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

Abstract

Background. Ice-ice disease is still a big challenge for seaweed farming that is characterized with “bleaching” symptom. Bacteria 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: Shewanella 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 Gammaproteobacteria group and O. anthropi is in the Alphaproteobacteria group, A. nicotianae and B. subtilis is in the of Proteobacteria 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.