Molecular identification of new bacterial causative agent of ice-ice disease on seaweed Kappaphycus alvarezii

Marlina Achmad, Alimuddin Alimuddin, Utut Widyastuti, Sukenda Sukenda, Emma Suryanti, Enang Harris

Background. Ice-ice disease is still a big challenge for seaweed farming that is characterized with “bleaching” symptom. are suspected as cause of ice-ice disease on seaweed Kappaphycus alvarezii. The 16S rRNA gene sequencing is current technique used for bacterial phylogeny and taxonomy studies. This study was aimed to identify bacterial onset of ice-ice disease on K. alvarezii.

Methods. Eight sequenced isolates from Indonesia were identified and characterized by biochemical tests and sequenced by 16S rRNA gene as target. The isolates sequence compared to the strains of bacteria from GenBank. DNA sequences are analyzed with ClustalW program and phylogeny were performed using the result generated by Mega v.5. The micropropagules (2-4 cm) was soaked in seawater containing 106 cfu/ml of bacteria to determine the pathogenicity. Onset of ice-ice symptoms was visually observed every day. Histology are analyzed to show tissue of micropropagule post-infection by bacteria.

Results. Identification of bacteria employed biochemical tests and 16 SrRNA gene sequence analysis. The results reveal eight species of bacteria, namely: haliotis strain DW01, 2 Vibrio alginolyticus strain ATCC 17749, Stenotrophomonas maltophilia strain IAM 12323, Arthrobacter nicotiannae strain DSM 20123, Pseudomonas aeruginosa strain SNP0614, Ochrobactrum anthropic strain ATCC 49188, Catenococcus thiocycli strain TG 5-3 and Bacillus subtilis subsp.spizizenii strain ATCC 6633. In term of groups, bacteria S. haliotis, V. alginolyticus, S. maltophilia, P. aeruginosa and C. thiocycli are the in group and O. anthropi is in the Alphaproteobacteria group, A. nicotianae and B. subtilis is in the of group both of are Actinobacteria and Firmicutes group Low GC respectively. The results showed that the fastest onset of ice-ice symptoms was caused by S. maltophilia (five hours post-infection), while the slowest it was caused by V. alginolyticus (44 hours post-infection). Other bacteria give rise to ice-ice symptoms for 15-21 hours post-infection. Thus, S. maltophilia also showed number of bleaching spot of higher than others. However, V. alginolyticus showed increased width of bleaching 2.29 mm2 greater than S. maltophilia.

Discussion. Bacteria S. haliotis and V. alginolyticus were found in healthy thallus, while others were found in bleaching thallus. Indicator of bleaching this is the first study shows S. maltophilia association to ice- ice disease on K. alvarezii. Interaction of temperature with different disease-causing pathogens ice-ice on seaweed is unreported and it is thought-provoking to examine in further research. In addition, the bacteria isolated in this study is potentially used to hold the assembly seaweed ice-ice disease through the challenge test.

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1 Molecular Identification of New Bacterial Causative Agent of Ice-Ice Disease 2 on Seaweed Kappaphycus alvarezii 3 4 Marlina Achmad12, Alimuddin3, Utut Widyastuti4, Sukenda3, Emma Suryanti5, Enang Harris3

5 1 Aquaculture Science, Graduate School, Bogor Agricultural University, Bogor, Indonesia 6 2 Fisheries Department, Marine Science and Fisheries Faculty, Hasanuddin University, 7 Makassar, Indonesia 8 3 Department of Aquaculture, Faculty of Fisheries and Marine Science, Bogor Agricultural 9 University, Bogor, Indonesia 10 4 Department of Biology, Faculty of Mathematics and Natural Science, Bogor Agricultural 11 University, Bogor, Indonesia 12 5 Centre of Brackishwater Research and Development, Maros Indonesia 13 14 Corresponding Author: 15 Alimuddin3 16 Agatis Street, Bogor, West Java, 16680, Indonesia 17 Email address: [email protected] 18 19 ABSTRACT

20 Background. Ice-ice disease is still a big challenge for seaweed farming that is characterized

21 with “bleaching” symptom. Bacteria are suspected as cause of ice-ice disease on seaweed

22 Kappaphycus alvarezii. The 16S rRNA gene sequencing is current technique used for bacterial

23 phylogeny and taxonomy studies. This study was aimed to identify bacterial onset of ice-ice

24 disease on K. alvarezii.

25 Methods. Eight sequenced isolates from Indonesia were identified and characterized by

26 biochemical tests and sequenced by 16S rRNA gene as target. The isolates sequence compared

27 to the strains of bacteria from GenBank. DNA sequences are analyzed with ClustalW program

28 and phylogeny were performed using the result generated by Mega v.5. The micropropagules

29 (2-4 cm) was soaked in seawater containing 106 cfu/ml of bacteria to determine the

30 pathogenicity. Onset of ice-ice symptoms was visually observed every day. Histology are

31 analyzed to show tissue of micropropagule post-infection by bacteria.

32 Results. Identification of bacteria employed biochemical tests and 16 SrRNA gene sequence

33 analysis. The results reveal eight species of bacteria, namely: Shewanella haliotis strain DW01,

34 Vibrio alginolyticus strain ATCC 17749, Stenotrophomonas maltophilia strain IAM 12323,

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35 Arthrobacter nicotiannae strain DSM 20123, Pseudomonas aeruginosa strain SNP0614,

36 Ochrobactrum anthropic strain ATCC 49188, Catenococcus thiocycli strain TG 5-3 and

37 Bacillus subtilis subsp.spizizenii strain ATCC 6633. In term of groups, bacteria S. haliotis, V.

38 alginolyticus, S. maltophilia, P. aeruginosa and C. thiocycli are the in Gammaproteobacteria

39 group and O. anthropi is in the Alphaproteobacteria group, A. nicotianae and B. subtilis is in

40 the of Proteobacteria group both of are Actinobacteria and Firmicutes group Low GC

41 respectively. The results showed that the fastest onset of ice-ice symptoms was caused by S.

42 maltophilia (five hours post-infection), while the slowest it was caused by V. alginolyticus (44

43 hours post-infection). Other bacteria give rise to ice-ice symptoms for 15-21 hours post-

44 infection. Thus, S. maltophilia also showed number of bleaching spot of higher than others.

45 However, V. alginolyticus showed increased width of bleaching 2.29 mm2 greater than S.

46 maltophilia.

47 Discussion. Bacteria S. haliotis and V. alginolyticus were found in healthy thallus, while others

48 were found in bleaching thallus. Indicator of bleaching this is the first study shows S.

49 maltophilia association to ice-ice disease on K. alvarezii.

50

51 Keywords: bacteria, ice-ice, Kappaphycus alvarezii, micropropagule, pathogenicity

52 53 Introduction 54 55 Seaweed, especially, Kappaphycus alvarezii is an important commodity for aquaculture in

56 Indonesia. The K. alvarezii is a source of kappa-carrageenan used for foods, cosmetics,

57 pharmaceuticals and photographies (Yu et al. 2002).

58 Ice-ice disease is still a big challenge for seaweed farming, for certain season. This disease is

59 characterized with white symptom “bleaching or whitening” on upper tissue of the seaweed and

60 it can be caused from different opportunistic pathogenic bacteria (OPB) (Largo et al. 1995a;

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61 Vairappan et al. 2001; Aris 2011). The difference of the source bacterial isolate can be cause

62 variation species different founded it.

63 Pathogenicity test against disease-free seaweed has not been done. The OPB existed in K.

64 alvarezii thallus are Vibrio sp. (P11) and Cytophaga sp. (P25) (Largo et al. 1999) and caused

65 pathogenic in the aseptic K. alvarezii. K. alvarezii disease-free can be produced through tissue

66 culture. The success of tissue culture has been reported by Sulistiani and Yani (2014).

67 Maintaining callus for two months can generate mikropropagule (Reddy et al., 2008). The

68 mikropropagule is potentially used to test candidate disease-causing bacterial pathogenicity ice-

69 ice.

70 The development of bacterial species identification techniques is rapidly grew. The 16S rRNA

71 gene sequencing is current technique used for bacterial phylogeny and taxonomy studies. There

72 are three main reasons using this gene: a) exist in almost every bacterium, b) steady function

73 over time has not changed, suggesting that random change of sequence is more accurate and c)

74 the 16S rRNA gene (1,500 bp) is large enough for an information purpose (Patel 2001; Widow

75 and Abott 2007). The aim of this research was to identify biochemical, pathogenic and

76 molecular bacterial candidate cause ice-ice disease through 16S rRNA gene sequencing as the

77 target.

78 79 Materials and Methods 80 81 Collection of seaweed sample

82 K. alvarezii was collected from seaweed farming at Bulukumba Beach, South Sulawesi,

83 Indonesia (Figure 1) on January 2015, salinity and temperature ranges of location were 30-32

84 ppt and 28-29oC, respectively. Samples were chilled in a cooler box and transported to the

85 laboratory within twelve hours.

86

87 Isolation and identification of bacteria using biochemical test

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88 Thallus of K. alvarezii with and without ice-ice disease were used as sample for isolation of

89 bacteria. One gram of thallus crushed to produce liquid, then 0.1 ml of the liquid distributed

90 into petridishes containing solid media of sea water complex (SWC) consists of 5 g bacto-

91 peptone, 5 g yeast extract, 3 ml glycerol, 250 ml distilled water, 750 ml sterile seawater and 20

92 g bactoagar. The bacteria were cultured at 28°C for 24 hours. Isolates were scratched several

93 times to obtain pure isolates. Then, colony types were biochemically evaluated and identified.

94

95 Extraction of DNA genome

96 Bacterial genomic DNA was extracted using Presto ™ mini kit gDNA bacteria (Geneaid,

97 Taiwan). Gram-negative bacteria cell were lysed using proteinase K-contained GT buffer (20

98 mL) and incubated at 60°C for 10 minutes. Gram-positive bacteria cell were lysed using

99 lysozyme-contained GT buffer (4 mg/ml) and incubated at 37°C for 30 minutes. Then, it was

100 added proteinase K (20 mL) and incubated at 60°C for 10 minutes.

101 DNA was dissolved using 100 mL elution buffer. DNA concentration was measured and

102 confirmed using Genquant (Teare et al. 1997) and separated using 1% agarose gel

103 electrophoresis. Visualization of DNA was obtained using stained ultraviolet light and dye

104 ethidium bromide stain. Finally, DNA solution were stored at -20°C until next processing.

105

106 PCR amplification and nucleotide analysis

107 Amplification of 16S rRNA gene was performed using a universal primer (Marchesi et al.

108 1998), which is 63F (5'-CAG GCC CAC TAA GTC ATG CAA-3 ') and 1387R (5'-GGG CGG

109 GTA WGT CAA GGC-3'). The PCR program used pre-denaturation of 94°C for two minutes,

110 30 cycles of amplification at a denaturation of 92°C for 30 seconds, annealing of 55°C for 30

111 seconds, extension of 72°C for 1 minute and a final extension of 75°C for 20 minutes. PCR

112 results were separated using 1% agarose gel electrophoresis to confirm the amplification

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113 product. Then, PCR results were purified using a PCR clean-up and gel extraction (Geneaid),

114 then products were sequenced using ABI3730XL machine.

115 Sequencing results were edited using Bioedit v 7.4 program (Tamura et al. 2011; Azanza et al.

116 2013). Later, sequences were analyzed using the basic local alignment search tool (Altschul et

117 al. 1990; Azanza et al. 2013) for identification of bacteria isolates and related other organisms

118 sequences. 16S rRNA sequences were associated to bacteria strain in the Gene Bank.

120.199 oE

120.193 oE

119

120 Figure 1. Sampling sites in South Sulawesi, Indonesia. 121 122 Phylogeny analysis

123 The results of DNA sequences, previously be aligned (alignment) with nucleotide sequences,

124 were performed using ClustalW program. Phylogeny analysis was made using the MEGA v.5,

125 0 program (Azanza et al. 2013) with sample repetition bootstrap 1000 times.

126

127 Pathogenicity test

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128 Pathogenicity test was conducted using micropropagule samples produced by SEAMEO

129 Biotrop. The micropropagule regenerates embryogenic callus and it is produced from tissue

130 culture technology (Sulistiani and Yani 2014). Before pathogenicity test, according to

131 Vairappan et al. (2001), media and micropropagule are held for three days to ensure free

132 contamination with microorganisms. The bacterial suspension (106 cfu/ml) of 0.3 ml was

133 inserted to micropropagule media at a bottle chicken. Each bottle was consisted two parts of 2-

134 4 cm MP length. Each type of bacteria is made for three bottles. Bacteria and MP inoculations

135 were aseptically performed at laminar air flow (LAF). Then It was shaken using a shaker bottle,

136 100 rpm speed and incubated for 7 days at 20°C, 14:10 hours (light: dark) photoperiod. Daily

137 MP conditions were observed to determine the initial appearance of ice-ice disease symptoms.

138 Histological MP was made according to Aris (2011) with: twice fixation in neutral buffered

139 formalin (BNF), then five times of dehydration in different alcohol (70, 90 and 100%.), then,

140 2-3 times of cleaning in toluene, impregnation liquid paraffin and planting in paraffin for final

141 dyeing preparations. Preparates were stained using hematoxylin and eosin stains. Data were

142 analyzed descriptively.

143 144 RESULT 145 146 Identification of bacteria isolates

147 Algal-surface bacteria were successfully removed and cultured in SWC media. Table 1. shows

148 biochemical and physiological characteristics of bacteria isolates. There are six bacteria species

149 were group of Gram-negative bacteria, such as Shewanella sp., Vibrio sp., Stenotrophomonas

150 sp., Pseudomonas sp. and Ochrobactrum sp., while two isolates were group of Gram-positive

151 bacteria, namely Arthrobacter sp. and Bacillus sp. These bacteria belong to characteristics of

152 rod shape and motile.

153

154 Phylogenetic diversity of bacteria isolates

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155 Nucleotide sequences of 16S rRNA gene analyses were correlated with biochemical characters.

156 The bacteria were identified to species level among strains of Shewanella haliotis DW01, Vibrio

157 alginolyticus strain ATCC 17749, Stenotrophomonas maltophilia strain IAM 12323,

158 Pseudomonas aeruginosa strain SNP 0614, Ochrobactrum anthropi strain ATCC 49188,

159 Arthrobacter nicotianae strain DSM 20123 and Bacillus subtilis subsp. spizizenii strain ATCC

160 6633. The similarity of 16S rRNA gene nucleotide sequence between bacterial isolates with

161 Gene Bank database are more than 90% (see Table 2.).

162 Table 1. Biochemical and physiological characteristics of bacteria isolates in seaweed K. 163 alvarezii 164 Characteristics Shw Vib Ste Arb Psd Ocb Cac Bal Gram stain - - - + - - - + Cell shape r r r c r r c r Colony Form Circular + - - + - - + - Irregular - + + - + + - + Margin Entire + - - + - - + - Undulate - - + - - + - - Filamentous ------+ Curled - + - - + - - - SIM + + + + + + - + Catalase + + + - + + + + Oxidase - + - - - - - + OF test - O - - O - - - 165 Notes: c: Coccus; r: Rod; O: Oxidation; F: Fermentation; +: Positive; −: Negative. Shw: Shewanella sp.; Vib: Vibrio sp., Ste: 166 Stenotrophomonas sp., Arb: Arthrobacter sp., Psd: Pseudomonas sp., Ocb: Ochrobactrum sp., Ctc: Catenococcus sp., Bal: Bacillus sp. 167 168 169 170 171 Table 2. The 16S rRNA gene nucleotide sequences of bacterial isolates on ice-ice disease at 172 seaweed K. alvarezii with similarity percentage to Gene Bank database. 173 174

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Identity Query/ Accession Isolate Bacteria % Subject Number NA1 Shewanella haliotis strain DW01 98% 1197/1227 NR_117770.1 NB2 Vibrio alginolyticus strain ATCC 17749 97% 1196/1229 NR_117895.1 TA8 Stenotrophomonas maltophilia strain IAM 12323 97% 1102/1136 NR_041577.1 TA9 Arthrobacter nicotianae strain DSM 20123 96% 1183/1231 NR_026190.1 TA2 Pseudomonas aeruginosa strain SNP0614 97% 1277/1316 NR_118644.1 TA4 Ochrobactrum anthropi strain ATCC 49188 95% 1075/1134 NR_074243.1 UB3 Catenococcus thiocycli strain TG 5-3 98% 1195/1220 NR_104870.1 UA5 Bacillus subtilis subsp. spizizenii strain ATCC 6633 95% 1234/1294 NR_118486.1 175 176 Notes: NA1 as bacteria were isolated from healthy thallus (N), site of sampling (A), number of sample(1); TA8 as bacteria 177 were isolated from surface of the ice-ice thallus (T), site of sampling (A), number of sample (8); UB3 as bacteria were 178 isolated from tip of the ice-ice thallus (U), site location (B), number of sample (3) 179

180 This study showed that molecular identification of bacteria isolates identified interesting

181 species that have not been reported from previous study. The bacteria species showed a high

182 similarity accordance to Gene Bank database and supported by bootstrap values for phylogeny

183 tree (Figure 2). Majority of the bacteria isolates were group of Proteobacteria division, gamma

184 subdivision, i.e Shewanella haliotis DW01, Vibrio alginolyticus strain ATCC 17749,

185 Stenotrophomonas maltophilia strain IAM 12323, Pseudomonas aeruginosa strain SNP 0614

186 and Catenococcus thiocycli strain TG 5-3 whereas alpha subdivision is O. anthropi strain

187 ATCC 49188. However, there are two species of bacteria outside the division Proteobacteria

188 both of are B. subtilis subsp. spizizenii strain ATCC 6633 (Firmicutes) and A. nicotiannae strain

189 DSM 20123 (Actinobacteria).

190 Identified putative species from this study include bacteria related to Stenotrophomonas sp.,

191 Pseudomonas sp., Shewanella sp., Ochrobactrum sp., Bacillus sp. Athrobacter sp., and 2

192 species allied to Vibrio sp.

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AJ293474.1| Stenotrophomonas maltophilia strain e-a22

100 DQ077704.1| Stenotrophomonas maltophilia isolate FLX Isolat 3 | Stenotrophomonas maltophilia strain IAM 12323

44 M59158.1 Xanthomonas maltophilia

100 Isolat 5 | Pseudomonas aeruginosa strain SNP0614 AY665977.1| Pseudomonas aeruginosa

100 99 KC495571.1| Pseudomonas fluorescens strain KP6 I 99 AF438148.1| Pseudomonas sp. AMSN

100 Isolat 1 | Shewanella haliotis strain DW01 NR 044453.1| Shewanella marina strain C4

100 NR 036790.1| Vibrio fluvialis strain VL 5125

99 Isolat 2 | Vibrio alginolyticus strain ATCC 17749 83 Isolat 7 | Catenococcus thiocycli strain TG 5-3 AF526515.2| Ochrobactrum intermedium isolate ADV24

100 Isolat 6 | Ochrobactrum anthropi strain ATCC 49188 II 70 NR 115043.1| Ochrobactrum pituitosum strain CCUG 50899

100 Isolat 8 | Bacillus subtilis subsp. spizizenii strain ATCC 6 III EU350371.1| Bacillus pumilus strain ST277

99 Isolat 4 | Arthrobacter nicotianae strain DSM 20123

100 KU200946.1| Arthrobacter arilaitensis strain Abk7 IV 83 KP296222.1| Arthrobacter nicotianae strain Na44RA-2

0.02 193

194 Figure 2. A maximum likelihood tree based on the 16S rRNA gene fragments of the bacterial 195 isolates with bootstrap supports generated with 1000 times resampling. Accession 196 numbers precede the identity of the reference sequences. Based on phylogeny tree, 197 bacteria ice-ice disease on seaweed from Indonesia divided into four classification: I. 198 Gammaproteobacteria; II. Alphaproteobacteria; III. Firmicutes; IV. Actinobacteria

199

200 Pathogenicity test

201 Pathogenicity test showed that when initial symptoms are different from bacterial

202 bleaching, see Figure 3. The bacteria cause rapid bleaching is S. maltophilia (5

203 hourspostinfection, hpi), followed by O. Anthropi (15 hpi) and P. aeruginosa at 17 hpi. The

204 bacteria S. maltophilia showed number of spots and wide of bleaching, which are 17 spots and

205 1.87 mm2 respectively, higher compared to other isolates (see Figure 4).

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206 On the other hand, Stenotrophomonas maltophilia isolate from disease thallus showed

207 the fastest time of beginning symptom within five hours which total spot and wide bleaching

208 significantly affected on K. alvarezii.

209

210 Figure 3. Time early emergence of bleaching on seaweed K. alvarezii. This graphic compared 211 among bacterial of emergence the bleaching on seaweed.

spot mm2 18 2.5

16

14 2

12 1.5 10

8 1 6

4 0.5 2

0 0

Number of bleaching (spot) width of bleaching (mm2) 212

213 Figure 4. Indicators of ice-ice symptom on seaweed K. alvarezii. There are two axis that 214 showed number of bleaching (spot) as primary axis and width of bleaching (mm2) as 215 secondary axis. 216

217 Histology

218 Morphology (Figure 5) and histology (Figure 6) showed different micropropagules that are

219 challenged with the bacteria. Cell walls and protoplasm in healthy-looking tissue were intact

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220 without apparent damage (Figs. 6A, 6B and 6C). The micropropagule showed less severe

221 symptoms generally have a habit of attacks at one place, namely one (on the tip of

222 micropropagule), and severe bleaching is indicated with symptoms at almost of surface the

223 micropropagule. Histologically also showed difference micropropagules networks indicated

224 light worse to heavy worse after treatment pathogenicity test (Figure 4D-4F).

(B) (C (A) 225

226 Figure 5 Morphology of micropropagule K. alvarezii pascainfection by bacteria. (A) Before 227 experimented, and post-infection by bacterial with not severed areas (B), and 228 severed areas (C). Black arrow showed the bleaching symptom. 229

230 Discussion

231 Bacterial identification results are generally in the group of Gammaproteobacteria, a

232 group of bacteria associated with a cause of a disease. Bacteria Gammaproteobacteria group is

233 bacteria essential for health study. A number of genome studies have been conducted to identify

234 unique proteins in bacterial species Gammaproteobacteria associated with virulence (Van

235 Sluys et al., 2002; Gao et al., 2009).

236 V. alginolyticus bacterium has a close relationship with the bacterium S. haliotis and

237 this is in line with the Kita-Tsukamoto et al. (1993) and Thompson et al. (2004) who found that

238 based on partial 16S rRNA sequences selected from 50 species, included in the family

239 , are generally species of Vibrio and Aeromonas species, Deleya, Escherichia,

240 Marinomonas, Pseudomonas and Shewanella. Isolates of C. thiocycli also is in one branch of

241 V. alginolyticus. Based on the taxonomic level C. thiocycli goes into family of Vibrionaceae

242 (GBIF 2016).

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243 Thus, S. maltophilia is a primary bacterium candidate cause a disease of ice-ice on K.

244 alvarezii. This type of bacteria is the first reported disease associated with ice-ice. Bacteria

245 Stenotrophomonas sp are microorganisms that have widely spread (Brooke 2012) and been

246 reported as opportunistic pathogens (Falagas et al. 2009). However, S. maltophilia isolated from

247 the seaweed Laminaria saccharina is a source of active antibiotics (Wiese et al. 2009).

248 Other bacteria were isolated, in this study, and reported in several species of seaweed.

249 Epibiotic bacterium P. aeruginosa as the brown alga Padina tetrastromatica (Ravisankar et al.

250 2013). Type O. anthropi discovered a bacterium degrade alginate in brown algae (Zhou et al.,

251 2008), C. thiocycli and B. subtilis has been isolated from seaweed Sargassum spp. (Susilowati

252 et al. 2015), A. nicotiannae reported has been isolated from biofilm on surface of marine

253 organisms and algae (Lee et al. 2003). However, these bacteria have not been reported as a

254 major trigger bacterial ice-ice disease in seaweed K. alvarezii.

255 Also, in this study, bacteria O.anthropi and P.aeruginosa are the second and third

256 bacterium after S. maltophilia cause ice-ice. Yet, other bacteria that have been reported to be

257 the cause of ice-ice disease, namely Cytophaga-Flavobacterium (Largo et al. 1995a), Vibrio sp.

258 (Largo et al. 1995a; Aris 2011), Pseudomonas sp., Plesiomonas sp. and Flavobacterium sp.

259 (Aris 2011). Pseudomonas sp. also has been isolated from the seaweed K. alvarezii and known

260 cause disease ice-ice (Largo et al. 1995b; Aris 2011). However, S. maltophilia and O. anthropi

261 have not been found in seaweed K. alvarezii troubled ice-ice.

262 The gentlest bacterium is V. alginolyticus with an average time of beginning to appear

263 bleaching is 44 hours post-infection. This time is much longer than control showing early

264 symptoms of bleaching, 23 hours post-infection. The emergence of symptoms of bleaching is

265 on control allegedly due to stress during handling of treatment. Generally, the color changes to

266 white seaweed originated from former talus end cutting (Sulistiani and Yani 2014).

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267 Bacteria S. haliotis and V. alginolyticus were isolated from healthy thallus, so both of

268 these bacteria were not suspected to be associated with ice-ice disease, they are good bacteria

269 and this result similar with Zadeh et al. (2010) that Shewanella algae found in healthy digestive

270 system of black tiger shrimp Penaeus monodon, it is a probiotics candidate. Adding probiotic

271 S. haliotis on feed could improve growth and disease resistance on white spots over vaname

272 shrimp Litopenaeus vannamei (Hao et al. 2014). Species of V. alginolyticus are also reported

273 as effective bio control of diseases caused by pathogenic bacteria on tiger shrimp larvae (Austin

274 et al. 1995; Widanarni et al. 2003) and shrimp vanname (Gomez-Gil et al. 2000). Unfortunately,

275 this bacterium has a different story on seaweed. It is found as pathogens in K. alvarezii. It has

276 been identified by Aris (2011) on seaweed thallus, length with 1 mm, bleaching phenomenon

277 first seen in 12 hours post-infection with the bacterium V. alginolyticus PNGK 1. Yet, V.

278 alginolyticus strain isolated from K. alvarezii was apparently different with V. alginolyticus

279 strain reported by Aris (2011).

280 The protoplasm content is higher in a healthy micropropagule and did not seriously than

281 severe micropropagule. Protoplasm indicates occurrence of severe chronic the micropropagule.

282 This is in line with those reported by Quere et al. (2015) that algae disease characterized by

283 reduced number of cell protoplasm.

284

285

286

287

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A

288 289 B C

P CW 290 291 292 Figure 6 Longitudinal histological sections showing of micropropagule, K. alvarezii. (A) 293 Display without infection (control), post-infection by bacteria, overview with not 294 severed areas (B), and severed areas (C). P, content protoplasm; CW, cell wall 295

296 297 298 Figure 7 Scanning electron micrographs illustrating the presence of surface bacteria and the 299 condition of the surface cellular layers of the control thallus of K. alvarezii. Magnify 300 5,000x 301

302 On ecological component, the occurrence of ice-ice on seaweed is predominantly influenced by

303 unsteady temperature, salinity and light intensity (Largo et al. 1995b). In this study,

304 micropropagule as product of tissue culture disease-free use and challenge test carried out under

305 the same conditions at the indoor laboratory. The result is negative and control micropropagule

306 also showed symptoms of bleaching at 23 hours post-infection. It came out that stress during

307 the preparation of the micropropagule cause of bleaching. Interaction of pathogenic bacteria

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308 with environmental stress-related illnesses ice-ice on K. alvarezii was conducted with 20 ppt

309 salinity and it decreased concentration of bacteria 103 and 104 cfu/ml (Largo et al. 1995a).

310 However, interaction of temperature with different disease-causing pathogens ice-ice on

311 seaweed is unreported and it is thought-provoking to examine in further research. In addition,

312 the bacteria isolated in this study is potentially used to hold the assembly seaweed ice-ice

313 disease through the challenge test.

314 315 CONCLUSION 316 317 Among eight isolates of seaweed ice-ice disease tested on pathogenicity concluded that

318 the leading candidate Stenotrophomonas maltophilia triggers ice-ice disease on K. alvarezii.

319 320 321 322 AKNOWLEDGEMENT 323 324 We wish to thank the Ministry of Research and Technology Republic of Indonesia for the

325 Scholarship of doctoral program 2011. Seameo Biotrop Indonesia for micropropagule

326 materials. Amalia Nur Anshary, Dendi Hidayatullah and Hasan Nasrullah kindly assisted with

327 identification of invading bacteria and interpretation of the dendogram.

328 REFERENCE

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Table 2 - Figure of sequencing

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Figure 2 - figure of phylogeny tree

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Figure 3 - Figure of time early emergence of bleaching

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Figure 4 - figure of others indicator of bleaching

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