| Hamful algal blooms have become a critical world-renowned problem.Algicidal activities of fungus against the bloom-forming algae have attracted increasing attention.However,there are relatively few studies on the inhibition of fungi on cyanobacteria.The aim of this study was to isolate the fungi with the algicidal ability against the bloom-forming cyanobacterium Microcystis,and to further investigate the algicidal mechanism.28 fungal strains were isolated from Lake Taihu of China,and 4 of them were found to have algicidal activity against M.wesenbergii NJ-24 on agar plate.Based on the result of molecular identification,the four strains were Trichoderma asperellum SHS3,T.asperellum SHS4,T.brevicompactum F3302,and uncultured Penicillium F20.The morphology of T.asperellum SHS3 and T.asperellum SHS4 was quite different.To evaluate the algicidal activity the freeze-dried fungal mycelia pellicle was inoculated into liquid algal culture.After incubation of 5 days,the algicidal rate of 3 fungus strains T.asperellum SHS3,T.asperellum SHS4 and uncultured Penicllium F20 were 96.0%,96.4%and 95.5%.T.asperellum SHS3 exhibited significant inhibitory effects on the three species of cyanobacteria.After incubation of 5 days,compared with the controls,the chlorophyll a(Chi a)contents of the treated cultures of M.wesenbergii NJ-24,M.aeruginosa PCC7820 and Synechocystis sp.PCC6803 were decreased by 96.0%,98.5%and 88.2%,respectively.On the contrary,T.asperellum SHS3 significantly increased the growth of the green alga C.pyrenoidosa FACHB-5.After co-culture with T.asperellum SHS3 for 5 days,the Chi a content of the culture of C.pyrenoidosa FACHB-5 was increased by 28.8%compared with the control.The isolated T.asperellum SHS3 exhibited significant inhibitory effects on the growth of Microcystis flos-aquae NJ-183.After co-culture with T.asperellum SHS3 for 5 days,the Chi a content of the culture of M.flos-aquae NJ-183 was decreased by 45.3%,and the colony surface areas decreased by 64.0%compared with the control.Also,the arrangement of cells in colonies became loosen.The hydrophobicity and autoaggregation ability of M.flos-aquae NJ-183 treated with T.asperellum SHS3 decreased significantly compared with the control.The algicidal activity of extracellular products from T.asperellum SHS3 was further investigated.The fresh fungal filtrate of Trichoderma asperellum SHS3 had strong algicidal activity,but the heat-treated fungal filtrate had no algicidal activity.The filtrate of the fungal culture which was dialyzed with 3.5 kD pore size dialysis tubes significantly inhibited the cyanobacterial growth,indicating that some macromolecules secreted by the fungus inhibited the cyanobacterial growth.Finally,the algicidal activity of the fungal extracellular proteins was determined.Ammonium sulfate was added to the filtrate of the fungal culture to precipitate the protein.The extracellular proteins of the fungus exhibited strong algicidal activity.The above results indicated that the algicidal substances of T.asperellum SHS3 may be heat-sensitive extracellular proteins.Microscopic observation of the cyanobacteria M.wesenbergii NJ-24,M.aeruginosa PCC7820 and Synechocystis sp.PCC6803 after negative staining showed that the sheath of most of cyanobacterial cells treated by T.asperellum SHS3 disappeared.The chlorophyll fluorescence confocal images of the Microcystis cells showed that some fluorescence dots were observed outside the cells in the cyanobacterial culture treated by the fungus.The above results indicated that T.asperellum SHS3 might degrade the polysaccharide material of the cyanobacterial sheath through producing extracellular proteins,and in turn broke cyanobacterial cells.In this study,it was found that T.asperellum SHS3 had strong algicidal activity and had the potential to control hamful algal blooms.It was the first time to demonstrate the algicidal activity of fungal extracellular proteins. |
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