Seed Protection of Solanum lycopersicum with Pythium oligandrum against Alternaria brassicicola and Verticillium albo-atrum

Abstract

Pythium oligandrum, strain M1, is a soil oomycete successfully used as a biological control agent (BCA), protecting plants against fungal, yeast, and oomycete pathogens through mycoparasitism and elicitor-dependent plant priming. The not yet described Pythium strains, X42 and 00X48, have shown potential as BCAs given the high activity of their secreted proteases, endoglycosidases, and tryptamine. Here, Solanum lycopersicum L. cv. Micro-Tom seeds were coated with Pythium strains, and seedlings were exposed to fungal pathogens, either Alternaria brassicicola or Verticillium albo-atrum. The effects of both infection and seed-coating on plant metabolism were assessed by determining the activity and isoforms of antioxidant enzymes and endoglycosidases and the content of tryptamine, amino acids, and heat shock proteins. Dual culture competition testing and microscopy analysis confirmed mycoparasitism in all three Pythium strains. In turn, seed treatment significantly increased the total free amino acid content, changing their abundance in both non-infected and infected plants. In response to pathogens, plant Hsp70 and Hsp90 isoform levels also varied among Pythium strains, most likely as a strategy for priming the plant against infection. Overall, our results show in vitro mycoparasitism between Pythium strains and fungal pathogens and in planta involvement of heat shock proteins in priming.

Keywords: antioxidants; capillary electrophoresis; fungal diseases; plant protection; seed-coating.

Figure 1

Interactions between Pythium strains and fungal pathogens A. brassicicola (A–D) and V. albo-atrum (E–H) after 24 h on a scale of 100 μm. Control growth of fungal pathogens (A,E); P. oligandrum M1 (B,F); Pythium X42 (C,G); P. oligandrum 00X48 (D,H). Arrows point at the sites where Pythium strains coil around the fungal hyphae.

Figure 2

Dual culture plate competition tests between Pythium strains and fungal pathogens A. brassicicola (A–D) and V. albo-atrum (E–H). Control growth of fungal pathogens (A,E); P. oligandrum M1 (B,F); Pythium X42 (C,G); P. oligandrum 00X48 (D,H).

Figure 3

Relative content of A. brassicicola (black columns) and V. albo-atrum (grey columns) determined by RT-qPCR (A); content of total proteins (B); total free amino acids (C); tryptamine (D); total phenolics (E); antioxidant capacity (F) of tomato leaf extracts from plants grown from seeds treated with a Pythium strain (i.e., M1, X42, and 00X48) and from untreated control seeds (Control). Relative pathogen content (A) was expressed to the standard gene actin. Different letters above each bar denote significant differences (p ≤ 0.05) between plant groups according to one-way ANOVA (Holm–Sidak). The same letters above a bar indicate that no significant differences were found among the groups. Each column bar represents the mean ± SD. The degrees of freedom, F-value, and p-values can be found in Table S2. Abbreviations: asc. acid, ascorbic acid; D.W., dry weight; F.W., fresh weight.

Figure 4

Antioxidant enzyme activity in tomato leaf (A,C,E,G) and stem (B,D,F,H) extracts from plants grown from seeds treated with a Pythium strain (i.e., M1, X42, and 00X48) and from untreated control seeds (Control): soluble peroxidases (A,B); bound peroxidases (C,D); ascorbate peroxidase (E,F); glutathione reductase (G,H). Plants were infected with A. brassicicola (black columns) and V. albo-atrum (grey columns) and compared with non-infected control plants (white columns). Different letters above each bar denote significant differences (p ≤ 0.05) among plant groups according to one-way ANOVA (Holm–Sidak). The same letters above a bar indicate that no significant differences were found between groups. Each column bar represents the mean ± SD. The degrees of freedom, F-values, and p-values can be found in Table S4. APOD, ascorbate peroxidase; F.W., fresh weight; GR, glutathione reductase; PODs, peroxidases.

Figure 5

Detection of the activity of peroxidases (A); ascorbate peroxidase (B); superoxide dismutase (C); glutathione-S-transferase (D) in leaves (left) and stems (right) after electrophoretic separation under native conditions in 10% polyacrylamide gel. SOD was measured only in leaves. The activity of individual SOD isoenzymes was determined in inhibition studies using H₂O₂ and KCN (data not shown). Arrows indicate the main isoforms that are discussed in this study. Abbreviations: A-C, control plants infected with A. brassicicola; A-M1, plants treated with M1 and infected with A. brassicicola; A-X42, plants treated with X42 and infected with A. brassicicola; A-00X48, plants treated with 00X48 and infected with A. brassicicola; V-C, control plants infected with V. albo-atrum; V-M1, plants treated with M1 and infected with V. albo-atrum; V-X42, plants treated with X42 and infected with V. albo-atrum; V-00X48, plants treated with 00X48 and infected with V. albo-atrum; APOD, ascorbate peroxidase; GST, glutathione-S-transferase; PODs, peroxidases; SOD, superoxide dismutase.

Figure 6

Enzyme activities in tomato leaf extracts from plants grown from seeds treated with a Pythium strain (i.e., M1, X42, or 00X48) and from untreated control seeds (Control): CAT (A); G6PDH (B); NADP-ME (C); SDH (D). Plants were infected with A. brassicicola (black columns) and V. albo-atrum (grey columns), and the results were compared with those of non-infected plants (white columns). Different letters above each bar denote significant differences (p ≤ 0.05) among plant groups according to one-way ANOVA (Holm–Sidak). The same letters above a bar indicate that no significant differences were found among groups. Each column bar represents the mean ± SD. The degrees of freedom, F-values, and p-values can be found in Table S5. Abbreviations: CAT, catalase; F.W., fresh weight; G6PDH, glucose-6-phosphate dehydrogenase; NADP-ME, NADP-malic enzyme; SDH, shikimate dehydrogenase.

Figure 7

Endoglycosidase activities in tomato leaf (A,C,E) and stem (B,D,F) extracts from plants grown from seeds treated with a Pythium strain (i.e., M1, X42, or 00X48) and from untreated control seeds (Control): cellulase (A,B); chitinase (C,D); endo-β-1,3-glucanase (E,F). Plants were infected with A. brassicicola (black columns) and V. albo-atrum (grey columns), and the results were compared with those of non-infected plants (white columns). Different letters above each bar denote significant differences (p ≤ 0.05) among plant groups according to one-way ANOVA (Holm–Sidak). The same letters above a bar indicate that no significant differences were found among the groups. Each column bar represents the mean ± SD. The degrees of freedom, F-values, and p-values can be found in Table S6. Abbreviations: F.W., fresh weight.

Figure 8

Relative content of cytosolic Hsp70 (A) and Hsp90 (B) determined by immunoblotting after SDS-electrophoresis in tomato leaf extracts from plants grown from seeds treated with a Pythium strain (i.e., M1, X42, or 00X48) and from untreated control seeds (Control). The relative intensity of bands (C–H) was evaluated in GelAnalyzer 19.1. Abbreviations: A-C, control plants infected with A. brassicicola; A-M1, plants treated with M1 and infected with A. brassicicola; A-X42, plants treated with X42 and infected with A. brassicicola; A-00X48, plants treated with 00X48 and infected with A. brassicicola; V-C, control plants infected with V. albo-atrum; V-M1, plants treated with M1 and infected with V. albo-atrum; V-X42, plants treated with X42 and infected with V. albo-atrum; V-00X48, plants treated with 00X48 and infected with V. albo-atrum; ST, Precision Plus Protein™ Kaleidoscope™ by Bio-Rad. Arrows point at proteins discussed in the text.