Identification of F. o. cucumerinum and F. o. luffae by RAPD-generated DNA probes

AIMS: To create a fast, sensitive and specific method for identifying Fusarium oxysporum f. sp. cucumerinum and F. o. luffae. METHODS AND RESULTS: Specific DNA bands were selected as probes from RAPD profiles of 13 formae speciales of F. oxysporum. The forma specialis-specific probe OPC18300c and OPC18520f could be used to identify F. o. cucumerinum and F. o. luffae by RAPD-PCR followed dot blot hybridization, respectively. CONCLUSIONS: A specific method for identifying F. o. cucumerinum and F. o. luffae was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: F. oxysporum formae speciales identification with a DNA probe can be relatively rapid and provides a method to identify the pathogen without host inoculation tests.     

The structure and interrelationship of fungal populations in native and cultivated soils

Mitochondrial DNA haplotypes have been characterized for 120 isolates of the asexual fungus Fusarium oxysporum. Sixty of these isolates were obtained from soil in a native grassland in the San Joaquin Valley of California, including 20 isolates from each of six different sampling locations. The same sampling strategy was used to obtain 60 additional isolates from an agricultural field of the same soil type directly adjacent to the native soil. Twenty-three different mitochondrial DNA haplotypes were identified among the 120 isolates, including 11 haplotypes represented by two or more isolates and 12 that were unique. The five most common mitochondrial DNA haplotypes accounted for 93 (78%) of the 120 isolates. Isolates representing each of these five mitochondrial DNA haplotypes were found both in the cultivated and in the native soil. Seventy-two per cent of the isolates found in the cultivated soil were associated with the same mitochondrial DNA haplotype as one or more isolates in the native soil. The remaining isolates in the cultivated soil were associated with comparatively rare mitochondrial DNA haplotypes, most of which showed a close relationship to one of the haplotypes found in the native soil. Hierarchial gene diversity analysis indicated that a significant proportion of the mitochondrial DNA haplotype diversity was attributable to differences between sampling sites in the native soil but not in the cultivated soil. This may reflect significant spatial structuring of genetic diversity in populations of F. oxysporum in a native soil. The proportion of mtDNA haplotype diversity attributable to differences between populations in the native and cultivated soils was not significant. This suggests that our entire collection, encompassing strains from both native and cultivated soils, is representative of a single population of F. oxysporum.     

Linear plasmidlike DNA in the plant pathogenic fungus Fusarium oxysporum f. sp. conglutinans.

Double-stranded, 1.9-kilobase-pair (kbp) DNA molecules were found in 18 strains representing three pathogenic races of Fusarium oxysporum f. sp. conglutinans. The DNA element (pFOXC1) from a race 1 strain and the DNA element (pFOXC2) from a race 2 strain were shown by restriction endonuclease mapping to be linear. pFOXC2 was found in mitochondrial preparations and appears to have blocked 5' termini, as it was sensitive to 3'----5' exonuclease III but insensitive to 5'----3' lambda exonuclease. The major 1.8-kbp BglII restriction endonuclease fragment of pFOXC2 was cloned in plasmid pUC12. The recombinant plasmid (pCK1) was not homologous to the mitochondrial or nuclear genomes from F. oxysporum f. sp. conglutinans. This suggests that pFOXC2 is self-replicating. pCK1 was homologous to all 1.9-kbp DNA elements of race 2 but was not homologous to those of race 1 or race 5. All race 1 and 5 elements were also shown to share common DNA sequences.     

Fusarium commune is a new species identified by morphological and molecular phylogenetic data

Fusarium commune sp. nov. was isolated from soil and Pisum sativum in Denmark and several widespread locations within the northern hemisphere from diverse substrates including white pine, Douglas fir, carnation, corn, carrot, barley and soil. Fusarium commune is characterized by and distinguished from its putative sister taxon, the F. oxysporum complex, in having long, slender monophialides and polyphialides when cultured in the dark. Based on the combined DNA sequence data from translation elongation factor 1α (EF-1α) and the mitochondrial small subunit ribosomal DNA (mtSSU rDNA), the 15 isolates of F. commune analyzed formed a strongly supported clade closely related to but independent of the F. oxysporum and Gibberella fujikuroi species complexes.     

Plant colonization by the vascular wilt fungus Fusarium oxysporum requires FOW1, a gene encoding a mitochondrial protein

The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. The pathogenicity mutant B60 of the melon wilt pathogen F. oxysporum f. sp. melonis was isolated previously by restriction enzyme-mediated DNA integration mutagenesis. Molecular analysis of B60 identified the affected gene, designated FOW1, which encodes a protein with strong similarity to mitochondrial carrier proteins of yeast. Although the FOW1 insertional mutant and gene-targeted mutants showed normal growth and conidiation in culture, they showed markedly reduced virulence as a result of a defect in the ability to colonize the plant tissue. Mitochondrial import of Fow1 was verified using strains expressing the Fow1-green fluorescent protein fusion proteins. The FOW1-targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also showed reduced virulence. These data strongly suggest that FOW1 encodes a mitochondrial carrier protein that is required specifically for colonization in the plant tissue by F. oxysporum.     

海南省香蕉枯萎菌生理小种的RAPD分析

利用随机扩增多态性DNA(RAPD)分子标记方法对海南省香蕉枯萎病菌2个生理小种(小种1和小种4)进行遗传多样性分析,以筛选出的15个随机引物对采自海南省各市县发病蕉区的分别属于1号生理小种和4号生理小种的16个代表菌株及广东省2个1号和4号生理小种对照菌株进行RAPD-PCR扩增,结果产生97个RAPD分子标记,其中多态性的条带有76条,通过聚类分析探讨了供试小种间的亲缘关系,并寻找到了1、4号生理小种的特异性条带,为在分子水平上进行香蕉枯萎病菌生理小种鉴定提供更为便利的手段。     

Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia- mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum nia- mutants at a frequency of up to ten transformants per microgram of DNA. Southern analysis of the DNA of the F. oxysporum transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.     

应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

以3株黄瓜尖镰孢菌(Fusarium oxysporum f.sp.cucumarinum)、23株镰孢菌属(Fusariumspp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料,采用化学裂解法提取总DNA,进行PCR-RFLP和巢式PCR检测,试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种,而巢式PCR对黄瓜尖镰孢菌具有特异性.运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测,结果表明,两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌,PCR-RFLP在感病品种接种后3d即可检测到病原菌,而巢式PCR在接种后5d才能检测到病原菌.     

The complete mitochondrial genome of Fusarium oxysporum: insights into fungal mitochondrial evolution

The complete mitochondrial DNA (mtDNA) sequence was determined for the phytopathogenic fungus Fusarium oxysporum. It is 34,477 bp long, maps circularly, and encodes for 14 protein-coding, 25 tRNA and 2 rRNA genes. The nucleotide and amino acid data sets from its 14 concatenated protein-coding mitochondrial (mt) genes were used along with gene order comparisons for an extensive phylogenetic study of the Subphylum Pezizomycotina. Our results are in agreement with current taxonomic treatments and additionally provide better statistical support for all relationships within Pezizomycotina when compared to analyses based on single or few gene data sets. The gene order of F. oxysporum was consistent with that established in the order Hypocreales (Class: Sordariomycetes) and enhanced previous suppositions on the ancestral state of Sordariomycetes. In comparison with mt genomes of the other orders it added further insights to the evolution of Pezizomycotina.     

Comparison of the levels of the 21S mitochondrial rRNA in derepressed and glucose-repressed Saccharomyces cerevisiae.

A cDNA preparation, synthesized by using Saccharomyces cerevisiae mitochondrial RNA as template and oligodeoxythymidylic acid as primer, was found to specifically hybridize to the mitochondrial 21S rRNA by the following criteria: (i) it hybridizes only to the 21S RNA species in mitochondrial RNA and not to RNA from a [rho0] mutant, and (ii) it hybridizes to fragments in restriction digests of mitochondrial DNA that contain the 21S rRNA gene but not to nuclear DNA. This cDNA was used as a probe to demonstrate that a 2.6-fold decrease in the cellular level of the mitochondrial large rRNA is associated with glucose repression of mitochondrial function in S. cerevisiae. A corresponding decrease in the level of mitochondrial DNA was not observed.     

新疆棉花枯萎病菌异核体及其不同表型分离子核DNA的差异

为探索丝状真菌异核体的产生机制,从新疆地区患枯萎病的棉杆中分离出棉花枯萎病菌Fusariumoxysporumf.sp.vasinfectum异核体菌株X515,并自其菌落出现的不同角变处分离出三个不同表型的分离子,X515-Ⅰ、X515-Ⅱ和X515-Ⅲ。选用200种随机引物对它们进行核DNA的RAPD分析,只在OPB-16引物对致病力强的分离子X515-Ⅰ核DNA的PCR扩增产物中出现了一条354bp的差异片段,其部分碱基序列与Fusariumoxysporumf.sp.lycopersici的短散布序列(Foxy)末端序列的相似性达88%。本研究结果提示,随着所采用引物数量的增加,可有望找出异核体及其不同分离子之间核DNA的差异,为最终阐明异核体形成的机制提供线索。     

A rapid screening technique for the detection of repeated DNA sequences in plant tissues

Summary DNA sequences cloned from nuclear and mitochondrial chromosomes have been used as hybridisation probes to distinguish different plant genotypes. The probes are hybridised to squashed segments of tissue e.g. root tips. The squash-dot method is rapid and suitable for screening large numbers of individual plants. One probe, specific for a rye repeated sequence family, enables rye chromosomes to be detected in wheat plants. A probe for ribosomal DNA enables plants with high or low numbers of ribosomal RNA genes to be distinguished. A maize mitochondrial DNA probe is used to distinguish plants with N, T or S cytoplasms.     

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.     

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.     

湖南地区人群中线粒体12S rRNA基因A1555G突变筛查

为建立快速、简便、准确筛查线粒体DNA 12S rRNA基因A1555G突变的基因检测技术平台,收集1 758例（女性808例,男性950例）正常人群样本,利用Bsm AⅠ酶切法筛查线粒体DNA A1555G突变以及通过实时荧光定量Taqman探针法和直接测序法对筛查结果进行验证,结果检测到2例A1555G阳性突变样本,其中1例为男性,1例为女性。实时荧光定量Taqman探针法与Bsm AⅠ酶切法、直接测序法检测结果完全相符,未发现假阳性和假阴性,该方法具有结果准确直观、简单省时,特异性强,敏感性高的优点,适用于对母系遗传性耳聋线粒体DNA A1555G突变的大规模筛查或氨基糖甙类抗生素应用前的预防性检测。     

The mitochondrial genome of Fusarium oxysporum

Physical and genetic maps have been constructed for mtDNA from strains of the fungus Fusarium oxysporum representing three pathogenically specialized forms. All three mtDNA maps are circular. Their sizes are 45 kb for F. oxysporum f.sp. raphani and 52 kb for both F. oxysporum f.sp. conglutinans and F. oxysporum f.sp. matthioli. The genetic loci for cytochrome b, the mitochondrial 25S ribosomal RNA and cytochrome oxidase subunit II, have been identified and are similarly arranged on the three genomes.     

Purification of mitochondrial DNA from total cellular DNA of small tissue samples

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.     

Gerbera jamesonii, a New Host of Fusarium oxysporum f.sp. tracheiphilum

The random amplified polymorphic DNA (RAPD) technique was used to analyze the total genomic DNA of pathogenic isolates of Fusarium oxysporum on Gerbera jamesonii by comparing them to representatives of the formae speciales chrysanthemi and tracheiphilum. A close genetic relationship was observed among most of the new isolates from G. jamesonii. They shared RAPD markers with the tested representatives of the forma specialis chrysanthemi . Some isolates of those tested from diseased G. jamesonii were placed in a different cluster, which included representative isolates of forma specialis tracheiphilum . This is the first report of F. oxysporum f.sp. tracheiphilum on G. jamesonii. A rapid protocol for DNA extraction directly from fungal colonies grown on potato dextrose agar allowed complete analysis in less than 4 h.     

Mitochondrial DNA in the mouse preimplantation embryo

Total DNA was extracted from mouse embryos that were collected from CD-1 random-bred females on Day 1 of pregnancy and cultured for up to 4 days in vitro, or from the reproductive tracts of pregnant females on Days 1, 3, 4 and 5 of pregnancy. Southern blot analyses with a cloned mouse mitochondrial DNA probe were performed to determine the relative levels of mitochondrial DNA in the zygote, morula, blastocyst and early egg cylinder stage embryos. The results indicated that the total amount of mitochondrial DNA does not change during development of the mouse embryo up to the egg cylinder stage and is not altered during in-vitro culture of the fertilized one-cell embryo to the blastocyst stage.