Fisheries Science 63(1), 94-98 (1997)

Analysis of Algicidal Ranges of the Bacteria Killing the Marine Dinoflagellate Gymnodinium mikimotoi Isolated from Tanabe Bay, Wakayama Pref., Japan

Ikuo Yoshinaga,*1 Takashi Kawai,*2 and Yuzaburo Ishida*l,•õ *1Labratory of Marine Microbiology , Department of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-01, Japan *2Patent Information Division , Central Research Laboratory, Takara Shuzo Co., Seta, Shiga 520-21, Japan (Received June 3, 1996)

Twenty eight strains of the marine bacteria which killed the marine dinoflagellate, Gymnodinium mikimotoi, were isolated during the occurrence of the huge red tide by G. mikimotoi in Tanabe Bay in 1990. Among them 22 strains belonged to the genus Vibrio, three to Flavobacterium, two to Acinetobac ter and one to Pseudomonas-Alteromonas. All of them grew in filter-sterilized natural seawater without any nutrient supplements, suggesting that the killing bacteria are ubiquitous in seawater and are related to the occurrence of G. mikimotoi opportunistically. Most of the killing bacteria did not affect the growth of three marine diatoms, Skeletonema costatum, Ditylum brightwellii and Thalassiosira sp., and some of them did not affect the growth of the marine dinoflagellate, Alexandrium catenella. These findings strongly suggest that the killing bacteria influence the change of microalgal community in a ma rine environment.

Key words: killing bacteria, growth inhibition, red tide, dinoflagellate, Gymnodinium mikimotoi, algicidal range

In an aquatic environment, microalgae (mainly of the bacteria which inhibit the growth of Gymnodinium phytoplankton) and heterotrophic bacteria are numerous mikimotoi, dinoflagellate, by the newly developed MPN and are considered to interact with each other, negatively (most probable number) method during the process of the or positively. Biomass, productivity, heterotrophic activi huge red tide by G. mikimotoi that occurred in Tanabe ty and species composition of bacterial population in Bay (Japan) in 1990.21) The fluctuation of the G. freshwaters and seawaters are regulated by the organic and mikimotoi's-growth inhibiting bacteria (Gm-GIB) correlat inorganic substances excreted from microalgal cells.1,2)On ed negatively with the fluctuation of G. mikimotoi, suggest the other hand, a bacterial population might be related to ing that Gm-GIB is strongly related with the development a change of microalgal community, through nutrient and decay process of the red tide by G. mikimotoi. regeneration, competition for nutrients, production of In this study, the taxonomic grouping of Gm-GIB isolat stimulative or inhibitory substances for algal growth and ed in 1990 was examined, to analyze the community struc other means.3-7) Hence, the development and decay pro ture of Gm-GIB. We also studied the effects of Gm-GIB cess of a microalgal bloom (a red tide), which can be de on the growth of several marine microalgae to discuss the fined as explosive growth of one or a few species of participation of Gm-GIB in the change of microalgal com microalgae on a usual change of microalgal community munity. structure, should be also influenced by a heterotrophic bac terial population. Materials and Methods Some researchers have reported that some bacteria iso lated from freshwaters and seawaters affect positively or Axenic cultures of G. mikimotoi, of three diatoms negatively the growth of various kinds of microalgae.8-13) (Skeletonema costatum, Ditylum brightwellii and Thalas In particular, the marine bacteria which possess an inhibi siosira sp.), and of one raphydophycean flagellate (Hetero tory activity on algal growth have been isolated from sigma akashiwo) used in this study were kindly supplied coastal seawaters, and considered to be related with the by Dr. M. Yamaguchi, Nansei National Fisheries Research decay process of red tides. 14-20)There is relatively little in Institute and Dr. I. Imai, Kyoto University. One dinoflagel formation on the distribution, fluctuation and community late, Alexandrium catenella, was isolated in our laborato structure of these bacterial populations in aquatic environ ry in 1987.22) All of them were cultivated in a SWM3 ments, except for the reports mentioned above. medium18) at 20•Ž with the L:D cycle of 14:10 under Previously we reported the distribution and fluctuation 8,000 lux.

•õ Present adress: Research Center on Environments, Research Organization of Science and Engineering, Ritsumeikan University, Shiga 525-77, Japan. Algicidal Ranges of Marine Bacteria 95

Strains of Gm-GIB were isolated from seawater and sedi ments at several sampling sites in Tanabe Bay, Wakayama Results Pref., Japan, in 1990.21) The sediment samples (1 g) collect ed with a K-K core sampler23) were suspended in 10 ml of We isolated 40 strains of Gm-GIB at various sampling sterilized SWM3 with 60ƒÊg/ml of Tween 80 and cen sites on various sampling dates in 1990, and all of them trifuged at 3,500 rpm for 10 min. The supernatants and killed and lysed G. mikimotoi perfectly within 2 weeks of seawater samples were inoculated into the algal culture ac co-cultivation.21) Only 28 strains were identified at the

cording to the MPN procedure, and incubated at the same genus level, because the rest (12 strains) could not be culti condition for algal cultivation. The MPN tubes in which vated after isolation. Of the 28 strains of Gm-GIB, 22 the algal growth was inhibited by the inoculation of natu strains belonged to the genus Vibrio and 3 strains to that ral bacterial assemblages on the process of counting proce of Flavobacterium followed them (Table 1). Two strains dure for Gm-GIB were selected, and subsamples from of Acinetobacter and one of Pseudomonas-Alteromonas each tube were spread onto a ST10-1 agar plate containing group were obtained. The vibrios were widely isolated 0.5 g trypticase peptone, 0.05 g yeast extract and 12 g agar from every sampling site and date. in aged seawater.24) Bacterial colonies on the agar plates Figure 1 shows the growth curves of two algicidal were picked up and inoculated into the log-phase culture vibrios (D26 and D32) in the filter-sterilized seawater and of G. mikimotoi in test tubes (130 mm •~ 13 mm) to in the culture filtrates of G. mikimotoi. Both isolates grew reconfirm their inhibitory activities on G. mikimotoi. in the seawater to ca 6-7 •~ 106 cells/ ml at maximum cell Strains of Gm-GIB were isolated and purified in the same density. Furthermore, D32 in the culture filtrate of G. manner as mentioned above. Each isolated strain was de mikimotoi grew to 6.2 •~ 107 cells/ml, one order of magni rived from separate MPN tubes, and preserved in the tude higher than in the filtered seawater, although the ST10-1 semiliquid medium (0.3% agar). growth of D26 was not enlarged in the culture filtrate of G. Several taxionomical characteristics of Gm-GIB isolates mikimotoi. The other algicidal-vibrios also grew in the sea were studied and tentatively identified at the genus level ac water without any nutrient supplements. cording to Simidu's scheme.25) Bacterial flagella shapes were observed by TEM (Hitachi Co.) after negative stain ing. The growth profiles of Gm-GIB isolates in filter-steri Table 1. List of Gm-GIB (G. mikimotoi's growth inhibiting bacter lized seawater and the culture filtrate of G. mikimotoi ia) isolated in Tanabe Bay, Wakayama, Pref., in 1990 were studied. The seawater for the former medium was col lected in Tanabe Bay in June 1990, and maintained at 20•Ž under dark condition after filtration with a GF/F filter (Whattman Co.). Just before the experiment, the sea water was sterilized with 0.2,ƒÊm membrane filter (Advan tech Co.). To prepare the latter medium, G. mikimotoi was cultivated in the SWM3 medium and filtrated with the GF/F filter and the 0.2ƒÊm membrane filter aseptically. Gm-GIB isolates cultivated in the ST10-1 liquid medium at 20•Ž for 12 h were harvested by centrifugation at 8,000 •~ g and washed twice with the filter-sterilized sea water. Then each isolate was inoculated into the test media at the initial cell density of 103 cells/m/ and incubated at 20•Ž in the dark. The bacterial cells were counted by direct microscopic observation after DAPI staining.26) To study the algicidal ranges of Gm-GIB isolates, each bacterium was cultivated with five species of marine microalgae except G. mikimotoi: three diatoms, S. costa tum, D. brightwellii and Thalassiosira sp.; one raphydophycean flagellate, H. akashiwo; and one dinoflagellate, A. catenella. These algal cultures were all cultivated in SWM3 axenically. Each strain of Gm-GIB

previously cultivated in the ST10-1 liquid medium was inoculated at the initial cell density of ca 103 cells/ ml into the log-phase culture (3.5 ml) of each alga individually. The experiment was performed in triplicate. The algal bacterial co-cultures were incubated at 20•Ž with the L:D cycle of 14:10 under 8,000 lux. The algal growth in each tube was estimated by in vivo auto-fluorescence with a Turner fluorometer.18,21) Bacterial cell count was deter mined by direct microscopic observation after DAPI stain ing. * Peudomonas -Alteromonas group. 96 Yoshinaga et al.

Fig. 1. Growth curves of Gm-GIB isolates (D26 and D32) in filter-steri lized seawater (•£) and the culture filtrate of Gymnodinium mikimotoi (•œ).

Figure 2 shows the inhibitory effects of four strains of Gm-GIB (D32, E41, E6 and T17) on the growth of G. mikimotoi, A. catenella, H. akashiwo and S. costatum. None of the four strains severely influenced the growth of S. costatum (Bacillariophyceae), although they killed G. mikimotoi within 2 weeks of co-cultivation. E6 and E41 killed H. akashiwo, and T17 suppressed its growth. E41 also inhibited the growth of A. catenella, which belongs to Dinophyceae. On the other hand, D32 did not inhibit the growth of the other algae except G. mikimotoi. In either case, the number of bacteria in the co-culture with prey Fig. 2. Effects of Gm-GIB isolates (D32, E41, E6 and T17) against ma algae increased from 103 cells/ml inoculum size, to 106 rine microalgae, Gymnodinium mikimotoi (Dinophyceae), Alexan -107 cells/ml. drium catenella (Dinophyceae), Heterosigma akashiwo Table 2 summarizes the killing activities and growth-in (Raphidophyceae) and Skeletonema costatum (Bacillariophyceae). hibitory effects of 28 strains of Gm-GIB against 5 marine Each algal growth was expressed by autofluorescence of chlo microalgae. None of the strains killed either of the two spe rophyll a. Error bars are shown. cies of marine diatoms (D. brightwellii and S. costatum) and only 4 strains killed Thalassiosira sp. or inhibited its growth. In contrast, all strains except six (T10, T16, D32, ment and decay process of the algal bloom by G. D35, E27 and G63) killed H. akashiwo or inhibited its mikimotoi in 1990. All of them should be called "algicidal growth. Fifteen strains of Gm-GIB inhibited the growth of bacteria" because they killed G. mikimotoi within 1 or 2 A. catenella and all of them inhibited the growth of H. weeks after bacterial inoculation at ca 103 cells/ Ml.21) akashiwo. There was no clear relationship between taxo The taxonomical studies on 28 strains of Gm-GIB nomic groups and their algicidal ranges and activities. showed that most of Gm-GIB (22 in 28 strains) belonged to the genus of Vibrio (Table 1). These algicidal-vibrios Discussion were obtained even from the seawater samples collected in Stn. 17 where bloom of G. mikimotoi did not occur (Table As we observed previously, at the end of July in 1990, 1). Moreover, all algicidal-vibrios were capable of growing G. mikimotoi cells firstly increased in the southern area in either the culture filtrate of G. mikimotoi or the filter (Stn. 6, 7 and 12) of Tanabe Bay and bloomed in the sterilized seawater without any organic nutrient supple southern and the eastern (Stn. 4) areas from August 7 to ments (Fig. 1). Hence, these algicidal-vibrios may common August 14 .21,27)On the other hand, Gm-GIB number esti ly inhabit seawaters of Tanabe bay, and be related to the mated by the MPN method with the algal culture development and decay of the red tide by G. mikimotoi op decreased from ca 103cells/ml to 101 cells/ml in accor portunistically. Imai et al.18) isolated an algicidal-cytopha dance with the occurrence of the red tide by G. ga sp. from coastal seawater in Hiroshima Bay (Japan). mikimotoi.21)The number of Gm-GIB subsequently recovered They speculated that this Cytophaga sp. might be ubiqui by September 4 when the red tide had already disappeared. tous in seawater because it grew in filter-sterilized seawater These observations strongly suggest that the population dy as did the algicidal-vibrios in the present study. Various namics of Gm-GIB influenced the development and decay killing bacteria against marine microalgae may commonly process of the red tide of G. mikimotoi in Tanabe Bay. inhabit coastal seawater and potentially regulate the We isolated 40 strains of Gm-GIB during the develop blooming and decay of marine microalgae. If the fluctua Algicidal Ranges of Marine Bacteria 97

Table 2. Algicidal ranges of Gm-GIB isolated in Tanabe Bay in 1990

tion of the killing bacteria and the intensity of their killing to G. mikimotoi in Tanabe Bay. activities in seawater can be estimated correctly, the impor tance of those bacteria in the development and decay pro Acknowledgments We thank Mr. T. Matsunaga and Ms. A. Tari for cess of red tide could be fairly assessed. their technical assistance, and Dr. A. Uchida, Kyoto University, for his Fukami et al. 16) also isolated the marine bacterium valuable advice. Also we thank Mr. T. Takeuchi, Wakayama Prefectural Fisheries Experimental Station, for providing information about the al (Flavobacterium sp.) that killed G. mikimotoi but had no gal community structure in Tanabe Bay. This study was supported by the effect on the growth of S. costatum, from the seawater in Fisheries Ground Preservation Division, Fisheries Agency of the Kochi Pref., Japan, and proposed that this bacterium Japanese Government. affects the change of microalgal species composition. Also in this study, most Gm-GIB did not inhibit the growth of References three diatoms (Table 2). Because Gm-GIB multiplied in co-culture with diatoms and yielded to the cell density of 1) R. H. Chrost and M. A. Faust: Organic carbon release by 106cells/ml sufficient to kill G. mikimotoi, Gm-GIB did phytoplankton: its composition and utilization by bac not have killing activities against the marine diatoms. Espe terioplankton. J. Plankton. Res., 5, 477-493 (1983). cially S. costatum and D. brightwellii were not affected by 2) S. Ohara, K. Fukami, and Y. Ishida: Algal effects on the bacterial assemblages in lake Biwa. Japan J. Limnol., 54, 261-268 (1993). Gm-GIB at all. During June and July in 1990, some kinds 3) C. E. Riquelme, K. Fukami, and Y. Ishida: Annual fluctuations of of diatoms (S. costatum, Chaetoceros spp., Nitzschia spp. phytoplankton and bacterial communities in Maizuru Bay and their and Thalassiosira spp.) were dominant in Tanabe Bay, interrelationship. Bull. Japan Soc. Microb. Ecol., 2, 29-37(1988). and they diminished as G. mikimotoi increased (T. 4) J. L. Romalde, A. E. Toranzo, and J. L. Barja: Changes in bacteri Takeuchi: Wakayama Pref. Fish. Exp. Station, pers. al population during red tides caused by Mesodinium rubrum and comm.). Gm-GIB isolates in this study that inhibit the Gymnodinium catenatum in north west coast of Spain. J. Appl. Bacteriol., 68, 123-132 (1990). growth of G. mikimotoi but not of diatoms might be one 5) J. L. Romalde, J. L. Barja, and. A. E. Toranzo: Vibrios associated of the agents regulating the algal succession from diatoms with red tides caused by Mesodinium rubrum. Appl. Environ. 98 Yoshinaga et al.

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