| Harmful algae blooms(HABs) have become global marine ecological disaster due to its enormous perniciousness. Phaeocystis globosa, as a preponderant HABs-causing species in the South China Sea, frequently outbroke and is associated with nuisance foam, oxygen depletion and toxic hemolytic substances, causing fish and shellfish mortality. Sulfurous organic compounds released by P. globosa have a negative effect on marine environment and speed up the formation of acid rain. Prevention of P. globosa red tide has become a problem demanding prompt solution. In the recent years, the control of HABs by algicidal bacteria has draw extensive attention. However, it is the basement of research to screen algicidal bacteria, and the inhibition mechanism is key issue of further elucidating red tide control by biological method. In this study, marine bacteria showing significant algicidal effct against P. globosa were isolated from Pearl Sea. The physiological properties and bacterial genus of algicidal bacteria were studied and identified, then algae-lytic characteristics of algicidal active substances were preliminary researched. Cell morphology, physiological and biochemical indexes were detected to explor algicidal mechanism from the view of programmed cell death. This study might provide theoretical foundation for finding new algicidal microbes and further understanding indirect algal-inhibition by algicidal bacteria. The main contents were as follows:A total of 23 strains were isolated from the water samples by streak plate method. Strain Ts-6 and Ts-12, exhibited siginificant inhibitory effect on P. globosa, and algicidal rate was 88.6% and 91.3%, respectively. The colonial morphology of strain Ts-6 was orange, and diameter was about 3-5 mm. A single bacterium was rod-shaped and gram-negative. However, the colonial morphology of strain Ts-12 was hoary, 2-3 mm. Ts-12 was also rod-shaped, but it was gram-positive. Strain Ts-6 could utilize glucose and starch, also utilize tryptophan to produce indole; while Ts-12 could utilize glucose, maltose, sucrose, also break down sulphur-containing amino acids to produce hydrothion. The bacteria were identified by 16 S rDNA sequencing analysis, and phylogenetic tree indicated that strain Ts-6 belonged to Shewanella, and strain Ts-12 belonged to Bacillus.Algicidal effect of both the strains was inhanced with the increase of bacterium solution volume and treatment time. Strain Ts-6 and Ts-12 indirectly inhibited P. globosa by secreting etracellular active substances. The mixture of Ts-6 supernatant and Ts-12 supernatant at a ratio of 1:3 showed optimal algicidal rate. Strain Ts-6 also showed strong inhibitory effect on Prorocentrum donghaiense, secondary effect on Heterosigam akashiwo, but inferior effect on Skeletonema costatum. However, strain Ts-12 showed modest effect on P. donghaiense and S. costatum, but weaker effect on H. akashiwo. Algicidal compound of Ts-6 supernatant showed better pH-stability, however, strain Ts-12 supernatant showed lower pH-stability, and the optimum pH range was 5.0-10.0. Algicidal substances from the two bacteria were polar and easily extracted by polar organic solvents, such as dichloromethane, ethyl acetate. However, main algicidal substances of the two bacteria were not biological macromolecular materials, such as protein and nucleic acid.Ts-6 supernatant caused oxidative stress, which further increased cellular ROS level and decreased photosynthetic pigment content, including chlorophyll and carotenoid. Some P. globosa cells treated with the supernatant underwent programmed cell death(PCD): in the early stage of PCD, phosphatidylserine of the cells translocated from the inner face of the plasma membrane to the outer surface. However, because of continuous cell death stimuli, most of the algal cells lost their membrane integrity, eventually necrosed. The activity of Caspase 3-like enzyme, as the final executor of apoptotic cell death, significantly increased, which probably regulated PCD of P. globosa. Meanwhile, the algal cells undergoing PCD showed chromatin condensation and DNA fragmentation. In addition, the chromatin structure of some necrotic or late apoptotic cells gradually disappeared. |
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