| Saccharina japonica is one of the significant economic seaweeds cultivated in China,and has very important economic and ecological values.With the development of kelp cultivation industry,diseases frequently occur due to environmental pollution,germplasm degradation,poor management and other reasons,causing serious economic losses.At present,compared with foreign scientists,there is not much attention paid on kelp diseases in China,with the focus is mostly on the research of pathogen isolation and identification.The present study investigated several kelp diseases occurred at the nursery and cultivation stage,and studied the etiology with green rot disease as the focus,in the hope of providing scientific basis for the research on the identification and detection of pathology,prevention of kelp pathogens,so as to effectively reduce risks and improve economic and ecological benefits from kelp cultivation.The present study firstly investigated the diseases that occurred during the nursery and cultivation periods of kelp,including sporeling malformation disease,green rot disease,rotten hole disease,yellow bubble disease,black spot disease,during which,the detection of aquaculture seawater quality;isolation and identification of culturable bacteria on diseased algae were both performed;Furthermore,the activity of some extracellular enzymes for the culturable bacteria isolated at seedling stage were also analyzed,which includes alginate-degrading enzymes,caseinase,pectinase,and cellulose.activities of culturable bacteria isolated at seedling stage;Making the green rot disease of kelp seedings as the primary object,the relationships between environmental factors,microorganisms and disease occurrence of kelp seedlings were investigated under laboratory conditions based on the data of water quality and the extracellular enzyme activity;The activity variations of immune defense enzymes during kelp seedling green rot.The research results will provide basic data and support for the kelp diseases investigation,and provide a scientific basis for effective monitoring and prevention of kelp seedling green rot.The specific research contents and results are as follows:1.Investigations on disease occurrence during kelp nursery and cultivation.From September 2018 to September 2019,investigations were conducted on diseases that occurred in kelp nursery farms or cultivated sea areas in the cities of Weihai,Qingdao,and Putian.During the nursery period,sporeling malformation disease occurred to the nursery curtains with various degrees in a seedling farm in Weihai,Shandong,in September 2018 and September 2019,respectively.The physicochemical factors of seawater within nursery ponds shows that temperature,p H,salinity,and nutrient contents(7.00?8.40?,7.10?7.90,32?33‰,N: 3.06?4.18 mg/L,P: 0.20?0.47 mg/L)are normal during the nursery stage.Green rot disease occurred in another nursery farm in Weihai,Shandong in October 2018,which spread rapidly and caused massive deaths of young sporophytes within a week.It is shown that temperature,p H,salinity and illumination indicators are within normal ranges(7.80?8.60?,8.00?8.10,34?35‰,4100?4300 lux),contrasting to the nutrient contents(total nitrogen: 30?35 mg/L,total phosphorus: 2.10-2.20 mg/L)which were significantly higher than the recommended dosage in the kelp nursery regulations.Hole rotten disease struck a cultivation area in Putian,Fujian in March 2019 during kelp cultivation.Detection of seawater quality showed that there were obvious salinity and temperature stratification,with the salinity was 2?3‰ lower than that of the normal sea area while the water temperature was about 2 ? higher than the same period of previous years,though p H(8.14?8.21),total nitrogen(0.32?0.34 mg/L),and total phosphorus(0.02?0.03 mg/L)indicators fell into normal ranges.Yellow bubble disease occurred in a cultivation area in Weihai,Shandong in April 2019.Black spot disease occurred in a cultivation area in Qingdao,Shandong in July 2019.Water quality factors were not detected in cultured water bodies for yellow bubble and black spot diseases.The hole rotten disease was relatively severe during kelp cultivation.2.Isolation and identification of cultureable bacteria.Culturable bacteria were isolated from samples of diseased kelp collected in 2018-2019,and phylogenetic tree analysis was performed on the bacteria isolated during the nursery stage.A total of 15 genera and 46 strains were isolated from kelp seedlings with sporeling malformation disease in the year of 2018,with the dominant genus determined to be Yangia.It is shown that these bacteria mainly distributed in 4 phyla/subphyla,most of which were affiliated with Alphaproteobacteria,accounting for 67.39% of the total isolated bacteria.This was followed by strains belonged to Gammaproteobacteria(19.56%),Firmicutes(8.70%),and Bacteroidetes(4.35%),respectively.In September 2019,a total of 126 strains of 11 genera were also isolated from such disease kelp sporelings in the same nursery farm,and a high proportion of Yangia bacteria were still determined.In addition,Thalassospira,Joostella,and Alteromonas bacteria also accounted for a high proportion.These bacteria mainly distributed in 3 phyla/subphyla,within which Alphaproteobacteria accounted for 61.11% of the total isolated bacteria,followed by Gammaproteobacteria for 26.98%,and Bacteroidetes for 11.91%.A total of 28 strains belonging to 7 genera were isolated from the kelp seedlings with green rot disease,and the dominant bacteria were determined to be Yangia,Pseudoalteromonas.A total of 31 strains belonging to 8 genera were isolated from the kelp seedlings with green rot disease,and the dominant bacteria were determined to be Yangia,Pseudoalteromonas,and Glaciecola,which mainly distributed in 3 phyla.Among them,most strains were assigned as Gammaproteobacteria(45.16% of the total isolated bacteria),followed by Alphaproteobacteria(38.71%),and Firmicutes(16.16%).A total of 78 strains belongin to 16 genera were isolated from kelp and the associated water samples regarding hole rotten disease,with the genus Cobetia dominated.A total of 36 strains within 11 genera were isolated from kelp with yellow bubble disease,and the dominant bacterial taxa was the genus Psychrobacter.A total of 41 strains within 5 genera were isolated from kelp struck by black bubble disease,and the dominant bacteria was determined to be the genus of Psychromonas.3.Detection of extracellular enzyme activity of cultured bacteria.Diseases occurred at the nursery stage are more serious due to the huge economy loss.In the present study,the activity of extracellular enzymes,such as alginase,caseinase,pectinase,and cellulose,were detected for bacteria isolated from kelp sporophytes struck by sporeling malformation disease and green rot disease.A total of 203 culturable bacteria strains were tested with no pectinase and cellulase activity were detected for all the strains.Regarding the bacterial strains isolated from malformation sporelings,38 strains distributed in 8 genera,were determined to occupy alginate-degrading activity,which was prevalent by 13 strains of Yangia bacteriaand followed by 8 strains of Thalassospira,5 strains of Alteromonas,5 strains of Joostella,4 strains of Halomonas,1 strain of Flavobacterium,1 strain of Roseobacter,and 1 strain of Pseudomonas,,respectively;14 strainswith caseinase activity were detected in 8 genera,including 3 strains of Bacillus,4 strains of Pseudoalteromonas,1 strain of Vibrio,1 strain of Yangia,2 strains of Thalassospira,1 strain of Marinobacter,1 strain of Idiomarina and 1 strain of Saccharospirillum.For bacteria isolated from kelp seedlings of green rot disease,alginic acid degrading enzyme activity were detected in a total of 10 strainswhich included 3 strains of pseudoalteromonas,6 strains of Yangia and 1 strain of Marinomonas;while 7 strains with caseinase activity were detected in 3 genera,including 5 strains of Pseudoalteromonas,1 strain of Yangia,and 1 strain of Tenacibaculum.4.Cause analysis of kelp seedling green rot.Green rot is a common disease during kelp nursery.In 2018,it is found that enrichment of nitrogen and phosphorus may be a possible cause of green rot disease through the investigation in a kelp nursery farm located in Shandong Province.In the present study,the analysis of water quality,simulation experiments of nitrogen-phosphorus enrichment,isolation and identification of dominant bacteria,and there re-inoculation and infection assay,and the orthogonal experiment of Yangia strain under different temperature,light and nutrient conditions were performed to analyze the cause of kelp seedling green rot.Simulation experiments showed that green rot occurred to kelp seedlings under the condition of 10 ?,2000?4000 lux,and nitrogen-phosphorus enrichment in terms of temperature,light and nutrient.The dominant epiphytic bacteria isolated from green rot kelp were affliated with the genus Yangia and Glaciecola.Re-inoculation and infection of such strains demonstrated that green rot occurred to kelp seedlings in the control group and experimental group with Glaciecola.DH-2-3,and no green rot in the experimental group with Yangia.SJ-H-12 under the condition of 10?,4000 lux,and nitrogen-phosphorus enrichment in terms of temperature,light and nutrient.And green rot occurred to kelp seedlings in the experimental group and the control group with Yangia.SJ-H-12 under the condition of 14?,2000 lux,and nitrogen-phosphorus enrichment in terms of temperature,light and nutrient.Based on these results,the causes of kelp seedling green rot were analyzed from the perspective of the interactions between environmental factors and microorganisms,it was found that excessive nutrient is the environmental stress condition that causes the seedlings to green rot,and Glaciecola.DH-2-3 may aggravate this kind of green rot,and Yangia.SJ-H-12 bacteria may inhibit the green rot of kelp seedlings at low temperature.The activity changes of immune defense enzymes in kelp seedlings were also detected during green rot.The results showed that the protein concentration displayed a decreasing trend firstly,followed by a increasing and then decreasing trend,which totally showed a downward trend;total antioxidant capacity increased and then decreased;the activity of superoxide dismutase increased firstly,and then increased again after a decrease,which resulted in an overall upward trend and was similar to that of polyphenol oxidase.The present study has certain significance for the monitoring and early warning for disease occurrence of kelp seedlings. |
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