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

植物寄生线虫(PPN)是一种全球性分布的土传害虫，给全球农业带来了巨大的经济负担。通过与控制和作物产量损失相关的成本，估计这些寄生虫每年造成的经济损失超过1000亿GB。PPN作物感染的诊断不可能基于对植物叶片的肉眼检查，需要对田间作物的根进行评估，收集PPN组织以进行基于实验室的分子诊断，和/或收集土壤样本，然后对包囊进行清洗和目视识别/计数。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即可进行的非破坏性诊断分析将是新颖和具有破坏性的。这种化验方法可快速/及早识别现场感染情况，并有助作出及时的管理决定，并提供有关PPN感染的基本生物过程的见解。这项建议将汇集一个多学科的研究小组，对称为快速蒸发电离质谱仪(REIMS)的环境电离质谱学方法进行彻底的改造。这项技术最初是作为临床医学中的一种方法开发的，用于在手术切除过程中实时识别癌组织边缘。然而，植物材料的分析在成分、结构异质性和物种间差异方面提供了完全不同的样本类型。这就需要RIMS方法的变革性发展，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中现有的独特专业知识组合来开发多种植物叶片加热方法，用于RIMS分析，并将最佳方法应用于PPN感染的诊断。工作包1：将开发使用二氧化碳激光来修改REMS，作为诊断PPN感染和更广泛的生化和代谢特征的植物材料分析的基于实验室的平台。我们将创建一种可以分析植物叶片的装置，以揭示代谢物的空间分布，并确定用于PPN诊断的最佳分析位置。我们还将创建一种能够安全地容纳二氧化碳激光的设备，该设备可以用于植物叶片的原位分析，而不需要收割。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。这一工作包将确定直接从植物叶片产生离子的替代加热方法--即使用与叶绿素的吸收曲线相匹配的激光和聚焦可见光波长装置的吸收曲线。它还将使用REMS确定可检测到的植物代谢特征的范围和识别，以向植物生物科学界提供更广泛的实用信息。工作包3：将使用番茄植株(S.lycopsicum cv.MoneyMaker)作为完成PPNS感染研究的模式物种。工作包1和2中开发的各种REIM模式将用于分析一系列PPN种类、时间点和初始感染负荷上的受感染植物。还将测量REMS分析对植物健康和适合度的影响，以确定REMS是否可以用作非破坏性的筛查工具。最后，将利用低分辨率质谱学信息的计算模型来探索便携式单四极质谱计的潜力。

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.