禾草内生真菌作为生防因子的潜力分析

早熟禾亚科多种禾草可与Neotyphodium内生真菌形成禾草-内生真菌共生体,这种植物-微生物共生体性状较为稳定,且在自然界中广泛存在。禾草-内生真菌共生体稳定的互利共生关系不但保证了内生真菌所需的全部营养物质,而且共生体产生的次生代谢物又可显著提高宿主禾草对生物胁迫的抗逆性。众多研究表明,内生真菌的侵染可显著提高宿主禾草对虫害、病害及伴生植物等多种生物胁迫的抗性。据不完全统计,禾草内生真菌对蛛形纲、线虫纲、昆虫纲3个纲至少79个种的害虫表现出较明显的抗性,对至少22个种的病原真菌表现出明显的抗性。尽管利用内生真菌进行禾草品种选育及其品质改良的技术日趋成熟,但是内生真菌在不同宿主禾草之间高效的替代转化技术,及其在宿主体内遗传的稳定性仍有待于进一步深入探索。研究者把禾草内生真菌作为生防手段,在未来的应用过程中不应只考虑其与宿主禾草之间的共生特异性,而应更全面地分析禾草-内生真菌-生态环境之间的相互关系,让内生真菌更好地为人类服务。     

Advances in research of Achnatherum inebrians-Epichloë endophyte symbionts

有关醉马草(Achnatherum inebrians)内生真菌(Epichloë gansuensis, E. inebrians)共生体的研究, 代表了我国禾草内生真菌研究领域的重要方向, 使中国的醉马草-内生真菌与美国的苇状羊茅(Festuca arundinacea)-内生真菌(E. coenophiana)和新西兰的多年生黑麦草(Lolium perenne)-内生真菌(E. festucae var. lolii)成为禾草内生真菌国际三大研究分支。该文综述了近30年来对醉马草内生真菌共生体的系统研究, 包括: 内生真菌的分布、带菌率、检测方法、多样性, 内生真菌提高宿主的抗旱、耐寒、耐盐碱、耐重金属、抗虫、抗病等抗逆性及其机理, 共生体产生的生物碱等次生代谢物, 对草食动物的毒性, 及其在草地生态系统中的作用等。研究者实验证实了醉马草本身无毒, 只有当内生真菌与醉马草共生并产生麦角新碱和麦角酰胺等麦角类生物碱后才能导致采食醉马草家畜中毒。文章展望了醉马草内生真菌基因组学和功能分析, 利用杀菌剂杀死内生真菌进行醉马草脱毒, 利用无毒内生真菌菌株进行饲用醉马草新品种选育, 利用有毒醉马草内生真菌共生体进行抗虫防鸟的机场绿化新品种选育及生物源农药与医药开发等。     

醉马草内生真菌共生体研究进展

有关醉马草(Achnatherum inebrians)内生真菌(Epichlo?gansuensis, E. inebrians)共生体的研究,代表了我国禾草内生真菌研究领域的重要方向,使中国的醉马草-内生真菌与美国的苇状羊茅(Festuca arundinacea)-内生真菌(E. coenophiana)和新西兰的多年生黑麦草(Lolium perenne)-内生真菌(E. festucae var. lolii)成为禾草内生真菌国际三大研究分支。该文综述了近30年来对醉马草内生真菌共生体的系统研究,包括:内生真菌的分布、带菌率、检测方法、多样性,内生真菌提高宿主的抗旱、耐寒、耐盐碱、耐重金属、抗虫、抗病等抗逆性及其机理,共生体产生的生物碱等次生代谢物,对草食动物的毒性,及其在草地生态系统中的作用等。研究者实验证实了醉马草本身无毒,只有当内生真菌与醉马草共生并产生麦角新碱和麦角酰胺等麦角类生物碱后才能导致采食醉马草家畜中毒。文章展望了醉马草内生真菌基因组学和功能分析,利用杀菌剂杀死内生真菌进行醉马草脱毒,利用无毒内生真菌菌株进行饲用醉马草新品种选育,利用有毒醉马草内生真菌共生体进行抗虫防鸟的机场绿化新品种选育及生物源农药与医药开发等。     

甘肃马先蒿寄生对禾草内生真菌共生体光合特性的影响

[目的]甘肃马先蒿与感染内生真菌的禾草(紫花针茅和麦宾草)建立根寄生关系,有关内生真菌对根寄生危害禾草光合作用调控方面的研究较少。[方法]本研究以紫花针茅和麦宾草带菌(E+)、不带菌(E-)植株为研究对象,研究甘肃马先蒿寄生和未寄生处理对紫花针茅和麦宾草E+、E-植株不同生长阶段光合特性影响的动态变化。[结果]甘肃马先蒿寄生显著降低紫花针茅和麦宾草的净光合速率、蒸腾速率和气孔导度,而胞间二氧化碳浓度和水分利用率却显著增加,这与禾草是否感染内生真菌无关。甘肃马先蒿寄生后E+紫花针茅的净光合速率、气孔导度和蒸腾速率高于E-植株,而麦宾草E+植株的净光合速率、气孔导度和蒸腾速率却低于E-植株;同时,根寄生条件下E+紫花针茅的胞间二氧化碳浓度和水分利用率低于E-植株;而E-麦宾草植株的胞间二氧化碳浓度和水分利用率却低于E+植株。[结论]内生真菌侵染影响甘肃马先蒿根寄生危害禾草的光合作用;甘肃马先蒿和内生真菌同时成为禾草营养消耗库时,内生真菌与禾草的共生关系处于一种互惠共生和相互拮抗的动态变化。     

甘肃马先蒿寄生对禾草内生真菌共生体光合特性的影响北大核心CSCD

[目的]甘肃马先蒿与感染内生真菌的禾草(紫花针茅和麦宾草)建立根寄生关系,有关内生真菌对根寄生危害禾草光合作用调控方面的研究较少。[方法]本研究以紫花针茅和麦宾草带菌(E+)、不带菌(E-)植株为研究对象,研究甘肃马先蒿寄生和未寄生处理对紫花针茅和麦宾草E+、E-植株不同生长阶段光合特性影响的动态变化。[结果]甘肃马先蒿寄生显著降低紫花针茅和麦宾草的净光合速率、蒸腾速率和气孔导度,而胞间二氧化碳浓度和水分利用率却显著增加,这与禾草是否感染内生真菌无关。甘肃马先蒿寄生后E+紫花针茅的净光合速率、气孔导度和蒸腾速率高于E-植株,而麦宾草E+植株的净光合速率、气孔导度和蒸腾速率却低于E-植株;同时,根寄生条件下E+紫花针茅的胞间二氧化碳浓度和水分利用率低于E-植株;而E-麦宾草植株的胞间二氧化碳浓度和水分利用率却低于E+植株。[结论]内生真菌侵染影响甘肃马先蒿根寄生危害禾草的光合作用;甘肃马先蒿和内生真菌同时成为禾草营养消耗库时,内生真菌与禾草的共生关系处于一种互惠共生和相互拮抗的动态变化。     

禾草内生真菌对其它微生物的影响研究进展

禾草内生真菌在宿主植物的茎叶等地上组织中普遍存在,不仅能够提高禾草对生物与非生物逆境的抗性,而且能够对周围环境中的不同微生物类群产生影响。主要总结了禾草Neotyphodium/Epichlo内生真菌对病原真菌、丛枝菌根真菌和土壤微生物的影响及其作用机理。发现禾草内生真菌普遍存在对病原真菌的抑制作用,而对丛枝菌根真菌存在不对称的竞争作用,且因种类而异。禾草内生真菌对土壤微生物群落的作用则会随着土壤类型和时间等外界因素发生变化。禾草内生真菌对不同类群微生物的影响机制主要包括:通过生态位竞争、抑菌物质分泌、诱导抗病性等对病原真菌造成影响;通过根系化学物质释放、营养元素调节、侵染条件差异等对丛枝菌根真菌造成影响;通过根际沉积物和凋落物等对土壤微生物群落造成影响。禾草内生真菌产生的生物碱能提高宿主植物对包括昆虫在内草食动物采食的抗性,影响病原菌的侵入、定殖和扩展;根组织分泌物中包含次生代谢产物能够抑制菌根真菌、土传病原真菌及其它土壤微生物的侵染与群落组成;也可能通过次生代谢物影响禾草的其它抗性。因此,禾草内生真菌在植物-微生物系统中的作用应该给予更多的关注和深入研究。     

禾草内生真菌生物碱的研究进展

综述了国内外近20a以来在禾草内生真菌生物碱方面的研究进展。目前,已发现至少4大类10余种生物碱与内生真菌有关。各类生物碱中典型代表物的分子结构已完全清楚,部分内生真菌在离体条件下可产生除黑麦草碱外的生物碱,但产碱量较其在植物体中所产生的低很多,可相差150余倍之多。随着生物技术的发展,美国、新西兰等国在波胺、麦角碱和loline的生物合成途径方面已有了初步的进展,对个别具有重要功能基因以及其所编码的酶已有了深入研究。各种生物碱的致毒机理尚未完全清楚,除饱和吡咯化合物为新陈代谢类毒素和神经性毒素外,其余3类生物碱均为神经性毒素。生物碱可增加禾草对40余种害虫的抗性,并可增加对某些线虫和病害的抗性。诸多因素均可影响寄主植株中生物碱的种类和浓度,包括寄主植株：种群和生态型,植物品种和基因型,植株的部位和生长期;环境：气候因素,土壤养分,季节和年度变化;内生真菌菌株和草地管理利用方式等。用于生物碱检测的主要方法为预分离检测法和直接检测法,其中高效液相色谱法以其分离能力强、选择性高、测定灵敏度高,操作简单,可在室温下进行,应用范围极广的优点而广泛应用。目前,国际关于禾草内生真菌生物碱研究的重点包括创造不含对家畜有毒素的有益禾草-内生真菌共生体,开展基因工程研究以及合理利用生物碱,使其成为新一代的“生物农药”。     

禾草内生真菌的遗传多样性及其共生关系

 近百年的禾草内生真菌研究历经了由浅入深的过程，从最初的家畜中毒事件认识到是一种共生内生真菌存在的缘故，到如今利用分子生物学技术揭示其共生机制，人类发现这类植物内生真菌并非想象中的对生态系统无足轻重。Epichloë及其无性型Neotyphodium与禾本科植物是系统发生的互利共生关系，尤其是Neotyphodium可提高宿主抵抗环境胁迫的能力和抵御动物的取食，增强植物的竞争力。禾草内生真菌有3种生活史：有性生活史、无性生活史和兼性生活史，后者表明真菌在不同的宿主及环境下既能营有性生殖也可营无性繁殖，是一种更灵活而有效的生活史对策。对内生真菌分子系统学、生活史以及与宿主禾草协同进化的研究发现，Neotyphodium起源于禾草致病真菌Epichloë的某些种，或是Epichloë与Neotyphodium的种间杂交后代。植物和内生真菌各异的生活史策略，真菌的种间杂交，两者的协同进化亦或种群间基因流的差异，都促成了共生体多样化的基因组合（Genetic combination ），也是其共生关系多样化的根源。内生真菌对宿主的有益作用只在特定基因型真菌、宿主和一定环境条件下才起作用，自然生态系统的共生关系要比农业系统复杂得多，是一个从互利共生至寄生关系的连续系统。未来对于更多共生体的遗传背景和基因与环境相互作用的阐明将有助于对禾草内生真菌共生关系本质更加深入的认识。     

小花棘豆(Oxytropis glabra DC)内生真菌的分布特点

[目的]为探明小花棘豆不同组织中内生真菌带菌率及其种属分布特点,对采自内蒙古阿拉善盟草原的小花棘豆各组织内生真菌进行检测与分离.[方法]制作小花棘豆不同组织临时装片,通过染色观察与分离鉴定方法研究小花棘豆各组织中内生真菌分布形态、带菌率以及内生真菌种属分布情况.[结果]通过临时装片染色技术从小花棘豆茎、叶、叶柄及种子中均检测到内生真菌并观察到其在各组织中的分布特点;采用普通分离方法从小花棘豆四种组织中分离出79株内生真菌,鉴定为5个种.比较小花棘豆各组织内生真菌带菌率与分离率发现种子＞叶＞茎＞叶柄,且小花棘豆内生真菌的主要优势种(相对分离频率)为疯草内内生真菌(Undifilum oxytropis)(77.32％),砖格孢属(Embellisia sp.)L12(64.00％),镰刀菌属(Fusarium equiseti)(50.00％).[结论]小花棘豆各组织中普遍存在内生真菌,其在各组织中的形态分布各异,内生真菌数量与种属分布存在组织差异性,种子和叶是内生真菌侵染和定殖的主要部位.     

白羊草Epichloë属内生真菌的分离鉴定及其重金属耐受性

通过对北方铜业铜矿峪矿十八河尾矿坝的优势植被白羊草进行叶鞘内生真菌分离,并对白羊草内生真菌的形态特征及孢子形态进行描述,同时,选取其中7个菌株进行act G、tef A和tub B扩增、测序和系统发育分析。结果表明,从白羊草中分离出的内生真菌属于为Epichlo3属,菌落正面呈白色,背面浅黄色,白羊草内生真菌生长速度较慢,孢子大小为1.208—8.048μm之间,孢子形态椭圆、球型。根据分子系统发育学分析结果,将白羊草内生真菌鉴定为Epichlo3 sibiria。通过对白羊草内生真菌进行不同浓度的重金属胁迫处理,发现其对重金属具有一定耐受性,具体表现为:Zn2+(120 mg/L)、Cu2+(160 mg/L)、Pb2+(240 mg/L)和Cd2+(8 mg/L)。对丰富天然禾草内生真菌资源,以及铜矿区生态修复过程中,白羊草内生真菌提高宿主抗性方面的应用具有重要意义。     

内生真菌对草坪植物病原真菌抑制作用的比较

分别从野生牧草羽茅(Achnatherum sibiricum (L.) Keng)、栽培种高羊茅(Festuca arundinacea Schreb.)(品种Millennium)、栽培种黑麦草(Lolium perenne L.) (品种Justus)中分离出内生真菌Neotyphodium sp.、N. coenophialum和N. lolli,通过体外培养法比较了这3种内生真菌对草坪植物病原真菌的抑制作用.结果表明,从羽茅中分离的内生真菌Neotyphodium sp.在两菌相交前对所有供试的病原真菌都有一定的抑制作用,其中对枝孢霉属(Cladosporium sp.)、弯孢霉属(Curvularia sp.)和拟茎点属(Phomopsis sp.)病原真菌的抑制效果尤为显著,对峙培养3d后的抑菌率分别达70.1%、52.3%和30.9%,营养竞争作用、重寄生作用是其主要的拮抗机制;从高羊茅中分离的内生真菌N. coenophialum对枝孢霉属病原真菌存在一定的抑制作用;而从黑麦草中分离的内生真菌N. lolli与病原真菌对峙培养时,病原真菌菌落慢慢侵占整个营养空间,内生真菌停止生长并逐渐褐变死亡.体外培养结果说明Neotyphodium sp.对供试病原真菌的拮抗效果优于N. coenophialum和N. lolli,由此推测Neotyphodium sp.与宿主植物羽茅的共生可能有利于宿主植物抵抗病原真菌的侵扰.     

禾本科植物内生真菌资源及其物种多样性

植物内生真菌是当代微生物资源研究的一个热门话题,对禾本科植物内生真菌资源研究进行了总结。与冷季型禾本科植物共生的麦角菌科epichlo内生真菌是目前的研究重点,包括偶尔在宿主植物体表形成子座的Epichlo属真菌和几乎完全不形成症状的Neotyphodium属真菌。具体介绍该真菌类群的物种多样性及其地理分布多样性,概括了它们在全球各大区域的分布特征。对近年来中国迅速发展的epichlo内生真菌的研究作了总结,最后展望了国际国内epichlo内生真菌资源探索的发展方向。特别指出:冷季型禾本科植物内生真菌在南美洲和亚洲,暖季型禾本科植物内生真菌在热带亚热带地区的研究还相对薄弱,值得今后继续加强探索和挖掘。     

内蒙古中东部草原羽茅内生真菌的遗传多样性

 该文从形态学和基因水平上对内蒙古中东部草原4个羽茅(Achnatherum sibiricum)种群所含的27个内生真菌(Neotyphodium)菌株的遗传多样性进行了研究,结果表明羽茅内生真菌在形态学和基因水平上均表现出较高的 遗传多样性。在PDA培养基上,根据菌落颜色、质地、生长速率和分生孢子外观等特征,可分为4个形态型;西乌旗、霍林郭勒和定位站3个种群所含的内生真菌菌落具有相似的生长特性和外观,而羊草(Leymus chinensis)样地种群无论从菌落外观和质地的多样性,还是遗传多样性指数均高于其它3个样地。用20个随机引物对这27株内生真菌基因组DNA 进行RAPD分析,共检测到463个位点,其中461个为多态位点,多态位点百分率达99.6％,特有位点93个,占20.1％。Nei基因多样性指数h和 Shannon多样性指数I分别为0.238 0和0.387 0;多数菌株间的遗传一致度较低而遗传距离较大,在DNA水平上存在较显著的遗传变异。基于Nei 无偏遗传距离和UPGMA法的聚类分析结果将27株内生真菌分为7组,并且与形态型的划分基本一致,RAPD分析方法提高了内生真菌类群划分的可靠性。     

Detection and quantification of three distinct Neotyphodium lolii endophytes in Lolium perenne by real time PCR of secondary metabolite genes

Perennial ryegrass (Lolium perenne) is a widely used pasture grass, which is frequently infected by Neotyphodium lolii endophytes that enhance grass performance but can produce alkaloids inducing toxicosis in livestock. Several selected endophyte strains with reduced livestock toxicity, but that confer insect resistance, are now in common use. Little is known regarding the survival and persistence of these endophytes when in competition with common toxic endophytes. This is mainly because there are currently no assays available to easily and reliably quantify different endophytes in pastures or in batches of seeds infected with multiple strains. We developed real time PCR assays, based on secondary metabolite genes known to differ between N. lolii endophyte strains, to quantify two selected endophytes, AR1 and AR37, and a common toxic ecotype used in New Zealand. A duplex PCR allowed assessment of endophyte:grass DNA ratios with high sensitivity, specificity and precision. Endophyte specific primers/probes could detect contamination of AR37 seeds with other endophytes down to a level of 3–25%. We demonstrated that it is possible to quantify different endophyte strains simultaneously using multiplex PCR. This method has potential applications in management of endophytes in pastures and in fundamental research into this important plant-microbe symbiosis.     

Widespread occurrence of Neotyphodium-like endophyte in populations of Bromus tomentellus Boiss. in Iran

Neotyphodium species occur as endophytic fungi in cool-season grasses around the world. The beneficial aspects of grass-Neotyphodium associations have provoked researchers to look for a novel association in plant species where this symbiotum has not been reported. We surveyed Russian bromegrass (Bromus tomentellus Boiss.) accessions from a germplasm collection for the presence of Neotyphodium spp. fungi and determined levels of endophyte infection in B. tomentellus populations in native rangelands of Iran. Among 50 collected accessions, symbiotic fungi were detected in 45 accessions without any symptoms of choke disease on host plants. In culture medium, fast-growing endophytes appeared from seeds after 7-14 days. Plants grown from seed collections were 80-100% infected. Based on morphological characteristics and PCR analysis, we concluded that this fungus is a member of the Neotyphodium group of endophytic fungi. Lack of apparent toxicity to grazing animals suggests a place for endophyte-infected B. tomentellus in rangeland renovation, providing this infected grass exhibits increased tolerance to abiotic stresses.     

Mutualistic asexual endophytes in a native grass are usually parasitic

Asexual systemic fungi that live symbiotically within grasses are viewed as strong mutualists on the basis of theory and empirical studies of introduced agronomic grasses. Evolutionary theory predicts that microbial symbionts that lose sexuality and rely on propagules of their hosts for transmission should evolve to benefit their hosts. Fungal endophytes of some cultivated turf and pasture grasses are well known for increasing plant performance and competitive abilities, especially under stress, and increasing resistance to herbivores, pathogens, and root-feeders by virtue of fungal alkaloids. The assumption of mutualism, however, has rarely been tested in native grasses, which often harbor high but variable frequencies of systemic asexual endophytes. We tested the effect of Neotyphodium infections for the native grass Arizona fescue in a 3-yr field experiment. We strictly controlled host genotype and manipulated soil moisture and nutrients. Infection generally decreased host growth in terms of plant volume, number of tillers, and dry mass of shoots and roots. Infected plants also showed decreased reproduction in terms of number and mass of seeds, and the seeds produced by infected plants had lower germination success than plants without their endophytes, suggesting that the negative effects of the symbiont are transferred to the next generation. Plant genotype strongly influenced host's growth and reproduction and interacted with the presence of the endophyte, but the interaction was usually in the direction of negative effects. Our results challenge the notion that systemic asexual endophytes must be plant mutualists for infections to persist in nature. We propose other hypotheses to explain the variable but usually high endophyte frequencies in natural populations of grasses.     

Fungal endosymbionts affect aphid population size by reduction of adult life span and fecundity

The symbiosis between grasses and endophytic fungi is a common phenomenon and can affect herbivore performance through acquired, chemical plant defence by fungal alkaloids. In laboratory experiments, two species of common grass aphids, Rhopalosiphum padi and Metopolophium dirhodum were tested, in a population experiment (on four plant cultivars) and individually (on one plant cultivar) for the effects of the endophyte, Neotyphodium lolii, that forms symbiotic associations with perennial ryegrass Lolium perenne. In the population experiment that lasted for four aphid generations both aphid species showed decreased population sizes when feeding on each of the four endophyte-infected cultivars. Individuals of R. padi tested individually showed reduced adult life span and fecundity when feeding on infected plants. Individuals of M. dirhodum showed no response in any of the traits measured. This suggests that R. padi individuals are more sensitive to endophyte infection than M. dirhodum individuals. However, all infected grass cultivars reduced population sizes of both aphid species over four generations. Therefore, fungal endophytes can reduce populations of aphid herbivores independent of plant cultivars.     

Fungal endophytes of a forage grass reduce faecal degradation rates

Mutualisms between fungal endophytes and forage grasses can exert broad-reaching effects on grassland communities and ecosystem processes. We hypothesised that endophytes of grasses would retard the process of faecal degradation since grazing animals consume primarily live plant material and excrete a large portion of the herbage they consume as faeces. We examined the degradation rates of faeces from sheep that had consumed pure swards of perennial ryegrass containing a range of unique strains (AR1, AR37, or Wild type) of the fungal endophyte, Neotyphodium lolii, or no endophyte. Ultimately, the presence of endophytes in perennial ryegrass resulted in slower faecal decay rates compared to the nil endophyte treatment, although only consistently for the C concentration decay rates that were approximately 2× to 4× slower in the endophyte-derived faecal matter. The decay rate of dry matter content was significantly slower (ca. 1.5×) in the novel endophyte-derived faeces (AR1 and AR37) compared to the nil endophyte-derived faeces. The N decay rates differed significantly only in the AR1 treatment that was approximately 4× slower than the nil endophyte group. The reduced decay rates are attributed to the presence of endophyte-derived alkaloids in the faeces, and a greater proportion of more easily degraded hemicellulose in faeces from sheep that consumed the endophyte-free grass. There were no significant differences in the faecal carbon and nitrogen decay rates among the three endophyte strain treatments. This suggests that all the strain-specific alkaloids might have similar effects, or that N. lolii has a general effect that is not strain-specific, such as altered fibre composition, as reported here. This is the first report of a fungal endophyte affecting the rate of faecal degradation, and the first report of the alkaloids peramine, lolitrem B and epoxy–janthitrems in faecal matter. This study shows that a common agronomic grass–endophyte mutualism can have effects on ecosystem processes that have not previously been considered.