Spontaneously Formed Ratio-Tunable Micro- and Nano-Capsule Coexist System for Precision and on-Demand Fungicide Delivery in Crop Leave
自发形成比例可调的微胶囊和纳米胶囊共存系统，用于作物叶子中的精确和按需杀菌剂输送
Abstract 抽象的
Multiscale particle size functional pesticide carriers can provide more efficient protection for plants, but this protection is difficult to achieve via single-scale formulation technology. This study presents a novel one-step method for the preparation of lignin-based micro/nanocapsules with controllable proportions within a unified system. This strategy enables the adjustment of the proportion of nanocapsules to between 18.81% and 85.21%. The microcapsules (MCs) vary in diameter from 2 to 3 µm, whereas the nanocapsules (NCs) span from 160 to 220 nm, with an encapsulation efficiency exceeding 90%. An increased proportion of NCs in the system leads to faster release, heightened sensitivity to UV light, and enhanced penetration into the leaves. During Phytophthora capsici (P. capsici) infection, the NCs in the leaves interact with the defensive enzymes of the plant to quickly respond. Moreover, an optimal balance of MCs and NCs is key to effective fungicide use, not just a higher concentration of NCs. A 65:35 ratio of NCs to MCs ensures effective inhibition of P. capsici outside leaves and a rapid response to leaf invasion. This study enhances fungicide efficiency and advances the development of nanoresponsive fungicides to promote sustainable agricultural practices.
多尺度粒径的功能性农药载体可以为植物提供更高效的保护，但这种保护很难通过单一尺度的制剂技术实现。这项研究提出了一种新颖的一步法，用于在统一系统内制备比例可控的木质素基微/纳米胶囊。该策略使得纳米胶囊的比例能够调整到18.81%至85.21%之间。微胶囊 (MC) 的直径范围为 2 至 3 µm，而纳米胶囊 (NC) 的直径范围为 160 至 220 nm，封装效率超过 90%。系统中 NC 比例的增加导致释放速度更快，对紫外线的敏感性更高，并增强对叶子的渗透。在辣椒疫霉 (P. capsici)感染期间，叶子中的 NC 与植物的防御酶相互作用以快速做出反应。此外，MC和NC的最佳平衡是有效使用杀菌剂的关键，而不仅仅是更高浓度的NC。 NC 与 MC 的比例为 65:35，可确保有效抑制叶外辣椒疫病菌，并对叶片入侵做出快速反应。这项研究提高了杀菌剂效率并促进了纳米响应杀菌剂的开发，以促进可持续农业实践。
1 Introduction 1 简介
Various plant diseases caused by Phytophthora pose significant threats to agriculture.[1] The early onset and prolonged duration of this disease present challenges for prevention and control. Sustained-release pesticide formulations are expected to maintain effective levels of active ingredients for a longer period, enhancing disease control.[2] Microcapsules (MCs), as representative slow-release pesticide products, have gained significant attention because of their advantages in reducing toxicity and extending the retention period. Nevertheless, despite decades of development, the application of this product remains limited. Currently, only ≈1% of pesticide registrations are for MCs formulations.[3, 4] This low rate is due to the poor match between the release rate and location of MCs and disease control needs, resulting in suboptimal application effectiveness that even falls short of that of unencapsulated formulations. The core of agricultural disease management revolves around prevention, aiming to swiftly reduce disease incidence at the initial stages. However, insufficient initial release could hinder early disease suppression if the release rate is too slow. Consequently, the advancement of pesticide microencapsulation technology, which offers both rapid and sustained effects, is crucial for enhancing MCs performance and managing diseases caused by Phytophthora.
由疫霉引起的各种植物病害对农业构成重大威胁。 1该病发病早、病程长，给防控带来挑战。缓释农药制剂有望在较长时间内保持活性成分的有效水平，从而加强疾病控制。 2微胶囊（MCs）作为缓释农药的代表产品，因其具有降低毒性、延长滞留期等优点而受到广泛关注。然而，尽管经过数十年的发展，该产品的应用仍然有限。目前，只有约 1% 的农药登记是针对中药配方的。 3 , 4这种低速率是由于 MC 的释放速率和位置与疾病控制需求之间的不匹配，导致应用效果不佳，甚至低于未封装制剂的效果。农业病害管理的核心是预防，力求在发病初期迅速降低病害发生率。然而，如果释放速率太慢，初始释放不足可能会阻碍早期疾病抑制。因此，农药微胶囊技术的进步，能够提供快速和持续的效果，对于提高MCs的性能和控制疫霉引起的疾病至关重要。
Considering both the rapid and sustained efficacy of MCs hinges on the implementation of an on-demand release mechanism and the precise regulation of their release rate.[5] To facilitate the on-demand release of active ingredients, several researchers have constructed various sophisticated carrier systems that can respond to specific conditions. The controlled release of pesticides in chewing mouthpiece pests, regulated by factors such as pH,[6-8] laccase,[9, 10] and glutathione,[11, 12] has been proven to be effective. The stable intestinal environment of these pests allows their response factors to interact with the carrier.[13] Simultaneously, intelligent responsive fungicides that respond to pH, enzymes, and temperature have been developed.[14-16] However, previous studies have confirmed that controlled release vectors respond to factors under controlled laboratory conditions, and the mechanism by which they interact with pathogens during plant infection is still unclear. Hence, there is a risk that fungicide carriers might not effectively engage with response factors in practical applications.
MCs的快速和持续疗效取决于按需释放机制的实施和对其释放速率的精确调节。 5为了促进活性成分的按需释放，一些研究人员构建了各种可以响应特定条件的复杂载体系统。通过诸如 pH、 6-8漆酶9 、 10和谷胱甘肽11 、 12等因素调节的咀嚼式害虫中杀虫剂的受控释放已被证明是有效的。这些害虫稳定的肠道环境允许它们的反应因子与载体相互作用。 13同时，还开发出了对 pH、酶和温度做出反应的智能响应型杀菌剂。 14 - 16然而，先前的研究已证实控释载体在受控实验室条件下对因子做出反应，并且它们在植物感染期间与病原体相互作用的机制仍不清楚。因此，在实际应用中，杀菌剂载体可能无法有效地与反应因素结合。
Furthermore, the release rate of MCs is significantly influenced by their particle size, and smaller MCs have a greater release rate. By utilizing the time difference between the release of small and large particles and compounding them, it is possible to achieve both rapid and sustained effectiveness of MCs.[17-19] Currently, particles are prepared individually and subsequently blended for utilization in at least three steps: making large particles and small particles and then combining them. In practice, there is a dearth of technology enabling rapid and one-step formation of multiscale particle combinations, which is crucial for boosting the productivity, efficiency, and efficacy of pesticides.
此外，MC的释放速率受其粒径的显着影响，较小的MC具有更大的释放速率。通过利用小颗粒和大颗粒释放之间的时间差并将它们混合，可以实现MC的快速和持续有效性。 17 - 19目前，颗粒是单独制备的，然后至少通过三个步骤混合使用：制备大颗粒和小颗粒，然后将它们组合。在实践中，缺乏能够快速、一步形成多尺度颗粒组合的技术，而这对于提高农药的生产率、效率和功效至关重要。
We developed a method for producing pesticide MCs using electrostatic attraction between sodium lignosulfonate (SL) and polyalkyl quaternary ammonium salts (PQAS) at the oil–water interface (Figure S2, Supporting Information). This process is simple and promising for industrial application, and the lignin-based carrier exhibits a specific responsive release behavior toward laccase secreted by pests and pathogens infecting woody plants. However, there are still some unclear mechanisms involved in the encapsulation process, such as why SL and PQAS, both in the aqueous phase, orient and deposit at the oil–water interface to form a shell instead of aggregating into disordered polymers in the continuous phase. Furthermore, the presence of particles ranging from 100 to 200 nm in diameter in microscale MCs systems has attracted our attention, as this system can naturally form a mixed system of multiscale pesticide-loaded particles and exhibit specific responsive release.
我们开发了一种利用油水界面上木质素磺酸钠（SL）和多烷基季铵盐（PQAS）之间的静电引力来生产农药MC的方法（图S2 ，支持信息）。该过程简单且具有工业应用前景，并且木质素基载体对感染木本植物的害虫和病原体分泌的漆酶表现出特定的响应释放行为。然而，包封过程中仍存在一些不清楚的机制，例如为什么SL和PQAS在水相中会在油水界面定向并沉积形成壳，而不是在连续相中聚集成无序聚合物。此外，微型MC系统中直径范围为100至200 nm的颗粒的存在引起了我们的注意，因为该系统可以自然地形成多尺度负载农药颗粒的混合系统并表现出特定的响应释放。
In this study, a pyraclostrobin delivery system was prepared using SL and PQAS as shell materials by electrostatic self-assembly. By examining how particle size affects antifungal efficacy, this study investigated the response release mechanism of pyraclostrobin-loaded lignin-based carriers against Phytophthora capsici (P. capsici) infection in Capsicum annuum L. This study elucidates the potential mechanism through which particle size at micro- and nanoscales intricately influences fungicidal efficacy, providing a theoretical foundation for the development of fungicide carriers.
本研究以SL和PQAS为壳材料，通过静电自组装制备了唑菌胺酯递送系统。通过研究粒径如何影响抗真菌功效，本研究研究了负载唑菌胺酯的木质素载体对辣椒疫霉（ P. capsici ）感染的反应释放机制。该研究阐明了微米和纳米尺度的粒径复杂影响杀菌效果的潜在机制，为杀菌剂载体的开发提供了理论基础。
2 Results and Discussion 2 结果与讨论
2.1 Characterization of SL Changes in a Solvent–Water Environment
2.1 溶剂-水环境中 SL 变化的表征
To investigate why SL and PQAS do not aggregate into disordered polymers in the continuous phase during encapsulation, the changes in the morphology of SL molecules in an aqueous solution containing cyclohexanone were studied by dissipative particle dynamics (DPD) simulation. In the simulation system, cyclohexanone and water were present in proportions of 2.5%, 8%, and 89.5% respectively. SL self-assembled into well-structured spherical micelles within an aqueous solution containing cyclohexanone (Figure 1A). As the simulation progressed, the spherical micelles became more distinct (additional simulation steps are shown in Figure S3, Supporting Information). Cyclohexanone beads were evenly dispersed within the core of these micelles, while SL was uniformly distributed in the outer shell.
为了研究为什么 SL 和 PQAS 在包封过程中不会在连续相中聚集成无序聚合物，通过耗散粒子动力学 (DPD) 模拟研究了 SL 分子在含有环己酮的水溶液中形态的变化。在模拟体系中，环己酮和水的比例分别为2.5%、8%和89.5%。 SL 在含有环己酮的水溶液中自组装成结构良好的球形胶束（图1A ）。随着模拟的进行，球形胶束变得更加明显（其他模拟步骤如图S3 ，支持信息所示）。环己酮珠均匀地分散在这些胶束的核心内，而SL均匀地分布在外壳中。
SL 的典型 DPD 形态 (A) 和廷德尔现象 (B)、电导率 (C)、动态光散射强度 (D)、 zeta电位 (E) 和 SL 在含有环己酮的水溶液中的形态 (F)。环己酮和 SL (G) 之间的相互作用较弱。数据显示为平均值±SE，并通过Tukey's检验在p <0.05水平上进行差异分析。
Furthermore, an additional investigation was conducted to determine whether SL could form spherical colloidal particles in an aqueous solution containing cyclohexanone. The conductivity of the SL solution without cyclohexanone was 5.75 mS m−1, and it decreased progressively with increasing cyclohexanone addition (Figure 1C). The conductivity of the SL solution was determined by the concentration of Na+. The presence of cyclohexanone led to the formation of spherical colloidal particles that encapsulated the core, hindering both the mobility and ionization of Na+.[20] The zeta potential, a measure of particle surface charge, decreased as cyclohexanone was added to the SL aqueous solution, suggesting the formation of spherical micelles with sulfonic acid groups (negatively charged) facing outward (Figure 1E). This configuration could form a spherical micelle structure where sulfonic acid and hydroxyl groups (hydrophilic groups) were oriented outward, while methoxy groups (hydrophobic groups) were facing inward, and this arrangement became more pronounced with increasing cyclohexanone concentration. Moreover, the absence of the Tyndall effect in an aqueous SL solution indicated its clear. However, as the addition of cyclohexanone increased, a distinct Tyndall phenomenon emerged due to the formation of spherical colloidal particles by SL in an aqueous solution containing cyclohexanone. The intensity of light scattering was linked to the Tyndall phenomenon, and the light scattering intensity of the SL aqueous solution gradually increased with increasing cyclohexanone addition (Figure 1D). After natural drying, the SL became amorphous after the addition of cyclohexanone (Figure 1F f-1). With increasing cyclohexanone addition, SL gradually formed a spherical colloidal state. However, the colloidal particles formed were fragile, and it was difficult to maintain their initial structure during the drying process. Notably, the shape of the spherical colloidal particles became clearer as the amount of cyclohexanone increased.
此外，还进行了一项额外的研究以确定SL是否可以在含有环己酮的水溶液中形成球形胶体颗粒。不含环己酮的SL溶液的电导率为5.75 mS m -1 ，并且随着环己酮添加量的增加而逐渐降低（图1C ）。 SL溶液的电导率由Na +的浓度确定。环己酮的存在导致形成封装核心的球形胶体颗粒，阻碍了Na +的迁移率和电离。 20当环己酮添加到 SL 水溶液中时， Zeta电位（颗粒表面电荷的量度）降低，这表明形成了磺酸基（带负电）朝外的球形胶束（图1E ）。这种构型可以形成球形胶束结构，其中磺酸和羟基（亲水基团）朝外，而甲氧基（疏水基团）朝内，并且这种排列随着环己酮浓度的增加而变得更加明显。此外，SL 水溶液不存在廷德尔效应，表明其澄清。然而，随着环己酮添加量的增加，由于SL在含有环己酮的水溶液中形成球形胶体颗粒，出现了明显的廷德尔现象。光散射的强度与廷德尔现象有关，并且SL水溶液的光散射强度随着环己酮添加量的增加而逐渐增加（图1D ）。 自然干燥后，加入环己酮后，SL 变成无定形（图1F f-1 ）。随着环己酮添加量的增加，SL逐渐形成球形胶体状态。然而，形成的胶体颗粒很脆弱，在干燥过程中很难保持其初始结构。值得注意的是，随着环己酮用量的增加，球形胶体颗粒的形状变得更加清晰。
To assess whether SL forms micelles in various solvents, methanol (completely soluble in water) and n-hexane (completely insoluble in water) were used as controls. The addition of methanol and n-hexane to the aqueous SL solution did not induce the Tyndall phenomenon (Figure S4C c-2, Supporting Information), and dynamic light scattering showed minimal changes in the solution intensity upon their addition (Figure S4D, Supporting Information). The addition of methanol and n-hexane to the aqueous solution of SL did not significantly alter its zeta potential, indicating no change in the morphology of SL (Figure S4B, Supporting Information). After the addition of n-hexane, the conductivity of the SL solution remained unchanged, indicating that n-hexane did not affect the ionization of SL (Figure S4A a-1, Supporting Information). Conversely, the conductivity decreased with methanol addition, likely due to increased hydrophilic solvent content, which enhanced water–SL interactions and hindered ionization.[21](Figure S4A a-2, Supporting Information).
为了评估SL是否在各种溶剂中形成胶束，使用甲醇（完全溶于水）和正己烷（完全不溶于水）作为对照。向 SL 水溶液中添加甲醇和正己烷不会引起廷德尔现象（图S4C c-2 ，支持信息），并且动态光散射显示添加后溶液强度的变化最小（图S4D ，支持信息） ）。向 SL 的水溶液中添加甲醇和正己烷并没有显着改变其zeta电位，表明 SL 的形态没有变化（图S4B ，支持信息）。添加正己烷后，SL溶液的电导率保持不变，表明正己烷没有影响SL的电离（图S4A a-1 ，支持信息）。相反，电导率随着甲醇的添加而降低，这可能是由于亲水性溶剂含量增加，从而增强了水-SL相互作用并阻碍了电离。 21 （图S4A a-2 ，支持信息）。
Why do these results occur only in cyclohexanone and not in methanol or n-hexane? IGMH analysis was employed to explore the interactions between SL and these solvents. Following the DPD analysis approach, SL was divided into three structures (A, B, and C) for IGMH analysis (Figure S6, Supporting Information). This analysis suggested that structures A and C of the SL molecules interacted attractively with the cyclohexanone molecules (Figure 1G). The IGMH analysis results for methanol were similar to those for cyclohexanone, while the interaction between n-hexane and the SL structures was limited by weak van der Waals forces. In the presence of cyclohexanone, the SL molecule framework folded to form micelles. Methanol molecules escaped from SL attraction due to their affinity for hydrogen bonding with water, while the minimal intermolecular forces between n-hexane and SL resulted in their segregation. Therefore, the attraction of SL toward cyclohexanone is likely pivotal in the formation of spherical micelles in aqueous solutions containing cyclohexanone.
为什么这些结果仅出现在环己酮中，而不出现在甲醇或正己烷中？采用 IGMH 分析来探索 SL 与这些溶剂之间的相互作用。按照 DPD 分析方法，SL 被分为三个结构（A、B 和 C）用于 IGMH 分析（图S6 ，支持信息）。该分析表明 SL 分子的结构 A 和 C 与环己酮分子相互作用（图1G ）。甲醇的 IGMH 分析结果与环己酮的分析结果相似，而正己烷和 SL 结构之间的相互作用受到弱范德华力的限制。在环己酮存在的情况下，SL 分子框架折叠形成胶束。甲醇分子由于与水形成氢键的亲和力而逃脱了 SL 的吸引力，而正己烷和 SL 之间的最小分子间力导致了它们的分离。因此，SL 对环己酮的吸引力可能是在含有环己酮的水溶液中形成球形胶束的关键。
Notably, cyclohexanone has a solubility of 87 g L−1 in water at 20 °C. Consequently, during the preparation of MCs, the use of cyclohexanone as the organic phase allowed SL to partially deposit at the oil–water interface and to partially exist in aqueous solutions containing cyclohexanone. The presence of soluble cyclohexanone led to the formation of colloidal particles composed of SL. In the emulsion system, both microemulsion and nanometer-sized micelles coexisted. Upon the addition of PQAS, particles with two distinct size distributions were formed (Scheme 1).
值得注意的是，环己酮在20℃下在水中的溶解度为87g L -1 。因此，在MCs的制备过程中，使用环己酮作为有机相使得SL部分沉积在油水界面上并部分存在于含有环己酮的水溶液中。可溶性环己酮的存在导致形成由 SL 组成的胶体颗粒。在乳液体系中，微乳液和纳米胶束共存。添加 PQAS 后，形成了具有两种不同尺寸分布的颗粒（方案1 ）。
MCs和NCs共存系统的形成机制。
2.2 Preparation and Regulation of PyrNMCs
2.2 PyrNMCs 的制备和调控
The ratio of MCs and NCs in the pesticide delivery system can be regulated, enhancing the flexibility of the sustained-release formulation for achieving both rapid and sustained effects. By fixing the ratio of the organic phase within the entire formulation and adjusting the proportions of technical materials and solvents within the formulation, a gradual increase in the proportion of technical material led to a corresponding decrease in the proportion of NCs. The solubility of cyclohexanone in water plays a pivotal role in the formation of NCs. As the proportion of the technical materials in the organic phase increased, more cyclohexanone was utilized for dissolving the technical material, while the amount of cyclohexanone in water decreased, thereby reducing the formation of NCs. Furthermore, an increase in the proportion of technical material also led to a corresponding rise in the organic phase viscosity, potentially diminishing the efficiency of the emulsification process. Moreover, the system produced more emulsion droplets with large particle sizes.
农药释放系统中MC和NC的比例可以调节，增强缓释制剂的灵活性，实现快速和持续的效果。通过固定整个配方中有机相的比例，调整配方中原药和溶剂的比例，原药比例逐渐增加，NCs比例相应下降。环己酮在水中的溶解度在NCs的形成中起着关键作用。随着有机相中原药比例的增加，更多的环己酮被用于溶解原药，而水中环己酮的量减少，从而减少了NC的形成。此外，原药比例的增加也会导致有机相粘度相应上升，从而可能降低乳化过程的效率。此外，该系统产生了更多的大粒径乳液液滴。
Furthermore, the effects of fixing the proportions of technical materials and solvents (technical materials/solvent = 7.6%) and adjusting the ratio of the organic phase within the entire formulation on the ratio of PyrNMCs were also evaluated. The proportion of NCs gradually decreased as the organic phase ratio increased (Figure 2). As the organic phase expanded, the capacity for further dissolution of cyclohexanone decreased. Consequently, a significant amount of undissolved cyclohexanone was emulsified into micron-sized droplets, resulting in the formation of MCs. As the amount of cyclohexanone increased in the system, the number of NCs in the system gradually decreased when the proportion of the technical materials in the formulation was fixed. This is consistent with the fact that a significant amount of undissolved cyclohexanone formed micron-sized emulsions, resulting in the formation of MCs. Therefore, when pesticides were loaded, the formation of more spherical micelles led to an increase in nanocapsule production, and the formation of spherical micelles was determined by the solubility of cyclohexanone and the pesticide in water under the influence of SL. The organic phase, which contains cyclohexanone and technical materials, is crucial in the preparation of MCs.
此外，还评估了固定原药和溶剂的比例（原药/溶剂=7.6%）和调整整个配方中有机相的比例对PyrNMCs比例的影响。随着有机相比例的增加，NCs的比例逐渐降低（图2 ）。随着有机相膨胀，进一步溶解环己酮的能力下降。因此，大量未溶解的环己酮被乳化成微米大小的液滴，从而形成MC。当原药在配方中的比例一定时，随着体系中环己酮用量的增加，体系中NC的数量逐渐减少。这与大量未溶解的环己酮形成微米级乳液从而导致MC的形成的事实是一致的。因此，当负载农药时，更多球形胶束的形成导致纳米胶囊产量的增加，而球形胶束的形成由环己酮和农药在SL的影响下在水中的溶解度决定。含有环己酮和技术材料的有机相对于MC的制备至关重要。
通过有机相性质调节体系中MC和NC的比例。数据显示为平均值±SE，并通过Tukey's检验在p <0.05水平上进行差异分析。
Can enhancing the aqueous solubility of cyclohexanone potentially increase the production of NCs? The influence of temperature, a key factor affecting solubility (Figure S9, Supporting Information), was examined. Regrettably, the proportion of NCs in the system did not increase with increasing temperature during preparation (10-60 °C). High temperature can reduce the emulsifying properties of SL, weakening its interaction with the organic phase.[22] In addition, increasing the proportion of SL and PQAS can increase NCs, as more colloidal particles are formed during the oil-in-water (O/W) emulsion process. This increase in SL results in greater production of NCs. The particle size and encapsulation efficiency of both MCs and NCs discussed earlier are shown in Figures S8 and S10; Tables S7 and S8 (Supporting Information). MCs had a particle size distribution between 2 and 3 µm, whereas NCs ranged from 160 to 220 nm.
提高环己酮的水溶性是否有可能增加 NC 的产量？检查了温度的影响，这是影响溶解度的关键因素（图S9 ，支持信息）。遗憾的是，系统中NC的比例并没有随着制备过程中温度的升高（10-60℃）而增加。高温会降低SL的乳化性能，削弱其与有机相的相互作用。 22此外，增加 SL 和 PQAS 的比例可以增加 NC，因为在水包油 (O/W) 乳化过程中会形成更多的胶体颗粒。 SL 的增加导致 NC 产量增加。前面讨论的MC和NC的粒径和包封效率如图S8和S10所示；表S7和S8 （支持信息）。 MC 的粒径分布在 2 至 3 µm 之间，而 NC 的粒径分布在 160 至 220 nm 之间。
2.3 Characterization of PyrNMCs, PyrNCs and PyrMCs
2.3 PyrNMC、PyrNC 和 PyrMC 的表征
Both PyrMCs and PyrNCs contained C, N, O, S, and Cl (Figure 3B). Compared with PyrNCs, PyrMCs had a greater sulfur and nitrogen content on their surface (Table 1). Notably, the presence of the Cl element can be attributed to pyraclostrobin, and its content was almost the same in both PyrNCs and PyrMCs. And the presence of the N element can be attributed to PQAS and pyraclostrobin. It indicated that more SL and PQAS and was present in the shell of PyrMCs. The zeta potentials of PyrNCs (−24.12 mV) and PyrMCs (−24.22 mV) showed little difference (Figure 3C), indicating that more SL was involved in the formation of PyrMCs and that more DDBAC was attracted to form shells. This caused the shell of the PyrMCs to be thick, which was also confirmed by TEM (Figure 3D). The thermal stability of the MC shell surpassed that of the nanocapsule shell. This enhanced stability also increased the melting temperature of pyraclostrobin in the core. The difference in shell thickness can be attributed to the distinct encapsulation mechanisms of PyrMCs and PyrNCs, with the interfacial tension of SL favoring aggregation at the oil–water interface, leading to thicker PyrMCs than PyrNCs.
PyrMC 和 PyrNC 均含有 C、N、O、S 和 Cl（图3B ）。与PyrNCs相比，PyrMCs表面的硫和氮含量更高（表1 ）。值得注意的是，Cl元素的存在可归因于唑菌胺酯，并且其含量在PyrNCs和PyrMCs中几乎相同。 N元素的存在可归因于PQAS和唑菌胺酯。这表明PyrMCs的壳中存在更多的SL和PQAS。 PyrNCs (-24.12 mV) 和 PyrMCs (-24.22 mV) 的zeta电位几乎没有差异（图3C ），表明更多的 SL 参与了 PyrMCs 的形成，并且更多的 DDBAC 被吸引形成壳。这导致 PyrMC 的外壳变厚，TEM 也证实了这一点（图3D ）。 MC壳的热稳定性超过了纳米胶囊壳。这种增强的稳定性还提高了唑菌胺酯在核心中的熔化温度。壳厚度的差异可归因于 PyrMC 和 PyrNC 不同的封装机制，SL 的界面张力有利于在油水界面聚集，导致 PyrMC 比 PyrNC 更厚。
A) PyrNMCs@(85:15) (a-1)、PyrNMCs@(65:35) (a-2)、PyrNMCs@(50:50) (a-3) 和 PyrNMCs@(30: 70) (a-4)。 PyrNCs（蓝色）和 PyrMCs（红色）的 XPS (B) 和zeta电位 (C)。 PyrNCs (d-1) 和 PyrMCs (d-2) 的 TEM (D) 以及 PyrNCs、PyrMCs、唑菌胺酯及其壳材料的 TGA (E) 和 FTIR (F)。数据显示为平均值±SE，并通过Tukey's检验在p <0.05水平上进行差异分析。同一字母处理无显着性差异，不同字母间处理差异显着。
表 1. PyrNCs 和 PyrMCs 表面的 XPS 元素含量分析。
| Sample 样本 | Content [%] 内容 [％] | ||||
|---|---|---|---|---|---|
| C | N | O | Cl 氯 | S | |
| PyrMCs | 70.92 | 8.47 | 14.44 | 2.91 | 3.26 |
| PyrNCs 吡啶核糖核酸 | 77.12 | 6.19 | 13.16 | 3.02 | 0.51 |
The peaks at 2931, 2853, 1134, and 1031 cm−1 in the FTIR spectra of PyrNCs and PyrMCs correspond to ─CH2, ─CH3, ─SO3−, and C─N groups, respectively, with absorption intensities less than those of DDBAC and SL. Furthermore, the curves of pyraclostrobin exhibited a distinct peak at 1716 cm−1, corresponding to the stretching vibration of C═O, and this peak also appeared in the spectra of the PyrMCs and PyrNCs, although their absorption intensities were weakened. A similar trend was found in the FTIR and TGA results for the four kinds of PyrNMCs (Figures S13 and S14, Supporting Information). These results demonstrated the successful encapsulation of pyraclostrobin within the MCs and NCs without any chemical reactions.
PyrNCs和PyrMCs的FTIR光谱中2931、2853、1134和1031 cm -1处的峰分别对应于─CH 2 、─CH 3 、─SO 3 -和C─N基团，吸收强度小于DDBAC 和 SL 的那些。此外，唑菌胺酯的曲线在1716 cm -1处呈现出明显的峰，对应于C=O的伸缩振动，并且该峰也出现在PyrMCs和PyrNCs的光谱中，尽管它们的吸收强度减弱。四种 PyrNMC 的 FTIR 和 TGA 结果也发现了类似的趋势（图S13和S14 ，支持信息）。这些结果表明唑菌胺酯成功封装在 MC 和 NC 中，且没有任何化学反应。
2.4 Release Performance and Biological Activity Evaluation
2.4 释放性能及生物活性评价
The proportion of MCs and NCs in the PyrNMC system played a pivotal role in impacting the performance. PyrNCs exhibited a rapid release rate, while PyrMCs released more slowly due to differences in particle size and shell thickness. PyrMCs@polyurea exhibited significant sustained release, with a slower rate than all other PyrNMCs, PyrNCs, and PyrMCs (Figure 4A). Furthermore, increasing the proportion of NCs in a mixed system increased the release rate (Figure 4A a-2). Overall, due to their small particle size, large specific surface area, and thin shell, the NCs exhibited favorable quick release. Accordingly, in the PyrNMCs system, a higher nanocapsule ratio resulted in a faster release rate.
PyrNMC 系统中 MC 和 NC 的比例对性能的影响起着关键作用。 PyrNCs 表现出快速的释放速率，而 PyrMCs 由于粒径和壳厚度的差异而释放得更慢。 PyrMCs@聚脲表现出显着的持续释放，其速率比所有其他 PyrNMCs、PyrNCs 和 PyrMCs 慢（图4A ）。此外，增加混合系统中NC的比例会增加释放率（图4A a-2 ）。总体而言，由于NCs粒径小、比表面积大、壳薄，因此表现出良好的快速释放性。因此，在 PyrNMCs 系统中，较高的纳米胶囊比例导致较快的释放速率。
PyrNMC 的释放性能 (A) 及其在培养基中对抗辣椒疫霉的生物活性 (B) 以及在辣椒叶中对抗辣椒疫霉的生物活性 (C)。数据显示为平均值±SE，并通过Tukey's检验在p <0.05水平上进行差异分析。同一字母处理无显着性差异，不同字母间处理差异显着。
Moreover, the effectiveness of the sustained-release formulations was closely correlated with their release rate; faster release led to greater inhibition of P. capsici in the culture medium. Among all treatments, PyrNCs exhibited the greatest inhibitory effect on P. capsici (80.34%) at a concentration of 20 mg L−1 among all the treatments (Figure 4B). In the PyrNMCs system, increasing the proportion of NCs enhanced the inhibition rate against P. capsici.
此外，缓释制剂的有效性与其释放速率密切相关。更快的释放导致培养基中对辣椒疫病菌的更大抑制。在所有处理中，PyrNCs在20 mg L -1浓度下对辣椒疫病菌表现出最大的抑制效果（80.34%）（图4B ）。在PyrNMCs系统中，增加NCs的比例可以提高对辣椒疫病菌的抑制率。
To align with practical application scenarios more closely, the inhibition rate of these samples against P. capsici on pepper leaves was evaluated, and the results were consistent with earlier findings. Among the PyrNMCs treatments, higher nanocapsule proportions corresponded to a greater inhibition rate against P. capsici. The assessment of inhibition rates on both culture media and leaves revealed different release scenarios: in liquid and at the interface. These results demonstrated that in both environments, higher nanocapsule proportion led to greater rates of P. capsici inhibition in the PyrNMCs system.
为了更贴近实际应用场景，对这些样品对辣椒叶片上辣椒疫病菌的抑制率进行了评估，结果与前期研究结果一致。在 PyrNMCs 处理中，较高的纳米胶囊比例对应于对辣椒假单胞菌的较高抑制率。对培养基和叶子的抑制率的评估揭示了不同的释放情况：在液体中和在界面处。这些结果表明，在两种环境中，较高的纳米胶囊比例导致 PyrNMCs 系统中辣椒疫霉的抑制率较高。
2.5 Pot Experiment 2.5 盆栽实验
Effectiveness is the primary criterion for developing pesticide preparations. Based on the inhibition rates, a higher nanocapsule content should enhance PyrNMC performance. However, in practical applications, this was not the case. PyrMCs@polyurea showed only 25.30% control efficiency at 3 days after inoculation (DAI) due to its slow release, while that of PyrEC was 83.65%. PyrNMCs with higher nanocapsule ratios achieved 100% control efficiency at 3 DAI, except for PyrNMCs@(30:70) at 83.33%. At 6 DAI, the effectiveness of PyrMCs@polyurea and PyrNMCs (30:70) did not increase or decrease, respectively, against P. capsici. P. capsici infection involves mycelium invading leaf cells and spaces, while pyraclostrobin is poorly absorbed by pepper plants. Slow-release treatments, such as PyrMCs@polyurea and PyrNMCs (30:70), release little pyraclostrobin during the initial stages, thereby providing an opportunity for infection to enter the leaves. Furthermore, external pyraclostrobin is almost unable to exert an effect on the P. capsici inside the leaves (Figure 5).
有效性是开发农药制剂的首要标准。根据抑制率，较高的纳米胶囊含量应该会增强 PyrNMC 的性能。然而，在实际应用中，情况并非如此。由于释放缓慢，PyrMCs@polyurea 在接种后 3 天（DAI）的防效仅为 25.30%，而 PyrEC 的防效为 83.65%。具有较高纳米胶囊比例的 PyrNMC 在 3 DAI 时实现了 100% 的防治效率，但 PyrNMCs@(30:70) 的防治效率为 83.33%。在 6 DAI 时，PyrMCs@polyurea 和 PyrNMCs (30:70) 对抗辣椒疫病菌的有效性没有分别增加或减少。辣椒疫病菌感染涉及菌丝体侵入叶细胞和间隙，而唑菌胺酯很难被辣椒植物吸收。缓释处理，例如 PyrMCs@polyurea 和 PyrNMCs (30:70)，在初始阶段释放少量唑菌胺酯，从而为感染进入叶子提供了机会。此外，外用唑菌胺酯几乎无法对叶内的辣椒假单胞菌发挥作用(图5 )。
照片显示了接种后三天和六天吡唑醚菌酯制剂的防治效果（A）、盆内吡唑醚菌酯制剂对辣椒疫病菌的防治效果（B）以及实验期间的落叶数量（C）。数据显示为平均值±SE，并通过Tukey's检验在p <0.05水平上进行差异分析。同一字母处理无显着性差异，不同字母间处理差异显着。
PyrNMCs@(85:15) did not exhibit the highest efficacy among all treatments at 6 DAI, with its efficacy decreasing to 80.34%. Furthermore, plants shed infected leaves to prevent the spread of disease through their own defense mechanisms at 3 and 6 DAI in PyrNMCs@(85:15) (Figure 6C). However, PyrNMCs@(65:35) exhibited a consistent 100% control efficiency throughout the entire experimental duration, with no occurrence of falling leaves either. This finding indicates that factors beyond the bioactivity of pyraclostrobin toward P. capsici significantly affect its control efficacy. Factors such as UV light stability, release rate, and leaf permeation of pesticides, which are crucial for fungicide efficacy, remain largely unexplored.
PyrNMCs@(85:15) 在 6 DAI 时并未表现出所有治疗中最高的疗效，其疗效下降至 80.34%。此外，在 PyrNMCs@(85:15) 的 3 和 6 DAI 时，植物脱落受感染的叶子，通过其自身的防御机制来防止疾病传播（图6C ）。然而，PyrNMCs@(65:35) 在整个实验期间表现出一致的 100% 防治效率，也没有出现落叶现象。这一发现表明，除了唑菌胺酯对辣椒疫病菌的生物活性之外，其他因素也会显着影响其防治效果。杀虫剂的紫外线稳定性、释放速率和叶片渗透性等对于杀菌剂功效至关重要的因素在很大程度上仍未得到探索。
PyrNMCs在载玻片(A)和叶片(B)上的抗光解性能，PyrNMCs进入叶片的量(D)和荧光强度(E)，以及荧光观察方法(C)。数据显示为平均值±SE，并通过Tukey's检验在p <0.05水平上进行差异分析。同一字母处理无显着性差异，不同字母间处理差异显着。
2.6 Anti-Photolysis Performance and Leaf Surface Penetration of PyrNMCs
2.6 PyrNMCs的抗光解性能和叶面渗透能力
Figure 6A illustrates the anti-photolysis performance of these formulations. The anti-photolysis performance of the sustained-release formulations was better than that of the exposed formulations (PyrEC) due to the protection of the shell. MCs with SL-PQAS shells exhibit superior UV resistance to those with polyurea MCs, likely due to the well-known light absorption performance of SL.[23] Furthermore, the anti-photolysis performance of the four types of PyrNMCs was related to their release performance; the more rapid the release was, the greater the susceptibility to photolysis. However, the persistence effectiveness of PyrNMCs did not change regularly with the proportion of micro/nanocapsules. This indicated that the anti-photolysis performance of PyrNMCs was not the only factor determining their control efficiency. Similarly, to investigate the photolysis dynamics of different samples in a more practical context, their anti-photolysis performance on pepper leaves was also evaluated. Compared with those on slides, the PyrNMCs, especially PyrNMCs@(85:15), significantly improved the resistance of leaves to photolysis, with an 11.28% greater residual amount on leaves after 120 minutes (Figure 6B). PyrEC also improved the anti-photolysis performance of leaves. However, PyrMCs@(polyurea) exhibited minimal differences between the slides and leaves, suggesting that PyrNMCs and PyrEC may have penetrated into the leaves, improving their resistance to photolysis. Fluorescence imaging revealed that the amount of NCs in PyrNMCs correlated with their penetration into leaves (Figure 6C,E). Some fluorescence was also observed in the longitudinal section of PyrEC-treated leaves, likely due to its higher organic solvent and surfactant content, facilitating effective penetration. The amount of pyraclostrobin entering the leaves was measured, with PyrNMCs@(85:15) showing 18.15% penetration, which was significantly greater than that of the other treatments. PyrMCs@polyurea had negligible penetration (0.79%). This indicates that most samples, except for PyrMCs@polyurea, had the potential to penetrate leaves.
图6A说明了这些制剂的抗光解性能。由于外壳的保护，缓释制剂的抗光解性能优于暴露制剂（PyrEC）。具有 SL-PQAS 壳的 MC 比具有聚脲 MC 的抗紫外线性能更出色，这可能是由于 SL 众所周知的光吸收性能。 23此外，四种 PyrNMC 的抗光解性能与其释放性能有关；释放越快，光解作用的敏感性就越大。然而，PyrNMCs的持久有效性并不随微/纳米胶囊的比例发生规律性变化。这表明PyrNMCs的抗光解性能并不是决定其防治效率的唯一因素。同样，为了在更实际的背景下研究不同样品的光解动力学，还评估了它们对辣椒叶的抗光解性能。与玻片上的相比，PyrNMCs，尤其是PyrNMCs@(85:15)，显着提高了叶片的光解抗性，120分钟后叶片上的残留量增加了11.28%（图6B ）。 PyrEC还提高了叶片的抗光解性能。然而，PyrMCs@（聚脲）在载玻片和叶子之间表现出最小的差异，表明PyrNMCs和PyrEC可能已经渗透到叶子中，提高了它们的光解抗性。荧光成像显示 PyrNMC 中 NC 的数量与其对叶片的渗透相关（图6C、E ）。 在 PyrEC 处理的叶子的纵切面上也观察到了一些荧光，这可能是由于其较高的有机溶剂和表面活性剂含量，有利于有效渗透。测量了吡唑醚菌酯进入叶片的量，PyrNMCs@(85:15)的渗透率为18.15%，显着高于其他处理。 PyrMCs@聚脲的渗透率可以忽略不计（0.79%）。这表明除 PyrMCs@polyurea 之外的大多数样品都具有穿透叶子的潜力。
Notably, in pot experiments, PyrNMCs@(85:15), PyrNMCs@(65:35), and PyrNMCs@(50:50) achieved 100% control efficacy three days after vaccination. The pyraclostrobin of PyrNMCs was encapsulated, and even if they entered the leaves, they needed to be quickly released to maintain their effectiveness in controlling P. capsici. The rapid release mechanism after entering leaves is also an important factor in determining control efficiency and is not yet known.
值得注意的是，在盆栽实验中，PyrNMCs@(85:15)、PyrNMCs@(65:35)和PyrNMCs@(50:50)在接种疫苗三天后实现了100%的防治效果。 PyrNMCs的吡唑醚菌酯被封装起来，即使它们进入叶片，也需要快速释放以保持其防治辣椒疫病菌的有效性。进入叶片后的快速释放机制也是决定防治效率的重要因素，目前尚不清楚。
2.7 The Mechanism of PyrNMCs against P. capsici
2.7 PyrNMCs抗辣椒疫霉的作用机制
In fact, infection of pepper plants by P. capsici involves a complex process. When spores come into contact with leaves, they release hyphae that penetrate plant tissues by producing zoospores. Enzymes are secreted to break down cell walls, allowing the fungus to enter and reproduce.[24, 25] Furthermore, plants also secrete enzymes for defense. P. capsici and plant enzymes are present in intercellular spaces.[26] The PyrNMC shell made from SL and PQAS may interact with enzymes such as laccase, cellulase, and pectinase. The changes in three kinds of enzymes in the leaves of P. capsici during infection are shown in Figure 7A (a-1, a-2, and a-3), and the changes in P. capsici in the infested leaves are also shown in Figure S20A (Supporting Information). At 1 DAI, the amount of laccase increased significantly, the amount of cellulase increased slightly, and the amount of pectinase decreased. Laccase and cellulase gene expression levels indicated their involvement in plant defense. To further clarify the mechanisms of changes in laccases and cellulases in leaves, the relative expression levels of laccases and cellulases in leaves were investigated (Figure 7A a-4, a-5). The expression of Calac11 and Cacel1 initially decreased but then increased with infection duration, suggesting their involvement in pepper leaf resistance to P. capsici. As mentioned above, P. capsici also secretes enzymes that dissolve cell walls. After adding inducing factors (sterilized leaves and pectin) to the NPB media for 7 days (Figure 7B b-1), no significant changes were detected in the laccase levels within the mycelia. When cellulase levels increased, pectinase significantly increased. The relative expression of Pclac3 remained almost unchanged, while that of PcCel1 and Pcpme1 increased after inoculation, with Pcpme1 levels increasing fivefold two days later. This indicated that pectinase and cellulase were involved in P. capsici infection and that pectinase may play a dominant role in this process.
事实上，辣椒疫霉对辣椒植物的侵染涉及一个复杂的过程。当孢子与叶子接触时，它们会释放菌丝，通过产生游动孢子来穿透植物组织。分泌酶来分解细胞壁，使真菌进入并繁殖。 24 , 25此外，植物还分泌防御酶。辣椒假单胞菌和植物酶存在于细胞间隙中。 26由 SL 和 PQAS 制成的 PyrNMC 外壳可能与漆酶、纤维素酶和果胶酶等酶相互作用。辣椒疫霉侵染过程中叶片中三种酶的变化如图7A （a-1、a-2和a-3）所示，同时也示出了受侵染叶片中辣椒疫霉的变化在图S20A （支持信息）中。 1 DAI时，漆酶量显着增加，纤维素酶量略有增加，果胶酶量减少。漆酶和纤维素酶基因表达水平表明它们参与植物防御。为了进一步阐明叶片中漆酶和纤维素酶的变化机制，研究了叶片中漆酶和纤维素酶的相对表达水平(图7A a-4、a-5 )。 Calac11和Cacel1的表达最初下降，但随后随着感染时间的延长而增加，表明它们参与辣椒叶对辣椒疫病菌的抗性。如上所述，辣椒假单胞菌还分泌溶解细胞壁的酶。 将诱导因子（灭菌的叶子和果胶）添加到 NPB 培养基中 7 天后（图7B b-1 ），菌丝体内的漆酶水平没有检测到显着变化。当纤维素酶水平增加时，果胶酶显着增加。 Pclac3的相对表达量几乎保持不变，而PcCel1和Pcpme1的相对表达量在接种后增加，两天后Pcpme1水平增加了五倍。这表明果胶酶和纤维素酶参与了辣椒疫霉的感染，并且果胶酶可能在此过程中起主导作用。
感染过程中叶片(A)中3种酶(a-1、漆酶、a-2、纤维素酶、a-3果胶酶)的变化以及漆酶(a-4)和纤维素酶基因(a- 5）感染期间在叶子上。辣椒疫病菌生长过程中三种酶表达量的变化(B)以及辣椒疫霉中漆酶(a-6)、纤维素酶(a-7)和果胶酶(a-8)的相对表达水平感染期间。酶对 PyrMCs 和 PyrNCs 释放性能的影响 (C) 以及用三种酶处理的 PyrNMCs@(65:35) 的形态 (D)。酶对 PyrNMC 的影响 (E)。数据显示为平均值±SE，并通过Tukey's检验在p <0.05水平上进行差异分析。同一字母处理无显着性差异，不同字母间处理差异显着。
In addition, during the infection process, laccase, cellulase, and pectinase in the leaves were all inhibited after Na2CO3 treatment (Figure S20C, Supporting Information). The inhibition rate of PyrNMCs@(65:35) against P. capsici significantly decreased after Na2CO3 treatment; nearly half of the leaves remained infected with P. capsici, while no lesions were observed in the treatment without enzyme inhibitors at 80 mg L−1. This finding indicated that enzymes were key to opening PyrNMCs.
另外，在感染过程中，Na 2 CO 3处理后叶片中的漆酶、纤维素酶和果胶酶均受到抑制（图S20C ，支持信息）。 Na 2 CO 3处理后PyrNMCs@(65:35)对辣椒疫病菌的抑制率显着降低；近一半的叶子仍然受到辣椒疫霉的感染，而在没有酶抑制剂的80 mg L -1处理中没有观察到病变。这一发现表明酶是打开 PyrNMC 的关键。
To investigate which enzymes can mediate the release of PyrNMCs, three enzymes were added to the release medium (Figure 7C). Laccase and cellulase significantly increased the release rate, while pectinase had no effect. After laccase treatment, the MCs shell became porous and leaked its core, while complete release of the NCs was observed. Cellulase treatment completely disrupted the shells of the MCs, the NCs were completely broken, and the cores were released. The NCs and MCs released their contents (Figure 7D). Pectinase had little effect on the capsules. Furthermore, three enzymes had no effect on PyrMCs@polyurea (Figure S19, Supporting Information). The shell of PyrNMCs was made from SL and PQAS and is susceptible to degradation by laccase and cellulase, accelerating its release. Therefore, when pepper leaves are infected by P. capsici, the plant's defense system releases laccase and cellulase enzymes to degrade lignin-based nanocarriers and release active ingredients. This finding differs from current studies attributing the release of responsive fungicides to response factors released by pathogens.
为了研究哪些酶可以介导 PyrNMC 的释放，将三种酶添加到释放介质中（图7C ）。漆酶和纤维素酶显着提高了释放速率，而果胶酶则没有效果。经过漆酶处理后，MC 壳变得多孔并泄漏其核心，同时观察到 NC 完全释放。纤维素酶处理完全破坏了 MC 的壳，NC 完全破裂，核被释放。 NC和MC公布了他们的内容（图7D ）。果胶酶对胶囊影响不大。此外，三种酶对 PyrMCs@polyurea 没有影响（图S19 ，支持信息）。 PyrNMCs 的外壳由 SL 和 PQAS 制成，容易被漆酶和纤维素酶降解，从而加速其释放。因此，当辣椒叶被辣椒疫病菌感染时，植物的防御系统会释放漆酶和纤维素酶来降解木质素基纳米载体并释放活性成分。这一发现与目前的研究不同，目前的研究将响应性杀菌剂的释放归因于病原体释放的响应因子。
Since these enzymes are intercellular, only PyrNMCs that enter leaves can interact with them, providing a rapid response to P. capsici infection. When applied to pepper leaves (Figure 8), most of the MCs and NCs remain outside the leaves, with only a few NCs entering. Certainly, a small portion of the NCs entering the leaves play a crucial role in the control efficiency. Less residual pyraclostrobin outside leaves means more P. capsici-infected leaves, thus consuming more pyraclostrobin. Despite PyrNMCs@(85:15) penetrating deeply, its photolytic resistance led to increased P. capsici infection over time, as observed in fallen leaves at 3 and 6 DAI. PyrNMCs@(65:35) maintained lower P. capsici infection by preserving more pyraclostrobin outside the leaves.
由于这些酶是细胞间酶，因此只有进入叶子的 PyrNMC 才能与它们相互作用，从而对辣椒疫病菌感染做出快速反应。当应用于辣椒叶时（图8 ），大多数MC和NC留在叶外，只有少数NC进入。当然，进入叶片的一小部分NC对控制效率起着至关重要的作用。叶子外部残留的唑菌胺酯较少意味着更多的辣椒疫霉感染的叶子，从而消耗更多的唑菌胺酯。尽管 PyrNMCs@(85:15) 穿透很深，但其光解抗性导致辣椒疫病菌感染随着时间的推移而增加，正如在第 3 天和第 6 天的落叶中观察到的那样。 PyrNMCs@(65:35) 通过在叶子外保留更多的唑菌胺酯来维持较低的辣椒疫霉感染。
PyrNMCs 对抗辣椒疫病菌的机制。
3 Conclusion 3 结论
In this work, SL self-assembled into micelles in aqueous solutions containing cyclohexanone, offering a novel nanoscale template for the fabrication of pyraclostrobin-loaded MCs by electrostatic self-assembly. This innovative approach enables the creation of a sustained-release system that integrates both MCs and NCs in a single fabrication process. The proportions of MCs and NCs can be finely tuned by adjusting the properties of the organic phase and the quantity of shell-forming materials. Notably, an increased proportion of NCs correlates with a faster release rate and enhanced inhibitory effects against P. capsici in laboratory settings. These NCs entering leaves can be enzymatically released by defense enzymes (such as laccase and cellulase) secreted by infected leaves, challenging the conventional notion that the stimulating factors regulating fungicide release from carriers come from pathogens. Importantly, this study also suggested that a greater proportion of NCs does not invariably improve disease control efficacy. Instead, the presence of NCs and MCs at an optimal ratio ensures precise and on-demand delivery of fungicides to the leaf surface and interior, resulting in superior control effects. This research contributes significant theoretical insights for the advancement of intelligent and responsive fungicides and nanopesticides.
在这项工作中，SL 在含有环己酮的水溶液中自组装成胶束，为通过静电自组装制备唑菌胺酯负载的 MC 提供了一种新型纳米级模板。这种创新方法能够创建一个将 MC 和 NC 集成在单一制造过程中的缓释系统。 MCs和NCs的比例可以通过调整有机相的性质和成壳材料的数量来微调。值得注意的是，在实验室环境中，NC 比例的增加与更快的释放速率和对辣椒疫病菌的抑制作用增强相关。这些进入叶片的NC可以被受感染叶片分泌的防御酶（如漆酶和纤维素酶）酶促释放，挑战了调节载体释放杀菌剂的刺激因子来自病原体的传统观念。重要的是，这项研究还表明，更大比例的 NC 并不一定能提高疾病控制效果。相反，NC 和 MC 以最佳比例存在，可确保将杀菌剂精确且按需输送到叶表面和内部，从而产生优异的防治效果。这项研究为智能和响应性杀菌剂和纳米农药的发展提供了重要的理论见解。
4 Experimental Section 4 实验部分
Materials 材料
The sulfonation degree of sodium lignosulfonate (SL) was 0.8 mmol kg−1, with a molecular weight of 10 000 Daltons, and sulfonation occurred at the benzene ring, which was provided by MeadWestvaco Corp. (Illinois, USA). The cyclohexanone, N-hexane, methanol, xylene, dodecyl dimethyl benzyl ammonium chloride (DDBAC), fluorescein isothiocyanate isomer, sodium carbonate, ferric chloride, and ethylenediamine (EDA) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China), and were of analytical purity. MDI (4,4-methylenediphenyl diisocyanate) was obtained from Wanhua Chemical Group Co., Ltd., Shandong, China. The pyraclostrobin technical material (98.2%) was obtained from Shandong Kangqiao Biotechnology Co., Ltd. (Shandong, China). Calcium dodecyl benzene sulfonate (agricultural emulsifier No. 500) and styrene phenol polyoxyethylene ether (agricultural emulsifier No. 600) were purchased from Zibo Yunchuan Chemicals Co., Ltd. (Shandong, China). Solvent Blue 35 was purchased from Wuhan Kanos Technology Co., Ltd. (Hubei, China). A pectinase kit, cellulase kit, and laccase kit were all purchased from Beijing Solaibao Technology Co., Ltd. (Beijing China). Enzymes (laccase, pectinase, and cellulase) were obtained from Ningxia Xiasheng Industrial Group Co., Ltd. (Ningxia, China). Deionized water was used throughout the study.
木质素磺酸钠(SL)的磺化度为0.8mmol kg -1 ，分子量为10 000道尔顿，磺化发生在苯环处，由MeadWestvaco Corp.(Illinois, USA)提供。环己酮、正己烷、甲醇、二甲苯、十二烷基二甲基苄基氯化铵（DDBAC）、异硫氰酸荧光素异构体、碳酸钠、氯化铁和乙二胺（EDA）购自阿拉丁试剂有限公司（中国上海）。 ，并且具有分析纯。 MDI（4,4-亚甲基二苯基二异氰酸酯）购自中国山东万华化学集团有限公司。唑菌胺酯原药（98.2%）购自山东康桥生物科技有限公司（中国山东）。十二烷基苯磺酸钙（农用乳化剂500号）和苯乙烯酚聚氧乙烯醚（农用乳化剂600号）购自淄博云川化工有限公司（中国山东）。溶剂蓝35购自武汉卡诺斯科技有限公司（中国湖北）。果胶酶试剂盒、纤维素酶试剂盒、漆酶试剂盒均购自北京索莱宝科技有限公司（中国北京）。酶（漆酶、果胶酶和纤维素酶）购自宁夏夏盛实业集团有限公司（中国宁夏）。整个研究过程中使用去离子水。
Pepper Plant (Capsicum annuum L.)
辣椒植物（ Capsicum annuum L.）
The experimental variety Xianjiao No. 6 was obtained from the Chaofeng Seed Industry of Dezhou, Shandong Province, and cultivated under controlled indoor conditions throughout the entire growth cycle from routine management to the adult plant stage.
试验品种鲜椒6号购自山东省德州市超丰种业，从常规管理到成株期的整个生长周期均在室内可控条件下栽培。
P. capsici 辣椒假单胞菌
The wild-type P. capsici isolate was obtained from pepper plants in Taian, Shandong Province, and cultivated at a temperature of 25 °C under dark conditions on PDA agar medium. PDA medium was prepared by combining 200 g of potato, 20 g of glucose, 20 g of agar, and a liter of deionized water. Additionally, the agar used in the medium consisted of 2% agar and 98% deionized water.
野生型辣椒疫霉分离物从山东省泰安市的辣椒植株中获得，并在25℃、黑暗条件下在PDA琼脂培养基上培养。 PDA培养基由200g马铃薯、20g葡萄糖、20g琼脂和1升去离子水混合制备。此外，培养基中使用的琼脂由2%琼脂和98%去离子水组成。
Mycelia Acquisition 菌丝体采集
The mycelia of P. capsici cultured on PDA medium were transferred to NPB medium and incubated in a temperature-controlled oscillating incubator for 2 days at 25 °C. The NPB medium was prepared according to Table S1 (Supporting Information). The green bean extraction solution was prepared by boiling 125 g of green beans in 1 L of water and subsequently filtering the mixture.
将 PDA 培养基上培养的辣椒疫病菌菌丝体转移至 NPB 培养基中，并在控温振荡培养箱中于 25 °C 下培养 2 天。 NPB 培养基根据表S1 （支持信息）制备。通过将 125 g 绿豆在 1 L 水中煮沸并随后过滤混合物来制备绿豆提取溶液。
Characterization of SL in a Solvent–Water Environment—DPD simulation
溶剂-水环境中 SL 的表征 — DPD 模拟
Dissipative particle dynamics (DPD) were used to simulate the state of SL in a cyclohexanone-aqueous solution. The structure of the SL used for DPD simulation was determined according to Zhu's study[27] and modified according to the detailed parameters of SL provided by MeadWestvaco Corp. (Illinois, USA). As shown in Figure S3 (Supporting Information), SL was divided into three basic structures, and each was used as a bead based on the coarse-grained principle (A structure, pink bead; B structure, orange bead; and C structure, green bead). Zhu's research and the sulfonation degree and molecular weight of SL were obtained from MeadWestvaco Corp. (Illinois, USA). It could be inferred that the coarse-grained unit of SL consisted of AB2C2, and the selection of 8 coarse-grained units as the model for SL in DPD simulation corresponded to its actual molecular weight. Therefore, the proposed structure of SL in DPD simulations could be identified as (AB2C2)8. The number of beads of SL, cyclohexanone, and water were set in the DPD simulation according to the addition of SL in the system and the bead definition. Eight cyclohexanone molecules were coarse-grained into one bead of CYC (yellow ball), and ten water molecules were coarse-grained into one bead of H2O (blue ball). The Flory–Huggins parameter Xij and interaction parameter αij between the coarse-grained beads used in the DPD simulations were calculated, and the results are shown in Tables S2 and S3 (details of the procedure can be found in the Supporting Information).
使用耗散粒子动力学 (DPD) 来模拟 SL 在环己酮水溶液中的状态。用于DPD模拟的SL的结构是根据Zhu的研究27确定的，并根据MeadWestvaco Corp.（伊利诺伊州，美国）提供的SL的详细参数进行修改。如图S3 （支持信息）所示，SL被分为三种基本结构，并且根据粗粒度原则将每种结构用作珠子（A结构，粉色珠子；B结构，橙色珠子；C结构，绿色珠子）珠子）。 Zhu的研究以及SL的磺化度和分子量是从MeadWestvaco Corp.（伊利诺伊州，美国）获得的。可以推断SL的粗粒单元由AB 2 C 2组成，DPD模拟中选择8个粗粒单元作为SL的模型与其实际分子量相对应。因此，在 DPD 模拟中提出的 SL 结构可以被识别为 (AB 2 C 2 ) 8 。根据系统中SL的添加量和珠的定义，在DPD模拟中设置SL、环己酮和水的珠的数量。八个环己酮分子被粗粒化为一粒CYC（黄色球），十个水分子被粗粒化为一粒H 2 O（蓝色球）。计算了 DPD 模拟中使用的 Flory-Huggins 参数 X ij和粗粒珠之间的相互作用参数 α ij ，结果如表S2和S3所示（详细过程可在支持信息中找到）。
Characterization of SL in a Solvent–Water Environment
溶剂-水环境中 SL 的表征
The SL was dissolved in water to form a solution with an SL content of 2.5%, which was based on the amount used as shell material for MC and nanocapsule preparation. Cyclohexanone, n-hexane, and methanol were added to the solution. The SL content was maintained by changing the amount of deionized water. The zeta potential and conductivity of various SL solutions were measured using a zeta potential analyzer (90Plus PALS, Brookhaven, USA) and a conductivity meter (DDS-307A, INESA Scientific Instrument Co., Ltd., China), respectively. The scattered light intensity of the different SL solutions was determined by a nanolaser particle size analyzer (90Plus PALS, Brookhaven, USA). The morphology of SLs with different amounts of cyclohexanone was observed by transmission electron microscopy (Zeiss Libra 200FE, Carl Zeiss AG, Germany). Furthermore, to investigate the impact of cyclohexanone on the surface tension of an SL solution, a surface tensiometer (BZY-1, Shanghai Hengping Instrument and Meter Factory, China) was used to measure the surface tension in the SL solution with cyclohexanone. In addition, consistent with the above method, a 0.5% SL solution was prepared with deionized water, and cyclohexanone, n-hexane, and methanol were added to the solution. The amount of deionized water was adjusted to maintain the desired content of SL. The Tyndall phenomenon of the solutions was observed using a laser flashlight.
将SL溶解在水中形成SL含量为2.5%的溶液，该溶液基于用作MC和纳米胶囊制备的壳材料的量。将环己酮、正己烷和甲醇添加到溶液中。通过改变去离子水的量来维持SL含量。分别使用zeta电位分析仪（90Plus PALS，Brookhaven，美国）和电导仪（DDS-307A，中国仪电科学仪器有限公司）测量各种SL溶液的zeta电位和电导率。不同 SL 溶液的散射光强度由纳米激光粒度分析仪（90Plus PALS，Brookhaven，USA）测定。通过透射电子显微镜（Zeiss Libra 200FE，Carl Zeiss AG，德国）观察具有不同环己酮含量的SL的形貌。此外，为了研究环己酮对SL溶液表面张力的影响，使用表面张力计（BZY-1，上海横平仪器仪表厂，中国）测量环己酮SL溶液的表面张力。另外，按照上述方法，用去离子水制备0.5％SL溶液，并向该溶液中添加环己酮、正己烷和甲醇。调节去离子水的量以维持所需的SL含量。使用激光手电筒观察溶液的廷德尔现象。
Characterization of SL in a solvent–Water Environment—Independent Gradient Model Based on Hirshfeld Partition (IGMH) Analysis Between SL and Solvent Molecules
溶剂-水环境中 SL 的表征——基于 SL 和溶剂分子之间的赫什菲尔德分配 (IGMH) 分析的独立梯度模型
To clarify the mechanism of the morphological changes in SL in different solvent–aqueous solutions, the weak interactions between SL and methanol, cyclohexanone, and n-hexane molecules were investigated using IGMH analysis.[28, 29] The details of the procedure can be found in the Supporting Information.
为了阐明 SL 在不同溶剂-水溶液中形态变化的机制，使用 IGMH 分析研究了 SL 与甲醇、环己酮和正己烷分子之间的弱相互作用。 28 , 29该程序的详细信息可以在支持信息中找到。
Preparation and Regulation of PyrNMCs
PyrNMC 的制备和调控
The preparation method for PyrNMCs was modified according to previous research.[13] The process of preparation is shown in Figure S7 (Supporting Information). First, the organic phase was prepared by dissolving pyraclostrobin in cyclohexanone, while the aqueous phase was prepared with SL dissolved in water. The organic phase was carefully added to the water phase, and an O/W emulsion was formed through homogeneous shear at 10 000 rpm for 1 min. After stirring for 3 min at room temperature, a 0.3 mol L−1 DDBAC solution (3.00 g) was added dropwise. Subsequently, 0.4 g of FeCl3 solution (0.3 mol L−1) was added dropwise. PyrNMCs could be obtained by stirring at room temperature for 10 min. PyrNMCs in which PyrNMCs coexisted were named PyrNMCs. The proportion of PyrNMCs in the system was regulated by adjusting the properties of the organic phase (technical material/solvent, organic phase/formulation, and solvent/formulation), the temperature during preparation, and the addition of SL and DDBAC. In addition, the details of the preparation of the control formulations (PyrMCs@polyurea and PyrEC) can be found in the Supporting Information.
根据前期研究对PyrNMCs的制备方法进行了修改。 13准备过程如图S7 （支持信息）所示。首先，将唑菌胺酯溶解在环己酮中制备有机相，而将SL溶解在水中制备水相。将有机相小心地添加到水相中，并通过在10000rpm下均匀剪切1分钟形成O/W乳液。室温搅拌3分钟后，滴加0.3mol·L -1 DDBAC溶液(3.00g)。随后，滴加0.4g FeCl 3溶液(0.3mol L -1 )。室温搅拌10分钟即可得到PyrNMCs。 PyrNMCs共存的PyrNMCs被命名为PyrNMCs。通过调节有机相（原药/溶剂、有机相/制剂、溶剂/制剂）的性质、制备过程中的温度以及SL和DDBAC的添加量来调节体系中PyrNMCs的比例。此外，对照制剂（PyrMCs@聚脲和 PyrEC）的制备详细信息可以在支持信息中找到。
Characterization of PyrNMCs, PyrNCs, and PyrMCs
PyrNMC、PyrNC 和 PyrMC 的表征
The PyrNMCs could be centrifuged at 4000 rpm for 3 min to effectively separate them from the coexisting PyrNMCs. The determination of MCs and NCs in coexisting systems could be accomplished by quantifying the concentration of pyraclostrobin. A laser particle size analyzer (LS-POP 6, Zhuhai OMEC Instrument Co., Ltd., Guangdong, China) and a nanolaser particle size analyzer (90Plus PALS, Brookhaven Instruments Corporation, USA) were used for detecting different particle sizes. Therefore, the size of the PyrNCs was determined using a nanolaser particle size analyzer, while that of the PyrMCs was determined using a laser particle size analyzer. The morphology of PyrNMCs, PyrMCs, and PyrNCs was observed by using scanning electron microscopy (SEM; Phenom Pro, Phenom-World in Eindhoven, the Netherlands), while the elemental composition was determined through energy dispersive spectroscopy and X-ray photoelectron spectroscopy (XPS) analysis (ESCALAB Xi+, Thermo Scientific, USA). Fourier transform infrared spectroscopy (FTIR; TENSOR II, Bruker Optics, Germany) was used to determine the chemical composition of PyrNMCs, PyrMCs, PyrNCs, and PyrMCs@polyurea. A thermogravimetric analyzer (TGA, Discovery-TGA550, TA, USA) was used to determine the thermal stability of PyrNMCs, PyrMCs, PyrNCs, and PyrMCs@polyurea. The PyrNMCs, PyrMCs, PyrNCs, and PyrMCs@polyurea were washed three times and dried for the above characterization.
PyrNMCs 可以在 4000 rpm 下离心 3 分钟，以有效地将它们与共存的 PyrNMCs 分离。共存系统中 MC 和 NC 的测定可以通过量化唑菌胺酯的浓度来完成。使用激光粒度分析仪（LS-POP 6，珠海欧美克仪器有限公司，中国广东）和纳米激光粒度分析仪（90Plus PALS，布鲁克海文仪器公司，美国）来检测不同的粒度。因此，PyrNCs的尺寸使用纳米激光粒度分析仪测定，而PyrMCs的尺寸使用激光粒度分析仪测定。使用扫描电子显微镜（SEM；Phenom Pro，荷兰埃因霍温的 Phenom-World）观察 PyrNMCs、PyrMCs 和 PyrNCs 的形貌，同时通过能量色散谱和 X 射线光电子能谱（XPS）测定元素组成）分析（ESCALAB Xi+，Thermo Scientific，美国）。使用傅里叶变换红外光谱（FTIR；TENSOR II，Bruker Optics，德国）测定 PyrNMCs、PyrMCs、PyrNCs 和 PyrMCs@polyurea 的化学成分。使用热重分析仪（TGA，Discovery-TGA550，TA，美国）测定 PyrNMCs、PyrMCs、PyrNCs 和 PyrMCs@聚脲的热稳定性。将PyrNMCs、PyrMCs、PyrNCs和PyrMCs@聚脲洗涤3次并干燥以进行上述表征。
Release Profiles and Encapsulation Efficiency
释放曲线和封装效率
The release profiles and encapsulation efficiencies of PyrNMCs, PyrMCs, PyrNCs, and PyrMCs@polyurea were measured according to previous reports,[30] with minor modifications. The details of the procedure can be found in the Supporting Information.
PyrNMCs、PyrMCs、PyrNCs 和 PyrMCs@polyurea 的释放曲线和包封效率根据之前的报告进行测量， 30进行了较小的修改。该过程的详细信息可以在支持信息中找到。
Biological Activity Evaluation
生物活性评价
使用菌丝生长速率法测定不同制剂对辣椒疫病菌的功效。 31简而言之，辣椒疫霉分离物是使用 8 毫米打孔器从培养 3-4 天的边缘获得的。然后将菌丝体面向下的一面接种到含有不同浓度制剂的PDA平板上，并在25°C黑暗中培养5天。处理后5天检查菌落面积并在显微镜（Olympus cx23，日本）下观察其菌丝形态。处理后5天采用交叉法测定辣椒疫病菌的直径，每个处理重复3次。抑制率按下式计算：
选择同龄、同大小的辣椒叶，并去掉其叶柄。然后，将不同的制剂稀释至20、40、60、80和100mg L -1的浓度。将稀释剂均匀地施用到叶表面，确保完全蒸发，然后再接种到叶右上方的 5 毫米菌丝体塞上。叶子的叶柄用湿纸覆盖，以确保最佳的湿度水平。随后将叶子在光照和黑暗条件(16:8)下在水琼脂培养基中培养4天，之后检查病变区域。采用ImageJ软件测量病灶面积，每个治疗重复3次。抑制率按下式计算：
Pot Experiment 盆栽实验
To evaluate the application effect of PyrNMCs, a pot experiment was conducted in a controlled environment of an artificial greenhouse with a temperature set at 25 °C and light and dark conditions set at a ratio of 16:8. The soil was selected from the greenhouse of Dongdawu Town, Tai'an City, Shandong Province (116.9374141 °E 35.9858284 °N). Each pot was filled with 4000 g of soil, followed by the transplantation of pepper seedlings. When pepper seedlings reached the 8–10 leaf stage, plants exhibiting similar growth conditions were selected for a pot experiment. Subsequently, different formulations were diluted to 70 mg L−1, and 0.01% silicone additive was added to the diluent. The diluent was sprayed evenly on the pepper plants, which were allowed to dry naturally. Pepper leaves of the same size on the upper, middle, and lower parts of the plants were selected for inoculation with P. capsici. The leaf was inoculated on one side by mycelium inoculation and moisturized with a bag postinoculation. The lesion area of the leaves was recorded 3 days after inoculation. and the lesion area of the leaves was recorded on the sixth day after the first inoculation. The control group was sprayed with a solution containing 0.1% Silwet 903. In addition, the leaves were collected and cultured in a water agar medium due to defoliation caused by P. capsici infection. Fallen leaves were also treated via inoculation. Each treatment was repeated three times.
为了评价PyrNMCs的应用效果，在人工温室的受控环境中进行盆栽实验，温度设定为25℃，光暗条件设定为16:8。土壤选自山东省泰安市东大坞镇温室（116.9374141°E 35.9858284°N）。每盆装土4000克，然后移栽辣椒苗。当辣椒幼苗达到8-10叶期时，选择表现出相似生长条件的植株进行盆栽试验。随后，将不同配方稀释至70 mg L -1 ，并向稀释剂中添加0.01%有机硅添加剂。将稀释剂均匀喷洒在辣椒植株上，让其自然干燥。选择植株上、中、下部大小相同的辣椒叶接种辣椒疫病菌。通过菌丝体接种在叶子的一侧进行接种，并在接种后用袋子保湿。接种后3天记录叶子的病变面积。第一次接种后第六天记录叶片的病斑面积。对照组喷洒含有0.1％Silwet 903的溶液。此外，收集由于辣椒疫霉感染引起的落叶而在水琼脂培养基中培养的叶子。落叶也通过接种进行处理。每个处理重复3次。
UV Resistance of PyrNMCs PyrNMC 的抗紫外线性能
The UV resistance of different formulations was tested on pepper leaves and slides. The detailed procedures for determining the UV resistance of the slides and leaves can be found in the Supporting Information.
在辣椒叶和切片上测试了不同配方的抗紫外线能力。确定载玻片和叶片抗紫外线能力的详细程序可在支持信息中找到。
Enzyme Response of PyrNMCs
PyrNMC 的酶反应
To clarify the interaction between PyrNMCs and leaves and P. capsici, the changes in laccases, cellulases, and pectinases in leaves infected with P. capsici were investigated. The leaves were infected with mycelium, and the changes in laccase, cellulase, and pectinase at 0, 1, 2, 3, and 4 days after infection were investigated. Simultaneously, the changes in laccases, cellulases, and pectinases in P. capsici mycelia were investigated. At the same time, a Na2CO3 concentration of 50 mmol L−1 was used to explore the inhibition of enzymes. The activity of laccase, cellulase, and pectinase was determined using a commercial laccase assay kit, cellulase assay kit, and pectinase assay kit according to the manufacturer's instructions. Each reaction had three replicates.
为了阐明 PyrNMC 与叶片和辣椒疫霉之间的相互作用，研究了感染辣椒疫霉的叶片中漆酶、纤维素酶和果胶酶的变化。叶子被菌丝体感染，并调查感染后0、1、2、3和4天漆酶、纤维素酶和果胶酶的变化。同时，研究了辣椒假单胞菌菌丝体中漆酶、纤维素酶和果胶酶的变化。同时，采用50 mmol L -1浓度的Na 2 CO 3来探讨酶的抑制作用。根据制造商的说明，使用商业漆酶测定试剂盒、纤维素酶测定试剂盒和果胶酶测定试剂盒测定漆酶、纤维素酶和果胶酶的活性。每个反应重复三次。
To investigate the release speed of the PyrMCs and PyrNCs after immersion in a solution containing the three enzymes, 75 U g−1 of each enzyme was added to the water. The determination of release performance was performed as described above. Furthermore, PyrNMCs@65:35 and PyrMCs@polyurea were diluted to 300 ppm, 75 U g−1 of the three enzymes was added to the diluent, and the mixture was incubated under dark conditions at 25 °C for 2 h. The morphology of the samples was observed by SEM.
为了研究 PyrMC 和 PyrNC 浸入含有三种酶的溶液后的释放速度，将 75 U g -1的每种酶添加到水中。脱模性能的测定如上所述进行。此外，将PyrNMCs@65:35和PyrMCs@聚脲稀释至300ppm，将75Ug -1的三种酶添加到稀释液中，并将混合物在黑暗条件下25℃孵育2小时。通过SEM观察样品的形貌。
RNA Isolation and qRT‒PCR
RNA 分离和 qRT-PCR
The procedures related to gene domain prediction, RNA extraction, and qRT–PCR can be found in the Supporting Information.
与基因域预测、RNA 提取和 qRT-PCR 相关的程序可以在支持信息中找到。
Leaf Surface Penetration Test
叶面渗透测试
The penetration of PyrNMCs into the leaves was quantified through fluorescent labeling. Considering that pyraclostrobin was nonfluorescent, FITC was additionally added to the organic phase to prepare PyrNMCs, PyrMCs@polyurea, and PyrEC. The preparation method used for fluorescent PyrNMCs was the same as that described above. The fluorescent micro/nanocapsule was diluted to 70 mg L−1, and 0.01% silicone additive was added to the diluent. Then, the solution was evenly applied to the pepper leaves and moistened for 4 h. The samples were repeatedly rinsed with deionized water for three cycles, followed by drying. The leaves were cut into 3 × 10 mm segments, the leaves were cut into 3 × 10 mm segments, and the fluorescence of the longitudinal section of the leaves was observed by laser layer scanning microscopy system (ZEISS lightsheet Z.1, Oberkochen, Germany).
通过荧光标记对 PyrNMCs 渗透到叶子中进行定量。考虑到唑菌胺酯不具有荧光性，在有机相中额外添加FITC，制备PyrNMCs、PyrMCs@polyurea和PyrEC。荧光PyrNMCs的制备方法与上述相同。将荧光微纳米胶囊稀释至70 mg L -1 ，并在稀释液中添加0.01%有机硅添加剂。然后，将溶液均匀地涂抹在辣椒叶上，并湿润4小时。将样品用去离子水反复漂洗三个周期，然后干燥。将叶片切成3×10mm的段，将叶片切成3×10mm的段，用激光层扫描显微镜系统（ZEISS lightsheet Z.1，Oberkochen，德国）观察叶片纵切面的荧光。 ）。
In addition, PyrNMCs without FITC were treated with the same method as mentioned above on the leaves. The QuEChERS method was used to extract and purify pyraclostrobin from the leaves. The extraction method is described above, and HPLC was used to determine the amount of pyraclostrobin that entered the leaves. Each treatment was repeated three times.
此外，用与上述相同的方法在叶子上处理不含FITC的PyrNMC。采用 QuEChERS 方法从叶子中提取并纯化唑菌胺酯。提取方法如上所述，采用HPLC测定进入叶片中唑菌胺酯的量。每个处理重复3次。
Statistical Analysis 统计分析
HPLC analysis was quantified by the normalized method in this paper. The calculation and analysis of data in the paper were conducted by SPSS Statistics (version 16.0). The value of all experiments was represented as the mean ± SE. The SE was calculated by Tukey's multiple range test (P < 0.05). The data consisted of at least three independent experiments. Origin 2018 was used to make the charts and the curve-fitting analysis. The colors of SEM images were processed by PowerPoint 2019.
本文采用标准化方法对 HPLC 分析进行定量。论文中数据的计算和分析采用SPSSStatistics(16.0版本)进行。所有实验的值表示为平均值±SE。 SE 通过 Tukey 的多范围检验计算（ P < 0.05）。数据至少由三个独立实验组成。使用 Origin 2018 制作图表和曲线拟合分析。 SEM图像的颜色由PowerPoint 2019处理。
Acknowledgements 致谢
This work was supported by the National Natural Science Foundation of China (32272596), the National Key Research and Development Program of China (2022YFD1700500), Major Scientific and Technological Innovation Projects of Shandong Province (2022CXGCO20710), Taishan Industrial Leading Talent Project (tscx202211021).
该工作得到国家自然科学基金（32272596）、国家重点研发计划（2022YFD1700500）、山东省重大科技创新项目（2022CXGCO20710）、泰山市产业领军人才项目（tscx202211021）的支持。
Conflict of Interest 利益冲突
The authors declare no conflict of interest.
作者声明不存在利益冲突。
Supporting Information 支持信息
| Filename 文件名 | Description 描述 |
|---|---|
| adma202409839-sup-0001-SuppMat.docx9.2 MB adma202409839-sup-0001-SuppMat.docx 9.2 MB |
Supporting Information 支持信息 |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
请注意：出版商不对作者提供的任何支持信息的内容或功能负责。任何疑问（缺失内容除外）应直接联系文章的相应作者。
