Nano silver are injected into stems, fertilize leaves, and water citrus stump for prevention and treatment

Plant disease control is critical to the sustainable development of agriculture, with recent advances in nanotechnology offering a promising solution to this pressing problem. However, the effectiveness of nano silver distribution (AgNP) methods has not been fully explored and knowledge of the life cycle and mobility of NPs in plants is not well known. In this study, we evaluated the effectiveness of NP distribution methods and investigated the mobility and distribution of NP with different surface coatings (citrate (Ct), polyvinylpyrrolidone (PVP), and gum. Arabic (GA)) in the Mexican citrus tree. Contrary to the reported limited distribution effect for the foliar and root methods of distribution, petiole and stem injection can deliver large amounts of NPs into the plant, despite the timing of injection. petioles much longer than injected into the stem (7 days versus 2 hours in citrus). When NP enters the plant, repulsion interactions between the NPs and the duct surface are predicted to facilitate the transport of NP in the plant. Compared with PVP and Ct, GA is highly effective in inhibiting the aggregation of NPs in synthetic sap and enhancing NP mobility in plants. During the 7-day trial, the majority of nano-silver recovered from the plants (10 mL, 10 ppm GA-AgNP suspension) remained in the stem (average 81.0%), with a significant amount in the roots ( averaging 11.7%), some with branches (average 4.4%), and a few on leaves (average 2.9%). Furthermore, the NP concentration during injection and the incubation period after injection were found to influence the distribution of nano silver in the plant.

(Copyright by NanoCMM Technology)(Bản quyền thuộc NanoCMM Technology)

INTRODUCE

World demand for fruits and vegetables is steadily increasing, but production is growing

has decelerated over the same period. According to Siegel et al. global and fruit supplies

vegetables have decreased 22% compared to demand in 2009 and the shortage is likely to be exacerbated. Among the reasons for slow production, plant diseases, especially those caused by bacteria and fungi caused by insects, are a growing concern. For example, Huanglongbing (HLB), a deadly citrus disease, was responsible for $ 4.5 billion of total economic losses in Florida between 2006 and 2011. HLB caused by Candidatus Liberibacter asiaticus (C.Las) is caused by the spread of a leaf hopper and is currently incurable.

Pathogens reside mainly in the plant capillary system (eg, xylem / or phloem), where it is difficult to remove but can easily migrate throughout the plant. Challenges remain to characterize and cultivate these pathogens and manipulate them genetically. To date, progress has been reported on the control of pathogens through phytopathic resistance, antibiotic type, or the use of plant growth stimulants. However, the need for effective methods to control these diseases remains acute. Recent developments in nanotechnology offer a promising pathway towards a cure for plants. Several nanoparticles (NPs) have excellent antibacterial properties: copper nanoparticles and zinc nanoparticles are being evaluated for their effectiveness against C.Las or other microbial pathogens on citrus and nano silver (AgNPs) as well. has been evaluated for its ability to directly inhibit plant pathogens. In addition, NPs can efficiently migrate within plants (such as tomatoes, cucumbers, wheat), using the plant’s capillary system, where they can interact with invading pathogens. . However, some important issues must be addressed before actual agricultural applications can be feasible.

One of the key questions is how to efficiently introduce NP into the tree (distribution efficiency: the amount of NP entering the tree divided by the total quantity of NP quantified). Foliar (including spray / osmosis of an NP suspension, exposure to nano aerosols, vacuum infiltration, and pressure bath infusion) and NP Irrigation (i.e. wetting of soil) 22.23 are methods of Most widely reported. While foliar spray can provide NP to citrus chloroplasts or in the dermis of tobacco leaves, the NP delivered by this technique can be easily washed away from water (over 70% of the total NP). used on leaves). In addition, the epidermis of the leaves prevents the penetration of most NPs, with about 80% of the NP entering the remaining leaves in

First 200 nm below leaf epidermis after 7 days of exposure. In terms of root application, while most NPs are still in culture medium, the epidermis and Casparian strip (in their complete root form) prevent the penetration of NPs. 28 Therefore, it is very likely that the low fertilization efficiency of leaves and roots is highly effective, although the efficiency of foliar fertilization appears to be higher than that of basal application. To the best of our knowledge, little effort has been made systematically to investigate and compare the effectiveness of these methods on NP distribution.

Another distribution method

Direct injection into branches / stalks and stems has not been paid much attention. In a recent report, a direct injection method was used to effectively provide plant protection activators, antibiotics and plasmid DNA.

In addition to the chosen method of distribution, the mobility of NP is influenced internally by the vegetative environment and the manner in which water and solutes, including high concentrations of inorganic / organic and NP / substances. antimicrobial, its movement in vascular tissues, xylem (upward transport) and phloem (downward transport). Both the xylem and the phloem consist of shaped pipes (wood vessels in the xylem and sieve vessels in the phloem) for axial transport. Tube shape is made by cells arranged from start to finish whose end walls are perforated to facilitate transportation. On their side walls, adjoining circuits are connected by pits much smaller than perforated arrays. The pores lie between the cell walls of both vessels and retain a central membrane, known as the intervessel fovea. 35 Individual circuits have finite length and water continuity. Movement outside of the first circuit is ensured by superposition of the adjacent circuit. Therefore, these circuits relays relying on pit connections, including their intervessel membrane, through which the solutes must pass to enter a new circuit. 36 Once NP enters a plant, most still do not know how the plant’s internal complex environment (eg synthetic resin, resin flow rate 34.37, 38.39 and pores , the size of the membrane cavity, the laminate chamber and the sieve vessel 37–39) influence the life cycle and transport of NP. Based on previous reports, the high ionic strength of the sap can lead to NP agglutination, while the existence of multiple organic molecules may reduce agglutination through steric stabilization; 40–42 specific types of ions (eg, chlorides, 43 phosphates 44), or organic macromolecules (for example, humic acid 45 and extracellular polymers 46.47) can affect the dissolution rate of NPs; and, the resin flow rate and the pore size of a plant’s xylem pit membrane or the pore of the pholem sieve can influence the deposition and separation of NPs. Thus, the NP behavior in the sap needs to be assessed to better understand NP transport and its distribution in plant tissues. Importantly, most research exploring the use of the Park in agriculture has focused on small, easy (and fast) growing annual crops, such as tomatoes, cucumbers, wheat, and melons. Watermelon. 18,19,49,50 A few, if any, studies have looked at the fate and transport of NPs in general, perennial woody plants, such as trees.

Given the high value of perennials and vines, there is a strong economic incentive to develop plant-appropriate cures, which could justify the cost of NPs.

NP properties, such as their size and surface cover, also influence NP behavior in the tree. Based on previous studies, the small size is more suitable for penetrating the epidermis. 52.53 For example, only NPs less than 5.4 nm (PVP) are applied to a citrus leaf that penetrates the phloem; 54

Gold particles less than 10 nm in size pass through the epidermis of the wheat leaves after 2 weeks. 49 Aside from size, it seems that surface mulch may affect NP transport in plants.

The surface coatings change the hydrophobicity and surface charge, as well as give steric stability. 49.55–59 It has been reported that in foliar application, while the PVP coating enhances the absorption in wheat leaves compared with citrate coatings (same metal size for both coated NPs), the coating Citrate allows more efficient NP transport to the plant’s vascular system (after epidermal penetration) due to its hydrophilic properties. 49 However, in the original application, the PVP coating facilitates the transport of CdS (QD) quantum dots from root to shoot compared to bare QD in soybean. In terms of surface charges, the negative charges generated by the mulch facilitate NP transport in radishes, rye grass, rice plants and pumpkins, 60 and allow for faster transport of QDs. in the conduction system of Arabidopsis thaliana compared with the positively charged coating. 61 In addition, organic polymers can provide steric stability for NPs, 62 which is likely to play an important role in stabilizing NPs under high salinity conditions. 63 Therefore, variations in size and surface can be used to control NP transport in plants.

In this study, we evaluated the effectiveness of NP distribution methods (foliar fertilization, root application, fertilization, branch injection and stem injection) for citrus (Mexican lemon and clementine tangerine. Nour grafted on rootstock) Carrizo grafting), and testing the life cycle and motion of NPs in the plant. We use silver nanoparticles in this study for three main reasons: i) Silver nanoparticles have a specific plasmonic and make it easier to track them in complex plant substrates; ii) background

Nano silver concentration is relatively low, minimizing the possibility of interference; and iii) nano silver have become known as biocides, making them attractive candidates for treating citrus diseases, such as HLB.

In addition, we studied the effect of size and surface coating (polyvinylpyrrolidone (PVP), gum arabic (GA), and sodium citrate (Ct)) on NP transport. Our results demonstrate that the stem injection can efficiently introduce NP into the plant, and that NP can move systematically both zoologically and fundamentally through the plant’s vascular system, with the large amount of NPs remaining in the stem.

However, we cannot quantitatively distinguish the silver form (i.e., primary NP, protein halo formation, silver ions, chelate ions, and silver, precipitate. The NPs range from xylem to phloem (through the leaves), as well as the ability to excrete NPs from the roots of the plant This study provides insight into the effectiveness of the NP distribution method and the NP mobility in large plants, complex, such as woody plants.

RESULTS AND DISCUSSION

Characteristics of NP in synthetic resin

Because transporting NPs throughout the tree structure will likely involve migration through xylem and phloem, it is important to understand how NPs will behave in the sap that fills the vessels. We measured the concentration of inorganic solutes in the sap obtained from the veins of Mexican lemons (the extraction process did not distinguish between the sap xylem and the sap phloem, and we did not identify the compounds). resin in sap), and results are shown in Table 1. The pH of the sap is between 5.5 and 5.9, with the most abundant cations being K + and Ca 2+. In terms of anion, Cl -, SO42-, PO43- and NO3- are dominant ions, consistent with previous studies. A concentration test in Table 1 shows that i) some Mg2 + and Ca2 + can form a precipitate and ii) an imbalance between the total positive and negative charges in the sap, with an excess of electrical charges. Positive positives are obvious. It has long been recognized that a certain part of the mineral exists in insoluble form in the synthetic sap, 66 and that this fraction can vary depending on the roots and leaves and day to night. 67 In addition, there are many organic species in the citrus resin, mainly including sugars, short-chain carboxylic acids and amino acids (Table S1). 37 We speculate that precipitated minerals and missing organic anions (eg, carboxylic acid group) are responsible for the charge imbalance. The total ionic strength (binding to inorganic ions of the sap is determined to be over 500 mM. High salinity and abundant organic matter found in the sap can affect aggregation and transport). and resolution of NPs.

Note: Molecular masses of Ct, PVP and GA (g / mol): 294, 40000 and 250000. a, nanopure water measurements (pH 5.5, AgNP, 10 ppm); b, measurements in synthetic sap (pH 5.5; AgNP, 10 ppm); c, estimated from thermo-weight analysis in SI, Figure S4; d, a detailed description of the Estimated Polymer Layer Thickness (in the resinous resin) is provided in SI, Figure S5. (p> 0.05, insignificant difference between data from the same group).

The particle diameters of the PVP-, GA- and Ct-AgNPs metal cores, as measured using a transmission electron microscope (TEM), were 17.9 ± 7.5, 9.2 ± 4.2 and 28.7 ± 11.0 nm, respectively (Table 2, Figure S1), while the hydrodynamic (HD) diameters of these NPs (Table 2) are 85.1 ± 3.6, respectively, 40.5 ± 1.3 and 94.3 ± 5.4 nm in nano water. Each AgNP had a statistically different size distribution (Figure S2 ac). Furthermore, the importance of characterization of NPs in water conditions is related to the sap. On the basis of Table 1 and literature, 66 we have created a synthetic resin mainly consisting of soluble species (conductivity: 29.33 mS / cm; ionic strength: 467 mM; total carbon content Mechanical: 1.60 ± 0.05 × 104 ppm Composition details can be found in Table S1). When AgNP was exposed to synthetic resin, the HD diameters of PVP-, GA- and Ct-AgNP were 134.6 ± 2.5, 52.1 ± 8.9 and 428.2 ± 12 nm, and each AgNP remains the range of statistically different sizes (Figure S2 df) (The corresponding plots for the three DLS measurements are provided in Figure S3). Within 10 minutes, the size of GA-AgNP showed no significant change (89.8 ± 0.4 nm), while that of PVP-AgNP and Ct-AgNP increased by 208.7 ± 2, respectively. , 9 nm and 682 ± 12.4 nm (Figure 1a). This implied that GA successfully stabilized nano silver while citrate was ineffective in stabilizing AgNP compared to synthetic resin; PVP moderately stabilizes AgNP. Similar zeta potentials of the three AgNP types, are shown in Table 2 (PVP-, GA- and Ct-AgNP: -6.99 ± 0.28 mV, -10.13 ± 0.63 mV and -4, 24 ± 1.71 mV), which indicates that the high ionic strength of the electrophoresis compressed resin, this layer can limit the contribution of the electrostatic repulsion to the stability of NPs in the resin. .

Therefore, steric repulsion can play an important role in NP stabilization. A ct (molecular weight (M w), 294 g / mol) coating provides nano-silver with carboxylate functional groups, adding negative charges to the NP surface. GA (M w is 250000 g / mol), a natural excretion from acacia, is an organic mixture consisting of 80% by weight of polysaccharides (d-galactose, l-arabinose, l-rhamnose, d-glucuronic). acid) and 20% by weight protein. 68 High concentrations of GA have been shown to stabilize emulsions under challenging water conditions, with 25 mM CaCl2 PVP (M w of 40000 g / mol) being a non-ionic polymer with functional groups C = O, C – N and CH 2, contain a hydrophilic pyrrolidone base and a hydrophobic alkyl group.

PVP coatings have been shown to inhibit NP aggregation through steric interfering effects. Using thermal weight analysis (TGA) and polymer layer thickness model, 62.71.72 we estimate that the surface concentration of Ct, PVP and GA on AgNP is 9.6 ± 0.2 × 10. -4, 1.1 ± 0.1 × 10-3 and 5.3 ± 0.7 × 10-2 g / m 2, with PVP and GA forming a polymer layer with thicknesses 4.4 and 20.1 nm, respectively (a detailed description of the polymer surface concentration and calculated thickness is provided in SI, Figures S4 and S5). Given that the xylem pit membrane has a hole diameter in the range of 10-340 nm, it is possible that the pit membrane can block the transport of PVP- and Ct-AgNP synthesized between xylem vessel elements. In terms of the transport axis, since the average voids of the phloem sieves and xylem perforation plates are between 200 nm and 1.5 μm, 36.73.74.78 membrane-like structures are not pressurized. Significant size exclusion puts pressure on GA-AgNP transport, but may stop shipping most of the synthetic PVP- and Ct-AgNPs.

However, the individual (unconjugated) particles and especially the smaller sized GA-AgNPs, have the ability to pass through the pit membrane, increasing the overall conduct area available for these particles. In addition, it is worth noting that proteins in both xylem and resin phloem, especially sap from plants contaminated with pathogens or insects, 79,80 can replace the coating on nano silver and impact on Fate and mobility of nano-silver in plants. 81 It’s not clear if this is happening in our system and should be investigated in future studies.

To quantitatively explore the effect of zeta size and potential on in-circuit silver nanotransportation, we use the Derjaguin-Landau-Verwey-Overbeek (DLVO) model to calculate the interaction energy. between the NP and the surfaces of the vegetative vascular system (xylem and phloem); A detailed description of DLVO calculations can be found in SI. Our DLVO model shows that the negative charges on the surface PVP-, GA- and Ct-AgNP result in push interaction energies of 2.41, 2.29 and 3.58 kT, respectively. However, the total Lifshitz-van der Waals and the electrostatic interaction energies of these three AgNPs (both in xylem and phloem) are always negative, with the particle size resulting in a high gravitational force between the NP and the xylem / phloem surface ( Figure S6). This shows that electrostatic stability is not the main reason for NP stabilization, and there are other forces responsible for this stabilization that helps NP mobility in circuits.

With a layer of PVP or GA in addition to nano silver, it is important to take the steric interaction into account to explain NP mobility in plants. Assume that the inner surface of

The xylem / phloem circuits are uncoated and planar, and steric (U ste) interactions include osmosis, push interaction (U osm) and elastic (U ela) (in the range 0 <h <d, where h is the distance between the NP and the circuit surface, and d is the layer thickness). 62 Absorbent and elastic interaction

can be estimated through equations 1-3:

where r is the radius of the NP in the synthetic resin derived from the intensity size DLS (nm), M W and ρ is the volume fraction, molecular weight (g / mol) and density (1, 29 and 1.35 g / cm 3) of PVP or GA, respectively, N a is Avogadro number, Г max is the maximum surface concentration of PVP and GA, χ is the solvency parameter of Flory-Huggins for GA (0.47) and PVP (0.45), T is temperature (K), and kB is Boltzmann’s constant. It is determined that once h <d, U ste dominates the interactions between the NPs and the xylem / phloem surface. This is especially clear for U ste derived

Modified GA, which is two orders of magnitude higher than the sum of the Lifshitz-van der Waals and the electrostatic interaction energy (Figure S7). Therefore, it is very likely that steric interactions are the main reason for NP mobility in plants.

While the effects of particle size, pH, temperature, natural organic matter and common ions on the solubility of AgNPs have been thoroughly studied, the solubility of NPs in the sap remains undetermined. We studied the solubility of nano silver in the following three media: (1) the organic composition of the synthetic resin, (2) the inorganic component of the synthetic resin, and (3) the synthetic resin (Please refer to Table S1 for detailed composition). It was found that after 7 days on all Ag ion media accounted for less than 1% of the total Ag, with the lowest percentage (0.22%) found in sap organic matter (Figure 1c). This value is much lower than the solubility reported in deionized water (>> 5% 82,85,87), we used UV-Vis spectroscopy to investigate the composition of the AgNPs reaction. 88.89 The results showed that in reality, most still silver nanoparticles and AgCl appeared in the form of suspended solids after the reaction originating from the reaction system with synthetic tree resin 90 (detailed description of UV-Vis spectrum can be found in Figure S8). Since the mass fraction of dissolved Ag is the total amount of Ag in the inorganic sap and the synthetic resin solution are very close together (both approximately 0.7%), it is likely that inorganic solutes are present. In the sap promotes the solubility of nano silver , although relatively less solubility occurs. Based on a previous study, the 43 dissolution rates of AgNPs (mean diameter 32.9 nm) at the molar Cl / Ag ratio were 535, were 0.107 ± 0.020% / h, and the Cl rate increase / Ag increases the dissolution rate. However, in our study, while the Cl / Ag molar ratio was 610 and the size of the AgNPs was smaller (both increased the dissolution rate), the dissolution rate decreased. Therefore, we conclude that the presence of organic substances in the synthetic resin inhibited the degradation of AgNPs, 91 consistent with the earlier finding that CeO 2 can be transported from roots to shoots with very limited solubility.

The anti-microbial properties of nano silver are partly related to their solubility (as the silver ions are responsible for antimicrobial activity 93), the slow dissolution of nano silver in the sap can their antimicrobial performance is reduced, although it is not known what concentration of silver ions is actually needed to induce a satisfactory antimicrobial response. However, this limitation may potentially be addressed by increasing the amount of AgNP in the plant. Regardless, specific Experiments regarding the effectiveness of AgNPs as antimicrobial agents on plants are still needed.

Figure 1. (a) Size growth of AgNPs (10 ppm) in synthetic sap (pH = 5.5) within the initial 10 minutes, (b) sedimentation of AgNPs (100 ppm) in synthetic resin (pH = 5.5), and (c) dissolve AgNPs (100 ppm) in the inorganic components of the synthetic (Inorganic) sap, the organic constituents of the synthetic resin (Organic) and the sap Aggregate (Mixed) (pH = 5.5)

NP mobility is distributed according to different methods

Soil wetting, foliar application (drip irrigation) and branch irrigation were evaluated for the ability to introduce nano silver into Mexican lemons. As shown in Figure 2, the mean Ag concentration (defined as Ag weight / weight of leaf tissue) was highest in the leaves of the plants exposed to spray treatment after 7 days (149.97 ± 82). , 70 μg / kg dry tissue), followed by contact with foliar fertilization (55,183 ± 17.10 μg / kg dry tissue), and soil wetting (13.95 ± 8.13 μg / kg dry tissue) (The difference between the Ag content after 7 days in the branch fertilizing group and the foliar fertilizing / wetting group was significant, while the difference in Ag content between the foliar fertilizing group and the group injected for branches was not significant) . The One-way Analysis of variance (ANOVA) plus the Fisher Least Test for Difference (LSD) indicated that the difference between the overall mean nano silver content from the Soil Wetting Group (9, 97 ± 7.87 μg / kg) and the control group (8.26 ± 6.45 μg / kg) were insignificant, while the Ag content from the injection arm was significantly different from that in the control and soil drying groups. However, the mean nano silver content in the branch injection group (50.44 ± 67.39 μg / kg) while higher than in the foliar application group (31.78 ± 26.64 μg / kg), was not significant. statistical meaning. Therefore, our results imply that wetting the soil is the least effective method of NP distribution into the leaves, which is in agreement with previous studies reporting that the majority of NPs applied to the roots would absorption on their surface. 60.94–97 In the case of branching, the mean Ag concentrations in leaves of plants fed PVP-, GA-, and Ct-AgNP were 58.71 ± 69.15, 71.48 ± 108.81, and 34.48 ± 27.05 μg / kg, respectively; These results were not statistically significant different.

However, based on mean values, it is likely that PVP and GA are more effective in enhancing NP transport, compared to Ct. Besides the smaller sizes of GA-AgNP and PVP-AgNP (more than Ct-AgNP), steric repulsion is considered as another important factor for enhancing NP mobility in plants based on modeling work. our chemistry. In addition, while the surface concentration of GA was much higher than that of PVP, the mobility of NPs in the tree was not significantly affected by the identity of the mantle. In the 7-day solubility experiment in the synthetic resin, we found that PVP-AgNP absorbs a significant amount of organic matter from the solution (possibly through hydrogen bonding due to the presence of multiple C = groups). O 70), but not GA-AgNP and Ct-AgNP. It is possible that organic substances absorbed from tree sap provide additional steric repulsion for PVP-AgNP. In terms of Ct-AgNP, citrate desorption can easily occur under conditions of high salinity, 98 and organic matter in the sap, especially protein, can be attached (through interaction). hydrophobic) and thus stabilize some AgNP, 99 contributing to the transport of Ct-AgNP in the plant. In general, increasing transport time (the time between NP fertilization and tissue harvest) or NP dosage (between 20 ppm and 100 ppm) clearly increases the Ag content in the leaves, indicating that AgNP to be

The leaf is continuously transported to the leaf, and a higher amount of silver NPs results in the leaf with a higher Ag content. However, based on other research, it is possible to reach the load threshold of 100, which is beyond that

Increasing the NP concentration does not necessarily increase the NP content in the leaves; this is probably due to a blockage of a porous structure that prevents intercellular NP transport caused by NP aggregation.

Figure 2. Mean Ag content of six Mexican lemon leaves (from nearest leaf to farthest leaf from (> 50 cm) dosing area) as a function of NP coating, suspension concentration, and time. (a) soil has been eroded; (b) leaf application; (c) branch feeding. (Control: not exposed to AgNPs; 20 and 100: 20 and 100 ppm AgNP suspension; 1 day and 7 days after exposure 1 day and 7 days; PVP, GA and Ct: PVP-, GA- and Ct- AgNP). *, five samples are used for the chart because one pattern is recognized as abnormal data in the analysis of the box chart. One-way ANOVA test plus Fisher’s LSD test was used for statistical analysis (p <0.05)

We estimated the distribution effect of foliar fertilization by dividing the mass of Ag obtained from the entire plant by the total weight of Ag introduced (i.e. quantified) into the tree, as these plants were Ag concentrations in leaves were found to be significantly higher than those in the soil-exposed plants. Six weeks after leaf exposure (no adverse effect on plant growth), three plants (exposed to 0.5 ml 100 ppm PVP-, GA- or a Ct-AgNP suspension) were sampled for destroy and split into leaves (did not include the original three leaves with AgNP dosage), branches, stems and roots. We recovered from 1.5-3.0 μg Ag out of a total of 50 μg Ag (total weight of Ag added to plants), accounting for 3-6% of the total number of applied silver nanoparticles (Figure S9). This implies that silver nanoparticles can be transported to branches, stems and roots from leaves through phloem layers and that surface mantle can influence the

The ability of NP to move in plants like the distribution of different silver nanoparticles in leaves, stems and roots is different (Figure S9). In addition, it is possible that the distribution efficiency may be higher because (i) Ag supplemented leaves are not included in the leaf sample and (ii) the roots are capable of excreting NP into the soil in a similar manner. similar to wheat roots (details on this below). 98

However, this percentage is much higher than the rate of soil eroded, which has been reported to include 0.03-0.11% NP in crops. 101.102 In foliar applications, an amount of NP can enter the plant through the stomata without being trapped in the epidermis. In contrast, the epidermis on the roots prevents most of the NPs from entering the plant, and an intact Caspi strip prevents the NPs from transporting the property. 28 It has been reported that foliar applications can provide a greater amount of NP into plants than soil applications. 14.20 However, in foliar fertilization, even those NPs that enter the plant through the stomata, they still have to move through the mesodermic structure before reaching the vasculature. 32

The mesophyll structure can be a temporary storage space for NPs, hindering the transport of NPs to phloem. However, in the branching level, the nano-silver suspension can be completely absorbed by the plant, allowing the NP stream to flow directly from the feeding syringe to the phloem / xylem system. The method removes the interference of epidermal / mesodermal / mesenchymal structures to NP transport, and can efficiently introduce AgNP into the plant. However, in the process of nano-silver fertilization it was noted that the rate of AgNP suspension absorption was very different between plants (from 24 hours to 168 hours), making this form of application difficult to implement. Therefore, we decided to explore stem injection as a method to introduce 100% NP into plants in a shorter time.

In addition, it is worth mentioning that the difference in the original conductors of the NPs encountered shortly after application can affect the NP transport process. Specifically, after foliar fertilization (applied to the upper part of the tree) and branching (at the tip of the branch) the main path of the entrance will initially pass through the phloem, while after pumping the trunk and soil it will likely be is adopted xylem. However, in the long run, both xylem and phloem contribute to NP transport in the plant.

The effect of the surface mulch on the mobility of NP in the plant after injection

In the branches and trunks, only the closeness between xylem and phloem is found. inside the bark (Figure 3a). In cross sections, xylem occupies most of the central cylinder of the stem until a vascular cambium ring just below the bark. Cambium is a thin meristem that forms new xylem cells that are directed inside the limb, and new meristem is directed outward. The phloem occupies a very narrow area just outside the cambium, and it breaks easily. Therefore, distribution of NP through the body is targeted at xylem. 10 ml of 1,000 ppm nano silver suspension (Ct-AgNPs, PVP-AgNPs, GA-AgNPs) was injected into the 2.5-year-old tangerine tree with clementine via injection into the stem at 20-30 psi for a period of 2 hours. On day 1, day 7 and 42, local tricuspid (ie near the injection site) and tricuspid from a point farthest away from the injection site (called a “systemic” leaf) were collected and measured. Ag. On Day 42, plants were separated into leaves, branches, stems, and roots, and were demolished for the purpose of performing mass balance on Ag, and determining the distribution of Ag in plants. Interestingly, after the stalk was cut into 4-5 segments (depending on the total length of the stem), multiple brown spots in the secondary xylem region were observed in succession segments in several plants, including even in the segment below the injection point just above the root (Figure 3b). This brown color demonstrates that the stem injection can deliver NP throughout the stem, including towards the roots. Furthermore, the elemental mapping of the brownish region on the cross section of the trunk with an energy scanning electron microscope the scatter X-ray spectroscopy confirmed the presence of AgNP in xylem circuits, but not all. both the xylem circuit elements (a small area along the secondary xylem of the stem) used for transporting the NPs (Figure 3c)

Figure 3. (a) Light microscopic image of cross-section of mandarin trunk (sub-section is image of cross-section of tree); (b) injected GA-AgNPs into the stalk (NPs visible as brown staining in secondary xylem tissue near the phloem (marked with a red outline), and staining always on the stalk where the drug was injected) ; (c) elemental mapping of brown localization on cross sections of trunks by scanning electron microscopy with energy scatter X-rays (elemental color: blue-carbon; red-silver); (d) Ag concentrations in leaves were injected with Ct-AgNPs, PVP-AgNPs and GA-AgNPs on days 1, 7 and 42 after injection (LOC and SYS: local and systemic leaves); (e) Ag content in leaves, branches, stems and roots recovered on the 42nd day after injection; (f) Total Ag weight in Ag leaves, branches, stems and roots recovered on day 42 after injection. (c and d:, trunk;, Stump, Branch;, Leaf) (10 ml 1000 ppm AgNP, 2.5 year old mandarin clementine) (One-way ANOVA test plus Fisher’s LSD Test used for statistical analysis, p <0.05).

After injection, all three types of silver nanoparticles migrate from the injection point to the leaf, but the coating surface influences the transport to different degrees. Ag content in leaves injected with GA-AgNPs ranked highest, followed by plants injected with PVP-AgNPs, and then by Ct- AgNPs (GA-AgNP group vs Ct-AgNP / PVP-AgNP group, P <0.05; PVP-AgNP vs CT- AgNP, P> 0.05), indicated that GA-AgNPs had the greatest transport potential from stem to leaf (Figure 3d). Among the GA-AgNP-injected plants, the local leaf Ag concentration was always higher than in the systemic leaves, suggesting that transport may be limited due to AgNPs aggregation / deposition in xylem flasks, and May be screened by the factory. Interestingly, in plants receiving GA-AgNPs, both the local and systemic leaves exhibited the highest Ag concentrations on Day 1 (59.4 ± 52.5 μg / kg and 38.4 ± 28.9 μg. / kg), reduced to 27.01 ± 30.2 μg / kg and 19.6 ± 31.11 μg / kg, on Day 7 and up to 24.15 ± 20.4 μg / kg and 11.05 respectively ± 8.2 μg / kg, respectively, on Day 42 (Fig. 3d) (Day 1 local vs day 7/42 local, Day 1 systemic vs day 42 systemic, P <0.05). In addition, the amount of Ag changes throughout the leaf throughout

Experiment (Figure S10) demonstrated a similar trend as shown in Figure 3d. To estimate the possible contribution of bio-dilution to the change in mass / Ag content, we performed Pearson correlations to quantify the possible high leaf weight potential. resulted in high Ag mass in leaves (2-sided significance check was used). It was found that there was no significant correlation between them, suggesting that biological dilution may not be the main reason for the decrease in leaf Ag levels. Therefore, the decreasing weight over time implies that GA-AgNPs can be transported in the plant through both xylem (up transport) and phloem (down transport) and downward transport can remove AgNPs from the plant. and into the roots. The unclear decline of the Ag concentrations in the leaves in the plants injected with Ct-AgNPs and PVP-AgNPs, probably indicated that the NPs did not migrate efficiently in the plants. However, we cannot exclude the possibility that changes in the mass / Ag content of local / systemic leaves could be affected by (i) Ag + storage by proteins such as protecting plants from pollution. metal, 103 and (ii) one part nano silver are more mobile than others due to the heterogeneous NP coating density or size caused by nano silver

The distribution of Ag in the injection plant on day 42 after injection is shown in Figures 3e and 3f. For all three AgNP treatments, the Ag concentration in the stem was the largest, followed by the roots, branches and leaves. Ag concentrations in the stalks injected with Ct-AgNPs, PVP-AgNPs and GA-AgNPs were 40.468.5 ± 224.1, 88.360.1 ± 25.429.7 and 33.394 ± 24.575.7 μg / kg (data collected in each set P> 0.05), accounting for 99.9%, 69.0% and 66.5% of total Ag mass obtained from the whole plant, respectively. These results demonstrate that Ct-AgNPs cannot be transported within the plant (possibly due to their rapid agglomeration in the sap. Although the citrate on the surface of the Park can easily be replaced by macromolecules, contributing of this change to the mobility of Ct-AgNP is not clear). However, because PVP-AgNPs and GA-AgNPs are against synthesis, continuous transport is activated. While the Ag content in the roots of the plants injected with PVP-AgNPs or GA-AgNPs was similar (12,941.9 ± 9.125 μg / kg compared with 12,994.3 ± 2,084.9 μg / kg, P> 0.05), The amount of Ag in the branches injected with PVP-AgNPs (16,777.9 ± 11,254.4 μg / kg, P> 0.05) was much higher than the plants that received the GA-AgNPs injection (735.2 ± 464.5 μg / kg, P> 0.05) (PVP-AgNP vs. GA-AgNP, P <0.05). In contrast, Ag content in leaves injected with PVP-AgNPs (11.2 ± 7.9 μg / kg, P> 0.05) was slightly lower than in plants injected with GA-AgNPs (19.4 ± 6.7 μg / kg, P> 0.05) (PVP-AgNP vs GA-AgNP, P> 0.05). Low Ag concentrations were found in the leaves, the difference between these two coatings (on average 74% higher Ag content in the leaves of GA-AgNPs injected plants compared to PVP-AgNP-injected plants). It implies that GA-AgNPs can be transported to the leaves more easily than PVP-AgNPs. The distribution effect (i.e., the amount of silver recovered from the tree after 42 days compared to the initial weight introduced) of the Ct-AgNPs, PVP-AgNPs and GA-AgNPs via stem injection was estimated 19, respectively. , 4 ± 2.3%, 39.5 ± 4.5% and 22.8 ± 7.2%. Following the stem when injecting, we observed droplets containing Ct-AgNPs at the wound site on the tree (due to pruning) near the injection site; no such drops were observed after PVP-AgNP or GA-AgNP injection. Therefore, not all Ct-AgNPs were included in the tree, contributing to low total Ag recovered (only ~ 20% Ag). Based on the relatively high transport of PVP-AgNPs and GA-AgNPs compared with Ct-AgNPs (ie the Ag content in the stem injected with Ct-AgNPs, PVP-AgNPs and GA-AgNPs accounted for 99.9%, 69, 0% and 66.5% of total Ag recovered weight) and the majority of Ag is present in the roots, which may have been excreted by the roots, indicating that small NPs can be transported and excreted by the roots. plants are easier. Therefore, it is hypothesized that root excretion contributes to low Ag recovery in plants injected with PVP- or GA-AgNP.

Effect of concentration on NP transport after stalk injection

GA-AgNP exhibited the most intense transport behavior, moving from the injection site of the trunk to all parts of the plant. In particular, the higher Ag mass found in the roots shows that AgNP can be transported between xylem and phloem, although it is possible to inject NP into the root system itself. Therefore, to further investigate AgNP transport during the first seven days after injection, plants injected with GA-AgNP suspension were sampled for destruction on days 1, 3, and 7 and separated into leaf, branch, and leaf samples. trunk, roots.

Figure 4. Volume of recovered Ag (a & b) and Ag content (c & d) in different tissues of 2.5 year old mandarin clementine injected 10 ml of 10 ppm suspension (total 100 μg Ag) (a & c)

and 10 ml 100 ppm (1000 μg Ag total) (b & d) GA-AgNPs. Tissues were sampled on Days 1, 3, and 7 after injection (Stem; stem, branch; Leaf).

(*, 100 ppm GA- AgNPs suspension on Day 3 was unsuccessful and only 7 ml of suspension was injected within 2 hours while the remainder was 10 ml)

(One-way ANOVA test plus Fisher’s LSD test was used for statistical analysis (P <0.05). Significant differences between GA-AgNP group data and Ct-AgNP / PVP-AgNP; there was no significant difference between the PVP-AgNP group and the Ct-AgNP group)

The distribution of Ag in different plant segments on Dates 1, 3 and 7 after injection with 10 ppm or 100 ppm GA-AgNPs is shown in Figure 4. The total amount of Ag measured in the tree varied significantly between The times and no clear trends in Ag volume from Day 1 to Day 7 can be wise. Among plants injected with 10 ppm GA-AgNP suspension, the total Volume of Ag recovered ranged from 14.2 to 67.7 μg out of the total 100 μg injected (Figure 4a), while from plants injected with 100 ppm GA-AgNP suspension ranged from 150.0 to 722.3 μg out of the total 1,000 μg injected (Figure 4b). In all cases, the overwhelming majority (84.6 ± 3.4% of total Ag recovered in plants were injected with 10 ppm GA-AgNP suspension and 91.3 ± 5.5% in plants receiving the suspension. The GA-AgNP 100 ppm) silver remained in the stem over the 7-day trial. While roots and branches have a significant amount of Ag following when injected, the total leaf Ag mass was still small (<1.5 μg in both injections) and the increase in concentration of the GA-AgNP suspension did not lead to an increase in the proportion of leaf Ag Weight (Fig. 4 a & b). Total Ag mass in the arms increased from 0.2 ± 0.01 μg to 7.5 ± 4.7 μg in plants injected with 10 ppm GA-AgNP suspension (P <0.05) and from 20.1 ± 1.0 μg to 54.5 ± 48.3 μg in plants injected with 100 ppm GA-AgNP suspension (P> 0.05), as harvest times were extended from 1 day to 7 days. In contrast, root Ag mass decreased slightly from 33.8 ± 33.4 μg to 13.5 ± 7.2 μg in plants injected with 100 ppm GA-AgNP suspension (P> 0.05), despite the This decrease was not clear in plants injected with 10 ppm GA-Ag NP suspension (2.6 ± 2.1 μg versus 2.3 ± 2.0 μg on day 1 and day 7, respectively, without Significant difference )

That said, 1) an increase in Ag mass in the arm from day 1 to 7 could imply that GA-AgNP was continuously transported to the arm from the injection point, and 2) a decrease in the amount of Ag in the roots from day 1 to 7 may imply that GA-AgNPs were excreted from the roots. In addition, relatively constant Ag concentrations in the leaves of both groups with different injection concentrations may indicate certain physiological responses of citrus to AgNPs (such as Ag, detoxification and retention. NP), 103 should be further investigated. However, the silver mass we measured varies widely in each group (evident by the large time interval confidence), making any conclusions highly speculative.

To further analyze the Ag distribution in plants, we standardized the total Ag mass in plant tissues with their dry mass (Figures 4c, 4d) and calculated% Ag mass in each tissue, compared with with total weight of Ag recovered from the whole tree (Figure S11 a, b). In Figure 4c, Leaf Ag content increased from 6.6 ± 1.1 μg / kg on day 1 (mass% Ag = 2.9%) to 14.3 ± 13.4 μg / kg on Day 3 (% weight Ag = 6.3%) before decreasing slightly to 13.3 ± 4.0 μg / kg on Day 7, while the Ag concentration in the roots decreased from 35.8 ± 8.1 μg / kg (mass% Ag = 19.0%) to 21.2 ± 7.6 μg / kg (mass% Ag = 7.1%) from Day 1 to Day 3 before increasing to 50.4 ± 41.6 μg / kg (% mass amount of Ag = 8,9%) on day 7 (Figure 4c); The large confidence interval relative to the root composition between plants reflects the large difference between the sampled plants, which may be driven by natural physiological differences between plants. As the up and down transport of nano silver is responsible for the presence of Ag in the leaves and roots, respectively, the kinetic variation of the leaf and root composition implies that the up and down transport intensity is constantly changing. Similarly, in Figure 4d (plants injected with 100 ppm AgNP suspension), the mean total Ag content in the leaves of plants injected with 100 ppm AgNP suspension was 13.5 ± 10.9 μg / kg (% m / m). Ag = 0.1%), while in the plants injected with 10 ppm AgNP suspension was 11.0 ± 7.5 μg / kg (% mass fraction of Ag = 3%) (Figure 4c) (P> 0, 05). This showed that increasing concentration of AgNP suspension did not substantially increase the total AgNP content in leaves. As seen in Figure 3c, the AgNPs transport does not occupy the entire xylem cylinder, which is probably the result of our single-point injection method. It is very likely that if the injection is extended to multiple points, the transportability of xylem to the NP may be increased. In addition, on day 7, Ag content in branches was higher (1584.0 ± 1459.5 μg / kg, mass% Ag = 8.5%) compared to day 1 (430.8 ± 94.9 μg). / kg, mass% Ag = 3.7%) (P <0.05), but Ag content in roots (205.4 ± 187.0 μg / kg,% weight Ag = 3.7%) low than in day 1 (673.4 ± 639.1 μg / kg, mass% of Ag = 5.2%) (P> 0.05) (Figure 4d). This showed again that AgNPs were continuously transported to the branches from the stem through the xylem vessels, and presumably excreted from the plant by roots.

To verify whether AgNP could actually be transported under the pholem layer (eventually to the base) in the stalked plants, we measured the Ag content in phloem-rich tissue (bark) and rich tissue. xylem (tissue remaining without bark) on stalk 7 days after injection

(from plants injected with 10 ppm AgNP suspension). Although this is a rough estimate (e.g. we cannot rule out the possibility that some xylem vessels are still sticking to the bark), it helps to illustrate

transports NPs through the plant’s various conductive systems. It was found that the mean Ag Content in xylem-rich and phloem-rich stem tissue was 757.0 ± 512.0 μg / kg and 300.5 ± 170.8 μg / kg (injection was carried out in xylem-rich tissue) ( P <0.05), with an Ag content increased from 154.2 ± 38.7 μg / kg at the top of phloem-rich stem tissue (top 10 cm of total Stem 20 cm long) to 446, 8 ± 19.3 μg / kg in the bottom part of phloem-rich stem tissue (less than 10 cm of total stem 20 cm long) (P <0.05). This suggests that AgNPs are continuously transported from the stem to the roots via phloem, 51.52 although we cannot rule out radial transport of NPs between xylem and phloem, due to damage to the maintenance vessels. during injection.

In an attempt to understand the distribution of silver nanoparticles in the root system, we extracted the roots from the root hairs, and measured their Ag content. For plants injected with AgNP 10 ppm suspension, the mean Ag concentrations in the main roots and root hairs were 34.4 ± 10.1 μg / kg and 55.5 ± 3.0 μg / kg, respectively (P < 0.05); For plants injected with 100 ppm AgNP suspension, the mean Ag concentrations in the main roots and root hairs were 219.2 ± 61.8 μg / kg and 49.5 ± 33.7 μg / kg, respectively (P <0.05). The high Ag content in the root hairs (usually higher in the leaves and some branches) suggests that phloem transfers AgNPs from the main root to the hairy roots, where AgNPs can be removed from the plant, in a process similar to sugar. plants, amino acids, organic acids, nucleotides and enzyme secretions. 106–108 While this conclusion is speculative, a recent study has shown that the yellow NPs are excreted by the wheat roots, when the NPs are applied via foliar fertilizers.

In our study, since direct phloem distribution was not possible, xylem was the primary target for NP distribution during injection. However, the presence of nano-silver in stems and roots implies the potential application of nanotechnology to control C Las growth. Because C Las bacteria, phloem-reside, move down the roots and multiply even though they enter the plant by eating the aerial tissues (leaves, branches) by insects. 109

Effect of plant structure on silver nanotransportation from stem to leaf

In this study, we found that the Ag content in different leaves from different branches was significant, and we hypothesized that the nano silver content was high (μg Ag / kg dry tissue) in the branches. This will lead to a high amount of nano silver in the leaves on this branch. To test this hypothesis, we have determined the correlation between dry leaf weight (g), weight Ag in leaves (μg Ag recovered), Ag content in leaves (μg Ag / kg dried leaves), mass Ag content in branches (μg Ag recovered), Ag content in branches (μg Ag / kg dry branches), weight of dry branches (g), length of branches (cm), and spacing of branches from injection point (cm) ) using Pearson’s correlation analysis. We found no significant correlation between “leaf dry weight” and “leaf Ag weight” or “leaf Ag content”, indicating that leaf growth did not significantly affect transport. AgNP into leaves (from biologic dilution was not the main cause of the decline in Ag content in leaves observed from plant studies). However, we found that the Ag content in leaves was positively correlated with the weight / Ag content in one branch (r> 0.34, p = 0.01) (Table 3). In addition, the Ag content in leaves was inversely correlated with the total length of branches (r = -0,336, p = 0.01), while the inverse correlation between leaf Ag weight and total branch length was not significant. meaning (Table 4). This implies that a higher AgNP content in one branch results in a higher nano silver content in the leaf, and the longer the branch, the lower the Ag weight in the leaf. That is to say that there is a physical process (ie passing through the pit membrane) that reduces the mobility of the NP when transported over longer distances. Furthermore, as can be seen from Table 4, the weight of nano silver in one arm is positively correlated with the dry weight of the branch (r = 0.277, p = 0.05), and can be inversely correlated with the total length of the branch. (though the correlation has no meaning). Therefore, it is likely that a large diameter branch facilitates the transport of NP from stem to leaf. In addition, as can be seen from Figure 3, since not all xylem vessels are used to transport AgNP, the branches that occur connected to the AgNP transporters are likely to have higher AgNP concentrations.

The total amount of Ag recovered in 14 tangerines clementine (receiving 10 or 100 ppm GA-AgNP suspension injections) studied in mass balance experiments ranged from 14.22% to 72.23% of the total. Ag weight is input (Figure S12). Interestingly, low recovery (<30%) is often related to a high root dry weight (> 60 g dry weight, for 2-3 year old clementine mandarin) or a ratio of upper root weight to the root. If the whole plant is tall (> 0.25), the possibility of a high recovery rate (> 45%) is related to a low dry weight of the roots (<60 g dry weight for 2-3 year citrus trees) or low root-to-plant weight ratio (<0.25). This reinforces our hypothesis that AgNPs can be excreted by citrus roots, with larger root systems capable of faster excretion.

Table 3. Pearson correlation coefficient (r) between Ag weight in leaves / branches (ug), Ag content in dry leaves / branches (μg / kg), dry weight of leaves and branches (g), total dimension spike length (cm) and spike distance from injection site (cm) (samples from tangerines with clementine received 10 or 100 ppm GA-AgNP injection)

Note: a, a branch or in some rare cases two adjacent branches on the trunk are grouped and leaves in the same group of branches are grouped in one sample; **, correlation was significant at p = 0.01. *, correlation is significant at p = 0.05 level

Monitor nano silver in leaf tissue

To confirm the presence of nano silver on leaves, the mid-rib area from a systemic leaf was collected from clementine mandarin 1 day after injection of 10 ml 100 ppm GA-AgNP suspension was isolated, dipped and microscopic scanning of transmission electron microscopy (STEM. 23 nm, matching the distance of the crystal plane (1,1,1) of the AgNPs (Fig. 5b). 110 Additional STEM / TEM displays AgNPs in leaf samples (xylem area, membrane or extracellular space is shown in Figure S13

Figure 5. (a) NP (darker region of TEM image) found in xylem of a system leaf collected 1 day after 10 ml injection of 100 ppm GA-AgNP suspension, and (b) spatial configuration lattice of the particle (mean lattice space calculated from regions 1, 2, 3 and 4, d = 0.23 nm).

To further explore the leaf-based silver nanoparticle transport pathways, a 20 cm branch from mandarin clementine was placed in a 100 ppm GA-AgNP suspension for 24 hours (in that way we could increase the AgNP content on the leaves in a way. significantly, helping us to further define that AgNP may have a foliar transport pathway through microscope images). A leaf on the tip is collected and the area between the ribs is isolated, dipped and microtomed. AgNPs have been identified

in the foil using a super-glass image, allowing us to compare the relative abundance of nano silver at different locations. It was found that there are more silver nanoparticles in the extracellular space than the intracellular space of palisade cells and the mesenchyma near the stomata region (St area, Fig. 6 a & b). However, superfocal imaging is a semi-quantitative method of analysis (as most microscopy methods are), and these results must be considered in this context. In addition, we AgNPs observed in both the extracellular and intracellular spaces of the bundle cell as in the intracellular space next to the phloem particles (areas Ph and Bs, Fig. 6 c & d and Figure S14 ). Interestingly, very little AgNP was found in xylem. Therefore, we speculate that AgNPs migrate the aplasia from the xylem to the stomata through the mesenchymal cells by evapotranspiration. Coincidentally, sugars assimilated from photosynthesis in the mesoderm are continuously diffused into the phloem, and these accumulation lines absorb water (osmosis) from the adjacent xylem into the phloem. 111,112 Therefore, NPs are equipped in phloem with water. 32 However, it must be admitted that the NP transport path determined using hyperspectral imaging analysis may not be able to adequately demonstrate NP migration in leaves from AgNP-injected citrus plants, since bile The NP in leaves from cut branches was much higher, and we did not consider the physiological response of leaves to its pruning.

Figure 6. Analysis of hyperspectral image of AgNP distribution in a microtomed between the ribs of a clementine mandarin leaf (AgNP detected shown in purple rectangles) obtained by dipping a 20 cm clade into the suspension GA-AgNP 100 ppm for 24 hours: (a) and (b), the area of ​​the stomata; (NS) and (d) the xylem / phloem / bundle sheath area. The different components of the leaf, epidermis, accessory stomata, spongy tissue, xylem, phloem, and sheath are marked as Ep, Sc, St, Xy, Ph, and Bs, respectively. The scale bar in (a) applies to all tables

CONCLUSION

In this study, we demonstrated that injection into the stem, out of four tested methods for silver nanoparticles delivery into plants (other delivery methods tested were foliar fertilization, branch injection, and wetting soil), can easily provide large amounts of nano silver to citrus. Post-distribution, Ct-AgNPs tend to stay in the trunk because of the rapid incorporation caused by the high salinity of the sap, while PVP- and GA-AgNPs can be distributed across the entire plant through both transport up and down due to the strong steel thrust due to the surface coating. We demonstrated that the root system could be an NP reservoir as the amount of AgNP found there, and in addition, the root hairs were capable of excreting NP from the plant. Regarding the transport of AgNPs from stems to leaves, short branches near the injection site tend to have high Ag content, resulting in high Ag concentrations in leaves on these branches.

Furthermore, on the leaf, a potential transport pathway of silver nanoparticles from xylem to phloem near stomata was identified. This study shows the potential of using NPs as perennial crop antimicrobial agents or genes.

METHOD

Material

Sodium citrate, polyvinylpyrolidone (PVP, M = 40000), Arabic gum (GA, M = 250000),

silver nitrate solution (0.1 M), sodium borohydride, MgSO4, NH4 NO3, KCl, CaCl2, MgCl2, NaCl, boric acid, Fe (NO3) 3, ZnSO4, CuCl2, KH2PO4, fumaric acid, malic acid, proline, sucrose, glucose, fructose, citric acid, quinic acid, asparagin, glutaraldehyde, nitric acid and hydrochloric acid were purchased from Sigma Aldrich and used without further modification. Embed 812 is purchased from Fisher.

Synthesize nano silver

Nano silver has been synthesized according to a previous study. 113 Summary, 10 ml of AgNO3 stock solution was added to 270 ml of nanopure water. Then, 10 ml of GA (10% weight), PVP (10% weight), or sodium citrate (10% by weight) was added, and the solution was stirred at 1000 rpm for 15 min. ice tank.

Then, add 10 ml of sodium borohydride solution (1% by weight), and continue stirring the mixture for an additional 1 hour (in an ice bath). Vacuum filtration (PS 35 membrane, Solecta, Oceanside, CAI; at 50 psi) was used to separate the NPs from the aqueous medium, followed by triple rinsing with nanopure water. Nano silver deposited on membranes are then collected and used to make a 1000 ppm Aqueous Solution, and stored in a refrigerator (4 ° C).

Plant materials

Two different citrus species are used to study silver nanoparticles in plants: (1) 2-3 year old Mexican lime (Citrus aurantifolia (Christm.) Swingle) grown in a greenhouse at UC Riverside in 2017, and ( 2) 2-3 year old clementine mandarin (C. clementina hort. Ex Tanaka) grafted onto Carrizo rootstock (C. sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.) Developed in the same UC Riverside greenhouse in spring (February 26 to March 20) and fall (September 11 to November 5) of 2018. All citrus plants are manually watered with tap water or fertilizer water (NPK 21: 5 : 20 blend). Greenhouse temperature control includes a 3-stage cooling system, with an exhaust to turn the fan on at 85ºC, followed by the blower and the cooler evaporates when it reaches 89ºC.

NP characteristics (synthesis and dissolution analysis)

2-3 year old Mexican lime was used for all nano silver transport and distribution studies. Leaves were collected on May 5, 2016 and May 12, 2017 (ten leaves from each tree into two copies), and are immediately sent to Cal GAP Inc. (NovaCropControl, Netherlands) for resin extraction and inorganic solute analysis. 114 Based on this analysis and a recent study, 37 synthetic citrus sap included 995 mM KCl, 90 mM CaCl2, 20 mM MgCl2, 5.0 mM NaNO3, 6 mM KH2PO 4, 65.9 mM sucrose, 20,5 mM glucose, 10,3 mM fructose, 55,1 mM malic acid, 28,2 mM citric acid, 68,0 mM proline and 16,6 mM asparagine in deionized water, with adjusted pH to 5.5 with 0.1 NaOH and 0.01M HCl. The inorganic sap solution contains only inorganic solutes, while the organic sap only contains organic solutes in synthetic sap. 10 ppm PVP-, GA-, Ct-AgNPs (prepared from AgNP stock suspension) were used for HD size and zeta potential measurements by ZetaPlus equipment (BrookHaven, US). In order to measure the zeta potential of AgNP in the synthetic resin accurately, we condition the electrode prior to measurement (more information can be found in SI). AgNP synthesis is continuously monitored through changes in HD mode measured every 30 seconds over a period of 10 minutes. Sedimentation tests were performed on UV-Vis spectrometry (Thermo Scientific EvolutionTM 350, US) by measuring the absorbance at 395 nm every 30 min over an 8 hour period. A solubility test was performed in a 12 ml glass vial with a PTFE cap (Fisher Scientific, US). 10 ml of synthetic inorganic sap, organic sap, or sap, containing 100 ppm AgNPs, was added to the jars. After 7 days, the Ag ion concentration in each vial was measured by ICP-MS (NexION 2000, PerkinElmer, with limit of detection, 0.2 ppb) after the vials were centrifuged at 25000 g for 60 minutes (SorvallTM , Thermo Scientific), filtered through a 0.22 μm polyvinylidene fluorine filter and acidified in 5% nitric acid (Control experiments with fresh Ct-, PVP-, and GA-AgNP in these aqueous environments confirmed that centrifugation and filtration can successfully separate AgNP from the medium).

AgNP uptake and transport in Mexican lime plants after foliar fertilization, soil wetting, and branch injection

Suspensions of 20 ppm (0.5 ml, 10 μg) and 100 ppm (0.5 ml, 50 μg) PVP-, GA- and Ct-AgNP were applied to Mexican lime trees through foliar fertilization, soil loosening and injection of branches branches (Figure S15 a). In the foliar application, three well-grown leaves (on the same branch, top of the tree, about 75 cm above the ground) were lightly grinded with a fine sandpaper, and a 0.17 ml solution was added to each leaves in the number of three leaves (total of 0.5 ml). In eroded soils, a tiny silver nanoparticle (3 cm below the base of the stem) was removed to expose the roots, then 0.5 ml of AgNP suspension was dripped onto the roots, then covered. come back. In branch feeding, the end of a branch (length: more than 35 cm; height:> 15 cm above ground) was cut and a 5 ml syringe was connected to the cut branch with a rubber tube and used silicone sealing tape. 0.5 ml of solution was added to the syringe, and through gravity, was allowed to be absorbed by the plant. On 1 day and 7 days. After exposure to NP, 6 leaves were collected for Ag analysis. Leaf samples were dried at 80 ° C for 48 hours. After dry weight measurement, leaf samples were burned (at 550 ° C), and the ash was collected and decomposed with aqua regia (at 110 ° C for 1 hour). Ag concentration is measured by ICP-MS. After 6 weeks, six foliar-exposed plants (2 plants for each of the silver nanoparticles) were sacrificed, separated into leaves (the remainder after sampling on Day 1 and Day 7), branches , trunk and roots. The Ag content in these tissues was analyzed, and the total amount of Ag in the plants was obtained.

Nano silver transportation in mandarin clementine after injection into the stem

Nano silver suspension was injected into the stem of the tangerine tree with clementine (～ 5 cm above the ground; whole syringe diameter, ～ 1 cm) using a pneumatic spray device (Figure S15 b). Injections are performed at a pressure of 20–80 psi. While the majority of injections are completed within 2 hours, two injections with GA-AgNP suspension require a maximum of 24 hours to complete; It’s not clear why some injections take longer. However, it is unlikely that the xylem blockage is the cause of the longer injection time, as Ct-AgNPs never take more than 2 hours to administer. Two rounds of experiments were conducted. Day 1, to examine the effect of the surface modification on silver nanotransmission, 10 ml 1000 ppm (10 mg Ag in total) The Ct-, PVP- and GA-AgNP suspensions were injected. to tree (three trees per tree of AgNPs modified type) (starting February 26, 2018). Three sites (near the injection site) and systemic 3 leaves (farthest from the injection site) were collected on days 1, 7, and 42 after injection. On day 42, plants had separated leaves, branches, stems, and roots for weight Ag was mass distribution and balance analysis. Second, to test the effect of AgNP concentration on NP transport in plants (after injection), 10 ml of GA-AgNP suspension (10 ppm and 100 ppm, 0.1 and 1 mg Ag total) were was injected into a total of 18 plants (three repeated injections of three plants at each AgNP concentration) (starting September 17, 2018). On day 1, day 3, and day 7 after injection, the plants had their leaves, branches, stems, and roots removed. In general, the stalk is cut into 3-5 pieces and 4-7 branches are sampled, noting the distance between the branch insertion point on the stem and the injection point. Leaves from the same branch were collected together. To verify the presence of nano-silver in phloem-rich tissue, we carefully peel off the tissue outside the cambium layer (mainly the bark and phloem as illustrated in Figure 3a), and the rest of the stem is flaking is xylem-rich tissue. In addition, we also separate the root hairs from the main roots to quantify the amount of Ag in the root hairs. All plant tissue was weighed after drying at 80 ° C for 48-72 hours (until weight stopped changing), and then burned at 550 ° C. Ash was recovered and consumed. Chemical with aqua regia (at 110 ° C for 1 hour). Ag concentration in ash digestion was measured by ICP-MS. To check the reliability of the analytical method, small 10 μl. A 100 ppm AgNP suspension was applied to three leaves, three stem segments and the roots itself (from plants not injected with AgNPs). After burning and decomposition of acid, we recovered from 95% to 98% of the total amount of Ag added, demonstrating the stability of the analysis.

Visualize GA-AgNPs in clementine tangerine branches and leaves

To confirm the transport of GA-AgNPs from injection point to branch, the short branches are removed at a distance of approximately 2 cm above the injection point and cut to a length of 1 cm using the Model 650 Low Speed ​​Diamond Ring Saw. (South Bay Technology, Inc., San Clemente, CA). Glass knife polished surface mounted on the RMC MT-X microtome (Boeckeler Instruments, Inc., Tucson, AZ). Electron microscopy and micro EDX analysis were performed on a Tescan Mira3 SEM (Tescan, Brno, Czech Republic) equipped with a Bruker Quantax EDS system (Bruker, Billerica, MA) at the Central Facility of Soi and Advanced Micro Analysis

(CFAMM) at the University of California at Riverside. To explore the transport of nano-silver from injection point to leaf, foliar systems were collected 1 day after injection of 100 ppm GA-AgNP suspension. A 2 × 3 cm leaf tissue sample (mid-rib near the petiole) was cut and fixed in 5% glutaraldehyde for 24 hours at 4 ° C. The sample was then washed three times with 0.1 mol phosphate buffer. / L pH = 7.2, and dehydrate with a graded series of acetone (10%, 30%, 50%, 70%, 90% and 100%). Then the tissue was dipped in epoxy resin (ETON 812). 115 The embedded samples were microtomed (Leica, US) into 200 nm slices with a diamond knife on a standard microscope slide (Fixed, embedded, and microtoming performed in the Department of Pathology and Laboratories, California school in Los Angeles). The slide was then sent to Duke University and photographed using a hypermarket microscope (CytoViva, Auburn, Al). After CytoViva analysis, a defined sample area containing AgNPs was selected and cut to 60-80 nm for both TEM (T12 Refrigeration Electron Microscopy, FEI Tecnai) and STEM analysis (Titan 80- 300 kV, FEI).

To investigate the transport of nano-silver between xylem and phloem near and around the stomata of the leaf openings, we used ultraclassic images to determine AgNP on the entire textured leaf. A branch 20 cm long (with 10 leaves) was cut and placed in 100 ppm aqueous suspension in GA-AgNP nanoparticles. After 24 h, a leaf is obtained at the tip of the branch or leaf blade. embedded and microtomed to 200 nm-thick sections, and shaped using hyperspectral

microscope. A reference spectral library comprised of 38 spectra crafted from a 5 mg / L GA-AgNP control sample in nano water (Figure S16), was allowed to settle on the glass surface overnight. Manual spectrum selection has been made to ensure a high-quality, representative spectrum while minimizing the probability of false positives. Suitability of the reference library for the treated and reference leaf samples was made using the spectral angle.

The method maps to a threshold of 0.25 rad, thus revealing the individual and group AgNPs in the samples. This resulted in a false positive rate in the control leaf samples of <0.005% by pixel count (~ 5 pixels / image), and a positive match> 75% of the particles in the AgNP control image.

Detection in nano-silver treated samples is considered positive when at least 0.5% of the pixels in the image have matched the spectral library with the same 0.25 rad threshold. Any spectra that consistently resulted in false positives was removed from the spectrum library during optimization. Additional image showing mapped pixels in negative processed and controlled tissues is provided in SI, Figure S17.

Statistical analysis

All data are presented as mean ± SD (standard deviation). We do one way

The ANOVA test plus Fisher’s LSD test for statistical analysis. p <0.05 is considered significant.

LINKS CONTENT

Support Information.

Support Information is available free of charge over the Internet at http://pubs.acs.org/

The following files are available for free. Derjaguin-Landau-Verwey-Overbeek (DLVO)

Description of the model; Estimated polymer layer thickness; Electrode conditioning for Zeta potential; Figure S1 TEM image of AgNPs with different surface transformations: (a) Ct, (b) PVP and (c) GA. (Scale bars at a, b and c: 100 nm; scale bars in figure inserted, 50 nm); Figure S2. The size distribution of AgNP with different surface changes in water DI (ac) and synthetic resin (df); Figure S3. Corresponding to three DLS measurements in synthetic resin (10 ppm AgNP, pH = 5.5): (a) GA-AgNP, (b) PVP-AgNP and (c) Ct-AgNP. (the dots in the red oval are signals of data collected by the instrument); Figure S4. Change average weight from temperature for each coated AgNP and bare AgNP; Figure S5. Electrophoresis of ceiling coatings and surface AgNPs; Figure S6. DLVO interaction energy profile between AgNP (PVP-AgNP, GA-AgNP and Ct-AgNP) and xylem / phloem surface; Figure S7. Classic DLVO and steric interaction energy between PVP-AgNP (a) / GA-AgNP (b) and the xylem / phloem surface; Figure S8. UV-Vis spectra of the primary and reactive AgNP (7 days) with different surface changes (20 ppm in theory): (a) Ct, (b) PVP, and (c) GA. (Inorg: inorganic component of synthetic resin; Org: synthetic resin organic ingredient; Mix: synthetic resin); Figure S9. Ag mass was recovered in leaves, stems and roots from different plants in foliar fertilization with PVP-, GA-, Ct-AgNP after six weeks (Mexican lime, 0.5 ml was exposed to AgNP 100 ppm); Figure S10. % of Ag weight in dried leaves, branches, stems, and roots, including total weight of Ag recovered from plants injected with GA-AgNPs 10 ppm (a) and 100 ppm (b); Figure S11. Relationship between the ratio of the Ag mass recovered (the amount of Ag recovered from the plant to the theoretical value of the mass of Ag injected) and the dry root mass (or the ratio of the weight of the dry root to the whole plant) (2.5-year-old green mandarin, 10 ml of AgNPs were injected, but 100 ppm GA-AgNPs suspension on Day 3 was unsuccessful and only 7 ml of suspension was injected within 2 hours); Figure S12. Relationship between the rate of Ag mass recovery (Ag mass recovered from all 2.5-year-old mandarin plants reaching the theoretical value of the weight of Ag injected) and the dry root weight (or weight ratio of dry roots compared to the whole plant); Figure S13. TEM image of cross section of mid rib (mainly xylem area): (a, b) suspected NPs in xylem (discontinuous wall thickness is spiral thickening of a vessel element young; (c, d) Suspected AgNPs (red oval) in the membrane or intercellular space; Figure S14. Analysis of CytoViva on nAg distribution in the middle of the leaf is microscopic (AgNP is detected are shown in circles / rectangles): (a) AgNPs in the sheath (1, extracellular) and phloem (2, intracellular), (b) AgNPs in sponge tissue (1, extracellular; 2 , intracellular)); Figure S15. (a) Feeding branches and (b) pricking tree trunks into citrus trees; Figure S16. (a) Analysis of CytoViva of the nAg (20 nm) suspension and (b) its spectral library; Figure S17. Superglass images (a, c and e) and corresponding dark field images (d, d and f) of AgNPs distribution in a mid-rib portion of a clementine mandarin leaf obtained by dipping a branch 20 cm long into a 100 ppm GA-AgNP suspension for 24 hours (g, negative control image). Table S1 Synthetic resin composition of Mexican lime.

Reference source:

Delivery, Fate, and Mobility of Silver Nanoparticles in Citrus Trees