铁皮石斛疫病及其病原菌

铁皮石斛疫病2001年发现在浙江义乌栽培田间,是由疫霉菌引起的。在田间,疫霉菌侵染茎基部,引起当年移植苗根腐、植株枯萎和死亡,但侵染2-3年植株幼嫩顶部仅引起项枯症状。通过对病原菌形态学、交配型的观察,以及核糖体DNA ITS序列分析,侵染铁皮石斛的5个分离菌株被鉴定为烟草疫霉菌Phytophthora nicotianae。致病性试验表明,铁皮石斛是烟草疫霉菌的寄主。     

铁皮石斛疫病及其病原菌

铁皮石斛疫病2001年发现在浙江义乌栽培田间,是由疫霉菌引起的。在田间,疫霉菌侵染茎基部,引起当年移植苗根腐、植株枯萎和死亡,但侵染2-3年植株幼嫩顶部仅引起顶枯症状。通过对病原菌形态学、交配型的观察,以及核糖体DNAITS序列分析,侵染铁皮石斛的5个分离菌株被鉴定为烟草疫霉菌Phytophthora nicotianae。致病性试验表明,铁皮石斛是烟草疫霉菌的寄主。     

温度对铁皮石斛生长及生理特性的影响

通过不同温度下的控制实验,研究了铁皮石斛光合作用与生长对温度的响应,以期为铁皮石斛的栽培提供理论依据。温度对铁皮石斛的光合速率（Pn）有明显影响,30℃处理的植株具有最高的饱和光合速率（Pmax）,其较高的光合速率与RuBP电子传递速率与羧化速率间相对平衡有关。温度对铁皮石斛茎的生长及多糖含量有明显影响,20℃处理的石斛多糖含量显著性的高于其他两个处理,而茎长、茎节数、茎鲜重等则是在30℃下最高。结果表明,30℃的温度对铁皮石斛的光合作用较为适宜,但在20℃条件下植株具有更高的多糖含量。     

铁皮石斛内生真菌研究进展

目前已经从不同生境的铁皮石斛(Dendrobium officinale Kimura et Migo)中分离到内生真菌252株,这些内生真菌涵盖了真菌门（Eumycota;Mycobionta）5个亚门中的半知菌亚门(Deuterornycotina)、担子菌亚门(Basidiomycotina)和子囊菌亚门（Ascomycotina）等3个亚门,已经鉴定的内生真菌分属于48个属。有些内生真菌对铁皮石斛的种子萌发和植株生长均有促进作用。对铁皮石斛内生真菌的多样性和分类、铁皮石斛内生菌与宿主的关系进行了综述,探讨研究中存在的问题, 并对今后研究方向提出建议。     

硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响

利用盆栽的方式研究了不同硝酸镧水平(0、1.0、3.0、5.0和7.0mg·L-1)下接种兰科菌根真菌对铁皮石斛生物量、多糖和蛋白质合成的影响,并分析了叶绿素含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性的变化,以探讨硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响。结果表明:(1)添加适量的硝酸镧有利于菌根真菌侵染和菌根发育,提高铁皮石斛幼苗生物量。(2)在接种菌根真菌的同时添加5.0mg·L-1的硝酸镧,铁皮石斛的根重、茎叶重和总生物量均达到最大,分别是未添加硝酸镧以及未接种对照组的4.26倍、4.98倍和4.87倍,其菌根侵染率也高达92.8%;而且可显著提高叶片中叶绿素含量,并显著降低细胞内的丙二醛含量。(3)在适量(5.0mg·L-1)的硝酸镧水平下接种菌根真菌能促进铁皮石斛幼苗多糖和蛋白质的合成,并显著提高细胞内SOD、CAT和POD活性。研究认为,菌根真菌与适宜浓度硝酸镧(5.0 mg·L-1)联合使用能显著促进铁皮石斛菌根的形成,增强植株的生理活性和适应能力,提高其生物量和多糖等活性成分的积累,有效改善铁皮石斛的药用品质。     

同源四倍体铁皮石斛的生长及多糖积累

通过秋水仙素诱导铁皮石斛多倍体,以外观形态、气孔、染色体数目筛选多倍体植株,分析多倍化对铁皮石斛多糖累积及生长的影响。结果表明,同源四倍体铁皮石斛茎、叶及原球茎的多糖含量分别是二倍体的1.31、1．83及1．95倍,但生长速度显著或极显著低于对照。ISSR分析显示,同源四倍体植株产生较明显的基因变异。     

氮源和真菌诱导子对铁皮石斛原球茎悬浮培养的影响

利用正交实验设计研究不同种类和浓度的氮源对铁皮石斛原球茎生长的影响,利用完全随机实验设计研究不同真菌诱导子对铁皮石斛原球茎生长和多糖积累的影响.结果表明硝态氮促进铁皮石斛原球茎鲜重和干重的增加( P ＜0.05);铵态氮促进铁皮石斛原球茎鲜重增加( P ＜0.05).无论鲜重还是干重,硝态氮和铵态氮的影响均不存在互作,最佳组合为:67-V培养液 100 mg/L (NH4)2SO4 800 mg/L KNO3 30 g/L蔗糖 200 g/L马铃薯提取汁.F检验的结果表明,14种真菌诱导子对铁皮石斛原球茎生物量的增加(鲜重与干重)均无显著性影响( P ＞0.05).但与对照铁皮石斛原球茎的多糖含量相比,真菌诱导子g6、g14、g5、g4处理可使其多糖含量分别提高14%、9%、6%、4%.本研究获得了有利于铁皮石斛原球茎生长的硝态氮和铵态氮的最佳搭配方案以及有利于铁皮石斛原球茎积累多糖的四种真菌诱导子,表明通过液体悬浮培养生产铁皮石斛原球茎及其多糖成分具有较好的开发应用前景.     

广西铁皮石斛HPLC指纹图谱研究

采用HPLC法建立广西铁皮石斛提取液的指纹图谱,运用相似度评价药材质量。结果表明,在13个共有峰构成的铁皮石斛指纹图谱中,有11批药材相似度均在0.8以上,2批的相似度较差,未达到0.8,表明相似度大小与药材的产地有关。通过对栽培铁皮石斛不同部位药材的指纹图谱峰面积的比较,不提倡对药材提早采收。3批组织培养铁皮石斛的图谱相似度较低,说明培养到不同阶段的铁皮石斛材料组织培养物,其化学成分相差较大。     

兰科菌根真菌对干旱胁迫下铁皮石斛生长和抗氧化能力及相关基因表达的影响

利用盆栽的方式研究了干旱胁迫下接种兰科菌根真菌(OM)对铁皮石斛生长的影响,并分析了铁皮石斛叶片相对含水量、游离脯氨酸含量、电解质渗透率、丙二醛(MDA)含量、活性氧成分、抗氧化酶活性变化,用定量PCR技术分析了相关抗氧化酶基因的表达特性,以探讨菌根真菌对铁皮石斛干旱胁迫的缓解作用及其机制。结果表明：(1)与正常水分条件相比,干旱胁迫显著降低了铁皮石斛幼苗的生物量和叶片相对含水量,提高了叶片电解质渗透率、脯氨酸含量、MDA含量、O-·2产生速率和H2O2水平。(2)菌根真菌能显著提高干旱胁迫下铁皮石斛叶片相对含水量,降低叶片电解质渗透率、脯氨酸含量、MDA含量、O-·2产生速率和H2O2水平;在不同水分条件下,菌根真菌均能有效促进铁皮石斛幼苗生长,其株高、根重、茎叶重和总生物量均大于未接种组。(3)菌根真菌可诱导干旱胁迫下铁皮石斛超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)基因的表达,提高SOD、POD和CAT的活性,有效缓解干旱胁迫对质膜的过氧化伤害。研究认为,菌根真菌能提高干旱胁迫下铁皮石斛的抗氧化酶活性及其相关基因表达水平,增强铁皮石斛抗氧化防御能力,有效缓解干旱胁迫对铁皮石斛幼苗生长的抑制。     

铁皮石斛内生真菌分布

本研究从铁皮石斛(Dendrobium officinale)根、茎和叶中分离得到内生真菌67株,经形态学和分子生物学鉴定分别归属于16个属,Fusarium和Alternaria为铁皮石斛内生真菌的优势种群。研究发现,铁皮石斛根、茎、叶中内生真菌分布存在较大差别,具有一定的组织差异性,不同石斛植物中内生真菌的存在情况差别较大,具有一定的宿主差异性。本研究丰富了真菌资源,为石斛属药用植物内生真菌资源的开发利用提供了基础理论依据。     

植物根系复合共生体研究进展

植物根系与菌根真菌形成的互惠共生体,即菌根(mycorrhizas)是最常见、最广泛分布的共生体之一。自然条件下,一些植物的根系可同时形成由2种类型菌根构成的混合菌根,或菌根与细菌、菌根与放线菌、菌根与其他种类真菌构建的所谓复合共生体。文中从复合共生体的概念入手,简要介绍混合菌根、菌根与细菌、菌根与放线菌、菌根与其他真菌构建的复合共生体的多样性、形态解剖特征、生长发育及其功能等方面的研究概况,旨在为推进该领域的研究提供依据和借鉴的思路。     

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or “shiro”. We questioned whether this phenomenon could have occurred because of plant–microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant–fungal symbiosis.     

霍山石斛与内生真菌共培养体系的建立

将霍山石斛试管苗与内生真菌(SH-01菌株)共生培养,测定其生长状态,筛选最佳共生培养基。研究该培养体系下内生真菌对石斛试管苗生长和移栽成活率的影响。结果表明,适合共生培养基为1/2 MS 10 g/L蔗糖 10 g/L琼脂,内生真菌在共生培养过程中对石斛试管苗的生长有显著的促进作用。     

The use of vesicular-arbuscular mycorrhizal roots grown by the nutrient film technique as inoculum for field sites

Bean roots grown by the nutrient film technique and infected with vesicular-arbuscular mycorrhizal fungi were successfully used as inoculum for red clover growing in an unsterile field site. The endophyte introduced with the roots became well established in the presence of indigenous mycorrhizal fungi.     

铁皮石斛内生真菌的分离及其挥发性成分分析

铁皮石斛(Dendrobium officinale)是我国重要的濒危中药材,其药用价值和经济价值很高。以铁皮石斛为研究对象,采用组织块法,从铁皮石斛根、茎和叶中分离获得22株内生真菌。采用固相微萃取固定吸附(SPME),结合气相质谱(GCMS)解吸附分析技术对其中具有产香特性的3株内生真菌KLBMPD001、KLBMPD004和LBMPD009的挥发性成分进行分析。结果显示,这3株内生真菌均含有较高比例的棕榈酸,分别为37.94%、24.15%和48.55%,其中菌株KLBMPD001和KLBMPD009含有一定比例的硬脂酸,分别为12.64%和37.37%,只有菌株KLBMPD004含有8.74%的酞酸二丁酯。     

实验室条件下五唇兰菌根真菌专一性研究

利用从高原温带兰科植物菌根中获得的22个菌根真菌菌株, 对五唇兰(Doritis pulcherrima)进行了室内种子萌发、原球茎分化和组培苗回接试验, 从交叉回接的角度对附生兰科植物与菌根真菌的生理专一性进行了探讨。经过20周的共生培养, 只有编号为Cf1和Mm1的两个菌株使种子表现出种胚明显膨大的萌发迹象; 9个菌株能够促使原球茎较好地分化发育出根叶; 11个菌株处理苗的平均鲜重增长率高于对照组(156.25%), 其中Mm1的效果达到极显著水平(p = 0.01)。通过根切片显微观察, 在原球茎分化根和回接效果良好的处理苗的根皮层组织发现典型的菌丝团结构, 表明菌根体系已成功建立。温带地生兰菌根真菌对五唇兰种子萌发、原球茎发育和幼苗生长等3个重要生长阶段影响的试验显示, 五唇兰的种子和菌根真菌的共生萌发效果不佳, 而原球茎及幼株更容易与之建立良好的共生关系。同时, 也没有发现同一个真菌菌株能够对五唇兰的种子、原球茎和幼苗均产生促进作用。研究结果表明, 五唇兰的菌根真菌专一性因生理生长阶段的不同而存在差异。     

Balancing multiple mutualists: asymmetric interactions among plants, arbuscular mycorrhizal fungi, and fungal endophytes

Most organisms engage in beneficial interactions with other species; however, little is known regarding how individuals balance the competing demands of multiple mutualisms. Here we examine three-way interactions among a widespread grass, Schedonorus phoenix , a protective fungal endophyte aboveground, Neotyphodium coenophialum , and nutritional symbionts (arbuscular mycorrhizal fungi) belowground. In a greenhouse experiment, we manipulated the presence/absence of both fungi and applied a fertilizer treatment to individual plants. Endophyte presence in host plants strongly reduced mycorrhizal colonization of roots. Additionally, for plants with the endophyte, the density of endophyte hyphae was negatively correlated with mycorrhizal colonization, suggesting a novel role for endophyte abundance in the interaction between the symbionts. Endophyte presence increased plant biomass, and there was a positive correlation between endophyte hyphal density and plant biomass. The effects of mutualists were asymmetric: mycorrhizal fungi treatments had no significant impact on the endophyte and negligible effects on plant biomass. Fertilization affected all three species – increasing plant biomass and endophyte density, but diminishing mycorrhizal colonization. Mechanisms driving negative effects of endophytes on mycorrhizae may include inhibition via endophyte alkaloids, altered nutritional requirements of the host plant, and/or temporal and spatial priority effects in the interactions among plants and multiple symbionts.     

Neighboring Deschampsia flexuosa and Trientalis europaea harbor contrasting root fungal endophytic communities

Fungal endophytic communities and potential host preference of root-inhabiting fungi of boreal forest understory plants are poorly known. The objective of this study was to find out whether two neighboring plant species, Deschampsia flexuosa (Poaceae) and Trientalis europaea (Primulaceae), share similar root fungal endophytic communities and whether the communities differ between two sites. The study was carried out by analysis of pure culture isolates and root fungal colonization percentages. A total of 84 isolates from D. flexuosa and 27 isolates from T. europaea were obtained. The roots of D. flexuosa harbored 16 different isolate types based on macromorphological characteristics, whereas only 4 isolate types were found in T. europaea. The root colonization by dark septate and hyaline septate hyphae correlated with isolate numbers being higher in D. flexuosa compared to T. europaea. The different isolate types were further identified on the basis of internal transcribed spacer sequence and phylogenetic analysis. An isolate type identified as dark septate endophyte Phialocephala fortinii colonized 50 % of the T. europaea and 21 % of the D. flexuosa specimens. In addition, Meliniomyces variabilis, Phialocephala sphaeroides, and Umbelopsis isabellina were found colonizing the grass, D. flexuosa, for the first time and Mycena sp. was confirmed as an endophyte of D. flexuosa. Site-specific differences were observed in the abundance and diversity of endophytic fungi in the roots of both study plants, but the differences were not as predominant as those between plant species. It is concluded that D. flexuosa harbors both higher amount and more diverse community of endophytic fungi in its roots compared to T. europaea.     

Colonisation of barley roots by endophytic Fusarium equiseti and Pochonia chlamydosporia: Effects on plant growth and disease

Colonisation of plant roots by endophytic fungi may confer benefits to the host such as protection against abiotic or biotic stresses or plant growth promotion. The exploitation of these properties is of great relevance at an applied level, either to increase yields of agricultural crops or in reforestation activities. Fusarium equiseti is a naturally occurring endophyte in vegetation under stress in Mediterranean ecosystems. Pochonia chlamydosporia is a nematode egg-parasitic fungus with a worldwide distribution. Both fungi have the capacity to colonise roots of non-host plants endophytically and to protect them against phytopathogenic fungi under laboratory conditions. The aim of this study was to evaluate the root population dynamics of these fungi under non-axenic practical conditions. Both fungal species were inoculated into barley roots. Their presence in roots and effects on plant growth and incidence of disease caused by the pathogen Gaeumannomyces graminis var. tritici were monitored periodically. Both fungi colonised barley roots endophytically over the duration of the experiment and competed with other existing fungal root colonisers. Furthermore, colonisation of roots by P. chlamydosporia promoted plant growth. Although a clear suppressive effect on disease could not be detected, F. equiseti isolates reduced the mean root lesion length caused by the pathogen. Results of this work suggest that both F. equiseti and P. chlamydosporia are long-term root endophytes that confer beneficial effects to the host plant.