杨树溃疡病病原的多重PCR检测技术

利用真菌通用引物ITS1/ITS4,EF1-728F/EF1-986R对4种杨树溃疡病病原菌株和环境样本的核糖体DNA内部转录间隔区ITS序列和转录延伸因子EF-1α基因序列分别进行普通和多重PCR检测,再进行测序比对分析。结果表明:退火温度为55.6℃,各引物终浓度为0.2μmol·L-1,可以成功地扩增出菌株和环境样本的目的条带,并通过测序比对鉴定出多种来自培养和环境病害组织中的溃疡病病菌,多重PCR能够检测到1ng的基因组DNA。研究结果将为建立溃疡病病原多重PCR鉴定技术和以环境溃疡病病害样本为直接检测鉴定对象的高通量的基因芯片检测方法奠定基础。     

米槠叶片基因组DNA的提取及分析

以米槠(Castanopsis carlesii)群落的叶片为材料,采用改良的CTAB法提取米槠基因组DNA,并对获得的基因组DNA进行浓度检测、PCR扩增、琼脂糖凝胶电泳检测、含量测定分析以及扩增结果测序。结果表明:米槠叶片提取的基因组DNA质量和浓度较好,具有良好的完整性,PCR效果好,电泳图谱条带显示清晰,测序结果长度符合要求。利用该方法提取的DNA可用于米槠遗传多样性的分析,可进一步为米槠和格氏栲等其它栲属植物的DNA提取以及其它分子研究提供参考。     

利用简化的SDS法提取杨树基因组DNA

以杨树组培苗幼嫩叶片为材料,在室温下采用简化的SDS法快速制备其基因组DNA,并对制备DNA过程中的影响因子进行了初步研究。结果表明,采用此方法制备的基因组DNA在数量和纯度上可以满足聚合酶链式反应(PCR)的要求。另外,DNA提取缓冲液中添加一定浓度的PVP、β-巯基乙醇,有利于DNA提取。     

松赤枯病病原的快速分子检测

为实现对松针中可导致松赤枯病的枯斑拟盘多毛孢的早期检测,依据枯斑拟盘多毛孢ITS区保守序列设计特异性引物AF(R/F),建立了松赤枯病PCR检测体系。结果表明该方法可于枯斑拟盘多毛孢基因组DNA与松针基因组DNA中扩增出大小为480 bp的单一条带,可从无明显症状的组织中检测到枯斑拟盘多毛孢。建立的PCR检测体系适用于枯斑拟盘多毛孢的分子鉴定及松赤枯病的早期诊断。     

山核桃基因组DNA提取方法的比较研究

植物体内所含的蛋白质、单宁、酚类、多糖及色素等次生代谢物质影响TaqDNA聚合酶的活性,是导致PCR扩增反应失败的主要因素。以山核桃嫩叶为材料,对分别用经过改良的SDS法、改良的CTAB法、简易CTAB法及改良的高盐低pH法提取的基因组DNA进行检测比较,结果显示,改良的SDS法更适合于山核桃基因组DNA的提取,此方法提取的DNA能很好地用于PCR扩增。     

基于CTAB法提取毛竹基因组DNA的探讨

应用CTAB法、改良CTAB法、改良CTAB-高盐沉淀法对毛竹基因组DNA进行了提取,并用紫外分光光度计(UV3300)、琼脂糖凝胶电泳和PCR分析对提取的DNA进行了检测,对DNA的产量、质量、PCR效果等方面进行了综合比较.结果表明:改良CTAB-高盐沉淀法是提取高质量毛竹基因组DNA的较好方法.     

基于粪便DNA的中缅树鼩性别鉴定

指出了SRY基因是存在于哺乳动物雄性Y染色体上的性别决定基因,对该基因的检测可以快速准确地判定性别。利用分子粪便学方法从野外获取中缅树鼩粪便中提取到的基因组DNA,运用双重PCR同时扩增SRY基因和微卫星DNA后,经过电泳分析最终鉴定出了79只树鼩的性别。     

樟子松2种块菌属外生菌根形态描述和分子鉴定

利用外部形态和解剖结构特征进行对比,选出樟子松Pinus sylvestris var.mongolica 2个类似块菌属Tuber的外生菌根类型,再采用CTAB法对其进行基因组DNA提取,并利用真菌特有引物ITS1F和ITS4,在PCR基础上对其r DNA的ITS区段进行碱基序列测定,并与Gen Bank数据库中已知的相关序列进行Blast比对得出:类型1,类型2均为块菌属的外生菌根,类型1可鉴定到种,为辽东块菌Tuber liaotongense。     

茶油总DNA提取技术及扩增适用性

近年来茶油掺假问题日益突出,传统理化方法难以进行有效鉴定,开发基于特征DNA的茶油掺假的高效鉴定技术十分必要。本研究探索不同方法提取茶油总DNA的提取效果,通过优化提取过程,确立了茶油总DNA最优的改良SDS提取方法。该法可从40 m L茶油中提取出足量用于PCR实验的茶油总DNA。进一步将获得的茶油总DNA作为模板进行特征DNA的PCR扩增验证,经DNA测序分析表明其PCR扩增适用性较好。     

“松材线虫SCAR标记与系列分子检测技术及试剂盒研制”项目通过鉴定

2006年5月，由南京林业大学森林资源与环境学院叶建仁教授主持完成的“松材线虫SCAR标记与系列分子检测技术及试剂盒研制”通过了江苏省科技厅组织的科技成果鉴定。该项研究对松材线虫与拟松材线虫特异片段进行了系统筛选，共获得了7个松材线虫DNA特异片段与5个拟松材线虫DNA特异片段，为松材线虫与拟松材线虫的分子鉴定奠定了基础。首次将2个松材线虫与2个拟松材线虫的DNA特异片段成功转化为SCAR标记，丰富了松材线虫与拟松材线虫分子标记方法。首次采用SCAR标记方法成功构建了松材线虫检测试剂盒，PCR检测过程仅需2．2小时。首次成功标记了一个可用于检测松材线虫的非放射性探针DIG-F2／R1。用该探针对线虫基因组DNA点杂交，松材线虫均表现有较强的杂交信号，而拟松材线虫、霍夫曼尼伞滑刃线虫、大核滑刃线虫、长尾属线虫等均无杂交信号。     

Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real‐time PCR

A nested and real‐time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species‐specific primers, CF01/CF02, was designed. Whereas a 280‐bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25‐μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first‐round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25‐μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real‐time PCR with a detection limit of 0.1 pg genomic DNA per 20‐μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.     

Molecular identification of decay fungi in the wood of urban trees

Trees are valuable for urban areas, however, are also susceptible to wood rot fungi. For accurate and fast assessment of the severity and evolution of decay in standing trees, a molecular technique was used to identify the causal agents of wood rot. Fruit bodies of wood decay fungi were collected from infected trees in various stands in Germany. Thirty-six species were identified by traditional methods. The DNA of fruit bodies was extracted, ITS-rDNA amplified by PCR, and ITS regions sequenced. Wood samples from infected urban trees were collected, the entire DNA extracted from affected wood parts, and fungal ITS amplified and sequenced. Fungal species were identified by comparing sequence data with the fruit body data. The technique enables an accurate and rapid identification of causal rot fungi in urban trees.     

Diversity and identification of fungi associated with needles of Pinus radiata in Tasmania

Needle fungi have been extensively studied in conifers but rarely in Pinus radiata. Previous studies of P. radiata have been based on fungal isolation and not direct PCR detection from needles. This research was a component of a study examining factors linked to spring needle cast (SNC) in Tasmanian P. radiata plantations and aimed to identify as many as possible of the fungal species commonly associated with P. radiata needles in Tasmanian plantations. Needle samples were collected from 13 sites representative of the range of sites in which P. radiata is grown in Tasmania. Fungi were detected by a direct PCR approach and identified using barcode sequences from a reference collection of isolates from Tasmania, mainland Australia and New Zealand as well as sequences from the international nucleotide sequence databases. The total number of molecular operational taxonomic units (MOTUs) was 152, with 127 detected by direct PCR and sequencing, and only 35 operational taxonomic units (OTUs) isolated by culture-based methods. Dothideomycetes was the most diverse class detected in this study, with many MOTUs detected by direct PCR and not isolated. Leotiomycetes was the second-most diverse class and Sordariomycetes third, with several OTUs frequently isolated but rarely or not at all detected by direct PCR. DNA sequence data facilitated the discrimination and identification of OTUs, but some effort was required to avoid confusion caused by poorly identified isolates in public DNA databases. This is the most comprehensive study yet of fungi associated with pine needles in Australia and highlights the hitherto unrecognised diversity of Teratosphaeriaceae in P. radiata.     

The primer fITS9 prevents chimera formation during fungal DNA amplification in a bark beetle DNA background

Insects are frequently associated with fungi in natural habitats. Molecular detection of fungal species using the primer pair ITS1F and ITS4 to amplify the ribosomal ITS region has become standard for studies of fungal ecology. When addressing insect and fungal DNA together, sequencing of the ITS region often results in chimeras due to ITS4 binding to both insect and fungal DNA. Using the newly developed primer fITS9, placed in the 5.8S region in the middle of the ITS region, we show that chimeras (insect–fungal, fungal–fungal, fungal–plants or fungal–mammals) can be avoided and a shorter and less variable DNA sequence product can be obtained. This method allows a more targeted amplification of fungal DNA from mixed samples and decreases the necessity for a nested PCR approach.     

Use of fungal biosystematics and molecular genetics in detection and identification of wood‐decay fungi for improved forest management

Advances in fungal biosystematics and molecular genetics have clarified relationships among the wood‐decay fungi and are providing new tools for their detection and identification. Species complexes of forest pathogens, including those within Heterobasidion, Armillaria, Laetiporus, and Phellinus, are being resolved. The ability to isolate fungal DNA directly from wood without in intermediate culturing step will greatly facilitate sampling and disease detection and has applications in forest disease management, hazard tree assessment, invasive species detection, and carbon cycling, sequestration and climate change research. Recent changes in fungal nomenclature and their application to forest pathology are discussed.     

Detection to Pathogens of Poplar Cankers by Multiplex PCR Technique

Fungal pathogens causing poplar canker diseases are cosmopolitan in species,and have a wide range of hosts.These pathogens have diverse anamorphs and their morphology overlaps with each other;Their teleomorphs are hardly discovered in nature.Furthermore,the identification of these pathogens is usually limited by the geography,host and taxonomic knowledge.Therefore,a culture-independent method used to identify determine pathogens is expected to be developed for field diagnostics.Through amplifying,sequencing and analyzing multiplex nucleic acid templates and genetic marked target sequences,a multiplex PCR technique has been established and used to directly detect various environmental samples.In this study, four pathogen strains and environmental samples were amplified using fungal universal primers ITS1/ ITS4 and EF1-728F/EF1-986R by general PCR and multiplex PCR.The amplicons were sequenced,and then aligned in GenBank.The result showed that the multiplex PCR was able to successfully amplify the target gene and identify the pathogens from environmental samples in the condition of an annealing temperature of 55.6℃and primers final concentration of 0.2μmol·L^-1.The multiplex PCR could amplify the target at the concentration level of approximately lng of genomic DNA.This method would provide a useful tool for the identification of canker pathogens by the multiplex PCR and the high-throughput DNA microarray detection of environmental samples.     

Detection of Chalara fraxinea in common ash (Fraxinus excelsior) using real time PCR

A real time PCR assay was developed for the detection of Chalara fraxinea in common ash. PCR primers and Taqman probes, based on the internal transcribed spacer region of the multi‐copy gene rDNA, were tested for specificity and sensitivity. The primers amplified an 81 bp fragment for C. fraxinea but did not amplify DNA from other Chalara species or from other fungi isolated from ash, whether pathogenic or saprophytic. The limit of detection was 5 pg of genomic DNA per PCR. Moreover, naturally‐infected samples were correctly diagnosed. A procedure for DNA extraction from woody tissues using an electric drill yielded DNA of an appropriate quality for real time PCR. This molecular method could be useful for routine analysis of this emergent pathogen and for epidemiological studies.     

A molecular diagnostic assay for the detection and identification of wood decay fungi of conifers

Ten taxon‐specific primers were designed to amplify the Internal Transcribed Spacer of the rRNA operon of several important decay fungi of coniferous wood, including Armillaria spp., Echinodontium spp., Fomitopsis pinicola, Fuscoporia torulosa, Heterobasidion annosum sensu lato (s.l.), Onnia spp., Phaeolus schweinitzii, Phellinus weirii s.l., Pholiota spp. and Porodaedalea spp. Primers designed in this study and in a previous one for the identification of Laetiporus sulphureus and Stereum spp. were combined in two multiplex PCRs, which were tested for efficiency and specificity, and detected at least 1 pg of fungal target DNA. Target DNA at concentrations of 10^?1 pg or lower can be detected with this assay using SYBR^® Green Real‐Time PCR. Validation assays performed on 129 naturally infected wood samples or fruiting bodies confirmed the reliability of the multiplex PCR‐based diagnostic method. This method represents a simple and rapid diagnostic tool for the detection of a number of destructive wood decay fungi of conifer wood.