Fraunhofer Institute for Molecular Biology and Applied Ecology IME
Fraunhofer Institute for Molecular Biology and Applied Ecology IME

© Fraunhofer IME | Lena J. Freund

Pathogen diagnostics

Plant pests and pathogens are a major threat to crop production worldwide. This has increased dramatically in recent years. Globalization, trade and climate change play an important role. In 2019, the FAO estimated that between 20 and 40 percent of global crop production is lost to pests each year. Plant diseases, for example, cost the global economy around 220 billion US dollars each year. The damage is caused by pathogens from the group of bacteria, fungi, insects and viruses.

Traditional methods for the identification of plant pathogens are the observation of symptoms as well as the cultivation of the pathogens for morphological identification by microscopy. Nowadays, widely used diagnostic methods are based on immunological technologies such as enzyme-linked immunosorbent assay (ELISA) or lateral flow immunoassay (LFIA). In laboratories, nucleic acid-based methods for pathogen detection and identification are increasingly gaining acceptance. The polymerase chain reaction (PCR) and its variants are usually very specific and sensitive, and also relatively fast and inexpensive, but nevertheless have some disadvantages, as special equipment and trained personnel are required.

To control and, if possible, prevent plant diseases and the spread of pathogens to new areas, however, it is imperative to develop methods for early detection that can be used on-site in the field. For the realization of such point-of-care diagnostics, the interplay of the following properties is a prerequisite: High specificity, sensitivity, reproducibility, speed, cost-effectiveness, and multiplex detection capability paired/combined with ease of use so that low-trained personnel can perform the tests without having to use specialized laboratory equipment.

Technologies based on isothermal amplification of nucleic acids, such as loop-mediated isothermal amplification (LAMP), optimally fulfill these requirements for point-of-care diagnostics. Therefore, the Department of Functional and Applied Genomics focuses the R&D work on PoC diagnostics of plant pathogens on this technology.

Viral diagnostic

Viruses are a major cause of plant diseases, accounting for nearly 50 percent of the pathogens responsible for emerging or reemerging plant diseases worldwide. The economic impact is estimated at more than $30 billion annually. Plants, infected by viruses, cannot be controlled by crop protection products. When a crop area is heavily infested, the only option is typically to burn the affected plants. Control of plant viruses therefore focuses on preventive measures such as regular inspection of cultivated areas, clear quarantine regulations, interruption of infection chains and use of virus-free seed and planting material. Further advancing point-of-care diagnostics of plant viruses is therefore of particular importance.

Although several methods, mostly antibody-based, have already been developed for the diagnosis of plant viruses, the enormous variability of plant virus genomes even within a single virus family makes their accurate and reliable detection difficult. Moreover, single step detection of plants infected with several different viruses with commercially available diagnostic kits remains challenging, if not impossible, due to the limitations of the underlying biochemistry.  

Rapid test for potato viruses: Viruses threaten the potato harvest - innovations for easy detection are urgently needed.

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that affect tuber quality and reduce yields by up to 50 percent.

© BNA | Johannes Pawlik
Tuber infected with potato virus Y.
© BNA | Johannes Pawlik
Cross-section of a potato tuber infected with TRV.

One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV) and transmitted by nematodes. Necrotic spots appear on the surface of infected potato tubers. Inside the tuber necrotic lesions manifest as rust-like concentric patterns and/or mottling spots. The appearance of corky ringspot symptoms on tubers prior to commercialization results in about 45 percent of the affected tubers being downgraded in quality and value, while about 55 percent are declared unsaleable.

The detection of potato viruses typically requires field samples to be sent to a well-equipped laboratory for nucleic acid extraction, followed by RT-PCR (reverse transcription polymerase chain reaction) and melt curve analysis. To improve current disease management practices, we have developed a simple diagnostic method for the reliable detection of TRV and other important potato viruses.

It requires no prior RNA purification and only minimal sample preparation. The subsequent colorimetric loop-mediated isothermal amplification (LAMP) is finally visualized by lateral flow dipstick (LFD) analysis.

Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions.

A major advantage of the method: It can be carried out outside specialized laboratories without the need for complex technical equipment such as PCR cyclers.

Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

Selected Publication


Edgü, G., Freund, L.J. et al.
Fast, precise, and reliable multiplex detection of potato viruses by loop-mediated isothermal amplification (2020) International Journal of Molecular Sciences, 21 (22), art. no. 8741, 1-19. DOI

 

© Fraunhofer IME | Birgit Orthen

neoLAMP

 

We adapted the LAMP test for the detection of SARS-CoV-2.

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