Fungal infection caused cell death of H. pluvialis and the supernatant post infection (SPI) enhanced the infection process
When the H. pluvialis cells cultivated in 360 L panel photobioreactors were infected by P. sedebokerense, algal cells died rapidly and seriously (Fig. 1A-B). We collected the supernatant post infection (SPI) by removing both the host and fungal cells via centrifugation, and investigated the effects of SPI on the newly infection process. When the healthy algal cells were pre-treated with SPI and then challenged with the fungal swarmers, the infection process was significantly promoted (Fig. 1C). On the second day post inoculation, the color of the control cell culture was dark-green and a few algal cells were attached with the fungal swarmers. By contrast, a large number of dead algal cells were observed in the culture of algal cells pre-treated with SPI, which settled down to the bottom of flask. On the third day post inoculation, the color of the culture of algal cells pre-treated with SPI turned to brownish, whereas the control remained green. Pre-treating the algal cells with SPI prior to the pathogenic challenge caused significantly higher infection ratio after the second day post inoculation than the control (Fig. 1D). Additionally, the SPI remained the infection enhancing activity after pre-treated in 98oC water bath for 15 min, indicating that the activity of SPI was not eliminated by heating. It was further observed that in the 48h-SPI treated algal cells, the pigments were partially degraded and the cellular starch granules disappeared (Fig. 1E). Quantitative analysis revealed that when compared to the control, the contents of the total cellular carbohydrates and pigments (i.e. carotenoids and chlorophyll) were reduced in the algal cells treated with SPI by 50% and 20%, respectively (Fig. 1F). Additionally, after filtered with 3000 Da cut-off membrane, the activity of the filtrate did not significantly altered in degrading carbohydrates and pigments in the algal cells, suggesting that the activity in the SPI was most likely attributable to small molecules. These results together suggested that some heat-stable substances with small molecular weight produced during the infection process and were capable of enhancing the susceptibility of H. pluvialis cells to the pathogen by affecting the algal cell integrity.
SPI induced oxidative stresses within algal cells
Alterations in the subcellular structures of the algal cells challenged with both P. sedebokerense and SPI were observed with transmission electron microscopy (TEM) (Fig. 2A). Degradation of the subcellular membrane systems were observed in the algal cells treated with SPI without the involvement of fungus. Additionally, the algal cell walls were loosened after being treated with SPI. These results indicated that the substances in SPI degraded the algal cellular components and destructed membranes.
To uncover the identity of the secondary metabolites in SPI, transcriptomic analysis was conducted to facilitate understanding the effects of SPI on the algal cells. A total of 998 and 490 genes were up- and down-regulated in H. pluvialis, respectively, after the SPI treatment for 24 h. Expression of many genes involved in biotic stresses responses were significantly altered in the algal cells treated with SPI (Fig. 2B). Several genes coding for the anti-oxidative enzymes were found to be significantly up-regulated while genes coding for synthesis and transportation were down-regulated. Up-regulation of the genes involved in oxidative stress responses indicated that SPI may contain substances that can cause the generation of ROS (Hasanuzzaman et al., 2020; Torres et al., 2006). To test this hypothesis, the oxidative activities of SPI were measured by using the thiobarbituric acid (TBA) assay with fenton reagent as the positive control because it is a known reaction that generates oxidative stress through small molecules (Arantes et al., 2012; Eastwood et al., 2011). The results showed that the SPI possessed strong oxidative activity in vitro (Fig. 2C). In addition, the SPI showed lipid peroxidation activity when acting on the algal cellular membranes, leading to formation of malondialdehyde (Fig. 2D). To further identify the ROS produced by SPI, the dimethyl sulfoxide trapping method was used and the results suggested that SPI could produce hydroxyl radical in vitro (Fig. 2E).
Based on the transcriptomic results and a suite of observations and biochemical assays, it can be concluded that SPI contained substances that exerted oxidative stresses via generation of ROS in the algal cells. Oxidative degradation of the algal subcellular structures might be the cause of decreased resistance to fungal infection.
Secondary metabolites mediated fenton reaction facilitates the fungal infection
Metabolomic analysis was performed for identification of the small molecules causing the oxidative stresses. The SPIs were collected at different infection stages, i.e., 1, 3 and 5 day post inoculation of the fungus into the algal cell cultures. It was found that the degradation activity of SPI collected on Day (D) 5 was significantly higher than that on D1, suggesting that the concentration of the metabolites of target increased over 5 days. Based on this, 62 metabolites which showed over 2-fold increases on D5 than that on D1 were selected, most of which were organic acid, dipeptide, amino acid and derivatives. Ten metabolites, including tyramine, trimethoprim, indole-3-carboxylic acid, hordenine, deoxycytidine, 4-pyridoxic acid, lumichrome, 3-hydroxyanthranilic acid (3-HAA), baclofen and cyclohexylamine, with the phenol/quinone/aromatic structure, were retrieved manually (Fig. 3), since such types of compounds are known to be able to mediate the fenton reaction producing hydroxyl peroxide (Arantes et al., 2012; Eastwood et al., 2011) .
When these substances were added into the algal cell culture, it was firstly observed that hordenine and 3-HAA significantly reduced the contents of carbohydrates while tyramine, hordenine and cyclohexylamine caused degradation of the pigments in the treated algal cells (Fig. 4A). Secondly, the potential infection-prompting effect of the candidate metabolites was checked. On the 3rd dpi (day post infection), the algal cells pre-treated with either hordenine or 3-HAA showed significantly enhanced infection ratio than that of the control and other compounds (Fig. 4B).
The fenton reaction is initiated from the reduction of Fe3+ to Fe2+, which is the key factor for driving fenton reaction (Kameshwar & Qin, 2018). Thus the reducing activities of the 10 candidate metabolites were tested. Among them, 3-HAA showed the strongest activity in reducing Fe3+ to Fe2+ at 4 h (Fig. 4C). Additionally, 3-HAA and hordenine generated hydroxyl radical in the assay with DMSO as substrate (Fig. 4D). The intracellular concentration of hydrogen peroxide (H2O2), an important intermediate of the fenton reaction, was measured after staining with the fluorescence DCFH-DA. The result showed that in the hordenine-treating algal cells the concentration of H2O2 continued increasing during 48 h, while the level of H2O2 in the 3-HAA-treating algal cells transiently increased during 24 h and the gradually decreased (Fig. 4E).
These results taken together indicated 3-HAA and hordenine are the components in SPI which caused oxidative stresses in the algal cells through mediating fenton reaction. The metabolite 3-HAA is an intermediate of kynurenine pathway, and has been found in bacteria, yeast, fungi, plants and mammals and was reported to be a fungal producing mediator that was widely applied in fenton processes in dye decolorization due to its ability in reducing Fe3+ to Fe2+ (Santana & Aguiar, 2015; Santana et al., 2019). It is also a generator of free radicals through its auto-oxidation (Breton et al., 2000; Li et al., 2001). Thus, the generation of H2O2 from the auto-oxidation of 3-HAA could not be excluded here. Hordenine, originally detected in barley and also isolated from marine algal Phyllophora nervosa (Guven et al., 2010; Mann et al., 1963), is a phenethylamine alkaloid with various bioactivities, including antibiotic activity against microorganisms, inhibition of quorum sensing and biofilm formation (Rao, 1970; Zhou et al., 2018). It was reported that hordenine was responsible for the protective responses of plants to various stresses through jasmonate dependent defense pathway (Ishiai et al., 2016), and also acted as an plant allelochemical that can inhibit the growth of weed or defend against pathogens attack (Kotzamani et al., 2021; Lebecque et al., 2018).
Application of antioxidant to inhibit the fungal infection
As the oxidative stresses caused by the SPI impaired the algal cell structures and promoted the fungal infection process, an exogenous antioxidant was introduced to relieving such oxidative stress in the culture to inhibit the infection. BHA is one of the most commonly used synthetic antioxidants in food and biodiesel fuels to prevent oxidation for its low cost, high stability and effectiveness (Rodil et al., 2012; Ryu, 2010; Sahraee et al., 2019; Xu et al., 2021). Additionally, BHA is biosafe and environmental-friendly, rendering its application in the aquaculture industry (Additives et al., 2018; Williams et al., 1999). BHA was added into the infection system at different final concentrations (i.e. 2 ppm, 7 ppm and 12 ppm) (Fig. 5). The infection ratio of the newly infected algal cells was calculated to reflect the infection inhibitory effect. Compared to the untreated H. pluvialis culture, addition of BHA at 2 ppm delayed the complete fungal infection for 1 day. Elevating the concentration of BHA to 7 ppm decreased the infection greatly, and the infection was only about 30% on day 5, while the untreated group was 100% infected. Application of 12 ppm BHA to the system completely suppressed the fungal infection.
According to all the results described above, a model was proposed herein to illustrate the major findings of this study. Production of secondary metabolites such as 3-HAA and hordenine in the infection system mediated the generation of hydroxyl radical via the fenton reaction, the most reactive free radicals among various ROS, which disrupt the subcellular components of the H. pluvialis cells and make the algal cells more susceptible to the infection. However, by adding 12 ppm of the antioxidant BHA to the culture, the fungal infection was completely abolished, indicating the oxidative burst is essential for the pathogens to infest.