Differences in the Production of Extracellular Polymeric Substances (EPS) and Other Metabolites of Plenodomus (Leptosphaeria) Infecting Winter Oilseed Rape (Brassica napus L.)

Abstract

Species of the genus Plenodomus (Leptosphaeria) are phytopathogens of the Brassicaceae family, which includes oilseed rape. The spores of these fungi spread by airborne transmission, infect plants, and cause crop losses. The secondary metabolism of P. lingam and P. biglobosus was studied and compared, with the main focus being on the ability to produce Extracellular Polymeric Substances (EPS). In spite of the 1.5-2-fold faster growth rate of P. biglobosus on Czapek-Dox and other screening media, the average yield of EPS in this fungus was only 0.29 g/L, compared to that of P. lingam (0.43 g/L). In turn, P. biglobosus showed a higher capacity to synthesise IAA, i.e., 14 µg/mL, in contrast to <1.5 µg/mL produced by P. lingam. On the other hand, the P. lingam strains showed higher β-glucanase activity (350-400 mU/mL), compared to 50-100 mU/mL in P. biglobosus. Invertase levels were similar in both species (250 mU/mL). The positive correlation between invertase activity and EPS yield contrasted with the absence of a correlation of EPS with β-glucanase. Plenodomus neither solubilised phosphate nor used proteins from milk. All strains showed the ability to synthesise siderophores on CAS agar. P. biglobosus exhibited the highest efficiency of amylolytic and cellulolytic activity.

Keywords: IAA; Plenodomus (Leptosphaeria); enzyme activity; exopolysaccharide; extracellular polymeric substance (EPS); invertase; siderophore; winter oilseed rape; β-glucanase.

Figure 1

Dynamics of EPS (g/L) synthesis by Plenodomus lingam (PLIGR1, PLIGR2, PLIGR3) and Plenodomus biglobosus (PBIGR1, PBIGR2, PBIGR3) strains during the culture growth period of 4–17 days. Statistical data analysis: one-way ANOVA with post hoc Tukey’s HSD test, p < 0.05. Bars with the different letter are statistically significantly different from each other. Standard deviations are shown as deviation bars (n = 3).

Figure 2

Heat map presenting the differences in the EPS synthesis dynamics between Plenodomus lingam (PLIGR1, PLIGR2, PLIGR3) and Plenodomus biglobosus (PBIGR1, PBIGR2, PBIGR3) species during the 4–14-day culture growth. The colour intensity on the heat map corresponds to the EPS g/L synthesis efficiency.

Figure 3

Dynamics of fungal biomass growth and changes in the pH value in Plenodomus lingam (PLIGR1, PLIGR2, PLIGR3) and Plenodomus biglobosus (PBIGR1, PBIGR2, PBIGR3) strains during culture growth for 4–17 days. Statistical data analysis: one-way ANOVA with post hoc Tukey’s HSD test, p < 0.05. Bars with the different letter are statistically significantly different from each other. Bars with the different letter are statistically significantly different from each other. Standard deviations are shown as deviation bars (n = 3).

Figure 4

Heat map presenting the differences in the EPS yield and the biomass growth rate (mg/g) between Plenodomus lingam (PLIGR1, PLIGR2, PLIGR3) and Plenodomus biglobosus (PBIGR1, PBIGR2, PBIGR3) species during the 4–14-day culture growth. The colour intensity on the heat map corresponds to the EPS mg/g synthesis efficiency.

Figure 5

β-Glucanase activity in cultures of (A) Plenodomus lingam (PLIGR1, PLIGR2, PLIGR3) and (B) Plenodomus biglobosus (PBIGR1, PBIGR2, PBIGR3) strains. Statistical data analysis: one-way ANOVA with post hoc Tukey’s HSD test, p < 0.05. Bars with the different letter are statistically significantly different from each other. Standard deviations are shown as deviation bars (n = 3).

Figure 6

Invertase activity in cultures of (A) Plenodomus lingam (PLIGR1, PLIGR2, PLIGR3) and (B) Plenodomus biglobosus (PBIGR1, PBIGR2, PBIGR3) strains. Statistical data analysis: one-way ANOVA with post hoc Tukey’s HSD test, p < 0.05. Bars with the different letter are statistically significantly different from each other. Standard deviations are shown as deviation bars (n = 3).

Figure 7

Ability to synthesise the IAA phytohormone by (A) Plenodomus lingam (PLIGR1, PLIGR2, PLIGR3) and (B) Plenodomus biglobosus (PBIGR1, PBIGR2, PBIGR3) strains. Bars with the different letter are statistically significantly different from each other. Statistical data analysis: one-way ANOVA with post hoc Tukey’s HSD test, p < 0.05. Standard deviations are shown as deviation bars (n = 3).

Figure 8

Correlation between EPS synthesis efficiency (g/L) and β-glucanase and invertase activity in PLIGR1 (A,D), PLIGR2 (B,E), and PLIGR3 (C,F), and in PBIGR1 (G,J), PBIGR2 (H,K) and PBIGR3 (I,L). The correlations were analysed based on Pearson’s correlation coefficient R and the statistical significance p of this coefficient.

Figure 9

Correlation matrices between day, pH, biomass, EPS yield, β-glucanase, and invertase activity obtained in cultures of PLIGR1 (A), PLIGR2 (B) and PLIGR3 (C), and PBIGR1 (D), PBIGR2 (E) and PBIGR3 (F). The results are presented as Pearson’s correlation coefficient R.

Figure 10

Scree plot (A), biplot (B) and plot of points by day (C) of the Principal Component Analysis (PCA) describing the pH, amount of biomass, day of the culture, IAA and EPS yields, and β-glucanase and invertase activities in Plenodomus species.