Etiological Study On Pyropia/Porphyra Yezoensis And Screening Of Agarase-producing Bacteria

Pyropia/Porphyra yezoensis is one of the representative species of Rhodophyta,the concern of its origin, evolution and environmental stress adaptation mechanismsand other scientific issues making it one of the mode species of algae, P. yezoensishas important scientific value. P. yezoensis natural located in the intertidal zone of theNorthwest Pacific coast, is one of the dominant species in winter and spring withimportant ecological value. At present, P. yezoensis was large-scale cultivated inChina, Japan and South Korea, with an annual turnover of tens of billions of dollars,has an important economic value. But in recent years, along with the continuedexpansion of the cultivation scale, the frequency occurrence, size and harm degree ofdisease also increased year by year. The etiology and pathogenic conditions study ofP. yezoensis disease, not only provide the basis for the development of diseaseprevention and treatment measures, but will also provide the conditions for thescreening of the resistant lines. The study separated of P. yezoensis pathogens,in-depth study of its causes as well as the influence of environmental factors on theoccurrence of disease.Green spot disease is a common disease of P. yezoensis on gametophyte larvalperiod. The pathogen was isolated, cultured and corroborated by the artificialinfection experiment, the molecular biology analysis and physiological andbiochemical characterization results showed the pathogen was Pseudoalteromonascarrageenovora. the pathogen was Gram-negative rod-shaped bacterium with highprotease and lipase activities, in addition of a variety of aromatic amines enzymereaction were positive. The study found that the pathogenicity of the pathogen relatedwith environmental factors, the results showed that higher temperature and stockingdensity will accelerated the spread of the disease, but seawater proportion within acertain range changes does not affect the occurrence of the disease. The red rot disease is one of the most serious diseases of P. yezoensis production.The study material was P. yezoensis thallus infected by suspected red rot disease, andthe cytology and submicroscopic structure of the lesion organizations was observed.The pathogen was isolated, cultured and corroborated by the artificial infectionexperiment and identified by molecular biology analysis. There are a few spots ofdifferent sizes on the sick fronds, the central of the spot is green, arounding this partare red color, but at the edge of these spots there is a bright purple belt. Observedunder the optical microscope, It was found the infected cells had lost its normalstructures and penetrated by the mycelia which is similar with the red rot disease. Thepathogen was analyzed by the ITS sequence blast, results showed that the fungus wasAlternaria sp., It is not consistent with the typical red rot pathogen.The algae pathogens usually have the ability to degrade seaweed gel. In thisstudy, agarase-producing bacteria isolated from rotten seaweeds and holothurianintestinal tract, including identification of strains, the optimal culture conditions, theoptimum enzyme production conditions and agarase gene clone. Through screeningand rescreening, a total of four agarase-producing bacteria with a higher agardegradation capacity was isolated, respectively named HD, JL, QJ and HS. Relativecrude enzyme activity assay results showed that4agarase-producing bacteria culturedin the the2216E seawater medium had the best enzyme production state, the bestenzyme production conditions and the optimum growth conditions are uniform, Themaximum relative enzyme activity of the four agarase-producing bacteria are similarand extracellular enzyme activity is far greater than the intracellular enzyme andgenerally reached the maximum enzyme activity in36h. They are all rod andGram-negative bacteria. The16SrDNA sequence identification and physiological andbiochemical characterization results showed strains HD, JL and HS belongs toAgarivorans sp., the homeology between QJ and relatived sequences was less than95%, it may be a new genus or species. Strains HD, JL and HS can clone β-agaraseenzyme gene with a length of2868bp, but the strain QJ cannot. Theagarase-producing bacteria can be used to test the pathopoiesia ability to algae, on the other hand it had application prospect as a enzymatic tool for porphyrazymohydrolysis to make single-cell or protoplast.