Data_Sheet_1_Biopesticide Trunk Injection Into Apple Trees: A Proof of Concept for the Systemic Movement of Mint and Cinnamon Essential Oils.docx

The use of conventional pesticides is debated because of their multiple potential adverse effects on non-target organisms, human health, pest resistance development and environmental contaminations. In this setting, this study focused on developing alternatives, such as trunk-injected essential oil (EO)-based biopesticides. We analysed the ecophysiology of apple trees (Malus domestica) following the injection of Cinnamomum cassia and Mentha spicata nanoemulsions in the tree’s vascular system. Targeted and untargeted volatile organic compounds (VOCs) analyses were performed on leaf-contained and leaf-emitted VOCs and analysed through dynamic headspace–gas chromatography–mass spectrometry (DHS-GC-MS) and thermal desorption unit (TDU)-GC-MS. Our results showed that carvone, as a major constituent of the M. spicata EO, was contained in the leaves (mean concentrations ranging from 3.39 to 19.7 ng gDW–1) and emitted at a constant rate of approximately 0.2 ng gDW–1 h–1. Trans-cinnamaldehyde, C. cassia’s major component, accumulated in the leaves (mean concentrations of 83.46 and 350.54 ng gDW–1) without being emitted. Furthermore, our results highlighted the increase in various VOCs following EO injection, both in terms of leaf-contained VOCs, such as methyl salicylate, and in terms of leaf-emitted VOCs, such as caryophyllene. Principal component analysis (PCA) highlighted differences in terms of VOC profiles. In addition, an analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA) revealed that the VOC profiles were significantly impacted by the treatment. Maximum yields of photosystem II (Fv/Fm) were within the range of 0.80–0.85, indicating that the trees remained healthy throughout the experiment. Our targeted analysis demonstrated the systemic translocation of EOs through the plant’s vascular system. The untargeted analysis, on the other hand, highlighted the potential systemic acquired resistance (SAR) induction by these EOs. Lastly, C. cassia and M. spicata EOs did not appear phytotoxic to the treated trees, as demonstrated through chlorophyll fluorescence measurements. Hence, this work can be seen as a proof of concept for the use of trunk-injected EOs given the systemic translocation, increased production and release of biogenic VOCs (BVOCs) and absence of phytotoxicity. Further works should focus on the ecological impact of such treatments in orchards, as well as apple quality and production yields.

传统农药因其对非靶标生物、人类健康、害虫抗药性演化及环境污染存在多种潜在负面影响而备受争议。在此背景下，本研究聚焦于开发替代方案，例如树干注入型精油（essential oil, EO）基生物农药。我们针对苹果（Malus domestica）在其维管系统中注入肉桂（Cinnamomum cassia）与留兰香（Mentha spicata）纳米乳剂后的生理生态特性展开分析。针对叶片内源与叶片释放的挥发性有机化合物（volatile organic compounds, VOCs）分别开展靶向与非靶向分析，分析手段采用动态顶空-气相色谱-质谱联用（DHS-GC-MS）与热脱附单元-气相色谱-质谱联用（TDU-GC-MS）。

研究结果显示，作为留兰香精油主要成分的香芹酮可在叶片中检出（平均浓度范围为3.39~19.7 ng·gDW⁻¹），且以约0.2 ng·gDW⁻¹·h⁻¹的恒定速率释放。作为肉桂精油主要成分的反式肉桂醛则在叶片中累积（平均浓度分别为83.46与350.54 ng·gDW⁻¹），但未检测到释放。此外，研究结果表明，精油注入后，叶片内源VOCs（如水杨酸甲酯）与叶片释放VOCs（如石竹烯）的种类与含量均有所提升。主成分分析（PCA）结果显示不同处理组的VOC谱存在显著差异。此外，相似性分析（ANOSIM）与置换多变量方差分析（PERMANOVA）结果显示，处理组对VOC谱存在显著影响。

光系统II最大量子产额（Fv/Fm）的取值范围为0.80~0.85，表明实验全程苹果树均保持健康状态。靶向分析结果证实，精油可通过植物维管系统实现系统性转运。而非靶向分析则表明，上述精油可诱导植物产生潜在的系统获得性抗性（SAR）。最后，通过叶绿素荧光测定结果可知，肉桂与留兰香精油对处理苹果树未表现出植物毒性。

综上，鉴于精油可实现系统性转运、生物源挥发性有机化合物（biogenic VOCs, BVOCs）的产生与释放量提升，且无植物毒性，本研究可视为树干注入型精油应用的概念验证。未来研究可聚焦于该处理方式在果园中的生态影响，以及苹果品质与产量相关指标。