Algal Resources (2014) 7：95-105

Cultivable ephiphytic obtained from the unialgal culture strain of the red alga Gracilariopsis chorda (Holmes) Ohmi collected from the estuary of Katsuura River in Tokushima Prefecture in southwest Japan

Hirotaka KAKITA1 *, Hideki OBIKA1 and Hiroshi KAMISHIMA1

Abstract : To identify the cultivable epiphytic bacteria obtained from the unialgal culture strain of the red alga Gracilariopsis chorda (Holmes) Ohmi collected from the estuary of Katsuura River in Tokushima Prefecture in southwest Japan, morphological and phenotypic classification and genetic analysis were carried out. Eight kinds of cultivable bacteria were isolated. Isolate UGC1-1 (bacterial population: 7.5×107 cfu g-1), UGC1-2 (4.0× 107 cfu g-1)andUGC1-3(5.0× 106 cfu g-1) were isolated from Marine Agar plates. Isolate UGC1-4 (5.0×106 cfu g-1)andUGC1-5(1.0×106 cfu g-1) were isolated from 1/10 ORI and CAM plates, respectively. Isolate UGC1-6 (1.0×105 cfu g-1), UGC1-7 (6.0×104 cfu g-1)andUGC1-8 (2.0×104 cfu g-1) were isolated from CVT plates. The genetic analysis based on partial 16 S rRNA gene sequences (ca. 450 bp) revealed that the Isolate UGC1-1, UGC1-2, UGC1-3, UGC1-4, UGC1-5, UGC1-6, UGC1-7 and UGC1-8 related closely to Ascidianbacter aurantia- cus (Similarity 93.2 %), Ruegeria halocynthiae (99.5 %), Roseovarius sediminilitoris (97.2 %), Litoreibacter janthinus (99.3 %), Zobellia russellii (99.8 %), Marinobacter salarius (99.1 %), Granulosicoccus antarcticvs or G. coccoides (97.9 %) and Labrenzia aggregata (99.8 %), re- spectively. The Isolate UGC1-1 was suggested to be an undescribed taxon assigned to the family based on genetic analysis.

Keywords : Gracilariaceae; cultivable epiphytic bacteria; morphological classification; ge- netic analysis; Flavobacteriaceae

Introduction bacteria have already been identified in previ- ous studies (Shiba and Taga 1981; Cole 1982; Some bacterial species are known to associ- Brock and Clyne 1984; Coveney and Wetzel 1989; ate with particular macroalgal species. Positive Weinbweger et al. 1997; Dobretsov and Qian interactions between macroalgae and associ- 2002; Croft et al. 2005; Marshall et al. 2006; Rao ated bacteria include phytohormone produc- et al. 2006). Goecke et al. (2010) found out that tion, morphogenesis of macroalgae triggered by the beneficial relationship is based on the algal bacterial metabolites, specific antibiotic activi- activity to produce organic compounds and ox- ties affecting epibionts and elicitation of oxida- ygen which will be utilized by bacteria. Shiba tive burst mechanisms (Goecke et al. 2010). On and Taga (1981) reported that the bacterial flora the other hand, negative interactions induce or attached on a macroalga changes during the generate algal diseases (Goecke et al. 2010). repeatedly transferred batch culture of algae in Beneficial associations between macroalgae and a sterile medium. They pointed out that the

1 Health Research Institute, AIST Shikoku, National Institute of Advanced Industrial Science and Technology, Hayashi, Takamatsu, Kagawa 761-0395, Japan ＊Corresponding author : Tel: +81-878-693561, fax: +81-878-693553, e-mail: h-kakita@aist. go.jp

95 Hirotaka KAKITA, Hideki OBIKA and Hiroshi KAMISHIMA growth-stimulated bacteria become predominant Therefore, to characterize the cultivable epi- in the repeatedly transferred batch culture of phytic bacteria derived from Japanese Gracilar- algae in a sterile medium and the growth-in- iopsis chorda (Holmes) Ohmi, we investigated the hibited bacteria are washed out, because algal population and the generic composition of the exudates are the sole organic carbon sources in cultivable epiphytic bacteria derived from the batch culture of algae in a sterile medium. Japanese G. chorda using the repeatedly trans- Goecke et al. (2010) also reported that bacteria ferred batch culture of algae in a sterile medi- mineralize the organic substrates, supplying the um. In this paper, we conducted phenotypic and algae with carbon dioxide, minerals and‘growth generic characterization as for the epiphytic factors'. Several researchers have reported that bacterial isolates obtained from the unialgal bacteria supply macroalgae with growth facters culturestrainoftheredalgaG. chorda collect- relating to the production and turnover of var- ed from the estuary of Katsuura River in ious phytohormones and biostimulators of cell Tokushima Prefecture in southwest Japan. growth and development (Berland et al. 1972, Bolinches et al. 1988, Meusnier et al. 2001). Materials and Methods Dimitrieva et al. (2006) reported that a favora- ble growth-promoting effect by the bacterium Collection of materials Pseudoalteromonas porphyrae was observed on The algal specimens of the red alga Gracilar- Laminaria japonica. Freis (1975) clarified the iopsis chorda (Holmes) Ohmi were collected from ability of bacteria living on Enteromorpha spp. the estuary of Katsuura River [latitude 34.04 N to convert tryptophan into the phytohormone and longitude 134.58 E, the World Geodetic Sys- indole-3-acetic acid. tem 1984 (WGS 84)] in Tokushima Prefecture in Furthermore, an increased growth rate of an southwest Japan in June 2007. algal strain leads to an increase in its total production. The Japanese species of the red Unialgal culture strain algal family Gracilariaceae are important indus- The algal specimens were washed with ster- trial macroalgae, because they have been har- ile seawater and cultured in a sterile medium vested in Japan as commercial sources of agar for eight days, as described previously (Kakita and for food additives (Tokuda et al. 1987). et al. 2013). Thirty Erlenmeyer flasks were used However, no trials of the growth stimulation of for the repeatedly transferred batch culture of Japanese Gracilariaceae using epiphytic bacteria algae in a sterile medium. Three hundred mil- have ever been reported. In general, cultivable ligrams in wet weight of the washed alga were bacteria are easy to isolate, to increase, and to inoculated under a clean booth into each Er- store in comparison with unculturable bacteria. lenmeyer flask containing 400 mL of the sterile Therefore some kind of cultivable bacteria may medium and were incubated with aeration at be more applicable to algal growth stimulation 18℃, in a 14 hL:10 hD regime and at photon than unculturable bacteria. To utilize beneficial irradiances of 60 mol photons m-2s-1 under cool- bacteria for stimulating algal growth, the culti- white lamps. After the repeatedly transferred vable epiphytic bacteria derived from the red batch culture of algae in a sterile medium for algal family Gracilariaceae have to be isolated, eight days, the macroalga obtained was collect- characterized, and identified at the species level. ed and used for further study as a sample of a Sucha bacterial generic classification is a neces- cultured alga. sary prerequisite for the algal growth stimula- The unialgal stock culture of the red alga G. tion by using cultivable epiphytic bacteria. How- chorda was started from tetraspores released by ever the details of the generic composition, matured tetrasporophytes among the cultured which is based on the 16S ribosomal RNA gene algae. The establishment of a unialgal culture sequences of the cultivable epiphytic bacteria strain followed the methods of Yamamoto and derived from the Japanese Gracilariaceae, have Sasaki (1987). Spore inoculation, medium re- never been reported. newal, and upright body inoculation were car-

96 Cultivable bacteria from the culture strain of Gracilariopsis chorda collected in Katsuura River ried out under a clean booth. Ten spores were per one gram of a macroalga was calculated. inoculated into each 50 mL-volume screw tube Colonies with the same characteristics [i.e. containing 30 mL of the sterile medium and shape, color, size, the presence or absence of a were incubated without aeration at 18℃,ina14 hollow (a circular transparent zone)] were re- hL:10 hD regime, in salinity of about 33 ‰ and garded as equivalent colony types. Eight strains at a photon irradiance of 40 mol photons m-2s-1. in total were isolated from different types of In the static culture, the sterile medium was predominant colonies. The eight bacterial iso- replaced with a fresh one once every 3 weeks. lates were taken for morphological and pheno- Upright bodies obtained (about 10 mm length) typic classification and genetic analysis. were taken from the bottom of the screw tube with tweezers and inoculated into a flask to Morphological and phenotypic classification of perform the culturing of the upright bodies. The bacterial isolates unialgal stock culture of upright bodies was in- Gram stain was carried out using Hucker's cubated in 1000 mL round-shaped flasks con- modification (Conn et al. 1957). Motility, cell taining 800 mL of the sterile medium with shape, flagella staining, pigment production, aeration at 18 ℃, in a 14 hL:10 hD regime and oxidase activity, gelatin hydrolysis and DNA at a photon irradiance of 40 mol photons m-2s-1 hydrolysis were tested as described in Cowan under cool-white lamps. The macroalga was and Steel's Manual (Barrow and Feltham 1993). taken from each flask with a pair of tweezers Oxidation-fermentation on glucose was investi- and inoculated into new flasks containing a new gated by Leifson's method (1963). A salt re- sterile medium bi-weekly. After the unialgal quirement examination was carried out with stock culture in a sterile medium, the macroal- Hidaka and Sakai's basal medium (0.05 % of ga obtained was collected under a clean booth, polypeptone and 0.01 % of yeast extract) (Hida- referred to as unialgal culture strain UGC1, and ka and Sakai 1965). Medium A (0.05 % of polypep- used for bacterial experiments. tone and 0.01 % of yeast extract, pH7.6), medi- um B (the medium A containing 1 % NaCl), and Enumeration of bacteria medium C (the medium A dissolved in sterile Five grams in wet weight of the unialgal cul- seawater) were used. The bacteria grown in the ture strain UGC1 and 45 mL of a sterile solu- medium A were referred to as terrestrial type. tion (2.5 % concentration) of sodium chloride The bacteria, which did not grow in the medi- were put in a sterile stomacher bag and were um A but which did grow in the medium B, given stomaching treatment for 1 minute. The were referred to as halophilic type. The bacte- stomaching liquid was diluted ten times with a ria, which did not grow in the medium A and sterile solution (2.5 % concentration) of sodium B but which grew in the medium C, were chloride appropriately. Each 0.1 mL of the referred to as marine type (Hidaka and Sakai diluted-samples was spread on four kinds of 1965). Bacterial strains were identified accord- agar plates and the plates were incubated at ing to the scheme of Ezura et al. (1988). 20 ℃. Marine Agar (Marine Agar 2216, Becton Dickinson, MD, USA), 1/10 ORI, CAM and CVT Similarity analysis of the 16S rRNA gene se- were used as agar media (Simidu et al. 1983, quences of bacterial isolates Ezura et al. 1988, Olson 1963). Marine Agar, 1/10 The genomic DNA was extracted from each ORI, CAM and CVT are designed for cultiva- bacterial isolate with Proteinase K (TaKaRa, ting heterotrophic marine bacteria, oligotrophic Shiga, Japan) by heating conditions of 55 ℃,1h bacteria, polysaccharide-decomposing bacteria, and 95 ℃, 10 min. One microliter of the super- and psychrotrophic bacteria, respectively. CVT natant was used as a template for PCR ampli- was prepared with seawater instead of distilled fication. The partial 16S rRNA genes (ca. 450 water in this study. After 14 days of incuba- bp) of bacterial isolates UGC1-1, UGC1-2, UGC1- tion, the plates with between 10 and 100 colo- 3, UGC1-4, UGC1-5, UGC1-6, UGC1-7 and UGC nies were selected and the number of bacteria 1-8 were amplified using the MicroSEQ 500 16S

97 Hirotaka KAKITA, Hideki OBIKA and Hiroshi KAMISHIMA rDNA PCR kit (Applied Biosystems, CA, USA). 14 days of incubation on CAM. Type F (UGC The PCR-amplified products were sequenced 1-6) was white-colored, round-shaped, and ca. using the MicroSEQ 500 16S rDNA Sequencing 1 mm in diameter after 14 days of incubation kit and an automated DNA sequencer (ABI on CVT. Type G (UGC1-7) was creamy white- PRISM Model 310 Genetic analyzer) (Applied colored, round-shaped, and ca. 1 mm in diam- Biosystems, CA, USA) according to the manu- eter after 14 days of incubation on CVT. Type facture's protocol. The 16S rRNA gene se- H (UGC1-8) was white-colored, round-shaped, quences of the bacterial isolates were com- andca.2mmindiameterafter14daysof pared with sequences available in the public incubation on CVT. Table 1 summarizes the databases (DDBJ/EMBL/GenBank) using the morphological and other phenotypical charac- BLASTN algorithm (Altschul et al. 1990). teristics of the bacterial isolates obtained from the unialgal culture strain of the red alga G. Deposit of DNA sequence data chorda collected from the estuary of Katsuura The sequence data were deposited to DDBJ River in Tokushima Prefecture in southwest with accession numbers of LC011916 (data from Japan. Bacterial genera were determined in the UGC1-1), LC011917 (data from UGC1-2), LC011918 morphological manner according to the scheme (data from UGC1-3), LC011919 (data from UGC of Ezura et al. (1988). Among the cultivable 1-4), LC011920 (data from UGC1-5), LC011921 bacteria on Marine Agar plates, the Isolate UGC (data from UGC1-6), LC011922 (data from UGC 1-1 [Flavobacterium sp. (Phylum, ; 1-7) and LC011923 (data from UGC1-8). Class, Flavobacteria; Order, ; Family, Flavobacteriaceae) -Cytophaga sp. (Bacte- Results roidetes, Cytophagia, Cytophagales, Cytophagaceae) (marine type) (7.5×107 cfu g-1)] was predomi- Population of cultivable epiphytic bacteria nant (62.5 %). The remainder were the Isolate The populations of the cultivable epiphytic UGC1-2 [Moraxella sp. (Proteobacteria, Gamma- bacteria obtained from the unialgal culture strain proteobacteria, Psedomonadales, Moraxellaceae) of the red alga G. chorda collected from the (marine type) (4.0×107 cfu g-1, 33.3 %) and the estuary of Katsuura River were 1.2×108 cfu g-1 Isolate UGC1-3 [Moraxella sp. (marine type) (5.0 (Marine Agar), 5.0×106 cfu g-1 (1/10 ORI), 1.0×106 ×106 cfu g-1,4.2%)]. The predominant cultivable cfu g-1 (CAM) and 1.9 × 105 cfu g-1 (CVT). The bacterium on 1/10 ORI plates was the Isolate population of cultivable bacteria grown on Ma- UGC1-4 [Moraxella sp. (marine type) (5.0×106 rine Agar plate was higher more 20 times than cfu g-1)]. On the other hand, the Isolate UGC1-5 those grown on other agar media tested. [Flavobacterium sp. -Cytophaga sp. (marine type) (1.0×106 cfu g-1)] was predominant on CAM Morphological and phenotypic classification of plates. Among the cultivable bacteria on CVT bacterial isolates plates, the Isolate UGC1-6 [Pseudomonas sp. Type A (Representative strain: UGC1-1) was (Proteobacteria, Gammaproteobacteria, Pseudo- yellow-colored, round-shaped, and ca. 2 mm in monadales, Pseudomonadaceae) (marine type) diameter after 14 days of incubation on Ma- (1.0×105 cfu g-1)] was predominant (55.6 %). The rine Agar. Type B (UGC1-2) was cream-colored, remainder were the Isolate UGC1-7 [Alcaligenes round-shaped, and ca. 2 mm in diameter after sp. (Proteobacteria, Betaproteobacteria, Burk- 14 days of incubation on Marine Agar. Type C holderiales, Alcaligenaceae) (marine type) (6.0× (UGC1-3) was white-colored, round-shaped, and 104 cfu g-1, 33.3 %) and the Isolate UGC1-8 ca. 1 mm in diameter after 14 days of incuba- [Alcaligenes sp. (halophilic type) (2.0×104 cfu g-1, tion on Marine Agar. Type D (UGC1-4) was 11.1 %)]. The viable bacterial count of Vibrio sp. white-colored, round-shaped, and ca. 2 mm in (Proteobacteria, Gammaproteobacteria, Vibrion- diameter after 14 days of incubation on 1/10 ales, Vibrionaceae) was less than a detection ORI. Type E (UGC1-5) was yellow-colored, limit value (＜ 1×102 cfu g-1). round-shaped, and ca. 3 mm in diameter after

98 Cultivable bacteria from the culture strain of Gracilariopsis chorda collected in Katsuura River

Table 1. Morphological and other phenotypical characteristics of the bacterial isolates obtained from the unialgal culture strain of the red alga G. chorda collected from the estuary of Katsuura River in Tokushima Prefecture in southwest Japan

CVT*: CVT was prepared with seawater instead of distilled water in this study. NT**: not tested. Peri.***: Peritrichous. NP****: nonproductive. Salt requirement type*****: MT, marine-type; HT, halophilic-type. Genus******: FC, Flavobacterium sp. - Cytophaga sp.; M, Moraxella sp.; P, Pseudomonas sp.; A, Alcaligenes sp. Bacteria genera were determined in the morphological manner according to the scheme of Ezura et al. (1988). The colony color of the Isolate UGC1-6 was different from that of the Isolate UGC1-7.

Similarity analysis of the 16S rRNA gene se- Rhodobacterales, Rhodobacteriaceae, Litoreibact- quences of bacterial isolates er) (99.3 %) and its related species. The Isolate Table 2 summarizes the most closely related UGC1-5 belonged to a cluster which is con- species of the bacterial isolates obtained from the structed by Zobellia russellii (Flavobacteriaceae, unialgal culture strain of the red alga G. chorda Zobellia) (99.8 %) and its related species. The collected from the estuary of Katsuura river in Isolate UGC1-6 belonged to a cluster which is Tokushima Prefecture in southwest Japan. The constructed by Marinobacter salaries (Proteobac- Isolate UGC1-1 related closely to Ascidianbacter teria, Gammaproteobacteria, Alteromonadales, aurantiacus (Flavobacteriaceae, Ascidianbacter) Alteromonadaceae, Marinobacter) (99.1 %) and its (Similarity 93.2 %). The Isolate UGC1-2 belonged related species. The Isolate UGC1-7 belonged to to a cluster which is constructed by Ruegeria a cluster which is constructed by Granulosicoc- halocynthiae (Proteobacteria, Alphaproteobacte- cus antarcticus (Proteobacteria, Gammaproteobacte- ria, Rhodobacterales, Rhodobacteriaceae, Ruege- ria, Chromatiales, Granulosicoccaceae, Granulosic- ria) (99.5 %) and its related species. The Isolate occus) (97.9 %), G. coccoides (97.9 %) and its relat- UGC1-3 related closely to Roseovarius sedimilitoris ed species. The Isolate UGC1-8 related closely to (Proteobacteria, Alphaproteobacteria, Rhodobac- Labrenzia aggregata (Proteobacteria, Alphapro- teriales, Rhodobacteriaceae, Roseovarius)andOcean- teobacteria, Rhodobacterales, Rhodobacteriaceae, obacterium insulare (Rhodobacteraceae, Oceanobac- Labrenzia)(99.8%)andL. alba (98.8 %). terium) (97.2 %). The Isolate UGC1-4 belonged to a cluster which is constructed by Litoreibacter janthinus (Proteobacteria, Alphaproteobacteria,

99 Hirotaka KAKITA, Hideki OBIKA and Hiroshi KAMISHIMA

Table 2. Most closely related species of the bacterial isolates obtained from the unialgal culture strain of the red alga G. chorda collected from the estuary of Katsuura River in Tokushima Prefecture in southwest Japan

*: The length of the determined base sequence (bp) of 16S rRNA of the bacterial isolates. **: Bacterial strain was determined by the genetic analysis. Bacterial strain means the most closely related species of the each isolate. Accession numbers of the sequences of reference organisms are showninparenthesis.

bacteria were attached on the algal thallus and Discussion spores, and the cultivable epiphytic bacteria got into a static culture medium at spore inocula- Population of cultivable epiphytic bacteria tion. Kakita et al. (2013) reported that the popula- tions on Marine Agar plates of the heterotro- Morphological and phenotypic classification of phic bacteria attached to the naturally occur- bacterial isolates ring G. chorda collected in March, in June, the A unialgal culture strain means a strain which cultured G. chorda in March, in June, and the does not include any algae except a target alga. stocked G. chorda were 6.5×106,1.3×106,5.4× 107, Thus, the cultivable epiphytic bacteria obtained 3.4×107 and 2.6×107 cfu g-1, respectively. In our from a unialgal culture strain are expected to present study, the population on Marine Agar directly originate from a target alga, when plates of the cultivable epiphytic bacteria ob- compared with those on a naturally occurring tained from the unialgal culture strain of the alga and a cultured alga. The Isolate UGC1-1 red alga G. chorda was 1.2×108 cfu g-1 and was predominant among the eight isolates ob- abundant distribution of epiphytic bacteria was tained from the unialgal culture strain of the observed as well as the previous study by red alga G. chorda, and was identified as Fla- Kakita et al. (2013). We infer that the reason for vobacterium sp. -Cytophaga sp. by the morpho- the similarity in the large population density logical and phenotypic classification. The pre- between the unialgal culture strain and the cul- dominance of Flavobacterium sp. -Cytophaga sp. tured algae was that most cultivable epiphytic among the cultivable epiphytic bacteria ob-

100 Cultivable bacteria from the culture strain of Gracilariopsis chorda collected in Katsuura River tained from cultured algae in a sterile medium fer that the Isolate UGC1-5 may have high have been observed in the green alga Ulva linza enzymatic activities against polysaccharides, be- (Linne) J. Agardh (Shiba and Taga, 1981) and the cause a CAM medium contains large amounts red alga G. chorda (Kakita et al. 2013). Shiba and of polysaccharides such as CM-cellulose (1 g L-1) Taga (1981) pointed out that Flavobacterium and sodium alginate (4 g L-1). sp. -Cytophaga sp. is truly characteristic of the green alga U. linza, because they found the large Similarity analysis of the 16S rRNA gene se- and constant proportions of Flavobacterium sp. quences of bacterial isolates -Cytophaga sp. on the green alga U. linza dur- Stackebrandt and Goebel (1994) reported that ing the repeatedly transferred batch culture in the sequence analyses of 16S rRNA gene have a sterile medium. In our present study, the vi- been widely used for the prokaryote identifica- able bacterial count of Vibrio sp. derived from tion, because the primary structure of 16S rRNA the unialgal culture strain of the red alga G. gene is easier to determine than hybridization chorda was less than a detection limit value (＜ between DNA strands. They also reported that 1×102 cfu g-1), although Vibrio sp. and Pseudo- a 16S rRNA gene sequence similarity of 97.0 % monas sp. are generally predominant in the sea- should become the boundary for the delineation water of the oceans near Japan and the inner of prokaryotic species. Stackebrandt and Ebers bay areas (Simidu et al. 1977). The numerical (2006) renewed the threshold value for the de- inferiority of Vibrio sp. have been observed in lineation of prokaryotic species to 98.7 %-99.0 % cultured U. linza (Shiba and Taga, 1981) and G. from 97.0 %. Top three species in nucleotide chorda (Kakita et al. 2013). Shiba and Taga (1981) similarity values for the Isolate UGC1-1 were indicated that Vibrio sp. is not inhabitant of Ascidianbacter aurantiacus (Flavobacteriaceae, the green alga U. linza, because viable counts of Ascidianbacter)(Similarity93.2%),Croceitalea Vibrio sp. decreased with time in the repeated- eckloniae (Flavobacteriaceae, Croceitalea) (93.1 %), ly transferred batch culture in a sterile medi- and Muricauda aquimarina (Flavobacteriaceae, um. We are agreeable to Shiba and Taga's ex- Muricauda) (92.9 %). The details of Ascidianbact- planation for the predominance of Flavobacte- er aurantiacus have never been reported to date, rum sp. -Cytophaga sp. and the numerical in- although 16S rRNA gene sequence of this strain feriority of Vibrio sp. Three Isolates (UGC1-2, is deposited to the GenBank. Lee et al. (2008) UGC1-3 and UGC1-4) belonged to Moraxella sp. isolated Croceitalea eckloniae strain JCM (Japan Ishida and Kadota (1974) proved that Vibrio sp. Collection of Microorganisms) 13827T from the decreases and Acinetobacter group bacterium rhizosphere of the brown alga Ecklonia kurome, such as Moraxella sp. increases in number in andreportedthatthisstrainisanorange-col- response to increase in the concentration of the ored rod. They reported that this strain hydrol- organic substances in seawater. We infer that yses gelatin, but does not hydrolyse agar, ca- the eight isolates obtained may be epiphytic sein, starch and urea. They also reported that bacteria attached on the naturally occurring G. this strain produces acetoin, but does not pro- chorda in waters. Shiba and Taga (1981) point- duce H2S and indole. Yoon et al. (2005) isolated ed out that the growth-stimulated bacteria be- Muricauda aquimarina strain JCM11811T from a come predominant in the repeatedly transferred salt lake. This strain is golden yellow in color batch culture of algae in a sterile medium, be- on Marine Agar. Yoon et al. (2005) reported that cause algal exudates are the sole organic car- this strain does not hydrolyses gelatin, casein, bon sources in the batch culture of algae in a starch and urea. They also reported that this sterile medium. Therefore, we infer that the strain does not produce H2S and does not re- eight isolates, especially the Isolate UGC1-1, duce nitrate. Whether this strain can produce which obtained from the unialgal culture strain indole or not has never reported to date. The of the red alga G. chorda, may have a high colony color of the Isolate UGC1-1 was similar ability of utilizing algal exudates and degrada- to that of Muricauda aquimarina strain JCM tion products for bacterial growth. We also in- 11811T, but different from that of Croceitalea

101 Hirotaka KAKITA, Hideki OBIKA and Hiroshi KAMISHIMA eckloniae strain JCM13827T. However, sequence negative for nitrate reduction and indole pro- similarities between the Isolate UGC1-1 and three duction in API 20NE tests. members of genera in the family Flavobacteri- Both results of morphological and phenotypic aceae were less than 98.7 %. Both results of classification and genetic assay suggest that the morphological and phenotypic classification and Isolate UGC1-5 may belong to the family Fla- genetic assay suggest that the Isolate UGC1-1 vobacteriaceae. The closest relative of the Iso- may belong to the family Flavobacteriaceae and late UGC1-5 was Zobellia russellii. Nedashk- a separate taxon at species level. ovskaya et al. (2004) isolated Zobellia russellii We infer that the Isolate UGC1-2 may belong strain KMM3677T from the green alga Acrosi- to the genus Ruegeria, although the Isolate UGC phonia sonderi, and reported this strain decom- 1-2 was identified as Moraxella sp. (Proteobac- poses agar, gelatin, starch, alginate, DNA, Tween teria, Gammaproteobacteria, Pseudomonadales, 20, Tween 40 and Tween 80, but does not hydrol- Moraxellaceae) by the morphological and phe- yse casein, cellulose or chitin. They also indica- notypic classification. Kim et al. (2012) isolated ted that this strain reduces nitrate, but does not

Ruegeria halocynthiae KCTC (Korea Collection produce H2S, indole and acetoin. Therefore, for Type Cultures) 23463T strain from a sea we infer that the Isolate UGC1-5 may be a squirt (Halocynthia roretzi). This strain is a decomposer of agarophytes such as Gracilari- cream-colored non-motile rod. They reported that aceae and not be a supplier of the phytohor- this strain reduces nitrate, hydrolyses hypoxan- mone such as indole-3-acetic acid. 16S rRNA gene thine, L-tyrosine and xanthine, but not aescu- sequence studies showed that the Isolate UGC lin, casein, gelatin, starch, Tween 80 or urea. 1-6 clustered closely with Marinobacter salaries, The closest relatives of the Isolate UGC1-3 were although the Isolate UGC1-6 was determined as Roseovarius sediminilitoris and Oceanobacterium Pseudomonas sp. (Proteobacteria, Gammapro- insulare. Roseovarius sediminilitoris KCTC 23959T teobacteria, Pseudomonadales, Pseudomonadaceae, strain was isolated from a seashore sediment by Pseudomonas) by the morphorogical and phe- Park and Yoon (2013). The details of Ocean- notypic classification. Marinobacter salarius JCM obacterium insulare have never been reported to 19399T strain, which is isolated from seawater date, although 16S rRNA gene sequence of this from Chazhma Bay in the Sea of Japan, can- strain is deposited to the GenBank. The se- not produce indole (Ng et al. 2014). We infer that quence similarities between the Isolate UGC1-3 theIsolateUGC1-6maybelongtothegenus and other type species of the family Rhodobacter- Marinobacter, although the results from mor- aceae were less than 96.7 %. Thus, we infer that phological and phenotypic classification and ge- the Isolate UGC1-3 may belong to the family netic analysis for the Isolate UGC1-6 were dif- Rhodobacteraceae, although the Isolate UGC1-3 was ferent in bacterial order. identified as Moraxella sp. by the morphologi- The closest relatives of the Isolate UGC1-7 cal and phenotypic classification. 16S rRNA were Granulosicoccus antarcticus and G. coccoides, gene sequence studies showed that the Isolate although the Isolate UGC1-7 was identified as UGC1-4 clustered closely with Litoreibacter jan- Alcaligenes sp. (Proteobacteria, Betaproteobacte- thinus, although the Isolate UGC1-4 was deter- ria, Burkholderiales, Alcaligenaceae) by the mor- mined as Moraxella sp. by the morphorogical phological and phenotypic classification. The and phenotypic classification. Romanenko et al. genus Granulosicoccus was described in 2007 as (2011) isolated Litoreibacter janthinus JCM16492T a member of the newly proposed family Gran- strain from a shallow sediment sample collected ulosicoccaceae of the order Chromatiales (Lee et from the Sea of Japan. This strain produces al. 2007).Granulosicoccus antarcticus KCCM 42676T greyish-violet pigmented colonies on Marine strain was isolated from the Antarctic surface Agar. They reported that this strain is nega- seawater of King George Island, West Antarc- tive for H2S production and hydrolysis of casein, tica (Lee et al. 2007). Lee et al (2007) reported gelatin, DNA, Tween 80, chitin, starch and L- that this strain is a non-phototrophic, obligate- tyrosine. They also indicated that this strain is ly aerobic chemoheterotroph. They also repor-

102 Cultivable bacteria from the culture strain of Gracilariopsis chorda collected in Katsuura River ted that this strain is requiring NaCl for growth, UGC1-2, UGC1-3, UGC1-4, UGC1-5, UGC1-6, but neither halophilic nor halotolerant. Kurilenko UGC1-7 and UGC1-8 were closely related to et al. (2010) isolated Granulosicoccus coccoides Ascidianbacter aurantiacus, Ruegeria halocynthi- KMM6014T strain from the surface of leaves of ae, Roseovarius sediminilitoris, Litoreibacter jan- seagrass (Zostera marina) grown in Troitza Bay, thinus, Zobellia russellii, Marinobacter salaries, Gulf of Peter Great, Pacific Ocean, and repor- Granulosicoccus antarcticus or G. coccoides, and ted that this strain is chemoorganotrophic with Labrenzia aggregata, respectively. We also con- respiratory metabolism, negative for indole, clude that the predominant bacterium (the Iso- arginine dihydrolase, lysine decarboxylase, orni- late UGC1-1) obtained from the unialgal cul- thine decarboxylase and H2S production. The ture strain of the red alga G. chorda is proba- sequence similarities between the Isolate UGC1-7 bly an undescribed taxon which is assigned to and other type species of the class Gammapro- the family Flavobacteriaceae. teobacteria were less than 87.8 %. Therefore, we infer that the Isolate UGC1-7 may belong to the Acknowledgments genus Granulosicoccus, although the results from morphological and phenotypic classification and This research was partially supported by geneticanalysisfortheIsolateUGC1-7were Grant-in-Aid for Scientific Research (KAKENHI) different in bacterial class. (c), Grant Number 25340080, 2013. The Isolate UGC1-8 related closely to Labren- zia aggregata and L. alba, although the Isolate References UGC1-8 was identified as Alcaligenes sp. by the morphological and phenotypic classification. The Altschul SF, Gish W, Miller W, Myers EW, basonyms of Labrenzia aggregata and L. alba Lipman DJ. Basic local alignment search are Stappia aggregata and S. alba, repspectively tool.J.Mol.Biol.1990;215 : 403-410. (Biebl et al. 2007). Uchino et al. (1998) reported Barrow GI, Feltham RKA. Cowan and Steel's that Labrenzia aggregata ATCC 25650T strain manual for identification of medical bacte- does not reduce nitrate to nitrite. Pujalte et al. ria, 3rd ed. Cambridge University Press, (2005) isolated Stappia alba CIP (Collection de Cambridge. 1993. l'Institut Pasteur) 108402T strain from oysters Berland BR, Bonin DJ, Maestrini SY. Are some cultured at the Spanish Mediterranean coast, bacteria toxic for marine algae? Mar. Biol. near Vinaroz, and reported that this strain is a 1972 ; 12 : 189-193. chemoorganotrophic aerobe, does not have any BieblH,PukallR,Lu nsdorf H, Schulz S, All- pigments, and does not hydrolyse gelatin, case- gaier M, Tindall BJ, Wagner-Do bler I. De- in, starch, agar, alginate and lecithin. They al- scription of Labrenzia alexandrii gen. nov., so reported that this strain is negative for fol- sp. nov., a novel alphaproteobacterium con- lowing activities: indole production from tryp- taining bacteriochlorophyll a, and a propos- tophan, sulphide (H2S) production from thiosul- al for reclassification of Stappia aggregata phate, arginine dihydrolase, lysine decarboxy- as Labrenzia aggregata comb. nov., of Stap- lase and ornithine decarboxylase. We infer that pia marina as Labrenzia marina comb. nov. the Isolate UGC1-8 may belong to the genus and of Stappia alba as Labrenzia alba comb. Labrenzia, although the results from morpho- nov., and emended descriptions of the gen- logical and phenotypic classification based on era Pannonibacter, Stappia and Roseibium, classical were different. and of the species Roseibium denhamense We conclude, in this first report on the ge- and Roseibium hamelinense. Int. J. Syst. Evol. netic analysis of the cultivable epiphytic bacte- Microbiol. 2007 ; 57 : 1095-1107. ria obtained from the unialgal culture strain of BolinchesJ,LemosML,BarjaJL.Population the red algal family Japanese Gracilariaceae, that dynamics of heterotrophic bacterial com- genetic analysis based on partial 16S rRNA gene munities associated with Fucus vesiculosus sequences revealed that the Isolates UGC1-1, and Ulva rigida in an estuary. Microbial

103 Hirotaka KAKITA, Hideki OBIKA and Hiroshi KAMISHIMA

Ecol. 1988 ; 15 : 345-357. Ishida Y, Kadota H. Ecological studies on bac- Brock TD, Clyne J. Significance of algal excre- teria in the sea and lake waters polluted tory products for growth of epilimnetic with organic substances-I responses of bac- bacteria. Appl. Environ. Microbiol. 1984 ; 47 : teria to different concentrations of organic 731-734. substances. Bull. Jpn. Soc. Sci. Fish. 1974;40: Cole JJ. Interactions between bacteria and al- 999-1005. gae in aquatic ecosystems. Annu. Rev. Ecol. Kakita H, Obika H, Kamishima H. Bacteria at- Syst. 1982 ; 13 : 291-314. tached to the red alga Gracilariopsis chorda Conn HJ, Bartholomew JW, Jennison MW. Stai- (Holmes) Ohmi. Algal Resources 2013 ; 6 : 73- ning methods. In : the Society of American 80. Bacteriologist (ed.) Manual of microbiologi- Kim YO, Park S, Nam BH, Kang SJ, Hur YB, cal methods. McGraw-Hill, New York. 1957 ; Lee SJ, Oh TK, Yoon JH. Ruegeria halo- 10-36. cynthiae sp. nov., isolated from the sea Coveney MF, Wetzel RG. Bacterial metabolism squirt Halocynthia roretzi. Int. J. Syst. Evol. of algal extracellular carbon. Hydrobiologia Microbiol. 2012 ; 62 : 925-930. 1989 ; 173 : 141-149. Kurilenko VV, Christen R, Zhukova NV, Kali- Croft MT, Lawrence AD, Raux-Deery E, War- novskaya NI, Mikhailov VV, Crawford RJ, ren MJ, Smith AG. Algae acquire vitamin Ivanova EP. Granulosicoccus coccoides sp. B12 through a symbiotic relationship with nov., isolated from leaves of seagrass (Zos- bacteria. Nature 2005 ; 438 : 90-93. tera marina)Int.J.Syst.Evol.Microbiol. Dimitrieva GY, Crawford RL, Yuksel GU. The 2010 ; 60 : 972-976. nature of plant growth-promoting effects of Lee K, Lee HK, Choi TH, Kim KM, Cho JC. a pesudoalteromonad associated with the Granulosicoccaceae fam, nov. to include marine algae Laminaria japonica and linked Granulosicoccus antarcticus gen. nov., sp. to catalase excretion. J. Appl. Microbiol. nov., a non-phototrophic, obligately aerobic 2006 ; 100 : 1159-1169. chemoheterotroph in the order Chromatia- Dobretsov SV, Qian PY. Effect of bacteria as- les, isolated from Antarctic seawater. J. Mi- sociated with the green alga Ulva reticulata crobiol. Biotechnol. 2007 ; 17 : 1483-1490. on marine micro- and macrofouling. Bio- Lee HS, Kwon KK, Yang SH, Bae SS, Park CH, fouling 2002 ; 18 : 217-228. Kim SJ, Lee JH. Description of Croceitalea Ezura Y, Kawabata M, Miyashita H, Kimura T. gen. nov. in the family Flavobacteriaceae Change of bacterial population in the nurs- with two species, Croceitalea eckloniae sp. ery tanks for the forced cultivation of Ma- nov. and Croceitalea dokdonensis sp. nov. konbu Laminaria japonica. Nippon Suisan isolated from the rhizosphere of the ma- Gakkaishi 1988 ; 54 : 655-663 (in Japanese with rine alga Ecklonia kurome. Int. J. Syst. Evol. English abstract). Microbiol. 2008 ; 58 : 2505-2510. Freis L. Some observations on the morphology Leifson E. Determination of carbohydrate me- of Enteromorpha linza (L.) J. Ag. and En- tabolism of marine bacteria. J. Bacteriol. teromorpha compressa (L.) Grev. in axenic 1963 ; 85 : 1183-1184. culture. Bot. Mar. 1975 ; 18 : 251-253. Marshall K, Joint I, Callow ME, Callow JA. Goecke F, Labes A, Wiese J, Imhoff JF. Chem- Effect of marine bacterial isolates on the ical interactions between macroalgae and growth and morphology of axenic plantlets bacteria. Mar. Ecol. Prog. Ser. 2010 ; 409 : 267- of the green alga Ulva linza. Microbial 300. Ecology 2006 ; 52 : 302-310. Hidaka T, Sakai M. Comparative observations Meusnier I, Olsen JL, Stam WT, Destombe C, on the inorganic salt requirements of Vibrio Valero M. Phylogenetic analyses of Cauler- parahaemolyticus and marine or terrestrial pa taxifolia (Chlorophyta) and of its associ- bacteria. Shokuhin Eiseigaku Zasshi 1965 ; 6: ated bacterial microflora provide clues to 235-241 (in Japanese with English abstract). the origin of the Mediterranean introduc-

104 Cultivable bacteria from the culture strain of Gracilariopsis chorda collected in Katsuura River

tion. Mol. Ecol. 2001 ; 10 : 931-846. species definition in bacteriology. Int. J. Nedashkovskaya OL, Suzuki M, Vancanneyt M, Syst. Bacteriol. 1994 ; 44 : 846-849. Cleenwerck I, Lysenko AM, Mikhailov VV, Stackebrandt E, Ebers J. Taxonomic parame- Swings J. Zobellia amurskyensis sp. nov., ters revisited : tarnished gold standards. Mi- Zobellia laminariae sp. nov. and Zobellia crobiol. Today 2006 ; 33 : 152-155. russellii sp. nov., novel marine bacteria of Tokuda H. Ohno M, Ogawa H. The resources the family Flavobacteriaceae. Int. J. Syst. and cultivation of seaweeds. Midorishobo, Evol. Microbiol. 2004 ; 54 : 1643-1648. Tokyo. 1987. Ng HJ, Lo pez-Pe rez M, Webb HK, Gomez D, Uchino Y, Hirata A, Yokota A, Sugiyama J. Sawabe T, Ryan J, Vyssotski M, Bizet C, Reclassification of marine Agrobacterium Malherbe F, Mikhailov VV, Crawford RJ, species : Proposals of Stappia stellulata gen. Ivanova EP. Marinobacter salarius sp. nov. nov., comb.nov., Stappia aggregata sp. nov., and Marinobacter similis sp. nov., isolated nom. rev., Ruegeria atlantica gen. nov., comb. from sea water. PLoS ONE 2014 ; 9 : e106514. nov., Reuegeria gelatinovora comb. nov., Olson HC. Selective plating technique for de- Ruegeria algicola comb. nov., and Ahrensia tecting contamination in pasteurized milk. kieliense gen.nov.,sp.nov.,nom.rev.J.Gen. J. Dairy Sci. 1963 ; 46 : 362. Appl.Microbiol.1998;44 : 201-210. Park S, Yoon JH. Roseovarius sediminilitoris sp. Weinberger F, Hoppe HG, Friedlander M. nov., isolated from seashore sediment. Int. Bacterial induction and inhibition of a fast J. Syst. Evol. Microbiol. 2013 ; 63 : 1741-1745. necrotic response in Gracilaria conferta Pujalte MJ, Macia n MC, Arahal DR, Garay E. (Rhodophyta). J. Appl. Phycol. 1997 ; 9 : Stappia alba sp. nov., isolated from Medi- 277-285. terranean oysters. Syst. Appl. Microbiol. Yamamoto H, Sasaki J. Cross experiment be- 2005 ; 28 : 672-678. tween so-called Gracilaria verrucosa (Hud.) Rao D, Webb JS, Kjelleberg S. Microbial colo- Papenfuss from two localities, Shinori and nization and competition on the marine al- Kikonai in Hokkaido. Bull. Fac. Fish. Hok- ga Ulva australis. Appl. Environ. Microbiol. kaido Univ. 1987 ; 38 :1-4. 2006 ; 72 : 5547-5555. Yoon JH, Lee MH, Oh TK, Park YH. Muricauda Romanenko LA, Tanaka N, Frolova GM, flavescens sp.nov.andMuricauda aquima- Svetashev VI, Mikhailov VV. Litoreibacter rina sp. nov., isolated from a salt lake near albidus gen. nov., sp. nov. and Litoreibacter Hwajinpo beach of the east sea in Korea, janthinus sp. nov., members of the class and emended description of the genus Muri- Alphaproteobacteria isolated from the sea- cauda. Int. J. Syst. Evol. Microbiol. 2005 ; 55 : shore. Int. J. Syst. Evol. Microbiol. 2011 ; 61 : 1015-1019. 148-154. Shiba T, Taga N. Effects of the Extracellular Received 18 November 2014 products of Enteromorpha linza on its epi- Accepted 26 December 2014 phytic bacterial flora. Bull. Jpn. Soc. Sci. Fish. 1981 ; 47 : 1193-1197. Simidu U, Kaneko E, Taga N. Microbiological studies of Tokyo Bay. Microbial Ecol. 1977 ; 3 : 173-191. Simidu U, Lee WJ, Kogure K. Comparison of different techniques for determining plate counts of marine bacteria. Bull. Jpn. Soc. Sci. Fish. 1983 ; 49 : 1199-1203. Stackebrandt E, Goebel BM. Taxonomic note : a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present

105