Isolation and characterization of two marine algicidal bacteria against the Phaeocystis globosa
Rongjun Yana, b Pinghe Yinb
a College of Chemical Engineering and Materials，Zhejiang University of Technology, Hangzhou, Zhejiang Province 310014, China
b Institute of Hydrobiology, Jinan University, Guangzhou, Guangdong Province 510632, China
Abstract
During a red tide survey of the Zhuhai Estuary in southern China, we isolated two bacteria strain, designated Y01 and Y04, which had potent algicidal effects on harmful algal bloom (HAB) species Phaeocystis globosa. Genetic identification was achieved by polymerase chain reaction amplification and sequence analysis of 16SrDNA. Sequence analysis showed that the most probable affiliation of Y01 and Y04 were to the genus Bacillus. Bacterial isolate Y01 and Y04 showed algicidal activity through a contact attack. This is the first report of algicidal bacteria against P.globosa and the founding certain bacteria could play an important role in regulating the onset and development of HAB.
Keywords: Phaeocystis globosa; Algicidal bacteria; 16S rDNA; Bacillus; Harmful algal blooms
1. Introduction
The prymnesiophyte Phaeocystis globosa is a characterized of a harmful algal bloom (HAB) species with the potential to generate very high biomass, dominate the phytoplankton community for extended periods and be a nuisance to tourism, fisheries and the ecosystems (Brussaard et al, 1996). Lysis was identified as the major fate of ungrazed P. globosa in the North Sea and in the eastern part of the English Channel. Lysis can also be the result of viral infection such as a strain-specific virus for P. globosa (Brussaard et al., 2005). No bacterial strain algicidal to P. globosa has previously been isolated so far. In this study, two strains of bacteria algicidal to P. globosa were isolated from the red-tide area in the Zhuhai Estuary in southern China. Here we report identification, characteristics of the isolated bacteria algicidal activities.
2. Materials and methods
2.1 Isolation of algicidal bacteria and screening for algicidal activity.
A sea water sample was collected from a depth of 1m at a station (22°28’91”N and 113°58’82”E) in Zhuhai Estuary of southern China, during a bloom of P. globosa occurred on 25 February 2005. Samples were 10-fold serially diluted and 0.1mL of each dilution was spread onto Zobell 2216E agar plates followed by incubation for 7 days at 25°C. For screening of algicidal bacteria, bacterial isolates were grown in 5mL 2216E broth (25°C, 180 rpm) for 12–24 h. Bacteria were inoculated into P. globosa from colonies. The growth of P. globosa was monitored by measuring the fluorescence of the alga cultures at an excitation wavelength of 450nm and an emission wavelength of 680nm, and also by microscopic observation. Two bacterial strains were found to be algicidal against P. globosa. The bacteria were designed to be Y01 and Y04. These bacterial strains were kept at −80 °C in 10% (v/v) glycerol.
2.2 Sequencing and identification of phylogenetic analysis
Bacterial cells of the strains Y01 and Y04 were grown in 2216E broth and were collected by centrifugation (5000 × g for 10 min).PCR products were checked using 1% agarose gel electrophoresis. The amplified 16SrDNA was purified from agarose gel with a bacterial identification PCR Kit (Code No.D310) and ligated with TaKaRaAgarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A). PCR products of 16SrDNA were sequenced directly, the sequence was aligned and a phylogenetic tree was performed by DNAMAN 6.0 software with known algicidal bacterial sequences obtained from GenBank .
3. Results
3.1 Screening algal isolates for susceptibility to algicidal activity
The cytolytic effects of Y01 on P. globosa cells during the algicidal process were observed under a light microscopy (Fig.1). Normal axenic P. globosa cells are shown in Fig.1a. When the algicidal bacterium Y01 took effect, the swimming movements of P. globosa cells were inhibited and cell walls detached from the main cellular bodies (Fig.1b). With longer exposure time (2hours), P. globosa cells were bursted and cellular substance was released and decomposed (Fig.1c), resulting in the appearance of abundant broken fragments (Fig.1d).
3.2 Identification of strains Y01and Y04
The bacterial sequences obtained in this study were deposited in the GenBank database and were assigned accession numbers as follows: Y01 (DQ531607); Y04 (DQ531608). Fig.2 showed both Y01 (DQ531607) and Y04 (DQ531608) belonging to Bacillus.
4. Discussion
Most algicidal bacteria characterized belong to the Cytophaga/Flavobacterium/ Bacteroidetes(CFB) group or to the Gamma-Proteobacteria group (Imai et al., 1995; Yoshinaga et al., 1995). Of the 56 algicidal strains noted above, about 50% belong to the CFB group while about 45% are members of the Gamma -Proteobacteria, with the remaining strains representing the gram-positive genera Micrococcus, Bacillus, and Planomicrobium. Although there is no conclusive link between phylogeny and the lytic mechanism, the available data suggest that gram-positive bacteria and Gamma-Proteobacteria employ primarily an indirect mode of attack, while the lytic activity of the CFB group can involve either direct or indirect interactions with target cells(Roth et al, 2008). Given the frequent dominance of the CFB group and its inclusion of many known algicidal bacteria, it is likely that such bacteria will prove to be more abundant than previously thought. Fig.2 showed both Y01 (DQ531607) and Y04 (DQ531608) belonging to Bacillus.
Lysis was identified as the major fate of ungrazed P. globosa in the Dutch coastal waters of the North Sea (Brussaard et al., 2004). We were demonstrated that bacteria infecting the bloom forming alga P. globosa can be readily isolated from waters in which the bloom occurs. Based on the increase in putative several bacteria capable of inhibiting and killing various HAB species have been isolated, providing possible choices for controlling harmful algal blooms by human interference. Studies on the bacterial–algal interactions are also helpful for a better understanding of Phaeocystis-dominated ecosystems to regional and global marine production as well as to global C and S cycles in the marine ecosystem. Phylogenetic analysis of DNA sequences indicates and susceptibility to algicidal activity will stimulate research on algicidal mechanisms. When assessing the potential role of algicidal bacteria not only as natural regulators of bloom dynamics, but also as part of a HAB management and control strategy.
Acknowledgments
This work was supported by the National Science Foundation of China (No.200277016) and the 973 project (No. 2001CB409710). We would like to thank Professor Yuzao Qi of Jinan University for providing the P. globosa cultures.
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Fig.1Algicidal bacterium Y01 infecting the Phaeocystis globosal cell.
(a) Normal free-living cells of P.globosa of axenic culture;
(b) P.globosa cells after addition of the cells of bacterium strain Y01;
(c)(d) Broken cells of P.globosa by algicidal activities of bacterium strain Y01
Fig.2 Phylogenetic tree of known algicidal bacteriaThe scale bar indicates 5% sequence divergence.
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