Cite this paper:
YANG Huichao, YAN Yongwei, LI Jie, TANG Lei, MAO Yunxiang, MO Zhaolan. Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis[J]. HaiyangYuHuZhao, 2020, 38(1): 168-176

Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis

YANG Huichao1,2, YAN Yongwei1, LI Jie1, TANG Lei3, MAO Yunxiang3, MO Zhaolan1,2
1 Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture, Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
2 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;
3 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology;Key Laboratory of Marine Genetics and Breeding(Ocean University of China), Ministry of Education, Qingdao 266003, China
Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease (GSD) in Pyropia yezoensis. To prevent GSD from development and spread, an effective method to detect the pathogen at early GSD infection stages need to be established. In this research, PCR methods were established targeting the dnaA gene (encoding chromosome replication initiator protein) and the dnaN gene (encoding β sliding clamp of DNA polymerase Ⅲ protein) to detect P. marina with three primer pairs pws-dnaA2 (Forward, 5'-ACCGCATTAACGAACTACTCGTG-3'; Reverse, 5'-TGCCATTACCTACAGCATGG-3'), pcs-dnaN2 (Forward, 5'-CTTACAACGTTATCAGCGGC-3'; Reverse, 5'-GTTGAGTATTAAGTGATTGAGTAAGC-3') or pws-dnaN3 (Forward, 5'-ACTTACAACGTTATCAGCGGC-3'; Reverse, 5'-ACTGCTGTTTGAGTCTGCTAAC-3'). Three PCR methods corresponding to the three primer pairs sufficiently distinguished P. marina from 22 bacterial species, thus resulting in detection limits of 4 to 4×102 CFU cells or 2.37×101 to 2.37×103 fg of P. marina DNA per PCR reaction. In an artificial infection experiment of P. yezoensis infected with P. marina, all established PCRs successfully detected P. marina at early GSD infection stages. The results show that the established PCRs are specific and sensitive, and are potential for applications in early diagnosis of GSD in Pyropia.
Key words:    Pyropia yezoensis|green spot disease (GSD)|Pseudoalteromonas marina|PCR detection|early diagnosis   
Received: 2019-02-27   Revised:
PDF (1358 KB) Free
Print this page
Add to favorites
Email this article to others
Articles by YANG Huichao
Articles by YAN Yongwei
Articles by LI Jie
Articles by TANG Lei
Articles by MAO Yunxiang
Articles by MO Zhaolan
Arasaki S. 1947. Studies on red rot of Porphyra tenera. Nippon Suisan Gakkaishi, 13(3):74-90. (in Japanese)
Cao J, Wang S C, Yao C W, Xu Z, Xu X M. 2016. Hypolipidemic effect of porphyran extracted from Pyropia yezoensis in ICR mice with high fatty diet. Journal of Applied Phycology, 28(2):1 315-1 322,
Ding H Y, Ma J H. 2005. Simultaneous infection by red rot and chytrid diseases in Porphyra yezoensis Ueda. Journal of Applied Phycology, 17(1):51-56,
Ding H Y. 2008. Study on development process of green-spot disease in Porphyra yezoensis. Journal of Anhui Agricultural Sciences, 36(11):4 626-4 628, (in Chinese with English abstract)
Egan S, Gardiner M. 2016. Microbial dysbiosis:rethinking disease in marine ecosystems. Frontiers in Microbiology, 7:991,
Feng S Z, Xu H Y, Song Y Q, Zhang H P, Sun Z H. 2016.Phylogenetic analysis of closely related Leuconostoc mesenteroides species based on dnaA and rpoB gene homologues. China Dairy Industry, 44(2):8-11, (in Chinese with English abstract)
Fujita Y, Zenitani B. 1977. Studies on pathogenic Pythium of laver red rot in Ariake sea farm-II:experimental conditions and nutritional requirements for growth. Experimental conditions and nutritional requirements for growth.Nippon Suisan Gakkaishi, 43(1):89-95.
Fujita Y. 1990. Diseases of cultivated Porphyra in Japan. In:Akatsuka I ed. Introduction to Applied Phycology. SPB Academic Publishing, The Hague. p.177-190.
Gachon C M M, Sime-Ngando T, Strittmatter M, Chambouvet A, Kim G H. 2010. Algal diseases:spotlight on a black box. Trends in Plant Science, 15(11):633-640,
Hall T. 2011. BioEdit:an important software for molecular biology. GERF Bulletin of Biosciences, 2(1):60-61.
Han X J, Mao Y X, Li J, Li G Y, Li C, Liu L, Mo Z L. 2015.Identification and pathogenecity of a novel pathogen causing green rot disease in Porphyra haitanensis.Journal of Fisheries of China, 39(11):1 721-1 729, (in Chinese with English abstract)
Kerwin J L, Johnson L M, Whisler H C, Tuininga A R. 1992.Infection and morphogenesis of Pythium marinum in species of Porphyra and other red algae. Canadian Journal of Botany, 70(5):1 017-1 024,
Kim G H, Klochkova T A, Lee D J, Im S H. 2016. Chloroplast virus causes green-spot disease in cultivated Pyropia of Korea. Algal Research, 17:293-299,
Kim G H, Moon K H, Kim J Y, Shim J, Klochkova T A. 2014. A revaluation of algal diseases in Korean Pyropia (Porphyra)sea farms and their economic impact. Algae, 29(4):249-265,
Klochkova T A, Shim J B, Hwang M S, Kim G H. 2012. Hostparasite interactions and host species susceptibility of the marine oomycete parasite, Olpidiopsis sp., from Korea that infects red algae. Journal of Applied Phycology, 24(1):135-144,
Kumar V, Zozaya-Valdes E, Kjelleberg S, Thomas T, Egan S. 2016. Multiple opportunistic pathogens can cause a bleaching disease in the red seaweed Delisea pulchra.Environmental Microbiology, 18(11):3 962-3 975,
Kwak M S, Klochkova T A, Jeong S, Kim G H. 2017.Olpidiopsis porphyrae var. koreanae, an endemic endoparasite infecting cultivated Pyropia yezoensis in Korea. Journal of Applied Phycology, 29(4):2 003-2 012,
Lai P Y. 2009. Investigation and countermeasure of rot disease of Porphyra haitanensis in Fuding City in autumn 2008.Modern Fisheries Information, 24(7):6-9, (in Chinese)
Lee S J, Jee B Y, Son M H, Lee S R. 2017. Infection and cox2 sequence of Pythium chondricola (Oomycetes) causing red rot disease in Pyropia yezoensis (Rhodophyta) in Korea. Algae, 32(2):155-160,
Lee S J, Park S W, Lee J H, Kim Y S. 2012. Diseases of the cultivated Porphyra at seocheon area. Journal of Fish Pathology, 25(3):249-256,
Li J, Mou Z J, Yang H C, Mao Y X, Yan Y W, Mo Z L. 2018.Isolation and identification the pathogen of Pyropia yezoensis green spot disease. Progress in Fishery Science, published online First, August 2018. (in Chinese)
Liu Y M, Ma J H, Wen Q. 2012. Simultaneous infection by red rot and olpidiops disease in laver Porphyra haitanensis.Journal of Dalian Ocean University, 27(6):546-550, (in Chinese with English abstract)
Lu Q Q, Zhou W, Zhu J Y, Yan B L, Ni J Z, Yang L E. 2018.The history, status quo and development trend of Pyropia yezoensis industry of China. Marine Economy in China, (3):3-11. (in Chinese)
Ma J H. 1996. A preliminary study on the red rot disease of Porphyra yezoensis. Journal of Shanghai Fisheries University, 5(1):1-7. (in Chinese with English abstract)
Mohkam M, Nezafat N, Berenjian A, Mobasher M A, Ghasemi Y. 2016. Identification of Bacillus probiotics isolated from soil rhizosphere using 16S rRNA, recA, rpoB gene sequencing and RAPD-PCR. Probiotics and Antimicrobial Proteins, 8(1):8-18,
Mokrousov I, Vyazovaya A, Zhuravlev V, Otten T, Millet J, Jiao W W, Shen A D, Rastogi N, Vishnevsky B, Narvskaya O. 2014. Real-time PCR assay for rapid detection of epidemiologically and clinically significant Mycobacterium tuberculosis Beijing genotype isolates.Journal of Clinical Microbiology, 52(5):1 691-1 693,
Mou Z J. 2012. Etiological Study on Pyropia/Porphyra yezoensis and Screening of Agarase-Producing Bacteria.Ocean University of China, Qingdao. p.33-35. (in Chinese with English abstract)
Nakao Y, Onohara T, Matsubara T, Fujita Y, Zenitani B. 1972.Bacteriological studies on diseases of cultured Laver -Ⅰ.Green spot rotting-like deterioration of laver frond by bacteria, in vitro. Nippon Suisan Gakkaishi, 38(6):561-564, (in Japanese with English abstract)
Park C S, Kakinuma M, Amano H. 2006. Forecasting infections of the red rot disease on Porphyra yezoensis Ueda(Rhodophyta) cultivation farms. Journal of Applied Phycology, 18(3-5):295-299,
Provasoli L. 1968. Media and prospects for the cultivation of marine algae. In:Watanabe A, Hattori A eds. Cultures and Collections of Algae. Japan Society of Plant Physiology, Tokyo. p.63-75.
Saito Y, Matsusato T, Yoshikawa K. 1972. On the symptoms of "green spot" and "crape" in nori (Porphyra) culture.Bulletin of Nansei National Fisheries Research Institute, 5:1-9. Japanese)
Sandaa R A, Brunvold L, Magnesen T, Bergh Ø. 2008. Monitoring the opportunistic bacteria Pseudoalteromonas sp. LT-13 in a great scallop, Pecten maximus hatchery.Aquaculture, 276(1-4):14-21,
Sawabe T, Makino H, Tatsumi M, Nakano K, Tajima K, Iqbal M M, Yumoto I, Ezura Y, Christen R. 1998.Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica. International Journal of Systematic and Evolutionary Bacteriology, 48(3):769-774,
Sekimoto S, Klochkova T A, West J A, Beakes G W, Honda D. 2009. Olpidiopsis bostrychiae sp. nov.:an endoparasitic oomycete that infects Bostrychia and other red algae(Rhodophyta). Phycologia, 48(6):460-472,
Skovhus T L, Ramsing N B, Holmström C, Kjelleberg S, Dahllöf I. 2004. Real-time quantitative PCR for assessment of abundance of Pseudoalteromonas species in marine samples. Applied and Environmental Microbiology, 70(4):2 373-2 382,
Sunairi M, Tsuchiya H, Tsuchiya T, Omura Y, Koyanagi Y, Ozawa M, Iwabuchi N, Murooka H, Nakajima M. 1995.Isolation of a bacterium that causes Anaaki disease of the red algae Porphyra yezoensis. Journal of Applied Bacteriology, 79(2):225-229, 1365-2672.1995.tb00939.x.
Suto S, Umebayashi O. 1954. On the "perforating disease" in nori (Porphyra) culture. Nippon Suisan Gakkaishi, 19(12):1 176-1 178, (in Japanese with English abstract)
Wang Y N, Feng N S, Li Q, Ding J, Zhan Y Y, Chang Y Q. 2013. Isolation and characterization of bacteria associated with a syndrome disease of sea urchin Strongylocentrotus intermedius in North China. Aquaculture Research, 44(5):691-700,
Wei S, Chelliah R, Park B J, Park J H, Forghani F, Park Y S, Cho M S, Park D S, Oh D H. 2018. Molecular discrimination of Bacillus cereus group species in foods(lettuce, spinach, and kimbap) using quantitative realtime PCR targeting groEL and gyrB. Microbial Pathogenesis, 115:312-320,
Yan Y, Ma J H, Xu P, Sun Q H, Wang H Q. 2002.Pseudoalteromonas citrea, the causative agent of greenspot disease of Porphyrae yezoensis. Journal of Fishery Sciences of China, 9(4):353-358, (in Chinese with English abstract)