Ammonia activates pacC and patulin accumulation in an acidic environment during apple colonization by Penicillium expansum

Abstract

Penicillium expansum, the causal agent of blue mould rot, causes severe post-harvest fruit maceration simultaneously with the secretion of d-gluconic acid (GLA) and the mycotoxin patulin in colonized tissue. The factor(s) inducing patulin biosynthesis during colonization of the host acidic environment is unclear. During the colonization of apple fruit in vivo and growth in culture, P. expansum secretes pH-modulating GLA and ammonia. Although patulin and its possible opportunistic precursor GLA accumulate together during fungal development, ammonia is detected on the colonized tissue's leading edge and after extended culture, close to patulin accumulation. Here, we demonstrate ammonia-induced transcript activation of the global pH modulator PacC and patulin accumulation in the presence of GLA by: (i) direct exogenous treatment of P. expansum growing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the patulin biosynthesis cluster. Ammonia induced patulin accumulation concurrently with the transcript activation of pacC and patulin biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacC transcript expression under acidic conditions. Electrophoretic mobility shift assays using P. expansum PacC and antibodies to the different cleaved proteins showed that PacC is not protected against proteolytic signalling at pH 4.5 relative to pH 7.0, but NH4 addition did not further enhance its proteolytic cleavage. Ammonia enhanced the activation of palF transcript in the Pal pathway under acidic conditions. Ammonia accumulation in the host environment by the pathogen under acidic pH may be a regulatory cue for pacC activation, towards the accumulation of secondary metabolites, such as patulin.

Keywords: mycotoxin; pH modulation; post-harvest disease; secondary metabolite.

Figure 1

Accumulation of d‐gluconic acid (GLA), ammonia and patulin during the dynamic pH changes induced by P enicillium expansum. Solid medium (SM) at an initial pH 7.0 was inoculated with spore suspension (100 μL of 10⁶ spore/mL) and the different parameters were evaluated on different days post‐inoculation (PI) by sampling five 10‐mm‐diameter discs from five independent culture plates. Final pH (A), patulin and GLA accumulation (B) and ammonia accumulation (C). Average values of five replicates are reported for pH, GLA, ammonia and patulin accumulation [±standard error (SE)]. Experiments were repeated three times and results of a single representative experiment are shown. DW, dry weight; MDW, mycelium dry weight.

Figure 2

pH, d‐gluconic acid (GLA), patulin and ammonia accumulation in different areas of the decayed tissue of ‘Golden Delicious’ (A,B) and ammonia accumulation in ‘Granny Smith’ and ‘Gala’ apple fruits (C) inoculated with P enicillium expansum. The following parameters were measured in different regions of the decayed fruit 5 days after inoculation: (A) GLA and patulin accumulation; (B) ammonia accumulation and pH changes; (C) ammonia accumulation by different apple cultivars. Ammonia was measured as difference between infected tissue (IT) and healthy tissue. The present values were extracted from the leading edge of the colonized tissue (LECT), intermediate colonized tissue (ICT) and centre of the colonized tissue (CCT) (see photograph). Average values from three replicates of pH, total GLA and patulin accumulation [±standard error (SE)] are presented. Means of ammonia values with different letters (lower case for cv. ‘Golden Delicious’ (B) and ‘Gala’ (C) and upper case for ‘Granny Smith’ (C)) are significantly different at P < 0.05 according to one‐way analysis of variance (ANOVA) followed by Tukey–Kramer honestly significant difference (HSD) test. Experiments were repeated three times, and results of a single representative experiment are shown. FW, fresh weight.

Figure 3

Effect of ammonia concentration on final pH, d‐gluconic acid (GLA) and patulin accumulation, and gene expression in culture colonized by P enicillium expansum. Solid medium (SM) at an initial pH 7.0 was inoculated with 100 μL of a 10⁶ spore/mL suspension; at 24 h post‐inoculation, solid SM plates were placed in closed containers containing 5 M NaOH with 0, 15, 22 or 60 μm ammonia. Final pH, gene expression and patulin accumulation were evaluated 48 h after placing the plates in the containers. Five 10‐mm‐diameter discs were sampled from five independent culture plates. Final pH and GLA accumulation (A), pac C relative expression (RE) (B), patN RE (C) and patulin accumulation (D). Average values [±standard error (SE)] of five replicates are reported for pH. Means of GLA and patulin accumulation and RE values with different letters are significantly different at P < 0.05 according to one‐way analysis of variance (ANOVA) followed by Tukey–Kramer honestly significant difference (HSD) test. Experiments were repeated three times and results of a single representative experiment are shown. MDW, mycelium dry weight.

Figure 4

Effect of ammonia atmosphere on final pH, d‐gluconic acid (GLA) and patulin accumulation, and gene expression in apple fruits colonized by P enicillium expansum. Three ‘Golden Delicious’ apple fruits were inoculated at each infection site with 5 μL of a 10⁶ spore/mL suspension and transferred into closed containers containing 5 M NaOH with 0, 15, 22 and 60 μm NH₄Cl. Different parameters were evaluated at 5 days post‐inoculation: (A) final pH and GLA accumulation; (B) pac C relative expression (RE); (C) patN RE; (D) patulin accumulation. The presented RE values were extracted from the leading edge of the decayed tissue. Average values [±standard error (SE)] of five replicates are reported for pH. Means of GLA and patulin accumulation and RE values with different letters are significantly different at P < 0.05 according to one‐way analysis of variance (ANOVA) followed by Tukey–Kramer honestly significant difference (HSD) test. Experiments were repeated three times, and results of a single representative experiment are shown. FW, fresh weight.

Figure 5

Expression of 15 genes potentially involved in patulin biosynthesis in P enicillium expansum grown on solid medium (SM) at an initial pH 7.0 exposed to 15 μm NH₄Cl, as described previously in Fig. 3. Data were analysed using the 2^{−ΔΔ C T} method, and normalized using 28S rRNA as the housekeeping gene. Means with different letters are significantly different at P < 0.05 according to one‐way analysis of variance (ANOVA) followed by Tukey–Kramer honestly significant difference (HSD) test. RE, relative expression.

Figure 6

Effect of sucrose concentration on d‐gluconic acid (GLA), ammonia and patulin accumulation, final pH and pacC relative expression (RE) in P enicillium expansum in culture. Solid secondary medium at an initial pH 4.5 was amended with 15 or 175 mm sucrose and inoculated with 100 μL of a 10⁶ spore/mL suspension. The different parameters were evaluated at 72 h post‐inoculation. Five 10‐mm‐diameter discs were sampled from five independent culture plates. GLA and ammonia accumulation (A), patulin accumulation and final pH (B) and pacC  RE (C). Average values [±standard error (SE)] of five replicates are reported. Experiments were repeated three times and results of a single representative experiment are shown. DW, dry weight; MDW, mycelium dry weight.

Figure 7

Effect of ammonia concentration on relative expression (RE) of P enicillium expansum pac C at different periods after treatment. The fungus was grown first in liquid medium for 48 h and then transferred to 0.2 m phthalate‐buffered liquid SM medium at pH 4.5 for 3 h. The mycelia were then transferred to 0.2 m phthalate‐buffered liquid SM medium at pH 4.5 or 0.2 m phosphate‐buffered medium at pH 7.0 containing 0, 25, 50 or 100 μm NH₄Cl and, 15, 30 and 60 min later, samples were collected. pacC  RE at (A) pH 4.5 and (B) pH 7.0. Average values [±standard error (SE)] of five replicates are reported. The experiment was repeated three times and results of a single representative experiment are shown.

Figure 8

Detection of PacC forms in crude protein extracts of P enicillium expansum. Electrophoretic mobility shift assays (EMSAs) were performed using the ipnA2 probe and protein extracts from mycelia grown under the conditions indicated at the top of the figure. (A) Low‐mobility complexes (LMCs) formed by the binding of PacC ⁷² (primary product) and proteolysed PacC⁵³ are indicated on the left. Below is the high‐mobility complex (HMC) formed by the fully proteolysed PacC²⁷ form. The presence of antisera against different regions of PacC in the reaction is indicated at the top of the figure. (B) Charts below the EMSA images show the quantification of LMC (red) and HMC (blue) for each lane. See main text for the interpretation of mobilities and complexes. DBD, DNA‐binding domain; FP, free probe.

Figure 9

Comparison of the relative expression (RE) of pacC and palF of P enicillium expansum grown at pH 4.5 and pH 7.0, and amended with different ammonium chloride concentrations. RE was determined 30 min after ammonia treatment. Means of RE values with different letters are significantly different at P < 0.05 according to one‐way analysis of variance (ANOVA) followed by Tukey–Kramer honestly significant difference (HSD) test. The experiment was repeated three times and results of a single representative experiment are shown.