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。     

光照强度和光周期对醉马草内生真菌共生体幼苗生长和生物碱积累的影响

该研究通过测定不同光周期(8、12、14和16h·d~(-1))和光照强度(1 500、2 500和3 500lux)处理下醉马草内生真菌共生体幼苗的形态指标以及叶绿素、可溶性糖、麦角酰胺和麦角新碱的含量,以明确醉马草内生真菌共生体幼苗生长和生物碱积累的最适光照条件,为醉马草内生真菌共生体的应用提供理论依据。结果发现:(1)醉马草内生真菌共生体幼苗株高、叶绿素和可溶性糖含量随着光照时间的延长而增加,并在光周期为16h·d~(-1)时显著(P0.05)高于其他处理;根长、单株分蘖数和单株生物量均先升高后降低,且均在14h·d~(-1)处理下达到最大值。(2)随着光照强度的增加,醉马草内生真菌共生体幼苗4个形态指标以及叶绿素和可溶性糖含量均呈增加的趋势,并在光照强度为3 500lux时均达到最大值。(3)随着处理时间的延长,醉马草内生真菌共生体幼苗麦角酰胺和麦角新碱的含量在不同光周期处理下均呈上升的趋势,并在光周期为12h·d~(-1)和光照强度为2 500lux处理下麦角酰胺和麦角新碱的含量均显著高于其他处理(P0.05),且在处理第15天时达到最大值。研究表明,适宜的光照环境能显著促进醉马草内生真菌共生体幼苗生长和生物碱的积累,并以12h·d~(-1)光周期和2 500lux光照处理下最有利于麦角酰胺和的麦角新碱积累。     

刈割高度和频度对醉马草（Achnatherum inebrians）内生真菌共生体麦角生物碱的影响

在温室条件下,通过高效液相色谱法,测定了不同刈割高度（2.5,5.0和7.5cm）和刈割频度（1周1次和2周1次）对醉马草甘肃内生真菌共生体产生麦角酰胺和麦角新碱量的影响.前4个月醉马草中生物碱的含量基本成线性增加,在不同刈割高度处理中,7.5cm的刈割高度条件下生物碱含量要高于2.5cm的刈割高度;在刈割频度处理中,两周刈割一次生物碱含量要高于1周刈割1次.实验过程中检测到生物碱含量最高值可达最低值的2～3倍.上述结果表明,如果将醉马草内生真菌应用于植物保护,必须意识到潜在的植物管理因素（刈割频率或者播种密度等）的实施可能会降低或者提高植物保护的水平,相同的管理因素也可能影响其他禾草-内生真菌共生体中生物碱的含量,所以管理因素对禾草-内生真菌共生体产碱的影响将会是未来一个非常有趣的话题.     

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。     

不同pH对醉马草内生真菌共生体幼苗生长和产碱的影响

该研究考察了不同pH(3、5、7、9和11)处理对醉马草内生真菌共生体幼苗的形态指标、叶绿素、可溶性糖以及麦角酰胺和麦角新碱含量的影响,以明确共生体幼苗生长和产碱的最适pH条件。结果表明：（1）醉马草内生真菌共生体幼苗株高、根长、分蘖和生物量等均在pH 7时（达到最大）显著高于其他4个酸碱处理,而且4个生长指标在强碱性处理下均显著高于强酸处理。（2）幼苗叶绿素含量也在pH 7的中性条件下（达到最高）显著高于pH 3、pH 5和pH 11处理,且强碱性处理下叶绿素含量也显著高于强酸处理,但幼苗可溶性糖含量在不同pH处理之间没有显著变化。（3）在相同时间内,醉马草内生真菌共生体幼苗麦角酰胺和麦角新碱含量均随环境pH值升高而增加;在处理时间为15 d时,麦角酰胺在pH 9~11处理下积累量最多,麦角新碱含量在pH 11处理下达到最大值,酸性条件抑制醉马草内生真菌共生体幼苗产碱。研究发现,醉马草内生真菌共生体幼苗在中性环境条件下生长最佳,且在碱性条件下生长较酸性条件下生长良好,而体内麦角酰胺和麦角新碱的积累在强碱性条件下达到峰值,可将醉马草内生真菌共生体应用于盐碱地的栽培绿化。     

禾草-内生真菌共生体在草地农业系统中的作用

综述了国内外近年对禾草 -内生真菌 (N eotyphodium)的研究进展。全世界现已报道 14种禾草内生真菌 ,与 2 3个属的禾草形成共生体 ,我国已在 13属 2 5种天然草地禾草中发现内生真菌 ,其中发草属 (Descampsia)、大麦属 (H ord eum)和赖草属(L eymus)以往在国际文献中未曾报道。内生真菌在禾草体内产生的生物碱 ,致使采食带菌禾草的马、牛、羊、鹿等家畜产生中毒症状 ,每年给美国、新西兰等国造成的经济损失达 6.4亿美元之多 ,对其毒理研究取得了进展。已发现我国醉马草 (Achnatheruminebrians)对家畜的毒性与内生真菌的侵染有关。内生真菌侵染增加可使禾草对 42种害虫的抗性 ,并可增加对某些线虫和病害的抗性。与不带菌禾草相比 ,带菌禾草的另一特点是抗逆性强 ,牧草产量高。国际在该领域的研究主要集中在多年生黑麦草(L olium p erenne)和高羊茅 (Festuca arundinacea)。我国的研究发现 ,带菌布顿大麦草 (H ordeum bodg anii)和圆柱披碱草(Elymus cylind ricus)的牧草产量分别增加 3 3 .3 %和 2 78.8% ,分孽数分别增加 13 6.8%和 84.5%。目前 ,国际研究的重点包括大规模开展内生真菌生物学与生态学特性的研究 ,创造不含对家畜有害毒素的有益禾草 -内生真菌共生体 ,培育带内生真菌的草坪草品种 ,培育抗毒     

内生真菌感染对宿主羽茅及邻生植物抗病性的影响

Epichloë内生真菌能够影响宿主植物的抗病性, 但目前关于内生真菌感染对宿主邻生植物抗病性影响的研究很少。该研究首先调查了内蒙古呼伦贝尔天然草地中植物病害发生情况, 发现在常见牧草中禾本科植物病害最为严重, 其中感染内生真菌的羽茅(Achnatherum sibiricum)的总病情指数最低。进一步比较了与羽茅邻生及非邻生植物的病害差异, 结果显示羽茅显著降低了邻生羊草(Leymus chinensis)、狼针草(Stipa baicalensis)和冰草(Agropyron cristatum)的褐斑病病情指数。为探究羽茅对邻生植物病害的影响是否与其内生真菌有关, 分别从内生真菌发酵液、离体叶片和植株层面进行了室内验证试验。结果表明: 羽茅的2种内生真菌Epichloë gansuensis和Epichloë sibiricum发酵液显著抑制了新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)、核盘菌(Sclerotinia sclerotioru)和三叶草核盘菌(Sclerotinia trifoliorum)的生长; 对于离体叶片, E. gansuensis和E. sibiricum显著减小了宿主叶片受4种病原真菌侵染后的病斑长度。在新月弯孢和根腐离蠕孢的植株接种条件下, 内生真菌显著降低了宿主羽茅及邻生植物羊草的病害程度。该研究首次发现并验证了羽茅内生真菌对邻生禾草抵御褐斑病有一定的增益作用。     

内生真菌对羽茅抗病性的影响

为探讨不同种类内生真菌对宿主植物羽茅(Achnatherum sibiricum)抗病性的影响, 以感染不同内生真菌的天然禾草羽茅为实验材料, 进行了体外纯培养的内生真菌、感染内生真菌的离体叶片和在体叶片对3种植物病原真菌的抑菌实验。结果表明: 体外纯培养条件下, 分离自羽茅的内生真菌Neotyphodium sibiricum、Neotyphodium gansuensis和Epichloë gansuensis对新月弯孢霉(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)等3种病原真菌都具有抑制作用, 其中N. sibiricum的抑制作用最强, 对新月弯孢霉、根腐离蠕孢和枝孢霉的抑菌率分别为47.8%、40.1%、39.4%; 内生真菌培养滤液也可以有效抑制这3种病原真菌的孢子萌发, 其中N. gansuensis的抑制作用最强, 新月弯孢、根腐离蠕孢和枝孢霉的孢子萌发率分别为9.8%、8.7%、8.5%。对于离体叶片, N. sibiricum和N. gansuensis感染可以有效降低叶片受3种病原真菌侵染后的病斑数和孢子浓度, 其中N. sibiricum对根腐离蠕孢的抑制作用显著高于N. gansuensis, 而E. gansuensis只降低新月弯孢和枝孢霉侵染的病斑数以及枝孢霉侵染的孢子浓度。在体条件下, 内生真菌均可以显著降低病原真菌侵染羽茅后的病斑数、病斑长度和孢子浓度, 其中E. gansuensis的抑菌作用趋于最弱, 而N. sibiricum的抑菌作用趋于最强。     

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”。     

Mn、Zn和Fe对醉马草内生真菌共生体麦角酰胺和麦角新碱含量的影响

该研究通过温室砂培试验,考察了不同浓度Mn、Zn和Fe处理对醉马草内生真菌共生体麦角酰胺和麦角新碱含量的影响,以明确共生体产碱所需3种微量元素的最佳浓度范围。结果表明:(1)在短时间(3周)处理下,共生体麦角酰胺和麦角新碱含量均在Mn~(2+)浓度为5.0mmol·L~(-1)时达到最大值,且显著高于其余Mn~(2+)浓度梯度;在较长时间(6、9周)处理下,其麦角酰胺含量在Mn~(2+)浓度为5.0mmol·L~(-1)时最大但增幅不明显,麦角新碱在对照(0.01mmol·L~(-1) Mn~(2+))处理下积累量达到最大,且显著高于其余处理。(2)共生体麦角酰胺含量在处理时间为3、6和9周时,Zn~(2+)处理均在7.0mmol·L~(-1)下达到最大值,且处理3周的含量显著高于其余处理时间;其麦角新碱在3个处理时间下均于Zn~(2+)浓度为21.0mmol·L~(-1)时达到最大值。(3)在不同处理时间下,共生体麦角酰胺的含量均在Fe浓度为6.0~12.0mmol·L~(-1)下达到最大值,且在3周时的含量显著高于其余处理时间;麦角新碱含量在第3、6周时均于Fe浓度为0mmol·L~(-1)下达到最大值,在第9周时则于Fe浓度为0.03mmol·L~(-1)条件下达到最大值。研究认为,高浓度短时间的Mn、Zn和Fe元素处理有利于刺激醉马草内生真菌共生体2种麦角碱的积累,可通过控制3种微量元素的浓度和共生体的生长时间来提高2种麦角碱的含量,为今后能萃取更高浓度的麦角酰胺和麦角新碱作为临床药剂或生物农药提供较精确的外界条件。     

内生真菌种类和母本基因型对内生真菌-禾草共生体叶形状和叶面积的影响

Epichloë内生真菌感染能够影响宿主植物的生长发育, 但关于内生真菌感染对宿主植物叶形状和叶面积的研究很少。该研究以羽茅(Achnatherum sibiricum)为实验材料, 采用长宽系数计算和扫描测定叶面积相结合的方法探究内生真菌种类和羽茅母本基因型对羽茅-内生真菌共生体叶形状和叶面积的影响。结果表明: 内生真菌感染与否、内生真菌种类和宿主母本基因型对反映叶形状的叶校正系数、叶片长度、宽度和长宽比均无显著影响, 经计算与验证, 确定了羽茅叶片的校正系数为0.594 9。采用该校正系数及叶长宽计算的叶面积与实测叶面积无显著差异, 且二者均未受到内生真菌感染与否、内生真菌种类或宿主植物母本基因型的显著影响。     

Effects of endophyte fungal species and host plant genotype on the leaf shape and leaf area of endophyte-grass symbionts

Epichloë内生真菌感染能够影响宿主植物的生长发育, 但关于内生真菌感染对宿主植物叶形状和叶面积的研究很少。该研究以羽茅(Achnatherum sibiricum)为实验材料, 采用长宽系数计算和扫描测定叶面积相结合的方法探究内生真菌种类和羽茅母本基因型对羽茅-内生真菌共生体叶形状和叶面积的影响。结果表明: 内生真菌感染与否、内生真菌种类和宿主母本基因型对反映叶形状的叶校正系数、叶片长度、宽度和长宽比均无显著影响, 经计算与验证, 确定了羽茅叶片的校正系数为0.594 9。采用该校正系数及叶长宽计算的叶面积与实测叶面积无显著差异, 且二者均未受到内生真菌感染与否、内生真菌种类或宿主植物母本基因型的显著影响。     

Effects of grassland management,endophytic fungi and predators on aphid abundance in two distinct regions

Aims Fungal endophytes of cool-season grass species produce alkaloids toxic to herbivores, affecting food webs in agricultural and natural ecosystems. Field studies about the effects of endophytes on herbivores are rare and show contradictory results, leading to uncertain conclusions about the nature of endophyte–grass symbiosis. We asked whether the environmental contexts of local and regional scales and predation could modify the effects of endophytes on herbivores.Methods In a full factorial field experiment, we quantified the abundance of the aphid species Rhopalosiphum padi on the potted host grass Lolium perenne, which was either infected or uninfected with the endophytic fungus Neotyphodium lolii. Predators were either excluded or had free access to the pots with the aphids. One hundred and sixty grass pots were located in two regions on altogether 40 grassland sites, half of the sites intensively and half extensively managed. We tested the importance of endophyte infection, study region, management intensity of grasslands, predation and all two-way interactions on aphid abundance.Important findings Endophyte infection reduced aphid abundance significantly in one study region only. In both regions, we found that the impacts of aphidophagous predators and grassland management intensity on aphid abundance were substantially stronger and more consistent than that of endophytes on aphid abundance. Pots excluding predators and pots placed on extensive grasslands contained higher aphid abundance. The impact of predators and management on aphid abundance were not modified by the endophyte. We conclude that the effect of endophytes on herbivores can be weak in field experiments and depends on environmental context at a regional scale. Hence, more field research efforts are necessary to detect the relative importance of endophytes and the environmental context on biotic interactions in ecosystems.     

Arthropod abundance in tall fescue,Lolium arundinaceum,pastures containing novel ‘safe’ endophytes

Poor livestock performance on tall fescue is linked to infection by a fungal endophyte that enhances grass resistance to stress, including herbivory, while producing ergot alkaloids toxic to vertebrate grazers. Novel ‘safe’ endophyte/grass associations produce no ergot alkaloids yet retain stand persistence, but they could be more susceptible to insect outbreaks. We tested the hypothesis that grass‐feeding insects are more abundant in novel endophyte pastures compared with those containing common endophyte. Above‐ and below‐ground herbivores were sampled across two growing seasons in pastures containing common strain endophyte (KY31), novel endophytes (MaxQ or AR584) or endophyte free. We also sampled natural enemies as an indicator of possible tritrophic effects. With a few exceptions, numbers of predatory, chewing (grasshoppers, crickets, caterpillars) or sucking insects (leafhoppers, planthoppers) were similar in common and novel endophyte pastures. Contrary to our hypothesis, Aphrodes spp. leafhoppers were more abundant in KY31 than other pasture types in 2008; their nymphs also were more abundant in KY31 than in MaxQ in 2009. Adults (but not total numbers or nymphs) of another leafhopper, Psammotettix striatus, were less abundant in KY31 than other pasture types in 2009. Popillia japonica and Cyclocephala spp. grubs did not differ in density, weight or instar among the endophyte‐containing associations. In feeding assays, armyworm (Mythimna unipuncta) survival was higher on MaxQ and NE9301, and weight gain was higher on NE9301, suggesting armyworm outbreaks could be more severe on such grasses, but that result may not be attributable solely to alkaloids because common strain and endophyte‐free grasses did not differ in either parameter. Caterpillar abundance did not differ among pasture types in the field. Our results suggest that re‐seeding common strain endophytic pastures with livestock‐safe novel endophyte/grass associations to alleviate fescue toxicosis is unlikely to promote markedly higher populations of plant‐feeding insects.     

Contribution of ergot alkaloids to suppression of a grass-feeding caterpillar assessed with gene knockout endophytes in perennial ryegrass

Neotyphodium and Epichloë species (Ascomycota: Clavicipitaceae) are fungal symbionts (endophytes) of grasses. Many of these endophytes produce alkaloids that enhance their hosts’ resistance to insects or are toxic to grazing mammals. The goals of eliminating from forage grasses factors such as ergot alkaloids that are responsible for livestock disorders, while retaining pasture sustainability, and of developing resistant turf grasses, require better understanding of how particular alkaloids affect insect herbivores. We used perennial ryegrass Lolium perenne L. (Poaceae) symbiotic with Neotyphodium lolii × Epichloë typhina isolate Lp1 (a natural interspecific hybrid), as well as with genetically modified strains of Lp1 with altered ergot alkaloid profiles, to test effects of ergot alkaloids on feeding, growth, and survival of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), a generalist grass‐feeding caterpillar. Neonates or late instars were provided clippings from glasshouse‐grown plants in choice and rearing trials. Wild‐type endophytic grass showed strong antixenosis and antibiosis, especially to neonates. Plant‐endophyte symbiota from which complex ergot alkaloids (ergovaline and lysergic acid amides such as ergine) or all ergot alkaloids were eliminated by endophyte gene knockout retained significant resistance against neonates. However, this activity was reduced compared to that of wild‐type Lp1, providing the first direct genetic evidence that ergot alkaloids contribute to insect resistance of endophytic grasses. Similarity of larval response to the two mutants suggested that ergovaline and/or ergine account for the somewhat greater potency of wild‐type Lp1 compared to the knockouts, whereas simpler ergot alkaloids contribute little to that added resistance. All of the endophyte strains also produced peramine, which was probably their primary resistance component. This study suggests that ergot alkaloids can be eliminated from an endophyte of perennial ryegrass while retaining significant insect resistance.     

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.     

云南省可培养丝孢真菌资源的调查研究

本文报道曲霉属及其相关的有性型属、即散囊菌属和裸胞壳属的分类群共15个,其中新变种1个,我国新记录3个。它们是：日本曲霉小囊变种(新变种),赭曲霉,蜂蜜曲霉,孔曲霉,埋藏曲霉(新记录),佩特曲霉(新记录)、亮白曲霉,阿姆斯特丹散囊菌,谢瓦散囊菌,腊叶散囊菌,赤散囊菌,匍匐散囊菌原变种,构巢裸胞壳,无冠裸胞壳和刺孢裸胞壳(新记录)。