Microscopy of fluorescence and molecular

genetics applied to the follow-up and evaluation

of pathogenic bacteria of plants

We are going to talk about the participating, the entities, the objectives,
the materials, the methodology and the project and PIIISA conclusion.

1.Participating
First, this project has involved the researchers Carmen R. Beuzn, Javier
Ruiz-Albert and Jos Rufin Plaza. And the students and cordinators of
IES Pedro Espinosa, IES Isaac Albniz, IES Martn Rivero, IES Camilo
Jos Cela and IES Beln.

2.Entities
The entities which participate are:
-Department of Cell Biology, Genetics and Physiology. University of Malaga
(UMA)
-Institute of Horticulture and Subtropical Mediterranean (IHSM)
-Ministry of Economy and Competitiveness

3.Objectives
The project consists in inoculating bean plants with different bacteria in
order to observe and study how each type of bacteria affect the plant. The
bacteria are:
Pseudomonas Syringae Pathovar Phaseolicola (PPH)

 Pseudomonas Syringae Pathovar Tomato(PTO)

Derivative of Pseudomonas Syringae Pathovar Phaseolicola (RW60)

4.Materials
To make this project we have needed:
-Petri dishes, where we have cultivated the bacteria
- Micropipettes, which he allows us to use different liquids without having
to wash the device.
- Magnesium chloride
- Binocular microscope to observe the bacteria
-Tubes of 15mL
- Eppendorf tubes of 1 5 ml
- Syringe without needle to introduce the bacterium in the sheets of the
plant
- Plants of bean which we have inoculated
- Culture medium
In these photos we can see the materials that I have just quoted.

5.Methodology
Preparation
Firstly, the scientists explained us that we would work with three
pathovars of the bacterial specie, Pseudomona Syringae (a plants pathogean
specie). They were PPH (a bean plant pathogen), PTO (a tomato pathogen)
and RW60 ( a PPH derivate that lost part of its genetic information). So,
we wanted to know which pathovars cause the disease on bean plants and

what were the sintoms at a cellular level and in the leaf of the plant. The
first step was to prepare the cultive medium where bacteria could grow and
develop with yeast, salt, water, agar for the solid medium and we also
planted the beans

Development
Then, we could start to inoculate.
Inoculation is a method that consists in introducing a substance in an
organism, in this case the substance that contains the bacteria in the bean
plant.
Before inoculation, we needed to prepare the liquid with the bacteria. We
had to follow some steps to get it, but always in sterile conditions. What's
that? If we didn't work in sterile conditions, the bacteria would be
contaminate by air microorganisms. Then, we worked around a bunsen
lighter, what makes air to elevate with all the microorganisms, and that's
how we evited that bacteria were contaminate.
When we got the liquid, we injected the bacteria into the plant with a
syringe by the underside of leaves.

Take information
We observed the number of colonies and their size, we analyzed also the
form how they were associated. Then, we investigated the symptoms that
we can see to the naked eye in the leaf inoculated.

6.Project conclusions
Now Im going to talk about the conclusions:
In pph bacteria we can observe large colonies; some of them can be
associated. In the plant, we can see necrosis (that is the cells dead) and
chlorosis (that is the yellowish colour in the leaf). Finally, bacteria win.

In pto, we can observe smaller colonies than pph. We cant see any
symptoms because is plant who wins
In Rw60, when bacteria get into the plant, the plant swells the cell walls
accumulating compounds as a response to bacteria. We can see small
necrosis due to the high effort that plant makes to eliminate bacteria.

7.PIIISA Conclusions
In our opinion it is an innovative project and for us it has been an
extraordinary experience. We have conducted really interesting activities
and, of course, we have learnt a lot about science. We recommend its
continuation in next years.
We thank the following high-schools: IES Pedro Espinosa from Antequera
(Mlaga), IES Isaac Albniz from Mlaga, IES Martn Rivero from
Ronda (Mlaga), IES Camilo Jos Cela from Campillos (Mlaga), IES
Beln from Mlaga.

Finally, we are going to finish with an Albert Einsteins quote: Never

regard study as a duty but as an enviable opportunity to learn to know the
liberating influence of beauty in the realm of the spirit for your own
personal joy and to the profit of the community to which your later works
belong.
Albert Einstein