Alive or dead: that is the question

If plant material has been affected by harmful bacteria, fungi or insects, it needs to be disinfected. The identification of living and dead organisms following disinfection is important. If it appears during inspection that the plant material has been contaminated with a harmful organism but that this organism is dead, there is no further risk of harm.

In the application of biological plant protection or fertilisers, it is significant to be able to determine the vitality of the organisms used (bacteria, fungi, viruses). If the biological products do not contain any living organisms or contain too few living organisms, it will not be sufficiently effective.

Molecular techniques

Molecular techniques are increasingly used for the detection and quantifying of organisms. Molecular techniques have significant advantages when compared to more classical techniques such as luring, plating and biotesting. Molecular techniques are sensitive, specific and the results are quickly available. Moreover, these techniques can also detect dead organisms.

Furthermore, morphological detection techniques can also visually identify dead organisms, but these techniques are time consuming and less reliable.

RNA detection

The collaborative efforts of Wageningen University & Research and the inspection services has, in recent years, resulted in the development of several promising molecular techniques for the specific detection and quantifying of living and dead plant pathogens. These techniques are based on the detection of RNA that is degraded rapidly in dead organisms and on the possibility of measuring the permeability of the cell membrane in bacteria. In dead bacteria, this cell membrane becomes permeable. Several of these molecular techniques have since been validated and found to be suitable for routine use.

However, for the effective use of these techniques in practice, questions remain about the processes that occur when these organisms die. Further insight into these processes will determine whether the molecular methods that have been developed are universally applicable.

Objective(s)

• Understanding the processes of RNA degradation and cell membrane permeability that take place during the death of organisms.
• The integration of this knowledge in diagnostic protocols designed to detect living and dead organisms must lead to internationally accepted methodologies that can cost-effectively and quantitatively demonstrate the presence of living organisms.

Methodology

Well-characterised biological material is examined for the changes in molecularly determined aspects: the monitoring of the dynamics in the splicing/maturation and degradation of RNA and the penetration of PMA in dead cells. This concerns an integrated analysis of these processes using Next Generation Sequencing which results in a cost effective overview of the different stages of necrosis. Particular consideration will be given to the difference between RNA and DNA detection and the difference between PMA-DNA and DNA detection.

Further information

Would you like to find out more about what ‘dead’ or ‘living’ detection could mean for you? Then contact us!