Nanoprobe-based detection methods for plant diseases

When plants exhibit non-specific symptoms of disease, identifying the pathogen is labor-intensive and time-consuming, usually requiring laboratory diagnostics. The results are often only available after several days. This is problematic, as the risk of transmission is high, especially with highly contagious quarantine pathogens, and the entire plant population would then have to be destroyed.

In the project “Nanoprobe-based detection method for plant diseases” (NanoSPoD), the quarantine pest Tomato brown rugose fruit virus (ToBRFV) is the main focus, as it causes significant yield losses in tomato and pepper plants. To limit its spread, a rapid mobile testing system is being developed for regulatory authorities such as agricultural chambers. In our analysis method, viruses are specifically tagged with antibody-coupled magnetic nanosondes, and the virus quantity is determined using a mobile handheld device via the magnetic signal. After a brief sample preparation, on-site analyses can be completed within minutes, providing a major time advantage over conventional laboratory diagnostics.

To purify the sample and increase sensitivity, viruses can be separated and concentrated from homogenized plant material using magnetic enrichment. The viruses captured by the magnetic nanosondes are collected on the walls of the sample vessel with a magnet. The liquid is removed, and the nanosondes are resuspended in a smaller buffer volume. For measurement, the enriched sample is applied to a column matrix coated with virus-specific antibodies. The viruses marked by the magnetic nanosondes are then captured on the matrix surface, and the virus concentration is determined via a calibration using the magnetic signal. Further development of the magnetic measurement device aims to allow quantitative detection of multiple pathogens in a single sample in the future.

In addition to the mobile test system for defined pathogens, a second approach is being developed for the detection of unknown pathogens. This is based on Next Generation Sequencing (NGS) technologies, which in recent years have made decoding genetic information faster and more cost-effective. In our NGS screening, the RNA from the sample is first sequenced and then the pathogen is identified by matching sequences with the new “MorpheusFood” sequence database, developed by project partner Computomics GmbH.

Within the project, samples from five different pathogens were successfully identified, including the Tobacco mosaic virus (TMV), Tomato brown rugose fruit virus (ToBRFV), Beet mild yellowing virus, Potato virus Y, and Konjac mosaic virus, all of which infect agriculturally and economically valuable plants. TMV was detected even in plants that had been infected for only one day. ToBRFV could also be reliably detected in low-concentration samples.

In the future, the innovative NGS screening method will be expanded to detect other pathogen classes, such as fungal or bacterial agents, and optimized for speed. Potential applications of this powerful analysis approach in environmental monitoring and medical diagnostics are also being pursued. The NGS method offers the advantage of identifying not only known pathogens but also new, previously unknown ones. The availability of mobile sequencing devices makes the field use of our NGS screening feasible in the future.