茶树叶部内生真菌相互关系及抗菌活性初步研究

研究分离自健康茶树叶部的主要内生真菌之间的相互关系,以及内生真菌的抗菌性。结果表明：分布普遍、分离频率较高的茶叶内生真菌如芒果球座菌、拟盘多毛孢菌和炭疽菌,在PDA平板上、对峙培养条件下的抑菌性不强或者不明显,内生真菌相互之间能有限生长,生长到一定程度即达到平衡而共存于一个生态空间;分离频率低的内生真菌如木霉、菌株FHM等,则表现出较强的抑菌性。内生真菌菌株FHM和内生木霉代谢产物抑菌活性较强,经乙酸乙酯提取的FHM发酵液能对茶芽枯病菌、茶轮斑病菌和茶炭疽病菌等茶树的病原真菌,以及白色念珠菌、枯草芽孢杆菌等菌株起抑制作用。     

雷竹内生真菌分离及其功能性菌株筛选与鉴定

[目的]从雷竹根部分离其内生真菌并对其纤维素降解、植物病原菌抑制、铅的耐受能力等功能进行初步探究.[方法]采用组织块培养法分离内生真菌,刚果红平板培养基筛选纤维素降解菌,平板对峙法筛选植物病原拮抗菌.[结果]从雷竹根部共分离到35株内生真菌,其中具有纤维素降解功能的有7株;对油茶炭疽病原有抑制作用的有5株,对毛竹枯梢病原有抑制作用的有2株,对猕猴桃果腐病原有抑制作用的有2株;对铅具有强耐受性的有11株.利用形态特征和分子生物学方法对两株具有良好潜在应用价值的内生真菌LZ031、LZ033进行鉴定,分别为腐皮镰刀菌(Fusarium solani)和芦竹节菱孢(Arthrinium arundinis).[结论]雷竹根部具有丰富的内生真菌资源,且内生真菌功能多样,具有潜在的应用价值.     

干旱区不同品种苹果树叶片内生真菌群落组成和功能特征

分析新疆典型干旱区轮台国家果树资源圃不同品种苹果树叶片内生真菌的分布特征及差异,为病害防治、品种选育和筛选功能微生物菌株提供参考。利用ITS基因高通量测序技术对6个品种苹果树叶片内生真菌进行测序,并比较其群落组成和功能特性。研究表明,苹果树叶片蕴含着丰富多样的内生真菌资源,共检测到真菌序列566453条,鉴定到5个门、15个纲、30个目、42个科及43个属。不同品种间内生真菌群落多样性存在一定差异,野苹果叶片内生真菌的多样性指数和丰度指数均较高。在门水平上的优势真菌菌群为子囊菌门(85.22%～98.68%),不同品种间差异不大。在纲和属上不同品种间有一定的差异,子囊菌门中的锤舌菌纲和座囊菌纲为优势纲,相对丰度分别在17.48%～69.69%和28.21%～67.59%之间;可归类的优势真菌属包括小粉孢属(17.47%～69.68%)、链格孢属(13.36%～39.35%)、枝孢霉属(2.94%～8.80%)和隐球菌属(0.20%～2.36%)。不同品种苹果树叶片真菌共有属的OTU数有42个,占各样品相对丰度的99%以上,而独有属的丰度各品种均低于1%。由于长期适应干旱区环境,在属水平...     

东省不同地区玉米内生真菌的群落组成和多样性分析

为探究山东省玉米内生真菌的资源分布、群落组成及多样性,采用叶片组织分离法对山东省52个市县的玉米内生真菌进行分离纯化,通过内转录间隔区（internal transcribed spacer,ITS）序列分析技术进行内生真菌的种属鉴定,并分析内生真菌的群落组成和多样性。结果表明,共分离到857株内生真菌,隶属25个属,其中链格孢属Alternaria的菌株数量最多,为583株,占总菌株数的68.03%,其次为镰刀菌属Fusarium有71株,占总菌株数的8.28%;大部分地区的优势属为链格孢属,少数地区的优势属为镰刀菌属、炭疽菌属Colletotrichum和弯孢属Curvularia。不同地区的内生真菌定殖率变化很大,在12.50%~100.00%之间,平均定殖率为76.15%;陵城的多样性指数和均匀度指数最高,分别为1.74和0.97;莱芜的丰富度指数最高为2.22。山东省玉米内生真菌可以分为5个类群,其中黄河流域地区内生真菌定殖率相对较高。表明山东省玉米内生真菌种类丰富,不同地区间多样性水平存在差异。     

当归内生真菌抗植物病原菌的活性研究

[目的]从当归内生真菌中筛选抗小麦赤霉病菌、番茄早疫病菌、番茄灰霉病菌、水稻纹枯病菌等植物病原菌的有益菌株,并筛选代谢产物中的抑菌活性部位.[方法]采用平板拮抗法对抗性菌株进行初步筛选,采用抑制菌丝生长法以及抑制孢子萌发法筛选有益内生真菌代谢产物中的抑菌活性部位.[结果]抗小麦赤霉病菌、番茄早疫病菌的内生真菌较多,分别占24.3％和28.6％.对小麦赤霉病菌、番茄早疫病菌、番茄灰霉病菌以及水稻纹枯病菌生长抑制作用较强的内生真菌包括5个属8个菌株,其中6个菌株只对1种病原菌菌丝生长有抑制作用.此外,对4种植物病原真菌孢子萌发具有抑制作用的内生真菌超过20％,其中内生真菌Myxormia sp.2和Myxormia sp.4的提取物对4种植物病原真菌孢子萌发的抑制作用显著,抑制率达到90％以上.通过对有益内生真菌代谢产物的分析表明,乙酸乙酯提取部位对植物病原真菌菌丝体和孢子萌发的抑制作用相对较强.[结论]具有抗菌活性的当归内生真菌及其次生代谢产物具有丰富的多样性.     

山东省不同地区玉米内生真菌的群落组成和多样性分析

为探究山东省玉米内生真菌的资源分布、群落组成及多样性,采用叶片组织分离法对山东省52个市县的玉米内生真菌进行分离纯化,通过内转录间隔区(internal transcribed spacer,ITS)序列分析技术进行内生真菌的种属鉴定,并分析内生真菌的群落组成和多样性。结果表明,共分离到857株内生真菌,隶属25个属,其中链格孢属Alternaria的菌株数量最多,为583株,占总菌株数的68.03%,其次为镰刀菌属Fusarium有71株,占总菌株数的8.28%;大部分地区的优势属为链格孢属,少数地区的优势属为镰刀菌属、炭疽菌属Colletotrichum和弯孢属Curvularia。不同地区的内生真菌定殖率变化很大,在12.50%～100.00%之间,平均定殖率为76.15%;陵城的多样性指数和均匀度指数最高,分别为1.74和0.97;莱芜的丰富度指数最高为2.22。山东省玉米内生真菌可以分为5个类群,其中黄河流域地区内生真菌定殖率相对较高。表明山东省玉米内生真菌种类丰富,不同地区间多样性水平存在差异。     

耕作方式对玉米田土壤真菌群落及多样性的影响

为明确不同耕作方式对玉米田土壤真菌区系的影响,2011-2015年选取全国13个主要玉米种植区不同保护性耕作处理的土壤465份,进行了玉米田土壤真菌多样性的研究。采用稀释平板法和水稻秸秆组织定殖法分离出真菌3 167株,利用形态学鉴定出43属71种。其中,接合菌6属8种,占总菌株数的9.66%;子囊菌3属4种,占2.05%;无性型真菌36属59种,占85.54%。除无孢类群外,玉米田中的优势菌分别为镰孢菌属(Fusarium)、木霉属(Trichoderma)、青霉属(Penicillium)、曲霉属(Aspergillus),分别占总菌株数的15.57%、15.50%、14.34%、12.53%。13个地区中吉林洮南地区玉米田土壤真菌多样性指数和丰富度指数最高,分别为R=5.772 7和H′=2.730 0;陕西杨凌地区玉米田土壤真菌均匀度指数最高(J=0.970 2);辽宁沈阳和吉林洮南玉米田土壤真菌相似度指数最高(C_j=0.806 5),云南曲靖、四川绵阳地区玉米田土壤真菌相似度指数最低(C_j=0.045 5)。研究表明,深松处理使玉米田土壤有益真菌数量逐年增加,且高于旋耕处理;致病菌数量显著下降,降低水平低于旋耕处理;中性菌数量年际间变化不显著;秸秆还田处理玉米田土壤有益菌、致病菌和中性菌数量均高于旋耕处理;随耕作年限增加有益菌和中性菌数量平稳上升,致病菌数量逐年下降,但不排除有回升的可能性。     

宁夏地区苦豆子内生真菌的分离与初步鉴定

为了探讨宁夏地区苦豆子内生真菌类群的多样性,以宁夏地区苦豆子草为试验材料,采用组织分离法从其根、茎、叶中分离内生真菌,依据形态进行初步鉴定。结果表明,从宁夏地区苦豆子875个表面消毒的根、茎、叶组织块中,共分离出内生真菌115株。其中根中分离出18株,占15.7%,茎中分离出41株,占35.7%,叶中分离出56株,占48.6%,分布规律为叶、茎、根依次递减,叶组织中的内生真菌多样性较高。对产孢的113株进行初步鉴定,分类为2纲3目5科7属,其中青霉属(Penicillium)3株,占2.6%,黑葱花霉属(Periconia)4株,占3.6%;链格孢属(Alternaria)45株,占39.1%;镰孢霉属(Fusarium)9株,占7.8%,曲霉属(Aspergillus)10株,占8.7%,枝梗茎点霉属(Dendrophoma)30株,占26.1%,根霉属(Rhizopus)9株,占7.8%;其中链格孢属(Alternaria),枝梗茎点霉属(Dendrophoma)为优势种群。宁夏地区苦豆子内生真菌多样性丰富,同时不同部位内生菌的数量、种类及分布存在差异。     

我国东北地区玉米苗枯病致病真菌的种类与分布研究

采集东北地区(黑龙江、吉林和辽宁省)玉米苗枯病的病株800株,采用组织分离法共分离组织8 000块,得到真菌7 049株,真菌分离频率为88.11％.其中镰孢菌共4 034株,占分离所得真菌总株数的57.23％.黑龙江、吉林和辽宁各省分离所得的苗枯病致病镰孢菌株数在各省分离所得的真菌总株数中所占比例基本一致,分别为56.89％、61.26％和55.07％.表明镰孢菌在3省均为玉米苗枯病的主要致病菌.同时,在分离出的真菌中有丝核菌1 099株,占分离真菌总株数的15.59％.但是,3省所分离到的菌株比率存在明显差异,分别为16.09％、9.47％和18.87％.     

手掌参内生真菌及根际土壤真菌的群落组成

为筛选促生菌,采用高通量测序技术对手掌参茎、叶、果荚和根4种组织内生真菌及根际土壤真菌的群落组成进行分析。结果表明:从手掌参所有组织样品及根际土壤中共获得4 192个操作分类单元(operational taxonomic unit,OTU),其中茎中OTU数量最多,为1 096个,其次为根际土壤和叶中,分别为1 048个和983个,根中最少,为397个。所有样品中OTU注释后隶属于4门15纲,优势菌群为子囊菌门,相对丰度为47.25%～81.58%。属水平上主要菌群为枝孢霉属Cladosporium、粉褶蕈属Entoloma、背芽突霉属Cadophora和赤霉菌属Gibberella,分别为果荚、土壤、根、叶和茎中优势菌群。多样性指数表明茎中真菌群落的丰富度及多样性最高,而根部最低,不同样品之间真菌群落的多样性和丰富度差异显著。热图分析和主坐标分析均表明茎、叶和果荚中内生真菌的群落组成较相似,但与根部内生真菌和土壤中真菌的群落组成存在一定差异。     

Protective effects of a wheat cultivar mixture against splash‐dispersed septoria tritici blotch epidemics

The use of cultivar mixtures to control foliar fungal diseases is well documented for windborne diseases, but remains controversial for splash‐dispersed diseases. To try to improve this strategy, a cultivar mixture was designed consisting of two wheat cultivars with contrasted resistance to Mycosphaerella graminicola , responsible for the rainborne disease septoria tritici blotch (STB), in a 1:3 susceptible:resistant ratio rather than the 1:1 ratio commonly used in previous studies. The impact of natural STB epidemics in this cultivar mixture was studied in field experiments over 4 years. Weekly assessments of the number of sporulating lesions, pycnidial leaf area and green leaf area were carried out on the susceptible cultivar. In years with sufficient STB pressure, disease impacts on the susceptible cultivar in the mixture were always significantly lower than in the pure stand (e.g. 42% reduction of pycnidial leaf area for the three upper leaves in 2008 and 41% in 2009). In years with low STB pressure (2010 and 2011), a reduction of disease impacts was also shown but was not always significant. After major rainfall events, the number of sporulating lesions observed on the susceptible cultivar after one latent period was reduced on average by 45% in the mixture compared to the pure stand. All the measurements showed that a susceptible cultivar was consistently protected, at least moderately, in a mixture under low to moderate STB pressure. Therefore, the results prove that the design of an efficient cultivar mixture can include the control of STB, among other foliar diseases.     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

Effect of cold‐induced changes in physical and chemical leaf properties on the resistance of winter triticale (×Triticosecale) to the fungal pathogen Microdochium nivale

This study showed that several mechanisms of the basal resistance of winter triticale to Microdochium nivale are cultivar‐dependent and can be induced specifically during plant hardening. Experiments and microscopic observations were conducted on triticale cvs Hewo (able to develop resistance after cold treatment) and Magnat (susceptible to infection despite hardening). In cv. Hewo, cold hardening altered the physical and chemical properties of the leaf surface and prevented both adhesion of M. nivale hyphae to the leaves and direct penetration of the epidermis. Cold‐induced submicron‐ and micron‐scale roughness on the leaf epidermis resulted in superhydrophobicity, restricting fungal adhesion and growth, while the lower permeability and altered chemical composition of the host cell wall protected against tissue digestion by the fungus. The fungal strategy to access the nutrient resources of resistant hosts is the penetration of leaf tissues through stomata, followed by biotrophic intercellular growth of individual hyphae and the formation of haustoria‐like structures within mesophyll cells. In contrast, a destructive necrotrophic fungal lifestyle occurs in susceptible seedlings, despite cold hardening of the plants, with the host epidermis, mesophyll and vascular tissues being digested and becoming disorganized as a result of the low chemical and mechanical stability of the cell wall matrix. This work indicates that specific genetically encoded physical and mechanical properties of the cell wall and leaf tissues that depend on cold hardening are factors that can determine plant resistance against fungal diseases.     

Histological investigation of stripe rust (Puccinia striiformis f.sp. tritici) development in resistant and susceptible wheat cultivars

The wheat cultivar Kariega expresses complete adult plant resistance against stripe rust, whereas cv. Avocet S is susceptible. Using confocal laser scanning microscopy, initial fungal penetration into flag leaves was identical in both cultivars, with directional germ-tube growth towards stomata that were penetrated without the formation of an appressorium, followed by differentiation of a substomatal vesicle, infection hyphae, haustorial mother cells and haustoria. During the following 4 days, further fungal development occurred more quickly in the resistant than in the susceptible cultivar. However, by 7 days postinoculation (dpi) the situation changed, with exponential growth of the pathogen occurring only in the susceptible line. Induced cellular lignification, a typical defence reaction of cereals, was observed at 4 dpi in the resistant cultivar, and 2 days later lignified tissue completely surrounded the fungal colonies. In the susceptible cultivar, isolated lignified host cells occurred at 6 dpi, and long, unbranched fungal hyphae outgrowing the resistance reaction were observed.     

我国北方玉米上平脐蠕孢属和弯孢属真菌及其所致叶斑病

平脐蠕孢属(Bipolaris)和弯孢属(Curvularia)真菌可引起多种玉米叶斑病。为了解当前玉米生产上此类病害的发生情况,2014年8-9月对我国玉米主产区北京、河北、河南、黑龙江和吉林5省市玉米上疑似由该两属真菌引起的叶部病斑样品进行了采集,随后进行了真菌的分离和鉴定。共采集样品42份,根据其形状特点归为4类:长条形、椭圆形、小点状和梭形病斑。经组织分离获得平脐蠕孢属和弯孢属真菌28株,基于形态学和rDNA-ITS序列的系统发育分析共鉴定出5个种:玉蜀黍平脐蠕孢(B.maydis)、玉米平脐蠕孢(B.zeae)、玉米生平脐蠕孢(B.zeicola)、新月弯孢(C.lunata)和穂状弯孢(C.spicifera)。从长条形病斑和椭圆形病斑上分离到的主要是B.maydis和B.zeicola,从小点状病斑分离到的主要是C.lunata,其次是B.zeae。分离出C.lunata的样品病斑较为稀疏、颜色略浅、呈苍白色,分离出B.zeae的样品病斑更为密集、颜色较深。从梭型病斑分离到的是C.spicifera。有少数样品可分离到上述两种菌。采用孢子悬浮液喷雾法对温室玉米苗接种,上述5种真菌均可致病。以接种B.maydis发病最快,发病最重;接种B.zeicola、C.lunata或C.spicifera发病较慢,症状明显;接种B.zeae发病最慢,仅引起小点状病斑。研究结果可为玉米叶斑病的正确诊断提供资料和依据。     

Impact of carrot resistance on development of the <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight pathogen (<Emphasis Type="Italic">Alternaria dauci</Emphasis>)

The interaction between Alternaria dauci and two carrot cultivars differing in their resistance to leaf blight was investigated by microscopy. The fungal development between 1 and 15 days post-inoculation was quite similar in the susceptible cv. Presto and the partially resistant cv. Texto: After conidial germination, leaf adhesion of the pathogen was achieved with mucilaginous filaments; hyphae penetrated the leaves directly with/without the formation of appressoria-like structures or via stomata; the fungus spread by epiphytic hyphae with hyphopodia and subcuticular mycelia. Intense necrotic plant cell reactions occurred under the fungal structures. At 21 days post-inoculation, typical features of fungal development were noted for each cultivar: growing hyphae emerged from stomata in cv. Presto, whereas conidiophores without conidia were observed in cv. Texto. Leaf tissues of both cultivars were strongly damaged and vesicle-like structures (assumed to be plant phenolics) were abundantly present between mesophyll cells. A real-time PCR method was developed for in planta quantification of A. dauci. Between 1 and 15 days post-inoculation, the fungal biomass was equivalent in the two cultivars and was about fourfold higher in cv. Presto than cv. Texto at 21 and 25 days post-inoculation. Taken together, our results indicated that A. dauci was able to colonize both cultivars in a similar manner during the first steps of the interaction, then fungal development in the partially resistant cultivar was restricted due to putative plant defence reactions. The results of this study enhance the overall understanding of infection processes in the A. dauci-carrot pathosystem.     

云南疣粒野生稻抗白叶枯病鉴定及叶片组织学观察

 本研究采用水稻白叶枯病菌强致病性代表菌株,评价了云南省自然分布的疣粒野生稻代表生态居群18份材料的抗病性,观察叶片组织学结构,初步分析了疣粒野生稻的抗性基因。结果表明,16份材料表现中抗、高抗甚至免疫;9份材料对4个菌株的抗性有一定差异;同时发现云南地方代表菌株X1和CN9404的致病性比C1和BD8438强。叶片组织学结构表明,高抗材料与感病的栽培稻米泉黑芒都无蜡质层,二者的叶肉组织、薄壁细胞和表皮细胞相同,表皮毛、维管束、厚壁组织等的差异也不明显,不足以引起抗病性差异。疣粒野生稻高抗或免疫白叶枯病并非借助其特殊叶片结构的物理作用,而主要是抗病基因的作用。参试疣粒野生稻中没有与Xa1、Xa21同源的基因,加之独立进化,推断其具有新的优异抗白叶枯病基因,值得深入研究和发掘利用。     

Leaf morpho-physiology and phytochemistry of olive trees as affected by cultivar type and increasing aridity

The olive species (Olea europaea L.) is an ancient traditional crop grown under rainfed conditions in the Mediterranean Basin. In response to the growing national and international demand for olive oil, the olive cultivars are introduced into highly arid new bioclimatic areas. Subsequently, the morpho-physiology and phytochemistry of olive trees are potentially changing among cultivar types and geographical conditions. In the present work, we have undertaken an assessment on the impacts of geographical location and cultivar types on the leaf morpho-physiology and phytochemistry of olive trees. Thus, leaves of the two most cultivated olive tree varieties, Chemlal and Sigoise, were collected from three geographical regions (Setif, Batna, and Eloued) with increasing aridity in Algeria. Leaf samples from the geographical regions were analyzed using the standard physiological experiment, colorimetric method, and a chromatography assay. Leaves of both cultivars exhibited a significant variance in terms of the leaf shape index but not for the leaf tissue density, specific leaf weight, and specific leaf area. Photosynthetic pigment contents were affected by both cultivar type and geographical location, with the lowest pigment content recorded in the Sigoise cultivar from the Setif region. Compared with the Setif and Batna regions, dried leaves of both cultivars from the Eloued region showed the higher levels of the total polyphenol, total flavonoid, and total tannin, as well as a better antioxidant capacity. Liquid chromatography-mass spectrometry analysis of all leaf extracts identified the following phenolic acids as major compounds: oleuropein, naringin, apigenin-7-O-glucoside, kaempferol, quercetin, quercitrin, luteolin-7-O-naringenin, and quinic acid. Lower contents were found for p-Coumaric acid, trans-Ferulic acid, hyperoside, rutin, apigenin, caffeic acid, protocatechuic acid, o-Coumaric acid, and gallic acid. Also, epicatechin and catechin+ were not found in the leaf extracts of the Sigoise cultivar. The leaf organic extracts in both cultivars displayed promising anti-cancer activity that was affected by geographical location and organic solvent polarity. Briefly, although increasing aridity and soil organic and mineral deficiency affected the leaf morpho-physiological parameters, both cultivars sustained a chemical richness, a good antioxidant, and an anti-tumoral capacity in leaves. Furthermore, the findings revealed that regardless the olive tree genotype, there was a significant impact of geographical location on the leaf morpho-physiology, bioactivity, and chemical composition, which may consequently modulate the growth and oil production of olive trees.     

Susceptibility of European pear cultivars to Pseudomonas syringae pv. syringae using immature fruit and detached leaf assays

The susceptibility of thirty-three pear cultivars and two pear rootstocks to four virulent strains of Pseudomonas syringae pv. syringae was evaluated by inoculating detached immature fruits and young leaves. The four strains were similarly virulent and did not show cultivar specificity although they were isolated from different pear cultivars and exhibited different biochemical profiles. The most frequently planted pear cultivars, Conference, Abate Fetel, General Leclerc, Williams, D. Comice, El Dorado, Alexandrine, B. Anjou, Passe Crassane and the rootstock OHxF 333 were susceptible to P. syringae pv. syringae. Maximal severity values were obtained on 'Preguystar' leaves (about 90%). The rootstock Winter Nelis was less susceptible. Results with immature fruit and detached leaf assays agreed with field observations on cultivar susceptibility to bacterial blast. However, the detached leaf test gave a more accurate prediction and has the advantages that symptoms develop quickly (48 h), and leaves are available for a longer period of time than fruits. This method is proposed as a rapid and reproducible screening system of cultivar susceptibility to bacterial blast of pear.