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

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.

Plant parasitic nematodes (PPNs) exert a significant economic burden on global agriculture - estimated at over £100 billion per year. Diagnosis of PPN 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 analysis. As a result, current PPN diagnostics lack throughput, convenience, and sensitivity. To address this, across three Work Packages, assembles a multidisciplinary team to undertake the radical transformation of rapid evaporative ionisation mass spectrometry (REIMS) to allow in situ and non-destructive analysis of plant material.

Work Package 1: will develop the use of a CO2 laser to modify REIMS as a laboratory-based platform for analysis of plant material. This will allow analysis of the spatial distribution of metabolites and identify the optimal location for PPN diagnostic analysis, a device that is capable of the safe containment of a CO2 laser for the in situ analysis of plant leaves, and explore alternative post-desorption matrices to enhance detection of plant metabolites.

Work Package 2: will explore the potential of REIMS as an in-field tool for analysis of plant material. We will identify alternative methods of heating for ion generation directly from plant leaves - namely the use of alternative wavelength lasers and 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) to complete PPNs infection studies. The REIMS modalities developed will analyse infected plants across a range of PPN species, timepoints, and infection load. The effect of REIMS analysis on plant health and fitness will also be measured and the potential of portable single quadrupole instruments using computational modelling explored.