RESEARCH ARTICLE

Sebastian Engel Æ Melany P. Puglisi Æ Paul R. JensenWilliam Fenical

Antimicrobial activities of extracts from tropical Atlantic marine plantsagainst marine pathogens and saprophytes

Received: 7 June 2005 / Accepted: 24 January 2006 / Published online: 15 March 2006� Springer-Verlag 2006

Abstract Studies investigating disease resistance inmarine plants have indicated that secondary metabolitesmay have important defensive functions against harmfulmarine microorganisms. The goal of this study was tosystematically screen extracts from marine plants forantimicrobial effects against marine pathogens andsaprophytes. Lipophilic and hydrophilic extracts fromspecies of 49 marine algae and 3 seagrasses collected inthe tropical Atlantic were screened for antimicrobialactivity against five ecologically relevant marine micro-organisms from three separate kingdoms. These assaymicrobes consisted of the pathogenic fungus Lindrathalassiae, the saprophytic fungus Dendryphiella salina,the saprophytic stramenopiles, Halophytophthora spin-osa and Schizochytrium aggregatum, and the pathogenicbacterium Pseudoaltermonas bacteriolytica. Overall,90% of all species surveyed yielded extracts that wereactive against one or more, and 77% yielded extractsthat were active against two or more assay microor-ganisms. Broad-spectrum activity against three or fourassay microorganisms was observed in the extracts from48 and 27% of all species, respectively. The green algaeHalimeda copiosa and Penicillus capitatus (Chlorophyta)were the only species to yield extracts active against allassay microorganisms. Among all assay microorgan-isms, both fungi were the most resistant to the extractstested, with less than 21% of all extracts inhibiting thegrowth of either L. thalassiae or D. salina. In contrast,over half of all lipophylic extracts were active against thestramenopiles H. spinosa and S. aggregatum, and thebacterium P. bacteriolytica. Growth sensitivity tohydrophilic extracts varied considerably between indi-

vidual assay microorganisms. While 48% of all hydro-philic extracts were active against H. spinosa, 27% wereactive against P. bacteriolytica, and only 14% were ac-tive against S. aggregatum. Overall, more lipophilic ex-tracts inhibited microbial growth than hydrophilicextracts. The variability observed in the antimicrobialeffects of individual extracts against each assay micro-organism reflects the importance of choosing appropri-ate test microbes in assays from which ecologicallyrelevant information is sought. Results from this surveydemonstrate that antimicrobial activities are prevalentamong extracts from marine algae and seagrasses, sug-gesting that antimicrobial chemical defenses are wide-spread among marine plants.

Introduction

Extensive chemical investigations of extracts from mar-ine organisms have led to the discovery of a variety ofsecondary metabolites with antimicrobial activitiesagainst human pathogens (Rinehart et al. 1981; Reicheltand Borowitzka 1984; Pesando 1990). However, theseactivities provide little indication of the extent to whichthese compounds serve the host organism as defensesagainst harmful marine microorganisms (Paul 1992; Hay1996; Engel et al. 2002). While chemically mediateddisease resistance is well documented among terrestrialplants (Ingham 1972, 1973; Hammerschmidt 1999), littleis known about the antimicrobial functions of secondarymetabolites produced by marine plants. This lack ofknowledge is partly attributed to our limited under-standing of the etiology of marine diseases, as well as theconsiderable difficulties associated with developing eco-logically relevant experiments to address questionsregarding antimicrobial chemical defenses.

Our knowledge of marine pathogens comes primarilyfrom the aquaculture industry where periodic massmortalities coupled with large economic losses haveinitiated rigorous investigations to identify and control

Communicated by P.W. Sammarco, Chauvin

S. Engel Æ M. P. Puglisi Æ P. R. Jensen Æ W. Fenical (&)Center for Marine Biotechnology and Biomedicine,Scripps Institution of Oceanography, University of California,La Jolla, San Diego, CA, 92093-0204, USAE-mail: wfenical@ucsd.eduTel.: +1-858-5342133Fax: +1-858-5583702

Marine Biology (2006) 149: 991–1002DOI 10.1007/s00227-006-0264-x

the causative agents of a variety of marine infectiousdiseases (Andrews 1976, 1979; Sparks 1985; Correa1997; Muroga 2001). Examples include ‘Vibriosis’ inabalone caused by the bacteria of the genus Vibrio(Elston and Lockwood 1983), ‘lobster fusariumosis’caused by the ascomycete Fusarium cf. solanii (Lightnerand Fontaine 1975), and ‘Porphyra disease’ caused bythe oomycete Pythium marinum (Kazama 1979). There isa wealth of literature indicating that marine animals andplants are susceptible to infection by taxonomically di-verse microorganisms (Kohlmeyer and Kohlmeyer 1979;Sparks 1985; Porter 1986; Richardson 1998); however,the mechanisms of disease resistance in healthy marineplant populations are not well understood.

Although reports on disease epidemics in marinecommunities are rare compared to those from theaquaculture industry, periodic outbreaks of infectiousdiseases in marine plant and animal populations haveraised concerns regarding the health of the marine biota(Harvell et al. 1999, 2002; Martin et al. 2002). Amongthese marine diseases are fungal infections such as ‘raisindisease’ in Sargassum spp. (Andrews 1976), ‘Thalassiadisease’ in Thalassia testudinum (Porter 1986), which arecaused by Lindra thalassiae, and ‘aspergillosis’ inCaribbean sea fans, which is caused by Aspergillus syd-owii (Alker et al. 2001). Some zoosporic fungi (Stra-menopiles) are responsible for widespread epidemicssuch as ‘eelgrass wasting disease’ in Zostera marina,which is caused by Labyrinthula zosterae (Short et al.1987; Muehlstein et al. 1991). Known bacterial infec-tions include ‘coralline lethal orange disease’ (CLOD) incoralline algae, which is caused by an as yet undescribedbacterial pathogen (Littler and Littler 1995) and ‘redspot disease’ in the kelp Laminaria japonica, which iscaused by the marine bacterium Pseudoalteromonasbacteriolytica (Sawabe et al. 1998). While the etiology ofnew marine epidemics has been addressed, few studieshave examined the foundation of disease resistance inhealthy marine organisms. Given the large numbers ofsecondary metabolites that have been reported frommarine organisms (Blunt et al. 2003, 2004, 2005;Faulkner 2002 and previous reviews), it seems reason-able to hypothesize that biologically active secondarymetabolites have evolved to provide resistance to infec-tious diseases.

To date, only a handful of studies have providedevidence for antimicrobial chemical defenses amongmarine organisms. Studies have demonstrated that bac-teria associated with surface tissues of crustacean eggsproduce antifungal compounds that protect the eggsfrom fungal infection (Gil-Turnes et al. 1989; Gil-Turnesand Fenical 1992). Jensen et al. (1998) proposed thatthalassiolin (Fig. 1) protects the seagrass T. testudinumfrom infestation by the co-occurring thraustochytridSchizochytrium aggregatum. Studies investigating halo-genated furanones (Fig. 1) from the red alga Delisiapulchra demonstrated that these compounds inhibitbacterial settlement, attachment, and growth (Maximi-lien et al. 1998; Steinberg and de Nys 2002; Steinberg

et al. 2002). More recently, Kubanek et al. (2003) de-signed a fungal growth inhibition assay using two marinefungi, Dendryphiella salina and L. thalassiae, to guide theisolation of a potent antifungal metabolite, lobophoro-lide (Fig. 1), from extracts of the brown alga Lobophoravariegata. Using this same assay, Puglisi et al. (2004)characterized two antifungal metabolites, capisterones Aand B (Fig. 1) from extracts of the green alga Penicilluscapitatus. By demonstrating significant inhibitory effectsof purified compounds against ecologically relevantmarine microorganisms, these studies have provided thefirst line of evidence that secondary metabolites functionas antimicrobial chemical defenses.

In this study, we investigated the hypothesis thatmarine algae and seagrasses maintain antimicrobialchemical defenses. To address this hypothesis, weexamined antimicrobial activities of extracts from trop-ical Atlantic algae and seagrasses against a panel of well-characterized pathogenic and saprophytic marinemicroorganisms. This panel was composed of two fungi,two stramenopiles, and one bacterium, representingmembers of three microbial kingdoms that parasitizeand decompose marine plants. Three separate growthinhibition assays were used to assess the antimicrobialeffects of algal and seagrass extracts against the panelmicroorganisms. Given that the defensive secondarymetabolites can either be water-borne or tissue-bound,the antimicrobial effects of both hydrophilic (water-soluble) and lipophilic (ethyl acetate-soluble) extractswere tested. The results from this survey provide afoundation for future chemical investigations designedto identify and characterize the secondary metabolitesresponsible for antimicrobial activities in marine plants.

Materials and methods

Collection of algae and seagrasses

In June 2002, 31 species of green algae from nine families,8 species of red algae from six families, 10 species of brownalgae from two families, and 3 species of seagrasses fromtwo families (Table 1) were collected by SCUBA andsnorkeling in reef and mangrove habitats near the fol-lowing tropical Atlantic islands in the Bahamas: Swee-tings Cay (26�33.721¢N, 77�52.973¢W), Little SanSalvador (24�34.730¢N, 75�57.663¢W), Long Island(23�38.730¢N, 75�21.416¢W), San Salvador (24�01.121¢N,74�32.600¢W), Cat Island (24�10.810¢N, 75�33.296¢W),and Stirrup Cay (25�48.350¢N, 77�58.057¢W). All speciesof algae and seagrasses were identified by visual com-parison with the current literature (Littler and Littler2000).

Extraction of algae and seagrasses

For each of the 52 species collected, 10 ml of fresh tissuewas measured by volumetric displacement in a gradu-

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ated cylinder and individually extracted for 24 h in a 1:1solution of dichloromethane and methanol. The result-ing organic extract was filtered, reduced in vacuo, andpartitioned between ethyl acetate and water to separatelipophilic (ethyl acetate-soluble) from hydrophilic (wa-ter-soluble) constituents. All extracts were dried in va-cuo, weighed, and stored under nitrogen at �20�C untiltested.

Assay microorganisms

All extracts were screened for antimicrobial effectsagainst the following panel microorganisms:

Lindra thalassiae

This marine fungus belongs to the Ascomycota and hasbeen described as one of the most indiscriminatepathogens in the ocean, causing ‘raisin disease’ in thebrown algae Sargassum spp. and ‘Thalassia disease’ inthe seagrass T. testudinum (Andrews 1976; Porter 1986).The strain used in this study was purchased from theAmerican Type Culture Collection (ATCC 56663).

Dendryphiella salina

This marine fungus belongs to the Ascomycota and is acommon saprophyte on decomposing marine plants(Kohlmeyer and Kohlmeyer 1979). Being ubiquitous intropical oceans, this filamentous fungus has also beenisolated from cold waters of the Antarctic and Arctic

(Pugh and Jones 1986). The strain used in this study wasprovided by Professor D. Porter, University of Georgia.

Halophytophthora spinosa

This stramenopile (zoosporic fungus) belongs to theOomycota and is a common saprophyte on tropicalmarine plants (Porter 1986; Hyde et al. 1998). Halo-phytophthora species are considered as the primary col-onizers of submerged marine plants and the dominantmycelial decomposers in mangrove habitats (Nakagiri2002; Leano 2002). The strain used in this study wasprovided by Professor D. Porter, University of Georgia.

Schizochytrium aggregatum

This stramenopile (zoosporic fungus) belongs to theLabyrinthulomycota and is a common saprophyte andparasite on marine plants (Kohlmeyer and Kohlmeyer1979; Moss 1986; Hyde et al. 1998). S. aggregatum re-sides within the family Thraustochytriidae, which hasbeen shown to play important roles in biofilm formation(Raghukumar et al. 2000) and degradation processes inmarine environments (Bremer 1995; Leano 2002). Thestrain used in this study was provided by ProfessorD. Porter, University of Georgia.

Pseudoaltermonas bacteriolytica

This bacterium belongs within the gamma 3 subclass ofthe Proteobacteria and is the causative agent of red spotdisease in the kelp L. japonica (Sawabe et al. 1998). The

O

OH

HO

OSO3Na

O

HO

O

O

OHOH

HO O

O

R2

BrBr

R1

Furanones

2a, R1 = H, R2 = Br

2b, R1 = H, R2 = H

2c, R1 = OAc, R2 = H

2d, R1 = OH, R2 = H

O OMe

O

OMe

OMe

O

OMe

OHO

OOMe

OH

O

OH

H

NaO3SO OR

Thalassiolin1

Lobophorolide3

4a: R = COCH34b: R = H

Capisterones

Fig. 1 Secondary metabolites from marine algae that have been hypothesized to function as chemical defenses against harmful marinemicroorganisms

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strain used in this study was provided by ProfessorT. Sawabe, Hokkaido University.

Antifungal assay

The growth inhibition assay described by Kubanek et al.(2003) was used to assess the antifungal effects of

extracts against the ascomycetes L. thalassiae andD. salina, and the oomycete H. spinosa. Workingcultures of these three test strains were maintained onYPM P/S media (2 g yeast extract, 2 g peptone, 4 gD-mannitol, 16 g agar, 250 mg of both penicillin G andstreptomycin sulfate in 1 l natural seawater). All assayswere conducted in sterile 24-well microtiter plates withthree replicate treatment and respective solvent control

Table 1 Species of algae andseagrasses collected fromtropical Atlantic reefs andmangroves and screened forantimicrobial activity

Order Family Species

ChlorophytaBryopsidales Bryopsidaceae Bryopsis ramulosa

Caulerpaceae Caulerpa cupressoidesCaulerpa cupressoides var. lycopodiumCaulerpa cupressoides var. mamillosaCaulerpa fastigiataCaulerpa paspaloidesCaulerpa paspaloides var. laxaCaulerpa sertularioides

Udoteaceae Avrainvillea longicaulisBoodleopsis verticillataHalimeda copiosaHalimeda monileHalimeda opuntiaHalimeda tunaPenicillus capitatusPenicillus dumetosusPenicillus lamourouxiiRhipocephalus phoenixRhipocephalus phoenix f. brevifoliusUdotea cyathiformisUdotea flabellumUdotea looensisUdotea verticillosa

Cladophorales Anadyomenaceae Microdictyon boergeseniiMicrodictyon marinum

Cladophoraceae Cladophora proliferaSiphonocladaceae Dictyosphaeria cavernosaValoniaceae Valonia macrophysa

Dasycladales Dasycladaceae Batophora sp.Dasycladus vermicularis

Ulvales Ulvaceae Enteromorpha sp.

RhodophytaCeramiales Ceramiaceae Ceramium cimbricum

Rhodomelaceae Laurencia sp.Corallinales Corallinaceae Amphiroa brasiliana

Amphiroa rigidaGracilariales Gracilariaceae Gracilaria sp.

Gracilaria tikvahiaeRhodogorgonales Rhodogorgonaceae Rhodogorgon ramosissimaRhodymeniales Champiaceae Champia salicornioides

PhaeophytaDictyotales Dictyotaceae Dictyota cervicornis

Dictyota menstrualisLobophora variegata crust formLobophora variegata ruffled formPadina sanctae-crucisStypopodium zonale

Fucales Sargassaceae Sargassum filipendulaSargassum hystrixSargassum platycarpumSargassum pteropleuron

MagnoliophytaHydrocharitales Hydrocharitaceae Thalassia testudinumPotamogetonales Cymodoceaceae Halodule beaudettei

Syringodium filiforme

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wells. Treatments were prepared by homogenouslymixing measured amounts of extract dissolved in aminimal amount of methanol into warm (50�C) YPMmedia. The amount of extract added was such that thefinal concentration equaled the natural volumetric con-centration found in the sample (e.g., the amount of ex-tract obtained from 2 ml of algal material was mixedinto a final volume of 2 ml YPM media). Solvent con-trols were prepared by adding the amount of methanolused to dissolve the extracts for respective treatments.Subsequently, 400 ll aliquots of treatment and controlmedia were dispensed into each of three treatment andcontrol wells. Solidified media were inoculated using asterile needle to transfer a small amount of mycelium(ca. 1 mm2) from a working culture onto the center ofeach treatment and control well. All assay plates weresealed with Parafilm and incubated at 27�C. Fungalgrowth was monitored for 3 days and growth inhibitionwas assessed as the percentage mycelial coverage ontreatment wells relative to that on respective solventcontrol wells. Extracts were considered active if themean fungal growth was inhibited by more than 50%.

Disk-diffusion assay

Growth inhibition of S. aggregatum was assessed using adisk-diffusion assay described by Jensen et al. (1998).Working cultures of S. aggregatum were maintained onB1 media (2.5 g peptone, 1.5 g yeast extract, 16 g agar,3 ml 50% glycerol, in 1 l natural seawater). A sterilecotton-tipped applicator was used to transfer S. aggreg-atum cells from a working culture onto B1 assay plates.Treatment disks were prepared by adding 25 ll of extractat a natural volumetric concentration onto a 6 mm sterilepaper disk. Negative controls using 25 ll of solvent, andpositive controls using 25 ll of nystatin (100 lg ml�1)were prepared for each assay. All disks were allowed toair dry and were subsequently placed equidistantly ontothe surface of the inoculated assay plate and incubated at27�C. The inoculum was sufficient to produce confluentgrowth on the assay plates and growth inhibition wasassessed as the diameter of the zone of inhibited micro-bial growth (mm) minus the diameter of the paper disk(6 mm). Extracts were considered active if the calculatedzone of inhibition was greater than 5 mm.

Antibacterial assay

A bacterial growth inhibition assay described byKubanek et al. (2003) was used to assess the antibioticeffects of extracts against the pathogenic bacteriumP. bacteriolytica. A 24-h liquid culture of P. bacterioly-tica in B1 medium was diluted 1:160 with additionalsterile B1 media and 100 ll added to all the wells of asterile 96-well microtiter plate. Triplicate treatmentswere prepared by adding 100 ll of extract dissolved in aminimal amount of methanol and B1 media to the top

well in each row so that the final concentration wasequal to the natural volumetric concentration found inthe sample. Treatments were then serially diluted 1:1down the plate so that the volumetric IC50 could bedetermined. Negative solvent controls and positivecontrols containing vancomycin (250 lg ml�1) wereused in each assay. All assay plates were placed on ashaker table for 12 h and incubated at 27�C. The initial(t=0 h) and final (t=12 h) turbidity of the solutionswas determined with a spectrophotometer at 600 nmand growth inhibition was assessed as the percentagechange in turbidity in treatments relative to solventcontrols. Extracts were considered active if bacterialgrowth was inhibited by more than 50% and the volu-metric IC50 values were recorded for all active extracts.

Results

In total, the extracts from 49 species of marine algae andthree species of seagrasses collected from tropicalAtlantic reef and mangrove habitats (Table 1) werescreened for antimicrobial activity against five ecologi-cally relevant marine microorganisms. Overall, the ex-tracts from 90% of all species surveyed were activeagainst one or more assay microorganism, while extractsfrom 77% were active against two or more test strains.Broad-spectrum activity against three or four assay mi-crobes was observed in the extracts from 48 and 27% ofall species, respectively. The green algae Halimeda copi-osa and P. capitatuswere the only species to yield extractsactive against all assay microorganisms. The only speciesthat did not produce extracts active against any of thepanel microorganisms were the green algae Batophorasp., Boodleopsis verticulata, Champia salicornioides,Dasycladales vermicularis, and Dictyosphaeria cavernosa.

Among all assay microorganisms, both fungi wereresistant to more extracts than the stramenopiles or thebacterium (Fig. 2). Only 21% of all lipophilic and 19%of all hydrophilic extracts were active against L. thal-assiae, and 17 and 10%, respectively, were active againstD. salina (Fig. 2). In contrast, over 50% of all lipophilicextracts were active against both stramenopiles. While48% of all hydrophilic extracts were active against H.spinosa, only 14% were active against S. aggregatum(Fig. 2). Activity against the bacterium P. bacteriolyticawas observed in 58% of all lipophilic and 27% of allhydrophilic extracts (Fig. 2). Overall, antimicrobialactivity was more prevalent in lipophilic extracts butvaried considerably among assay microorganisms andbetween algal and seagrass species.

Antimicrobial activity of extracts from green algae(Chlorophyta)

Green algae were the most abundant in this study,comprising 60% of all species surveyed (Table 1).Overall, 40% of all green algae yielded extracts that were

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active against H. spinosa and P. bacteriolytica, and 35%yielded extracts that were active against S. aggregatum(Fig. 3a, Table 2). In comparison, the extracts from21% of all green algae were active against L. thalassiae,and only 15% yielded extracts active against D. salina(Fig. 3a). While most extracts from green algae wereactive against multiple assay microorganisms, a fewspecies yielded extracts that were only active against oneassay microorganism. For example, the extracts fromValonia macrophysa were only active against H. spinosa(Fig. 3a). Selective activity against S. aggregatum wasobserved in the extracts from Enteromorpha sp. andUdotea verticillosa (Table 2).

All other algae yielded extracts, which were activeagainst multiple assay microorganisms.

Among these, the extracts from H. copiosa andP. capitatus were the only to inhibit the growth of allassay microorganisms. Broad-spectrum activity againstthree or four assay microorganisms was observed in theextracts from Bryopsis ramulosa, Cladophora prolifera,Mycrodictyon boergesenii, and most species belonging tothe families Caulerpaceae and Udotaceae (Fig. 3a,Table 2).

Among those members of the family Caulerpaceae,antimicrobial activity was most prevalent in the extractsfrom Caulerpa fastigiata and C. paspaloides, inhibitingthe growth of L. thalassiae, H. spinosa, S. aggregatum,and P. bacteriolytica (Fig. 3a, Table 2). While theextracts from C. cupressoides var. lycopodium were activeagainst the fungus L. thalassiae and the stramenopileS. aggregatum, the extracts from all other species withinthe family Caulerpaceae were active against one or bothstramenopiles and the bacterium P. bacteriolytica(Fig. 3a, Table 2).

Among the family Udotaceae, five of the six repre-sentative genera contained species that yielded extractswith broad-spectrum activity against fungi, strameno-piles, and the bacterium. For example, the extracts fromAvrainvillea longicaulis, Udotea flabellum, and U. looen-sis were active against L. thalassiae, H. spinosa, S. ag-gregatum and P. bacteriolytica. Similarly, the extracts

from Rhipocephalus phoenix f. brevifolius were activeagainst D. salina, both stramenopiles, and P. bacteri-olytica. The extracts from all species of Halimeda andPenicillus were active against multiple assay microor-ganisms (Fig. 3a, Table 2).

Antimicrobial activity of extracts from red algae(Rhodophyta)

Red algae comprised 15% of all species surveyed in thisstudy (Table 1). Overall, 63% of all red algae yieldedextracts that were active against H. spinosa, while 38%yielded extracts with activity against L. thalassiae,S. aggregatum, and P. bacteriolytica (Fig. 3b, Table 2).In comparison, only 13% of all red algae yielded ex-tracts that were active against D. salina (Fig. 3b).Selective activity was only observed in the lipophilicextract from Amphiroa rigida against S. aggregatum, andin the hydrophilic extract from Rhodogorgon ramosiss-ima against P. bacteriolytica (Table 2). All other extractsfrom red algae were active against multiple assaymicroorganisms.

Among all extracts with broad-spectrum activity, theextracts from Gracilaria sp. were the most active,inhibiting the growth of L. thalassiae, D. salina,H. spinosa, and P. bacteriolytica (Fig. 3b, Table 2). Incomparison, extracts from the closely related speciesG. tikvahiae were only active against the stramenopiles.While the extracts from Laurencia sp. lacked antifungalactivity, they inhibited the growth of both stramenopilesand P. bacteriolytica. Extracts from Ceramium cimbri-cum and Amphiroa brasiliana were active againstL. thalassiae and H. spinosa (Fig. 3b).

Antimicrobial activity of extracts from brown algae(Phaeophyta)

Brown algae comprised 19% of all species surveyed(Table 1). Overall, 80% of all brown algae yielded

0

20

40

60

80

L. thalassiae D. salina H. spinosa S. aggregatum P. bacteriolytica

Assay Microorganism

% A

ctiv

e E

xtra

cts

EtOAc

WaterN = 52

Fig. 2 Percentage of lipophilic(EtOAc) and hydrophilic(water) extracts from allsamples (N=52) inhibitingmicrobial growth by more than50% for each assaymicroorganism

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0 25 50 75 1000 25 50 75 1000 25 50 75 100

Valonia macrophysaUdotea looensis

Udotea flabellumUdotea cyathiformis

R. phoenix f. brevifoliusRhipocephalus phoenix

Penicillus lamourouxiiPenicillus dumetosus

Penicillus capitatusMicrodyction marinum

Microdyction boergesenii

Halimeda tunaHalimeda opuntia

Halimeda monileHalimeda copiosa

Enteromorpha sp.Cladophora prolifera

Caulerpa sertularioidesC. paspaloides var. laxa

Caulerpa paspaloidesCaulerpa fastigiata

C. cupressoides var. m.C. cupressoides var. l.

Caulerpa cupressoidesBryopsis ramulosa

Avrainvillea longicaulis

Chlorophyta

Lindra thalassiae Dendryphiella salina Halophytophthora spinosa

% Growth Inhibition

0 25 50 75 1000 25 50 75 1000 25 50 75 100

Thalassia testudinumSyringodium filiforme

Halodule beaudettei

Stypopodium zonale

Sargassum pteropleuronSargassum platycarpum

Sargassum hystrixSargassum filipendula

Padina sanctae-crucis

Lobophora variegata r.Lobophora variegata c.

Dictyota menstrualisDictyota cervicornis

Rhodogorgon ramosissima

Laurencia sp.Gracilaria sp.

Gracilaria tikvahiaeChampia salicornioides

Ceramium cimbricumAmphiroa rigida

Amphiroa brasiliana

Lindra thalassiae Dendryphiella salina Halophytophthora spinosa

Rhodophyta

Phaeophyta

Magnoliophyta

% Growth Inhibition

a

b

Fig. 3 Antifungal activity of lipophilic (black bars) and hydrophilic (white bars) extracts from samples active against the ascomycetes,Lindra thalassiae and Dendryphiella salina, and the oomycete, Halophytophthora spinosa. a Chlorophyta, b Rhodophyta, Phaeophyta, andMagnoliophyta

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extracts that were active against H. spinosa and P. bac-teriolytica, and 50% yielded extracts that were activeagainst S. aggregatum (Fig. 3b, Table 2). In comparison,extracts from 40% of all brown algae were active againstL. thalassiae, and only 30% yielded extracts activeagainst D. salina (Fig. 3b). While the lipophilic extractfrom Dictyota menstrualis was selectively active againstS. aggregatum, all other extracts from species of thefamilies Dictyotaceae and Sargassaceae were activeagainst multiple assay microorganisms.

Among the family Dictyotaceae, extracts fromL. variegata (crust form) and Stypopodium zonale werethe most active, inhibiting the growth of one fungus,both stramenopiles, and the bacterium (Fig 3b,

Table 2). Similarly, the extracts from Padina sanctae-crucis were active against L. thalassiae, H. spinosa, andP. bacteriolytica. While the extracts from L. variegata(ruffled form) were active against P. bacteriolytica andboth stramenopiles, the extracts from Dictyotacervicornis were active against both fungi and thebacterium (Fig 3b, Table 2). While none of the ex-tracts from any species within the family Sargassaceaewere active against L. thalassiae, extracts from allspecies inhibited the growth of H. spinosa (Fig. 3b).Among these, the extracts from S. hystrix were themost active, inhibiting the growth of D. salina,H. spinosa, S. aggregatum, and P. bacteriolytica(Fig. 3b, Table 2).

Table 2 Antimicrobial activityof extracts active againstSchizochytrium aggregatum(zone of inhibition in mm) andPseudoalteromonasbacteriolytica (volumetricIC50 = Fraction of naturalvolumetric concentrationyielding 50% growth inhibition)

Species Schizochytriumaggregatum

Pseudoalteromonasbacteriolytica

Lipophilic Hydrophilic Lipophilic Hydrophilic

ChlorophytaAvrainvillea longicaulis 10 6 0.13 –Bryopsis ramulosa – – 0.37 1Caulerpa cupressoides 12 8 0.25 –Caulerpa cupressoides var. lycopodium 9 6 – –Caulerpa cupressoides var. mamillosa – – 0.58 –Caulerpa fastigiata 14 – 0.43 –Caulerpa paspaloides 10 – 0.18 –Caulerpa paspaloides var. laxa 9 – 0.25 –Caulerpa sertularioides – – 0.13 –Cladophora prolifera 9 – 0.45 0.40Enteromorpha sp. 6 – – –Halimeda copiosa 12 – 0.50 –Halimeda monile – – 0.71 1Halimeda opuntia 6 – 0.29 –Microdictyon boergesenii – – – 0.46Microdictyon marinum 13 9 0.51 –Penicillus capitatus 6 – 0.35 –Penicillus dumetosus – – 0.60 –Penicillus lamourouxii – – 0.34 –Rhipocephalus phoenix 10 – 0.54 –Rhipocephalus phoenix f. brevifolius 9 – 0.49 –Udota cyathiformis 7 – – –Udota flabellum 15 – 0.56 –Udota looensis 10 – 0.52 –Udotea verticillosa 6 – – –

RhodophytaAmphiroa rigida 13 – – –Champia salicornis 6 – – –Gracilaria sp. – – 0.34 0.63Gracilaria tikvahiae 8 – – –Laurencia sp. 14 – 0.47 –Rhodogorgon ramosissima – – – 1

PhaeophytaDictyota cervicornis – – 0.40 –Dictyota menstrualis 12 – – –Lobophora variegata crust form 10 8 1 0.51Lobophora variegata ruffled form 7 – 0.24 0.67Padina santae-crucis – – 0.83 0.66Sargassum filipendula – – – 1Sargassum histrix 15 8 – 1Sargassum palycarpum – – 0.60 1Stypopodium zonale 9 11 0.43 –

MagnoliophytaHalodule beaudetti 11 – 0.50 0.55Syringodium filiforme 8 – 0.40 0.67

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Antimicrobial activity of extracts from seagrasses(Magnoliophyta)

Halodule beaudettei, Syringodium filiforme, and T. te-studinum were the only species of seagrasses collected inthis survey (Table 1). While none of the extracts fromthese seagrasses were active against fungi, they were allactive against stramenopiles (Fig. 3b, Table 2). Amongthese, the extracts from H. beaudettei were the mostactive, inhibiting the growth of H. spinosa, S. aggrega-tum, and P. bacteriolytica. The extracts from S. filiformewere active against S. aggregatum and P. bacteriolytica(Table 2), while the extracts from T. testudinum wereonly active against H. spinosa (Fig. 3b).

Discussion and conclusions

Over the last 50 years, numerous studies have reportedon the antimicrobial activities of extracts from marineplants against biomedically important microorganisms(Pratt et al. 1951; Burkholder et al. 1960; Sieburth 1964;Hornsey and Hide 1974; Caccamese et al. 1980, 1981,1985; Rao and Parekh 1981; Rinehart et al. 1981; Rei-chelt and Borowitzka 1984; Ballantine et al. 1987; Bal-lesteros et al. 1992; Vlachos et al. 1996). Subsequentchemical investigations of bioactive extracts led to thediscovery of many structurally diverse antimicrobialmetabolites from marine plants (Blunt et al. 2003, 2004,2005; Faulkner 2002, and previous reviews). Whilemarine plant metabolites have been studied extensivelyfor their biomedical potential (see references above),their activities against human pathogens provide littleinformation about their ecological role in antimicrobialchemical defenses.

From an ecological perspective, antimicrobial de-fenses of marine plants may reduce fouling, inhibitpremature decomposition, or directly provide resistanceto infectious diseases. To date, most studies haveinvestigated antimicrobial defenses with regard to theprevention of biofouling. Sieburth and Conover (1965)were among the first to demonstrate that phlorotanninsfrom two Sargassum sp. inhibit two species of foulingbacteria. Subsequent studies have continued to showthat the extracts and purified metabolites from marineplants can inhibit microorganisms associated with bio-film formation (Devi et al. 1997; Konig and Wright1997; Hellio et al. 2000, 2001, 2004; Steinberg and deNys 2002; Da Gama et al. 2002; Echigoya et al. 2005).While these studies have shown that the antimicrobialchemical defenses of marine plants can reduce fouling,their roles in resisting decomposition and infection re-main elusive. In this study, we explored the antimicro-bial effects of extracts from marine plants against plantsaprophytes, parasites, and pathogens by systematicallyscreening their extracts for inhibitory activities against acarefully chosen panel of well-described marine micro-organisms. This panel included two known phytopath-ogens and three saprophytes that have been described to

parasitize and decompose marine plants (Andrews 1976;Kohlmeyer and Kohlmeyer 1979; Moss 1986; Porter1986; Bremer 1995; Sawabe et al. 1998; Hyde et al. 1998;Nakagiri 2002; Leano 2002).

The results from this survey clearly demonstrate thatalgal and seagrass extracts, tested at natural volumetricconcentrations, can inhibit the growth of harmful mar-ine microorganisms. Nearly all of the species surveyed(90%) yielded extracts that were active against one ormore assay microorganisms. Further, over 50% of alllipophilic extracts were active against the parasiticstramenopiles H. spinosa and S. aggregatum, and thepathogenic bacterium P. bacteriolytica. Similarly, stud-ies investigating antifouling effects of marine plant ex-tracts reported that over 50% of all extracts were activeagainst one or more fouling microbes (Devi et al. 1997;Hellio et al. 2000, 2001). This high degree of antimi-crobial activity among extracts from marine plants isunprecedented when compared to the relatively lowpercentages of active extracts reported in surveys testingtheir effects against biomedically important microor-ganisms. For example, a large-scale screening programtested over 500 extracts from 159 species of marineplants against 12 human pathogens and reported thatonly 36% of all extracts showed antimicrobial activity(Reichelt and Borowitzka 1984). Given that this level ofactivity is consistent with the results of other biomedicalsurveys (Burkholder et al. 1960; Caccamese et al. 1980;Rao and Parekh 1981; Ballesteros et al. 1992), the rela-tively high percentage of active extracts observed in thisand other ecological studies suggest that the antimicro-bial metabolites may selectively target marine microor-ganisms and therefore may not be active against humanpathogens. Further, the prevalence of extracts withantimicrobial activity against phytopathogens, parasites,saprophytes, and fouling microbes indicates that marineplants have adapted to selective pressures from harmfulmicroorganisms by maintaining chemical defenses thattarget ecologically relevant microbes.

The results of this survey did not show taxonomictrends in the activities of the lipophilic or hydrophilicextracts against the panel microorganisms. Antimicro-bial activities of extracts varied considerably among allalgal and seagrass species, and between assay microor-ganisms (Table 2, Fig. 3a, b), suggesting that microbialgrowth inhibition is mediated by a variety of antimi-crobial metabolites. Overall, fewer extracts were activeagainst fungi than stramenopiles or the bacterium(Fig. 2). In addition, more lipophilic than hydrophilicextracts inhibited the growth of all assay microbes(Fig. 2) indicating that lipophilic secondary metabolitesmay play a critical role in defending host tissues fromharmful microorganisms.

While the data presented in this survey provide evi-dence to support the hypothesis that marine plantsmaintain antimicrobial chemical defenses, caution mustbe exercised about drawing early conclusions about therole of secondary metabolites in the observed activities.Crude organic extracts are complex mixtures of primary

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and secondary metabolites, including fatty acids that canexhibit antimicrobial activities. Further, given that mostmarine plants contain epi- and endophytic microorgan-isms, it is not possible to rule out that microbialmetabolites may contribute to the overall activity of theextracts tested. In addition, it has been demonstratedthat extraction procedures and the method of storagecan significantly alter the composition of a crude extractand may result in false negative or positive assay results(Cronin et al. 1995). Thus, we stress that the isolationand characterization of secondary metabolites respon-sible for the observed antimicrobial activities is essentialbefore it will be possible to understand their biosyntheticorigin and hypothesize on their adaptive role in anti-microbial defenses.

Few studies have investigated the antimicrobialfunctions of purified metabolites from marine algae andseagrasses. Our examination of extracts from the sea-grass T. testudinum demonstrated the presence of anantibiotic flavone glycoside, thalassiolin (Fig. 1), inhibitsthe growth of S. aggregatum at 1/10th of the naturalvolumetric concentration (Jensen et al. 1998). Lowabundances of S. aggregatum on the surfaces of healthyseagrass blades suggested that thalassiolin is an effectivedefense against decomposition by this saprophyte. Al-though the extracts from T. testudinum screened in thissurvey were not active against S. aggregatum, thehydrophilic extract selectively inhibited the growth ofH. spinosa (Fig. 3b). One explanation for this discrep-ancy is that the antimicrobial chemical defenses ofT. testudinum are not constitutive but, rather, are in-duced by the presence of specific microorganisms. Fur-ther, it is possible that T. testudinum populations haveadapted to specific microbial pressures, resulting inpopulation-specific antimicrobial defenses.

Of the many active extracts identified in this survey,those from the brown alga L. variegata and the greenalga P. capitatus have thus far been chosen for furtherstudy. Using a bioassay-guided approach, chemicalinvestigations of extracts from L. variegata resulted inthe isolation of a 22-membered cyclic lactone, lobop-horolide (Fig. 1) that appears to provide resistanceagainst fungal infection (Kubanek et al. 2003). Surfaceextractions demonstrated that lobophorolide is presenton the algal surface at concentrations sufficient to inhibitthe growth of the saprophyte D. salina and the pathogenL. thalassiae (Kubanek et al. 2003). In the second study,bioassay-guided fractionation of the extracts fromP. capitatus resulted in the isolation of two potentantifungal triterpene sulfate esters, capisterones A and B(Fig. 1) (Puglisi et al. 2004). Lobophorolide and capis-terones exhibit antifungal activities at concentrationswell below those found in the host alga, suggesting thatthese metabolites may function as effective antimicrobialchemical defenses.

The results of this survey have shown that antimi-crobial activities are prevalent and widespread amongextracts from marine plants. Further, Kubanek et al.(2003) and Puglisi et al. (2004) demonstrated that these

activities could be traced to specific secondary metabo-lites. Thus, using ecologically relevant microbes in as-says such as those that led to the isolation of thalassolin(Jensen et al. 1998), lobophorolide (Kubanek et al.2003), and capisterones (Puglisi et al. 2004) provides auseful approach for the discovery of novel antimicrobialmetabolites from marine plants. The extent to whichisolated metabolites function as antimicrobial chemicaldefenses in the host plant can then be further examinedby correlating antimicrobial activities with in vivometabolite concentrations and levels of microbial infes-tation. It will also be important to address the locationof antimicrobial metabolites within the host tissues andtheir geographic distribution among different host pop-ulations to develop a better understanding of chemicallymediated disease resistance in marine plants.

Acknowledgements We thank Professor Joseph R. Pawlik, Uni-versity of North Carolina at Wilmington, for his generous invita-tion to participate in his research expedition aboard the R/VSeward Johnson (NSF OCE97-11255 and OCE00-95724). We alsothank the government of the Bahamas for allowing us to conductresearch in their territorial waters. We thank ProfessorsE. B. Gareth Jones, David Porter, and Tomoo Sawabe for pro-viding assay microorganisms. We thank Chris Kauffman, SaraKelly, Tatum Neely, Allan Spyere, Tracy Mincer, Craig Fairchild,and Micha Ilan for their assistance. This research was supported bya grant from the National Science Foundation NSF grant CHE01-11370 to WF and conducted in accordance with the current envi-ronmental regulations of the Bahamas.

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