ABSTRACT
Prorocentrum donghaiense blooms occur frequently in the Yangtze River estuary and the adjacent East China Sea. These blooms have damaged marine ecosystems and caused enormous economic losses over the past 2 decades. Thus, highly efficient, low-cost, ecofriendly approaches must be developed to control P. donghaiense blooms. In this study, a bacterial strain (strain Y42) was identified as Paracoccus sp. and was used to lyse P. donghaiense. The supernatant of the strain Y42 culture was able to lyse P. donghaiense, and the algicidal activity of this Y42 supernatant was stable with different temperatures and durations of light exposure and over a wide pH range. In addition to P. donghaiense, Y42 showed high algicidal activity against Alexandrium minutum, Scrippsiella trochoidea, and Skeletonema costatum, suggesting that it targets primarily Pyrrophyta. To clarify the algicidal effects of Y42, we assessed algal lysis and determined the chlorophyll a contents, photosynthetic activity, and malondialdehyde contents of P. donghaiense after exposure to the Y42 supernatant. Scanning electron microscopy and transmission electron microscopy analyses showed that the Y42 supernatant disrupted membrane integrity and caused algal cell breakage at the megacytic zone. Photosynthetic pigment loss and significant declines in both photosynthetic efficiency and the electron transport rate indicated that the Y42 supernatant damaged the photosynthetic system of P. donghaiense. Malondialdehyde overproduction indicated that the Y42 supernatant caused lipid peroxidation and oxidative damage to membrane systems in the algal cell, ultimately leading to death. The findings of this study reveal the potential of Y42 to remove algal cells from P. donghaiense blooms.
IMPORTANCE P. donghaiense is one of the most common dinoflagellate species that form harmful algal blooms, which frequently cause serious ecological pollution and pose health hazards to humans and other animals. Screening for bacteria with high algicidal activity against P. donghaiense and studying their algicidal processes and characteristics will contribute to an understanding of their algicidal effects and provide a theoretical basis for preventing algal blooms and reducing their harm to the environment. This study reports the algicidal activity and characteristics of Paracoccus against P. donghaiense. The stability of the algicidal activity of Paracoccus in different environments (including different temperature, pH, and sunlight conditions) indicates its potential for use in the control of P. donghaiense blooms.
INTRODUCTION
In recent decades, harmful algal blooms (HABs) have frequently occurred in eutrophic water. These HABs have had serious economic impacts on fisheries, and they pose considerable threats to public health and the aquatic environment throughout coastal areas of the world (1). Many approaches have been implemented to prevent and to remove HABs, including physical and chemical methods (2). However, these approaches are costly and may lead to secondary pollution (3). Consequently, economical, effective, and environmentally friendly methods of controlling HABs must be developed. Biological methods, especially those involving the use of microorganisms with algicidal activity, such as bacteria, fungi, and viruses, have attracted widespread attention (4). Algicidal bacteria are the main producers of extracellular active substances, and they have been widely studied for their ability to inhibit the growth of harmful algae. Many algicidal bacteria, such as Acinetobacter (5), Alcaligenes (6), Bacillus (7, 8), Deinococcus (9), Hahella (10, 11), Mangrovimonas (12), Pseudoalteromonas (13), Pseudomonas (14, 15), Streptomyces (16, 17), and Vibrio (18, 19) species, have the potential to decrease the intensity of HABs. Furthermore, a few algicidal bacteria exhibit broad host ranges and exert effects on a variety of algal species (11, 20–22).
Prorocentrum donghaiense is a major cause of HABs in the Yangtze River estuary and the adjacent East China Sea and is responsible for frequent large-scale HABs and serious damage to marine ecosystems and mariculture, resulting in enormous economic losses over the past 2 decades (23, 24). Considerable efforts have been devoted to the investigation of environmental conditions that regulate the occurrence and maintenance of P. donghaiense blooms and the cellular responses of this dinoflagellate to different conditions, such as N/P repletion, N/P depletion, different N/P substrates, acidification, and eutrophication (25–29). Although many algicidal bacteria have been identified and exploited in the control of Microcystis aeruginosa (5, 7, 14, 17, 30), Alexandrium tamarense (9, 12, 18, 19), Phaeocystis globosa (8, 11, 16, 31–33), and Skeletonema costatum (34–37), few studies have reported bacteria with algicidal effects against P. donghaiense. Therefore, an effective biocontrol method for the prevention and dissolution of P. donghaiense algal blooms must be developed.
In this study, we isolated and identified a bacterium, strain Y42, with algicidal activity against P. donghaiense. To estimate the potential use of strain Y42 to control P. donghaiense blooms, we investigated its algicidal activities and features. To explore the algicidal effect of strain Y42, we observed the P. donghaiense cell death process, examined the photosynthetic performance of P. donghaiense, and analyzed oxidative stress in the dinoflagellate after exposure to a cell-free supernatant isolated from a strain Y42 culture. Finally, we evaluated the algicidal activities of strain Y42 against other species that cause HABs.
RESULTS
Characterization and identification of the algicidal strain.After isolation and purification, 137 strains of bacteria were obtained from the surface sediment sample, of which 12 strains were found to exert antialgal activity against P. donghaiense. Among these 12 strains, isolate Y42 showed the strongest alga-lysing activity when 5% (vol/vol) of the strain culture was added to a P. donghaiense culture for 3 days. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses showed strain Y42 to be rod shaped (approximately 0.4 to 0.7 μm by 0.8 to 1.2 μm), with a smooth cell surface. PCR amplification of the 16S rRNA gene (1,425 bp) and sequencing showed that the strain Y42 sequence exhibited the greatest similarity (97%) to that of Paracoccus sp. Lp91 (GenBank accession no. KU693337), with the shortest genetic distance among the Paracoccus species and related species analyzed (Fig. 1). These results indicated that strain Y42 belonged to the genus Paracoccus, and it was termed Paracoccus sp. strain Y42.
Morphological characteristics and phylogenetic identification of bacterial strain Y42. (a) SEM image. (b) TEM image. (c) Phylogenetic tree based on 16S rRNA gene sequences, showing the position of strain Y42, representatives of Paracoccus, and other related species. Bootstrap values (expressed as percentages of 1,000 replications) are shown at the branch points. The scale bar represents 0.01 substitutions per nucleotide position (evolutionary distance).
Algicidal mode and activity of strain Y42.To explore the algicidal mode of strain Y42, cell-free supernatant and bacterial cells from a strain Y42 culture were collected by centrifugation and filtration, respectively. The different fractions were separately added to algal cultures to monitor their algicidal effects on P. donghaiense. Figure 2a and b show that the Y42 culture lysed 75% of the algal cells after 48 h of treatment and 94% of the algal cells after 72 h of treatment. The cell-free supernatant from the Y42 culture demonstrated algicidal rates of 61% and 90% after treatment for 48 h and 72 h, respectively, and these rates approximated those for the whole culture. In contrast, no algicidal activity was observed with the bacterial cells alone. The growth rate of the algal culture treated with bacterial cells was similar to that of an algal culture without any treatment. These results suggested that strain Y42 lysed P. donghaiense indirectly and the substance contributing to algal cell death was present in the cell-free supernatant.
Algicidal modes and activities of strain Y42 against P. donghaiense. (a) Growth status of algal cultures after exposure to different fractions of Y42 cultures. (b) Algicidal modes of Y42 cultures. (c) Growth status of algal cultures after exposure to different concentrations of Y42 supernatant. (d) Algicidal activities of Y42 supernatant. Values are means ± standard deviations (SDs) (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ******, P < 0.000001, significant differences from control values.
To assess the algicidal activity of strain Y42 against P. donghaiense, different concentrations (1%, 3%, 5%, 7%, and 10% [vol/vol]) of the Y42 supernatant were added to algal cultures. Although the treatment groups that received 1% Y42 supernatant did not show evidence of algicidal activity, algal cell growth was inhibited (Fig. 2c and d). In the treatment groups that received 3% Y42 supernatant, 55% of the algal cells had been lysed after exposure for 72 h; the algicidal effects were enhanced with increasing Y42 supernatant concentrations and treatment times. For example, 5% Y42 supernatant resulted in 61% of the cells being lysed after treatment for 48 h and 90% of the cells being lysed after treatment for 72 h. When the concentrations reached 7% and 10%, the Y42 supernatant displayed greater algicidal activity, with the algicidal rates increasing to 69% and 74%, respectively, after treatment for 48 h and to 94% after treatment for 72 h (Fig. 2c and d). Thus, the algicidal activity of the Y42 supernatant was concentration dependent. Based on the algicidal effects and the dose of Y42 supernatant, 5% Y42 supernatant, which demonstrated high algicidal activity, was chosen as the concentration to use for further study.
Algicidal stability of the Y42 culture supernatant.To clarify the effects of temperature and pH on algicidal activity, the algicidal activity of the cell-free supernatant from Y42 cultures treated with different temperatures or pH levels was measured. Figure 3a shows that, after incubation at −20, 0, 10, 40, 50, 60, 80, or 100°C for 2 h, the supernatants continued to exhibit high algicidal activity, and the algicidal rates of the treatment groups were similar to that of the reference group (25°C; P > 0.05). These results indicated that the algicidal activity was stable across different temperatures. Figure 3b shows that the algicidal activity of the Y42 supernatant was insensitive to changes in pH. Compared with the reference value (pH 8), the algicidal activity remained stable over a wide pH range (from pH 3 to pH 12; P > 0.05). To understand the effect of light exposure on algicidal activity, we exposed the Y42 supernatant to the sun for 1, 2, 4, 6, 8, or 10 h, and the Y42 supernatant continued to show high algicidal activity following constant exposure to the sun for 10 h (Fig. 3c). These results showed that the algicidal activity of the Y42 supernatant was not affected by light exposure (P > 0.05).
Algicidal stability of the Y42 supernatant treated with different temperatures (a), pH values (b), and durations of natural light exposure (c). Values are means ± SDs (n = 3). None of the treatment groups showed a significant difference from the reference group (25°C in panel a, pH 8 in panel b, and 0 h of sunlight exposure in panel c) (P > 0.05).
Algicidal activity of Y42 toward other algal species.Strain Y42 showed no algicidal activity toward the following algal species within Chlorophyta, Chrysophyta, and Xanthophyta: Platymonas subcordiformis, Dunaliella salina, Platymonas helgolandica, Chlorella vulgaris, Nannochloropsis sp., Phaeocystis globose, and Heterosigma akashiwo (Table 1). In addition to exhibiting high algicidal activity toward P. donghaiense, Y42 showed high algicidal activity toward Alexandrium minutum (85.85%) and Scrippsiella trochoidea (82.64%) within Pyrrophyta. Interestingly, Y42 did not lyse Alexandrium tamarense, which also belongs to Pyrrophyta. Regarding algal species of Bacillariophyta, Y42 exhibited high algicidal activity toward Skeletonema costatum (91.20%) and had no lytic activity toward Thalassiosira pseudonana, Phaeodactylum tricornutum, Amphiprora alata, or Nitzschia closterium. The results strongly suggested that Y42 primarily target Pyrrophyta.
Lytic abilities of the Y42 supernatant for different algal species
Algal lysis.Algal lysis was observed using a microscope (Fig. 4). The shape of P. donghaiense is asymmetric and elongated, as described by Lu and Goebel (38). The anterior end has a slight extension, and the posterior end is rounded (Fig. 4a). The algal cells treated with Y42 supernatant swelled and became rounded (Fig. 4b), and the cells subsequently burst, which led to leakage of the cell contents, leaving only cell wall debris (Fig. 4c to e). With penetration of the Y42 supernatant, the densely stained nucleus of the algal cells diffused throughout the cell and was finally released (Fig. 4f to j).
Changes in the morphology and nucleus of P. donghaiense cells during cell death. Algal cells were harvested by centrifugation after 0 h (a and f), 12 h (b and g), 24 h (c and h), 48 h (d and i), or 72 h (e and j) of exposure to 5% of the Y42 strain supernatant, stained with DAPI (5 μg/ml) for 5 min at room temperature in the dark, and viewed with a fluorescence microscope. Morphological changes were visualized using bright-field illumination (a to e), and nucleus changes were visualized using UV illumination (f to j).
Effects of the strain Y42 supernatant on the ultrastructure of P. donghaiense.To monitor the events involved in the algicidal bacterium-induced cell damage in P. donghaiense, the ultrastructure of the algal cells was observed by SEM and TEM. Control cells appeared healthy, with intact cellular characteristics, including cell membrane integrity, homogeneous cytoplasm, obvious lipid droplets, regularly arranged chloroplasts, and a large nucleus with compact chromosomes (Fig. 5a and f). Compared with these control cells, most of the cells treated with the Y42 supernatant displayed morphological differences and structural damage. The treated cells lost plasma membrane integrity, and the cell wall was broken at the megacytic zone, which resulted in leakage of the cellular contents. Eventually, the intact cell structure disappeared, and only disintegrative cell walls were observed (Fig. 5b to e). TEM analysis revealed a decrease in cellular contents and formation of a number of membrane vesicles; the cells were eventually lysed after treatment with the Y42 supernatant (Fig. 5g to j).
Cell death process in P. donghaiense, visualized by SEM (a to e) and TEM (f to j). A damaged P. donghaiense cell treated with the Y42 culture supernatant for 0 h (a and f), 12 h (b and g), 24 h (c and h), 48 h (d and i), and 72 h (e and j) is shown. M, megacytic zone; C, chromosome; Chl, chloroplast; L, lipid droplet; N, nucleolus. Red arrows show where the cell membrane was disrupted. Bars in TEM images = 2 μm; bars in SEM images = 5 μm.
Effects of the strain Y42 supernatant on the photosynthetic system of P. donghaiense.To investigate the effects of the Y42 supernatant on the photosynthetic system of the algal cells, the chlorophyll a (Chl a) content, maximum quantum yield of photosystem (PS) II (variable fluorescence [Fv]/maximum fluorescence [Fm]), and relative photosynthetic electron transport rate (rETR) were measured. Figure 6a shows that the Chl a contents of algal cells treated with the Y42 supernatant were significantly decreased, relative to those of the control cells (P < 0.001). Compared with the Chl a contents of the control group, the Chl a contents of the 3% and 5% Y42 treatment groups decreased to 77% and 72%, respectively, after 12 h of exposure. With prolonged treatment times, the percentages continued to decrease, reaching 45% in the 3% treatment group and 17% in the 5% treatment group. These results indicated that the Y42 supernatant was capable of damaging Chl a.
Effects of the Y42 culture supernatant on the Chl a contents (a), Fv/Fm (b), and rETR (12 h) (c) of P. donghaiense. PAR, photosynthetically active radiation. Values are means ± SDs (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001, significant differences from control values.
The Fv/Fm values of cells exposed to 3% and 5% Y42 supernatant for 12 h decreased significantly (P < 0.01), to values 48% and 71% lower, respectively, than the control value. The Fv/Fm values continued to decrease with increasing treatment times, reaching 41% in the 3% treatment group and 17% in the 5% treatment group within 24 h; thereafter, the values remained stable and significantly lower than those of the control group (P < 0.01). These findings suggested that the photosynthetic capacity of the treated cells was inhibited (Fig. 6b). Similarly, the rETR value decreased significantly after treatment with Y42 supernatant for 12 h, suggesting that the photosynthetic rate of the algal cells was inhibited by the Y42 supernatant (Fig. 6c).
Effects of the strain Y42 supernatant on lipid peroxidation in P. donghaiense.Malondialdehyde (MDA) is a by-product of lipid peroxidation, and levels typically increase upon membrane lipid peroxidation. MDA contents were significantly increased in treatment groups and within 12 h of exposure reached maximum levels, which were 3.7 times those of control cells (P < 0.01) (Fig. 7). Although the MDA contents in both treatment groups declined slightly after 12 h of exposure, they remained high, compared to the control levels. The increases in MDA contents with treatment indicated that the Y42 supernatant induced peroxidation of membrane lipids and caused oxidative damage to the membrane system of P. donghaiense.
Effects of the Y42 culture supernatant on the MDA contents of P. donghaiense. Values are means ± SDs (n = 3). *, P < 0.05; **, P < 0.01, significant differences from control values.
DISCUSSION
Bacterial and HAB species are closely linked and interact in the marine environment. Certain marine bacteria are capable of promoting or inhibiting the growth of HAB species (39, 40), and such killing of HAB species by marine bacteria may contribute to the termination or disappearance of HABs. Treatment methods that exploit the relationship between bacteria and HAB species for HAB control are environmentally friendly (41) and are attracting increasing attention. P. donghaiense has caused the largest and most frequent red tides in the Yangtze River estuary and the East China Sea over the past 2 decades. Paracoccus sp. strain Y42 exhibited high algicidal activity toward P. donghaiense (Fig. 2). Although Paracoccus has been used in wastewater treatment (42) and compound biodegradation (43, 44), it has not yet been applied to control the occurrence of HABs. This study evaluated the algicidal activity and application potential of Paracoccus in controlling P. donghaiense blooms.
In general, bacteria exert algicidal effects by directly or indirectly attacking algal cells (34). A direct attack requires direct contact between the algicidal bacteria and algal cells, whereas an indirect attack does not require cell-to-cell contact. Algicidal bacteria can produce algicidal substances to kill algal cells (40). Our algicidal mode analysis showed that the Y42 supernatant had algicidal activity similar to that of the Y42 culture (Fig. 2), suggesting that strain Y42 indirectly attacks P. donghaiense by excreting an algicidal substance.
The stability of the Y42 supernatant algicidal activity under different temperature and sunlight conditions and in a wide pH range (pH 3 to 12) indicates the potential of this strain for use in the control of P. donghaiense blooms (Fig. 3). The pH of the surface seawater in the East China Sea is relatively constant, remaining in the range of 7.9 to 8.3, and the temperature changes seasonally from 16°C to 31°C (45). Furthermore, the strong stability of the algicidal activity of the Y42 supernatant with regard to pH and temperature suggests that the algicidal substances of the Y42 supernatant are not peptides or polysaccharides.
P. donghaiense is a species of marine photoautotrophic phytoplankton, and microalgal cell photosynthesis greatly contributes to global primary production (46). Photosynthesis plays an important role in photoautotrophic organisms, and Chl a, which is present in the thylakoid membrane, is an essential photosynthetic pigment that plays a central role in light harvesting and energy conversion. Fv/Fm represents the photochemical efficiency of algal cells, and rETR represents the relative photosynthetic electron transport rate of algal cells. The significant reductions in Chl a contents and the inhibition of both Fv/Fm and rETR in this study indicated that the Y42 supernatant disrupted the photosynthetic system of P. donghaiense (Fig. 6). Because the primary photochemical reaction and electron transport are accompanied by the movement of electron carriers and/or related enzymes within the thylakoid membrane, photosynthesis is affected mainly by the structure and fluidity of this membrane (47). The significant reductions in Fv/Fm and rETR in the Y42 supernatant-treated cells suggest that algicidal substances excreted by strain Y42 can disrupt the thylakoid membrane. The increase in MDA contents after treatment also suggests that algicidal substances can initiate membrane lipid peroxidation and cause oxidative damage to the membrane systems of P. donghaiense, including the plasma and thylakoid membranes.
Although this research revealed the algicidal activities and characteristics of Paracoccus sp. strain Y42, the mechanism underlying the algicidal ability of Y42 warrants further investigation; such investigation could involve determining the factors that contribute to the temperature- or pH-independent features of the algicidal activity, as well as the mechanism by which Y42 exerts lytic activity at the molecular level. In addition, identifying algicidal substances is important. Algicidal bacteria might kill algae via certain types of ectoenzymes (34, 35); however, the insensitivity of the activity of the Y42 supernatant to temperature suggests that ectoenzymes are not likely candidates as the algicidal molecules excreted by strain Y42. Other algicidal agents, including isatin and the red pigments of prodigiosins (11), have also been identified in marine algicidal bacteria. A recent study on Paracoccus contaminans LMG 29738T provided insight into the nature of algicidal compounds. Aurass and colleagues identified four clusters for secondary metabolite generation in the genome sequence of P. contaminans LMG 29738T, specifically, two microcin clusters, a lassopeptide cluster, and a homoserinlactone cluster (48). Based on their findings, the algicidal substance excreted by Paracoccus sp. strain Y42 is likely a small antibiotic-type secondary metabolite. Accordingly, we aim to identify lytic agents through size fractionation and mass spectral analyses.
In conclusion, Paracoccus sp. strain Y42 is a novel marine algicidal bacterium that was isolated in this study and was used to lyse P. donghaiense. Strain Y42 can damage membrane integrity and the photosynthetic system, lysing P. donghaiense cells by excreting algicidal substances. The algicidal substances caused oxidative damage to the membrane system of P. donghaiense, including the plasma membrane and the thylakoid membrane, which ultimately resulted in cell death. Because the algicidal activity of Y42 is stable under different temperature and sunlight conditions and over a wide pH range (pH 3 to 12), this organism is a promising candidate for removing algal cells from P. donghaiense blooms.
MATERIALS AND METHODS
Prorocentrum donghaiense cultures.P. donghaiense was obtained from the Culture Collection Center of Marine Algae of the State Key Laboratory of Marine Environmental Science, Xiamen University (China). The axenic algal culture was maintained at 20 ± 1°C in sterile f/2 medium prepared with 0.45-μm-filtered natural seawater, with illumination at a light intensity of approximately 50 μmol photons m−2 s−1 under a 12-h/12-h light/dark cycle. Cell numbers were counted under a light microscope. Exponential-phase algal cultures were used for experiments.
Isolation and screening of alga-lysing bacteria.A surface sediment sample was collected at a depth of 30 cm from the Yunxiao Mangrove National Natural Reserve (Fujian Province, China). A 1-ml aliquot of the water sample was serially diluted 10-fold in sterile seawater, and 100-μl aliquots of each dilution were plated onto Zobell 2216E agar plates, followed by incubation at 28°C for 5 days. Colonies with different morphologies were further purified several times until individual colony morphologies were obtained. To screen for algicidal bacteria, the isolated strains were separately inoculated into liquid Zobell 2216E medium at 28°C for 3 days, with shaking at 150 rpm. For each bacterial culture, 1 ml of culture was added to 20 ml of P. donghaiense culture. The mixed algal-bacterial cultures were incubated under the algal culture conditions described above. An algal culture with addition of the same volume of sterile 2216E medium served as the control group. Algicidal activity was monitored by counting cell numbers using a microscope (BX41; Olympus, Tokyo, Japan) and was calculated according to the following formula: algicidal activity (%) = [(N0 − Nt)/N0] × 100%, where N0 represents the cell number of the algal cultures measured immediately after treatment and Nt represents the cell number of the algal cultures at different treatment times (49). All experiments were performed in triplicate.
Identification of algicidal bacteria.Genomic DNA was extracted according to the method described by Ausubel et al. (50). The 16S rRNA gene was amplified by PCR using primers 27F and 1492R (51). The PCR product was purified using the TaKaRa Minibest DNA purification kit (TaKaRa Bio Inc., Dalian, China) and cloned into vector pMD-T for sequencing (Illumina; BGI Genomics, Wuhan, China). Sequences of related taxa were obtained from the GenBank database and the EzTaxon server (http://eztaxon-e.ezbiocloud.net) (52). Phylogenetic analysis was carried out using MEGA version 5.0, based on the neighbor-joining and maximum-likelihood algorithms.
Algicidal assay.Strain Y42 was inoculated into 50 ml of 2216E broth and grown to stationary phase at 28°C for 48 h, on a shaker at 150 rpm. The cell-free supernatant of the Y42 culture was collected by centrifugation at 15,000 rpm for 10 min and then passed through a 0.22-μm Millipore membrane. The cell pellets were washed twice with sterile 2216E medium and resuspended in the same volume of sterile f/2 medium. The different fractions of the Y42 cultures were added to the algal cultures at a concentration of 5% (vol/vol), to assess their algicidal activities. An algal culture with addition of the same volume of sterile 2216E medium served as the control group.
Algicidal stability assay.The cell-free supernatant of the Y42 culture was incubated at −20, 0, 10, 40, 50, 60, 80, or 100°C for 2 h and, after returning to room temperature (25°C), the treated supernatants were added to algal cultures to examine the effect of temperature on algicidal activity. To determine the effect of pH on the stability of the Y42 supernatant, the pH was adjusted to 3, 4, 5, 6, 7, 9, 10, 11, or 12 using 2 M HCl or 2 M NaOH. The pH-adjusted supernatants of the Y42 culture were stored at room temperature for 2 h, readjusted back to their initial pH (pH 8), and then added to algal cultures. Additionally, to investigate the effect of light on algicidal activity, the Y42 supernatant was exposed to natural sunlight for 1, 2, 4, 6, 8, or 10 h and then added to algal cultures. The 2216E medium with the same treatments as those of the Y42 supernatants served as the control groups. The initial Y42 supernatant (25°C, pH 8) without any treatment served as the reference group.
Algicidal specificity assay.To investigate the algicidal specificity of strain Y42, 15 algal species in addition to P. donghaiense were tested by adding 5% (vol/vol) fresh Y42 culture, followed by culturing at 20 ± 1°C for 72 h under the conditions described above. These additional algal species were Platymonas subcordiformis, Dunaliella salina, Platymonas helgolandica, Chlorella vulgaris, Nannochloropsis sp., Phaeocystis globose, Heterosigma akashiwo, Alexandrium tamarense, Alexandrium minutum, Scrippsiella trochoidea, Thalassiosira pseudonana, Phaeodactylum tricornutum, Amphiprora alata, Skeletonema costatum, and Nitzschia closterium, which were provided by the State Key Laboratory of Marine Environmental Science at Xiamen University. The algal cultures were maintained at 20 ± 1°C in sterile f/2 medium prepared with natural seawater, with illumination at a light intensity of 50 μmol photons m−2 s−1 under a 12-h/12-h light/dark cycle.
Nucleus analysis.After being treated with cell-free supernatant, algal cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellets were fixed for 10 min at room temperature with 4% (vol/vol) paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). After being washed twice with PBS, the fixed cells were stained with 5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 5 min in the dark at room temperature. Fluorescence was evaluated by fluorescence microscopy.
TEM and SEM.Algal cells were harvested by centrifugation at 3,500 rpm for 10 min after cultures were treated with 5% Y42 supernatant for 12, 24, 48, or 72 h. The collected cells were fixed overnight at 4°C in 0.1 M PBS (pH 7.4) containing 2.5% (vol/vol) glutaraldehyde and then were washed three times with PBS. The cells were postfixed for 2 h in 1% (vol/vol) OsO4 in 0.1 M PBS and then were dehydrated in a graded ethanol series (concentrations of 30, 50, 70, 90, and 100%). The samples were embedded in LR white. Ultrathin sections (60 to 80 nm) were stained with 3% acetic acid uranium-citric acid and observed with a transmission electron microscope (JEM-2100HC; JEOL Co., Japan).
SEM samples were collected and fixed as described above. The fixed cells were attached to a coverslip, dehydrated in a graded ethanol series (30, 50, 70, 90, 95, and 100%), and then critical point dried. The dry cells were sputter coated with gold and viewed using a scanning electron microscope (JSM-6390; JEOL Co.) (53, 54).
Chlorophyll fluorescence measurements.To assay the photosynthetic response of algal cells exposed to the Y42 supernatant, the maximum quantum yield of PS II (Fv/Fm) and the rETR were investigated using fast chlorophyll fluorescence with a pulse-amplitude-modulation fluorometer (XE-PAM; Walz, Effeltrich, Germany). Fv/Fm was measured after the algal cells were incubated in the dark for 15 min (55).
Determination of MDA contents.MDA levels were assessed using the Microscale MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China). The algal cultures were treated with different concentrations of the Y42 supernatant (3% and 5%), and samples were collected by centrifugation at 4,000 rpm for 5 min after 0, 12, 24, 36, 48, 60, or 72 h of exposure; algal cultures mixed with the same volume of 2216E medium served as the control. Algal cells were collected by centrifugation at 4,000 rpm for 5 min and then washed twice with 0.1 M PBS (pH 7.4). The cells were resuspended in PBS (1.5 ml) and sonicated on ice using the Ultrasonic cell disruption system (NingBo Scientiz Biotechnological Co., Ltd., China) (80 W; 5 s on and 5 s off 100 times). The remaining debris was removed by centrifugation at 15,000 rpm for 10 min at 4°C. The supernatant was collected for MDA measurements according to the MDA assay kit manual. The protein contents were measured using a total protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute), with bovine serum albumin as the standard.
Accession number(s).The partial sequence of the 16S rRNA gene (1,425 bp) from strain Y42 was deposited in GenBank under accession no. KY977433.
ACKNOWLEDGMENTS
This research was financially supported by the National Natural Science Foundation of China (grants 41676101, 41476095, 41576109, and J1310027) and XMU Undergraduate Innovation and Entrepreneurship Training Programs.
Q.Y., K.Y., and H.X. conceived and designed the experiment, F.Z., Q.Y., Q.C., K.Y., D.Z., Z.C., S.L., X.S., Y.F., L.Y., and L.K. conducted the experiments, T.Z. and H.X. analyzed the data, and H.X. wrote the paper. All authors reviewed the manuscript.
We declare no conflicts of interest.
FOOTNOTES
- Received 1 May 2018.
- Accepted 12 July 2018.
- Accepted manuscript posted online 27 July 2018.
- Address correspondence to Hong Xu, hxu{at}xmu.edu.cn.
F.Z. and Q.Y. contributed equally to this work.
Citation Zhang F, Ye Q, Chen Q, Yang K, Zhang D, Chen Z, Lu S, Shao X, Fan Y, Yao L, Ke L, Zheng T, Xu H. 2018. Algicidal activity of novel marine bacterium Paracoccus sp. strain Y42 against a harmful algal-bloom-causing dinoflagellate, Prorocentrum donghaiense. Appl Environ Microbiol 84:e01015-18. https://doi.org/10.1128/AEM.01015-18.
REFERENCES
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