Oxidative stress facilitates infection of the unicellular alga Haematococcus pluvialis by the fungus Paraphysoderma sedebokerense

Abstract

Background: The green microalga Haematococcus pluvialis is used as a cell factory for producing astaxanthin, the high-value carotenoid with multiple biological functions. However, H. pluvialis is prone to the infection by a parasitic fungus Paraphysoderma sedebokerense, which is the most devastating threat to the mass culture of H. pluvialis all over the world. Through dissecting the mechanisms underlying the infection process, effective measures could be developed to mitigate the pathogen threatening for the natural astaxanthin industry. By far, understanding about the interaction between the algal host and fungal pathogen remains very limited.

Results: We observed that there were heat-stable substances with small molecular weight produced during the infection process and enhanced the susceptibility of H. pluvialis cells to the pathogen. The infection ratio increased from 10.2% (for the algal cells treated with the BG11 medium as the control) to 52.9% (for the algal cells treated with supernatant contained such substances) on the second day post-infection, indicating the yet unknown substances in the supernatant stimulated the parasitism process. Systematic approaches including multi-omics, biochemical and imaging analysis were deployed to uncover the identity of the metabolites and the underlying mechanisms. Two metabolites, 3-hydroxyanthranilic acid and hordenine were identified and proved to stimulate the infection via driving oxidative stress to the algal cells. These metabolites generated hydroxyl radicals to disrupt the subcellular components of the algal cells and to make the algal cells more susceptible to the infection. Based on these findings, a biosafe and environment-friendly antioxidant butylated hydroxyanisole (BHA) was selected to inhibit the fungal infection, which completely abolished the infection at 12 ppm. By applying 7 ppm BHA every 2 days to the algal cell culture infected with P. sedebokerense in the 100 L open raceway ponds, the biomass of H. pluvialis reached 0.448 g/L, which was comparable to that of the control (0.473 g/L).

Conclusions: This study provides for the first time, a framework to dissect the functions of secondary metabolites in the interaction between the unicellular alga H. pluvialis and its fungal parasite, indicating that oxidative degradation is a strategy used for the fungal infest. Eliminating the oxidative burst through adding antioxidant BHA could be an effective measure to reduce parasitic infection in H. pluvialis mass culture.

Keywords: Antioxidant; Fungal pathogen; Haematococcus pluvialis; Oxidative stress; Secondary metabolites.

Fig. 1

Fungal contamination crashed the H. pluvialis cell culture and the supernatant post-infection (SPI) enhanced the fungal infection process. A Crash of outdoor 360 L H. pluvialis algal culture after been contaminated by the fungal parasite P. sedebokerense. B Parasitic process of fungus on the algal cells. C SPI-treatment enhanced the fungal infection on the algal cells. D Infection ratio of P. sedebokerense on the H. pluvialis cells treated by SPI. E Morphological changes of algal cells treated with SPI. The pyrenoids were stained with the Lugol’s reagent (arrows point). F Changes in the algal cellular components after SPI treatment. The contents of the pigments and carbohydrates of the algal cells treated with SPI were normalized to that of the control, i.e., the algal cells treated with the BG11 medium. The quantitative data were presented as mean ± S.D. (n = 3). **, p < 0.01 (Student’s t test). Scale bar, 20 μm

Fig. 2

SPI caused algal structure degradation and exhibited oxidative activities both in vivo and in vitro. A TEM observation of the algal cells with different treatment. N, nuclear. W, cell wall. Ch, chloroplast. F, fungal cell. B Subset of the annotated and differentially expressed genes following SPI treatment. C Oxidative activities of the SPI determined by thiobarbituric acid (TBA) assay. D Lipid peroxidation in algal cells treated with SPI. E Hydroxyl radical detection. For the biochemical assays, the Fenton reagents (0.83 mM ferrous ions and 30 mM hydrogen peroxide) and BG11 medium was used as positive and negative control, respectively. The quantitative data were presented as mean ± S.D. (n = 3). **, p < 0.01 (Student’s t test). Scale bar, 5 μm

Fig. 3

Identification of the metabolites with putative functions in causing oxidative stresses in the algal cells through comparative metabolomics analysis. A Relative fold changes (FC) and the number of annotated metabolites in the SPIs collected on 1, 3 and 5 day post-infection. B Ten metabolites (D5/D1 FC > 2) with putative functions involved in driving Fenton reaction were screened

Fig. 4

Effects of the screened metabolites on the algal cellular composition and infection process. A Degradation of the algal cellular components by the screened metabolites. The control and the mixture was the algal cells treated with BG11 medium and 10 reagents that mixed in equal volume, respectively. B Effects of the screened metabolites on the infection ratio. C Fe³⁺ reducing ability of 3-HAA and hordenine. D Production of hydroxyl radicals by hordenine and 3-HAA. E Relative intracellular ROS intensity of algal cells after treated with hordenine or 3-HAA, determined by DCFH-DA staining. The fluorescence intensity of the BG11 medium-treated algal cells (control) was considered as 100%. The concentration of cyclohexylamine was 2% (v/v), and the concentration of the other 9 reagents were 0.2% (w/v) in the assays. The solutions were prepared with the BG11 medium and heated in the 95 °C water bath for 15 min, followed by ultrasonic treatment for 5 min and filtrated with 3000 Da cut off membrane prior to use. The quantitative data were presented as mean ± S.D. (n = 3). *, p < 0.05, **, p < 0.01 (Student’s t test). The Fe³⁺ reducing ability and hydroxyl radical productivity of the other 8 metabolites can be referred to Figures S3 and S4 (Additional file 1)

Fig. 5

Inhibitory effect of BHA on the fungal infection. A Infection process was inhibited by the addition of BHA at various concentrations. Microscopic images showed the untreated and 12 ppm BHA-treated cell cultures on the 3^rd day post-infection, respectively. B Dry weight of H. pluvialis cell culture in the 100 L open raceway pond was rescued by applying 7 ppm BHA every 2 days. Control, the uninfected and untreated cell culture. Fungal infection, the infected  and untreated cell culture. 7 ppm every 2 days, the infected cell culture treated with 7 ppm BHA every 2 days. Scale bar, 20 μm