禾草内生真菌检测方法研究进展

从最初发现黑麦草(Lolium perenne)和高羊茅(Festuca arundinacea)引起家畜中毒到后来其携带的内生真菌被发现,以及进一步的研究证实,内生真菌的存在不但导致家畜中毒而且能显著提高宿主在群落中的竞争力,也因禾草内生真菌的这种重要生理和生态作用,使其逐步成为国内外研究的热点之一,这也给内生真菌检测技术的发展提供了契机。初期主要是借鉴病原真菌等微生物检测的一些较成熟的方法,对禾草内生真菌进行染色镜检或分离检测,但容易受宿主种类、物候期、检测部位等因素的影响,造成检测结果的不准确。随着分子生物学、遗传学等学科的快速发展,酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)、聚合酶链式反应法(polymerase chain reaction,PCR)、实时荧光PCR法(realtime PCR)等现代分子技术在禾草内生真菌方面的应用和改进,使得内生真菌的检测方法不断推陈出新,这些方法能在一定程度上对传统检测方法给予有效的补充。快速有效的确定禾草内生真菌的存在、分布规律、分类地位等,需要准确合理地选择特异性较强的检测方法,例如定性或定量检测,并结合经典的染色镜检和分离法对禾草内生真菌进行确定。本研究对以上内容进行了综述,以期更深层次的借鉴和发展关于其它微生物的经典检测方法,同时开发具有禾草内生真菌特异性的检测技术,这些技术不仅能够鉴定内生真菌存在与否,而且能对内生真菌进行定量和活性的检测,是发展的重要方向和热点。对内生真菌定性、定量和活性的检测是未来值得研究的重点。

Research progress of methods on grass fungal endophyte detection

After the initial discovery of perennial ryegrass and tall fescue lead poisoning of farm animals, the responsible endophytic fungi in the grass was identified;further study confirmed the presence of endophytic fungi not only led to livestock poisoning, but significantly improved the competitive ability of the host in the community.The ecological and physiological functions of grass endophytic fungi have established this field as a popular research topic worldwide, which has provided opportunities for the development of endophytic fungi detection technology.Generally, well-established detection methods for pathogenic fungi have been used for the detection grass endophytic fungi, such as microscopic detection with staining.However, the accuracy of results was easily influenced by different host species, phenophases, and tissues.The rapid development and application of molecular biology, genetics, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), real-time PCR methods, and other modern molecular techniques has enabled the continuous improvement of the detection methods of endophytic fungi, which can compensate for the shortage of traditional methods.The rapid and efficient determination of the existence, distribution, classification, and position of the endophytic fungi, requires an accurate and reasonable selection of specific detection methods, such as qualitative or quantitative detection combined with classical microscopy staining to determine the endophytic fungi.In this paper, we reported recent research on the detection methods of endophytic fungi and also discuss the development of classical detection method of other microbes, which are important for the development of specific detection technologies for endophytic fungi and can quantitatively determine the existence and the activity of endophytic fungi.Specific endophytic fungi detection technologies, which are no longer limited to only the identification of the presence of endophytic fungi, but can simultaneous quantify the activity of endophytic fungi detection, are an important focus for the technological development.