Team:Toulouse/Result/experimental-results

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The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br>
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br>
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After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in a model plant </b> : <i>Nicotiana benthamiana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain.  
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After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain.  
<p class="title2">3. <i> In planta </i> tests with SubtiTree
<p class="title2">3. <i> In planta </i> tests with SubtiTree
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<p class="title3">Purpose
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  The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br>
  The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br>
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SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. The absence of leaves degradation by the fungus is a sign of a functional antifungal module. </br>
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SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br>
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br>
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br>
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<p class="texte">24 hours after SubtiTree inoculation, no modification of leaves' phenotype  can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effect.</br>
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<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br>
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Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i>  expressing DE41 or GAFP1-D4E1, unlike with the WT bacteria. A second experiment is expected to be more statistically precise.</br>
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Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i>  expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br>
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br>
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br>

Revision as of 14:35, 12 October 2014