食线虫真菌28S rDNA PCR扩增片段的RFLPs分析*

本文对16个食线虫真菌(节丛孢属、隔指孢属和单顶孢属)菌株和3个其他相关丝孢菌(顶辐孢属和单端孢属)菌株的28S rDNA 片段进行了扩增, 并用限制性内切酶Sau3A I, Msp I, Rsa I and Hae III对PCR产物进行了消化。采用UPGMA法对 PCR 产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA 的ITS区间、5.8S和18S的序列分析结果一致。     

食线虫真菌28S rDNA PCR扩增片段的RFLPs分析

本文对16个食线虫真菌（节丛孢属,隔指孢属和单顶孢属）菌株和3个其他相关丝孢菌（顶辐孢属和单端孢属）菌株的28S rDNA片段进行了扩增,并用限制性内切酶Sau3 A,I,Msp I,RsaIand HaeⅢ对PCR产物进行了消化,采用UPGMA法对PCR产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA的ITS区间,5．8S和18S的序列分析结果一致。     

节丛孢属丝孢菌对松材线虫和拟松材线虫的捕食*

利用节丛孢属捕食线虫真菌的 8个种 ,1 1个菌株 ,对松材线虫 (Bx)和拟松材线虫(Bm)进行了捕食力的测定。结果显示节丛孢属真菌对这两种线虫的捕食能力既存在种间差异 ,也存在同种不同菌株间的差异。指状节丛孢的 3个菌株Ad 1 ,Ad 2 ,Ad 3菌株对两种线虫均表现出较高的捕食率 ,接种 7d后对Bx和Bm的捕食率分别达到了 98.08%,91.16 %,86.3 %和 96.28%,90.45 %,85.38%;同一菌株对松材线虫和拟松材线虫的捕食没有选择性。此外 ,通过对松材线虫和拟松材线虫     

弯孢节丛孢和厚皮单顶孢菌株的RAPD分析

对来自不同地方的7个弯孢节丛孢（ArthrobotrysmusiformisDrechsler）和9个厚皮单顶孢（Monacrosporiumeudermatum（Drechsler）Subram）标准菌株用4个随机引物（OPW-18、OPW－16、OPM-1、OPK-19）进行了随机扩增多态性DNA（RAPD）分析。扩增谱带聚类分析结果不能将弯孢节丛孢和厚皮单顶孢的菌株分开,具有附属丝的菌株没有被聚在一起,菌株间地理起源上的亲缘关系也没有体现,证明RAPD分析不能作为节丛孢属和单顶孢属的属间鉴别分析,附属丝地捕食线虫真菌不具分类学意义,不能以之作为成立小掘氏霉属的证据,系统聚类关系与地理来源基质也没有相关性。     

北细辛内生真菌的分离鉴定及代谢产物的生物活性

采用表面消毒法分别从3种北细辛中分离获得10株形态特征不同的优势内生真菌,经形态学和18S rDNA ITS分子分类学分析鉴定为小丛壳属(Glomerella sp.)、叶点霉属(Phyllosticta sp.)、柄孢壳菌属(Po-dospora sp.)、刺盘孢属(Colletotrichum sp.)和镰孢属(Fusarium sp.)。对北细辛优势内生真菌的发酵产物进行体外抗肿瘤和抗菌活性检测,结果表明:除菌株E3、E4和E10外,其余菌株均有不同程度的抗肿瘤和抗菌活性;镰孢属(Fusarium sp.)菌株E9对A549、MDA-MB-231和PANC-1肿瘤细胞抑制率达75%以上;小丛壳属(Glomerella sp.)菌株E1和叶点霉属(Phyllosticta sp.)菌株E2靶向FabI的抗菌活性较强,抑制率达59%。     

节丛孢属丝孢菌对松材线虫和拟松材线虫的捕食

利用节丛孢属捕食线虫真菌的8个种,11个菌株,对松材线虫（Bx）和拟松材线虫（Bm）进行了捕食力的测定。结果显示节丛孢属真菌对这两种线虫的捕食能力既存在种间差异,也存在同种不同菌株间的差异。指状节丛孢的3个菌株Ad－1,Ad－2,Ad－3菌株对两种线虫均表现出较高的捕食率,接川7d后对Bx和Bm的捕食率分别达到了98．08％,91．16％,86．3％和96．28％,90．45％,85．38％;同一菌株对松材线虫和拟松材线虫的捕食没有选择性。此外,通过对松材线虫和拟松材线虫繁殖真菌的筛选实验,肯定了利用拟松材线虫代替松材线虫进行生防菌株筛选的可行性。     

大叶醉鱼草内生真菌LL3026菌株杀虫活性及ITS-5.8S rDNA序列分析

从大叶醉鱼草的叶子中分离得到一株内生真菌LL3026,以卤虫模型测稀释后发酵液的杀虫活性,结果表明LL3026发酵液杀虫活性较强,且温度、光照及紫外照射对LL3026发酵液杀虫活性影响不显著;采用分子生物学方法对LL3026菌株rDNA的ITS基因(ITS-5.8S rDNA)进行PCR扩增、测序,构建系统发育树。ITS基因显示其属于刺盘孢属真菌。     

用AFLP技术和16S rDNA PCR\|RFLP分析毛苜蓿根瘤菌的遗传多样性

采用选择性扩增片断长度多态性(简称AFLP)DNA指纹技术对采自我国云南省与西藏交界的高山地区的野生型豆科植物毛苜蓿根际土样分离的291株毛苜蓿(Medicago edgeworthii)根瘤菌进行遗传多样性的研究。从AFLP图谱中,揭示出毛苜蓿根瘤菌有较显著的遗传多样性,从291株中选择出90个代表株用计算机进行树状图的分析。结果表明,所分析的菌株在79%的相似性水平上聚类成3个群。对这90个代表株进行多聚酶链反应(PCR)扩增的16S rDNA的4种限制性内切酶长度多态(简称16S rDNA PCR\|RFLP)分析,得出2个不同的16S rDNA PCR\|RFLP类型的菌株。分别选出这2个类型的代表菌株与各种根瘤菌的参比菌株进行16S rDNA PCR\|RFLP分析,再进行树状图的分析,初步得出了它们在根瘤菌系统分类中的地位。分析结果表明：毛苜蓿根瘤菌与根瘤菌属中的Rhizobium mongolense的相似性很高。     

多重PCR对真菌性角膜炎主要致病菌的菌属鉴定

目的:建立多重PCR体系对真菌性角膜炎主要致病真菌进行快速诊断并同时进行菌属鉴定的方法。方法:建立两个多重PCR体系(体系1和体系2),对真菌性角膜炎九种主要致病真菌DNA进行检测,观察该体系对真菌临床菌株、人类基因组及其他眼部常见致病微生物DNA的检测结果。结果:体系1对镰孢菌属扩增均产生约360bp的特异产物,对曲霉菌属、牵连青霉菌和新月弯孢菌扩增均产生约470bp的特异产物。体系2对镰孢菌属、曲霉菌属均无特异产物,而对牵连青霉菌产生了360bp的特异产物,对新月弯孢霉产生了300bp的特异产物。根据DNA模板在两个多重PCR体系中扩增出的不同特异条带可将九种真菌分为四个菌属。57株真菌临床菌株中55株的鉴定结果与常规鉴定结果一致。两体系对人类基因组及其他眼部常见致病微生物DNA的扩增结果均为阴性。结论:通过两个多重PCR体系检测可将真菌性角膜炎在菌属水平进行诊断及鉴定。该方法具有快速、简便、特异、灵敏的特点,具有较好的临床应用前景。     

不同覆土基质微生物结构特征研究及其对双孢蘑菇产量的影响

覆土是影响双孢蘑菇产量、质量和出菇整齐度的重要因子,利用现代分子生态学的方法快速、准确地对不同覆土基质微生物结构特征进行检测,以进一步了解微生物群落与双孢蘑菇相互作用关系。测定了不同覆土的理化特性,应用PCR技术对不同覆土材料提取总DNA,扩增细菌16S rDNA和真菌28S rDNA,运用变性梯度凝胶电泳技术对PCR产物进行分析,研究双孢蘑菇不同覆土基质微生物结构特征。结果表明：不同处理的覆土材料微生物群落的基因具有多样性,其中细菌群落基因多样性存在差异,使用纯泥炭与粉碎稻草处理差异最大,相似性仅为62%;通过真菌28S rDNA变性梯度凝胶电泳结果显示,粉碎稻草处理多样性指数最高,达3.576,但随着泥炭比例的提高,覆土处理中真菌群落的多样性相对减少;栽培试验发现,双孢蘑菇子实体形成量、总产量可能与覆土中的真菌群落多样性呈负相关。     

Identification of fungi associated with rotylenchulus reniformis

The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.     

RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria isolated from soil of India: Identification and detection of genetic variability

178 bacterial strains were isolated from the soil samples collected from different regions of India out of which, 20 bacterial isolates were selected for alkaline protease production. The alkaline protease production efficiency of organisms was monitored at regular intervals (24 h) upto 7 days at 37 °C, pH 10. The 16S rDNA sequencing and RAPD-PCR based technique were used to identify the genetic variability among the 20 isolates of alkaline protease producing bacteria. The phylogenetic analysis indicated that the isolates can be separated into two clusters which could be further subdivided into five groups. Group 1 and 5 represented the family Bacillaceae, Groups 2 represented the Micrococcaceae family while Group 3 included the Arthrobacter bacterial group (family Micrococcaceae) from different geographical locations, respectively. Group 4 was identified as Pseudomonadaceae which was gram (−) bacteria. 21 different oligonucleotide primers were used to amplify approximately 261 fragments from each DNA sample. The bands were scored on the basis of their presence and absence and similarity between DNA samples was checked using Jaccard’s coefficient. Isolates were distinguished into distinct groups based on RAPD profiles from different geographical locations, morphological features and enzyme production efficiency. For cluster analysis the dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA). The results indicated that 16S rDNA and RAPD-PCR are suitable methods for rapid identification and differentiation of alkaline protease producing bacteria.     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys.     

RFLP analysis of the PCR-amplified 28S rDNA inRhizoctonia solani

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.     

Isomucor (Mucoromycotina): a new genus from a Cerrado reserve in state of Sao Paulo, Brazil

During a survey of mucoralean fungi from a Cerrado reserve (Brazilian savanna) some isolates of a Mucor-like fungus were isolated from soil plates. Characterization based on morphological, physiological and molecular data from translation elongation factor (EF-1α), 28S (D1/D2) and ITS1-5.8S-ITS2 rDNA sequences was made. The isolates produce lateral branches bearing multispored sporangiola in addition to the multispored sporangia and a uniformly septate mycelium as the main differentiating characteristics. To our surprise this fungus possesses two EF-1α genes 1.4 and 1.5 kb long. Evidence from the analyzed datasets supports the delimitation of a new genus and the inclusion of Mucor fuscus based on 28S and ITS rDNA data.     

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.     

日本血吸虫中国大陆株基因多态性研究

对日本血吸虫中国大陆株湖南、湖北、江西、安徽、四川、云南隔离群以及一个实验室传代品系从基因水平进行了多态性研究。首先,在用ＰＣＲ－ＳＳＷＣＰ技术分析了２８Ｓ ｒＤＮＡ－Ｄ２高变区基础上,测定了该区安徽和云南隔离群的ＤＮＡ序列;其次,用ＰＣＲ获得了含有ＩＴＳ的ｒＤＮＡ片段,并对其进行了酶切点重复序列的多态性分析;最后,用ＲＡＰＤ技术分析了全基因组ＤＮＡ的多态性。结果表明,安徽与云南隔离群的２８Ｓ ｒ     

攀枝花市银合欢根瘤菌遗传多样性

【目的】研究分离自四川攀枝花的银合欢根瘤菌的遗传多样性。【方法】采用联合16S rDNA RFLP和IGS RFLP的综合聚类分析(16S-IGS RFLP)、AFLP及多位点持家基因(16S rDNA,atpD,recA)序列的联合分析对供试银合欢根瘤菌进行研究。【结果】31株未知菌具有15种16S-IGS遗传图谱类型、27种AFLP类型。16S-IGS RFLP结果表明,没有未知菌与Bradyrhizobium的参比菌株聚在一起。在71.4%的相似水平上,31个未知菌按属的水平分成3个分支:S、M和R,分别分布在Sinorhizobium属(28株)、Mesorhizobium属(2株)和Rhizobium属(1株)。S分支的28个菌在84%的相似水平上,16S-IGS RFLP聚类图中构成3个群:群S1、群S2、群S3;在AFLP聚类图中构成9个AFLP群:S1–S9。多位点基因序列表明,代表菌株SCAU215、SCAU231分别与M.Plurifarium、R.huautlense亲缘关系最近。而分布于Sinorhizobium属SCAU222和SCAU228、SCAU213、SCAU216可能代表Sinorhizobium的3个新类群。【结论】攀枝花市银合欢根瘤菌遗传多样性丰富,分布于Sinorhizobium、Mesorhizobium和Rhizobium三个属,且优势类群为Sinorhizobium。