Parallel to the GFP plate induction assays it was attempted to induce dothistromin production of D. septosporum using NZE7 and NZE10 wild type strains with extracts from competitors’ growth media. As discussed in Chapter 3, it was speculated that the higher expression of the dot genes and the higher dothistromin production in the DB media compared to PDB was caused by the presence of the components of fungal origin in DB. Therefore it was tested if adding an elicitor made from yeast could induce dothistromin production and dothistromin gene expression in PDB media. PDB media was chosen as the dothistromin genes are expressed at a relatively low and constant level (Section 3.2.2) and therefore an induction should be detectable.
“Elicitor” broths were created using similar methods as described by Kim et al. (2001). The elicitor broth created from yeast was obtained by dissolving 10 g yeast extract (BD) in 80 ml H2O, 320 ml of 95% ethanol were subsequently added and the solution was left overnight at 4ºC. The liquid was poured off and the precipitate that formed was allowed to air dry and dissolved in a minimal amount of distilled water. The mixture was lyophilized and re-dissolved in 50 ml distilled water. For each sample 5 ml of the solution was filter sterilized before adding to the growth media.
Flasks containing 20 ml PDB media were inoculated with 106 wild-type D. septosporum spores/ml. After 6 days growth with shaking (180 rpm) at 22°C a 5 ml volume of “yeast elicitor” broth was added to each flask. As controls 5 ml sterile water and 5 ml PDB were added to three replicated flasks. Mycelium was harvested after 48 h and growth, dothistromin production (Section 2.2.2.2) and dothistromin gene expression (Section 2.13) were determined. The results were compared to control samples taken at day 6 and shown in Figure A.6.1 and A6.2. The other used “elicitor” broths consisted of autoclaved and filtered PDB media in which fungal competitor species had been inoculated and grown for 5 days with shaking (180 rpm) at 22°C. Three replicate D. septosporum flasks were harvested for each type of “elicitor” broth 3 h after addition and a further three flasks harvested 24 hours after addition of the “elicitor” broth. Liquid broth from each D. septosporum flask was assayed for dothistromin concentration using an ELISA assay as described previously (Section 2.13). Mycelium was harvested and divided to calculate the dry weight and to extract RNA as previously described in Section 2.2.2.2. Gene expression of dotA, dotC, pksA and vbsA was assessed by real time RT-PCR as described in Section 2.10.4.
0.01 0.1 1
Control + water + YE + PDA
0.001 0.01 0.1 1
Control + water + YE + PDA
0.1 1 10
Control + water + YE + PDA
dotA
dotC
vbsA
pksA
tub1
0.001 0.01 0.1 1Control + water + YE + PDA
0.01 0.1
Figure A6.2: Relative gene expression of dothistromin genes in yeast elicitor trial.
Shown are gene expression levels relative to ribosomal 18S rDNA (Y-axis) for the control (before adding media) and 48h after adding water, yeast elicitor (YE), and potato dextrose media (PDA). Although the toxin concentration was highest in the YE media (Figure A6.1) no induction by YE of gene expression for dotA, pksA, vbsA or dotC was detected. The β-tubulin gene expression (tub1) was included as a constitutive control. Each data point is the mean ± standard deviation of three replicate samples.
0 5 10 15 20 25
Control + water + YE + PDA
µµµµ g D O T H / m g D W
Figure A6.1: Dothistromin synthesis in yeast elicitor trial.
Shown is the dothistromin synthesis in µg dothistromin per mg DW for the control (before adding media) and 48h after adding water, yeast elicitor (YE), and potato dextrose media (PDA). The toxin concentration was highest in the YE media and control flasks, although an increase between the YE and control sample was not seen. The addition of water and PDA to the samples resulted in lower level of dothistromin production than with YE. Each data point is the mean ± standard deviation of three replicate samples.
An additional experiment was performed, this time using “yeast elicitor” and “ elicitor broth” consisting out of autoclaved PDB media in which A. alternata and Phoma glomerata had been grown for 6 days. P. glomerata was a contaminating fungus which frequently grew on needles of seedlings used in the pathogenicity trials (Barron 2006). In this trial only the toxin production was determined. Samples were taken 3 h and 24 h after adding the elicitor broth, as the high toxin production in the “yeast elicitor” media in the previous experiment (Figure A6.1) and the relatively low gene expression in those samples (Figure A6.2) suggested that an induction might have occurred at an earlier time. Results are shown and discussed in Figure A6.3.
These preliminary results did not allow detection of induction of dothistromin synthesis or gene expression by the yeast extract used by Kim et al. (2001) to induce secondary metabolites in ginseng cell cultures. This suggests that the higher dothistromin production in DB media (Chapter 3) is not due to the presence of material of fungal origin. Further no significant increase of dothistromin production was seen using an elicitor broth generated from P. glomerata. However, the A. alternata broth might have induced the production of dothistromin (Figure A6.3)
The liquid culture assay also had several limitations. As for the plate induction assay discussed in Chapter 5, a temporary increase of gene expression could have been missed as only a few time points were tested. However, an increased dothistromin concentration would reveal an earlier induction of dothistromin gene expression or secretion of the toxin. Further as discussed in Section 5.3, the PDB media might restrict the synthesis of some metabolites. Therefore the elicitor broths might not contain the substances which induce toxin production in D. septosporum. Furthermore the elicitor media were autoclaved and certain substances might have been destroyed; volatile compounds, which have been shown to play a major role in interactions of micro-organisms and their environment (Wheatley 2002), would have been lost in the process.
0 20 40 60 1 2 3 4 5 6 7 8 9
*
*
*
3h ♦ 24h Yeast elicitor 3h ♦ 24h P. glomerata 3h 24h PDB C 3h ♦ 24h A. alternata m g D W 3 6 9 12*
µµµµ g D O T H /m g D W + 0 5 10 15 20 1 2 3 4 5 6 7 8 9 3h ♦ 24h Yeast elicitor 3h 24h P. glomerata 3h 24h PDB C 3h ♦ 24h A. alternata +,*
µµµµ g D O T H /m lA
B
C
Figure A.6.3: Growth and dothistromin production of D. septosporum in elicitor media.
Shown are the values obtained from the control flasks (before adding elicitor broth or additional PDB media) C, and after addition of PDB, elicitor broth of A. alternata, elicitor broth of P. glomerata and yeast elicitor.
The first column of each pair represents values obtained 3h and the second column values obtained 24h after addition of media.
Significant different values from the control flask C are indicated with an * (n=3, p<0.05). Significant differences between each pair are indicated with an ♦ in the legend (n=3, p<0.05). and a significant difference between the elicitor sample and the PDB control is indicated with an + (n=3, p<0.05).
(A) Biomass per flask in mg DW. A significant increase in growth compared to the control C and between the 3h and 24h time point is seen for all three tested elicitor media.
(B) The concentration of dothistromin in the media is shown in µg DOTH/ml. Only the 24h yeast elicitor sample showed significant difference between the control C and its equivalent 24h PDB control. A significant increase of dothistromin concentration is seen between the 3h and 24h samples of both the yeast elicitor and A. alternata elicitor media.
(C) The dothistromin synthesis is shown in µg DOTH/mg DW. A significant increase for each pair is only seen for the 3h and 24h sample of the A. alternata elicitor. The 24h A. alternata elicitor sample showed also significant difference between the equivalent 24h PDB control. Further, those data suggest the significant increase of dothistromin concentration of the 24h yeast