小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性(英文)

应用一对寡核苷酸引物ITS1与ITS4对核DNAG＋C百分数小于30％的小克银汉霉（Cunninghamella）属的核糖体DNA（rDNA）内转录间区（YTS）进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为;第一组4种1变种,小于764碱基对（bp）;第二组2种,765～824bp;第三组2种1变种,大于825bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了RsaI,TaqI;Tru9I,和HinfI4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性（RFLPs）的分析。在雅致小克银汉霉（Celegans）、巴西玉蕊小克银汉霉（Cbertholletiae）、和布拉氏霉（Cblakesleeana）各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR－RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.     

红壤中VA菌根真菌(球囊霉目)的种类和生态分布

本文报道了发育于第四纪红色粘土母质上的红壤中的4属13种VA菌根真菌(球囊霉目):1.细齿无梗囊霉 Acaulospora denticulata Sieverding & Toro;2.丽孢无梗囊霉Acaulospora elegans Trappe & Gerdemann:3.光壁无梗囊霉Acaulospora laevis Gerdeman D & Trappe:4.巨大巨孢囊霉Gigaspora gigantea(Nicol.& Gerd.)Gerdemann & Trappe;5.珍珠巨孢囊霉Gigaspora margarita Becket & Hall;6.聚球囊霉Glomus aggregatum Schenck & Smith;7.明球囊霉Glomus clarum Nicolson & Schenck;8.集球囊霉Glomus fasiculatum(Thaxter)Gerdemann & Trappe;9.地球囊霉Glomus geosporum(Nicol.& Gerd.)Walker;10.木薯球囊霉Glomus manihot Howeler,Sieverding & Schenck;11.变形球囊霉Glomus versiforme(Karsten)Berch;12.美丽盾孢囊霉Scutellospora calospora(Nicol.& Gerd.)Walker & Sanders;13.异配盾孢囊霉Scutellospora heterogama(Nicol.& Gerd.)Walker & Sanders。其中细齿无梗囊霉(Acaulospora denticulata)、巨大巨孢囊霉(Gigaspora gigantea)、木薯球囊霉(Glomus manihot)和异配盾孢囊霉(Scutellospora heterogama)4种为国内新记录种。对这13种VA菌根真菌的形态进行了描述讨论,并对这些种群的出现频度、不同利用方式     

贵州烟草根围AM真菌多样性的初步研究

从贵州省内烟区不同土壤生态环境下采集烟草根际土样,湿筛离心法分离丛枝菌根（AM）真菌孢子,鉴定出烟草AM真菌4属20种,其中球囊霉属9种,无梗囊霉属7种,巨孢囊霉属3种,盾巨孢囊霉属1种。从土壤样品DNA中扩增AM真菌特异性片段并采用DGGE技术对AM真菌多样性进行分析。测序结果显示烟草根际土壤中菌根真菌主要菌群为球囊霉属,与湿筛离心法的鉴定结果一致。为进一步研究贵州地区AM真菌多样性以及开发应用提供了依据。     

山东省不同植被区内野生植物根围AM菌的生态分布

ＡＭ菌是土壤习居菌 ,生态适应性强 ,可发生在各种生态环境。寄主范围也十分广泛 ,除少量植物如莎草科、苋科、灯心草科、藜科、石竹科等 2 0余科植物不能或不易形成ＡＭ外 ,大多数植物包括苔藓、蕨类、裸子植物、被子植物都能被菌根菌侵染。当前人们十分重视对野生植物上ＡＭ菌的调查[3 ,4 ,1 0 ,1 1 ,1 4 ]。研究发现 ,野生植物上可能有比栽培作物更多的ＡＭ菌种类[1 ]。我国野生植物资源丰富 ,开发和利用野生寄主植物上的ＡＭ菌潜力巨大。由于ＡＭ菌对寄主植物的选择性及对环境条件的适应性不同 ,或进化过程中的历史原因 ,造成了自然生态…     

Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

Colonization of roots by arbuscular mycorrhizal fungi using different sources of inoculum

Arbuscular mycorrhizal fungi (AMF) form a number of different infective propagules that are used to form new mycorrhizal associations. These are spores, extraradical hyphae and infected roots. However, not all fungi are equally capable of colonizing roots with all of the above-mentioned propagules and there is conflicting evidence of major differences in colonization strategy between members of the Glomineae and Gigasporineae. In this study, we tested the abilities of eight fungal species from four different genera to colonize roots using three different types of inoculum. Glomus and Acaulospora isolates colonized from all inoculum types, whereas Gigaspora and Scutellospora isolates colonized mainly from spores and to a limited degree from root fragments. Extraradical hyphae were not suitable propagules for the species of Gigaspora and Scutellospora tested. This indicates that AMF have different colonization strategies and that this is largely differentiated at the suborder level. It is unclear why there is such a difference among the fungi in inoculum types. Future research should examine differences in the anatomy and physiology to discern a mechanism for such differences in life-history strategies.     

渤海湾岛屿的丛枝菌根真菌*

从渤海湾岛屿上的天然植被根围土壤中分离到丛枝菌根真菌7属35种, 其中无柄囊霉属Acaulospora5种,原囊霉属Archaespora1种,内养囊霉属 Entrophospora 1种,球囊霉属Glomus属18种,巨孢囊霉属 Gigaspora3种, 类球囊霉属Paraglomus属1种,盾巨孢囊霉属Scutellospora6种。群生盾巨孢囊霉Scutellospora gregaria和易误巨孢囊霉Gigaspora decipiens为我国的新记录种。标本保藏于莱阳农学院菌根生物技术实验室。     

五指山常见热带树种的丛枝菌根真菌多样性

采用野外调查的方法,分析了五指山不同海拔高度7个科10种常见热带树种形成丛枝菌根(Arbuscular Mycorrhizal,AM)的状况及其根际土壤中AM真菌的多样性。结果表明,所调查的10种热带常见树种都能形成AM共生体,其菌根侵染率随寄主植物的不同,从21.8%～90.5%变化不等,同时,在10种常见植物的根系中也都观察到了AM真菌的典型结构——丛枝和泡囊。从10种植物的根际土壤中共分离到36种AM真菌,隶属于Acaulospora,Glomus,Gigaspora和Scutellospora4个属,其中,Glomus属的真菌是该地区的优势类群,其出现频度和相对多度分别为84%和56%。在所调查的10种热带常见树种中,Swietenia macrophylla根际AM真菌的孢子最丰富,密度高达7.32;Machilus namu根际的AM真菌种类则最为丰富,多样性指数达到1.6548。通过对不同海拔高度Swietenia macrophylla根际AM真菌分布的分析表明,海拔高度显著影响着AM真菌的分布,Gigaspora属的真菌随海拔高度的增加显著升高,Scutellospora属的真菌则显著降低。     

Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR

The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon-discriminating molecular probes were developed. The 5' end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote-specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon-discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon-discriminating primers for the second, was performed on individual trypan blue-stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co-existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual and mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.     

福建漳州常见药用植物根围的丛枝菌根真菌

为了解闽南地区药用植物根围丛枝菌根真菌多样性分布,作者调查了福建省漳州市20种常见药用植物根围的AM真菌。从福建省漳州市小溪镇、国强乡等地共分离出无梗囊霉属Acaulospora 12种、原囊霉属Archaeospora 1种、巨孢囊霉属Gigaspora 2种、球囊霉属Glomus 42种、盾巨孢囊霉属Scutellospora 9种,其中沙生球囊霉Glomus arenarium、金黄球囊霉Glomus aureum和厚皮球囊霉Glomus callosum等3种为我国新记录种。     

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.     

rDNA-RFLP identification of Candida species in immunocompromised and seriously diseased patients

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from S?o Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8 s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.     

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP

 In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300–900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon’s index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease. Received: 25 August 1997 / Accepted: 19 September 1997     

中国的内囊霉科菌根真菌

本文报道了25种采集于我国广州、昆明、武汉、北京和哈尔滨等地农业土壤中的内囊霉科真菌。其中球内囊霉属(Glonus)16种,无柄内囊霉属(Acaulospora)4种,楯孢内囊霉属(Scutellospora)3种,巨孢内囊霉属(Giga-spora)1种,内养内囊霉属(Entrophospora)1种。此外还发现了硬内囊霉属(Sc-kerocystis)的孢子果。其中20种和除球内囊霉属外的另外5属为我国的新记录。     

土壤因子对野生植物AM真菌的影响

1　引　　言关于生态因子对ＡＭ真菌发生和分布的影响 ,国内外均有报道[1,2 ,7] ,张美庆等[6] 比较系统地研究了我国东、南沿海七省ＡＭ真菌的生态分布 ;吴铁航[5] 报道了几种土壤因子对栽培作物根围内ＡＭ真菌分布的影响 .本文主要介绍土壤类型、质地、营养状况、ｐＨ等对野生植物根围ＡＭ真菌的侵染、孢子密度和种属分布的影响 .2　研究地区与研究方法2 1　研究地区概况山东省位于 33°2 5′～ 38°2 3′Ｎ ,114°36′～ 12 2°4 3′Ｅ之间 ,属暖温带大陆性季风气候 ,年平均气温 11～ 14℃ ,年平均降水量为 5 5 0～ 95 0ｍｍ .根据地貌不…     

Differentiation of Chlamydia Species by Combined Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis

To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.     

Comparison of restriction fragment length polymorphisms of ribosomal DNA between Diphyllobothrium nihonkaiense and D. latum.

Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA) were compared between Diphyllobothrium latum and D. nihonkaiense using seven kinds of restriction endonucleases. No intra-specific variation in restriction fragment profiles was shown within both species of Diphyllobothrium. Digestion of the genomic DNA with three endonucleases. SmaI, HinfI and HhaI, provided one or two different bands between two species, although the hybridization patterns generated with the others. HindIII, XbaI, StyI and HaeIII, were the same in both. RFLPs in the digested profiles with SmaI, HinfI and HhaI could be used as species-specific markers even if only fragments of strobilae with morphological similarity were available. Other cestodes, Spirometra erinacei and Taenia saginata, used as controls showed quite different restriction fragment patterns with all the enzymes used.     

Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.     

Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.