引用本文
陈振江, 魏学凯, 曹莹, 田沛, 赵晓静, 李春杰. 禾草内生真菌检测方法研究进展. 草业科学, 2017,34(7):1419-1433
Chen Zhen-jiang, Wei Xue-kai, Cao Ying, Tian Pei, Zhao Xiao-jing, Li Chun-jie. Research progress of methods on grass fungal endophyte detection. Pratacultural Science,2017,34(7): 1419-1433  
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《草业科学》编辑部
禾草内生真菌检测方法研究进展
陈振江, 魏学凯, 曹莹, 田沛, 赵晓静, 李春杰
草地农业生态系统国家重点实验室 兰州大学草地农业科技学院,甘肃 兰州 730020
通信作者：李春杰(1968-),男,甘肃镇原人,教授,博士,研究方向为禾草内生真菌共生体。E-mail:[email protected]

第一作者:陈振江(1991-),男,甘肃定西人,在读博士生,研究方向为禾草内生真菌共生体。E-mail:[email protected]

收稿日期: 2016-12-29
基金: 基金项目:国家“973”项目(2014CB138702); 国家自然科学基金项目(31372366); 教育部创新团队发展计划项目(IRT13019)
摘要

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。

关键词: 禾草内生真菌; 分离检测; 显微观察法; 酶联免疫吸附法; 生物碱高效液相色谱法; 分子检测法
中图分类号:S432.4+4 文献标志码:A 文章编号:1001-0629(2017)07-1419-15 doi: 10.11829/j.issn.1001-0629.2016-0386
Research progress of methods on grass fungal endophyte detection
Chen Zhen-jiang, Wei Xue-kai, Cao Ying, Tian Pei, Zhao Xiao-jing, Li Chun-jie
State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou 730020, China
Corresponding author: Li Chun-jie E-mail:[email protected]
Abstract

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.

Keyword: endophyte; isolation; microscopic observation; ELISA; HPLC; molecular biology

禾草内生真菌(grass endophyte)是指在禾草体内度过大部分或者全部生命周期, 但却不会引起禾草外部显示任何病害症状的一大类真菌[1]。目前, 关于禾草内生真菌的研究主要集中在与冷季型早熟禾亚科(Pooideae)植物共生的Epichloё 属[2]。禾草与内生真菌形成的共生体对宿主和内生真菌均具有一定增益作用:提高了宿主的生物抗性[3, 4, 5]和非生物抗性[6, 7, 8, 9, 10], 促进禾草的根系发育、分蘖能力等[11, 12, 13], 提高宿主植物的竞争力[14, 15]; 宿主禾草为内生真菌的传播提供了营养及繁殖场所, 增加了草地的寿命, 提高了生态服务价值[16]; 但此共生体也会产生有毒物质, 家畜采食后会引起家畜中毒, 对其生长性能、受孕率、神经系统和免疫系统等会产生影响[17, 18, 19], 从而对草地畜牧业产生了不利的影响[17]。

除部分内生真菌有广泛的宿主范围和部分寄主能被多种内生真菌感染外, 绝大多数禾草常被一种真菌感染, 导致内生真菌与宿主之间有着比较严格的宿主特异性[18], 因此分离自不同宿主的禾草内生真菌数量和种类不同。而且由于禾草的种类和基因型以及禾草生长的环境因素影响, 与之共生的禾草内生真菌在种类和数量上具有很大的差异性和多样性[20], 已经发表的内生真菌有43种, 尚有部分菌株未鉴定[2, 21, 22]或未发表[23]。

禾草内生真菌主要分布在植株的茎髓、叶鞘、叶片和种子等地上部分, 与宿主植物协同生长; 内生真菌菌丝在细胞间隙分布不规则且在禾草不同组织中的菌丝形态存在一定差异[22, 24]。Epichloё 属内生真菌种类和数量较多, 其不同内生真菌种类的菌落颜色、质地、分生孢子及分生孢子梗的形态和大小存在差异, 明显显示出丰富的形态多样性[19, 25]。禾草内生真菌分子系统发育的研究表明, Epichloё 的起源有3种途径[26]:直接起源于Epichloё 的某个种, Epichloё 的种间杂交, Epichloё 与其无性态Neotyphodium属之间杂交; 通过杂交重组产生了新的遗传特性, 从而出现了许多新的种、亚种及变种[2]。因此, 检测和鉴定禾草中是否存在内生真菌, 确定禾草内生真菌的种属对研究禾草内生真菌共生体多样性、内生真菌提高禾草抗逆性及筛选有益内生真菌从而培育优良品种十分重要。

内生真菌的研究最基础的方法是分离检测, 从形态学上鉴定内生真菌的形态; 但其检测周期长, 且无法控制环境等因素对菌种的保存和鉴定产生的影响[16]。镜检法与分离检测法常相结合应用于禾草内生真菌的分离鉴定上, 在显微镜下通过苯胺蓝染色快速、直观地检测禾草内生真菌的存在, 对分离菌落的分生孢子等进行观测, 弥补了分离检测法的不足。对高羊茅(Festuca arundinacea)内生真菌的亚显微结构进行观察发现, 禾草的叶片、叶鞘、茎髓和种子中普遍存在内生真菌, 叶鞘中带菌率最高[27]。免疫学上检测内生真菌的方法有免疫印迹法、酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)等, 通过酶的高效催化作用与底物发生显色反应, 从而定性或者定量鉴定不同禾草的内生真菌, Musgrave应用ELISA法检测出多年生黑麦草(Lolium perenne)内生真菌, 其菌丝量浓度为 0.1 mg· mL-1。相对于镜检测法, 此法特异性强, 在一定程度上提高了检测的速度和灵敏性[28]。此外普通PCR标记和实时荧光PCR标记等分子生物学技术也被用作检测禾草内生真菌, 利用Tub-2基因可设计出Epichloё 及无性态通用Taqman探针及引物, 建立双色荧光实时定量PCR检测醉马草(Achnatherum inebrians)内生真菌的方法, 可准确快速地检测出醉马草内生真菌DNA片段的含量, 进而计算出醉马草内生真菌的含量[29]。因此, 正确合理地选择定性或定量检测禾草内生真菌的方法(形态学法、镜检法、分子法、免疫学法等)对确定禾草内生真菌的有无、分布规律、种属等至关重要。

1 镜检和分离检测

禾草内生真菌研究初期以镜检和分离检测两种检测方法结合使用为主, 通过对组织进行染色, 观察内生真菌在宿主中的形态特征和分离培养的菌落形态来鉴定种间的区别。

禾草内生真菌镜检法根据宿主不同组织特异性常采用不同的染色试剂(表1), 主要使叶鞘的内表皮、茎的髓质和种子着色, 然后在光学显微镜下检测禾草内生真菌菌丝, 统计禾草带菌率。不同的染剂可使不同的共生体着色:乳酚-棉蓝使Danthonia spicata和多年生黑麦草的种子着色[37, 38]; 乳酚-酞酚蓝可染色多年生黑麦草内生真菌[39]; 苯胺蓝使高羊茅和黑麦草的菌丝着色[40, 41]; 龙胆紫溶液和革兰氏染剂也可使菌丝染色[42]; 玫瑰红染剂可将牧草和草坪草的茎髓、种子等组织中的内生真菌染成红色在显微镜下观察菌丝形态[43]; 苯胺蓝染色高羊茅种子后在硝酸中浸泡过夜使其软化, 可在显微镜下观察到糊粉层中被染成蓝色的菌丝[44]。除了试剂染色镜检法外, 荧光素双醋酸盐(FDA)溶液作为染剂, 在荧光显微镜下观察禾草内生真菌, 镜下亮蓝色的丝状体就是具有活性的禾草内生真菌菌丝体。荧光素双醋酸盐(FDA)溶液也可以作为载体在荧光显微镜下观察多年生黑麦草内生真菌并检测其菌丝体的活性[45](表2)。目前禾草内生真菌镜检法主要使用的是苯胺蓝染色剂, 多使用1%的苯胺蓝染液进行染色效果最佳[16]。此方法只能检测禾草是否带菌, 但无法准确鉴定禾草内生真菌的活性。李春杰等对醉马草不同生育期和不同组织内生真菌菌丝体检测对比, 建立了适合醉马草内生真菌检测的方法[45, 46]。

除了光学显微镜的使用外, 一些更加先进的显微镜也被应用到内生真菌的检测中。如利用光学显微镜、电子显微镜和荧光显微镜对黑麦草叶片细胞间隙的Epichloё 及无性态内生真菌进行观察, 发现内生真菌的侵染寄主是沿着禾草叶片延伸生长的方向分裂和扩展, 同时证明了禾草内生真菌具有居间生长的特点[47]。透射电子显微镜只能观察禾草组织超薄切片标本, 而有可能禾草内生真菌表面的结构与内生真菌内部的结构不同, 使检测复杂化; 超薄切片标本, 制样过程复杂, 制作标本有损伤; 此外电子显微镜购买和维护的价格都比较高[16]。镜检法与分离检测法常相结合应用于禾草内生真菌的分离鉴定上, 在显微镜下观察禾草显微结构快速、直观, 弥补了分离检测法仅依靠形态学特征鉴定菌种的不足。Christensen在E. festucae真菌和寄主基因型对高羊茅内生真菌共生体兼容性和维管束影响的研究中使用光学显微镜和透射电子显微镜观察寄主细胞的变化以及叶片维管束胞间菌丝, 并通过观察母体和后代禾草内生真菌共生体分离培养的菌落形态学特征, 发现维管束胞间菌丝浓度最高[48]。

通过显微镜镜检, 发现内生真菌的菌丝后进行分离培养, 根据菌落形态来鉴定种间的差异。禾草内生真菌分离培养(形态学鉴定)的步骤一般是:首先对禾草表面进行消毒灭菌处理, 然后将禾草组织切成1 cm小节放在培养基中培养, 最后通过分离培养的菌株形态特征来鉴定菌种[16]。但Epichloё 属内生真菌在体外分离培养时生长缓慢, 容易受到培养条件、培养基类型及自身遗传组成的影响, 且有的菌株在常规培养基上不易产生孢子, 而孢子是禾草内生真菌形态学鉴定的一个重要因子[49]。不产孢的菌株一般无法用形态学特征来鉴定, 需要和其它方法相结合来检测; 对夏季采自黄山景区海拔500~1 600 m处的23个属631株禾草镜检发现, Epichloё 属内生真菌的确存在, 并得出禾草内生真菌的分布与寄主植物的种类、寄主植物的地理分布类型等有关, 通过分离培养观察发现其子座等形态特征与欧洲菌株有较大的差异性[50]。将禾草组织中的内生真菌在不同的培养基中进行分离培养[51], 通过观察其菌落形态特征(颜色、形状)、菌落生长速率及产孢量、产孢特征、孢子特性等鉴定指标, 并通过显微镜下观察菌丝及分生孢子的形态学特征[52], 从而鉴定禾草内生真菌的种、属[53, 54]。已知的内生真菌通常采用镜检和分离结合的方法进行鉴定, 如分离培养多年生黑麦草内生真菌获得了多年生黑麦草植株Gliocladium-like和Acremonium loliae两个内生真菌菌株[55, 56]; 通过观察亚利桑那羊茅(Festuca arizonica)内生真菌分离培养的菌株孢子和菌落的形态学特征, 建立了新种E. typhina var. huerfanum[57]; 通过比较醉马草分生孢子大小、分生孢子梗长宽及醉马草内生真菌菌落的形态, 确定了新种E. gansuensis[58]。

表2 不同染色剂溶液的使用 Table 2 Uses of stain solutionss
2 免疫检测法

酶联免疫吸附法(ELISA)是采用抗原与抗体的特异反应将待测物与酶连接然后通过酶的高效催化作用与底物发生显色反应, 对受检物质进行定性或定量分析的一种检测方法[27]。因ELISA技术准确度高、特异性强、适用范围广、检测样本多速度快, 并且ELISA技术的条件要求比较低, 方便操作, 所以ELISA技术经常被制成试剂盒的形式, 被广泛运用在禾草内生真菌的检测分析上[59, 60, 61, 62]。

ELISA最初被应用于定量检测高羊茅种子和叶鞘组织中的内生真菌[63, 64, 65, 66]。使用ELISA法检测高羊茅体内的内生真菌, 发现每毫升冰冻干燥的菌丝体中至少含100 ng的量[67]。从兔子(Leporidae)体中提取多克隆抗血清来检测高羊茅种子和组织中的内生真菌, 在种子检测时需要让种子吸水过夜保证其新鲜性, 较费时[68]。镜检法和单克隆抗体免疫印迹试剂法对比检测高羊茅内生真菌的带菌率, 免疫印迹法在大样品检测中准确率更高, 更容易操作[69]。Koh和Hik借助免疫法验证了内生真菌在天然草地中的分布规律以及在生态环境中所起的作用[70], 之后免疫技术法被广泛应用到禾草内生真菌的检测及阐述禾草内生真菌的分配模式[71, 72, 73], 禾草内生真菌共生体动力学的季节性倾向[74, 75]和食草动物、内生真菌侵染禾草的动力学关系中[70, 76, 77, 78]。但是由于各种禾草内生真菌的理化性质、形态特性等方面的不同, 寄主植物不同, 抗体生产、测定、酶标记物的制备不能标准化, 检测时受到特异性和非特异性的干扰, 使得ELISA技术有其局限性和不足。而且随着分子生物技术的发展, 测序等分子检法方法成本的降低, ELISA使用率下降。

3 利用次生代谢物间接检测

产生生物碱类次生代谢物是禾草与内生真菌共生最重要的特征, 也是采用这种方法检测内生真菌的重要依据。目前, 已发现至少十余种与禾草内生真菌相关的生物碱, 主要有4种Epichloё 内生真菌生成的不同植物保护性生物碱:吲哚双萜类(indolditerpene)、吡咯并吡嗪类(pyrrolopyazine)、麦角碱类(ergot alkaloids)和饱和吡咯化合物(pyrroliziline)[5, 79]。每种禾草内生真菌共生体可以产生多种生物碱, 同一种生物碱也可由不同的共生体产生[80]。一般在禾草内生真菌共生体中可检测到一种或多种生物碱, 但绝大多数含有多种生物碱[81]。其中高羊茅和黑麦草中最常见的是吡咯并吡嗪类生物碱(peramine), 黑麦草碱(loline)和麦角碱次之, 吲哚双萜类生物碱(lolitrem)最少[82, 83]。不同禾草内生真菌共生体生成不同的生物碱, 主要是由功能产碱基因在内生真菌基因组中的有无决定, 有时候也因基因表达水平的不同[84]。除生物碱类物质之外, 有关禾草内生真菌共生体其它代谢产物的研究也越来越多[85, 86, 87], 如从麦宾草(Elymus tangutorum)内生真菌次生代谢产物中可分离得到17种化合物, 其中cyclosporin T具有较强的灭菌活性[87]。

禾草内生真菌生物碱研究的初期以简单的显色反应和色谱法来检测产碱量。Dimenna和Turner等曾用VanUrk反应检测麦角碱[88, 89]。由于此检测技术的灵敏度低, 无法进行定量检测。生物碱检测方法的不断改进, 从最初的简单比色法[90]、薄层析法(TLC)[91, 92]发展为ELISA法[93]、气相(GC) [94, 95]、质谱方法(MS)[96]以及高效液相色谱法(HPLC)[97, 98] (表3)。Najafabadi等利用HPLC-MS方法检测高羊茅内生真菌不同生长期麦角瓦灵的含量[108]。Roberts等建立了NIR检测高羊茅内生真菌中麦角碱含量的方法[109]。

近年来, 国内外开展了对禾草内生真菌生物碱合成基因、基因组、比较基因组及分子系统发育关系等分子学的深入研究, 证实了其实用性和精确性[110, 111, 112, 113]。已有研究证实可以从分子层面修饰和敲除有毒生物碱基因, 如从多年黑麦草分离出的Epichloё lolii和Epichloё sp.菌株中敲除dmaW基因使其共生体不产生麦角缬氨酸(ergovaline)[114]。陈丽通过对醉马草内生真菌Epichloё gansuensis (Eg)和Epichloё gansuensis var. inebrians (Ei)菌株进行了PCR分子检测和鉴定, 建立了适用于醉马草内生真菌的多基因 PCR 检测体系, 得到了醉马草内生真菌dmaW、idtG、mtA和mtB基因特异性引物[115]。多基因PCR检测材料用菌株或共生体 DNA均可, 既可检测禾草内生真菌共生体产生的生物碱, 从而证实内生真菌是否入侵禾草。除此之外, 可以对生物碱进行测序来间接地检测内生真菌, 如对草地羊茅(Festuca elati)内生真菌共生体中分离的麦角菌科(Clavicipitaceae)生物碱基因组通过测序分析, 发现了Ergoline麦角菌科生物碱生物合成途径[116]。这些禾草内生真菌共生体次生代谢产物的检测方法也间接证明了内生真菌的存在。

表3 禾草内生真菌生物碱含量的主要检测方法 Table 3 Main methods of alkaloid detection of grass endophytes
4 分子检测法

从1996年起Glenn和Bacon利用分子生物学技术建立真菌属以来[117], 利用分子生物学进行内生真菌检测的研究不断发展进步。

4.1 保守基因测序进行内生真菌种类鉴定

将基因序列分析等高识别率的方法用于内生真菌亲缘关系的比较, 将更加准确地鉴定禾草内生真菌的种类。常用的多基因拷贝技术有同功酶、微卫星等。在内生真菌分类初期用核糖体DNA内转录间隔区rDNA-ITS的多态性的序列进行分析, 检测此序列是否可以用来分析遗传关系, 如ITS1-4的4条通用引物可用于内生真菌的ITS序列检测[118]。除了分析内转录间隔区(ITS)序列外, 肌动蛋白(act1)、β -微管蛋白(tub2)和翻译延伸因子1-α (tef1)特异基因序列也可用于内生真菌DNA的扩增, 如利用tub2、tef1和act1基因扩增, 建立了E. aotearoae、E. melicicola、E. australiensis[119], 也可以跟系统发育分析结合进行禾草内生真菌的鉴定[120]。

4.2 各种分子标记技术进行内生真菌基因型分析

微卫星(microsatellite)也称为简单序列重复(simple sequences repeats, SSR), 因数量多、分布广、多态性丰富、检测快速方便等优点而被应用于内生真菌个体识别和遗传多样性分析等[121, 122]。Jong等利用该技术对寄生宿主禾草体内的Neptyphodium coenophialum和Neotyphodium lolii进行了检测并分析了二者之间的遗传变异特征[123]。对宿主和内生真菌DNA用SSR引物进行扩增可以得到区别二者DNA的条带, 从而可以检测禾草是否被内生真菌侵染[124]。尽管微卫星技术在禾草内生真菌的检测方面具有较明显的优势, 但仍有部分缺陷。在进行微卫星标记应用时, 需要设计出特异性较强的引物进行扩增, 然而这项工作通常费时费力, 给微卫星技术的广泛应用带来一定的困难[125]。

实时PCR(real-time quantitative polymerase chain reaction, Rt-PCR)又称为实时荧光定量PCR, 实时荧光 PCR 标记法灵敏度高、特异性强, 能快速准确地定量检测禾草内生真菌[126, 127, 128]。采用实时荧光PCR方法可以快速检测E. coenophiala和E. festucae var. lolii内生真菌, 并可检测到单粒种子上[129]。定量PCR技术可以用于阐述不同禾草内生真菌和寄主基因型在禾草内生真菌共生体相互作用中的影响[130], 但实时荧光定量PCR因检测光源和荧光素种类的局限及高成本限制了其被广泛的应用。

随机扩增多态DNA(RAPD)标记法不受环境、发育、数量性状遗传等的影响, 能够客观地提示供试材料之间DNA的差异, 可以检测出RFLP标记不能检测的重复顺序区。对E. festucae var. lolii和E. Coenophiala用不同的引物扩增, 发现引物5'-CCGAGGGGGGGTGAC-3'可以区分二者[131]。RAPD技术可用来验证两种禾草内生真菌是否可以同时侵染同一种宿主植物[132]。用RAPD技术对雀麦属(Bromus)植物中的Epichloё typhina进行标记检测, 发现在遗传过程中存在着变异[133]。该技术使用的效果因生物种类而定, 但因其具有快速、简便、分辨率高和样本少等特点, 被广泛应用在禾草内生真菌的检测中[134, 135]。利用分子生物学技术检测禾草内生真菌, 可以弥补传统禾草内生真菌检测方法的不足, 并结合遗传学确定了禾草内生真菌的种属。

5 问题与展望

Petrini 对内生真菌与宿主的专一性进行分析, 得出每种宿主平均被4~5种专性内生真菌侵染, 按地球上目前已知的25万种植物估算, 全世界内生真菌种类在100万种以上[136]。目前, 人们只发现80属290种禾本科植物中含有内生真菌; 而我国幅员辽阔, 生物物种资源丰富, 有190余属禾本科植物, 目前只在早熟禾属、羊茅属、披碱草属等14属43种(种群)天然草地禾草中发现内生真菌[17], 且明确其分类地位及命名的很少。传统方法容易受主观因素的影响, 单凭传统方法分类鉴定的结果存在一定的误差, 需要结合其分子手段进一步鉴定[137]。普通的分子标记需要特定的特异性引物, 且成本高, 目前已很难满足一般的分子检测的需要。

随着生物技术和生物信息学的快速发展, 真菌的检测技术也在不断进步。新一代测序技术的发展和推广, 如全基因组测序技术的引进, 对内生真菌的系统进化, 深入分析生物碱合成相关基因, 寻找真菌与寄主相互作用的基因奠定了重要的基础。美国肯塔基大学植物病理学系和澳大利亚维多利亚农业生物研究中心(AgriBio, Victoria) German Spangenberg实验室相继完成了对不同禾草内生真菌的全基因组测序, 有助于全球科学家共同开展禾草内生真菌的基因图谱构建等工作[116, 138, 139]。也可以用更多的长片段基因(比如线粒体基因)进行真菌系统发育的研究, 更清楚地阐明了基因的结构变异、拷贝数目、碱基缺失等与真菌生态功能的关系, 加速分子标记的开发, 更准确地追溯真菌起源, 对以前分类模糊的种进行精确的定性[140]。

生物芯片是近十几年迅速发展起来的一项基于基因表达和基因功能研究的高新技术, 综合了分子生物学、化学染料、激光、半导体微电子等领域的最新科学技术, 它具有高通量、多参数同步分析, 全自动、快速分析, 高准确度、灵敏度分析的优点[141], 该方法在真菌毒素监测等其它真菌及植物病原真菌检测中已被广泛应用[142, 143], 而在禾草内生真菌检测中的应用仍是空白, 若将此方法用于禾草内生真菌田间检测, 可大大缩短天然禾草内生真菌共生体筛选和培育含有高密度内生真菌草坪草的工作量, 提高工作效率。总之, 不同领域新科学技术的不断成熟和融合, 更加成熟的新技术将会应用到禾草内生真菌的检测中, 为开发和挖掘新的禾草内生真菌共生体提供基础。

随着内生真菌在育种等领域的不断深入, 其在子代中的稳定性成为困扰研究者的一个主要问题。结合传统和分子生物学技术建立针对种子中内生真菌存在及活力的高效检测方法势在必行。越来越多的研究发现, 内生真菌基因组中存在较多的对家畜等其它动物的毒性基因, 这给内生真菌的检测带了来新的挑战。内生真菌的检测不应仅停留在表面的带菌率方面的检测, 深入检测其基因组的产碱基因特征, 分析其对家畜和食物链中其它动物及天敌甚至人类的潜在威胁, 并进行较全面的风险评估, 以期利用内生真菌更好地为人类服务。传统的内生真菌检测方法只是针对其在宿主禾草体内的存在性进行分析, 但是并不能对内生真菌的活力状况进行准确判定, 因此发展针对这一方面的检测技术具有较大的潜力。目前新的真菌检测技术不断涌现, 给内生真菌的检测提供了较好的借鉴, 但是也给使用者的选择带来不少的困惑。因此, 应结合传统和新一代的检测方法, 制定出一套切实可行的检测技术体系; 针对共生体的生长时期、形态特征和具体的物种特异性, 分别选择出较为适用的技术方法进行检测, 这样才能快速有效地对禾草内生真菌进行检测。

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