Halogenated metabolites with antibacterial activity fromthe Okinawan Laurencia species

Charles Santhanaraju Vairappana,1, Minoru Suzukia,*, Tsuyoshi Abeb, Michio Masudac

aDivision of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, JapanbThe Hokkaido University Museum, Sapporo 060-0810, Japan

cDivision of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan

Received 4 December 2000; received in revised form 19 April 2001

Abstract

The chemical compositions of ve species of the red algal genus Laurencia from coastal waters of Okinawa Prefecture, Japan, havebeen investigated. A halogenated C15 acetogenin, (12E)-lembyne-A, was isolated fromL. mariannensis, and a halogenated sesquiterpene,(6R,9R,10S)-10-bromo-9-hydroxy-chamigra-2,7(14)-diene, was rst found from L. majuscula as a naturally occurring compound.Laurencia nidica yielded previously known laurinterol and isolaurinterol. Samples of L. cartilaginea and L. concreta aorded no

halogenated metabolites. The structures of these halogenated metabolites as well as their antibacterial activity against some marinebacteria are reported. # 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Laurencia; Rhodomelaceae; Red alga; Sesquiterpene; C15 acetogenin; Halogenated compound; Chemotaxonomy; Antibacterial activity

1. Introduction

The red algal genus Laurencia is unique due to itsability to produce a wide variety of halogenated second-ary metabolites with diverse structural features depend-ing on the species and localities. Although the roles ofthese halogenated compounds are still a matter of spec-ulation, their importance as chemical defense substancesagainst marine herbivores is undeniable (Hay et al.,1987; Kurata et al., 1998).Some halogenated metabolites have been shown to

possess antibacterial activities against terrestrial bacteria(Waraszkiewicz and Erickson, 1974; Wratten and Faul-kner, 1977; Carter et al., 1978; Caccamese et al., 1980;Konig and Wright, 1997a). More recently, we reportedon the antibacterial potential of halogenated com-pounds isolated from the Malaysian Laurencia againstmarine bacteria from the algal habitat (Vairappan et al.,2001), suggesting the possible role played by thesehalogenated compounds as the seaweeds chemicaldefense substances.

As part of our continuing study on the chemical com-positions of Laurencia species from the Okinawanwaters, we harvested ve species of Laurencia; L. mar-iannensis Yamada, L. majuscula (Harvey) Lucas, L. nidi-ca J. Agardh, L. cartilaginea Yamada, and L. concretaCribb from four dierent sites. Laurencia mariannensiscontained a new C15 acetogenin, (12E)-lembyne-A (1),along with 2-bromo-3-chloro-5-acetoxy-chamigra-7(14),9-dien-8-one (2) that was previously isolated from theOkinawan sea hare Aplysia dactylomela (Sakai et al.,1986) and from the Okinawan L. majuscula collected atTaketomi (Masuda et al., 1997). Laurencia majusculacontained a brominated sesquiterpene, (6R,9R,10S)-10-bromo-9-hydroxy-chamigra-2,7(14)-diene (3), togetherwith previously known (Z)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (4) and (E)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (5) from thisspecies collected at Kochi Prefecture, Japan (Suzuki andKurosawa, 1978; Suzuki et al., 1979). Metabolite 3 hasnot been previously found as a natural source. Further-more, L. nidica produced laurinterol (6) and iso-laurinterol (7), characteristic metabolites of the JapaneseL. okamurae Yamada (Suzuki and Kurosawa, 1979),while L. cartilaginea and L. concreta produced no halo-genated secondary metabolites.We report herein the chemical compositions of these

Laurencia species, structures of halogenated compounds,

0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.PI I : S0031-9422(01 )00260-6

Phytochemistry 58 (2001) 517523

www.elsevier.com/locate/phytochem

* Corresponding author. Tel.: +81-11-706-2272; fax: +81-11-706-

4862.

E-mail address: misu@ees.hokudai.ac.jp (M. Suzuki).1 Present address: Borneo Marine Research Institute, University

Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia.

and their antibacterial activity against marine bacteriaisolated from the algal habitat.

2. Results and discussion

2.1. Laurencia mariannensis

A sample collected from Teruma, Yonashiro, wasextracted with methanol. The methanol extract was frac-tionated by CC over silica gel with a stepped gradient(hexane and EtOAc). The fraction eluted with hexaneEtOAc (9:1) was further subjected to prep. TLC withhexaneEtOAc (3:1) to give 2-bromo-3-chloro-5-acet-oxy-chamigra-7(14),9-dien-8-one (2) (Sakai et al., 1986;Masuda et al., 1997) in 14% yield based on the weightof the extracts. The fraction eluted with hexaneEtOAc(4:1) was also separated using prep. TLC with hexaneEtOAc (3:1) to give compound 1 (6%).Compound 1, oil, 24D +42 (CHCl3), was assigned the

molecular formula C15H16BrClO2 (HRFDMS). Its IRspectrum revealed absorptions due to a terminal acet-ylenic group (vmax 3310 cm

1), a vinyl ether stretching(vmax 1680 cm

1), and CO bands (vmax 1110 and 1050cm1). In addition, since there were no hydroxyl orcarbonyl absorptions, both oxygens were suggested tobe involved in ether links. The presence of a terminalconjugated enyne moiety, which is frequently encoun-tered in Laurencia C15 acetogenins, was evident fromthe 1H NMR spectrum (Table 1) [H 3.31 (1H, br s),5.58 (1H, dd, J=10.7, 1.5 Hz) and 6.06 (1H, dd,J=10.7, 10.7 Hz)]. Interpretation of the 1H1H COSYspectrum showed the presence of a partial structure 1a

(Fig. 1) together with an isolated ethyl group. The J-value (10.7 Hz) between H-3 and H-4 as well as thechemical shift value (H 3.31) of the acetylenic protonindicated the geometry of the double bond at C-3 to beZ (Suzuki and Kurosawa, 1987).The 13C NMR spectrum (Table 1) revealed the pre-

sence of 14 carbon atoms, 12 of which were hydrogen-bearing carbons. Among 14 carbon atoms, three oxy-gen-bearing carbons were demonstrated by the signalsat C 77.9, 81.5 and 84.2 and further attributed to C-7,C-9 and C-10, respectively, by HSQC spectrum. Fur-thermore, based on the chemical shift values of H-5 (H4.72) and C-5 (C 54.9), a halogen atom was deduced tobe attached to C-5 position. The planar formula forcompound 1 could be assigned by the HMBC spectrumwhose results are summarized in Table 1. In the HMBCspectrum, cross peaks of C-7/H-10 and C-10/H-7established the ether ring closure between C-7 and C-10.In addition, a cross peak between the terminal methylgroup and the vinyl carbon at C 105.8 indicated thatthe ethyl group was attached to the tetrasubstituteddouble bond. The chemical shift value (C 105.8) of thisvinyl carbon atom suggested that another substituent isa halogen atom (Suzuki et al., 1983). Therefore, thisdouble bond had to be inserted between C-9 and C-11 togive a planar formula 1b or 1c. Facile loss of a chlorineatom on the FDMS is evident from the fragment ion ofm/z 309, 307 [M-Cl]+ which strongly suggested the presenceof a vinyl bromide moiety in 1. Hence the chlorine atomwas linked to C-5 and the bromine atom to C-13.The 1H and 13C NMR spectral data of 1 were very

similar to those of lembyne-A (10), which has recentlybeen isolated from the Malaysian Laurencia sp. (Vair-appan et al., 2001). The chemical shifts and couplingconstants of the protons fromC-1 to C-10 in the 1HNMRspectrum of 1 are almost identical to those of 10. However,

Table 113C NMR (100 MHz, DEPT), 1H NMR (400 MHz), and HMBC

spectral dataa for 1

Cb 13C

()

1H

()Multiplicity,

J (Hz)

HMBC

correlations

1 78.7 3.31 br s

2 85.8

3 110.9 5.58 dd, J=10.7, 1.5 H-1, H-4, H-5

4 142.2 6.06 dd, J=10.7, 10.7 H-3, H-5

5 54.9 4.72 dd, J=10.7, 10.7 H-3, H-4, H-7

6 54.2 2.74 ddd, J=10.7, 9.3, 4.4 H-4, H-5, H-7, H-10, H-11

7 77.9 4.29 dd, J=4.4, 4.4 H-6, H-9, H-10, H-11

8 36.8 1.92 d, J=13.6 H-6, H-7, H-10

1.71 m

9 81.5 4.67 dd, J=6.8, 4.9 H-7, H-10, H-11

10 84.2 5.13 dd, J=4.9, 4.9 H-7, H-9, H-11

11 45.2 3.46 dd, J=9.3, 4.9 H-5, H-6, H-7, H-9, H-10

12

13 105.8 H2-14, H3-15

14 28.1 2.64 dq, J=14.6, 7.3 H3-15

2.38 dq, J=14.6, 7.3

15 13.8 1.10 dd, J=7.3, 7.3 H2-14

a Measured in chloroform-d1.b Assignments were made with the aid of the HSQC spectrum.

Fig. 1. Partial and planar structures for 1.

518 C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523

distinct dierences are observed in the chemical shiftvalues of H-11 and H2-14. The low-eld chemical shiftof the H-11 in 1 is due to the deshielding being causedby the bromine atom at C-13 that is situated close to theH-11. Furthermore, the large magnetic nonequivalence ofthe H2-14 in 1 is attributed to the electronic inuence ofthe oxygen atom that is cis to the ethyl group. Thus thestereochemistry of the vinyl ether moiety in 1 is estab-lished as E-conguration. In consequence, the structure ofcompound 1 must be represented by formula 1, (12E)-lembyne-A, including the same relative stereochemistry,5S*, 6R*, 7R*, 9R*, 10R*, and 11S*, as lembyne-A (10).(12E)-Lembyne-A (1) and lembyne-A (10) have struc-

tures very similar to cis-maneonene-C (100) and (12Z)-cis-maneonene-C (1000), respectively, the latter of whichwas obtained from cis-maneonene-C by treatment withp-toluenesulfonic acid (Waraszkiewicz et al., 1978).However, there are some obvious dierences between our1H and 13C NMR spectral data (in C6D6) and the pub-lished data (Waraszkiewicz et al., 1978) along with theiroptical rotations (1: [a]D +42; 10: [a]D +198, 1b00: [a]D+336, 1000: [a]D +137 C). Thus cis-maneonene-C and(12Z)-cis-maneonene-C may be a stereoisomer at C-5 of(12E)-lembyne-A and lembyne-A, respectively.

2.2. Laurencia majuscula

The methanol extract of a sample collected fromYagachi, Nago, was subjected to a combination of col-umn and thick-layer chromatography to give 10-bromo-9-hydroxy-chamigra-2,7(14)-diene (3) (9%) along withpreviously known (Z)-10,15-dibromo-9-hydroxy-chami-gra-1,3(15),7(14)-triene (4) (3%) and (E)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (5) (4%), iso-lated from this species which had been collected atKochi Prefecture, Japan (Suzuki and Kurosawa, 1978;Suzuki et al., 1979).Compound 3, oil, C15H23OBr, 25D 110 (CHCl3),

was readily assigned the planar structure by its 1D and2D (1H1H COSY, HSQC, and HMBC) NMR spectra.The relative conguration was determined by theNOESY spectrum. A NOE between H-10 and Hb-5showed that the H-10 adopts an axial conguration andhence the bromine atom is equatorial. Since based onthe J-value (J9,10=2.9 Hz), the vicinal bromine andhydroxyl groups are in cis-conguration as in the caseof compounds 4 and 5, the C-9 hydroxyl group is axial.Furthermore, NOEs between Ha-14/Ha-1 and H3-13(one of gem-dimethyl)/Ha-4 also conrmed the stereo-chemistry of the spiro carbon at C-6. Thus the relativestereochemistry of 3 would be represented by formula3a as shown in Fig. 2.A literature survey indicated that the 1H and 13C

NMR spectral data of 3 were almost identical withthose of deschloroelatol (30), which has previouslybeen isolated from Laurencia obtusa (Kennedy et al.,1988) and L. rigida (Konig and Wright, 1997b). How-ever, judging from the signs of the optical rotations,compound 3 must be an enantiomer of deschloroelatol.Compound 3 had previously been obtained fromobtusol as the partially dehalogenated compound bytreatment with lithium aluminum hydride in ether(Gonzalez et al., 1976). Co-occurrence of compound 3with halochamigrenes 4 and 5 in the same alga alsosuggested that the absolute congurations of chiralcenters at C-6, C-9, and C-10 are the same as those of 4and 5.

Fig. 2. Stereochemistry of 10-bromo-9-hydroxy-chamigra-2,7(14)-diene (3).

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523 519

Accordingly, the structure of compound 3 should be(6R,9R,10S) -10 -bromo-9-hydroxy-chamigra -2,7(14) -diene, which has not previously been found as a naturallyoccurring compound.The dierence of the chemical compositions in the

samples from Yagachi, Nago (present study) and Take-tomi island (Masuda et al., 1997) suggests that chemicalraces (Abe et al., 1999) are also present in Laurenciamajuscula.

2.3. Laurencia nidica

The methanol extract of a sample collected fromToyohara, Nago, gave laurinterol (6) (22%) and iso-laurinterol (7) (4%) (Suzuki and Kurosawa, 1979). Thechemical composition of Laurencia nidica collected attwo sites (Teruma and Toyohara) is dierent from thatof a sample harvested from Goza, Mie Prefecture, Japan(Shizuri et al., 1984); it is, however, rather similar to thatof a Hawaiian sample (a clumpy pink variety) from Oahuisland (Waraszkiewicz and Erickson, 1974, 1975).

2.4. Laurencia cartilaginea and Laurencia concreta

No halogenated secondary metabolites were found inboth extracts of Laurencia cartilaginea and L. concreta.As reported in a previous paper (Suzuki et al., 1987), a

sample of L. cartilaginea collected at Sakurajima,Kagoshima Prefecture in Japan, close to the type locality,Moji, Fukuoka Prefecture, also showed the absence ofhalogenated compounds in its extract. Both Laurenciaspecies have no corps en cerise (our unpublishedobservations). Corps en cerise are considered to bethe sites of synthesis and/or storage for the halogenatedsecondary metabolites of Laurencia (Young et al.,1980). Although the Hawaiian L. cartilaginea wasreported to produce halogenated chamigrane-type ses-quiterpenes (Juagdan et al., 1997), the identication oftheir material seems to be questionable.

2.5. Antibacterial activity

The antibacterial activities of the isolated halogenatedmetabolites of Okinawan Laurencia were tested againsteight species of marine bacteria. These were Alcaligenesaquamarinus, Alteromonas sp., Azomonas agilis, Azoto-bacter beijerinckii, Erwinia amylovora, Escherichia coli,Halobacterium sp., and Halococcus sp. All these bacteriawere Gram-negative strains. The results of the paperdisc diusion assays are shown in Table 2. Prominentantibacterial activities were seen in compounds 1, 3, 6and 7, while 4 and 5 only showed very marginal activity.Compound 2 was inactive towards the bacteria tested.Compound 1 showed activity against Alcaligenes aqua-marinus, Azomonas agilis, Erwinia amylovora, andEscherichia coli. Their minimum inhibitory concentra-tion (MIC) values are in the range of 2030 mg/disc asshown in Table 3. Compound 3 showed antibacterialactivity against ve species of bacteria, Alcaligenes aqua-marinus, Azomonas agilis, Azotobacter beijerinckii, Erwi-nia amylovora, and Escherichia coli. Its MIC values werein the range of 1030 mg/disc (Table 3). Compounds 6and 7 showed better antibacterial activity than com-pounds 1 and 3. Their antibacterial activities and respec-tive MIC values are shown in Tables 2 and 3, respectively.

Table 2

Antibacterial activity of the halogenated compounds against marine

bacteria isolated from algal habitats in the Japanese coastal waters

Test bacteria Compoundsa

1 3 6 7

Alcaligenes aquamarinus ++ ++

Alteromonas sp. ++ +++

Azomonas agilis ++ ++ +++ +++

Azobacter beijerinckii ++ ++

Erwinia amylovora ++ ++ +++ +++

Escherichia coli ++ ++ ++ +++

Halobacterium sp.

Halococcus sp.

a Inhibition zone diameter; +++: 1924 mm, ++: 1218 mm, +:

712 mm, : no inhibition. Concentration: 90 mg/disc.

520 C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523

The possibility that these halogenated compounds areinvolved in protecting these Laurencia species againstbacterial intrusion is real when we look at the distributionof these compounds. Basically, halogenated compoundsare reported to be contained in corps en cercise (Younget al., 1980), an unusually swollen refractile inclusion,that are located in the outer cortical layer and tricho-blast cells of Laurencia. Corps en cercise are absent inthe inner cortical layers, and their specic distributionpattern could be suggestive of their importance as bar-ricades to withhold any bacterial intrusion.

3. Experimental

3.1. General

1H (400 MHz) and 13C NMR (100 MHz); CDCl3 orC6D6, TMS as int. standard (coupling constant, J in Hz);low and high resolution MS: 70 eV; optical rotations:CHCl3; CC: silica gel (Merck, Kieselgel 60, 70230 mesh);prep. TLC: silica gel 60 F254 (Merck). All known metabo-lites were identied by comparison of the spectral datawith those of the authentic specimens. Yields are basedon the weights of the extracts.

3.2. Collection

Five Laurencia samples were collected from four sites inOkinawa Prefecture, Japan: L. mariannensis Yamada atTeruma, Yonashiro, on 4 June 1997; L. majuscula (Har-vey) Lucas at Yagachi, Nago, on 6 June 1997; L. nidicaJ. Agardh at Teruma,Yonashiro, on 4 June 1997, andToyohara, Nago, on 5 June, 1997; L. cartilagineaYamadaat Yagachi, Nago, on 6 June 1997; L. concreta Cribb atHedomisaki, Kunigami, on 6 June 1997. The voucherspecimens are deposited in the Herbarium (SAP) of theGraduate School of Science, Hokkaido University.

3.3. Extraction and separation of Laurencia mariannensis

The partially dried alga (4 g) was soaked in methanolfor one week. The MeOH soln was concentrated in vacuoand partitioned between Et2O and H2O. The Et2O soln.was washed with water, dried over anhydrous Na2SO4,and evaporated to leave a dark green oil (65 mg). Theextract was fractionated by Si gel column chromato-graphy with a stepped gradient (hexane and EtOAc).The fraction eluted with hexaneEtOAc (9:1) was fur-ther submitted to prep. TLC with hexaneEtOAc (3:1)to give 2-bromo-3-chloro-5-acetoxy-chamigra-7(14),9-dien-8-one (2) (14%). Further separation of the fractioneluated with hexaneEtOAc (4:1) under the above-mentioned TLC yielded compound 1 (6%).

3.4. Compound 1

Oil; 24D +42 (CHCl3; c 0.02); IR vmax (neat) cm1:3310, 1680, 1110, 1050, 930, 880; 1H and 13C NMRspectra, Table 1; 1H NMR spectrum (400 MHz, C6D6), 1.21 (3H, dd, J=7.3, 7.3 Hz, H3-15), 1.28 (1H, m,Ha-8), 1.78 (1H, d, J=13.6 Hz, Hb-8), 2.41 (1H, ddd,J=10.7, 9.3, 4.4 Hz, H-6), 2.50 (1H, dq, J=14.6, 7.3 Hz,Ha-14), 2.71 (1H, dd, J=2.4, 0.9 Hz, H-1), 2.84 (1H, dq,J=14.6, 7.3 Hz, Hb-14), 3.39 (1H, dd, J=9.3, 4.9 Hz,H-11), 3.85 (1H, dd, J=4.9, 4.4 Hz, H-7), 4.18 (1H, dd,J=6.8, 4.9 Hz, H-9), 4.52 (1H, dd, J=4.9, 4.9 Hz, H-10), 4.86 (1H, dd, J=10.7, 10.7 Hz, H-5), 5.02 (1H, dd,J=10.7, 2.4 Hz, H-3), 5.43 (1H, ddd, J=10.7, 10.7, 0.9Hz, H-4); LR-FDMS m/z (rel. int.): 346, 344, 342(30:90:76) [M]+, 309, 307 (11:10) [MCl]+; HRFDMSm/z: 342.0013. Calc. for C15H16

79Br35ClO2, 342.0022 [M].

3.5. Extraction and separation of Laurencia majuscula

Extraction of partially dried alga (8 g) was carried outby conventional methods described above to yield abrownish green extract (95 mg). A combination of columnand thick-layer chromatography of the MeOH extractgave three halochamigrenes, 10-bromo-9-hydroxy-chamigra-2,7(14)-diene (3) (9%), (Z)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (4) (3%), and(E)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (5) (4%).

3.6. 10-Bromo-9-hydroxy-chamigra-2,7(14)-diene (3)

Oil; 25D 110 (CHCl3; c 0.20); IR vmax (neat) cm1:3550, 3450, 1640, 1395, 1365, 1220, 1200, 1180, 1085,1070, 1035, 895, 830, 810; 1H NMR (400 MHz, CDCl3), 1.10 (6H, s, H3-12 and H313), 1.50 (3H, br s, H3-15),1.67 (1H, m, Hb-4), 1.75 (1H, m, Hb-5), 1.80 (1H, m,Ha-5), 1.84 (1H, m, Ha-4), 2.19 (1H, m, Hb-1), 2.20(1H, m, Ha-1), 2.48 (1H, dd, J=14.2, 2.9 Hz, Hb-8),2.71 (1H, dddd, J=14.2, 2.9, 2.9, 1.5 Hz, Ha-8), 4.16

Table 3

Minimal inhibitory concentration (MICa) of the halogenated com-

pounds against marine bacteria isolated from algal habitats in the

Japanese coastal waters

Test bacteria Compoundsa

1 3 6 7

Alcaligenes aquamarinus 20 20

Alteromonas sp. 5 10

Azomonas agilis 30 20 5 5

Azobacter beijerinckii 15 15

Erwinia amylovora 30 15 5 5

Escherichia coli 30 10 5 10

Halobacterium sp.

Halococcus sp.

a Concentration: mg/disc. : no inhibition.

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523 521

(1H, dd, J=2.9, 2.9 Hz, H-9), 4.68 (1H, d, J=2.9 Hz,H-10), 4.80 (1H, br s, Hb-14), 5.10 (1H, dd, J=1.5, 1.5Hz, Ha-14), 5.29 (1H, br s, H-2); 13C NMR (100 MHz,CDCl3), CH3: 21.5 (C-13), 23.8 (C-15), 25.0 (C-12), CH2: 26.6 (C-5), 28.5 (C-4), 30.9 (C-1), 38.8 (C-8), 116.5 (C-14), CH: 72.6 (C-10), 73.2 (C-9), 120.2 (C-2), C: 43.9(C-11), 47.8 (C-6), 133.4 (C-3), 141.9 (C-7); LREIMSm/z (rel.int.): 300, 298 (14:15) [M]+, 285, 283 (12:12) [M-CH3]

+, 219 (21) [MBr]+, 217 (32) [MBrH2]+, 201

(100) [MBrH2O]+, 173 (27), 119 (64), 105 (54), 69 (52),

55 (45), 41 (44); HREIMS m/z: 298.0927. Calc. forC15H23

79BrO, 298.0933 [M]. The 1H and 13C NMRspectral data were almost identical with those reportedin the literature for deschloroelatol (Konig and Wright,1997b).

3.7. Extraction and separation of Laurencia nidica

Extraction of partially dried alga (10 g) was carriedout by conventional methods as described above to givea green oily extract (110 mg). A combination of columnand thick-layer chromatography of the MeOH extractsgave laurinterol (6) (22%) and isolaurinterol (7) (4%).

3.8. Extraction and separation of Laurencia cartilagineaand Laurencia concreta

The MeOH extracts of both samples contained nohalogenated compounds.

3.9. Antibacterial bioassay

Antibacterial bioassay was carried out using eight spe-cies of marine bacteria isolated from algal surfaces col-lected from the Japanese waters. These bacteria areAlcaligenes aquamarinus, Alteromonas sp., Azomonas agi-lis, Azotobacter beijerinckii, Erwinia amylovora, Escher-ichia coli, Halobacterium sp., and Halococcus sp. Oneloopful of each organism was precultured in 20 ml ofpeptone water (3% NaCl) overnight. The turbidity ofthe culture was adjusted to an optical density (OD)McFarland 0.5 (Sonnerwirth and Jarett, 1980; Hindleret al., 1990). Then 0.1 ml of the precultured bacterialsuspension was used to seed Nutrient Agar plates (3%NaCl). Paper discs (Whatman, 6 mm) impregnated withvarious amounts of the respective pure compounds wereplaced on the seeded agar plates and the diameters ofthe inhibitory zones were measured after the plates wereincubated at 28 C for 24 h.

Acknowledgements

This study was supported in part by a Grant-in Aid forScientic Research (No. 09839001) from the Ministry ofEducation, Science, Sports and Culture of Japan and

also by the Sasakawa Scientic Research Grant fromthe Japan Science Society (No. 12-300).

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