Biosensor for catching a cereal killer

Background: Wheat is a staple food for about two billion people in the world. The fungal pathogen Zymoseptoria tritici, causes one of the most damaging foliar diseases in wheat (Septoria tritici blotch, STB), resulting in reduced crop yield and increased production costs because of fungicide applications (with an average cost of ~€1 billion per year). Fungal infection is characterised by an unusually long latent infection phase, where infected plants remain symptomless for 10-14 days. Following this there is a rapid transition to necrotrophic growth characterised by the death of infected leaves and appearance of fungal fruiting bodies in infected tissue.

Control of fungal infections is heavily reliant on the azole, quinone outside inhibitors (QoI) and succinate dehydrogenase inhibitor (SHDI) classes of fungicides. The extreme diversity of Z. tritici field populations (and sexual lifestyle under field conditions) mean that emergence of fungicide resistance is a rapidly growing threat to global wheat productivity [1]. Resistance breaking strains frequently exhibit overexpression of chemical efflux pumps, increase the expression of or accumulate site mutation in fungicide target genes (e.g. CYP51) thus allowing them to escape effective chemical control [2].

To maximise plant productivity, farmers must know (i) when their crops are under threat from pathogens, and (ii) the efficacy of chemical protectants before application. Knowing the presence of fungal infection as early as possible along with determining their fungicide sensitivity profile would allow farmers to make better-informed and timely decisions of fungicide applications in their crops. This project aims to develop a close to real-time sensor technology allowing farmers and agronomists to better assess the pathogen threat to wheat crops.

Objectives: The objectives of the project are as follows:
(1) To enable early and accurate diagnosis of fungal-infected wheat.
(2) To identify fungicide resistance strains.
(3) To develop an easy-to-use and rapid bioassay that is suitable for in-field detection of infected wheat and the fungicide sensitivity of the pathogens.

Methodology:
The wheat plants will be inoculated with Z. tritici field isolates (or GM strains) to generate biological samples for developing the sensor technology. The method is readily adaptable for other phytopathogens, where development of fungicide resistance is widespread.
We will develop a waveguide-excited fluorescence microscopy method for determining the sensitivity of fungi to different fungicides. Using waveguide excitation will result in higher signal-to-noise ratios because the excitation intensity will be higher than in conventional fluoresce microscopy. This allows the use of uncooled cameras and short excitation times to reduce photobleaching.

We will develop a portable instrument using 3D printing of carbon-fibre composite material using techniques we have already used to produce optical instrumentation.We will develop a hydrogel waveguide based sensor [3] where antibodies to Z. tritici surface antigens will be immobilized, allowing capture of the fungal cells from samples. As the waveguide is of low index, the penetration depth of the evanescent field is much greater than that of techniques such as SPR, which generates a much stronger interaction between the optical mode and the captured cells [4]. In one region of the surface, the cells with be treated for a suitable period with a fungicide, after which both areas will be treated with a viability stain such as FUN-1. When excited with blue light at ~488 nm, live cells produce red fluorescence, while dead cells fluoresce green. The ratio of red to green fluorescence in the two regions will indicate the pathogen’s susceptibility to the fungicide. This technique could be extended to multiple regions for screening many fungicides simultaneously.