应用高通量测序技术对进境澳大利亚小麦病原真菌的检测研究

相比较其他检测技术,高通量测序技术具有高效、快速等特点,并拥有全面分析复杂核苷酸群体的功能,本文运用高通量测序技术对进境澳大利亚4批次小麦种子和筛下物上的病原真菌进行检测,通过基因组DNA提取、高通量测序、生物信息学分析后得到分类OTU共622个,基于不同分类水平,分属于5个门、64个目、201个属。检测显示,种子与筛下物携带的病原真菌的种类和比例有较大差异,并且筛下物携带的病原真菌数量和种类远多于种子携带的病原真菌。     

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...     

不同生长状态祁连圆柏针叶内生真菌群落结构与多样性分析

为掌握西北干旱地区祁连圆柏内生真菌群落结构以及多样性,探索内生真菌与祁连圆柏针叶生长状态的联系,通过高通量测序技术对健康和枯黄的圆柏针叶进行了分析。研究结果显示：6组样品测序共得到2,338,753条有效序列,683个操作分类单元(Operational Taxonomic Units, OTUs)。ACE、Chao1、Shannon指数表明发病组样品的物种丰富度与多样性略高于健康组。门分类水平上,两组样品的优势菌群均为子囊菌门,在属分类水平上,枝孢属Cladosporium是健康组样品中的优势属,球腔菌属Mycosphaerella是发病组样品中的优势属。在群落结构组成上,健康组样品中枝孢属Cladosporium、球腔菌属Mycosphaerella、丝状担子菌属Filobasidium相对丰度均高于发病组,组成占比也较为均匀,发病组样品中除Mycosphaerella之外其余属真菌占比均较低,没有明显的层次结构。Beta多样性显示6组样品大致分为两个分布区,各样品组间差异较小,两组样品差异较为明显。属分类水平上大多数属间呈正相关,负相关主要集中球腔菌属Mycosphaerella...     

北苍术根区土壤中AM真菌多样性及其与土壤养分相关性分析

本研究采用Illumina MiSeq高通量测序技术,对河北承德栽培和野生北苍术根区土壤AM真菌群落多样性的差异和群落组成进行了检测,同时解析了土壤养分因子对AM真菌群落多样性的影响。结果显示,北苍术根区土壤中检测的AM-OTU分属于1门1纲5目6科8属AM真菌。野生北苍术AM真菌群落由球囊霉属（77.20%）等组成;栽培北苍术由巨孢囊霉属（34.48%~47.20%）、球囊霉属（30.05%~53.7%）等组成;土壤有机质、pH、有效磷、有效钾与AM真菌多样性指数Shannon、Simpsoneven呈正相关,其中有机质对AM真菌群落多样性影响最大。硼与AM真菌多样性指数Shannon、Simpsoneven、Sobs均呈正相关,铁、铜、锰、锌与之呈负相关。本研究阐明了野生与栽培北苍术根区AM真菌多样性及优势菌群差异,揭示了土壤养分因子与其多样性间的关系,为AM真菌在北苍术仿野生栽培中的应用提供了理论依据。     

短期玉米秸秆还田对冷凉地区土壤真菌多样性的影响

在玉米吐丝期,利用传统涂布平板法和高通量测序技术,设置常规秸秆不还田(CK)、秸秆深翻还田(SF)和秸秆旋耕还田(XG)3个处理,研究0~10 cm、10~20 cm和20~30 cm土层真菌群落丰富度、多样性和群落结构变化,揭示冷凉地区玉米秸秆还田真菌群落结构及其差异。结果表明:冷凉地区玉米秸秆还田对土壤真菌多样性的影响较大,秸秆还田土壤中可培养真菌数为SFXGCK;秸秆还田后土壤真菌优势菌群发生变化,CK优势菌为马拉色氏霉菌属、被孢霉属和葡萄孢属,XG优势菌为被孢霉属;XG和SF表层土壤中耐冷酵母菌属和被孢霉属是优势菌;SF处理20~30 cm土层优势菌为葡萄孢属。真菌菌群Alpha多样性、群落组成丰度、RDA(Redundancy analysis)和菌群相似性综合分析可知,SF和XG 0~20 cm土层菌群差异小;SF和XG均能增加10~30 cm土层真菌群落丰度。     

健康与感染青枯病烟株根际土壤与茎秆真菌群落结构与多样性

为研究感染青枯病后烟株根际土壤与茎秆真菌群落结构与多样性的变化,对健康和感染青枯病烟株的根际土壤、病株茎秆发病组织和健株茎秆健康组织等样品中真菌ITS区的rDNA进行了PCR扩增、用Illumina MiSeq测序技术对扩增DNA片段进行高通量测序,并分析不同样品的真菌群落组成与多样性。结果表明,所有烟株根际土壤中优势门为子囊菌门Ascomycota和接合菌门Zygomycota;所有茎秆样品中优势门为担子菌门Basidiomycota和子囊菌门。在属水平,被孢霉属Mortierella、镰刀菌属Fusarium和隐球菌属Cryptococcus为所有土壤中的主要菌属,Boeremia主要存在于发病烟株根际土壤中,而木霉属Trichoderma主要存在于健康烟株根际土壤。发病茎秆病害组织中优势属为小画线壳属Monographella、隐球菌属、鬼伞属Coprinopsis和赤霉属Gibberella;发病茎秆病健交界处组织中优势属为隐球菌属、红酵母属Rhodotorula和小画线壳属。健康烟株茎秆组织中优势属为隐球菌属、链格孢属Alternaria和红酵母属;健康烟株中与发病茎秆病健交界处组织等高茎秆中优势属为镰刀菌属、隐球菌属、链格孢属和Gibellulopsis。青枯菌侵染烟株后根际土壤、发病茎秆病害组织和发病茎秆病健交界处组织的真菌群落中物种丰富度与多样性均显著提高,且发病茎秆病害组织与发病茎秆病健交界处组织真菌群落的变化大于根际土壤。研究结果为烟草青枯病的生物防治提供了参考。     

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。     

免耕对不同麦玉轮作方式土壤真菌群落结构的影响

为探讨耕作及轮作方式对农田土壤真菌群落结构的影响,设置免耕、传统耕作2种耕作方式,以及小麦-玉米轮作、小麦/玉米间作、小麦-冬油菜-玉米轮作3种种植模式,共形成6个处理,测定土壤理化性质并采用ITsDNA高通量测序技术分析不同处理对土壤真菌群落的影响.研究结果表明:与传统耕作相比,免耕显著增加了土壤有机碳、全磷、全氮、...     

高通量测序技术在鉴定木本植物双生病毒中的应用

高通量测序技术(next-generation sequencing,NGS)平台提供了一种高效、快速、低成本、深度测序DNA的解决方案,2009年该技术开始被应用于植物病毒学领域,包括新病毒的发现,病害病原的鉴定,病毒基因组多样性及进化的研究,显著加快了植物病毒学的发展进程。迄今,应用高通量测序技术已经成功鉴定了上百种新的植物病毒和类病毒。双生病毒是一类对多种作物造成毁灭性危害的DNA病毒,多发生于草本作物。然而利用高通量测序技术,从柑橘、葡萄、苹果和桑树等多种多年生木本植物中检测到了新的双生病毒,显示出了高通量测序技术所独有的、传统检测技术所不具备的优势。本文围绕高通量测度技术在植物病毒学领域的应用进行概述,重点阐述NGS用于检测木本植物双生病毒的几个实例。     

复合微生物菌肥对苹果再植病害调控及对根围土壤真菌群落结构的影响

 为研究复合微生物菌肥对再植苹果幼树的影响,从再植土壤酶活性、真菌群落结构和功能的方面分析,探究复合微生物菌肥的作用机理。本研究设置了KMM和对照CK两个处理,再植苹果树于春秋两次分别施用复合微生物菌肥(KMM)和有机肥(CK)。通过土壤常规农业化学分析法和Illumma MiSeq高通量测序技术,系统研究了两个处理再植苹果树的生长、根围土壤酶活性以及真菌群落结构和功能的变化。试验结果表明,施用复合微生物菌肥显著提高了再植苹果幼树株高、干径、叶绿素、分枝个数和分枝长度;提高再植土壤酶活性(中性磷酸酶、脲酶、蔗糖酶和过氧化氢酶);降低再植土壤真菌群落丰富度,改变真菌群落结构。相对丰度高于1%的优势真菌属中有21个菌属与再植果树的5项生长参数具有相关性,并且在不同分类水平上具有一定的差异,缝壳菌属(Lophiostoma)、织球壳菌属(Plectosphaerella)、赤壳菌属(Nectria)和维希尼克氏属(Vishniacozyma)显著增加,丝孢菌属(Anguillospora)和假丝酵母菌属(Pseudeurotium) 显著降低。通过对测序片段功能水平的分析,在二级功能水平上显著降低了真菌群落氨基酸、核苷和核苷酸的生物合成、电子转移、发酵、呼吸、CTP生物合成嘧啶脱氧核糖核酸功能序列的丰度值,共6种功能类群均与土壤酶活性呈负相关。施用复合微生物菌肥,改变了真菌群落结构,减弱真菌群落相关代谢的功能,提高土壤酶活性,促进再植苹果幼树的生长。     

Molecular Identification and Phylogenetic Grouping of Diaporthe phaseolorum and Phomopsis longicolla Isolates from Soybean

ABSTRACT Diaporthe phaseolorum and Phomopsis longicolla isolates from soybean were examined using traditional mycological characteristics and molecular methods. Cultural characteristics including types of fruiting bodies and conidia were assessed for isolates collected from soybean stems and seeds. Cultures were identified as P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, or D. phaseolorum var. sojae. Molecular markers for these groups were developed and analyzed using polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLP) and DNA sequencing in the internal transcribed spacer (ITS) and the 5.8S ribosomal DNA. The ITS(4) and ITS(5) primers amplified PCR products for all isolates studied. Gel electrophoresis of undigested PCR products and DNA sequencing produced various fragment lengths including 604 bp for P. longicolla, 602 and 603 bp for D. phaseolorum var. caulivora, 603 bp for D. phaseolorum var. meridionalis, and from 597 to 609 bp for D. phaseolorum var. sojae. Digestion of these PCR products with enzymes AluI, HhaI, MseI, RsaI, and ScrFI resulted in distinct bands for identification of P. longicolla and the varieties of D. phaseolorum I. All P. longicolla, D. phaseolorum var. caulivora, and D. phaseolorum var. meridionalis isolates were distinguished using AluI and HhaI with RsaI or ScrFI. The banding patterns of D. phaseolorum var. sojae isolates were complex and were separated into 11 subgroups after digestion with AluI, HhaI, MseI, RsaI, and ScrFI. Phylogenetic analysis of 20 isolates of D. phaseolorum and P. longicolla based on the DNA sequence of the ITS region resolved six clades termed A, B, C, D, E, and F. Clade A included all sequenced D. phaseolorum var. caulivora isolates, two from Italy and one from the United States. Isolates in clade B were exclusively associated with D. phaseolorum var. meridionalis. Clades A and B formed a well-supported monophyletic group. Isolates in clades C, D, E, and F were morphologically defined as isolates of P. longicolla, D. phaseolorum var. sojae, and Diaporthe spp. The ITS sequences similarity of seven geographically diverse P. longi-colla isolates illustrated that P. longicolla isolates have a similar genetic background, with some affiliations to some D. phaseolorum var. sojae isolates. Morphological characteristics of the isolates along with the terminal clades of the ITS phylogeny suggest that P. longicolla is an individual species, D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis are varieties of D. phaseolorum, and D. phaseolorum var. sojae is either several varieties of D. phaseolorum or possibly several distinct species.     

Molecular Detection of Diaporthe phaseolorum and Phomopsis longicolla from Soybean Seeds

ABSTRACT Species-specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break the cells. DNA fragment lengths ranged from 200 to 1,200 base pairs (bp), with the majority of fragments <500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA, three TaqMan primer/probe sets were designed. Primer/probe set PL-5 amplified a 96-bp fragment within the ITS1 region of P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, and D. phaseolorum var. sojae. Set PL-3 amplified a 86-bp DNA fragment within the ITS2 region of P. longicolla. Set DPC-3 amplified a 151-bp DNA fragment within the ITS2 region of D. phaseolorum var. caulivora. TaqMan primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA. When using PCR-RFLP for Diaporthe and Phomopsis detection, the sensitivity was as low as 100 pg of pure DNA. Among 13 soybean seed lots from Italy and the United States, the total Diaporthe and Phomopsis detected using a traditional seed-plating technique ranged from 0 to 32%. P. longicolla was most prevalent, followed by D. phaseolorum var. sojae. D. phaseolorum var. caulivora, which only occurred in 0.5% of the Italian seed lots, was not detected in the U.S. seed lots. D. phaseolorum var. meridionalis was not detected in either the U.S. or Italian seed lots. Using TaqMan primer/probe set PL-3, the frequency of P. longicolla was 18% in seed lot I3, similar to the frequency obtained from PCR-RFLP and potato dextrose agar plating detection. The frequencies of D. phaseolorum and P. longicolla in each seed lot obtained by the different detection methods were comparable with respect to total infection and individual species detection. However, TaqMan detection provided the fastest results of all the methods tested.     

Seed-borne fungi detected in consignments of soybean seeds (Glycine max) imported into India

Inspection of 16 017 samples of soybean seeds imported into India from 1978 to 2004 resulted in the detection of 21 pathogens, including Peronospora manshurica which is not present in India. Seed-borne fungi of high economic significance included: Ascochyta sojicola , Botryotinia fuckeliana , Cercospora kikuchii , Colletotrichum dematium , Corynespora cassicola , Diaporthe phaseolorum var . sojae , Fusarium oxysporum, Glomerella cingulata , Glomerella glycines , Macrophomina phaseolina , Nectria haematococca , Passalora sojina , Thanatephorus cucumeris as well as other fungal pathogens for which soybean is not a host such as Alternaria padwickii , Cochliobolus sativus , Fusarium culmorum , Fusarium poae , Glomerella graminicola , Setosphaeria rostrata, Verticillium albo-atrum , etc. Some of the fungi detected have very wide host range. Details are presented on the fungi detected, the countries from which the imported consignments originated, and phytosanitary significance.     

A Preliminary Investigation of Pathogenic Fungi from Lotus (Nelumbo nucifera Gaertn)in Nanchang City

[Objectives] Lotus (Nelumbo nucifera Gaertn) is an economically important aquatic plant in China. Fungaldisease is a serious problem in lotus cultivation. In this study, the pathogenic fungi on lotus in Nanchang City were investigated to lay the foundation for the disease control. [Methods] Lotus leaves and stems in ponds of Nanchang City were collected, the fungi on leave/stem spots were isolated and purified. Colonies morphological characters and ITS sequences were used to identify the strains. [Results] 49 strains were isolated and identified to 20 species, belonging to 12 genera. [Discussion] 15 species may firstly be reported on lotus in this study, i.e., Alternaria angustiovoidea, Alternaria compacta, Alternaria ricini, Alternaria tenuissima, Arthrinium arundinis, Botryosphaeria dothidea, Curvularia spicifera, Diaporthe australiana, Diaporthe eres, Diaporthe tectonae, Epicoccum nigrum, Fusarium fujikuroi, Neofusicoccum parvum, Nigrospora sphaerica, and Phomopsis eucommii.     

Morphologic, Molecular, and Pathogenic Characterization of Diaporthe phaseolorum Variability in the Core Soybean-Producing Area of Argentina

ABSTRACT Isolates of the Diaporthe/Phomopsis (D/P) complex were collected in the main soybean producing area of Argentina during the 1996-97, 1997-98, and 1998-99 growing seasons. Twenty-three morphologic characters related to type of colonies, stroma, pycnidia and conidia, presence of perithecia, and asci length were studied by principal component analysis (PCA). Genomic DNA were analyzed by the random amplified polymorphic DNA (RAPD) technique. From both studies, 18 isolates were identified as D/P complex and grouped in four major taxa: (i) Diaporthe phaseolorum var. meridionalis, (ii) D. phaseolorum var. caulivora, (iii) D. phaseolorum var. sojae, and (iv) Phomopsis longicolla. In addition to distinguishing interspecific and intraspecific variability, molecular markers allowed the detection of differences among isolates within the same variety. Pathogenicity was assayed in the greenhouse, by the toothpick method, inoculating the D/P isolates to soybean genotypes carrying different resistance genes (Rdc1, Rdc2, Rdc3, and Rdc4) against soybean stem canker (SSC). Pathogenic analysis distinguished two main groups: (i) the SSC-producing isolates, including D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora, and (ii) the non-SSC-producing isolates, including D. phaseolorum var. sojae and P. longicolla. Cultivar RA-702 (susceptible control) was compatible with both D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora isolates; meanwhile, Tracy-M (Rdc1 and Rdc 2 genes) was incompatible with D. phaseolorum var. meridionalis but compatible with D. phaseolorum var. caulivora isolates. The fact that Rdc1 and Rdc2 together (as in Tracy-M) confer an almost immune reaction to all assayed isolates of D. phaseolorum var. meridionalis but were ineffective against the D. phaseolorum var. caulivora isolates evaluated suggests that the virulence or avirulence genes in D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora are different. Moreover, physiological races of D. phaseolorum var. meridionalis were detected by using differential soybean genotypes carrying distinct single Rdc genes. As far as we know, this is the first report on the existence of physiological races of D. phaseolorum var. meridionalis in South America. Selective pressure due to deployment of resistant host cultivars may have changed the frequency of the virulence or avirulence genes within the population of D. phaseolorum var. meridionalis. On the whole, our results show that pathogenic variability of D. phaseolorum in the core soybean-producing area of Argentina is higher than previously recognized.     

美国进境大豆北方茎溃疡病菌的分离与鉴定

在对美国进境大豆检疫过程中,对大豆籽粒和夹带茎秆进行病原真菌的分离培养,使用形态学和分子生物学相结合的方法对可疑菌株进行鉴定,确定该菌为Diaporthe phaseolorum(Cke.&; Ell.)Sacc.var.caulivora Athow &; Caldwell。该病原菌是进境植物检疫潜在危险性病原真菌,在国内属首次截获。     

大豆南方茎溃疡病菌的分离与鉴定

在对美国进境大豆进行检疫时，从混杂的豆秆上发现并分离到了大豆南方茎溃疡病菌，通过形态学和分子生物学鉴定，确定该菌为Diaporthe phaseolorum（Cke．＆ Ell.）Sacc．var．meridionalis Morgan-jones。该病原菌是国外近年来在大豆生产中新发生的危险性病原真菌，国内未见报道。     

CLIMEX-GIS预测大豆北方茎溃疡病菌在中国的潜在分布

大豆北方茎溃疡病菌是大豆的重要病原菌,广泛分布于世界主要大豆产区,造成严重的产量和品质损失。本文应用生物模型CLIMEX结合GIS软件预测大豆北方茎溃疡病菌在中国的适生区,并根据EI值划分相应的适生等级。结果表明,大豆北方茎溃疡病菌在我国绝大部分地区适合生长,其中东北地区、华北地区和云贵高原地区处于中适生区或高适生区。该菌在我国还未报道,通过分析其在我国潜在分布区对于防止病菌的传入、传播和蔓延有重要的检疫意义。     

大豆北方茎溃疡病菌分离鉴定技术的改进

本文对产生大豆北方茎溃疡病菌子囊壳的豆秆分别进行冷冻和水浸处理,发现2种处理均能促进成熟的子囊壳产孢,并且同一子囊壳经冷冻和水浸处理能够多次喷出子囊,释放子囊孢子,利用此特性有利于在不损失鉴定材料的前提下,多次获得分离物以完成病菌的分离鉴定,特别利于伴生其他真菌和子囊壳数量很少情况下,分离纯化大豆北方茎溃疡病菌。     

甘肃省临夏州小麦脚腐病病原鉴定

对甘肃省临夏州小麦脚腐病病原进行了分离鉴定,从3块病田的37株罹病小麦上分离得到56个真菌菌株,分别被鉴定为雪腐镰刀菌[Fusarium nivale(Fr.)Ces.]、燕麦镰刀菌[Fusarium avenaceum(Corda et Fr.)Sacc.]、麦斑点附球霉[Epicoccum triticiP.Henn.]、小壳色单隔孢菌[Diplodiellasp.]、交链孢菌[Alternariaspp.]、芽枝霉[Cladosporiumsp.]、黑孢霉[Nigrosporasp.]。其中,雪腐镰刀菌致病性最强,燕麦镰刀菌致病性中强,麦斑点附球霉致病性弱,其余菌不致病。