Etiology Analysis Of Bacterial Diseases In Porphyra Yezoensis And Laminaria Digitata

Seaweed culture is an important part of China's mariculture industry.The total output of seaweed aquaculture in 2018 reached 2.35 million tons,accounting for 8.72%of the total annual aquaculture products.The algal aquaculture brings considerable economic benefits while the algae diseases gradually become an important bottleneck restricting the healthy development of algae aquaculture.Furthermore,the research on algae diseases is still in the initial stage.In this study,the Porphyra yezoensi"yellow spots"and Laminaria digitata"green rot"were investigated.The etiology,pathology,infection microecology and other technical methods were used to analyze the pathological characteristics of P.yezoensis and L.digitata pathogen,and the surface microflora structure of pathogen infected algae was analysis.The results were further enhance the research foundation of algae diseases in mariculture,and provide theoretical support for the development of prevention and control technology of algae diseases and the guarantee of healthy cultivation of algae.The results are as follows:1. The etiology analysis of P.yezoensis conchocelis"yellow spot symptom"P.yezoensis conchocelis were collected from Lanshan,Shandong Province,and the clinical symptoms of the disease P.yezoensis conchocelis was present different sizes of yellow spots in the attachment.With the aggravation of infection,the yellow brown gradually deepened and finally ulcerated and died.The P.yezoensis conchocelis with typical yellow spot symptoms were collected and cultured in vitro,the dominant strain PZ02 was isolated and identified by Gram straining,morphological observation,physiological and biochemical identification and 16Sr DNA sequence analysis.Furthermore,the pathogenicity of PZ02 was confirmed by regression infection analysis.The results show that:?1?PZ02 is a gram-negative bacterium with short rod-shaped and a single flagellum,the cell size was?0.5-1.0?um×?1.5-5.0?um.PZ02 is similar with Pseudomonas luteola based on 16S r DNA analysis,and the similarily is arrived at 100%.?2?The physiological and biochemical results showed that PZ02 could grow in the0.0%,6%and 10%Na Cl tryptone water,and with positive reaction for oxidase,3%Na Cl ONPG,ornithine decarboxylase,lysine decarboxylase and arginine double hydrolase.PZ02 could assimilate and utilize glucose and inositol,but not grow in excepted sucrose,lactose,arabinose,rhamnose,xylose,dense disaccharide and sorbitol.The physiological and biochemical characteristics indicated that PZ02 was consistent with Pseudomonas.Furthermore,the drug sensitivity test showed that PZ02 was highly sensitive to lomefloxacin,fleroxacin,chloramphenicol,penicillim,gentamicin,kanamycin,haloperic acid,etc.,and resistant to pipemidic acid,nalidixic acid,tetracycline,cefazolin,cefalexin,cefradine,florfenicol,azithromycin,neomycin,polymyxin,ampicillin,oxacillin.?3?Artificial infection showed that healthy P.yezoensis conchocelis emerging typical symptoms of natural macular disease with different sizes of yellow spots after 13d infection with 10⁸ CFU/m L of PZ02 suspension,but the 10⁶CFU/m L and 10⁷CFU/m L groups were noneffective.The reidentification of etiology show that the same strains were obtained from the filaments of P.yezoensis conchocelis.So the results indicated that PZ02was the pathogen of"yellow spot"symptom of P.yezoensis.2. The etiology and histopathology analysis of L.digitata"green rot symptom"The etiology and histopathology of L.digitata breeding in Rongcheng,Shandong Province with green rot symptom were collected and analyzed.The clinical symptoms was the color of L.digitata changed from brown to light and rot leaves.LD01 was isolated from the rot leaves,furthermore,the morphology,regression infection test,biochemical identification and 16S r DNA were analysis.?1?LD01 was identified as Vibrio alginolyticus,with the similarily was 99.34%.Artificial infection showed that healthy L.digitata emerging typical symptoms of green rot symptom after infection with 10⁸ CFU/m L of LD01 suspension,but no obvious symptom in experimental groups with concentration less than the 10⁸ CFU/m L group.The reidentification of etiology show that the same strains were obtained from the artificial infected L.digitata.The results indicated that LD01 was the pathogen of green rot symptom of L.digitata.?2?The physiological and biochemical results showed that LD01 could grow in the3%,6%and 10%Na Cl tryptone water,and with positive reaction for oxidase,ornithine decarboxylase,lysine decarboxylase.LD01could assimilate and utilize sucrose and mannitol,but not grow in0.0%Na Cl tryptone water,negative reaction in arginine dihydrolase,3%Na Cl MR-VP,3%Na Cl ONPG,and not useglucose,arabinose,lactose,inositol,amygdalin,muscle Alcohol,rhamnose,honey disaccharide.Furthermore,the drug sensitivity test showed that LD01 was highly sensitive to ofloxacin,fleroxacin,nalidixic acid,tetracycline,doxycycline,florfenicol,mycobacterium,azithromycin,amikacin,ceftazidime,cefotaxime.And resistant to ciprofloxacin,acetylspiramycin,polymyxin,neomycin,streptomycin,kanamycin,ampicillin,penicillin,oxacillin,co-trimoxazole,cefalexin,cefradine pipemidic acid.?3?The results of histopathological and ultrastructural studies showed that the cell wall of the endothelial layer of the algal body was broken,followed by the outer layer and the medulla.Under the scanning electron microscope,it was found that many kinds of organelles in algal cells were affected.A large number of bacteria were adhered to the surface of the algal.The cell wall was broken with the passage of time.The results of transmission electron microscopy?TEM?showed that chloroplasts were the most sensitive organelles after the infection of V.alginolyticus,followed by the nucleus.The nucleoli dissolved with the extension of time.?4?Chlorophyll fluorescence spectrum detection showed that compared with the control group,the maximum fluorescence yield(F_v/F_m)and the potential activity?F_v/F₀?of photosystem?reaction center in the infected group decreased with the V.alginolyticus concentration,which indicated that the activity of both the donor side and the reaction center of photosystem in the leaves of the infected group could be affected by the pathogens.3. Microbial community structure characteristic of L.digitata based on high throughput sequencingHigh-throughput sequencing technology was used to analyze the microbial community structure of L.digitata,the results was as follow:A total of 91784 optimized sequences were obtained and 790 OTUs could be classified into 10 phylum and 9 genera.Based on the phylum levels,Cyanobacteria?>90%?was the dominant phylum in healthy groups.For diseased groups,there are two dominant phylum,Cyanobacteria?>46%?and Proteobacteria?>50%?,followed by Bacteroides?>1%?and Planctomycetes?>1%?.Based on the genus levels,the dominant genera in healthy groups were Psychromonas?>1%?and Blastopirillula?>1%?,the relative abundance of unknown species in healthy group which can not be detected sequence was more than 90%.The dominant bacteria in the affected group were Vibrio?>30%?,followed by Psychromonas,Pseudoalteromonas,Marinemononas,and Sulfitobacte.The results indicated that there were similar microbial community but significant abundance between healthy and disease samples.In addition,at the genus level,Vibrio is the primary dominant bacteria in the diseased L.digtata group.