TPsiC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.

Structural characterization of an intermolecular RNA-RNA interaction involved in the transcription regulation element of a bipartite plant virus.

The 34-nucleotide trans-activator (TA) located within the RNA-2 of Red clover necrotic mosaic virus folds into a simple hairpin. The eight-nucleotide TA loop base pairs with eight complementary nucleotides in the TA binding sequence (TABS) of the capsid protein subgenomic promoter on RNA-1 and trans-activates subgenomic RNA synthesis. Short synthetic oligoribonucleotide mimics of the RNA-1 TABS and the RNA-2 TA form a weak 1:1 bimolecular complex in vitro with a K(a) of 5.3 x 10(4) M(-1). K(a) determination for a series of RNA-1 and RNA-2 mimic variants indicated optimum stability is obtained with seven-base complementarity. Thermal denaturation and NMR show that the RNA-1 TABS 8mers are weakly ordered in solution while RNA-2 TA oligomers form the predicted hairpin. NMR diffusion studies confirmed RNA-1 and RNA-2 oligomer complex formation in vitro. MC-Sym generated structural models suggest that the bimolecular complex is composed of two stacked helices, one being the stem of the RNA-2 TA hairpin and the other formed by the intermolecular base pairing between RNA-1 and RNA-2. The RCNMV TA structural model is similar to those for the Simian retrovirus frameshifting element and the Human immunodeficiency virus-1 dimerization kissing hairpins, suggesting a conservation of form and function.

Development of abamectin loaded plant virus nanoparticles for efficacious plant parasitic nematode control.

Plant parasitic nematodes are one of the world's major agricultural pests, causing in excess of $157 billion in worldwide crop damage annually. Abamectin (Abm) is a biological pesticide with a strong activity against a wide variety of plant parasitic nematodes. However, Abm's poor mobility in the soil compromises its nematicide performance because of the limited zone of protection surrounding the growing root system of the plant. In this study, we manipulated Abm's soil physical chemistry by encapsulating Abm within the Red clover necrotic mosaic virus (RCNMV) to produce a plant virus nanoparticle (PVN) delivery system for Abm. The transmission electron microscopic and dynamic light scattering characterization of Abm-loaded PVN (PVN(Abm)) indicated the resultant viral capsid integrity and morphology comparable to native RCNMV. In addition, the PVN(Abm) significantly increased Abm's soil mobility while enabling a controlled release strategy for Abm's bioavailability to nematodes. As a result, PVN(Abm) enlarged the zone of protection from Meloidogyne hapla root knot nematodes in the soil as compared to treating with free Abm molecules. Tomato seedlings treated with PVN(Abm) had healthier root growth and a reduction in root galling demonstrating the success of this delivery system for the increased efficacy of Abm to control nematode damage in crops.

Polymeric systems incorporating plant viral nanoparticles for tailored release of therapeutics.

Therapeutic polylactide (PLA) nanofibrous matrices are fabricated by incorporating plant viral nanoparticles (PVNs) infused with fluorescent agents ethidium bromide (EtBr) and rhodamine (Rho), and cancer therapeutic doxorubicin (Dox). The native virus, Red clover necrotic mosaic virus (RCNMV), reversibly opens and closes upon exposure to the appropriate environmental stimuli. Infusing RCNMV with small molecules allows the incorporation of PVN(Active) into fibrous matrices via two methods: direct processing by in situ electrospinning of a polymer and PVNs solution or immersion of the matrix into a viral nanoparticle solution. Five organic solvents commonly in-use for electrospinning are evaluated for potential negative impact on RCNMV stability. In addition, leakage of rhodamine from the corresponding PVN(Rho) upon solvent exposure is determined. Incorporation of the PVN into the matrices are evaluated via transmission electron, scanning electron and fluorescent microscopies. Finally, the percent cumulative release of doxorubicin from both PLA nanofibers and PLA and polyethylene oxide (PEO) hybrid nanofibers demonstrate tailored release due to the incorporation of PVN(Dox) as compared to the control nanofibers with free Dox. Preliminary kinetic analysis results suggest a two-phase release profile with the first phase following a hindered Fickian transport mechanism for the release of Dox for the polymer-embedded PVNs. In contrast, the nanofiber matrices that incorporate PVNs through the immersion processing method followed a pseudo-first order kinetic transport mechanism.

Encapsidation of nanoparticles by red clover necrotic mosaic virus.

Icosahedral virus capsids demonstrate a high degree of selectivity in packaging cognate nucleic acid genome components during virion assembly. The 36 nm icosahedral plant virus Red clover necrotic mosaic virus (RCNMV) packages its two genomic ssRNAs via a specific capsid protein (CP) genomic RNA interaction. A 20-nucleotide hairpin structure within the genomic RNA-2 hybridizes with RNA-1 to form a bimolecular complex, which is the origin of assembly (OAS) in RCNMV that selectively recruits and orients CP subunits initiating virion assembly. In this Article, an oligonucleotide mimic of the OAS sequence was attached to Au, CoFe2O4, and CdSe nanoparticles ranging from 3 to 15 nm, followed by addition of RNA-1 to form a synthetic OAS to direct the virion-like assembly by RCNMV CP. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements were consistent with the formation of virus-like particles (VLPs) comparable in size to native RCNMV. Attempts to encapsidate nanoparticles with diameters larger than 17 nm did not result in well-formed viral capsids. These results are consistent with the presence of a 17 nm cavity in native RCNMV. Covalent linkage of the OAS to nanoparticles directs RNA-dependent encapsidation and demonstrates that foreign cargo can be packaged into RCNMV virions. The flexibility of the RCNMV CP to encapsidate different materials, as long as it is within encapsidation constraint, is a critical factor to be considered as a drug delivery and diagnostic vehicle in biomedical applications.

Controlled encapsidation of gold nanoparticles by a viral protein shell.

Icosahedral virus capsids demonstrate a high degree of selectivity in packaging cognate nucleic acid components during assembly. This packaging specificity, when integrated as part of a nanotechnological protocol, has the potential to encapsidate a wide array of foreign materials for delivery of therapeutics or biosensors into target cells. Red clover necrotic mosaic virus (RCNMV) exclusively packages two genomic ssRNAs initiated by a specific protein:RNA interaction between the RCNMV coat protein (CP) and the viral RNA origin of assembly (OAS) element. In the present work, an oligonucleotide mimic of the RCNMV OAS sequences is attached to Au nanoparticles as a recognition signal to initiate the virion-like assembly by RCNMV CP. Covalent linkage of the OAS to Au functions as a trigger for specific encapsidation and demonstrates that foreign cargo can be packaged into RCNMV virions.

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release.

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 +/- 30 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.