Allelopathic Inhibition by the Bacteria Bacillus cereus ...

Journal of

Marine Science and Engineering

Article

Allelopathic Inhibition by the BacteriaBacillus cereus BE23 on Growth andPhotosynthesis of the Macroalga Ulva prolifera

Naicheng Li 1 Jingyao Zhang 1 Xinyu Zhao 2 Pengbin Wang 3 Mengmeng Tong 1and Patricia M Glibert 45

1 Ocean College Zhejiang University Zhoushan 316021 China linaicheng23163com (NL)jyzhang96zjueducn (JZ)

2 College of Marine Life Sciences Ocean University of China Qingdao 266003 China xyzhao331gmailcom3 Key Laboratory of Marine Ecosystem Dynamics Second Institute of Oceanography

Ministry of Natural Resources Hangzhou 310012 China algaesioorgcn4 University of Maryland Center for Environmental Science Horn Point Laboratory

Cambridge MD 21613 USA glibertumcesedu5 School of Oceanography Shanghai Jiao Tong University 1954 Huashan Rd Shanghai 200204 China Correspondence mengmengtongzjueducn

Received 27 August 2020 Accepted 13 September 2020 Published 16 September 2020

Abstract Bacteria-derived allelopathic effects on microalgae blooms have been studied with an aimto develop algicidal products that may have field applications However few such studies have beenconducted on macroalgae Therefore a series of experiments was conducted to investigate the impactsof different concentrations of cell-free filtrate of the bacteria Bacillus cereus BE23 on Ulva proliferaExcessive reactive oxygen species (ROS) were produced when these cells were exposed to highconcentrations of filtrate relative to f2 medium In such conditions the antioxidative defense systemof the macroalga was activated as shown by activities of the enzymes superoxide dismutase (SOD) andcatalase (CAT) and upregulation of the associated genes upMnSOD and upCAT High concentrationsof filtrate also inhibited growth of U prolifera and reduced chlorophyll a and b the photosyntheticefficiency (FvFm) and the electron transport rate (rETR) Non-photochemical quenching (NPQ) wasalso inhibited as evidenced by the downregulation of the photoprotective genes PsbS and LhcSRCollectively this evidence indicates that the alteration of energy dissipation caused excess cellularROS accumulation that further induced oxidative damage on the photosynthesis apparatus of the D1protein The potential allelochemicals were further isolated by five steps of extraction and insolation(solid phasendashliquid phasendashopen columnndashUPLCndashpreHPLC) and identified as N-phenethylacetamidecyclo (L-Pro-L-Val) and cyclo (L-Pro-L-Pro) by HR-ESI-MS and NMR spectra The diketopiperazinesderivative cyclo (L-Pro-L-Pro) exhibited the highest inhibition on U prolifera and may be a goodcandidate as an algicidal product for green algae bloom control

Keywords Ulva prolifera Bacillus sp allelopathy photosynthetic system reactive oxygen species(ROS) antioxidative system

1 Introduction

Allelopathic interactions are considered to be important factors that affect the growth or survivalof organisms within the same ecological habit Allelochemicals are secondary metabolites from plantsalgae or bacteria [1] They may have positive benefits (positive allelopathy) or may be detrimental(negative allelopathy) [2] Allelopathy has been considered to be one potential control mechanismfor harmful algae blooms (HABs) [3] The inhibition effects of allelopathic compounds on algae

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J Mar Sci Eng 2020 8 718 2 of 18

include destroying the cell structure [45] altering production of the reactive oxygen species (ROS) [6]impacting intracellular enzymatic activities [7] or altering the photosynthesis system [8] and relatedgene expression [9] External stress can induce the production of ROS ie hydrogen peroxide (H2O2)and superoxide radical (O2

bullminus) and can induce the regulation of the antioxidative defense or thephotoprotection system [1011]

A number of bacteria-derived algicidal compounds have drawn wide attention as a control forHABs [12ndash14] and the algicidal compounds belonging to the Cytophaga-Flavobacterium-Bacteroides (CFB)phylum have been identified [15] Among this phylogenetic profile the genus of Bacillus shows promisein controlling HABs as negative effects have been demonstrated on the diatom Skeletonema costatumthe raphidophyte Heterosigma akashiwo the dinoflagellate Prorocentrum donghaiense [16] the prymnesiophytePhaeocystis globosa [1617] and the cyanobacterium Microcystis aeruginosa [18] The potential allelochemicalsthat have been isolated and identified from Bacillus sp include terpene steroids and alkaloids [1920]The active compounds and mechanisms remain to be identified due to the species-specific response toalgicidal bacteria [21]

The green tides caused by blooms of Ulva prolifera have occurred in the Yellow Sea of China since2007 [22ndash26] These massive blooms negatively impact the local communities aquaculture operationsand tourism causing great damage to the local ecosystem service and enormous economic loss [27]The rapid growth of U prolifera on the other hand makes it the strongest competitor for nutrientsand light [2829] in the bloom area thereby driving the great impact on the marine biodiversity andstructure of the community [30ndash32] There are currently no effective measures to control these blooms

The Bacillus sp-derived control of HABs is promising but limited exploration has been undertakenin mitigating the green tides As a complicating factor the life stage of thalli has been reported to be animportant factor in green tide development [27] Therefore a series of experiments were performedto understand the extent to which bacterial allelopathy may be effective in controlling the thalli ofU prolifera Specifically the following questions were addressed (1) does the cell-free filtrate ofBacillus sp inhibit the growth of U prolifera and if so what is the effective dose (2) What is themechanism by which negative allelopathy occurs particularly with respect to the antioxidative defensesystem and the photosynthetic system II (PSII) response (3) What are the potential allelochemicals inthe filtrate of Bacillus sp that cause negative effects on U prolifera

2 Materials and Methods

21 Algal Culture and Identification

Asexual isolates of Ulva prolifera were provided by Zhejiang Xiangshan Xuwen Algal ExploitationCompany China in October 2018 Specimens were subsequently transferred to the laboratory onice sterilized with 07 potassium iodide (KI) for 5 min and then rinsed with autoclaved seawaterThe pre-sterilized thalli were maintained in sterilized f2 medium [33] with salinity of 30 temperature of20 C and light of 60 micromolmiddotm2

middotsminus1 (1212 h of lightdark cycle) The media were replaced every 5 daysTo minimize the interference of carry-over epiphytic bacteria in U prolifera cultures were pretreated

before each exposure experiment by antibiotic mixtures of penicillin (100 mgL) polymixin (075 mgL)and neomycin (09 mgL) for 48 h [34]

The macroalga was identified using the method described in Li et al [35] Total DNA was extractedwith a commercial Plant DNA Mini Kit (TaKaRa China) ITS and 5S sequences were amplified by thecorresponding PCR primers (Table 1) and the conducted BLAST analyses in the NCBI database

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Table 1 Sequences of primer pairs for Ulva prolifera analysis

Primer Sequence (5primendash3prime)

5SF 5prime-GGTTGGGCAGGATTAGTA-3prime

R 5prime-AGGCTTAAGTTGCGAGTT-3prime

ITSF 5prime-TCGTAACAAGGTTTCCGTAGG-3prime

R 5prime-GCTGCGTTCTTCATCGWTG-3prime

22 Experiment 1 Bacteria-Derived Allelopathic Inhibition on U prolifera

221 Preparation of Cell-Free Filtrate from Bacillus cereus

The bacterium strain Bacillus cereus BE23 was previously isolated from the mangrove area inHainan province China and maintained in Luria Bertani (LB) broth (peptone 100 gL yeast extract50 gL sea salt 32 gL dissolved in dH2O) at 28 C with shaking at 180 rpmmin The strain wasidentified by the 16S rDNA gene and 1439 bp sequence that was acquired by PCR amplificationThe bacteria were transferred from stock culture with the initial concentration of 1010mL in 500 mLof LB medium In 5 days cell density of Bacillus cereus BE23 reached approximately 1 times 1012mLthen cell-free filtrates were prepared by centrifuging 450 mL of the culture and filtering the supernatantthrough a Milliporetrade (Burlington MA USA) Membrane Filter 022 microm pore size

222 Preparation of the Exposure Treatment

Triplicate intact macroalga thalli (approximately 125 gL) were cultured in bacterial-free conditionswith different ratios of Bacillus cereus BE23 filtrate to total media (filtrate + seawater in volumes of01 1100 180 160 140 120 and 110 hereafter identified as Control T1100 T180 T160 T140 T120and T110 respectively) to a total of 400 mL each in 500 mL flasks Then stock f2 medium was addedto each flask All final media were at f2 levels assuming that no or low nutrients were carried over bythe filtrate The concentration of bacteria cells in each treatment was 25 times 109 125 times 1010 165 times 101025 times 1010 5 times 1010 and 1 times 1011 respectively The control treatment of U prolifera was cultured inf2 medium only without a bacterial filtrate All experiments were conducted in the same cultureenvironment under a light intensity of 60 micromolmiddotm2

middotsminus1 and with a lightdark cycle of 1212 h salinity of30 and temperature of 20 C The experiments were conducted in 500 mL flasks containing 400 mL ofculture medium Nutrients (equivalent to the nitrogen and phosphate level in f2 media) were addedevery 48 h to exclude any effects of nutrient limitation and pH values were monitored simultaneouslyThe culture flasks were randomly changed in terms of incubator position every day to balance theeffect of illumination Sterile conditions were used throughout

Specimens of macroalga were harvested after 192 h (8 days) of exposure for biomass photosynthesisand antioxidant analysis

223 Growth

The wet weight biomass of the macroalga was determined (plusmn00001 g) at 0 and 192 h respectivelySamples were treated by blotting with 3 layers of filter paper and conditioning for 10 min at roomtemperature The relative growth rates (G) were calculated as

Gx = (Wx minusWc)Wc

where Wc is the initial wet weight (g) of thalli and Wx is the fresh thalli wet weight (g) after treatment XThe inhibition rate (IR) by the bacterium filtrates was calculated as

IR = (Gc minus Gx)Gc

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where Gx is the relative growth rate () of U prolifera after treatment X and Gc is the relative growthrate () after 192 h in control

224 The Antioxidant Defense System

Macroalgal samples (02~03 g wet weight) were homogenized in a bath of liquid nitrogenand extracted with commercial potassium phosphate buffer (pH = 72~74 Solarbio China) Thenthe extract was centrifuged at 10000 rpmmin for 10 min yielding material for further analysis oftotal soluble protein (TSP) H2O2 and the enzymes superoxide dismutase (SOD) and catalase (CAT)Genes associated antioxidant activity manganese superoxide dismutase (upMnSOD) and catalase(upCAT) were also quantified

The TSP content was measured using the Coomassie blue dye binding assay [36] Fifty microlitersof extracts was homogenized with the Coomassie blue dye for 10 min and absorbance was measured at595 nm The results of TSP were expressed as g protein per liter (protmiddotgL) One hundred microliterswas mixed with the reaction reagents and detected at 405 nm The concentration of ROS wasmeasured as hydrogen peroxide (H2O2) and measured with a commercial assay kit (Jiancheng NanjingChina) following the manufacturerrsquos protocols Concentrations of H2O2 were determined based on thedecomposition of H2O2 by peroxidase and the results were expressed as mmol H2O2 per g of TSP (mmolgprot) The activity of SOD was measured according to the method of Sun et al [37] Samples (20 microL) andreaction reagents were mixed in the microliter 96-well flat-bottom plates and put into the plate reader(Tecan Switzerland) for incubation at 37 C After 20 min incubation the mixtures were detected at 450 nmOne unit of SOD was defined as the amount of enzyme required to generate 50 inhibition of reductionof WST-1 [2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(24-disulfophenyl)-2H-tetrazolium monosodium salt]The activity of CAT was assayed with the method described by Dhindsa et al [38] Briefly a reactionmixture was composed of 50 microL extracts 15 mM hydrogen peroxide and 50 mM phosphate bufferAfter addition of the enzyme extract absorbance at 240 nm was recorded for 1 min One unit of CATactivity is the amount of enzyme necessary to degrade 1 micromol H2O2 per mg of protein per sec

The antioxidant enzyme coding genes (upMnSOD and upCAT) were amplified with gene-specificprimer pairs (Table 2) RNA extraction and real-time PCR were performed the same as thephotosynthetic genes

Table 2 Sequences of primer pairs in Ulva prolifera for real-time PCR

Primer Sequence (5prime-3prime) Product Length

TubulinF 5prime-CAAGGATGTCAATGCTGCTGT-3prime

112R 5prime-GACCGTAGGTGGCTGGTAGTT-3prime

PsbSF 5prime-AACAGGTTCATCCATCACGG-3prime

121R 5prime-TTGCCTCAAACTCATCCTCTG-3prime

LhcSRF 5prime-CTATGCGAAGACTCTCAACG-3prime

83R 5prime-CCTCGCGGTAGCGCTTAACT-3prime

PsbAF 5prime- CTTTATGGGCTCGCTTTTGT-3prime

103R 5prime- TGGAACTACAGCACCAGAAA-3prime

PsbDF 5prime- CAGGAAGTGTTCAACCAGTA-3prime

167R 5prime- AGCAGCGATGTGATGAGACG-3prime

upMnSOD F 5prime-ATCACCAGGCGTATGTCACC-3prime94R 5prime-TTCAAGTGCCCTCCACCGTT-3prime

upCAT F 5prime-CTCTCAAGCCCAATCCTCGT-3prime95R 5prime-AGTTCAGTGGGATGCCAACA-3prime

225 Photosynthesis System

Concentrations of chlorophyll a (Chl a) and b (Chl b) were determined according to Zhao et al [39]Macroalgae (02 g) were grounded in liquid nitrogen and extracted in 90 vv) acetone buffer (5 mL)for 12 h Then the mixture was centrifuged at 4 C 10000 rpmmin for 10 min The supernatant wascollected for chlorophyll analyses and optical densities were measured with an ultravioletndashvisible

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spectrophotometer (HITACHI U2900 Japan) at 663 and 645 nm wavelength Concentrations of Chl aand b were then calculated as follows and reported as units of mgg fresh weight (mgg FW)

Chl a = 127 OD663 minus 269 OD645

Chl b = 229 OD645 minus 468 OD663

Parameters associated the photosynthesis system II (PSII) were measured using an Imaging-PAM(Walz Germany) These parameters included the effective quantum yield (Y(II)) non-photochemicalquenching (NPQ) relative electron transport rate (rETR) and photochemical quenching (qP) The actiniclight was set to be similar to the cultivation light (56 micromolmiddotmminus2

middotsminus1) Subsamples of U prolifera weredark-acclimated for 20 min prior to all measurements All parameters were calculated according to therelationships in Table 3

Table 3 Fluorescence parameters calculated from PAM in Ulva prolifera after exposure

Parameter Definition Equation

FvFm maximum quantum yield of PSII (Fm minus F0)FmY(II) effective quantum yield of PSII (Frsquom minus Ft)FrsquomNPQ non-photochemical quenching (Fm minus Frsquom)FrsquomrETR relative electron transport rate 05 times Y(II) times PAR times IA

qP photochemical quenching (Frsquom minus Ft)(Frsquom minus Frsquo0)

Four genes were selected for characterization PsbS LhcSR PsbA and PsbD PsbS and LhcSRare associated with photoprotection and non-photochemical quenching (NPQ) PsbA and PsbD areindicators of the D1 and D2 protein of the PSII apparatus respectively The tubulin gene was deployedas a housekeeping gene to standardize the expression variations of target genes [39]

These genes were amplified with gene-specific primer pairs (Table 2) Samples of U proliferawere quickly frozen in liquid nitrogen and stored at minus80 C until RNA extraction Total RNA wasextracted by a commercial MiniBEST Plant Total RNA Extraction Kit (TaKaRa Dalian China) andthe reverse transcripts cDNA were analyzed using a Prime Scripttrade II 1st stand cDNA Synthesis kit(TaKaRa Dalian China) Real-time PCR was performed using the ldquoTB GreenTM Fast qPCR Mixrdquokit (TaKaRa Dalian China) The amplification program of real-time PCR was set at 94 C for 30 sfollowing 40 cycles of 94 C for 5 s and 60 C for 10 s in Light Cyclerreg 480 System (Roche Germany)Dissociation curve analysis of the amplification products was carried out to verify the single PCRproduction at the end of each thermal program

23 Experiment 2 Isolation and Identification the Potential Allelopathic Compounds from Cell-Free Filtrate ofBacillus cereus BE23

231 Step 1 Solid Phase and Liquid Phase Extraction of Potential Allelopathic Compounds

Cell-free filtrate (10 L approximately 1 times 1016 bacteria cells) of the Bacillus cereus BE23 culture wascollected after 5 days of growth by centrifuging at 10000 rpmmin for 10 min and filtering with a 022micrommembrane The filtrate was eluted by solid phase extraction (SPE) with the resin DiaionregHP20 (particlesize of 20ndash60 mesh) and the remaining residuals were rinsed off by methanol After resuspendingthe residuals in Milli-Q water they were used for liquid phase extraction (LPE) Three extractingagents cyclohexane ethyl acetate and 1-butanol were considered as selection agents for differentpolarity fragments Sub-residuals of LPE were extracted from each agent 3 times and concentrated in arotary evaporator (IKA RV8V Germany) in a 30~40 C water bath (Figure 1) The sub-residuals wereidentified as cyclohexane (Ech) ethyl acetate (Eea) and 1-butanol seriatim (Ebs) These sub-residualsEch Eea and Ebs were weighted with an electron balance (plusmn00001 g) dissolved in 20 mL dimethylsulfoxide (DMSO) and stored at 4 C for further bioassay experimentation

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Figure 1 Isolation and bioassay program for potential allelopathic compounds from crude extraction

of cell‐free filtrate of Bacillus cereus BE23

234 Structure Identification

The three potential allelochemicals Ech5‐4 Eea2‐5 and Eea3‐2 were preliminarily analyzed by an

Agilent 6230 time‐of‐flight liquid chromatographyndashmass spectrometer (TOF LC‐MS) (Agilent CA

USA) to determine the molecular weight Then structures were identified by a pulse Fourier

transform nuclear magnetic resonance spectroscope (NMR 600 MHz JNM‐ECZR JEOL Japan)

Deutero methanol or deutero dimethyl sulfoxide solutions containing trimethylsilyl were used as

reference substances and acted as solvents to record 1H and 13C NMR spectra All chemical shifts were

exhibited as relative values

24 Statistical Analysis

All data were presented as mean plusmn standard error and were analyzed by one‐way ANOVA with

a significant level of 005 (Sigma plot 125 Systat Software Inc London UK) A phylogenetic tree

was constructed using the neighbor‐joining algorithm with the MEGA 70 program Relative gene

expression levels were analyzed following the 2minusΔΔCt method

3 Results

31 Identification of Macroalga and Bacteria

The 5S sequence of the macroalga 418 bp was 100 identical to Ulva prolifera

(GenBankIDHM5847721) and the ITS sequence 614 bp was 99 identical to U prolifera

(GenBankIDKF1308701) Thus the macroalga deployed in the present study was identified as U

prolifera

The 16S rDNA sequence of the bacterial strain BE23 (GenBank accession number MN814015)

was 100 identical with few genetic distance differences to that of Bacillus cereus strain ATCC14597

(Supplementary Figure S1) Thus bacterial strain BE23 was identified as Bacillus cereus

32 Inhibition on the Growth of U prolifera

To simplify the treatment and response analysis of U prolifera two major treatment groups of B

cereus filtrates were classified They are herein separated as high‐concentration (HC) ie the T110 and

T120 treatments and low‐concentration (LC) ie the T140 T160 T180 and T1100 treatments

SPE LLE

Cyclohexane

Ethyl acetate

1-butanol seriatim

First bioassay

Open column chromatography

cyclohexaneethyl acetate

2001(Ech1)

1001(Ech2)

501 (Ech3)

251 (Ech4)

101 (Ech5)

51 (Ech6)

01 (Ech7)

dichloromethanemethanol

501 (Eea1)

251 (Eea2)

101 (Eea3)

51 (Eea4)

21 (Eea5)

11 (Eea6)

01 (Eea7)

Second bioassay

UPLC pre-HPLC

Third bioassay

Mobile Phase

2352min

1343min

1625min

Ech5-4

Eea2-5

Eea3-2

HR-ESI-MS NMR

Figure 1 Isolation and bioassay program for potential allelopathic compounds from crude extractionof cell-free filtrate of Bacillus cereus BE23

The first U prolifera bioassay experiment was performed in 6-well plates by filling them withmacroalgae (approximately 005 g) and crude extraction (5 mgL) or DMSO (control) in 10 mL f2medium Each treatment was conducted in triplicate for 192 h under the same environmental conditionsas the primary U prolifera culture Growth and inhibition rates were used to determine the potentialallelopathic activities in each treatment (Supplementary Figure S2) Of the three extracting agentsextractions in cyclohexane (Ech) and in ethyl acetate (Eea) had an inhibition effect (SupplementaryFigure S2) therefore these extractions were used for further investigation

232 Step 2 Open Column Chromatography to Select the Potential Allelopathic Compounds

To further purify the potential allelopathic compounds Ech and Eea were eluted through anopen silica gel column chromatography (170 times 30 mm in dimension and with a silica particle size of200ndash300 mesh) respectively and the eluents from each mobile phase were collected As for extractionsin cyclohexane (Ech) the mobile phase was cyclohexane and ethyl acetate with ratios of 2001 1001501 251 101 51 and 01 (hereafter named as Ech1 Ech2 etc) For extraction in ethyl acetate (Eea)the mobile phase was dichloromethane and methanol with ratios of 501(Eea1) 251(Eea2) 101(Eea3)51(Eea4) 21(Eea5) 11(Eea6) and 01(Eea7) respectively

Then a second bioassay was performed in 6-well plates by adding 005 g of U prolifera (wet weight)and the corresponding extracted compounds (5 mgL) in 10 mL of f2 medium Each treatment wasconducted in triplicate for 192 h under the same environmental conditions as the primary U proliferaculture The extractions with significant inhibition Ech5 Eea2 and Eea3 (Supplementary Figure S3)were collected for further detection

233 Step 3 Ultra- and High-Performance Liquid Chromatography to Select the PotentialAllelopathic Compounds

The bioactive fractions were collected separately and analyzed by analytical ultra-performanceliquid chromatography (UPLC ultimate 3000 Thermo Fisher Scientific USA) with a C18 column(250 times 46 mm 5 microm Agilent China) at a flow rate of 1 mLmin and the UV detection at 210 nmThe mobile phase was methanol or acetonitrilewater (1090 vv) minus100 methanol with an elutiontime of 35 min The dominant components (highest peaks) including 5 components from Ech57 components from Eea2 and 8 components from Eea3 were chosen and the optimal UPLC conditionswere retrieved for a further preparative step

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The fractions were then purified and collected by preparative high-performance liquidchromatography (HPLC Shimadzu AP20 Japan) with a C18 column (250times 212 mm 5 microm NanoMicroChina) at a flow rate of 10 mLmin for different times up to 35 min for Ech5 Eea2 and Eea3 separatelyusing the recorded optimized mobile phase (Figure 1)

The third bioassay was conducted with the 20 components Three compounds Ech5-4 Eea2-5and Eea3-2 were collected at 2352 1343 and 1625 min in each extraction run (Supplementary Figure S4)

The three potential allelochemicals Ech5-4 Eea2-5 and Eea3-2 were preliminarily analyzed byan Agilent 6230 time-of-flight liquid chromatographyndashmass spectrometer (TOF LC-MS) (AgilentCA USA) to determine the molecular weight Then structures were identified by a pulse Fouriertransform nuclear magnetic resonance spectroscope (NMR 600 MHz JNM-ECZR JEOL Japan)Deutero methanol or deutero dimethyl sulfoxide solutions containing trimethylsilyl were used asreference substances and acted as solvents to record 1H and 13C NMR spectra All chemical shifts wereexhibited as relative values

All data were presented as mean plusmn standard error and were analyzed by one-way ANOVA witha significant level of 005 (Sigma plot 125 Systat Software Inc London UK) A phylogenetic treewas constructed using the neighbor-joining algorithm with the MEGA 70 program Relative geneexpression levels were analyzed following the 2minus∆∆Ct method

3 Results

The 5S sequence of the macroalga 418 bp was 100 identical to Ulva prolifera (GenBankIDHM5847721)and the ITS sequence 614 bp was 99 identical to U prolifera (GenBankIDKF1308701) Thus the macroalgadeployed in the present study was identified as U prolifera

The 16S rDNA sequence of the bacterial strain BE23 (GenBank accession number MN814015)was 100 identical with few genetic distance differences to that of Bacillus cereus strain ATCC14597(Supplementary Figure S1) Thus bacterial strain BE23 was identified as Bacillus cereus

To simplify the treatment and response analysis of U prolifera two major treatment groups of Bcereus filtrates were classified They are herein separated as high-concentration (HC) ie the T110 andT120 treatments and low-concentration (LC) ie the T140 T160 T180 and T1100 treatments

Cell-free filtrates of Bacillus cereus BE23 were used as the source of the allelopathic compoundstested on U prolifera These cell-free filtrates induced growth of U prolifera at LC ie T1100~T140

(ANOVA p lt 005) with growth rates of 105 plusmn 11 on average (n = 12) but inhibited growth at HCtreatments (T120 and T110) with inhibition rates of 67 and 75 respectively (Figure 2) Values of pHwere monitored during the exposure in all treatments (Supplementary Table S1) and variation of thepH value was within the optimal range for U prolifera growth [40]

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J Mar Sci Eng 2020 8 x FOR PEER REVIEW 8 of 18

Cell‐free filtrates of Bacillus cereus BE23 were used as the source of the allelopathic compounds

tested on U prolifera These cell‐free filtrates induced growth of U prolifera at LC ie T1100~T140

(ANOVA p lt 005) with growth rates of 105 plusmn 11 on average (n = 12) but inhibited growth at HC

treatments (T120 and T110) with inhibition rates of 67 and 75 respectively (Figure 2) Values of pH

were monitored during the exposure in all treatments (Supplementary Table S1) and variation of the

pH value was within the optimal range for U prolifera growth [40]

Figure 2 Relative growth rates and inhibition rates of Ulva prolifera under the exposure of different

amounts of cell‐free filtrate of Bacillus cereus BE23 T1100 and T180 ~T110 indicate the treatments of

volume ratio of cell‐free filtrate of Bacillus cereus BE23 to f2 medium Values are means plusmn SD (n = 3)

indicates a significant difference (p lt 005) and indicates a significant difference (p lt 0001) compared

to control

33 Response of Antioxidant System of U prolifera

A significant amount of H2O2 (ANOVA p lt 0001) was produced in the HC treatments ranging

from 3821 to 5033 mmolgprot (Figure 3) after 192 h of exposure The production of ROS was

associated with changes in activities of SOD (ANOVA p lt 005) and CAT (ANOVA p lt 0001) with

concentrations of T140 eliciting a response in SOD activity (Figure 4a) but only the highest dosage

T110 elicited a response in CAT (Figure 4b) The antioxidant enzyme genes upCAT and upMnSOD

were upregulated gradually in response to the increased dosage of cell‐free extracts (Figure 4ab)

indicating the initiation of the antioxidant defense system under the stress of the filtrate of Bacillus

cereus BE23

Figure 3 H2O2 content of Ulva prolifera under the exposure of different amounts of cell‐free filtrate of

Bacillus cereus BE23 T1100 and T180~T110 indicate the treatments of volume ratio of cell‐free filtrate of

Figure 2 Relative growth rates and inhibition rates of Ulva prolifera under the exposure of differentamounts of cell-free filtrate of Bacillus cereus BE23 T1100 and T180~T110 indicate the treatments ofvolume ratio of cell-free filtrate of Bacillus cereus BE23 to f2 medium Values are means plusmn SD (n = 3) indicates a significant difference (p lt 005) and indicates a significant difference (p lt 0001) comparedto control

A significant amount of H2O2 (ANOVA p lt 0001) was produced in the HC treatmentsranging from 3821 to 5033 mmolgprot (Figure 3) after 192 h of exposure The production ofROS was associated with changes in activities of SOD (ANOVA p lt 005) and CAT (ANOVA p lt 0001)with concentrations of T140 eliciting a response in SOD activity (Figure 4a) but only the highestdosage T110 elicited a response in CAT (Figure 4b) The antioxidant enzyme genes upCAT andupMnSOD were upregulated gradually in response to the increased dosage of cell-free extracts(Figure 4ab) indicating the initiation of the antioxidant defense system under the stress of the filtrateof Bacillus cereus BE23

to control

cereus BE23

Figure 3 H2O2 content of Ulva prolifera under the exposure of different amounts of cell-free filtrate ofBacillus cereus BE23 T1100 and T180~T110 indicate the treatments of volume ratio of cell-free filtrate ofBacillus cereus BE23 relative to f2 medium Values are means plusmn SD (n = 3) indicates a significantdifference (p lt 005) and indicates a significant difference (p lt 0001) compared to control

J Mar Sci Eng 2020 8 718 9 of 18

Bacillus cereus BE23 relative to f2 medium Values are means plusmn SD (n = 3) indicates a significant

difference (p lt 005) and indicates a significant difference (p lt 0001) compared to control

Figure 4 (a) Superoxide dismutase (SOD) activity and relative gene expression of manganese

superoxide dismutase (upMnSOD) and (b) catalase (CAT) activity and catalase gene expression

(upCAT) of Ulva prolifera under the exposure of different amounts of cell‐free filtrate of Bacillus cereus

BE23 T1100 and T180 ~T110 indicate the treatments of volume ratio of cell‐free filtrate of Bacillus cereus

BE23 relative to f2 medium Values are means plusmn SD (n = 3) indicates a significant difference (p lt

005) and indicates a significant difference (p lt 0001) compared to control

34 Response of PSII System of U prolifera

To investigate the effects of the Bacillus cereus BE23 filtrate on the photosynthetic pigments of the

macroalga Chl a and b contents were quantified (Figure 5a) No significant changes of either Chl a or

b were observed in the LC treatments but significant decreases were observed (ANOVA p lt 0001)

in the HC exposures from 041 to ~013 mgg FW for Chl a and from 057 to ~024 mgg FW for Chl b

(Figure 5a)

The photosynthetic response of U prolifera under the stress of cell‐free filtrate of B cereus BE23

was significant (Figures 5b 6 and 7) The maximum photochemical quantum yields of PSII (FvFm)

were reduced in the HC treatments from 080 to ~029 (n = 6 Figure 5b) Accordingly values of Y(II)

the effective quantum yield of PSII were significantly downregulated (ANOVA p lt 0001) from 022

to 015 in the HC treatments (Figure 6a) Similar responses were found in the relative electron

transport rates (rETR) coincident with a sharp reduction in photochemical quenching (qP) (Figure

6b) A significant enhancement of NPQ activity (Figure 6b) (ANOVA p lt 0001) was recorded in the

LC treatments from 018 to 044 However high doses of the filtrate of Bacillus cereus BE23 induced a

downregulation of NPQ (ANOVA p lt 0001) indicating photoinhibition damage

Figure 4 (a) Superoxide dismutase (SOD) activity and relative gene expression of manganesesuperoxide dismutase (upMnSOD) and (b) catalase (CAT) activity and catalase gene expression(upCAT) of Ulva prolifera under the exposure of different amounts of cell-free filtrate of Bacillus cereusBE23 T1100 and T180~T110 indicate the treatments of volume ratio of cell-free filtrate of Bacillus cereusBE23 relative to f2 medium Values are means plusmn SD (n = 3) indicates a significant difference (p lt 005)and indicates a significant difference (p lt 0001) compared to control

To investigate the effects of the Bacillus cereus BE23 filtrate on the photosynthetic pigments of themacroalga Chl a and b contents were quantified (Figure 5a) No significant changes of either Chl a or bwere observed in the LC treatments but significant decreases were observed (ANOVA p lt 0001) inthe HC exposures from 041 to ~013 mgg FW for Chl a and from 057 to ~024 mgg FW for Chl b(Figure 5a)

(Figure 5a)

Figure 5 (a) The chlorophyll a and b content and (b) the maximum quantum yields of PSII (FvFm)of Ulva prolifera under the exposure of different amounts of cell-free filtrate of Bacillus cereus BE23Values are means plusmn SD (n = 3) indicates a significant difference (p lt 0001) compared to control

The photosynthetic response of U prolifera under the stress of cell-free filtrate of B cereus BE23was significant (Figure 5b Figure 6 Figure 7) The maximum photochemical quantum yields of PSII(FvFm) were reduced in the HC treatments from 080 to ~029 (n = 6 Figure 5b) Accordingly values ofY(II) the effective quantum yield of PSII were significantly downregulated (ANOVA p lt 0001)from 022 to 015 in the HC treatments (Figure 6a) Similar responses were found in the relative electrontransport rates (rETR) coincident with a sharp reduction in photochemical quenching (qP) (Figure 6b)A significant enhancement of NPQ activity (Figure 6b) (ANOVA p lt 0001) was recorded in the LCtreatments from 018 to 044 However high doses of the filtrate of Bacillus cereus BE23 induced adownregulation of NPQ (ANOVA p lt 0001) indicating photoinhibition damage

J Mar Sci Eng 2020 8 718 10 of 18

Figure 5 (a) The chlorophyll a and b content and (b) the maximum quantum yields of PSII (FvFm)

of Ulva prolifera under the exposure of different amounts of cell‐free filtrate of Bacillus cereus BE23

Values are means plusmn SD (n = 3) indicates a significant difference (p lt 0001) compared to control

Figure 6 Photosynthetic system II parameters of Ulva prolifera under the exposure of different

amounts of cell‐free filtrate of Bacillus cereus BE23 (a) quantum yield (Y(II)) and relative electron

transport rate (rETR) and (b) non‐photochemical quenching (NPQ) and photochemical (qP) T1100

and T180 ~T110 indicate the volume ratio of cell‐free filtrate of Bacillus cereus BE23 relative to f2 medium

in the different treatments Values are means plusmn SD (n = 3) indicates a significant difference (p lt

0001) compared to control

The expression of the two assayed photoprotection‐related genes PsbS and LhcSR varied in

response to cell‐free filtrate exposure (Figure 7a) The relative expressions of both genes increased

with the bacterial filtrate dosage from 1100 (T1100) to 140 (T140) but were significantly downregulated

in the HC treatments (T120 and T110) The highest PsbS and LhcSR were in treatments of T140 reaching

266 and 529 times that of the control and the lowest value was in the T110 treatment at 075 and 072

of the control (Figure 7a) The response of PsbA and PsbD was not as clear but a substantial

degradation of PsbA was observed in the HC treatment with a value of 059 of the control in T110

(Figure 7b)

Figure 7 Relative expression of the genes (a) PsbS and LhcSR and (b) PsbA and PsbD of Ulva prolifera

under the exposure of different amounts of cell‐free filtrate of Bacillus cereus BE23 T1100 and T180 ~T110

indicate the treatments of volume ratio of cell‐free filtrate of Bacillus cereus BE23 relative to f2

medium Values are means plusmn SD (n = 3)

35 Identification of Allelochemicals from Bacillus cereus BE23 Filtrate

To isolate the bioactive compounds five steps of extraction and insolation (solid phasendashliquid

phasendashopen columnndashUPLCndashpreHPLC) were conducted After each isolation the separated groups

were tested for bioactivity (Figures S2ndashS4) Three bioactive compounds in the cell‐free filtrates of

(b)(a)

Figure 6 Photosynthetic system II parameters of Ulva prolifera under the exposure of different amountsof cell-free filtrate of Bacillus cereus BE23 (a) quantum yield (Y(II)) and relative electron transport rate(rETR) and (b) non-photochemical quenching (NPQ) and photochemical (qP) T1100 and T180~T110

indicate the volume ratio of cell-free filtrate of Bacillus cereus BE23 relative to f2 medium in the differenttreatments Values are means plusmn SD (n = 3) indicates a significant difference (p lt 0001) comparedto control

The expression of the two assayed photoprotection-related genes PsbS and LhcSR varied inresponse to cell-free filtrate exposure (Figure 7a) The relative expressions of both genes increased withthe bacterial filtrate dosage from 1100 (T1100) to 140 (T140) but were significantly downregulated inthe HC treatments (T120 and T110) The highest PsbS and LhcSR were in treatments of T140 reaching266 and 529 times that of the control and the lowest value was in the T110 treatment at 075 and072 of the control (Figure 7a) The response of PsbA and PsbD was not as clear but a substantialdegradation of PsbA was observed in the HC treatment with a value of 059 of the control in T110

(Figure 7b)

(b)(a)

Figure 7 Relative expression of the genes (a) PsbS and LhcSR and (b) PsbA and PsbD of Ulva proliferaunder the exposure of different amounts of cell-free filtrate of Bacillus cereus BE23 T1100 and T180~T110

indicate the treatments of volume ratio of cell-free filtrate of Bacillus cereus BE23 relative to f2 mediumValues are means plusmn SD (n = 3)

To isolate the bioactive compounds five steps of extraction and insolation (solid phasendashliquidphasendashopen columnndashUPLCndashpreHPLC) were conducted After each isolation the separated groupswere tested for bioactivity (Figures S2ndashS4) Three bioactive compounds in the cell-free filtrates ofBacillus cereus BE23 were identified by high-resolution mass spectrometric data and NMR spectroscopicanalysis The molecular formula C10H13NO of compound Ech5-4 was deduced from its ion at mz1641072 [M+H]+ (Supplementary Figure S5a calculated for C10H14NO 1641075) and its 13C dataThe 13C-NMR spectrum (600 MHz DMSO-d6) of Ech5-4 displayed signals at δC 1695 (C=O) 1400 (C

J Mar Sci Eng 2020 8 718 11 of 18

C-1) 1291 (CH C-3 C-5) 1288 (CH C-2 C-6) 1265 (CH C-4) 407 (CH2 C-7) 357 (CH2 C-8)and 2309 (CH3) (Supplementary Figure S5bc) The 1H-NMR signals were observed at δH 792 (1Hbrs NH) 727ndash730 (2H t J = 80 Hz Ar-H) 718ndash720 (3H m Ar-H) 322ndash326 (2H m H-7) 269 (2H tJ = 75 Hz H-8) and 178 (3H s -CH3) Based on these data and the comparison with the reporteddata [41] compound Ech5-4 was identified as N-phenethylacetamide (Figure 8a)

Bacillus cereus BE23 were identified by high‐resolution mass spectrometric data and NMR

spectroscopic analysis The molecular formula C10H13NO of compound Ech5‐4 was deduced from its

ion at mz 1641072 [M+H]+ (Supplementary Figure S5a calculated for C10H14NO 1641075) and its 13C

data The 13C‐NMR spectrum (600 MHz DMSO‐d6) of Ech5‐4 displayed signals at δC 1695 (C=O) 1400

(C C‐1) 1291 (CH C‐3 C‐5) 1288 (CH C‐2 C‐6) 1265 (CH C‐4) 407 (CH2 C‐7) 357 (CH2 C‐8)

and 2309 (CH3) (Supplementary Figs S5b and S5c) The 1H‐NMR signals were observed at δH 792

(1H brs NH) 727ndash730 (2H t J = 80 Hz Ar‐H) 718ndash720 (3H m Ar‐H) 322ndash326 (2H m H‐7) 269

(2H t J = 75 Hz H‐8) and 178 (3H s ‐CH3) Based on these data and the comparison with the

reported data [41] compound Ech5‐4 was identified as N‐phenethylacetamide (Figure 8a)

The molecular formula of C10H14N2O2 for compound Eea2‐5 was determined based on its mz

2170953 [M+Na]+ (Supplementary Figure S6a calculated for C10H14N2NaO2 2170953) The 13C and 1H NMR spectra of Eea2‐5 showed signals for the functional groups of carbonyl (δC 1681) methine

(δC 612 δH 434 1H t J = 90 Hz) and methelene (δC 457 282 237 δH 345ndash353 2H m 225ndash230

1H m 199ndash209 2H m 191ndash197 1H m) (Supplementary Figure S6bc) These data and comparison

with the reference data [42] indicated that compound Eea2‐5 was cyclo (L‐Pro‐L‐Pro) (Figure 8b)

The compound Eea3‐2 has the molecular formula of C10H10N2O2 deduced from its mz 2191103

[M+Na] (Supplementary Figure S7a calculated for C10H10N2NaO2 2191109) The 13C‐NMR spectrum

(600 MHz Methanol‐d4) of Eea3‐2 exhibited 10 carbon signals resonating at δC1728 (C C‐1) 1678 (C

C‐6) 618 (CH C‐7) 603 (CH C‐2) 464 (CH2 C‐5) 301 (CH C‐8) 298 (CH2 C‐3) 235 (CH2 C‐4)

191a (CH3 C‐10) and 169 (CH3 C‐9) The 1H NMR spectrum displayed signals at δH 420 (1H t J

= 86 Hz H‐2) 405 (1H br t H‐7) 356 (1H m H‐5a) 348 (1H m H‐5b) 248 (1H m H‐3a) 231 (1H

m H‐8) 202 (1H m H‐3b) 191ndash196 (2H m H‐4) 108b (3H d J = 73 Hz H‐9) and 095b (3H d J

= 73 Hz H‐10) Thus the compound Eea3‐2 was identified as cyclo (L‐Pro‐L‐Val) (Figure 8c) [43]

Figure 8 Structures of the compounds Ech5‐4 (a) Eea2‐5 (b) and Eea3‐2 (c) isolated from the crude

extract of Bacillus cereus BE23 filtrate

4 Discussion

Bacteria‐derived interactions play important roles in species distribution and abundance [44]

succession of algal blooms [45] and biomass control of microorganisms [46] and macroalgae [47]

Such allelopathic interactions consist of two pathways direct (bacterial and algal cell contact) and

indirect (release of natural products) [1232] The present study demonstrated the potential

mechanisms of allelopathic stress on U prolifera by products of B cereus BE23 in indirect ways

The low dosage (ie T1100~T140) of B cereus BE23 filtrate promoted the growth of U prolifera

whereas the high dosage (T120 and T110) inhibited biomass production (Figure 2) The response of the

macroalgae in the LC treatments may have resulted from a hormesis effect [48] and adaption to the

low concentrations of allelochemicals [49] The upregulation of physiological activity of U prolifera

(Figures 4ndash6) in the LC treatments contributed to the growth‐promotive effect Meanwhile the

nutrients including the inorganic nutrient from f2 + artificial seawater and the nutrient carrying over

by the B cereus BE23 filtrate (4~40 mL) contributed to the growth of macroalga Inorganic nitrogen

ie nitrate or ammonium has been reported to be rapidly taken up by Ulva [28] and within 192 h

the addition of inorganic nutrient of f2 medium was calculated to be sufficient to the thalli of U

prolifera [5051] The carried-over inorganic nutrient was low (less than 10) therefore the effects of

nutrients in B cereus BE23 filtrate were minimal to the growth of Ulva in the present study

Figure 8 Structures of the compounds Ech5-4 (a) Eea2-5 (b) and Eea3-2 (c) isolated from the crudeextract of Bacillus cereus BE23 filtrate

The molecular formula of C10H14N2O2 for compound Eea2-5 was determined based on its mz2170953 [M+Na]+ (Supplementary Figure S6a calculated for C10H14N2NaO2 2170953) The 13C and1H NMR spectra of Eea2-5 showed signals for the functional groups of carbonyl (δC 1681) methine(δC 612 δH 434 1H t J = 90 Hz) and methelene (δC 457 282 237 δH 345ndash353 2H m 225ndash2301H m 199ndash209 2H m 191ndash197 1H m) (Supplementary Figure S6bc) These data and comparisonwith the reference data [42] indicated that compound Eea2-5 was cyclo (L-Pro-L-Pro) (Figure 8b)

The compound Eea3-2 has the molecular formula of C10H10N2O2 deduced from its mz 2191103[M+Na] (Supplementary Figure S7a calculated for C10H10N2NaO2 2191109) The 13C-NMR spectrum(600 MHz Methanol-d4) of Eea3-2 exhibited 10 carbon signals resonating at δC1728 (C C-1) 1678 (CC-6) 618 (CH C-7) 603 (CH C-2) 464 (CH2 C-5) 301 (CH C-8) 298 (CH2 C-3) 235 (CH2 C-4)191a (CH3 C-10) and 169 (CH3 C-9) The 1H NMR spectrum displayed signals at δH 420 (1H tJ = 86 Hz H-2) 405 (1H br t H-7) 356 (1H m H-5a) 348 (1H m H-5b) 248 (1H m H-3a) 231 (1Hm H-8) 202 (1H m H-3b) 191ndash196 (2H m H-4) 108b (3H d J = 73 Hz H-9) and 095b (3H dJ = 73 Hz H-10) Thus the compound Eea3-2 was identified as cyclo (L-Pro-L-Val) (Figure 8c) [43]

4 Discussion

Bacteria-derived interactions play important roles in species distribution and abundance [44]succession of algal blooms [45] and biomass control of microorganisms [46] and macroalgae [47]Such allelopathic interactions consist of two pathways direct (bacterial and algal cell contact) andindirect (release of natural products) [1232] The present study demonstrated the potential mechanismsof allelopathic stress on U prolifera by products of B cereus BE23 in indirect ways

The low dosage (ie T1100~T140) of B cereus BE23 filtrate promoted the growth of U proliferawhereas the high dosage (T120 and T110) inhibited biomass production (Figure 2) The response of themacroalgae in the LC treatments may have resulted from a hormesis effect [48] and adaption to thelow concentrations of allelochemicals [49] The upregulation of physiological activity of U prolifera(Figures 4ndash6) in the LC treatments contributed to the growth-promotive effect Meanwhile the nutrientsincluding the inorganic nutrient from f2 + artificial seawater and the nutrient carrying over by theB cereus BE23 filtrate (4~40 mL) contributed to the growth of macroalga Inorganic nitrogen ie nitrateor ammonium has been reported to be rapidly taken up by Ulva [28] and within 192 h the additionof inorganic nutrient of f2 medium was calculated to be sufficient to the thalli of U prolifera [5051]The carried-over inorganic nutrient was low (less than 10) therefore the effects of nutrients inB cereus BE23 filtrate were minimal to the growth of Ulva in the present study

A general stress response in algae is the production of ROS [5253] and it can be produced inresponse to abiotic and allelopathic stresses [54ndash56] Here ROS was produced in response to BE23cell-free filtrates (Figure 3) The source of ROS may include two main pathways the intrinsic oxidization

J Mar Sci Eng 2020 8 718 12 of 18

by allelochemicals and inactivation of the electron transport in the PSII systems The production ofROS is also a signal of the pressure from the excitation energy collected by the PSII light-harvestingcomplex [5758] To regulate the extra ROS algae have a series of antioxidant defense mechanismsincluding the ability to vary antioxidant enzymes or genes Variations in activities of the enzymes SODand CAT are important in alleviating oxidative damage [5960] In general SOD scavenges the cellularROS first catalyzing O2bullminus to H2O2 Then the CAT enzyme decomposes H2O2 to O2 and H2O [61]MnSOD one of the total SODs was selected as the representative enzyme it is mostly detected in thecytosol and thylakoid membrane [62]

Here a small amount of ROS (H2O2) was produced in the LC treatments ie T160 and T140but no significant variation was observed in the quantum efficiency of photosynthesis (FvFm)indicating U prolifera may activate photoprotection to defend against such allelopathic stress Howevera significant increase in ROS concentration (ANOVA p lt 0001) was recorded in the HC treatmentsaccompanied by the decline in rETR indicating normal electron transport in PSII was disturbed andexcess energy likely contributed to the ROS generation in HC treatments High production of ROSinduced oxidative stress in the algae and finally inhibited the photosynthesis systems To moderate theoxidative damage U prolifera upregulated the activity of SOD and CAT supported herein by the geneexpression level of upMnSOD and upCAT in the LC treatments (Figure 5) Similar responses have beennoted in Cylindrospermopsis raciborskii under hyper-salinity or light-stress conditions [6364] and linoleicacid stress [65] The upregulation of the transcript levels of FeSOD and CAT genes in U proliferahave also been reported in response to salicylic acid and hyper-temperature [66] In the present studyhowever the enhanced CAT activities were not sufficient to scavenge the sudden increased H2O2 andthis likely caused extensive oxidative stress in this macroalga

External stresses including allelopathic stressors can alter the algal energy flux of PSII by reducingthe photosynthetic efficiency [67ndash69] and by enhancing non-photochemical quenching (NPQ) [65]The maximum quantum yield (FvFm) is an effective indicator of the efficiency of photochemical stressIn Ulva sp changes in FvFm have been observed when the algae are exposed to internal or externalstresses [70] such as light [71] desiccation [72] salinity [73] and allelopathy [50]

Significant declines in FvFm (Figure 5b) growth rate (Figure 2) and Chl a and b (Figure 5a) wereshown after 192 h exposure to high concentrations of B cereus BE23 filtrate suggesting disruption of thePSII reaction centersrsquo (RCs) complexes [67] including the electron transport chain [74] Reduced rETRand Y(II) indicate a reduction in the electron transport rate and CO2 assimilative capacity [75] Thereforeone mechanism by which U prolifera responds to allelopathic stress is a lowering of the photosyntheticperformance which directly impacts carbon fixation and therefore the growth rate [76] The significantdecreases in the Chl a and b concentrations in the HC treatments may also be considered as an adaptivestrategy which decreases the absorption of photons thereby leading to less ROS production [67]

The NPQ pathways are photoprotective mechanisms for phototrophs [77] In the present studyno significant variation in FvFm (Figure 5b) or rETR (Figure 5a) was observed in the LC treatmentshowever a significant increase in NPQ was recorded as the concentrations of the LC treatmentsincreased namely T140 and T160 Under the HC treatments a substantial decrease in NPQ wasobserved indicating that allelopathic stress may hinder the operation of photoprotective mechanismsand thus the macroalgae dissipated excess energy through non-regulated pathways [78] At high levelsof bacterial filtrate U prolifera was unable to self-protect against photodamage [39] The significantdecrease in qP in the treatments with high concentrations of filtrate indicated a high level of energydissipation and potential damage to the PSII reaction centers Thus the decrease in the efficiencyof PSII was associated with a simultaneous decrease in the photochemical and non-photochemicalpathways in the HC treatments reflecting a complete disruption of normal energy pathways

Previous studies have suggested that Ulva sp can modulate NPQ levels by adjusting the copynumber of LhcSR or PsbS and regulation of the xanthophyll cycle [7980] It thus appears that low levelsof exposure to B cereus BE23 filtrate induced an upregulation of LhcSR and PsbS in U prolifera andactivated the photoprotection mechanism that enables the self-regulation of external allelopathic stress

J Mar Sci Eng 2020 8 718 13 of 18

without loss of electron transfer efficiency of photosynthesis and growth An upregulated transcriptlevel of both selected genes and a triggering of LhcSR-dependent NPQ was also previously reportedin Ulva sp [80] High amounts of filtrate in contrast inhibited the photosynthetic efficiency and thecapability of self-regulation of U prolifera as evidenced by the downregulation of FvFm qP and NPQactivity and finally the inhibition of growth Therefore the low value of NPQ was a result of the lossof the photoprotection of U prolifera and a failure of self-regulation under allelopathic stress [81]

Allelopathic damage to the PSII systems is also suggested by the responses of the genes locatedin the D1-D2 protein [5482] PsbA and PsbD encoding the D1 and D2 subunits of the PSII complexconstitute the heterodimeric photochemical reaction center [80] Here no clear variation in PsbA andPsbD gene expression was observed after 192 h exposure in the LC treatments (Figure 7b) suggestingthe excess absorbed electrons (Figure 4a) were dissipated by the upregulated NPQ together with theupregulation of LhcSR and PsbS transcript levels (Figure 7a) In contrast clear downregulation ofPsbA expression levels was recorded in the HC treatments suggesting that the B cereus BE23 filtratesuppressed PsbA expression and may have blocked the elector transport on the PSII receptor side fromQA to QB [81]

In summary the inhibition effect on the PSII of Ulva due to bacteria-derived stress may go throughtwo main steps (1) the inhibition of the electron transport chain and (2) the deleterious effects on PSIIRCsrsquo complexes [8384] In the present study the upregulated expression of PsbS and LhcSR under LClevels of cell-free filtrate might indicate the successful regulation of stress via regulated NPQ [8586]but failure in the HC treatments The depletion of the transcript pools of LhcSR and PsbS contributeddirectly to the decrease in NPQ activity and likely inactivated the PSII RCsrsquo complexes Downregulationof Chl a and b corresponded to the downregulation of PsbA expression levels suggesting the BE23filtrate degraded the absorption of light energy and blocked the electron transport on the PSII receptorside [6580] Surplus electrons exceeded the electron transport chain capacity of U prolifera and inducedadditional ROS production (Figure 3) that in turn damaged the PSII systems [16] Together these dataclearly document the photooxidative stress in U prolifera upon allelopahtic stress in HC treatments

Using ESI and NMR three potential allelopathic chemicals were isolated and identified from thecell-free filtrate of B cereus BE23 The chemical cyclo (L-Pro-L-Pro) (Figure 8b) extracted from Eea2displayed the largest inhibitory effect on U prolifera (Supplementary Figure S6) and has previouslybeen shown to yield a strong algicidal effect on Microcystis aeruginosa [55] and Phaeocystis globosa [54] byinhibiting the operation of the photosynthesis and antioxidant systems of target algae In the presentstudy the diketopiperazine derivatives decreased the gene expression of PsbA [5487] directly impactingthe PSII electron acceptor sides resulting in the failure of the photosynthetic process Given that cyclo(L-Pro-L-Pro) is easily biodegradable [88] it may be a good candidate as an environmentally friendlyalgicide for green algae bloom control

5 Conclusions

The high concentration of the cell-free filtrate of B cereus BE23 (approximately 1 times 1011mL)yielded significant inhibition of growth of U prolifera via degradation of the photosynthetic system asshown by changes in biomass accumulation photosynthetic responses gene regulation and enzymeactivities The potential allelopathic compounds inhibited growth by means of reduction of FvFmrETR and NPQ resulting in U proliferarsquos failure to dissipate the excess energy through regulated NPQpathways This alteration of energy dissipation caused excess cellular ROS accumulation and theantioxidative defense system was generated This ROS production also inhibited the PSII reaction centerapparatus The potential allelochemicals were further isolated and identified as N-phenethylacetamidecyclo (L-Pro-L-Val) and cyclo (L-Pro-L-Pro) The diketopiperazines derivative cyclo (L-Pro-L-Pro)exhibited the highest inhibition effect on U prolifera and further study on its potential as an algicidalproduct for green algae bloom control is warranted

Supplementary Materials The following are available online at httpwwwmdpicom2077-131289718s1Figure S1 Phylogenetic tree of Bacillus cereus BE23 Figure S2 Relative growth rates and inhibition rates of

J Mar Sci Eng 2020 8 718 14 of 18

Ulva prolifera of the first bioassay test Figure S3 Relative growth rates and inhibition rates of Ulva prolifera in thesecond bioassay test Figure S4 Relative growth rates and inhibition rates of Ulva prolifera in the third bioassaytest Figure S5 High-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum (a) 13C NMRspectrum (b) and 1H NMR spectrum (c) of compound Ech5-4 Figure S6 High-resolution electrospray ionizationmass spectrometry (HRESIMS) spectrum (a) 13C NMR spectrum (b) and 1H NMR spectrum (c) of compoundEea2-5 Figure S7 High-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum (a) 13C NMRspectrum (b) and 1H NMR spectrum (c) of compound Eea3-2 Table S1 Changes of pH values with culture timein exposed experiments

Author Contributions Conceptualization NL and MT methodology XZ and NL software NL validationNL JZ XZ PW PMG and MT formal analysis MT and PMG investigation NL JZ and XZ resourcesMT data curation NL and JZ writingmdashoriginal draft preparation NL writingmdashreview and editing MTPMG and PW visualization MT supervision MT project administration MT funding acquisition MTAll authors have read and agreed to the published version of the manuscript

Funding This research was supported by a National Key RampD Program of China NO 2016YFC1402104Key Laboratory of Integrated Marine Monitoring and Applied Technologies for Harmful Algal Blooms Ministryof Natural Resources of the Peoplersquos Republic of China (MNR) MATHAB201803 and Funding for Tang Scholar toMT

Acknowledgments The authors are grateful to Zhizhen Zhang of Zhejiang University for helping identify thenatural products and Min Wu for providing the bacteria Bacillus cereus BE23 strain

Conflicts of Interest The authors declare that they have no conflict of interest

References

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2 Gross EM Hilt S Lombardo P Mulderij G Searching for allelopathic effects of submerged macrophyteson phytoplanktonmdashState of the art and open questions Hydrobiologia 2007 584 77ndash88 [CrossRef]

3 Zhang YW Wang JT Tan LJ Characterization of allelochemicals of the diatom Chaetoceros curvisetus andthe effects on the growth of Skeletonema costatum Sci Total Environ 2019 660 269ndash276 [CrossRef] [PubMed]

4 Zhang H Peng Y Zhang S Cai G Li Y Yang X Yang K Chen Z Zhang J Wang H et al Algicidaleffects of prodigiosin on the harmful algae Phaeocystis globosa Front Microbiol 2016 7 602 [CrossRef][PubMed]

5 Zhou S Yin H Tang SY Peng H Yin DG Yang YX Liu ZH Ding Z Physiological responses ofMicrocystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa Ecotoxicol Environ Saf 2016127 214ndash221 [CrossRef]

6 Zhang FX Ye Q Chen QL Yang K Zhang DY Chen ZR Lu SS Shao XP Fan XY Yao LM et alAlgicidal Activity of novel marine bacterium Paracoccus sp Strain Y42 against a harmful algal-bloom-causingdinoflagellate Prorocentrum donghaiense Appl Environ Microbiol 2018 84 [CrossRef]

7 Qian HF Xu JH Lu T Zhang Q Qu Q Yang ZP Pan XL Responses of unicellular alga Chlorellapyrenoidosa to allelochemical linoleic acid Sci Total Environ 2018 625 1415ndash1422 [CrossRef]

8 Zhao W Zheng Z Zhang JL Roger SF Luo XZ Allelopathically inhibitory effects of eucalyptusextracts on the growth of Microcystis aeruginosa Chemosphere 2019 225 424ndash433 [CrossRef]

9 Yu Y Zeng YD Li J Yang CY Zhang XH Luo F Dai XZ An algicidal Streptomyces amritsarensisstrain against Microcystis aeruginosa strongly inhibits microcystin synthesis simultaneously Sci Total Environ2019 650 34ndash43 [CrossRef]

10 Arora A Sairam RK Srivastava GC Oxidative stress and antioxidative system in plants Curr Sci 200282 1227ndash1239

11 Apel K Hirt H Reactive oxygen species Metabolism oxidative stress and signal transduction Annu RevPlant Biol 2004 55 373ndash399 [CrossRef] [PubMed]

12 Mayali X Azam F Algicidal bacteria in the sea and their impact on algal blooms J Eukaryot Microbiol2004 51 139ndash144 [CrossRef] [PubMed]

13 Zheng NN Ding N Gao PK Han MX Liu XX Wang JG Li S Fu BY Wang RJ Zhou J Diversealgicidal bacteria associated with harmful bloom-forming Karenia mikimotoi in estuarine soil and seawaterSci Total Environ 2018 631 1415ndash1420 [CrossRef]

J Mar Sci Eng 2020 8 718 15 of 18

14 Sun R Sun P Zhang J Esquivel-Elizondo S Wu Y Microorganisms-based methods for harmful algalblooms control A review Bioresour Technol 2018 248 12ndash20 [CrossRef] [PubMed]

15 Lu XH Zhou B Xu L Liu LL Wang GY Liu XD Tang XX A marine algicidal Thalassospira and itsactive substance against the harmful algal bloom species Karenia mikimotoi Appl Microbiol Biotechnol 2016100 5131ndash5139 [CrossRef]

16 Hou SL Shu WJ Tan S Zhao L Yin PH Exploration of the antioxidant system and photosyntheticsystem of a marine algicidal Bacillus and its effect on four harmful algal bloom species Can J Microbiol2016 62 49ndash59 [CrossRef]

17 Hu XL Yin PH Zhao L Yu QM Characterization of cell viability in Phaeocystis globosa cultures exposedto marine algicidal bacteria Biotechnol Bioprocess Eng 2015 20 58ndash66 [CrossRef]

18 Shao JH He YX Chen AW Peng L Luo S Wu GY Zou HL Li RH Interactive effects of algicidalefficiency of Bacillus sp B50 and bacterial community on susceptibility of Microcystis aeruginosa with differentgrowth rates Int Biodeterior Biodegrad 2015 97 1ndash6 [CrossRef]

19 Jeong SY Ishida K Ito Y Okada S Murakami M Bacillamide a novel algicide from the marinebacterium Bacillus sp SY-1 against the harmful dinoflagellate Cochlodinium polykrikoides Tetrahedron Lett2003 44 8005ndash8007 [CrossRef]

20 Wu LM Wu HJ Chen LN Xie SS Zang HY Borriss R Gao XW Bacilysin fromBacillus amyloliquefaciens FZB42 has specific bactericidal activity against harmful algal bloom speciesAppl Environ Microbiol 2014 80 7512ndash7520 [CrossRef]

21 Skerratt JH Bowman JP Hallegraeff G James S Nichols PD Algicidal bacteria associated with bloomsof a toxic dinoflagellate in a temperate Australian estuary Mar Ecol Prog Ser 2002 244 1ndash15 [CrossRef]

22 Liu DY Keesing JK Xing QG Shi P Worldrsquos largest macroalgal bloom caused by expansion of seaweedaquaculture in China Mar Pollut Bull 2009 58 888ndash895 [CrossRef] [PubMed]

23 Wang ZL Xiao J Fan SL Li Y Liu XQ Liu DY Who made the worldrsquos largest green tide inChinamdashAn integrated study on the initiation and early development of the green tide in Yellow SeaLimnol Oceanogr 2015 60 1105ndash1117 [CrossRef]

24 Ye NH Zhuang ZZ Jin X Wang Q Zhang X Li DM Wang HX Mao YZ Jiang ZJ Li B et alChina is on the track tackling Enteromorpha spp forming green tide Nat Preced 2008 [CrossRef]

25 Ye NH Zhang XW Mao YZ Liang CW Xu D Zou J Zhuang ZZ Wang QY lsquoGreen tidesrsquoare overwhelming the coastline of our blue planet Taking the worldrsquos largest example Ecol Res 201126 477ndash485 [CrossRef]

26 Huo YZ Han HB Shi HH Wu HL Zhang JH Yu KF Xu R Liu CC Zhang ZL Liu KF et alChanges to the biomass and species composition of Ulva sp on Porphyra aquaculture rafts along the coastalradial sandbank of the Southern Yellow Sea Mar Pollut Bull 2015 93 210ndash216 [CrossRef]

27 Zhang JH Huo YZ Wu H Yu K Kim JK Yarish C Qin YT Liu CC Xu R He PM The origin ofthe Ulva macroalgal blooms in the Yellow Sea in 2013 Mar Pollut Bull 2014 89 276ndash283 [CrossRef]

28 Li HM Zhang YY Chen J Zheng X Liu F Jiao NZ Nitrogen uptake and assimilation preferences ofthe main green tide alga Ulva prolifera in the Yellow Sea China J Appl Phycol 2018 31 625ndash635 [CrossRef]

29 Xiao J Zhang XH Gao CL Jiang MJ Li RX Wang ZL Li Y Fan SL Zhang XL Effect oftemperature salinity and irradiance on growth and photosynthesis of Ulva prolifera Acta Oceanol Sin 201635 114ndash121 [CrossRef]

30 Liu Q Yan T Yu RC Zhang QC Zhou MJ Interactions between selected microalgae and microscopicpropagules of Ulva prolifera J Mar Biol Assoc UK 2017 98 1571ndash1580 [CrossRef]

31 Fan X Xu D Wang YT Zhang XW Cao SN Mou SL Ye NH The effect of nutrient concentrationsnutrient ratios and temperature on photosynthesis and nutrient uptake by Ulva prolifera Implications for theexplosion in green tides J Appl Phycol 2014 26 537ndash544 [CrossRef]

32 Sun X Wu MQ Xing QG Song XD Zhao DH Han QQ Zhang GZ Spatio-temporal patterns ofUlva prolifera blooms and the corresponding influence on chlorophyll-a concentration in the Southern YellowSea China Sci Total Environ 2018 640 807ndash820 [CrossRef] [PubMed]

33 Guillard RRL Culture of Phytoplankton for Feeding Marine Invertebrates In Culture of Marine InvertebrateAnimals Springer Boston MA USA 1975

34 Jin Q Dong SL Wang CY Allelopathic growth inhibition of Prorocentrum micans (Dinophyta) by Ulvapertusa and Ulva linza (Chlorophyta) in laboratory cultures Eur J Phycol 2005 40 31ndash37 [CrossRef]

J Mar Sci Eng 2020 8 718 16 of 18

35 Li H Huang HJ Li HY Liu JS Yang WD Genetic diversity of Ulva prolifera population in Qingdaocoastal water during the green algal blooms revealed by Microsatellite Mar Pollut Bull 2016 111 237ndash246[CrossRef] [PubMed]

36 Bradford MM A rapid method for the quantitation of microgram quantities of protein utilizing the principleof protein-dye binding Anal Biochem 1976 72 248ndash254 [CrossRef]

37 Sun X Lu Z Liu B Zhou Q Zhang Y Wu Z Allelopathic effects of pyrogallic acid secreted bysubmerged macrophytes on Microcystis aeruginosa Role of ROS generation Allelopath J 2014 33 121ndash130

38 Dhindsa RS Plumb-Dhindsa P Thorpe TA Leaf senescence Correlated with increased levels ofmembrane permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalaseJ Exp Bot 1981 32 93ndash101 [CrossRef]

39 Zhao XY Tang XX Zhang H Qu TF Wang Y Photosynthetic adaptation strategy of Ulva proliferafloating on the sea surface to environmental changes Plant Physiol Biochem 2016 107 116ndash125 [CrossRef]

40 Wang JW Yan BL Lin AP Hu JP Shen SD Ecological factor research on the growth and induction ofspores release in Enteromorpha Prolifera (Chlorophyta) Mar Sci Bull 2007 26 60ndash66

41 Zhao PJ Wang HX Li GH Li HD Liu J Shen YM Secondary metabolites from endophyticStreptomyces sp Lz531 Chem Biodivers 2007 4 899ndash904 [CrossRef]

42 Li T Wang GC Huang XJ Ye WC ChemInform Abstract Whitmanoside A (I) a New α-PyroneGlycoside from the Leech Whitmania pigra J Cheminform 2013 44 [CrossRef]

43 Furtado NAJC Pupo MT Carvalho I Campo VL Duarte MCT Bastos JK Diketopiperazinesproduced by an Aspergillus fumigatus Brazilian strain J Braz Chem Soc 2005 16 1448ndash1453 [CrossRef]

44 Tilney CL Pokrzywinski KL Coyne KJ Warner ME Effects of a bacterial algicide IRI-160AA ondinoflagellates and the microbial community in microcosm experiments Harmful Algae 2014 39 210ndash222[CrossRef]

45 Meyer N Bigalke A Kaulfuss A Pohnert G Strategies and ecological roles of algicidal bacteriaFEMS Microbiol Rev 2017 41 880ndash899 [CrossRef]

46 Hare CE Demir E Coyne KJ Craig Cary S Kirchman DL Hutchins DA A bacterium that inhibitsthe growth of Pfiesteria piscicida and other dinoflagellates Harmful Algae 2005 4 221ndash234 [CrossRef]

47 Zozaya-Valdes E Egan S Thomas T A comprehensive analysis of the microbial communities of healthy anddiseased marine macroalgae and the detection of known and potential bacterial pathogens Front Microbiol2015 6 9ndash18 [CrossRef]

48 Perveen S Mushtaq MN Yousaf M Sarwar N Allelopathic hormesis and potent allelochemicals frommultipurpose tree Moringa oleifera leaf extract Plant Biosyst 2020 18 1ndash6 [CrossRef]

49 Wang CX Zhu MX Chen XH Qu B Review on allelopathy of exotic invasive plants Procedia Eng2011 18 240ndash246

50 Li NC Tong MM Glibert PM Effect of allelochemicals on photosynthetic and antioxidant defensesystem of Ulva prolifera Aquat Toxicol 2020 224 105513 [CrossRef]

51 Xu D Gao ZQ Zhang XW Fan X Wang YT Li DM Wang W Zhuang Z Ye N Allelopathicinteractions between the opportunistic species Ulva prolifera and the native macroalga Gracilaria lichvoidesPLoS ONE 2012 7 e33648 [CrossRef]

52 Zhou QX Hu XG Systemic stress and recovery patterns of rice roots in response to graphene oxidenanosheets Environ Sci Technol 2017 51 2022ndash2030 [CrossRef] [PubMed]

53 Wang Y Zhao XY Tang XX Antioxidant system responses in two co-occurring green-tide algae understress conditions J Ocean Univ 2016 34 102ndash108 [CrossRef]

54 Tan S Hu XL Yin PH Zhao L Photosynthetic inhibition and oxidative stress to the toxic Phaeocystisglobosa caused by a diketopiperazine isolated from products of algicidal bacterium metabolism J Microbiol2016 54 364ndash375 [CrossRef] [PubMed]

55 Guo XL Liu XL Pan JL Yang H Synergistic algicidal effect and mechanism of two diketopiperazinesproduced by Chryseobacterium sp strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosaSci Rep 2015 5 14720 [CrossRef] [PubMed]

56 Zhou QX Xu JR Cheng Y Quantitative analyses of relationships between ecotoxicological effects andcombined pollution Plant Soil 2004 261 155ndash162 [CrossRef]

57 Hess FD Light-dependent herbicides An overview Weed Sci 2000 48 160ndash170 [CrossRef]

J Mar Sci Eng 2020 8 718 17 of 18

58 Ni LT Rong SY Gu GX Hu LL Wang PF Li DY Yue FF Wang N Wu HQ Li SY Inhibitoryeffect and mechanism of linoleic acid sustained-release microspheres on Microcystis aeruginosa at differentgrowth phases Chemosphere 2018 212 654ndash661 [CrossRef]

59 Wang GX Zhang Q Li JL Chen XY Lang QL Kuang SP Combined effects of erythromycin andenrofloxacin on antioxidant enzymes and photosynthesis-related gene transcription in Chlorella vulgarisAquat Toxicol 2019 212 138ndash145 [CrossRef]

60 Zhou QX Yue ZK Li QZ Zhou RR Liu L Exposure to PbSe nanoparticles and male reproductivedamage in a rat model Environ Sci Technol 2019 53 13408ndash13416 [CrossRef]

61 Kurama EE Fenille RC Rosa VE Jr Rosa DD Ulian EC Mining the enzymes involved in thedetoxification of reactive oxygen species (ROS) in sugarcane Mol Plant Pathol 2010 3 251ndash259 [CrossRef]

62 Fan MH Sun X Xu NJ Liao Z Wang RX cDNA cloning characterization and expression analysis ofmanganese superoxide dismutase in Ulva prolifera J Appl Phycol 2015 28 1391ndash1401 [CrossRef]

63 Cruces E Rautenberger R Cubillos VM Ramirez-Kushel E Rojas-Lillo Y Lara C Montory JAGomez I Interaction of photoprotective and acclimation mechanisms in Ulva rigida (Chlorophyta) in responseto diurnal changes in solar radiation in Southern Chile J Phycol 2019 55 1011ndash1027 [CrossRef]

64 Sung MS Hsu YT Wu TM Lee TM Hypersalinity and hydrogen peroxide upregulation of geneexpression of antioxidant enzymes in Ulva fasciata against oxidative stress Mar Biotechnol 2009 11 199ndash209[CrossRef]

65 Xu S Yang SQ Yang YJ Xu JZ Shi JQ Wu ZX Influence of linoleic acid on growth oxidative stressand photosynthesis of the cyanobacterium Cylindrospermopsis raciborskii N Z J Mar Freshw Res 201751 223ndash236 [CrossRef]

66 Fan MH Sun X Liao Z Wang JX Cui DL Xu NJ Full-length cDNA cloning characterizationof catalase from Ulva prolifera and antioxidant response to diphenyliodonium J Appl Phycol 201830 3361ndash3372 [CrossRef]

67 Long M Tallec K Soudant P Le Grand F Donval A Lambert C Sarthou G Jolley DF Heacutegaret HAllelochemicals from Alexandrium minutum induce rapid inhibition of metabolism and modify the membranesfrom Chaetoceros muelleri Algal Res 2018 35 508ndash518 [CrossRef]

68 Wang X Szeto YT Jiang C Wang X Tao Y Tu J Chen J Effects of Dracontomelon duperreanum leaf litteron the growth and photosynthesis of Microcystis aeruginosa Bull Environ Contam Toxicol 2018 100 690ndash694[CrossRef]

69 Yu SM Li C Xu CC Effiong K Xiao X Understanding the inhibitory mechanism of antialgalallelochemical flavonoids from genetic variations Photosynthesis toxin synthesis and nutrient utility EcotoxEnviron Saf 2019 177 18ndash24 [CrossRef]

70 Maxwell K Johnson GN Chlorophyll fluorescencemdashA practical guide J Exp Bot 2000 51 659ndash668[CrossRef]

71 Zheng ZZ Gao S Wang GC Far red light induces the expression of LHCSR to trigger nonphotochemicalquenching in the intertidal green macroalgae Ulva prolifera Algal Res 2019 40 101512 [CrossRef]

72 Gao S Shen SD Wang GC Niu JF Lin AP Pan GH PSI-driven cyclic electron flow allows intertidalmacro-algae Ulva sp (Chlorophyta) to survive in desiccated conditions Plant Cell Physiol 2011 52 885ndash893[CrossRef] [PubMed]

73 Gao S Chi Z Chen HL Zheng ZB Weng YX Wang GC A Supercomplex of approximately 720 kDaand composed of both photosystem reaction centers dissipates excess energy by PSI in green macroalgaeunder salt stress Plant Cell Physiol 2019 60 166ndash175 [CrossRef] [PubMed]

74 Lelong A Haberkorn H Le Goiumlc N Heacutegaret H Soudant P A new insight into allelopathic effectsof Alexandrium minutum on photosynthesis and respiration of the diatom Chaetoceros neogracile revealedby photosynthetic-performance analysis and flow cytometry Microb Ecol 2011 62 919ndash930 [CrossRef][PubMed]

75 Genty B Briantais JM Baker NR The relationship between the quantum yield of photosynthetic electrontransport and quenching of chlorophyll fluorescence Biochim Biophys Acta Gen Subj 1989 990 87ndash92[CrossRef]

76 Mhatre A Patil S Agarwal A Pandit R Lali AM Influence of nitrogen source on photochemistryand antenna size of the photosystems in marine green macroalgae Ulva lactuca Photosynth Res 2019139 539ndash551 [CrossRef]

J Mar Sci Eng 2020 8 718 18 of 18

77 Peers G Truong TB Ostendorf E Busch A Elrad D Grossman AR Hippler M Niyogi KKAn ancient light-harvesting protein is critical for the regulation of algal photosynthesis Nature 2009462 518ndash521 [CrossRef]

78 Figueroa FL Celis-Plaacute PSM Martiacutenez B Korbee N Trilla A Arenas F Yield losses and electrontransport rate as indicators of thermal stress in Fucus serratus (Ochrophyta) Algal Res 2019 41 101560[CrossRef]

79 Dong MT Zhang XW Zhuang ZZ Zou J Ye NH Xu D Mou SL Liang CW Wang WQCharacterization of the LhcSR gene under light and temperature stress in the green alga Ulva linza Plant MolBiol Rep 2011 30 10ndash16 [CrossRef]

80 Mou SL Zhang XW Dong M Fan X Xu J Cao S Xu D Wang W Ye NH Photoprotection in thegreen tidal alga Ulva prolifera Role of LhcSR and PsbS proteins in response to high light stress Plant Biol2013 15 1033ndash1039 [CrossRef]

81 Kommalapati M Hwang HJ Wang HL Burnap RL Engineered ectopic expression of the psbA geneencoding the photosystem II D1 protein in Synechocystis sp PCC6803 Photosynth Res 2007 92 315ndash325[CrossRef]

82 Barati B Lim PE Gan SY Poong SW Phang SM Gene expression profile of marine Chlorella strainsfrom different latitudes Stress and recovery under elevated temperatures J Appl Phycol 2018 30 3121ndash3130[CrossRef]

83 Ohnishi N Allakhverdiev SI Takahashi S Higashi S Watanabe M Nishiyama Y Norio M Two-stepmechanism of photodamage to photosystem II Step 1 occurs at the oxygen-evolving complex and step 2occurs at the photochemical reaction center Biochemistry 2005 44 8494ndash8499 [CrossRef] [PubMed]

84 Hakala M Tuominen I Keraumlnen M Tyystjaumlrvi T Tyystjaumlrvi E Evidence for the role of the oxygen-evolvingmanganese complex in photoinhibition of Photosystem II Biochim Biophys Acta Bioenergy 2005 1706 68ndash80[CrossRef] [PubMed]

85 Correa-Galvis V Redekop P Guan K Griess A Truong TB Wakao S Niyogi KK Jahns PPhotosystem II Subunit PsbS is involved in the induction of LHCSR protein-dependent energy dissipation inChlamydomonas reinhardtii J Biol Chem 2016 291 17478ndash17487 [CrossRef]

86 Pinnola A Cazzaniga S Alboresi A Nevo R Levin-Zaidman S Reich Z Bassi R Light-HarvestingComplex stress-eelated proteins catalyze excess energy dissipation in both photosystems of physcomitrella patensPlant Cell 2015 27 3213ndash3227 [CrossRef] [PubMed]

87 Li Y Zhu H Lei X Zhang H Cai G Chen Z Fu L Xu H Zheng TL The death mechanism ofthe harmful algal bloom species Alexandrium tamarense induced by algicidal bacterium deinococcus sp Y35Front Microbiol 2015 6 992ndash997 [CrossRef]

88 Perzborn M Syldatk C Rudat J Enzymatical and microbial degradation of cyclic dipeptides(diketopiperazines) AMB Express 2013 3 51 [CrossRef] [PubMed]

copy 2020 by the authors Licensee MDPI Basel Switzerland This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (httpcreativecommonsorglicensesby40)

Introduction

Materials and Methods

Algal Culture and Identification
Experiment 1 Bacteria-Derived Allelopathic Inhibition on U prolifera
Preparation of Cell-Free Filtrate from Bacillus cereus
Preparation of the Exposure Treatment
Growth
The Antioxidant Defense System
Photosynthesis System
Experiment 2 Isolation and Identification the Potential Allelopathic Compounds from Cell-Free Filtrate of Bacillus cereus BE23
Step 1 Solid Phase and Liquid Phase Extraction of Potential Allelopathic Compounds
Step 2 Open Column Chromatography to Select the Potential Allelopathic Compounds
Step 3 Ultra- and High-Performance Liquid Chromatography to Select the Potential Allelopathic Compounds
Structure Identification
Statistical Analysis
Results
Identification of Macroalga and Bacteria
Inhibition on the Growth of U prolifera
Response of Antioxidant System of U prolifera
Response of PSII System of U prolifera
Identification of Allelochemicals from Bacillus cereus BE23 Filtrate
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 2 of 18

include destroying the cell structure [45] altering production of the reactive oxygen species (ROS) [6]impacting intracellular enzymatic activities [7] or altering the photosynthesis system [8] and relatedgene expression [9] External stress can induce the production of ROS ie hydrogen peroxide (H2O2)and superoxide radical (O2

bullminus) and can induce the regulation of the antioxidative defense or thephotoprotection system [1011]

A number of bacteria-derived algicidal compounds have drawn wide attention as a control forHABs [12ndash14] and the algicidal compounds belonging to the Cytophaga-Flavobacterium-Bacteroides (CFB)phylum have been identified [15] Among this phylogenetic profile the genus of Bacillus shows promisein controlling HABs as negative effects have been demonstrated on the diatom Skeletonema costatumthe raphidophyte Heterosigma akashiwo the dinoflagellate Prorocentrum donghaiense [16] the prymnesiophytePhaeocystis globosa [1617] and the cyanobacterium Microcystis aeruginosa [18] The potential allelochemicalsthat have been isolated and identified from Bacillus sp include terpene steroids and alkaloids [1920]The active compounds and mechanisms remain to be identified due to the species-specific response toalgicidal bacteria [21]

The green tides caused by blooms of Ulva prolifera have occurred in the Yellow Sea of China since2007 [22ndash26] These massive blooms negatively impact the local communities aquaculture operationsand tourism causing great damage to the local ecosystem service and enormous economic loss [27]The rapid growth of U prolifera on the other hand makes it the strongest competitor for nutrientsand light [2829] in the bloom area thereby driving the great impact on the marine biodiversity andstructure of the community [30ndash32] There are currently no effective measures to control these blooms

The Bacillus sp-derived control of HABs is promising but limited exploration has been undertakenin mitigating the green tides As a complicating factor the life stage of thalli has been reported to be animportant factor in green tide development [27] Therefore a series of experiments were performedto understand the extent to which bacterial allelopathy may be effective in controlling the thalli ofU prolifera Specifically the following questions were addressed (1) does the cell-free filtrate ofBacillus sp inhibit the growth of U prolifera and if so what is the effective dose (2) What is themechanism by which negative allelopathy occurs particularly with respect to the antioxidative defensesystem and the photosynthetic system II (PSII) response (3) What are the potential allelochemicals inthe filtrate of Bacillus sp that cause negative effects on U prolifera

2 Materials and Methods

21 Algal Culture and Identification

Asexual isolates of Ulva prolifera were provided by Zhejiang Xiangshan Xuwen Algal ExploitationCompany China in October 2018 Specimens were subsequently transferred to the laboratory onice sterilized with 07 potassium iodide (KI) for 5 min and then rinsed with autoclaved seawaterThe pre-sterilized thalli were maintained in sterilized f2 medium [33] with salinity of 30 temperature of20 C and light of 60 micromolmiddotm2

middotsminus1 (1212 h of lightdark cycle) The media were replaced every 5 daysTo minimize the interference of carry-over epiphytic bacteria in U prolifera cultures were pretreated

before each exposure experiment by antibiotic mixtures of penicillin (100 mgL) polymixin (075 mgL)and neomycin (09 mgL) for 48 h [34]

The macroalga was identified using the method described in Li et al [35] Total DNA was extractedwith a commercial Plant DNA Mini Kit (TaKaRa China) ITS and 5S sequences were amplified by thecorresponding PCR primers (Table 1) and the conducted BLAST analyses in the NCBI database

J Mar Sci Eng 2020 8 718 3 of 18

223 Growth

Gx = (Wx minusWc)Wc

J Mar Sci Eng 2020 8 718 4 of 18

J Mar Sci Eng 2020 8 718 5 of 18

Chl a = 127 OD663 minus 269 OD645

Chl b = 229 OD645 minus 468 OD663

3 Results

prolifera

SPE LLE

Cyclohexane

Ethyl acetate

1-butanol seriatim

First bioassay

2001(Ech1)

1001(Ech2)

501 (Ech3)

251 (Ech4)

101 (Ech5)

51 (Ech6)

01 (Ech7)

501 (Eea1)

251 (Eea2)

101 (Eea3)

51 (Eea4)

21 (Eea5)

11 (Eea6)

01 (Eea7)

Second bioassay

UPLC pre-HPLC

Third bioassay

Mobile Phase

2352min

1343min

1625min

Ech5-4

Eea2-5

Eea3-2

HR-ESI-MS NMR

J Mar Sci Eng 2020 8 718 7 of 18

3 Results

J Mar Sci Eng 2020 8 718 8 of 18

to control

cereus BE23

to control

cereus BE23

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 3 of 18

223 Growth

Gx = (Wx minusWc)Wc

J Mar Sci Eng 2020 8 718 4 of 18

J Mar Sci Eng 2020 8 718 5 of 18

Chl a = 127 OD663 minus 269 OD645

Chl b = 229 OD645 minus 468 OD663

3 Results

prolifera

SPE LLE

Cyclohexane

Ethyl acetate

1-butanol seriatim

First bioassay

2001(Ech1)

1001(Ech2)

501 (Ech3)

251 (Ech4)

101 (Ech5)

51 (Ech6)

01 (Ech7)

501 (Eea1)

251 (Eea2)

101 (Eea3)

51 (Eea4)

21 (Eea5)

11 (Eea6)

01 (Eea7)

Second bioassay

UPLC pre-HPLC

Third bioassay

Mobile Phase

2352min

1343min

1625min

Ech5-4

Eea2-5

Eea3-2

HR-ESI-MS NMR

J Mar Sci Eng 2020 8 718 7 of 18

3 Results

J Mar Sci Eng 2020 8 718 8 of 18

to control

cereus BE23

to control

cereus BE23

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 4 of 18

J Mar Sci Eng 2020 8 718 5 of 18

Chl a = 127 OD663 minus 269 OD645

Chl b = 229 OD645 minus 468 OD663

3 Results

prolifera

SPE LLE

Cyclohexane

Ethyl acetate

1-butanol seriatim

First bioassay

2001(Ech1)

1001(Ech2)

501 (Ech3)

251 (Ech4)

101 (Ech5)

51 (Ech6)

01 (Ech7)

501 (Eea1)

251 (Eea2)

101 (Eea3)

51 (Eea4)

21 (Eea5)

11 (Eea6)

01 (Eea7)

Second bioassay

UPLC pre-HPLC

Third bioassay

Mobile Phase

2352min

1343min

1625min

Ech5-4

Eea2-5

Eea3-2

HR-ESI-MS NMR

J Mar Sci Eng 2020 8 718 7 of 18

3 Results

J Mar Sci Eng 2020 8 718 8 of 18

to control

cereus BE23

to control

cereus BE23

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 5 of 18

Chl a = 127 OD663 minus 269 OD645

Chl b = 229 OD645 minus 468 OD663

3 Results

prolifera

SPE LLE

Cyclohexane

Ethyl acetate

1-butanol seriatim

First bioassay

2001(Ech1)

1001(Ech2)

501 (Ech3)

251 (Ech4)

101 (Ech5)

51 (Ech6)

01 (Ech7)

501 (Eea1)

251 (Eea2)

101 (Eea3)

51 (Eea4)

21 (Eea5)

11 (Eea6)

01 (Eea7)

Second bioassay

UPLC pre-HPLC

Third bioassay

Mobile Phase

2352min

1343min

1625min

Ech5-4

Eea2-5

Eea3-2

HR-ESI-MS NMR

J Mar Sci Eng 2020 8 718 7 of 18

3 Results

J Mar Sci Eng 2020 8 718 8 of 18

to control

cereus BE23

to control

cereus BE23

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

3 Results

prolifera

SPE LLE

Cyclohexane

Ethyl acetate

1-butanol seriatim

First bioassay

2001(Ech1)

1001(Ech2)

501 (Ech3)

251 (Ech4)

101 (Ech5)

51 (Ech6)

01 (Ech7)

501 (Eea1)

251 (Eea2)

101 (Eea3)

51 (Eea4)

21 (Eea5)

11 (Eea6)

01 (Eea7)

Second bioassay

UPLC pre-HPLC

Third bioassay

Mobile Phase

2352min

1343min

1625min

Ech5-4

Eea2-5

Eea3-2

HR-ESI-MS NMR

J Mar Sci Eng 2020 8 718 7 of 18

3 Results

J Mar Sci Eng 2020 8 718 8 of 18

to control

cereus BE23

to control

cereus BE23

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 7 of 18

3 Results

J Mar Sci Eng 2020 8 718 8 of 18

to control

cereus BE23

to control

cereus BE23

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 8 of 18

to control

cereus BE23

to control

cereus BE23

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 9 of 18

(Figure 5a)

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 10 of 18

(Figure 7b)

(b)(a)

(Figure 7b)

(b)(a)

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 11 of 18

4 Discussion

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 12 of 18

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 13 of 18

5 Conclusions

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 14 of 18

References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 15 of 18

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 16 of 18

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 17 of 18

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

J Mar Sci Eng 2020 8 718 18 of 18

Introduction

Growth
Results
Discussion
Conclusions
References

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