Team:Toulouse/Result/experimental-results

The first experiment deals with the culture conditions to see if Bacillus subtilis can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different Bacillus subtilis solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell was performed.

The bacterial solutions could not be counted because of two main problems : the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focudes on the intermediate values (Figure 1).
First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Morever, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.
Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.

Figure 1 : CBB presence has no effect on bacterial. The bacterial concentration was measured regarding the presence () or the absence of CBB () for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).

Transformed Bacillus subtilis with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of Vibrio cholerae allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specificaly attached to the chitin are put on plates and counted.

The first observation is that both bacterial solutions of wild type Bacillus subtilis and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more WT bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module.
Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.

Figure 2 : Attachment of Bacillus subtilis with binding module to chitin. The WT bacteria concentration () or the bacteria with the binding system () has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p < 0.05.

Tests with commercial peptides

Results
The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with Ceratocystis Platani.
As Ceratocystis Platani is pathogenic, we could not perform tests directly with this fungus.
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.
FIGURE
An inhibiton halo was noticeable with commercial D4E1 peptide at 100µM on Aspergillus brasiliensis. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.
As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.

FIGURE


Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.
Test with SubtiTree


In order to test Bacillus subtilis mutants, it was essential to find the right balance between the fungal growth and the bacterial one.This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.

The transformed Bacillus subtilis strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads disposed plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of
Trichoderma reesei's growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1. However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides. Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: A number of post-transcriptional modification are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.
PHOTO
Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. Another effect was noted with the same strains expriming D4E1 and GAFP-1 + D4E1 on another fungus Aspergillus brasiliansis. This effect is comparable to the one previously noted with low concentration of sulfate copper.

The choice of our chassis appears to be optimal as we noted that wild type Bacillus subtilis disturbs the hyphae growth of the fungi. Some strains of Bacillus subtilis (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.

After this set of experiments, the strain expressing the operon GAFP-1 + D4E1 is the best candidate to play a major role in the fight against fungal diseases such as Canker stain. Our team decided to follow the experiments on a model plant. Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity...

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