Non-Destructive Detection of Below-Ground Plant Pathogens: VOC Profiling by Frequency Comb Spectroscopy

Research into pests and diseases is essential to fulfil the goal of ensuring food security. Aerial pests and disease are easier to quantify and identify. However, the detection and determination of the pathogen burden is far more difficult for soil-borne organisms. An example are the plant parasitic nematodes that have devastating impacts on plant productivity throughout the world. This is an issue both in research and strategically in the field. Currently sampling methods are time consuming, destructive and costly. New methods of detection are urgently required to speed up research into these pathogens.

Plant parasitic nematodes represent an important global threat to agricultural production. Despite this, as soil-borne pathogens plant parasitic nematodes are classically under-diagnosed. The nematodes display a wide range of parasitic strategies. Some feed destructively as they move through plant roots whilst others reprogramme root cells to form a novel nutrient sink from which they feed for many weeks. These diverse parasitic strategies result in differing levels of direct plant damage and changes to plant physiology and thus are likely to elicit different plant responses. Plant parasitic nematodes are therefore an excellent patho-system around which to develop and test a novel system for detection and diagnosis of plant disease.

Volatile organic compounds (VOCs) are abundant chemicals that are emitted by organisms in all terrestrial and marine ecosystems. The chemical composition of plant-emitted VOCs and their abundance can carry information about the plants' physiological status and the stresses to which they have been subjected. VOCs are an essential part of plant defense systems and are emitted in response to pest and pathogen attack. We have established that plants release VOCs in response to infection by plant-parasitic nematodes and propose to develop new technology to detect and monitor VOCs in order to establish a VOC 'fingerprint' for nematode infection.

Infrared spectroscopy acts as a chemical 'fingerprinting' mechanism since every molecule that can absorb infrared light does so at specific, unique frequencies. However, VOCs emitted by plants are oftentimes in the low parts-per-billion (ppb) levels, which can test the limits of instrumentation sensitivity, and the overlapping infrared spectra of different VOCs can be an issue for low spectral resolution techniques. A new frequency comb laser-Fourier transform infrared spectrometer (FC-FTIR) will be built to reach the necessary ppb level molecular sensitivities by using a long interaction path length, with the molecular selectivity being a result of the simultaneously broadband and high resolution infrared laser. Thus, the infrared spectra collected with FC-FTIR can yield information about an overall profile of plant VOCs emitted, and even the identity and quantity of the VOCs. Statistical analysis of healthy versus infected plant VOC emission profiles will aid in determining the relative health of the plant and potentially linking the emission profile to a specific plant pathogen.

This work addresses the current need to move towards a technology that is both sensitive and selective in order to identify a profile of plant VOCs emitted under different pathogen burdens, while still being general enough to detect all possible emitted VOCs (i.e. not targeting a specific VOC composition based on an assumed plant disease). This technology potentially allows the identification of a broad range of plant pathogens in a non-destructive manner, in real time. It therefore has impact across a wide range of biological disciplines in plant science and beyond. Ideally, this instrument would also be able to monitor VOC profiles in the field in order to locate infected areas within crops because of the highly directional nature of using a coherent light source, ensuring a rapid response for remedial action thus minimizing infection impact and mitigating crop loss.

Research into plant pests and diseases is essential to fulfil the goal of ensuring food security, however detection and diagnosis is difficult for soil-borne organisms. Plant parasitic nematodes that have devastating impacts on plant productivity throughout the world are one good example. Current sampling methods are time consuming, destructive and costly. New methods of detection are urgently required to speed up research into these pathogens.

Volatile organic compounds (VOCs) are emitted by plants in response to pest and pathogen attack. We have established that plants release VOCs in response to infection by nematodes. In order to measure trace amounts of VOCs and to be able to differentiate potentially small changes in VOC profiles, a sensitive detection technique must be used. Long-path optical measurements, such as differential optical absorption spectroscopy (DOAS), are proven techniques to measuring trace gasses in the atmosphere. In this project, we will be coupling a unique light source with a cavity-enhanced DOAS style measurement: a frequency comb laser, which is a simultaneously broadband and high resolution coherent light source. A new frequency comb-Fourier transform infrared spectrometer (FC-FTIR) will be built and coupled to an off-axis re-entrant optical cavity, reaching the necessary parts-per-billion level molecular sensitivities for plant VOC emission detection, with the molecular selectivity being a result of the infrared light source. We will infect potato plants with one of three species of nematode that each has a different mode of parasitism. We will then measure plant VOC emission from infected and uninfected plants using on-line FC-FTIR analysis in addition to off-line analysis by PTR-MS. Principal component analysis will be used in the interpretation of the infrared spectra and mass spectra of healthy and infected potato plants.