The annual economic loss caused by plant viruses exceeds 10 billion dollars due to the lack of ideal control measures. Quercetin is a flavonol compound that exerts a control effect on plant virus diseases, but its poor solubility and stability limit the control efficiency. Fortunately, the development of nanopesticides has led to new ideas. In this study, 117 nm quercetin nanoliposomes with excellent stability were prepared from biomaterials, and few surfactants and stabilizers were added to optimize the formula. Nbhsp70er-1 and Nbhsp70c-A were found to be the target genes of quercetin, through abiotic and biotic stress, and the nanoliposomes improved the inhibitory effect at the gene and protein levels by 33.6% and 42%, respectively. Finally, the results of field experiment showed that the control efficiency was 38% higher than that of the conventional quercetin formulation and higher than those of other antiviral agents. This research is the first to report the combination of biological antiviral agents and nanotechnology to control plant virus diseases, and it significantly improved the control efficiency and reduced the use of traditional chemical pesticides.
Research
Field Application of Nanoliposomes Delivered Quercetin by Inhibiting Specific hsp70 Gene Expression Against Plant Virus Disease
https://doi.org/10.21203/rs.3.rs-956244/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 04 Jan, 2022
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Plant virus disease
Quercetin
Nanoliposomes
Field application
Plant virus diseases known as plant cancers, accounting for nearly half of all plant diseases [1, 3]. Plant viruses affect 118 genera and 1516 species in 26 families [4]. Plant viruses are unlike COVID-19 that can infect humans and other higher animals. However, once the main crops are infected by plant viruses, the yield and quality will be seriously affected [5, 6]. According to statistics, economic losses caused by tomato spotted wilt virus alone exceed $1 billion per year [7], while another two single-stranded DNA viruses, tomato yellow leaf curl virus and African cassava mosaic virus, cause $1.9-2.7 billion losses [8]. The most important plant virus worldwide, tobacco mosaic virus (TMV), cause plants to fail to grow normally because of leaf tissue necrosis [9, 10]. Research on TMV dates back to 1901, and in 1946, Stanley won the Nobel Prize in Chemistry for the elucidation of the molecular structure of TMV. Billions of dollars are spent each year to prevent and control plant virus diseases [11]. In crop production, the major precautions to prevent plant virus diseases are as follows: growth of a virus-free propagule [12], induction pf cross protection [13] and development of virus-resistant plants by editing genes1 and attenuated vaccines [14, 15]. However, once a plant is infected with viruses, antiviral agents, which can effectively reduce the impact of infextion on the growth of the host plants, become the most important protection measure [16, 17]. In recent years, due to their environmentally friendly natural, natural antiviral agents have been widely used in the control of plant virus diseases.
Quercetin (3,5,7-trihydroxy-2(3,4-dihydroxyphenyl)-4H-benzopyran-4-ktone) is a flavonol found widely in plant [18, 19]. It is widely used in the fields of food and medical treatment because of its antioxidant function [20, 21] and antitumor activity and its capacity to inhibit a variety of liver and influenza viruses [22]. Similarly, many studies have reported its inhibitory effect on various plant viruses [23], hence, based on its low cost, quercetin has considerable potential for control of plant virus diseases. For example, Wang found that 1 mol/L quercetin could effectively inhibit the proliferation of cucumber necrosis virus (CNV), turnip crinkle virus (TCV) and TMV in leaf cells of the model plant nicotiana benthamiana (Nb), and the inhibition rate to viral RNA was up to 90% [24]. As a planar molecule with compact stacking structure and strong intermolecular attraction, quercetin would not be dissolved by solvents [25]. Moreover, quercetin and its derivatives are prone to degrade under natural conditions [26], although quick degradation of active ingredients means an advantage for the environment and nontarget organisms, it reduces the control efficiency for plant virus [27]. Therefore, there is an urgent need to develop a carrier with excellent solubility and stability to deliver quercetin.
In recent years, nanotechnology has been actively utilized in various fields, especially in nanomedicine [28, 29], nanobiology [30, 31], etc. In particular, the active ingredients of hydrophobic drugs for the treatment of cancer have been encapsulated via nanotechnology: the biocompatibility of nanoparticle promoted the transport of foreign materials through the cell membrane, and by modifying their surface, the targeting ability of the delivery system will be enhanced [32]. In general, the majority of the active ingredients of traditional chemical pesticides are hydrophobic [33]. Similarly, encapsulating these ingredients of pesticides in nanoparticles is superior to traditional formulation: the dispersion and stability of active ingredients has effectively improved [34, 35]. Nanoparticles could also increase the coverage, adhesion, and penetration of the active ingredients [36].
Lipid, a general term for fats and lipids, belongs to the three categories of nutrients, with the advantages of being renewable and biodegradable, so they are an ideal delivery system for active ingredients [37]. More specifically, the hydroxyl groups in the lipid structure are lipophilic, while the ester chain are hydrophilic; such an amphiphilic structure endows lipids with the potential to be utilized in the delivery system of carrier material [38]. Nanoliposomes, consisting of lipid nanomaterials, are one of the potential options considered in the delivery system of pesticides. Because they have a larger specific surface area and greater solubility and bioavailability than traditional materials, nanoliposomes are regarded as a promising carrier material [39]. With lecithin working as an amphiphilic compound and cholesterol as a shape stabilizer, nanoliposomes have been prepared through ultrasonic homogenization technology by Bang to encapsulate etofenprox [40]. The size of liposome could be reduced to 150 nm by adjusting the proportion of lecithin. To date, however, the use of antiviral agents delivered via nano delivery system to control plant virus diseases has not been reported. This study attempts to develop a nanoliposomes biopesticide delivery system aimed at the control of plant virus diseases in which nanoliposomes are utilized as a carrier to encapsulate quercetin, the environment-friendly antiviral agent.
In this study, quercetin nano-liposome (H-TQ-NPs) was successfully made by thin-film ultrasonic method. This process selected quercetin, lecithin and cholesterol as materials, Tween 80 as the surfactant and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) as the stabilizer. The morphology, particle size, polydispersity index (PDI), zeta potential and encapsulation efficiency of H-TQ-NPs were determined. Then the structure changes of the liposome before and after modification were studied by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM), and the stability of H-TQ-NPs was studied under different conditions. In this study, with Nb-TMV as the research model, the mechanism of quercetin was also explored: the inhibition effect of free quercetin and H-TQ-NPs on TMV were compared at the gene and protein levels. Thus, the antiviral activity of H-TQ-NPs was verified at the individual level. Finally, the efficacy of two formulations of quercetin against TMV was compared in the field (Figure 1).
Materials.Anhydrous quercetin, Dimethyl sulfoxide (DMSO), methanol, trichloromethane and tween-80 were purchased from China National Pharmaceutical Group Co., Ltd. Phosphate buffer (PBS), cholesterol and lecithin were got from Beijing Solarbio Science & Technology Co., Ltd. Chitosan quaternary ammonium salt was purchased from Shanghai yuanye Bio-Technology Co., Ltd. An 8% Ningnanmycin soluble concentrate (8% NL) was purchased from Deqiang Biological Co., Ltd, a 5% Aminooligosaccharin soluble concentrate (5% AL) was purchased from Hainan Zhengye Zhongnong Hi-Tech Co., Ltd, and a 0.5% Lentinan soluble concentrate (0.5% LL) was purchased from Beijing Yanhua Yongle Biological Technology Co., Ltd.
Preparation and Characterization of H-TQ-NPs. Anhydrous quercetin (10 mg) was accurately weighed and then dissolved in a 10 mL methanol solution with magnetic stirring for completely dissolution (220 rpm); lecithin (100 mg) and cholesterol (20 mg) were dissolved in 20 ml trichloromethane and magnetic stirring (220 rpm). The methanol solution of quercetin was introduced into the trichloromethane solution, Tween 80 (0.2 mL) was added, and the solute was uniformly dispersed via magnetic stirring. The solution was dried by means of rotation evaporation; the appropriate amount of deionized water was added to dissolve the solute, and a light-yellow film was obtained. After 30 minutes of setting, an aqueous solution of H-TQ-NPs was obtained through ultrasonic dispersion. An HACC solution (0.5 mg/mL) was prepared and diluted, and then the HACC solution was mixed with the H-TQ-NPs solution in a volume ratio of 1:2. The microstructure of H-TQ-NPs was observed by TEM and its particle size, PDI, Zeta potential were determined.
Loading Efficiency of H-TQ-NPs. The loading efficiency of H-TQ-NPs was determined by indirect method [41]. Firstly, the prepared H-TQ-NPs solution was centrifuged at high speed. The concentration of free quercetin in the supernatant was determined by ultraviolet spectrophotometer at 371 nm. The loading efficiency was calculated according to the following formula (1):
Stability Test. The prepared H-TQ-NP solutions were placed separately at different temperatures, and samples were removed regularly to determine the zeta potential, particle size, PDI and quercetin concentration changes at 1 day and 20 days. Then, the pH value of the H-TQ-NPs was adjusted with newly prepared 1 M HCl and 1 M NaOH. The solutions were set for a period, and samples were removed regularly to measure the zeta potential, particle size, PDI and quercetin concentration changes.
Virus Inoculation. The upper new leaves of ordinary tobacco inoculated with TMV were ground with PBS buffer (0.01 M, pH 7.2–7.4) and filtered. The filtrate was centrifuged at 10000×g for 20 minutes, and the obtained supernatant was centrifuged at 10000×g for 30 minutes and then resuspended to precipitate with 0.01 mol/L PBS buffer (pH 7.2–7.4) to obtain a TMV inoculation solution [42].
A plasmid containing TMV-GFP was transferred into the agrobacterium GV3101 by the CaCl2-mediated freeze-thaw method. Agrobacterium cells were placed in LB solid medium (50 µg/ml kanamycin and 100 µg/ml rifampicin) and cultured at 28 ℃ for 48 hours. A single colony of agrobacterium was verified by PCR and then placed in LB liquid medium (50 µg/ml kanamycin and 100 µg/ml rifampicin) at 28 ℃ and 200 rpm until its OD600 reached 1.0–2.0. The OD600 of the cell solution was adjusted to 0.3–0.5 with an infiltrating solution (10 mM MgCl2, 1 mM 2-(N-morpholino) and 0.15 mM acetosyringone). Finally, the resuspended agrobacterium infiltrating solution was placed at 28°C for 3 hours to perform the inoculation experiment.
Nb tobacco blades at the 6- to 8-leaf stage were evenly sprinkled with 60-mesh-sieved quartz sand, and the obtained virus inoculation solution was evenly coated on the leaf surface with cotton swabs. Two hours after inoculation, the quartz sand on the surface of the leaves was rinsed with sterile water.
Abiotic Stress Treatment. After normal culture at 25 ℃, Nb was cultured in an incubator at 42 ℃ with artificial light for 2 hours. The test leaves and normal leaves were stored at -80 ℃, and the experiments were repeated three times. The relative expression of Nbhsp70 mRNA and hsp70 protein was determined.
Before being placed into the incubator at 42 ℃, five groups of Nb were treated as follows: sprayed with water (CK), sprayed with no-load nanoliposomes (K-NPs), sprayed with quercetin solution (QT), soaked in H-TQ-NP solution, and sprayed with H-TQ-NP solution. Three hours after the treatment, these five groups were placed in incubators at 42 ℃ with light for 2 hours. The leaves were stored at -80 ℃, and the experiments were repeated three times. The relative expression of Nbhsp70 mRNA and hsp70 protein was determined.
Pot Experiment. A certain amount of Nb inoculated with TMV or TMV-GFP was sprayed with H-TQ-NPs, CK, K-NPs, and QT twice at an interval of 12 hours, and the experiments were repeated three times. The distribution and quantity of TMV-GFP in Nb was observed regularly. After 60 hours of TMV inoculation, the leaves were collected and stored at -80 ℃, and the relative accumulation of TMV coat protein (TMV-CP) mRNA and the protein expression of TMV-CP were determined.
Field Experiment. Field experiments were carried out on May 21, 2020, in Huiping Green Tobacco Base, Mianning County, Xichang city, Liangshan Prefecture, Sichuan Province. The soil was loam soil with above-average fertility and good drainage and irrigation systems. Three commercially available antiviral agents were selected and used in the control group. A total of five treatment groups with identical concentrations were prepared: H-TQ-NPs, QT, 8% NL, 5% AL and 0.5% LL. The block arrangement was randomized four times. The plant spacing and row spacing were 50 cm and 110 cm, respectively. During the field experiment, the tobacco was in the root-extended period, which was the initial stage of disease. The incidence rate of plants was 2%, and the highest degree of disease was grade 0. The first pesticide application occurred on June 10, the second application was performed 7 days later, and the third application was performed on July 2 due to the weather.
The application method was spray application with a Xinxiu-16-type electric sprayer, whose working pressure was 3–4 kg/cm2 and spray volume was approximately 800 mL/min. The water consumption at three measured times was 225, 300, and 450 L/hm2. Five points were randomly sampled in each block, and 6 plants at each point were investigated. The total number of plants and the number of diseased plants at all levels were recorded. Disease progression was investigated before application, and the control efficiency was investigated 7 days after each application. Furthermore, the growth of each treated tobacco was observed to determine whether there was any drug injury.
Statistical Analysis. The least significant difference method (LSD) and Q test method in SPSS software were applied to analyze the significance of the differences in the data from the laboratory experiment. Duncan's new multiple range method (DMRT) was used to analyze the significance of the control effect in the field experiment. All graphical data are reported as the mean ± standard deviation (SD). Significance levels were set at * p < 0.05.
Preparation and Characterization of H-TQ-NPs. In this study, quercetin nanoliposomes were prepared by a thin-film ultrasonic method. The microstructure of the H-TQ-NPs was observed by TEM. The sample that was not loaded with quercetin failed to form nanoliposomes. As shown in Figure 2A, the quercetin nanoliposomes (Q-NPs), the simplest liposome obtained by rotatory evaporation, were multicompartment liposomes with uneven particles with sizes of 100–350 nm, and their double membrane structure was clearly observed. After adding HACC, a small amount of irregular nanoparticles was formed (Figure 2B), and no double-layer film structure was observed. In the image of the TQ-NPs (Figure 2C), the liposome aggregate disappeared, and the nanoliposomes were distributed in a single chamber, with a stable bilayer structure and a reduced particle size. In the H-TQ-NP sample group (Figure 2D), the nanoparticles were uniformly dispersed, and the nanoliposome size in the field of vision was mostly approximately 100 nm. It was concluded that the addition of HACC and Tween 80 was of great significance for stabilizing the nanoparticle structures. The Q-NP solution was colorless and turbid. The solution appeared yellow after the addition of HACC, and the transparency of the solution decreased after the addition of Tween 80. The final product (H-TQ-NPs) was a light-yellow turbid solution (Figure 2E). Figure 2F shows the Tyndall phenomenon of the Q-NP, TQ-NP and H-TQ-NP aqueous solutions. In the Q-NP solution, which was cloudy, the reflection and refraction of light were strong, and the light beam was not obvious. No light scattering occurred in the TQ-NP and H-TQ-NP solutions, and the light beam was clear.
Stability Test. The stability of the H-TQ-NPs was quantitatively characterized by measuring the changes in the zeta potential (Figure 3A), particle size (Figure 3B), PDI (Figure 3C) and quercetin concentration (Figure 3D) in each treatment group within 20 days. First, the zeta potentials of the newly prepared Q-NPs, TQ-NPs and H-TQ-NPs were -48.5 mV, -42.4 mV and +43.5 mV, respectively, and their absolute values were all greater than 30 mV, suggesting that the particles were stably dispersed in the medium. After 20 days of storage, the absolute value of the zeta potentials of the Q-NPs and TQ-NPs decreased, but that of the H-TQ-NPs supplemented with HACC remained stable, and the absolute value remained greater than 30 mV. Figure 3B shows the particle size changes of each treatment group. The average particle sizes of the newly prepared Q-NPs, TQ-NPs and H-TQ-NPs were 273±10.39 nm, 318.67±12.03 nm, and 117.50±7.11 nm, respectively. After storage for 20 days, the average particle size of the Q-NPs increased to approximately 500 nm, and there was no significant change in the sizes of the TQ-NPs and H-TQ-NPs, which indicated the good stability of the H-TQ-NPs. The changes in PDI of each treatment group are listed in Figure 3C. The PDI of the newly prepared Q-NPs, TQ-NPs and H-TQ-NPs were 0.48, 0.29 and 0.31, respectively, and changed to 0.55, 0.28 and 0.32, respectively, after 20 days of storage; this lack of a significant change in PDI in each group indicated that the particle distribution of the TQ-NPs and H-TQ-NPs was concentrated and remained stable during long-term storage. Finally, the changes in the concentrations of active ingredients in the system were measured to determine the protective effects of liposomes on quercetin. As shown in Figure 3D, the quercetin concentrations in the Q-NPs, TQ-NPs and H-TQ-NPs were 43.49±2.85 mg/L, 33.38 ±1.99 mg/L and 31.76 ±0.67 mg/L, respectively. After 20 days, the contents of the active ingredients in all systems decreased. The highest degree of active ingredient degradation occurred in the TQ-NP treatment group, with an active ingredient content of only 30%; the content in the Q-NP treatment group decreased by 50%; and the H-TQ-NP treatment group had the highest retention of active ingredients, which was more than 70%. The results showed that the H-TQ-NPs prepared in this study could effectively maintain the structural stability of quercetin and reduce its degradation, which was of great significance for improving the stability of quercetin.
Figures 3E and 3F show the appearance of each treated aqueous solution on day 1 and day 20, namely, K-NPs, Q-NPs, HACC nanoliposome solution (H-NPs), HACC quercetin nanoliposomes (H-Q-NPs), Tween 80 nanoliposomes (T-NPs), Tween 80 quercetin nanoliposomes (TQ-NPs), HACC Tween 80 solution, and H-TQ-NPs from left to right. The H-TQ-NP solution was clear and transparent with no precipitation, while flocculent precipitates appeared in the H-Q-NP aqueous solution, which was pale yellow. After 20 days of storage, the appearance of the solutions remained basically unchanged: in the H-Q-NP treatment group, the solutes aggregated into large particles; large-particle flocculent precipitates also appeared in the Q-NP and TQ-NP treatment groups; and the H-TQ-NP solution was stable and remained homogeneous and transparent without precipitation.
Loading Quercetin. The embedding of quercetin was further verified by FTIR analysis of the different treatment groups. In Figure 4, the characteristic absorption of the sample covered the entire region from 500–4000 cm−1. In the range of 500–2000 cm−1, the peaks at 1105 cm−1 and 1739 cm−1 corresponded to the stretching vibration of aromatic hydrocarbons, and the peak at 1546 cm−1 corresponded to the stretching vibration of -NH in the amino group. These peaks demonstrated that chitosan had successfully attached to the surface of the liposomes because these groups are characteristic structures of chitosan. The characteristic peak of the stretching vibration of the phenolic hydroxyl group (the characteristic group in the quercetin structure) disappeared in the range of 1200–1400 cm−1 compared with that of the original quercetin, which showed that quercetin was successfully encapsulated into the nanoparticles through hydrophobic interactions or hydrogen bonding.
The UV standard curve of quercetin in methanol appeared linear in the range of 0–0.06 mg/mL, and the regression equation was y=34.429x+0.074, R2=0.9989 (Figure S1). The concentration of free quercetin in the supernatant was determined to quantify the loading efficiency of the nanoliposomes. The loading efficiency of the TQ-NPs was 82.26%±0.30%, and that of the H-TQ-NPs with the addition of quaternary ammonium salt of chitosan was as high as 88.02%±0.10%.
Research on the Optimal Formula. With the addition of HACC, the stability and loading efficiency of H-TQ-NPs were both improved, and the amount of HACC added was further screened to optimize the formula. Different concentrations of HACC influenced the zeta potential (Figure 5A) and PDI (Figure 5C): with increasing HACC concentration, the absolute values of the zeta potential and PDI both showed an increasing trend; the addition of HACC enhanced the dispersion of the nanoparticles and expanded their particle size range (Figure 5B). The smallest particle size was observed when the HACC concentration was 0.2 g/L, and then it increased as the HACC concentration increased. After 20 days, the quercetin content in the 0.3 g/L treatment group decreased the least (Figure 5D). Based on the comprehensive results, the optimal formula contained 0.3 g/L HACC.
The Performance under Different Conditions. To study the performance of the H-TQ-NPs under different conditions, a series of pH, temperature and light conditions were set. In Figure 6, the color of the solution gradually deepened with increasing pH. The solution exhibited slight turbidity when the pH exceeded 6.0, which became more obvious as the pH continued to increase. The zeta potential, particle size, PDI and concentration of active ingredients at different pH values were measured. As shown in Figure 7A, with increasing pH value, the zeta potential exhibited a downward trend. Although the zeta potential decreased slightly, its absolute value was over 30 mV, regardless of whether it had been stored for 1 day or 20 days. After the pH increased, the particle size of the H-TQ-NPs gradually increased (Figure 7B), especially when the pH was greater than 6.5. Unlike the particle size, the PDI gradually decreased as the pH increased (Figure 7C). When the pH exceeded 5.0, the PDI tended to stabilize. After storage for 20 days, the concentration of quercetin in all groups decreased (Figure 7D), and the decrease was most obvious at pH 4.0. At pH 7.0, the amount of quercetin degradation was the least. In conclusion, the H-TQ-NPs had good stability in the pH range of 5.0–7.0, and at pH 6.0, the H-TQ-NPs were the most stable.
Next, the influence of different temperatures and light conditions on the H-TQ-NPs was explored (Table 1, Figure 8). First, the degradation results showed that light could significantly reduce the content of quercetin, some of which was degraded. Therefore, a low temperature without light was more suitable for the preservation of the H-TQ-NPs (Figure 8A). With increasing temperature, the zeta potential, particle size and PDI of the H-TQ-NP solution all decreased, which might be due to the influence of increasing temperature on the loading capacity of HACC. Additionally, the light conditions effectively reduced the particle size of the H-TQ-NPs, while zeta potential and PDI remained in a good state. Temperature and illumination had little influence on the appearance of the H-TQ-NP solution. There was no obvious change in the H-TQ-NPS solutions with different treatments, and the solution remained clear and transparent, without any precipitation (Figure 8B, C).
| Sunlight, 20 ℃ | Dark, 4 ℃ | Dark, 20 ℃ | Dark, 30 ℃ | Dark, 40 ℃ | |
|---|---|---|---|---|---|
| Zeta Potential (mV) | 34.63±0.81 | 30.72±0.37 | 28.64±1.68 | 29.94±0.28 | 28.71±1.43 |
| Particle Size (nm) | 110.35±2.44 | 116.85±2.37 | 124.82±2.71 | 125.62±1.81 | 127.19±3.64 |
| PDI | 0.45±0.03 | 0.46±0.04 | 0.39±0.03 | 0.41±0.05 | 0.39±0.02 |
Mechanism of Control TMV. The function of the plant hsp70 protein is similar to that of the animal hsp70 protein, which is highly expressed in response to stress. Previous studies have shown that Nbhsp70 plays a role in the process of virus inoculation in plants. Figure 9A shows that after heat treatment, the relative expression levels of Nbhsp70cp-1 and Nbhsp70c-A in the plants increased by 650 times and 16 times, respectively. The results of gray analysis of the protein quantification showed that the relative expression levels of the Nbhsp70 protein increased by 2.6 times after heat treatment (Figure 9B). The relative expression level of Nbhsp70 in Nb was measured after treatment with different concentrations of quercetin before heat treatment. The results are shown in Figure 9C. Compared with that int eh CK group, the relative expression level of Nbhsp70cp-1 in the H-TQ-NP-soaked group decreased by 74%, and that in the sprayed group decreased by 39%, which indicated that under the two application methods, quercetin could effectively inhibit the expression of Nbhsp70, and this effect was more obvious in the soaked group. The individual plant test also showed that the plants were scorched and shrunken after heat treatment, and these symptoms could be significantly reduced when the leaves were soaked and sprayed with the H-TQ-NPs (Figure 9D). Second, the symptoms of the leaves after QT treatment were also relieved but not as obviously as those of the H-TQ-NP treatment group. The leaves after K-NP treatment still showed symptoms of shrinkage and scorching, and there was no therapeutic effect.
After inoculation with TMV, the heart leaves of the Nb underwent obvious shrinkage and curling; the plant grew slowly, and the leaf margin of the whole plant rolled down. Five days later, the tender stem at the growth point of the heart leaves tipped to one side (Figure 10A). The results of western blot analysis indicated that TMV-CP protein accumulation gradually increased after inoculation, and the protein expression of Nbhsp70 also increased significantly with time (Figure 10B, 10C). In the first 2 days of inoculation, the expression levels of TMV-CP protein and Nbhsp70 protein were basically the same, and after 2 days, the levels increased, with the expression level of TMV-CP protein increasing more significantly. After that, the relative accumulation of TMV-CP in the inoculated leaves was detected by qRT-PCR. At 36 hours after inoculation, the relative accumulation of TMV-CP was upregulated more than one hundred-fold and increased by more than 15,000 times after 96 hours (Figure 10D). After TMV inoculation for 72 hours, the relative expression levels of Nbhsp70er-1 and Nbhsp70c-A were both increased by approximately 2-fold (Figure 10E). After 96 hours, the expression of Nbhsp70er-1 still showed the most significant change, with an increase of nearly 20 times. In addition, the relative gene expression levels of Nbhsp70 and Nbhsp70cp-1 increased by approximately 4-fold.
Consistent with abiotic stress treatment, TMV inoculation also resulted in upregulated expression of Nbhsp70cp-1 and Nbhsp70c-A, which means that Nbhsp70cp-1 and Nbhsp70c-A were the response factors of the host plants during TMV inoculation. Then, the plants inoculated with TMV were treated with quercetin, and the changes in the relative expression levels of Nbhsp70er-1 and Nbhsp70c-A were determined by qRT-PCR. The relative expression levels of Nbhsp70er-1 and Nbhsp70c-A after quercetin treatment were reduced by 49% and 72%, respectively, compared with those in the CK group (Figure 11B). These results demonstrated that the upregulation of Nbhsp70er-1 and Nbhsp70c-A was inhibited by quercetin treatment. The proliferation of TMV was inhibited by downregulating the expression of the two genes, thus achieving control of TMV. Therefore, increasing the transport and penetration efficiency of quercetin in plants is of great significance for improving the control efficiency.
Pot Experiment. Nanotechnology was applied to encapsulate the active ingredients in nanodelivery systems. The nanometer size not only improved the solubility of the active ingredients but also enhanced the transport and penetration efficiency. To determine whether the H-TQ-NPs prepared in this study could improve the efficiency of quercetin in controlling plant viral diseases, gene, protein, and individual plant tests were carried out. First, the expression levels of Nbhsp70er-1 and Nbhsp70c-A in the inoculated tobacco plant treated with different formulations of quercetin were decreased, while the relative expression levels of the two genes in the unloaded quercetin liposome group were consistent with those of the control group, which did not show an inhibitory effect. Compared with that of the free quercetin treatment, the inhibition effect of the H-TQ-NPs on Nbhsp70er-1 was 40.8% higher, and the inhibition effect on Nbhsp70c-A was increased by 26.4% (Figure 11B). The H-TQ-NPs could effectively improve the inhibitory effect of the active ingredients on the two target genes to inhibit TMV reproduction. Because the relative expression of Nbhsp70er-1 and Nbhsp70c-A was inhibited, the replication process of TMV in plants was also inhibited. The inhibitory effect of the nanopreparations on TMV was further compared by measuring the accumulation of TMV-CP in inoculated plants. After treatment for 72 hours, the relative expression of TMV-CP mRNA in the free quercetin and H-TQ-NPS groups was reduced by 83% and 93%, respectively (Figure 11C). Compared with that in the control group, the accumulation of TMV-CP was better inhibited after treatment with the H-TQ-NPs. Then, the protein accumulation of TMV-CP was analyzed. Although free quercetin treatment also downregulated the relative expression of Nbhsp70er-1 and Nbhsp70c-A and reduced the accumulation of TMV-CP, the relative accumulation of TMV-CP protein in the free quercetin treatment group was not significantly different from that in the control group. However, the relative accumulation of TMV-CP protein in the H-TQ-NP treatment group was 42% less than that in the control group, which indicated the inhibitory role of H-TQ-NPs on TMV at the protein level (Figure 11D, E).
To study the effects of quercetin on the proliferation of TMV more intuitively, Nb was inoculated with TMV-GFP, which exhibited green fluorescence, and then water, K-NPs, free quercetin and H-TQ-NPs were sprayed on the leaves. The quantity and distribution of TMV-GFP were observed under ultraviolet light. After TMV-GFP inoculation for 5 days (Figure 11F), the distribution of TMV-GFP green fluorescence in the inoculated leaves under UV light was poor after H-TQ-NP treatment, followed by free quercetin treatment, which indicated that quercetin could inhibit the proliferation of TMV-GFP at the initial site of virus inoculation, and the inhibitory effect of quercetin nanoliposomes was higher than that of free quercetin. Furthermore, the fluorescence accumulation of TMV-GFP in the K-NP group without quercetin was similar to that in the control group, showing no inhibitory effect on the proliferation of TMV-GFP. After 8 days (Figure 11G), the TMV-GFP distribution was dense in the control group, with high green fluorescence intensity and severe leaf deformity in comparison with those in the quercetin-treated group, where the TMV-GFP distribution was relatively low, the green fluorescence was sparse, and the leaf deformity was mild. The TMV-GFP distribution was the lowest in the H-TQ-NP-treated tobacco leaves. These results intuitively indicated that the H-TQ-NPs effectively reduced the initial proliferation of TMV-GFP in inoculated leaves, thereby reducing the amount of TMV-GFP and delaying the onset.
Field Experiment. Finally, the control efficiency of each formulation of quercetin against TMV was researched in the field (Figure 12); its safety on tobacco plants compared with that of common antiviral agents was verified: from the beginning of the experiment to the tobacco harvest, no phytotoxicity phenomena were observed; the growth of each plant was normal, and the treatment was safe for the growth of tobacco within the range of the test dose. At the same time, the field experiment results showed that all five formulations could play a certain role in controlling TMV. Among them, H-TQ-NPs showed the best control efficiency, with an average rate of 68.08%, which was significantly superior to those of the other three control formulations, namely, 5% for AL, 8% for NL and 0.5% for LL, with average control efficiencies of 59.52%, 57.91% and 57.31%, respectively. The lowest control rate was observed for the quercetin conventional formulation, with a value of 49.55%.
Recently, nanotechnology has become increasingly widely used in agriculture, especially in the production of crops; nanocarriers can be used to overcome environmental and biological obstacles to agricultural production and efficiently deliver active ingredients to destinations to improve the control efficiency on target organisms [43]. At present, what is used for delivery is not limited to traditional pesticides but also includes double-stranded RNA, viral DNA, Bt toxin, and so on to protect crop production [44–46]. Previous studies have shown that the application of quercetin for plant protection is associated with favorable antibacterial and antiviral activities. Therefore, nanoliposomes were used as carriers to deliver quercetin. There have been many studies using nanoliposomes as carriers for the delivery of herbicides, insecticides and other traditional pesticides [47]. In this study, the biomaterials lecithin and cholesterol with low cost and high compatibility were used as carriers; the production cost of the carrier was only US$ 0.5/g. The nanoliposomes were prepared by the thin-film ultrasonic method, and in the final stage of preparation, the organic solvent was removed by rotary evaporation to obtain the product, which not only further reduced the production costs but also eliminated the adverse effects of organic solvents on the environment during applications. Therefore, cost and environmental factors, the nanoliposomes prepared in this study were suitable for the delivery of active ingredients in the field considering. There have been no reports on the use of nanocarriers to deliver plant antiviral agents. The quercetin nanoliposomes prepared in this study for the control of plant virus diseases were investigated for the first time, and their optimal preparation conditions and storage environment were evaluated.
Lecithin and cholesterol were chosen to reduce the allergic reaction of plants to foreign and nonplant substances [48]. The initial embedding of quercetin was achieved as a result of the amphiphilicity of the phospholipid group, and the addition of Tween 80 helped to improve the interface stability of the liposomes and increase the amount of nanoliposomes attached to the surface of plant leaves. The permanent positive charge of the quaternary ammonium group in the HACC could increase the electrostatic interaction in the lecithin emulsions and the electrostatic repulsion in the droplets, thereby avoiding the aggregation of liposomes [49]. The H-TQ-NP solution was transparent without precipitation after the addition of HACC and Tween 80, the nanoparticles in the TEM image were uniformly dispersed, and the Tyndall effect was the most obvious for the H-TQ-NPs. A radius of dispersed particles less than 100 nm was less than the wavelength of the incident light, resulting in scattering and allowing the light column to be clearly seen. The particle size, zeta potential and PDI also confirmed this phenomenon, and the particle size was significantly reduced after adding HACC and Tween 80. The earlier definition of nanoagricultural products refers to agricultural products whose half particle size is less than 100 nm, and the particle size of the nanoliposomes prepared in this study was controlled at 100 nm, which met the standards for nanoagricultural products. Many properties, including water solubility, permeability, adhesion and so on, could be improved by preparing traditional chemical pesticides as nanoscale particles [50]. At the same time, the zeta potential of the nanoliposomes exceeded 50 mV, which meant that the particles had good dispersibility. PDI is an indicator for evaluating the uniformity of the particle size distribution, and the smaller the value is, the higher the concentration of the particle size distribution. The PDI was reduced by 35.4% compared to that of the Q-NPs; the particle size distribution was more concentrated, and there were basically no particles of other sizes generated. A more stable structure was formed to wrap quercetin, consistent with the TEM images, to effectively protect quercetin.
The results of the stability test showed that H-TQ-NPs had excellent stability, and there was no significant change in the zeta potential, particle size, or PDI after 20 days of storage. The best preparation conditions for the H-TQ-NPs at different concentrations of HACC and different pH values were explored, and the H-TQ-NPs prepared under the conditions of 0.3 g/L HACC and pH 6.0 had the best performance. The FTIR results showed that HACC was successfully attached to the surface of the H-TQ-NPs, and the positive charge carried by HACC increased the electrostatic repulsion between the nanoparticles. However, the addition of excessive HACC increased the number of groups attached to the surface and the particle size of the nanoliposomes. The uneven increase in particle size decreased the concentration of the particle size distribution, and the PDI value fluctuated; therefore, adding 0.3 mg/mL HACC led to the most stable performance. Since the surface of the H-TQ-NPs had several positive charges, the H-TQ-NPs were appropriately stabilized under weak acid conditions. Under alkaline conditions, the increase in anions reduced the positive repulsion on the surface and further reduced the stability and dispersibility of the nanoliposomes. Therefore, the solution could be adjusted to be weakly acidic during the preparation and storage of the H-TQ-NPs. Finally, to clarify the storage conditions of the H-TQ-NPs, the influence of different temperature and light conditions on the H-TQ-NPs was researched. The H-TQ-NPs had excellent stability under low temperature and dark conditions. The release rate was decreased because the activity of the nanoparticles was reduced at low temperature in the dark. At the same time, the quercetin released at low temperature in the dark degraded slowly, so the stability was better.
Quercetin could effectively reduce the number of viruses in the host plant, but studies have also shown that quercetin did not directly affect virus replication and did not induce host resistance [51, 52]. As a heat shock protein, the hsp70 protein is expressed in large quantities during biotic and abiotic stress and participates in protein assembly, transportation and degradation [53, 54]. The distribution of the hsp70 gene in the nucleus, cytoplasm, mitochondria, chloroplasts and endoplasmic reticulum has long been reported [55]. The viral replication complex is assembled from viral proteins and viral genomic RNA and necessary host factors [56], and it preferentially accumulates in the membrane structure of organelles such as the endoplasmic reticulum and chloroplasts. TMV forms a viral replication complex on the endoplasmic reticulum and may recruit Nbhsp70er-1 to participate in the folding of viral proteins. Under heat stress, the gene and protein expression of Nbhsp70cp-1 and Nbhsp70c-A in Nb was upregulated, and quercetin treatment inhibited Nbhsp70cp-1 and Nbhsp70c-A expression. Therefore, we speculated that the mechanism of quercetin against TMV also involved inhibiting the expression of the hsp70 gene. After Nb inoculation with TMV, the plants grew slowly, the leaves shrank and curled, the leaf edge curled down, the accumulation of TMV-CP and TMV-CP increased, and the relative expression of Nbhsp70er-1 and Nbhsp70c-A was upregulated. The treatment of inoculated plants with quercetin validated our conjecture that quercetin inhibited viral activity by downregulating the relative expression of Nbhsp70er-1 and Nbhsp70c-A.
The inhibition mechanism of quercetin was clarified, and it was considered that the low water solubility of quercetin was an important factor limiting the control effect. Based on the nanoscale particle size and amphiphilic nature of liposomes, the carrier could efficiently deliver quercetin to the inside of cells through osmosis or endocytosis. Research has shown that chitosan-coated liposomes were Higuchi models [57], indicating that the H-TQ-NPs prepared in this study permeated into the cell through penetration. The nanoparticles were destroyed by lipase in the cell, and quercetin could be released to reach the organelles. As expected, compared with free quercetin, the H-TQ-NPs increased the inhibitory effect of quercetin on Nbhsp70er-1 and Nbhsp70c-A, thereby inhibiting the replication of TMV and reducing the accumulation of TMV-CP mRNA and TMV-CP. Finally, the incidence of intact plants inoculated with TMV under laboratory conditions was significantly reduced, and the expression and distribution of fluorescent TMV-GFP was reduced. The field experiment also proved that the development of quercetin into nanoparticles had a better control effect than free quercetin. The nanoliposomes significantly improved the solubility of quercetin, which enabled efficient transport into plants and entrance into cells through penetration, thus inhibiting the expression of hsp70 protein and ultimately improving the prevention of TMV.
The preparation of high-efficiency and environmentally friendly antiviral agents to control plant virus diseases is of great significance to agricultural production and development. As a plant-derived agricultural antiviral agent, quercetin is widely used for plant protection. In previous studies, we verified that quercetin could be used for the control of TMV, but it had a short duration and was easy to degrade; the inhibitory effect on TMV was less than 7 days, so quercetin could be delivered into cells to exert its effect in a short time. However, the H-TQ-NPs prepared in this study significantly improved the transportation and delivery efficiency of quercetin, making the control efficiency on intact plants in the laboratory or in the field environment higher than that of free quercetin. In the future, H-TQ-NPs will continue to be used on a large scale in the field, and the safety of nanoliposomes should also be considered. The toxicity of lecithin and cholesterol in the plant could be ignored. This was also confirmed by the fact that there was no phytotoxicity in the field environment. Therefore, quercetin nanoliposomes could be considered a potential nano biopesticide to improve the control efficiency of antiviral agents.
In summary, a nanoliposome carrier was constructed using lecithin and cholesterol as materials and encapsulated the hydrophobic antiviral agent quercetin based on the amphiphilicity of the phospholipid group. The particle size of the quercetin nanoliposomes was controlled at approximately 110 nm, the absolute value of the zeta potential exceeded 30 mV, the PDI was less than 0.3, and the nanoliposomes showed good stability even after 20 days of storage. Quercetin downregulated the expression of hsp70 protein by inhibiting Nbhsp70er-1 and Nbhsp70c-A. hsp70 is a key protein involved in virus replication, and the replication of TMV was influenced, thereby achieving control of TMV. Because of their nanoscale particle size and hydrophilicity, nanoliposomes can efficiently transport quercetin in plants, penetrate the cell membrane through osmosis, and reach the vicinity of the organelle where the target gene exists. Therefore, quercetin nanoliposomes could effectively improve the inhibitory activity of quercetin on TMV. Under indoor conditions, the inhibition rates of quercetin nanoliposomes on the two main target genes increased by 40.8% and 26.4%, and the accumulation of TMV-CP decreased by 42.1%. Compared with that of free quercetin, the control effect was increased by 37.4% in the field environment. This was the first time that a nanodelivery system has been used for the control of plant virus diseases. The application of quercetin nanoliposomes was beneficial to reduce the use of conventional pesticides, and it has broad potential in the control of plant virus diseases.
Supplementary information
Additional file 1: Material in qRT-PCR, western and field application.
Additional file 2: Quantitative detection of NbHsp70 gene and TMV-CP gene.
Additional file 3: Table S1. Primer pairs used to detect gene RNA accumulation.
Additional file 4: Quantitative detection of Hsp70 protein and TMV-CP protein.
Additional file 5: Methods of disease grading.
Additional file 6: Figure S1. The standard curve of quercetin.
Authors’ contributions
Wang Jie, Yang Jinguang and Wang Fenglong contributed to conception. Su Chenyu, Hao Kaiqiang and Liu Fangfei contributed to study design. Shen Lili contributed to data acquisition. Su Chenyu Su and Wang jie contributed to original writing. Wang Fenglong, Yang Jinguang contributed to review and editing. All authors read and approved the final manuscript.
Funding
The authors are grateful for the financial support from Shandong Provincial Natural Science Foundation, China (No. ZR2019BC031), Science and Technology project of Guangxi (No.2020450000340001), Sichuan (No. SCYC202008), Green protection and control technology key project (110201901041(LS-04); 110202001033(LS-02)).
Availability of data and materials
All data generated or analyzed during this are included in this published article.
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable.
Competing interests
All authors declare that they have no competing interests.
Acknowledgements
The authors acknowledge the following people for their contributions: Liu Shanshan, Ge Ming.
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- Supportinginformation.docx
Supporting Information