壶瓶枣褐斑病病原菌的鉴定

 近几年来,山西红枣发生了1种严重的果实病害,症状表现为果顶或果肩部位形成红褐色的病斑。本研究以壶瓶枣为材料,对病菌进行分离。通过室内和田间致病性测定以及人工接种后再分离病菌,证明编号为CN535的真菌菌株为该病的致病菌。该病菌在PDA上7d菌落直径达69.2~73.5mm,基内菌丝和气生菌丝均发达,具明显的浅灰与墨绿色的同心轮纹;分生孢子单生或短链生,具纵横隔膜和短喙,大小为(22.5~40.0)μm×(8.0~13.5)μm,为典型的Alternaria属真菌特征。其rDNAITS序列分析结果表明该菌与A. alternata、A. tenuissima、A. longipes、A. mali和A. citri的同源性均为100%。用2对链格孢菌的专用引物AAF₂/AAR3和Aalt-F/Aalt-R分别扩增出相对应的341和450bp的片段。综合形态特征和分子分析结果,确定壶瓶枣褐斑病的病原菌为A. alternata (Fries) Keissler。     

香梨果萼黑斑病菌Alternaria alternata遗传转化体系的建立及GFP标记菌株的获得

 为建立香梨果萼黑斑病菌链格孢(Alternaria alternata)的原生质体遗传转化体系,本实验以香梨果萼黑斑病菌强致病性菌株LI1为供试材料,研究菌龄、酶系统、酶解时间等对链格孢菌原生质体制备的影响。链格孢菌菌丝在CM液体培养基中培养20 h,以0.7 mol·L^-1 NaCl为稳渗剂,1%裂解酶+1%崩溃酶+1%蜗牛酶的酶液组合下,28 ℃酶解4 h,原生质体制备效率最高。通过PEG/CaCl₂介导法将含有潮霉素B抗性基因和绿色荧光蛋白基因的质粒转入链格孢菌LI1,转化子生长表型及外源基因的PCR鉴定结果表明抗性基因已成功整合到香梨果萼黑斑病菌中。成功建立了香梨果萼黑斑病菌链格孢菌的原生质体遗传转化体系,并成功获得GFP标记菌株,为病原菌侵染定殖过程及致病机制研究奠定了基础。     

甜瓜种子携带的链格孢菌种类鉴定及其致病性研究

 采用洗涤检验法和马铃薯葡萄糖琼脂培养基法,从甜瓜种子上分离得到14个链格孢菌分离物。通过菌落形态、产孢表型、分生孢子以及分生孢子梗等形态学观察,结合分析ITS-rDNA和histone 3基因序列,对链格孢菌分离物的种类进行鉴定;甜瓜种子携带的链格孢菌种类分别为细极链格孢(Alternaria tenuissima)、交链格孢(A. alternata)和倒果链格孢(A. obovoidea)。还研究了这些链格孢菌分离物对甜瓜离体叶片的致病性及其对种子发芽的影响;结果表明,除A. obovoidea外,另外两种链格孢菌均对甜瓜离体叶片具有致病性;A. tenuissima和A. alternata的孢子悬浮液对甜瓜种子发芽均有一定的抑制作用。这是国内首次围绕甜瓜种子传带链格孢菌的种类及其致病性开展详细的研究。     

甜瓜种子携带的链格孢菌种类鉴定及其致病性研究

 采用洗涤检验法和马铃薯葡萄糖琼脂培养基法,从甜瓜种子上分离得到14个链格孢菌分离物。通过菌落形态、产孢表型、分生孢子以及分生孢子梗等形态学观察,结合分析ITS-rDNA和histone 3基因序列,对链格孢菌分离物的种类进行鉴定;甜瓜种子携带的链格孢菌种类分别为细极链格孢(Alternaria tenuissima)、交链格孢(A. alternata)和倒果链格孢(A. obovoidea)。还研究了这些链格孢菌分离物对甜瓜离体叶片的致病性及其对种子发芽的影响;结果表明,除A. obovoidea外,另外两种链格孢菌均对甜瓜离体叶片具有致病性;A. tenuissima和A. alternata的孢子悬浮液对甜瓜种子发芽均有一定的抑制作用。这是国内首次围绕甜瓜种子传带链格孢菌的种类及其致病性开展详细的研究。     

基于URP-PCR多态性片段的苦瓜枯萎病菌特异性检测技术的建立

 通过URP(Universal Rice Primers)-PCR分析尖孢镰孢菌苦瓜专化型基因组DNA扩增片段多态性,筛选检测尖孢镰孢菌苦瓜专化型的特异性引物,并建立了基于该引物的PCR检测方法。结果表明,特异性引物为FOMM-SPF/FOMM-SPR, PCR检测体系为25 SymbolmAL,包括2SymboltB Green Taq Master Mix 12.5 SymbolmAL,10 mmol·L^-1 的上下游引物各1 SymbolmAL,模板DNA 1 SymbolmAL,灭菌去离子水补足至25 SymbolmAL;PCR程序为95℃预变性3 min,94℃变性15 s,57℃退火30 s,72℃延伸20 s,共30个循环,循环结束后72℃延伸5 min;特异性扩增片段大小294 bp,检测灵敏度为2 ng·μL^-1 DNA或50个孢子·500 mg^-1土壤。该引物及其检测方法对尖孢镰孢菌苦瓜专化型的检测特异性好、灵敏度高,可以从土壤和植物样品中快速准确地检测出苦瓜枯萎病菌,无需病原菌的分离培养和致病性检测,对苦瓜枯萎病的早期诊断和预警及有效防控具有重要的指导意义。     

河南省小麦黑胚病优势病原菌链格孢菌(Alternaria spp.)遗传多样性分析

 采用RAPD技术对分离自河南省各地的43株小麦黑胚病优势病原菌(Alternaria spp.)菌株进行了遗传多样性分析。17个随机引物共扩增出151条清晰的DNA条带,所扩增出的DNA条带均为多态带,说明河南省小麦黑胚病菌存在着丰富的遗传多样性。利用NTSYS软件进行了病原菌的聚类分析,结果表明,河南省小麦黑胚病菌主要有2个种,即Alternaria alternata和A. tenuissima,种间的遗传相似系数的变化幅度为0.62~0.92。来自同一个地区的小麦黑胚病菌菌株基本上聚在了一起,表现出很近的亲缘关系,来自不同地区之间的菌株也可以交叉聚类。病原菌遗传多样性分析进一步验证了形态学的鉴定结果。     

大豆猝死综合症病原菌北美种的PCR鉴定

 应用等位基因特异性PCR(allele-specific PCR,AS-PCR)技术,建立了大豆猝死综合症(sudden death syndrome of soybean,SDS)病原菌北美种Fusarium virguliforme与其近似种Fusarium phaseoli(菜豆根腐病菌)的鉴别方法。针对翻译延长因子(EF-1α)基因上的3个SNP(单核苷酸多态性)位点,参照引物设计原则和等位基因特异引物(allele-specific primer,ASP)的要求设计了1对引物Fsg-α-1/2,能特异地扩增F.virguliforme,产生327 bp的电泳条带。另外,根据ITS区序列设计了1对通用引物Fu1/2,能扩增F.virguliforme和F.phaseoli,产生452 bp的电泳条带。本实验在传统的AS-PCR基础上进行改进,引入Fu1/2作为阳性质控引物,将ASP的特异性与双重PCR的严谨性相结合,建立了简便可靠的SDS病原菌鉴定方法。     

美国大豆中链格孢的分离鉴定研究

本文通过对美国进境大豆病害分离,共得到36个菌株,并对其中5株链格孢菌进行了形态学和分子生物学鉴定(其中2株),确认了它们分别是细极链格孢(Alternaria tenuissima(Kunze)Wiltshire)、樱桃链格孢(Alternaria cerasiPotebnia)、苘麻链格孢(Alternaria abutilonis(Speg.)Schwarze)、百日菊链格孢(Al-ternaria zinniaeH.Pape)、落葵链格孢(Alternaria basellaeT.Y.Zhang)。本研究证实了美国大豆中链格孢菌的多样性,不仅充实了正在构建的大豆病害数据库和菌种资源库,也可为港口的植物检疫工作提供借鉴。     

出口萝卜种子中芸薹生链格孢的分离与鉴定

本文对酒泉出境萝卜种子上发现的链格孢菌(Alternariasp.)进行了形态学和分子生物学鉴定,根据结果,将这种链格孢鉴定为芸薹生链格孢(Alternariabrassicicola);该种病原菌是危害十字花科蔬菜种子生产的世界性病原真菌,在甘肃酒泉繁种基地过去未见报道。     

云南葡萄产区葡萄炭疽病病原鉴定及致病力分析

为了明确引起云南葡萄产区炭疽病的病原种类,利用形态鉴定和特异性引物分子检测相结合的方法对从云南省主要葡萄产区采集的60株炭疽病菌菌株进行了鉴定。葡萄炭疽病菌菌株的菌落形态和生长速率与对照菌株尖孢炭疽菌Colletotrichum acutatum差异不明显,但其分生孢子大小显著小于尖孢炭疽菌,附着胞深褐色,球形或不规则形。胶孢炭疽菌Colletotrichum gloeosporioides特异性引物CgInt/ITS4从供试葡萄炭疽病菌菌株基因组DNA中扩增出1条约500 bp的特异性条带,而尖孢炭疽菌特异性引物CaInt2/ITS4对葡萄炭疽病菌无扩增条带。研究表明,引起云南葡萄主产区炭疽病的病原为胶孢炭疽菌;供试胶孢炭疽菌对红提葡萄均有致病力,但菌株致病力差异较大,对番茄和草莓存在交叉侵染的能力,且对多菌灵的敏感性较尖孢炭疽菌高。     

香蕉穿孔线虫(Radopholus similis)特异性分子检测技术研究

 香蕉穿孔线虫(Radopholus similis)是一种国际公认的极具毁灭性的有害线虫,也是我国重要的进境检疫性有害生物。利用线虫通用引物对香蕉穿孔线虫7个不同群体的ITS进行PCR扩增、克隆及测序,获得ITS片段序列长度为706bp。经与国外香蕉穿孔线虫种群及近缘种的ITS序列进行比对及同源性分析,构建设计了1对香蕉穿孔线虫的特异性引物RsF1/RsR1,特异性扩增片段长度为271 bp;同时引入D2A/D3B作内标,研究出特异性检测香蕉穿孔线虫的一步双重PCR分子技术和方法,该项检测技术特异性好,耗时较短、操作简便、可靠。     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

长岭发垫刃线虫(Trichotylenchus changlingensis)双重PCR检测方法研究

正长岭发垫刃线虫(Trichotylenchus changlingensis)是一种迁移性植物外寄生线虫,该线虫于2011年首次在我国吉林省长岭县发现,并根据其形态特征及最新分类系统将其归为发垫刃属[1,2]。该线虫能引起玉米叶片黄化,植株矮化,茎基部开裂,茎节缩短等症状,该病发生率普遍在21%～67%,给玉米生产造成了严重影响[3]。     

A PCR-based assay for the detection and identification of <Emphasis Type="Italic">Pyrenochaeta lycopersici</Emphasis>

The isolation of Pyrenochaeta lycopersici, causal agent of corky root of tomato, is difficult because of its slow growth and poor sporulation. Identification is complicated due the existence of two morphologically similar forms, Types 1 and 2, that differ in several physiological and molecular features. For the rapid and unambiguous identification of isolates, two oligonucleotide primer pairs were designed using ITS region sequences. Specific PCR products of 147 and 209 bp were obtained for isolates of Type 1 and Type 2, respectively. Specificity of both primer pairs was verified using several fungal and bacterial species. As little as 0.7 pg of target DNA could be detected with the protocol. A nested PCR procedure was necessary for the detection of the fungus in plant tissue. This technique will be of use in epidemiological studies and in the implementation of control strategies.     

PCR-based detection of Colletotrichum acutatum on strawberry

An oligonucleotide primer ( Ca Int 2) was synthesized from the variable internal transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) from Colletotrichum acutatum . PCR with primers Ca Int2 and ITS4 (from a conserved sequence of the rDNA) amplified a 490 bp fragment from several isolates of C. acutatum but not from other members of the genus Colletotrichum . Amplification of this fragment was achieved from 100 fg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from strawberry tissues infected by C. acutatum . Southern hybridization analysis confirmed the 490 bp fragment from C. acutatum DNA and infected strawberry to be identical. The species-specific primer ( Ca Int2) developed in this work could be used for the accurate identification of C. acutatum and its detection on other host plants.     

Specific Polymerase Chain Reaction Identification of Venturia nashicola Using Internally Transcribed Spacer Region in the Ribosomal DNA

ABSTRACT A technique based on the polymerase chain reaction (PCR) was developed for the identification of Venturia nashicola using nucleotide sequence information of the ribosomal DNA region. The complete internal transcribed spacer (ITS) region of V. nashicola strains and phylo-genetically related species was amplified with the two universal ITS1 and ITS4 primers, sequenced, and digested with five restriction enzymes. The alignment of nucleotide sequences and analyses of digestion patterns indicated constant polymorphisms between V. nashicola and related species at nucleotides 126 and 127, which overlapped a TaqI restriction site. An oligonucleotide primer named A126 was designed for identifying this variable region. A primer set (A126 and ITS4) that allowed the amplification of a 391-bp DNA fragment within the ITS region by PCR was specific to V. nashicola when it was checked against fungal genomic DNAs of related fungi. This primer set was a good candidate for a species-specific reagent in a procedure for identification of V. nashicola by PCR.     

香蕉炭疽菌rDNA ITS区的分子鉴定与检测

 香蕉炭疽病菌(Colletordchum muscat)是一种引起香蕉采后病害的最重要病原,本研究用真菌18S~28S间的内转录间隔区(internal transcribed spacer,ITS)通用引物18SF和28SR扩增香蕉炭疽菌和其它外群真菌的基因组DNA,扩增出约510bp的片段;通过克隆测序香蕉炭疽菌的ITS全序列并与GenBank中炭疽菌属其它种的ITS序列比对,设计出香蕉炭疽菌的特异性引物ColM1和ColM2。用此特异引物可以从香蕉炭疽菌株中扩增出382bp的特异性片段,而其余20个参试菌株和香蕉组织的PCR反应结果为阴性,灵敏度实验证明可以检测到目标DNA的浓度为0.1Pg。该方法可用于快速、准确和灵敏地检测香蕉炭疽菌,为快速监测组织中有无香蕉炭疽病菌潜伏侵染与及早采取防治措施提供积极的指导意义。     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Molecular Characterisation of Alternaria linicola and its Detection in Linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.     

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum- specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.