环境因子对一种病原真菌(<em>Amoeboaphelidium</em> sp.)感染微藻细胞能力的影响

彭新安; 丁奕; 张丹; 杜奎; 许岩; 温小斌; 耿亚洪; 李夜光

doi:10.11913/PSJ.2095-0837.2016.50798

环境因子对一种病原真菌(Amoeboaphelidium sp.)感染微藻细胞能力的影响

彭新安^1,2,
丁奕¹,
张丹¹,
杜奎^1,2,
许岩^1,2,
温小斌¹,
耿亚洪¹,
李夜光^1, ,

1.
中国科学院武汉植物园, 植物种质创新与特色农业重点实验室, 武汉 430074
2. 中国科学院大学, 北京 100049

基金项目: 中国石化集团公司“产油微藻病虫害控制技术开发及培养规程制定”（215104）。

详细信息

作者简介:
彭新安(1989-),男,硕士研究生,研究方向为微藻生物技术(E-mail:pengxinan6619@163.com)。

通讯作者:
李夜光(E-mail:yeguang@wbgcas.cn)。

中图分类号: Q949.2
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出版历程
- 收稿日期: 2016-03-21
- 修回日期: 2016-04-22
- 发布日期: 2016-10-27

Effects of Environmental Factors on Infectivity of a Pathogenic Fungus Amoeboaphelidium sp. Infecting Microalgal Cells

1.
Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
2. University of Chinese Academy of Sciences, Beijing 100049, China

Funds: This work was supported by grants from the China Petrochemical Corporation (No.215104:The development of pest control technology and establishment of cultivation rules on oleaginous microalgae).

摘要

摘要: 油球藻（Graesiella sp.WBG-1）是一株适合于开放池规模化培养的产油微藻，在室外规模培养过程中常因一种病原真菌（Amoeboaphelidium sp.）的污染严重影响其生长和油脂积累，甚至导致培养失败。本文利用MTT染色法借助光学显微镜对被病原真菌感染的油球藻细胞计数并统计感染率，设置不同温度、光照强度、pH以及通气量等培养条件，研究环境因子对病原真菌感染能力的影响。结果表明：MTT染色法简单易行，可用于油球藻规模培养中病原真菌的检测；温度、光照强度、通气量和pH值均能够显著影响病原真菌的感染能力，高温、高光照强度、弱酸性环境和藻液静止等培养条件不利于病原真菌对油球藻的感染；在温度30℃、光强140 μmol·m^-2·s^-1、通气量1.0 L·min^-1和pH 9.0±0.5的培养条件下成功建立了病原真菌感染油球藻的连续传代培养。本研究在实验室内模拟油球藻规模培养中被病原真菌感染的全过程，为深入研究该病原真菌对微藻细胞的感染行为及其感染机制提供了平台。
- 油球藻 /
- 病原真菌 /
- 环境因子 /
- MTT染色 /
- 连续传代培养
Abstract: Large-scale cultivation of microalgae is a core issue of microalgal biodiesel production, with contamination by biological pollutants difficult to avoid in outdoor cultivation. Graesiella sp. WBG-1 is an oleaginous microalga suitable for large-scale cultivation in outdoor open ponds. However, infection by the pathogenic fungus Amoeboaphelidium sp. can seriously affect growth and lipid accumulation, even leading to the entire collapse of the Graesiella sp. WBG-1 culture. In this study, MTT staining was used to stain sporangia of the pathogenic fungus, with the infected algal cells then detected and counted under microscopy, and the infection rate (ratio of infected cells to total cells) calculated to evaluate the infection ability of the fungus under different culture conditions of temperature, light intensity, pH and air flux (turbulence of algal liquid). The different culture conditions were set to investigate the effects of environmental factors on the infectivity of the pathogenic fungus. The results demonstrated that MTT staining was easy and suitable for the detection of pathogenic fungal infection in the mass culture of Graesiella sp. WBG-1. Temperature, light intensity, pH and air flux all had significant effects on the infectivity of the pathogenic fungus. High temperature, high light intensity, slight acid environments, and algal suspension without stirring had adverse effects on the infection of Graesiella sp. WBG-1. Successive transfer culture of the fungus infecting Graesiella sp. WBG-1 was successfully established at a temperature of 30℃, light intensity of 140 μmol·m^-2·s^-1, air flux of 1.0 L·min^-1 and pH of 9.0±0.5. After 3-4 d of transfer culture, the infection rate developed from 0 to above 90%, with almost all algal cells infected. The successive transfer culture of algae coupled with artificial infection by the pathogenic fungus simulated the infection procedure that occurs in the mass culture of Graesiella sp.WBG-1 in the field. Therefore, these results provide a good platform for studies on the infection behaviors of pathogenic fungi and the mechanism of infection.
- Graesiella /
- Pathogenic fungi /
- Environmental factors /
- MTT staining /
- Successive transfer culture

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参考文献(35)

[1]	李涛, 李爱芬, 桑敏, 吴洪, 尹顺吉, 张成武. 富油能源微藻的筛选及产油性能评价[J]. 中国生物工程杂志, 2011, 31(4):98-105. Li T, Li AF, Sang M, Wu H, Yi SJ, Zhang CW. Screening oleaginous microalgae and evaluation of the oil-producing charateristic[J]. China Biotechnology, 2011, 31(4):98-105.
[2]	梅洪, 张成武, 殷大聪, 耿亚红, 欧阳峥嵘, 李夜光. 利用微藻生产可再生能源研究概况[J]. 武汉植物学研究, 2008, 26(6):650-660. Mei H, Zhang CW, Yin DC, Geng YH, Ouyang ZR, Li YG. Survey of studies on renewable energy production by microalgae[J]. Journal of Wuhan Botanical Research, 2008, 26(6):650-660.
[3]	Brennan L, Owende P. Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[J]. Renew Sust Energ Rev, 2010, 14(2):557-577.
[4]	Pittman JK, Dean AP, Osundeko O. The potential of sustainable algal biofuel production using wastewater resources[J]. Bioresource Technol, 2011, 102(1):17-25.
[5]	Turner JA. A realizable renewable energy future[J]. Science, 1999, 285(5428):687-689.
[6]	嵇磊, 张利雄, 姚志龙, 闵恩泽. 利用藻类生物质制备生物燃料研究进展[J]. 石油学报(石油加工), 2007, 23(6):1-5. Ji L, Zhang LX, Yao ZL, Min EZ. Review on the progress of producing bio-fuel from microalgae[J]. Acta Petrolei Sinica(Petroleum Processing Section), 2007, 23(6):1-5.
[7]	康永锋, 史华进, 胡文振, 赵子琦, 周志刚. 利用海洋微藻制备生物柴油的研究进展[J]. 化工新型材料, 2013, 41(12):22-24. Kang YF, Shi HJ, Hu WZ, Zhao ZQ, Zhou ZG. Progress on biodiesel production by utilization of microalgae[J]. New Chemical Materials, 2013, 41(12):22-24.
[8]	Chisti Y. Biodiesel from microalgae[J]. Biotechnol Adv, 2007, 25(3):294-306.
[9]	Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications:a review[J]. Renew Sust Energ Rev, 2010, 14(1):217-232.
[10]	Carvalho AP, Meireles LA, Malcata FX. Microalgal reactors:a review of enclosed system designs and perfor-mances[J]. Biotechnol Prog, 2006, 22(6):1490-1506.
[11]	刘天中, 张维, 王俊峰, 郎莹, 张静. 微藻规模培养技术研究进展[J]. 生命科学, 2014, 26(5):509-522. Liu TZ, Zhang W, Wang JF, Lang Y, Zhang J. A review of mass cultivation technology for microalgae[J]. Chinese Bulletin of Life Sciences, 2014, 26(5):509-522.
[12]	Wang H, Zhang W, Chen L, Wang J, Liu T. The contamination and control of biological pollutants in mass cultivation of microalgae[J]. Bioresource Technol, 2013, 128:745-750.
[13]	Gutman J, Zarka A, Boussiba S. The host-range of Paraphysoderma sedebokerensis, a chytrid that infects Haematococcus pluvialis[J]. Eur J Phycol, 2009, 44(4):509-514.
[14]	Hoffman Y, Aflalo C, Zarka A, Gutman J, James TY, Boussiba S. Isolation and characterization of a novel chytrid species(phylum Blastocladiomycota), parasitic on the green alga Haematococcus[J]. Mycol Res, 2008, 112(Pt 1):70-81.
[15]	Donk EV, Ringelberg J. The effect of fungal parasitism on the succession of diatoms in Lake MaarsseveenⅠ(The Netherlands)[J]. Freshw Biol, 1983, 13(3):241-251.
[16]	Gons HJ, Ebert J, Hoogveld HL, van den Hove L, Pel R, Takkenberg W, Woldringh CJ. Observations on cyanobacterial population collapse in eutrophic lake water[J]. Anton Leeuw, 2002, 81(1-4):319-326.
[17]	Letcher PM, Lopez S, Schmieder R, Lee PA, Behnke C, Powell MJ, McBride RC. Characterization of Amoeboaphe-lidium protococcarum, an algal parasite new to the Cryptomycota isolated from an outdoor algal pond used for the production of biofuel[J]. PLoS One, 2013, 8(2):1-15.
[18]	欧阳峥嵘, 温小斌, 耿亚红, 梅洪, 胡鸿钧, 张桂艳, 李夜光. 光照强度, 温度, pH, 盐度对小球藻(Chlorella)光合作用的影响[J]. 武汉植物学研究, 2010, 28(1):49-55. Ouyang ZR, Wen XB, Geng YH, Mei H, Hu HJ, Zhang GY, Li YG. The effects of light intensities, tempretures, pH and salinities on photosynthesis of Chlorella[J]. Journal of Wuhan Botanical Research, 2010, 28(1):49-55.
[19]	Bruning K. Effects of phosphorus limitation on the epidemio-logy of a chytrid phytoplankton parasite[J]. Freshw Biol, 1991, 25(3):409-417.
[20]	Holfeld H. Infection of the single-celled diatom Stephanodiscus alpinus by the chytrid Zygorhizidium:Parasite distribution within host population, changes in host cell size, and host-parasite size relationship[J]. Limnol Oceanogr, 2000, 45(6):1440-1444.
[21]	Kagami M, Van Donk E, De Bruin A, Rijkeboer M, Ibelings BW. Daphnia can protect diatoms from fungal parasitism[J]. Limnol Oceanogr, 2004, 49(3):680-685.
[22]	Kairo SK, Bedwell J, Tyler PC, Carter A, Corbel MJ. Development of a tetrazolium salt assay for rapid determination of viability of BCG vaccines[J]. Vaccine, 1999, 17(19):2423-2428.
[23]	Stentelaire C, Antoine N, Cabrol C, Feron G, Durand A. Development of a rapid and highly sensitive biochemical method for the measurement of fungal spore viability. An alternative to the CFU method[J]. Enzyme Microb Tech, 2001, 29(8-9):560-566.
[24]	Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology:new insights into their cellular reduction[J]. Biotechnol Annu Rev, 2005, 11:127-152.
[25]	Mosmann T. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays[J]. J Immunol Methods, 1983, 65(1):55-63.
[26]	Maraldi NM, Zini N, Santi S, Scotlandi K, Serra M, Baldini N. P-glycoprotein subcellular localization and cell morphotype in MDR1 gene-transfected human osteosarcoma cells[J]. Biol Cell, 1999, 91(1):17-28.
[27]	Saravanan BC, Sreekumar C, Bansal GC, Ray D, Rao JR, Mishra AK. A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata[J]. Vet Parasitol, 2003, 113(3-4):211-216.
[28]	Seidl K, Zinkernagel AS. The MTT assay is a rapid and reliable quantitative method to assess Staphylococcus aureus induced endothelial cell damage[J]. J Microbiol Meth, 2013, 92(3):307-309.
[29]	Gaboriau F, Chéron M, Petit C, Bolard J. Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity:a new way to improve its therapeutic index[J]. Antimicrob Agents Ch, 1997, 41(11):2345-2351.
[30]	Bruning K. Infection of the diatom Asterionella by a chytrid.Ⅰ. Effects of light on reproduction and infectivity of the parasite[J]. J Plankton Res, 1991, 13(1):103-117.
[31]	Bruning K. Infection of the diatom Asterionella by a chytrid.Ⅱ. Effects of light on survival and epidemic development of the parasite[J]. J Plankton Res, 1991, 13(1):119-129.
[32]	Bruning K. Effects of temperature and light on the population dynamics of the Asterionella-Rhizophydium association[J]. J Plankton Res, 1991, 13(4):707-719.
[33]	Gutman J, Zarka A, Boussiba S. Evidence for the involvement of surface carbohydrates in the recognition of Haematococcus pluvialis by the parasitic blastoclad Paraphysoderma sedebokerensis[J]. Fungal Biol, 2011, 115(8):803-811.
[34]	Frénoy JP, Tran AT, Bourrillon R. Structure and stability of Ricinus communis haemagglutinin[J]. Biochem J, 1986, 240(1):227-231.
[35]	Kagami M, de Bruin A, Ibelings BW, Van Donk E. Parasi-tic chytrids:their effects on phytoplankton communities and food-web dynamics[J]. Hydrobiologia, 2007, 578(1):113-129.

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环境因子对一种病原真菌(Amoeboaphelidium sp.)感染微藻细胞能力的影响

作者简介: 彭新安(1989-),男,硕士研究生,研究方向为微藻生物技术(E-mail:pengxinan6619@163.com)。

通讯作者: 李夜光(E-mail:yeguang@wbgcas.cn)。

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出版历程

Effects of Environmental Factors on Infectivity of a Pathogenic Fungus Amoeboaphelidium sp. Infecting Microalgal Cells

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出版历程

目录

作者简介:
彭新安(1989-),男,硕士研究生,研究方向为微藻生物技术(E-mail:pengxinan6619@163.com)。

通讯作者:
李夜光(E-mail:yeguang@wbgcas.cn)。