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

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

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、p H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p H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140μmol·m^(-2)·s^(-1)、通气量1.0 L·min^(-1)和p H 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。

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,p H andair 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,p H 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° C,light intensity of 140 μmol·m-2·s-1,air flux of 1.0 L·min-1and p H 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.