Blog 1, Molecular Analysis: Possible research questions

Blog 1 Molecular Analysis

Possible research questions

Introduction

Just like humans, plants have various pathogenic bacteria’s which can lead to the death of the individuum. A well-known bacterium is Erwinia amylovora which causes fire blight. Another one is Pseudomonas syringae pv. syringae that is known to induce bacterial leaf blight. Especially fire blight is a huge problem in some regions in Switzerland. Even though several remedies are on the market to fight off the bacterium, a complete eradication is seen as impossible. (Arbeitsgruppe Feuerbrand)
Usually the plants can only get treated after the first negative effects of the disease are visible. Often whole branches, or even the complete tree need to be cut down. Some treatments are prophylactic but have a low efficiency rate e.g. slaked lime. (HJ Schärer, A. Häseli, J. Fuchs, L. Tamm).

If the bacteria’s presence could be identified before they are harmful to the plants, countermeasures would be more effective. Molecular biology has some techniques which allow to identify species solely on their specific DNA. The method that allows the multiplication of DNA is called Poly Chain Reaction, short PCR. With PCR it is theoretically possible to amplify one single DNA strand or a specific region in the DNA and receive up to thousands strands or more. Together with specially designed primers and polymerases, DNA regions, so called markers which are indicative for every species, can be amplified and the occurrence of different bacteria is possible to tell.(Madigan 579)

Different molecular techniques

Next to PCR, many methods are possible to identify bacteria species. The easiest one is with MALDI-TOF which isn’t a molecular technique. Anyhow, Flow Cytometry is a molecular method that allows to stain special compounds e.g. cell membrane of different species and analyse them in a fast and uncomplicated way. (Madigan). The cells are suspended in a fluid and then injected into the instrument. Ideally one cell at a time flows through a laser beam and the emitted light is characteristically to the cells and their components scattered. With the fluorescent markers a better differentiation is possible.

Time management and difficulties

PCR is a very fast and easy method that takes no longer than a few hours. Which makes the method difficult for soil or water samples is the fact, that some species are very rare (low densities), or live obligatory anaerobic. Additionally, soil samples first need to be purified, otherwise the PCR will give no clear results. For this step special kits are available. The sample taking at the plantation in Schinznach needs to be well prepared because it’s not clear at which regions of the site Erwinia amylovora and Pseudomonas syringae spp. can be expected. Fire blight that is caused by E. amylovora has only been observed at apple and pear trees. This means that those samples are best be taken at the region where those trees stand.(HJ Schärer, A. Häseli, J. Fuchs, L. Tamm) Pseudomonas syringae spp. can be expected on all kind of plants. (Hirano)

Flow cytometry is also a method which takes no longer than a few hours and gives accurate results. Procedures for analysing the samples are already existent.

Further research

For further research it would be necessary to find the optimal components for the PCR or the flow cytometry and their respective procedure protocols. It is also necessary to find the DNA sequence of Erwinia amylovora and Pseudomonas syringae spp. to match the perfect amplification markers.

Arbeitsgruppe Feuerbrand. Feuerbrand Grundlagen, Probleme und notwendige Massnahmen. Pro Natura, Schweizer Vogelschutz, WWF, 4 Mar. 2008, p. 13.

Hirano, Susan S. ‘Ecology and Physiology of Pseudomonas Syringae’. Nature Biotechnology, vol. 3, no. 12, Dec. 1985, pp. 1073–78. Crossref, doi:10.1038/nbt1285-1073.

HJ Schärer, A. Häseli, J. Fuchs, L. Tamm. Feuerbrand Praxisversuche Schlussbericht. Forschungsinstitut für biologischen Landbau, Nov. 2011, p. 30.

Madigan, Michael T. Brock Biology of Microorganisms. Fourteenth edition, Pearson, 2015.