Induced resistance is a systemically activated state of plant immunity against a broad range of pathogens (Flors et al., 2024; Kloepper et al., 2004; Pieterse et al., 2014). Induced systemic resistance is regulated by a complex network involving crosstalk between defense-related phytohormones, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) (Pieterse et al., 2014). Induced resistance is elicited by a variety of inducing agents, such as avirulent pathogens, plant growth-promoting rhizobacteria, or specific synthetic chemicals (Alström, 1991; Kuć, 1982; Ross, 1961; Van Peer et al., 1991; Wei et al., 1991; Zimmerli et al., 2000). Plants with activated induced systemic resistance by specific agents respond more quickly and strongly when encountering a pathogen, a phenomenon known as defense priming (Heil and Baldwin, 2002; Karasov et al., 2017; Van Hulten et al., 2006). Since a primed state requires less energy than a full defensive response, induced systemic resistance benefits both disease control and sustainable crop cultivation (Heil and Baldwin, 2002; Karasov et al., 2017; Van Hulten et al., 2006).
Chlorella is a genus of eukaryotic photosynthetic microorganisms present in a range of habitats, including fresh and sea water, soil, and plant tissue (Liu and Chen, 2016; Treves et al., 2016; Zhu et al., 2018). Chlorella cells synthesize many important raw materials that are used to produce foods, antibiotics, medicines, and biofuels (Kiani et al., 2022; Liu and Chen, 2016). The specific microalgal strains that are used in industrial biofuel production have been extensively studied; these include Chlorella spp. and Nannochloropsis spp. (Kiani et al., 2022; Yun et al., 2019, 2021; Zhang et al., 2014). Dried Chlorella extracts or culture solutions are applied to crop plants as bioprotectants to control diseases caused by pathogenic nematodes and fungi (Bileva, 2013; Choleva et al., 2005; Kim et al., 2018a, 2018b; Lee et al., 2016, 2017). In our previous study, we found that spraying Arabidopsis with Chlorella fusca supernatant elicits induced resistance, protecting plants against Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) (Lee et al., 2020); D-lactic acid was identified as the active component in the supernatant that triggered the immune response (Lee et al., 2020).
As industrial uses of Chlorella require only the harvested cells, microalgal cultivation generates large volumes of waste supernatant. We previously proposed a novel biocontrol strategy to recycle Chlorella waste supernatant with a model plant (Lee and Ryu, 2021). If such recycling is to be both economically efficient and agriculturally beneficial, however, supernatants from Chlorella strains that offer both high disease control activity and industrial advantages are required. Our objective was therefore screened industrial microalgal strains with a view to developing their supernatants as biocontrol agents for crop plants such as cucumber. Supernatant and its determinant D-lactic acid from Chlorella sp. HS2 and ABC001 efficiently reduced angular leaf spot disease caused by Pseudomonas syringae pv. lachrymans (Pla) in cucumber leaves by activating induced resistance without any antagonistic activity. Our results give an insight into agricultural application of industrial microalgae waste for plant disease management with minimizing the side-effect.
Materials and Methods
Plant materials and growth condition
Seeds from cucumber (Cucumis sativus L. cv. backdadagi, Nongwoobio, Suwon, Korea) were sown onto autoclaved soil-less potting medium (Punong, Co. Ltd., Gyeongju, Korea) in 50-hole plastic trays (28 × 54 × 5 cm). After 7 days, seedlings were transplanted into fresh pots (diameter 10 cm, height 8.5 cm) containing autoclaved soil-less potting medium. Cucumber plants were grown in an environmentally controlled growth room at 25°C with 7,000 lux light intensity and 12 h light/12 h dark cycles.
Preparation and application of Chlorella supernatants
Microalgae strains including Chlorella fusca strain CHK0059, Chlorella spp. strains ABC001 (previously known as Chlorella sp. KR1), HS2, C2, C8, and Nannochloropsis oceanica were cultivated under mixotrophic conditions, as described previously (Koh et al., 2023; Lee et al., 2020; Park et al., 2020; Sung et al., 1999; Yun et al., 2021; Zhang et al., 2014). Mixotrophic cultivation was conducted at 25°C under continuous illumination (100 μmol/m2/s) in 1× BG11 medium (Sigma-Aldrich, Burlington, MA, USA) containing 1 g/L glucose. To calculate the concentration of microalgal cells, cell numbers were counted with a hemocytometer (INCYTO, Cheonan, Korea). Supernatants were collected when cultures reached a concentration of 107 cells/mL using centrifugation at 4,000 × g for 10 min and were filtered using a 0.45 μm syringe filter (Lee et al., 2020).
To test biological control activity of microalgal supernatant against Pla, cucumber leaves were sprayed with 20 ml Chlorella supernatant, 0.5 mM acibenzolar-S-methyl (BTH) (Bion 50 WG, Syngenta, Basel, Switzerland), or 1× BG11 broth at 5 and 10 days post-transplantation. For D-lactic acid (Sigma-Aldrich) treatments, cucumber leaves were sprayed with 20 mL of serially diluted D-lactic acid (1 μM, 10 nM, and 100 pM) at 5 and 10 days post-transplantation. Sterilized distilled water (20 mL) was treated in the same manner as the negative control for D-lactic acid.
Assessment of induced systemic resistance by Chlorella supernatant
Pla were grown on King’s B (3% proteose peptone no. 3, 0.15% K2HPO4, 0.15% MgSO4·7H2O, 1% glycerol) medium containing 1% agar at 30°C for 2 days (Song et al., 2017). For the evaluation of induced systemic resistance, BG11 medium was treated as the negative control, and BTH as the positive control. At 7 days after second application of Chlorella supernatant, the seedling leaves were sprayed with suspension of Pla in sterilized distilled water. The optical density of the suspension was measured at an absorbance of 600 nm (OD600 = 1.0) using a spectrophotometer (Biochrom US, Holliston, MA, USA). To promote disease development, 20 mL of sterilized distilled water was applied to the cucumber root at 5 h before pathogen inoculation, and the relative humidity in the growth room was maintained at approximately 50%. Seedling leaves were sprayed with a culture of Pla suspension until run-off on cucumber seedling leaves. The numbers of symptomatic spots on cucumber leaves were counted at 7 days post-inoculation (dpi).
Assessment of plant growth promotion by Chlorella supernatant
To assess the effect of Chlorella supernatant on plant growth, cucumber leaves were sprayed with 20 mL Chlorella supernatant, 1× BG11 medium (negative control), 0.5 mM BTH at 5 and 10 days post-transplantation, following the same procedure as the assay for induced systemic resistance. The third leaf width and shoot weight of cucumber plants were measured 2 weeks after second application of Chlorella supernatant. To test the effect of D-lactic acid on cucumber growth, cucumber leaves were sprayed with 20 mL of serially diluted D-lactic acid (1 μM, 10 nM, and 100 pM), sterilized distilled water (negative control), and 0.5 mM BTH at 5 and 10 days post-transplantation. At 2 weeks after second application of D-lactic acid solution, the shoot weight of D-lactic acid-treated cucumber plants was measured.
Antagonism test
The antagonistic effects of HS2 and ABC001 supernatants against Pla were determined using the paper disc assay method. Two hundred microliters of Pla suspension (OD600 = 1.0) was plated onto King’s B medium containing 1% agar, and then paper discs, infiltrated with 50 μL of Chlorella supernatant, 1 μg/mL polymyxin B (an antibiotic), or BG11, were placed on the agar surface. After incubating the plates at 30°C for 2 days, we checked for the formation of an inhibition zone around the paper disc.
Analysis of defense gene expression through quantitative reverse transcription polymerase chain reaction
For quantitative reverse transcription polymerase chain reaction (qRT-PCR), cucumber leaves were harvested and immediately frozen in liquid nitrogen at 0 h and 6 h post-inoculation of Pla. Total RNA was extracted from cucumber leaf tissues using TRI Reagent (Sigma-Aldrich), and first-strand cDNA was synthesized from 2 mg of DNase-treated total RNA using oligo-dT primers and Moloney murine leukemia virus reverse transcriptase (MMLV-RT, Enzynomics, Daejeon, Korea), as described previously (Lee et al., 2020). qRT-PCR analysis was carried out using the Chromo4 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) using the cDNA template, iQTM SYBR Green Supermix (Bio-Rad), adhering to the manufacturer's instructions (Song et al., 2017). Gene expression levels of CsPATHOGENESIS-RELATED PROTEIN 2 (CsPR2), CsLIPOXYGENASE (CsLOX), and CsEHTYLENE-RESPONSE 1 (CsETR1) in cucumber were assessed using the primers listed in Supplementary Table 1. Relative RNA levels were calibrated and normalized relative to the level of CsActin mRNA.
Statistical analysis
Data were analyzed by analysis of variance (ANOVA) using JMP 4.0 software (SAS Institute Inc., Cary, NC, USA). Significant treatment effects were determined using the magnitude of the F-value (P < 0.05). When a significant F-value was obtained, the separation of means was analyzed by determining Fisher’s protected least significant difference at P < 0.05.
Results
Chlorella cell-free supernatant elicited induced systemic resistance in Arabidopsis and cucumber
As we previously showed that Chlorella supernatants mediate biological control (Lee and Ryu, 2021; Lee et al., 2020), we attempted to evaluate Chlorella strains that had both high biocontrol efficacy and industrial benefits. We initially tested the biological control activity of supernatant from ABC001, a high biofuel-producing strain, against Pto DC3000 in Arabidopsis (Supplementary Fig. 1) (Cho et al., 2020; Koh et al., 2023; Seon et al., 2020, 2023). At 7 dpi, the population of Pto DC3000 in Arabidopsis leaves pretreated with ABC001 supernatant was reduced by 6.6-fold compared with control-treated leaves (Supplementary Fig. 1). Similarly, the population of Pto DC3000 in Arabidopsis leaves pretreated with BTH, as positive control, was also reduced by 10.4-fold compared with control (Supplementary Fig. 1). These results implied that the supernatant of industrial strain can activate induced resistance in plant similar with previous study.
To find the optimum industrial strains for crop protection, cucumber leaves were sprayed with supernatants from C. fusca and the high biofuel-producing microalgal strains Chlorella spp. HS2, ABC001, C2, and C8, and N. oceanica (Fig. 1, Supplementary Figs. 2 and 3) (Kiani et al., 2022; Park et al., 2020; Seon et al., 2023; Yun et al., 2021; Zhang et al., 2014). The number of spots on the cucumber leaves significantly reduced by 4.4-fold, 2.4-fold, and 3.1-fold following treatment with C. fusca, HS2, and ABC001 supernatants, respectively, compared to control treatment (Fig. 1A-D). The number of spots on the cucumber leaves were slightly reduced following treatment with C2 supernatant, but not significant compared to control (Supplementary Fig. 2). By contrast, any reduction of disease symptoms was not observed in cucumber leaves treated with C8, and N. oceanica supernatants (Supplementary Figs. 2 and 3). These results indicated that supernatant from industrial strains Chlorella sp. HS2 and ABC001 protect cucumber against Pla.
Validation of direct antagonism of Chlorella culture supernatant against Pla
As the Chlorella supernatants and Pla were not spatially separated in our system, we determined whether the supernatants from HS2 and ABC001 directly inhibited Pla growth. Pla growth was inhibited by polymyxin B (Fig. 1E); however, inhibition zones were not observed around the edges of paper discs infiltrated with either HS2 or ABC001 supernatant (Fig. 1E).
Effect of Chlorella supernatants on cucumber growth
To test whether HS2 and ABC001 supernatants improve cucumber growth, the third leaf width and shoot weight of cucumber were measured in Chlorella supernatant treated cucumber (Fig. 2). BTH treatment reduced leaf width by 1.2-fold and shoot weight by 1.6-fold, compared with control treatment (Fig. 2B and C). By contrast, no changes in leaf width or shoot weight were found in seedlings treated with HS2 or ABC001 supernatants (Fig. 2B and C). This result showed that supernatant application did not require any growth penalty on cucumber.
D-lactic acid elicited induced resistance against Pla
To determine whether D-lactic acid also elicited induced resistance in cucumber, we sprayed cucumber leaves with D-lactic acid (Fig. 3). Low concentrations of D-lactic acid such as 1 μM, 10 nM, or 100 pM of D-lactic acid were used to avoid damage to leaves caused by its low pH (Supplementary Fig. 4). The numbers of symptomatic spots on leaves decreased by 54.6% and 86.2% following treatment with 1 μM D-lactic acid or BTH, respectively, compared with control treatment (Fig. 3B). Although spot number reduced slightly following 10 nM or 100 pM D-lactic acid applications, these treatments did not differ significantly from the control (Fig. 3B). Meanwhile, unlike BTH treatment, D-lactic acid application did not affect cucumber growth (Fig. 3C). This result indicated that spray treatment of D-lactic acid can activate induced resistance in cucumber.
Exogenous D-lactic acid application primed the marker genes for SA and JA signaling
To examine the activation of defense signaling by exogenous treatment of D-lactic acid in cucumber, we analyzed the expression of defense-related marker genes involved in SA, JA, and ET signaling at 0 and 6 h post-inoculation of Pla (Fig. 4). At 0 h post-inoculation, there was no significant difference in expression of CsPR2, which is involved in SA signaling, between treatments (Fig. 4). At 6 h post-inoculation, CsPR2 was upregulated by 1.97-fold following 1 μM D-lactic acid treatment, compared with the control treatment. The expression of this gene increased by 3.78-fold following BTH treatment, compared with the control (Fig. 4). Expression of CsLOX, a JA signaling marker, also increased by 2.98-fold following treatment with 1 μM D-lactic acid at 6 h post-inoculation, compared with the control, but not BTH treatment (Fig. 4). However, this D-lactic acid treatment did not alter the expression of CsETR1, an ET signaling marker. Exogenous D-lactic acid thus primed induced resistance against Pla by activation of SA and JA signaling pathways in cucumber.
Discussion
As biological control agents, C. fusca cultures act in cucumber, as their application reduces the severity of anthracnose caused by Colletotrichum orbiculare (Lee et al., 2016, 2017). However, as C. fusca cells directly inhibit appressorium formation, it is difficult to distinguish whether C. fusca elicits induced systemic resistance in cucumber plants (Lee et al., 2016, 2017). Meanwhile, foliar application of C. fusca supernatant activates induced resistance in Arabidopsis, protecting against Pto DC3000 without antibacterial activity (Lee et al., 2020). These findings led us to hypothesize that spraying plants with cell-free supernatants from industrial Chlorella strains also elicits induced systemic resistance rather than offering protection through antibiosis, not only in Arabidopsis but also in cucumber (Fig. 1, Supplementary Fig. 1). Since induced systemic resistance can protect crops against a broad range of pathogens, HS2 and ABC001 supernatants need to be tested on various crop plants under field conditions.
Diverse Chlorella species have been reported to primarily produce D-lactic acid, along with formic acid and acetic acid (Begum and Syrett, 1970; Gruber et al., 1974; Kessler and Soeder, 1962; Lee et al., 2020; Syrett and Wong, 1963; Vinayakumar and Kessler, 1975). Among 87 tested Chlorella strains, 64 strains produced D-lactic acid indicating that D-lactic acid is the common primary metabolic product in the Chlorella genus (Vinayakumar and Kessler, 1975). The biosynthesis of D-lactic acid is governed by expression of the D-lactate dehydrogenase (D-LDH) gene, which is conserved in higher plants and some microalgae like Chlorella but absent in animals and most microorganisms (An et al., 2017; Atlante et al., 2005; Gruber et al., 1974; Iatsenko et al., 2018; Monroe et al., 2019; Welchen et al., 2016). The previous transcriptome analysis revealed that the expression of D-LDH gene in ABC001 and HS2 was significantly upregulated under high lipid-accumulating conditions such as high salt and high CO2 levels (Koh et al., 2023; Yun et al., 2021; data not shown). However, most microalgae species such as N. oceanica used in our previous experiments generally lack D-LDH (Gruber et al., 1974; Koh et al., 2023; Yun et al., 2021). In our previous study, we identified D-lactic acid from cell-free supernatant of Chlorella and characterized the role of L-lactic acid on activation of plant immune response in Arabidopsis (Lee et al., 2020).
In the line, D-lactic acid in the Chlorella supernatant might play a significant role in the biocontrol of cucumber diseases (Fig. 3). D-lactic acid, produced through the detoxification of methylglyoxal, accumulates under biotic and abiotic stress and can damage plants by D-LDH (An et al., 2017; Jain et al., 2020; Wienstroer et al., 2012). Pre-treating tobacco plants (Nicotiana tabacum) with lactic acid not only reduces levels of endogenous D-lactic acid but also increases SA and JA biosynthesis and reactive oxygen species (ROS) burst (Yan et al., 2024). Similarly, spraying with D-lactic acid activated expression of SA and JA signaling components in cucumber (Fig. 4) and mitochondrial ROS burst in Arabidopsis (Lee et al., 2020). Therefore, D-lactic acid in Chlorella supernatant may not only have activated induced resistance but also reduced the damage caused by endogenous D-lactic acid in plants. Further investigations into the defense responses induced in cucumber by ABC001 and HS2 supernatants are required, as is quantification of D-lactic acid levels in the supernatants from different Chlorella strains.
Commercial application of Chlorella supernatant as plant protectants requires screening waste from cultures of industrial strains for suitability for reuse in an agricultural context. The Chlorella strains HS2 and ABC001 accumulate large amounts of lipids (Jin et al., 2017; Sung et al., 1999; Yun et al., 2019, 2021) and are more tolerant of various stresses, including oxidative stress, salt stress, and high CO2 levels, than other industrial strains; they are therefore cultivated on a large scale for biofuel production (Cho et al., 2020; Jin et al., 2017; Koh et al., 2023; Seon et al., 2020, 2023; Sung et al., 1999; Yun et al., 2019, 2021). Supernatants from HS2 and ABC001 cultures had high biocontrol activity against Pla in cucumber plants (Fig. 1C and D, Supplementary Figs. 2 and 3); thus recycling these supernatants in an agricultural setting may be both economical and sustainable.
Cucumbers are primarily cultivated indoors. Under such conditions, plants may be infected with pathogenic Pseudomonas species through insect vectors such as whiteflies or aphids (Lamichhane et al., 2015; Silva-Sanzana et al., 2023). Fertilizers and pesticides are typically applied through drip-irrigation, sprinkler, or hydroponic systems (Bhavsar et al., 2023; Ramakrishnam Raju et al., 2022). Cell-free supernatants from Chlorella cultures could be easily applied as a biological control agent through such systems. Treatment of pepper plants with HS2 and ABC001 supernatants reduced aphid infestation (data not shown); thus Chlorella supernatants might not only protect plants against Pla by eliciting induced resistance but also reduce the spread of Pla via insect vectors in greenhouse condition.
Our study supported our previous proposal that Chlorella supernatant could be used for crop protection. It showed that supernatants from cultures of the industrial strains HS2 and ABC001, whose cells are cultivated on a large scale for biofuel production, demonstrated high activity on induced resistance. These results suggest that a large-scale dual production system that combines culturing microalgal cells for biofuel production with recycling supernatant waste as a bioprotectant is feasible.
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