Adaptation of REIMS for in situ, non-destructive and potential on-site diagnosis of plant parasitic nematode infection

植物寄生线虫（plant parasitic nematodes，PPN）是一种全球性的土传害虫，对全球农业造成严重的经济负担。通过与控制相关的成本和作物产量损失，估计这些寄生虫每年造成的经济损失超过1000亿英镑。PPN作物感染的诊断不可能基于植物叶子的视觉检查，需要在田间评估作物根并收集PPN组织用于基于实验室的分子诊断，和/或收集土壤样品，然后洗涤和视觉识别/计数孢囊。因此，当前的PPN诊断缺乏吞吐量、便利性和灵敏度。开发一种不需要线虫学专业知识就可以进行的非破坏性诊断测定将是新颖的和破坏性的。这种检测将允许快速/早期识别现场感染状态，并将有助于及时的管理决策，并提供深入了解PPN infection. This proposal的基础生物过程将汇集一个多学科的研究团队进行彻底改造的敞开式电离质谱方法称为快速蒸发电离质谱（REIMS）。该技术最初是作为临床医学中的一种方法开发的，用于在手术切除期间实时识别癌组织边缘。然而，植物材料的分析提供了一种完全不同的样品类型，包括成分、结构异质性和种间变异。这需要REIMS方法的变革性发展，以使其适用于PPN感染诊断的植物材料分析以及更广泛的植物代谢研究和应用。在三个工作包的过程中，该项目将利用调查团队内部可用的专业知识的独特组合，开发用于REIMS分析的植物叶片加热的多种方法，并将最佳方法应用于PPN感染的诊断。将开发CO2激光器的使用，以修改REIMS作为一个实验室-该平台用于分析植物材料，以诊断PPN感染，并进行更广泛的生化和代谢分析。我们将创建一个可以分析植物叶片的设置，以揭示代谢物的空间分布，并确定用于PPN诊断分析的最佳位置。我们还将创建一个能够安全容纳CO2激光器的设备，该设备可用于植物叶片的原位分析，而无需收获。工作包2：将探索REIMS作为PPN和非寄生植物病原体诊断和植物代谢过程分析的现场工具的潜力。该工作包将确定直接从植物叶片产生离子的加热替代方法-即使用与叶绿素的吸收曲线相匹配的激光器和聚焦可见波长光设备。它还将使用REIMS确定可检测的植物代谢特征的范围和识别，为植物生物科学界提供更广泛的实用性。工作包3：将使用番茄植物（S。番茄Moneymaker）作为完成PPN感染研究的模式物种。工作包1和2中开发的各种REIMS模式将用于分析一系列PPN物种、时间点和初始感染负荷的受感染植物。还将测量REIMS分析对植物健康和适应性的影响，以确定REIMS是否可用作非破坏性的筛选工具。最后，将利用低分辨率质谱信息的计算建模来探索便携式单四极杆质谱仪的潜力。

Plant parasitic nematodes (PPNs) are globally distributed, soil-borne pests, which exert a significant economic burden on global agriculture. Through costs associated with control, and crop yield loss, it has been estimated that these parasites are responsible for economic losses greater than £100 billion per year. Diagnosis of PPN crop infection is impossible based on visual inspection of plant foliage, requiring assessment of crop roots in the field and collection of PPN tissue for laboratory-based molecular diagnostics, and/or collection of soil samples, followed by washing and visual identification / counting of cysts. As a result, current PPN diagnostics lack throughput, convenience, and sensitivity. The development of a non-destructive diagnostic assay that could be conducted without the need for nematology expertise would be novel and disruptive. Such an assay would allow rapid / early identification of field infection status and would facilitate timely management decisions and provide insights into the underpinning biological processes of PPN infection.This proposal will bring together a multidisciplinary research team to undertake the radical transformation of the ambient ionisation mass spectrometry method known as rapid evaporative ionisation mass spectrometry (REIMS). This technology was originally developed as a method in clinical medicine for the real-time identification of cancerous tissue margins during surgical resection. The analysis of plant material, however, provides an entirely different sample type with regards to composition, structural heterogeneity, and inter-species variation. This necessitates the transformative development of the REIMS method to adapt it for the analysis of plant material for PPN infection diagnosis and for wider plant metabolic studies and applications. Over the course of three work packages, this project will utilise the unique combination of expertise available within the Investigatory team to develop multiple methods of plant leaf heating for REIMS analysis and apply the optimal method to the diagnosis of PPN infection.Work Package 1: will develop the use of a CO2 laser to modify REIMS as a laboratory-based platform for analysis of plant material for the diagnosis of PPN infections and wider biochemical and metabolic profiling. We will create a set-up by which plant leaves can be analysed to reveal the spatial distribution of metabolites and identify the optimal location for analysis for PPN diagnosis. We will also create a device that is capable of the safe containment of a CO2 laser that can be used for the in situ analysis of plant leaves without the requirement for their harvest.Work Package 2: will explore the potential of REIMS as an in-field tool for PPN and non-parasitic plant pathogen diagnosis and analysis of plant metabolic processes. This work package will identify alternative methods of heating for ion generation directly from plant leaves - namely the use of lasers that match the absorbance profile of Chlorophyll and that of a focussed visible wavelength light device. It will also determine the range and identification of detectable plant metabolic features using REIMS to inform wider utility for the plant bioscience community. Work Package 3: will use the tomato plant (S. lycopersicum cv. Moneymaker) as a model species on which to complete PPNs infection studies. The various REIMS modalities developed in Work Packages 1 and 2 will be used to analyse infected plants across a range of PPN species, timepoints, and initial infection load. The effect of REIMS analysis on plant health and fitness will also be measured to determine whether REIMS can be used as a screening tool that is non-destructive. Finally, the potential of portable single quadrupole mass spectrometers will be explored using computational modelling of low-resolution mass spectral information.