Abscisic Acid Metabolizing Rhodococcus sp. Counteracts Phytopathogenic Effects of Abscisic Acid Producing Botrytis sp. on Sunflower Seedlings

Abstract

One of the important traits of many plant growth-promoting rhizobacteria (PGPR) is the biocontrol of phytopathogens. Some PGPR metabolize phytohormone abscisic acid (ABA); however, the role of this trait in plant-microbe interactions is scarcely understood. Phytopathogenic fungi produce ABA and use this property as a negative regulator of plant resistance. Therefore, interactions between ABA-producing necrotrophic phytopathogen Botrytis sp. BA3 with ABA-metabolizing rhizobacterium Rhodococcus sp. P1Y were studied in a batch culture and in gnotobiotic hydroponics with sunflower seedlings. Rhizobacterium P1Y possessed no antifungal activity against BA3 and metabolized ABA, which was synthesized by BA3 in vitro and in associations with sunflower plants infected with this fungus. Inoculation with BA3 and the application of exogenous ABA increased the root ABA concentration and inhibited root and shoot growth, suggesting the involvement of this phytohormone in the pathogenesis process. Strain P1Y eliminated negative effects of BA3 and exogenous ABA on root ABA concentration and plant growth. Both microorganisms significantly modulated the hormonal status of plants, affecting indole-3-acetic, salicylic, jasmonic and gibberellic acids, as well as cytokinins concentrations in sunflower roots and/or shoots. The hormonal effects were complex and could be due to the production of phytohormones by microorganisms, changes in ABA concentrations and multiple levels of crosstalk in hormone networks regulating plant defense. The results suggest the counteraction of rhizobacteria to ABA-producing phytopathogenic fungi through the metabolism of fungal ABA. This expands our understanding of the mechanisms related to the biocontrol of phytopathogens by PGPR.

Keywords: Botrytis; PGPR; abscisic acid; biocontrol; phytohormones; phytopathogens.

Figure 1

Number of Rhodococcus sp. P1Y (A), biomass of Botrytis sp. BA3 (B) and abscisic acid (ABA) concentration in the nutrient solution (C) in batch culture with pure cultures and a mixture of microorganisms. Vertical bars show confidence intervals (p = 0.05, n = 6).

Figure 2

Effect of exogenous abscisic acid (ABA) on biomass of sunflower roots (A) and shoots (B). Mean data of two independent experiments. Different letters indicate significant differences between treatments (Fisher’s LSD test, p < 0.05, n = 20 for treatments from 0 to 400 nM ABA and n = 10 for treatments from 2000 to 50,000 nM ABA).

Figure 3

Biomass of sunflower roots (A) and shoots (B) inoculated with Rhodococcus sp. P1Y (P1Y) and Botrytis sp. BA3 (BA3) and treated with 400 nM of abscisic acid (ABA). Mean data of three independent experiments. UC stands for un-inoculated control. Different letters indicate significant differences between treatments (Fisher’s LSD test, p < 0.05, n = 45).

Figure 4

Sunflower plants uninoculated (A,E) or inoculated with Rhodococcus sp. P1Y (B,F), Botrytis sp. BA3 (C,G) and a mixture of Rhodococcus sp. P1Y and Botrytis sp. BA3 (D,H) at the end of the experiment. The scale on G is the same for all parts of the figure.

Figure 5

Tissue damage of sunflower roots (A) and shoots (B–F) inoculated with Botrytis sp. BA3. (A) A root system with inhibited growth and maceration of the main root. (B,C) Damaged shoots. (D) The place of mycelium formation, indicated by the red arrow in (B). (E) Greenish-brown damaged tissue with the residual amount of chlorophyll, indicated by the left red arrow in (C). (F) Black damaged tissue containing fungal mycelium indicated by the right red arrow in (C).

Figure 6

Concentration of abscisic acid (ABA) in the nutrient solution inoculated with Rhodococcus sp. P1Y (P1Y) and Botrytis sp. BA3 (BA3) and treated with 400 nM of ABA at the end of experiments with sunflower plants. Mean data of three independent experiments. UC stands for un-inoculated control. Nd stands for not detected. Different letters indicate significant differences between treatments (Fisher’s LSD test, p < 0.05, n = 9).

Figure 7

Concentration of abscisic acid (ABA) in roots (A) and shoots (B) of sunflowers inoculated with Rhodococcus sp. P1Y (P1Y) and Botrytis sp. BA3 (BA3) and treated with 400 nM of ABA. Mean data of three independent experiments. UC stands for un-inoculated control. Different letters indicate significant differences between treatments (Fisher’s LSD test, p < 0.05, n = 9 for roots, and n = 4 for shoots, respectively).

Figure 8

Heat maps of the effects of treatments on phytohormone concentrations and the relationship between phytohormones in roots (A) and shoots (B). Hierarchical clustering was conducted using Ward’s method (ward.D) based on squared Euclidean distances. Color intensity represents the percent change from control values, where 0% corresponds to the untreated control. Clusters are indicated by the letter C followed by a number.