| Pyropia(Bangiales,Rhodophyta),also known as laver,is an important proportion of aquaculture industry in China.This species has been suffered from several diseases due to the increasing of culture quantity density,which seriously affects sustainable development of the laver industry.Research on Pyropia diseases are relatively late and weak in China.A lot of work needs to be started to clarify the diseases,the relevant pathogens,the disease pathogenesis and the disease prevention and control methods.In this paper,we first start a preliminary investigation on diseases occurrence of Pyropia yezoensis from seeding stage to sea cultivation period.Then,we focused on the yellow spot disease,a high incidence of shell filamentous disease at seeding stage,by characterization of the cultivable bacteria from normal and diseased samples of filamentous laver,and identification of the potential pathogenic bacterial species through artificial infection tests.Finally,we developed a PCR method to detect green spot pathogen in P.yezoensis.These results will provide basic data and support for epidemiological study of Pyropia in China.Following is the contents and results.1.Investigation on diseases of seedling and cultivation period.From August 2017 to January 2019,diseases occurrences were investigated in more than ten farms in Jiangsu,Zhejiang and Shandong province.During seedling period,most of the farms suffered from different diseases,such as yellow spot disease,white spot disease,shark skin disease,etc..Yellow spot disease was the most common disease,followed by white spot disease.Some ponds suffered from the two diseases,which could lead to the whole lost of the farm.There was no significant difference in water physical and chemical indexes between normal and diseased ponds.During cultivation period,red rot disease and Olpidiopsis disease were the most common.Two kinds of oomycetes were identified in two epidemic areas,namely,Pythium chondricola and Olpidiopsis pyropia.2.Analysis of cultivable microorganisms isolated from P.yezoensis seedling.AtSeptember 2017 and October 2018,samples of water and shell-boring conchocelis respectively were collected from yellow spot disease ponds and normal ponds,and proceed to microorganism isolation and identification.A total of 571 bacterial strains belonged to 79 genus,and 8 actinomycete strains belonged to 5 genus were isolated and identified..Proteobacteria(81.28%)was the most predominant in abundance,followed by Bacteroidetes(10.25%),Firmicutes(6.55%)and Actinobacteria(1.92%).By compared the diseased and the normal samples,Vibrionaceae,Rhodobacteraceae,Flavobacteriaceae,Halomonadaceae,Pseudoalteromonadaceae and Oceanospirillaceae from the diseased samples were obviously higher in abundance than those from the normal samples.While Rhodospirillaceae and Erythrobacteraceae from the normal samples were obviously higher in abundance than those from the diseased samples.The isolated bacterial strains were assayed for the extracellular enzyme activity.The result showed that 319 strains had caseinase activity,288 strains had amylase activity,377 strains had gelatinase activity,35 strains had lipase activity and 5 strains had agarose enzyme activity,and no pectinase and cellulase activity were found.Majority strains isolated from yellow spot disease samples presented caseinase,amylase,gelatinase and agarase enzyme activity.3.Optimization condition of shell-dissolving of Pyropia yezoensis conchocelis.In seedling stage,shell-dissolving agents are commonly used to dissolve the shell to obtain the conchocelis,which can be used to observe the growth and development of the shell-boring conchocelis.In order to reduce the effect of shell-dissolving agents on conchocelis,we analysed the effects of two kinds of shell-dissolving agents,the Perenyi’s solution and the acetic acid solution,on the shell-boring conchocelis at different concentration and different immersion time.The results showed that Perenyi’s solution was better than the acetic acid solution.The optimum working time for Perenyi’s solution was 90 s of immersion,when the conchocelis was undergo the least morphological damage.4.Identification and pathogenesis of bacterial pathogens of yellow spot disease.When the diseased shell-boring conchocelis were co-cultured with the normal shell-boring conchocelis,several needle spots appeared as yellow-color were observed on the shell after 7 days of co-infection.With the development of the course,the number of the lesion spots increased,and diameter of the lesion became wider gradually.Generally,the lesion number increased explosively after 5-7 weeks of co-infection,andthe lesion increased to about 2 mm in diameter.Under the microscope,color of the disease conchocelis turned from brown to orange,then to yellow,and finally disintegrated.For determination the potential bacterial pathogens,17 bacterial strains were selected as pathogen candidates and each bacterial strain was respectively added into the shell-boring conchocelis and the free-living conchocelis of P.yezoensis.The results showed that 5 strains belonged to Ruegeria,Roseovarius,Marinomonas,Maribacter and Flavobacterium could cause the symptoms of yellow spot disease.5.Development of PCR method for detecting green spot disease pathogen of P.yezoensis.Pseudoalteromonas tbzcY1 had been demonstrated as a pathogen of P.yezoensis green spot disease.In this research,we targeted Pseudoalteromonas genus conserved region of dnaA and dnaN gene to design degenerate primers pw-dnaA and pw-dnaN1 for amplifying fragment 1258 and 1054 bp,respectively.Phylogenetic analysis based on 16 S rRNA,dnaA and dnaN genes revealed that the tbzcY1 was clustered with Pseudoalteromonas marina,being 99% sequence similar deposited in GenBank.we established a PCR method to detect P.marina with three species-specific primer pairs(pws-dnaA2,pcs-dnaN2 or pws-dnaN3)targeting dnaA or dnaN gene to amplify fragment 386,253 or 721,respectively.This method could sufficiently distinguish P.marina from 22 species of bacteria.The sensitivity of this method was 4Colony-Forming Units(CFU)of bacteria or 23.7 fg of bacterial genomic DNA per PCR reaction with primer pws-dnaA2.Using this method,we could distinguish the experimental infection laver at the early stage.This method could be used to detect GSD in the Pyropia cultivation,and may provide opportunity for the prevention and treatment of GSD at the early stage. |
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