A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates

A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates
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  Harmful Algae xxx (2004) xxx–xxx A bacterium that inhibits the growth of   Pfiesteria piscicida and other dinoflagellates Clinton E. Hare, Elif Demir, Kathryn J. Coyne, S. Craig Cary,David L. Kirchman, David A. Hutchins ∗ College of Marine Studies, University of Delaware, 700 Pilottown Road, Lewes, DE 19958, USA Received 15 October 2003; received in revised form 4 January 2004; accepted 15 March 2004 Abstract Toxic dinoflagellate blooms have increased in estuaries of the east coast of the United States in recent years, and thediscovery of   Pfiesteria piscicida  has brought renewed attention to the problem of harmful algal blooms (HAB) in general.Manybacteriaandviruseshavebeenisolatedthathavealgicidaloralgistaticeffectsonphytoplankton,includingHABspecies.Twenty-two bacterial isolates from the Delaware Inland Bays were screened for algicidal activity. One isolate ( Shewanella IRI-160) had a growth-inhibiting effect on all three dinoflagellate species tested, including  P. piscicida  (potentially toxiczoospores),  Prorocentrum minimum , and  Gyrodinium uncatenum . This bacterium did not have a negative effect on the growthof any of the other four common estuarine non-dinoflagellate species tested, and in fact had a slight stimulatory effect on adiatom, a prasinophyte, a cryptophyte, and a raphidophyte.  Shewanella  IRI-160 is the first non-microzooplankton example of a microbe with the ability to control and inhibit the growth of   P. piscicida , suggesting that bacteria in the natural environmentcould play a role in controlling the growth and abundance of   P. piscicida  and other dinoflagellates. Such bacteria could alsopotentially be used as management tools to prevent the proliferation of potentially harmful dinoflagellates in estuaries andcoastal waters.© 2004 Elsevier B.V. All rights reserved. Keywords:  Algicidal bacteria; Algistatic; Biological controls; Dinoflagellates; Harmful algal blooms;  Pfiesteria ;  Shewanella 1. Introduction The consequences of eutrophication in coastalembayments frequently include an increase in phyto-plankton biomass and a shift to non-siliceous species,especially dinoflagellates (Smayda, 1990; Justic et al.,1995; Richardson, 1997; Burkholder, 2001). Approx-imately 75% of known toxic marine phytoplanktonspecies are dinoflagellates (Sournia, 1995), and in ∗ Corresponding author. Tel.:  + 1-302-645-4079;fax:  + 1-302-645-4007.  E-mail address: (D.A. Hutchins). the last 15 years the number of identified specieshas increased from  ∼ 20 to around 60 (Burkholder,1998). As conditions in many nutrient-enriched es-tuaries become more suitable for dinoflagellates tooutcompete other classes of phytoplankton (Boesch,2000; Anderson et al., 2002), it is reasonable toexpect that there would be an increase in HABevents (Smayda, 1990; Richardson, 1997; Paerl,1998). Pfiesteria piscicida  has brought a great deal of at-tention to research on harmful dinoflagellate species(Burkholder and Glasgow, 2001; Magnien, 2001). Atthe same time, many studies have focused on the need 1568-9883/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.hal.2004.03.001  2  C.E. Hare et al./Harmful Algae xxx (2004) xxx–xxx to find both short-term and long-term methods to con-trol and mitigate the growth of HAB species (Doucetteet al., 1998). Long-term solutions like decreasing an-thropogenic nutrient inputs should be the main goalof any management plan (Boesch et al., 1996), buttake years to achieve results, limiting their ability toimmediately control HAB events. Potential environ-mentally sound short-term solutions to the problemsof HAB species in coastal waters need to receivemore serious attention (Boesch et al., 1996; Anderson,1997; Cembella, 1998). One such research area is thesearch for members of the microbial community thatare host-specific controls for HAB species.Bacteria and viruses may play major roles in theprevention, regulation, and termination of HAB events(Doucette, 1995; Doucette et al., 1998). Certain bac-teria and viruses isolated from HAB-affected watershave an algicidal effect on the growth of HAB species(Doucette, 1995; Yoshinaga et al., 1997; Doucetteet al., 1998). In many cases these microbial controlsare species- or genus-specific (Fukami et al., 1992;Imai et al., 1995; Doucette et al., 1999), while oth-ers attack many different algal classes (Yoshinagaet al., 1997; Kato et al., 1998; Lovejoy et al., 1998).Frequently, the abundance of these algicidal bacteriaincreases during the decline of an algal bloom, andthey may be involved in the collapse of blooms in na-ture (Fukami et al., 1991; Kim et al., 1998; Yoshinagaet al., 1998).Few studies have examined control of   P. piscicida by biological means. Some zooplankton, including ro-tifers and copepods, have been shown to feed on manyof the life stages of this dinoflagellate (Burkholderand Glasgow, 1995; Mallin et al., 1995; Stoeckerand Gustafson, 2002; Stoecker et al., 2002). Stoeckerand Gustafson (2002) suggested that microzooplank-ton grazing could potentially regulate densities of non-inducible strains of   P. piscicida  (defined as in-capable of killing fish with toxin; Burkholder et al.,2001). Little is known, however, about the potentialof bacteria and viruses to regulate the abundance of  P. piscicida .The goal of this study was to isolate bacteria fromthe Delaware Inland Bays that have an algicidal oralgistatic effect on laboratory cultures of   P. piscicida (potentially toxic strain). We discovered one possiblebacterial biological control that stopped the growthof three dinoflagellate species, including  P. piscicida ,while having no apparent negative effect on severalalgal species from other classes. 2. Material and methods 2.1. Algal cultures All stock and experimental cultures were main-tained on a 12:12h light:dark cycle at approximately90  molphotonsm − 2 s − 1 . Media were made using0.2  m filtered seawater collected at the mouth of theDelaware Bay, Delaware, USA. Seawater was mi-crowave sterilized and amended with f/2 enrichmentsconsisting of 883  M nitrate, 36.3  M phosphate,and trace metals and vitamins solutions (Guillardand Ryther, 1962; Guillard, 1975). L1 medium wasamended with similar major nutrients and vitamins,but has a modified trace metal solution (Guillard andHargraves, 1993). For diatoms only, 107  M silicatewas also included in the L1 medium (Guillard andHargraves, 1993).Potentially toxic  P. piscicida  cultures (Dino-phyceae, Burkholder et al., 2001) were obtained fromJ. Burkholder (N.C. State University). Cultures wereincubated at 24 ◦ C at a salinity of 15psu using f/2media and were fed with a unialgal culture of thecryptophyte  Rhodomonas  sp. (Crytophyceae, CCMP757).  Rhodomonas  cultures were grown at 24 ◦ C at asalinity of 33psu using L1 medium. Chattonella subsalsa  (Raphidophyceae),  Prorocen-trum minimum  (dinoflagellate, Desmophyceae), and Tetraselmis  sp. (green flagellate, Prasinophyceae) cul-tures were isolated from the Delaware Inland Bays.  P.minimum  and  C. subsalsa  have been deposited withthe Provasoli-Guillard National Center for Cultureof Marine Phytoplankton (CCMP) under the culturecodes CCMP 2233 and CCMP 2191, respectively.Botharepotentiallyharmfulspecies,buttoxicityisun-known for these isolates.  C. subsalsa  and  Gyrodiniumuncatenum  (nontoxic dinoflagellate, Dinophyceae,CCMP 1310) cultures were grown at 24 ◦ C and  P.minimum  and  Tetraselmis  cultures were maintainedat 18 ◦ C at a salinity of 30psu using L1–Si medium. Thalassiosira weissflogii  (non-toxic diatom, Bacillar-iophyceae, CCMP 1336) cultures were grown at 18 ◦ Cusing L1 + Si medium. All cultures were non-axenicand contained over 10 6 bacterialcellsml − 1 .  C.E. Hare et al./Harmful Algae xxx (2004) xxx–xxx  3 2.2. Bacterial isolation and maintenance Bacterial isolates were obtained from the inletof Indian River Bay, Delaware, where potentiallytoxic strains of   P. piscicida  have also been doc-umented (Coyne et al., 2001; Humphries, 2002).Single colonies of each bacterium were transferredfrom R 2 A agar plates (0.5gl − 1 proteose peptone,0.5gl − 1 yeast extract, 0.5gl − 1 dextrose anhydrous,0.3gl − 1 pyruvic acid sodium salt, 15gl − 1 agar,and 3:1 ratio of seawater to MilliQ, Atlas, 1993)to liquid LM medium (modified version of LuriaBertani medium, 20gl − 1 NaCl, 10gl − 1 tryptone,5gl − 1 yeast extract, Sambrook et al., 1989) andgrown overnight at 225rpm, 22 ◦ C. Bacterial isolateswere then transferred to LM agar plates (as abovebut including 15gl − 1 agar) for a culture library.Twenty-two bacterial isolates were screened for al-gicidal effects on  P. piscicida . All bacterial isolateswere frozen at  − 80 ◦ C after initial screening for usein later experiments by adding liquid bacterial cul-ture to a 50% freezing medium (65% glycerol, 0.1MMgSO 4  and 25mM Tris–Cl, pH 8.0, Sambrook et al.,1989). 2.3. Screening of bacterial isolates Initial screening for algicidal effects was only per-formed on  P. piscicida  cultures. A single colony of each bacterium was transferred to liquid LM mediumand grown for 8h before being transferred into freshLM medium for 6–8h. Cultures were then centrifugedat 4000rpm for 8min and rinsed with sterile seawaterthree times before being harvested in sterile 15 salin-ity seawater. P. piscicida  culture (10ml) at an initial concentra-tion of approximately 6000cellsml − 1 was placed in13ml culture tubes. In duplicate, 1ml each of the 22bacterial cultures was added to the  P. piscicida  cul-tures. For controls, an equal amount of sterile sea-water was added. Cultures were incubated at 20 ◦ Cfor 8 days. One milliliter was preserved with 1% glu-taraldehyde on days 0, 4 and 8 for  P. piscicida  cellcounts. All cultures were given 1ml of   Rhodomonas sp. (CCMP 757) as prey after sampling on day 4. Ex-periments were repeated in triplicate for any isolatesthat showed an apparent negative effect on the growthof   P. piscicida  cultures. 2.4. Bacterium IRI-160 additions Bacterial isolate IRI-160 had the greatest negativeeffect on  P. piscicida  cultures in preliminary exper-iments. The bacterial isolate IRI-106 (unidentifiedgamma- Proteobacterium ) was used as a bacterialcontrol because it did not affect  P. piscicida  growthduring initial screening. Both bacterial isolates wereremoved from  − 80 ◦ C storage, transferred to LMagar plates, and incubated at room temperature. Asingle colony was picked, transferred to a new LMplate, and incubated at room temperature for 2–3 daysbefore a single colony was transferred to 100ml of liquid LM medium. Bacteria cultures were incubatedon a shaker table at 225rpm, 24 ◦ C. Bacteria weretransferred in late exponential phase (approximately8–10h) to new sterile LM medium (100ml) andincubated as described above until mid to late expo-nential phase before being harvested (approximately7h). The culture was then centrifuged for 10min at4000rpm and rinsed with 100ml of sterile L1 mediathree times before being re-centrifuged and harvestedin 50–100ml of L1 media. One milliliter was diluted1000:1 with 0.2  m filtered seawater and counted asdescribed below to determine bacterial density.Bacteria were added in triplicate (except for  G.uncatenum  where duplicates were used) to 50mlof algal culture at a concentration of approximately10 8 cellsml − 1 in the final incubation volume. Bacte-rial concentrations used in the  P. minimum  experimentwere higher, at nearly 10 9 ml − 1 . These total bacterialconcentrations are similar to those typically observedin the Indian River Bay, Delaware, USA (CEH andDAH, unpublished data). An equal volume of ster-ile L1 medium was added to controls. An additionalcontrol treatment was included for  P. piscicida  usingthe harmless bacterial isolate IRI-106. The controlbacterium was added at a similar concentration torule out any possibility that negative effects seen in P. piscicida  cultures in the IRI-160 addition was dueto the addition of high bacterial concentrations andcompetition for nutrients.Cultures were incubated at the appropriate tempera-ture for either 8 ( P. piscicida  and all non-dinoflagellatealgal species) or 16 days ( P. minimum  and  G. un-catenum ). Samples for phytoplankton cell counts werepreserved on days 0, 2, 4, 6, 8, 10, 12, and 16 and forbacterial cell counts on days 0, 4, 8, 12 and 16.  4  C.E. Hare et al./Harmful Algae xxx (2004) xxx–xxx 2.5. Phytoplankton cell counts Phytoplankton cell count samples were preservedwith 1% glutaraldehyde and stored in the dark at4 ◦ C until analysis. Samples from each of the trip-licate experimental bottles in each treatment werecounted using the average of 10 fields at 100 × ( C. subsalsa ,  P. minimum ,  Rhodomonas  sp.,  G. un-catenum ,  Tetraselmis  sp., and  T. weissflogii ) or 400 × ( P. piscicida ) magnification using epifluorescence mi-croscopy.  P. piscicida  counts included only flagellatedzoospores, and encysted cells were not enumerated.For all species except  P. piscicida , only fluorescingcells were considered viable and were counted. Celldensities and standard deviations were calculatedfrom the average of the three replicates, significantdifferences were determined with a single factorANOVA test. 2.6. Bacterial cell counts Bacteria were enumerated using epifluorescencecounts of 0.1–1ml DAPI-stained samples (Hobbieet al., 1977; Porter and Feig, 1980). Samples werepreserved with 4% filtered formaldehyde, stored at4 ◦ C in the dark, and filtered onto 25mm 0.2  m black polycarbonate filters within 3 days before mountingon microscope slides. Slides were stored in the dark at − 20 ◦ C until counting.Bacteria were enumerated by semi-automated mi-croscopy and image analysis (Cottrell and Kirchman,2003). The microscope setup consisted of a SPOT-RTcolor camera (Diagnostics Instruments) mounted onan Olympus BX51 microscope. Image acquisition andprocessing were accomplished using ImagePro plussoftware (Media Cybernetics) and numerical calcula-tions and data storage were done in Microsoft Excel.A graphical user interface designed with Visual Basic6 (Microsoft) was used to coordinate microscope con-trol, image acquisition, image processing and numer-ical calculations. Ten random images were acquiredfor each slide using a 100 × oil immersion objective. 2.7. PCR and DGGE analysis Samples were collected on days 0, 4, and 8 toevaluate the diversity of the bacterial community incontrols and bacterial addition cultures of IRI-160 to P. piscicida . A subsample (100  l) was added to 0.6mlof CTAB extraction buffer (100mM Tris–HCl (pH8.0), 1.4M NaCl, 2% (w/v) cetyltrimethylammoniumbromide (CTAB), 0.4% (v/v)   -mercaptoethanol, 1%(w/v) polyvinylpyrollidone, 20mM EDTA; Dempsteret al., 1999), heated to 50 ◦ C for 20min, vortexed, andstoredat − 80 ◦ CuntilDNAwasextracted.16Sriboso-mal DNA was amplified by PCR using primers 519RC(5 ′ -ATT ACC GCG GCT GCT GG-3 ′ ) and 338FGC(5 ′ -CGC CCG CCG CGC CCC GCG CCC GTC CCGCCG CCC CCG CCC TCC TAC GGG AGG CAGCAG-3 ′ ) in 50  l reactions containing 67ng templateDNA, 0.4mM BSA, 0.2mM dNTPs, 1.0  M of eachprimer, 2.0mM MgCl 2 , 1X  Taq  polymerase buffer(Sigma, St. Louis, MO) and 0.5 units of Jumpstart  Taq DNA polymerase (Sigma). The PCR reaction con-sisted of 22 cycles of 1min at 94 ◦ C, 1min at 65 ◦ Cminus 0.5 ◦ C per cycle, and 1min at 72 ◦ C, followedby 13 cycles of 1min at 94 ◦ C, 1min at 55 ◦ C, and1min at 72 ◦ C, followed by a 5min extension at 72 ◦ C.Denaturing gradient gel electrophoresis (DGGE)was performed as described in Muyzer et al. (1993).PCR reactions were loaded directly onto an 8% poly-acrylamide gel with a gradient of 35–60% denaturant(100% denaturant corresponds to 7M urea and 40%formamide). Electrophoresis was carried out for 5hand 40min at 130V at 60 ◦ C using a Dcode Univer-sal Mutation Detection System (Bio-Rad, Hercules,CA). Fractionated PCR products were stained with0.5mgml − 1 ethidium bromide and visualized on aUV transluminator. The image was captured usingan Alpha-Imager system (Alpha Innotech Corp., SanLeandro, CA). 2.8. Sequencing and phylogenetic analysis A culture of the bacterium IRI-160 was filteredonto a 47mm 0.2  m Supor filter and lysed in 5mlof sucrose lysis buffer (40mM EDTA, 400mMNaCl, 0.75M sucrose, 50nM Tris–HCl at pH 9.0;Giovannoni et al., 1990). The sample was frozenat  − 20 ◦ C and extracted using a modified sucroselysis extraction method (Giovannoni et al., 1990).DNA was diluted to approximately 25ng  l − 1 insterile water. 16S rDNA was amplified using PCRwith eubacteria specific primers (Giovannoni, 1991)EubA (5 ′ -AAG GAG GTG ATC CA(ACGT) CC(AG)  C.E. Hare et al./Harmful Algae xxx (2004) xxx–xxx  5 CA-3 ′ ) and EubB (5 ′ -AGA GTT TGA TC(AC) TGGCTC AG-3 ′ ) in a 50  l reaction containing 25ng tem-plate DNA, 0.2mM BSA, 0.2mM dNTPs, 0.25  Mof each primer, 1.25mM MgCl2, 1X  Taq  polymerasebuffer (Sigma, St. Louis, MO) and 0.4 units of Jump-start  Taq  DNA polymerase (Sigma, St. Louis, MO).The PCR reaction consisted of 35 cycles of 30s at94 ◦ C, 30s at 55 ◦ C, and 2min at 72 ◦ C, followed bya 5min extension at 72 ◦ C.PCR product was purified using a Qiagen PCRpurification column (Qiagen, Valecia, CA). DNA wassequenced using Big Dye Terminator Cycle Sequenc-ing Ready Reaction Kit (PE Applied BiosystemsInc.) using primers EubA, EubB and 519F (5 ′ -CAGC(AC)G CCG CGG TAA T(AT)C-3 ′ ). The sequencewas aligned using MegAlign 5.01 software (DNAS-TAR, Inc., Madison, WI) with sequences obtainedfrom GenBank. Sequences were placed into geneticdata environment (Smith et al., 1994) and a phyloge-netic tree was constructed using Phylip distance treetool, consisting of known algicidal bacteria and com-mon bacteria from both the gamma- Proteobacteria and Cytophaga-Flavobacterium-Bacteroides groups. 3. Results 3.1. Preliminary screening of bacteria One out of the 22 bacterial isolates screened,IRI-160, had an apparent negative effect on the growthof   P. piscicida . When this isolate was tested a secondtime, there was little effect after 4 days, but by day8 the density of   P. piscicida  in the bacterial addition(2460  ±  1120cellsml − 1 ) was half than that of thecontrol (4690 ± 900cellsml − 1 , data not shown). 3.2. Bacterium IRI-160 additions to P. piscicida The bacterial isolate IRI-160 was tested again intriplicate against  P. piscicida  cultures, along with twocontrols. The first control was the addition of 0.2  mfiltered medium with no bacteria. The second con-trol was the addition of a bacterial isolate (IRI-106)that had no effect on  P. piscicida  growth during ini-tial screening (the bacterial control). There was nosignificant difference between the two controls over8 days ( P >  0 . 05, Fig. 1). After an initial lag phase Time (days)02468    P .  p   i  s  c   i  c   i   d  a    (   1   0   3   c  e   l   l  s  m   l  -   1   ) 05101520253035No-addition ControlIRI-160Bacterial Control ****** Fig. 1. Impact of bacterial strain IRI-160 (10 8 cellsml − 1 ) on  P. piscicida  cultures. Controls include both the addition of 0.2  mfiltered sterile medium (control) and the addition of a harmlessbacterium at 10 8 cellsml − 1 (bacterial control, IRI-106).  P. piscicida counts included only flagellated zoospores, and encysted cellswere not enumerated. Data points represent triplicate means ± 1S.D. Significant differences between the control treatments andIRI-160 addition are indicated by  ∗ :  P <  0 . 05;  ∗∗ :  P <  0 . 01;  ∗∗∗ : P <  0 . 001. of 2 days,  P. piscicida  density in the controls in-creased from about 5640 ± 290cellsml − 1 on day 2to 31,720 ± 1140cellsml − 1 in the no-addition controland 30,750 ± 1980cellsml − 1 in the bacterial additioncontrol by day 8.There was a significant difference between bothcontrols and the IRI-160 addition (Fig. 1). The densityof  P.piscicida remainedrelativelyconstantover8daysat around 10,000cellsml − 1 (11,350 ± 3950cellsml − 1 on day 8). By day 6 there was a significant differencein  P. piscicida  density between the IRI-160 additionand the controls ( P <  0 . 01). Following the additionof IRI-160,  P. piscicida  grew exponentially for only 2days (between days 2 and 4) before quickly enteringstationary phase for the remainder of the incubation,while in both controls  P. piscicida  grew exponentiallyfor6daysandneverenteredstationaryphase(Table1).Since  P. piscicida  is heterotrophic and was fed al-gal prey during the experiments, prey limitation wasexamined. Though the abundance of prey was lowerin the bacterial treatment than in the controls, prey didnot appear to become limiting in any of the treatmentsuntil day 8 (Fig. 2). On day 8, there was a significantdifference ( P <  0 . 01) between the prey abundance inthe control and bacteria addition treatment. Despite
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