Nanopore technologies for Septoria Surveillance

Septoria tritici blotch (STB) of wheat is the most economically important disease affecting wheat production in Northern Europe. STB is controlled using a combination of agronomy, genetic resistance and fungicides however, selection and subsequent adaptation of the underlying pathogen populations can lead to breakdowns in host resistance and resistance to chemical controls. As the pathogen adapts we will need to understand the composition of the pest population (or pathotypes) to be able to describe specific differences between members of the same species and how these differences affect the range of wheat varieties affected by STB. An effective and cost-efficient solution for STB will directly depend on our understanding of the underlying pathogen (Zymoseptoria tritici).

The use of advanced sequencing technologies has the potential to quickly and efficiently screen many individual pathogen isolates, providing a wealth of information at a low cost. This type of approach has already been demonstrated to be a powerful tool in yellow rust of wheat. However, the cost of purchasing machinery required to conduct sequencing is often prohibitive and runs can take between 24-48 hours to complete. There are also requirements for high-powered computing facilities and experienced operators to conduct the relevant analyses to arrange sequencing data in to a practical format.

NanoPath will employ the MinION sequencer from Oxford Nanopore Technologies as a surveillance tool to improve the speed and ease at which researchers can identify and survey new pests in the field. This will first assess the utility of the MinION and help to develop a model and computational platform to apply nanopore sequencing to a wider range of crop pathogens. The MinION device is portable and able to generate sequencing data following a relatively easy-to-use process. We also propose to implement a data analysis pipeline that will make use of a number of open-source primary data software tools optimised for the discovery and characterisation of genetic variants this crop pathogen.

Septoria tritici blotch (STB) of wheat, caused by the plant pathogen Zymoseptoria tritici, is the most economically important disease affecting wheat production in Northern Europe. STB is controlled using a combination of agronomy, genetic resistance and fungicides however, selection and subsequent adaptation in pathogen populations can lead to erosion of host resistance. These adaptations are associated to the occurrence of new pathotypes. Understanding the composition of Z. tritici populations and occurrence of different pathotypes is important when attempting to manage the disease effectively, especially when changes in virulence are apparent. Recently, changes in virulence in Z. tritici isolates have been reported in the UK. There is a large significant increase in the severity of disease symptoms when plants with partial resistance were inoculated with the new Zt isolate resulting in a higher frequency of dead leaves and a greater number of lesions than compared to the historic isolate. As suggested by experiments we conducted the new isolates are able to reproduce asexually having eroded the resistance in the host. NanoPath will employ the MinION sequencer from Oxford Nanopore Technologies as a surveillance tool to improve the speed and ease at which researchers can identify new Z. tritici isolates in the field. We will first assess the utility of the MinION for STB pathotyping and help develop a model and computational platform to apply nanopore sequencing to a wider range of crop pathogens. We also propose to implement a data analysis pipeline that will make use of a number of open-source primary data software tools optimised for the discovery and characterisation of genetic variants in Z. tritici. Concurrently, Illumina short read chemistry (HiSeq 4000) will be used to sequence the same isolates (10-20x coverage) as a strategy to validate the quality of reads produced on the MinION.

The delivery of the NanoPath's objectives is focused on current diagnostics and surveillance needs for which suitable tools are lacking. In this way achieving impact is an integral part of the proposal. The recent advances in sequencing technologies have opened up new opportunities for the implementation of targeted approaches for the characterisation and surveillance of pathogens in general. The Programme's objectives will directly contribute solutions in areas of research relevant to the BBSRC scientific priority in food security and integrate novel methods in genomics with emerging data analysis protocols.

Academic, Economic and Commercial Impacts
The development of the NanoPath platform will generate new opportunities for collaborative work with R&D groups in industry and academic institutions. The characterisation of the genetic diversity in Septoria, for instance, will enable and support the design of novel approaches to control this crop disease which is one of the most important in Northern Europe in terms of economical losses.

Similarly the infrastructure and technology advances described in the objectives will have a direct effect in key UK R&D areas such small biotechnology industries and academic labs with limited access to compute power. The use of the MinION device coupled with the development of bioinformatics analysis pipelines that can be executed in cloud-based systems will be accessible to a great number of researchers with limited requirement for laboratories or computing facilities.

The transformative effect of the availability of large datasets in the development of technologies and services is undoubtedly one of the main drivers supporting the biotechnology industry. One example is the effect that genomics assisted methods will have the understanding of the genetic diversity in crop pests. The availability of pre-competitive data generated by NanoPath will have a direct impact on research in the crop pathogens area.

Societal impacts
The implementation of NanoPath will directly impact the agricultural sector with the potential to indirectly support improved yields by increasing productivity.

It is widely recognised that the shortage of expertise and skill in biomathematics and informatics in the UK and across the world is a major risks for a future development of data-driven science. In this context and in a modest measure we hope to support the training of talented computational biologists by demonstrating the value the MinION (or more general nanopore technologies) to implement effective surveillance strategies.

Policy: BBSRC, research councils and UK
NanoPath will contribute to reinforce the UK's reputation of leaders in the agri-food sector by supporting the use of cutting-edge technologies to monitor plant crops. The availability of reliable monitoring and surveillance tools will be one of the key pillars to support the most modern crop disease controls.