scholarly journals Two Groups of Phytoplasmas from Japan Distinguished on the Basis of Amplification and Restriction Analysis of 16S rDNA

Plant Disease ◽  
1997 ◽  
Vol 81 (3) ◽  
pp. 301-305 ◽  
Author(s):  
Seiichi Okuda ◽  
James P. Prince ◽  
Robert E. Davis ◽  
Ellen L. Dally ◽  
Ing-Ming Lee ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Rrna Gene ◽  
Gene Group ◽  
Beet Leafhopper ◽  
Pcr Products ◽  
Elm Yellows

Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron

Parasitology ◽  
2002 ◽  
Vol 124 (2) ◽  
pp. 177-184 ◽  
Author(s):  
M. B. A. MENDONÇA ◽  
N. S. NEHME ◽  
S. S. SANTOS ◽  
E. CUPOLILLO ◽  
N. VARGAS ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Ribosomal Rna ◽  
Southern Blotting ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Pcr Products ◽  
Phylogenetic Lineages ◽  
Definition Of ◽  
Ribosomal Cistron ◽  
Panstrongylus Geniculatus

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3–Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Sα rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5·8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Sα rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.

scholarly journals Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR

2006 ◽  
Vol 72 (2) ◽  
pp. 1072-1078 ◽  
Author(s):  
Isabelle Robène-Soustrade ◽  
Philippe Laurent ◽  
Lionel Gagnevin ◽  
Emmanuel Jouen ◽  
Olivier Pruvost
Keyword(s):  
Diagnostic Tool ◽  
Nested Pcr ◽  
Restriction Analysis ◽  
Detection Threshold ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Amplification Product ◽  
Xanthomonas Axonopodis ◽  
Sequence Characterized Amplified Region ◽  
Pcr Test

ABSTRACT Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (103 CFU ml−1) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.

Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’

2004 ◽  
Vol 54 (4) ◽  
pp. 1349-1353 ◽  
Author(s):  
Chuji Hiruki ◽  
Keri Wang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Reference Strain ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Beet Leafhopper ◽  
Signature Sequences ◽  
Elm Yellows ◽  
The 16S Rrna Gene

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S–23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S–23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name ‘Candidatus Phytoplasma trifolii’ is proposed for the CP group.

Analysis of mitochondrial DNA, chloroplast DNA, and double-stranded RNA in fertile and cytoplasmic male-sterile sunflower (Helianthus annuus)

1986 ◽  
Vol 28 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Gregory G. Brown ◽  
Howard Bussey ◽  
Lee J. DesRosiers
Keyword(s):  
Male Sterility ◽  
Restriction Analysis ◽  
Nuclear Gene ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Male Sterile ◽  
Gene Effects ◽  
Mitochondrial Dnas ◽  
Specific Association ◽  
Chloroplast Dnas

The extent of variation in the mitochondrial DNAs (mtDNAs), chloroplast DNAs (ctDNAs), and double-stranded RNAs (dsRNAs) of sunflower lines carrying fertile and male-sterility conferring cytoplasms was examined. To minimize nuclear gene effects, efforts were concentrated on two chromosomally isogenic lines, CM400 (fertile) and cmsCM400 (male sterile), which differ only in their cytogenes. A circular 1.45 kilobases (kb) plasmid DNA was found in the mitochondria of the four fertile lines examined, but was absent in the male-sterile line. Restriction enzyme analysis of mtDNAs of the fertile and male-sterile cytoplasms with BamHI, EcoRI, and HindIII revealed no fragment mobility differences between them other than those which could be ascribed to the 1.45-kb circle. Similar restriction analysis of ctDNA showed no differences between fertile and male-sterile cytoplasms. Both CM400 and cmsCM400 contain dsRNA molecules. The number and sizes of these dsRNAs varied from preparation to preparation in both lines. Species of 3.3 and 1.5 kb, which were the only dsRNAs common to all preparations from CM400, were also the only species common to all preparations from cmsCM400. Thus, no consistent differences between the fertile and male-sterile cytoplasms were seen in these molecules. The specific association of the 1.45-kb plasmid with fertile cytoplasm together with the absence of variation in ctDNA and dsRNA, suggests the involvement of mtDNA in sunflower cytoplasmic male sterility.Key words: DNA (mitochondrial), sterility (male), sterility (cytoplasmic), Helianthus, sunflower, DNA chloroplast.

scholarly journals Restriction enzyme analysis of the chloroplast DNA of Phaseolus vulgaris L. vr. Rio Negro

1996 ◽  
Vol 26 (3) ◽  
pp. 507-509
Author(s):  
Sergio Echeverrigaray ◽  
Maria Tereza Vitral Carvalho ◽  
Eric Derbyshire
Keyword(s):  
Phaseolus Vulgaris ◽  
Chloroplast Dna ◽  
Sucrose Gradient ◽  
Restriction Analysis ◽  
Gradient Centrifugation ◽  
Repeat Sequence ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Phaseolus Vulgaris L ◽  
Rio Negro

The chloroplast DNA of Phaseolus vulgaris L. vr. Rio Negro was isola ted from chloroplasts obtained by descontiuous sucrose gradient centrifugation. The restriction analysis with the enzymes HindIII, EcoRI and BamHI and their combination, allowed to identified more than 20 fragments of 18 to 0.65kb. The size of Phaseolus vulgaris L. cp DNA was estimated in 140kb with the presence of a repeat sequence of about 22kb.

scholarly journals Molecular Detection and Identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in Spoiled Wines

2004 ◽  
Vol 70 (3) ◽  
pp. 1347-1355 ◽  
Author(s):  
Luca Cocolin ◽  
Kalliopi Rantsiou ◽  
Lucilla Iacumin ◽  
Roberto Zironi ◽  
Giuseppe Comi
Keyword(s):  
Direct Detection ◽  
Molecular Methods ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Rrna Gene ◽  
Brettanomyces Bruxellensis ◽  
Dna And Rna ◽  
Detection And Identification ◽  
26S Rrna ◽  
Total Agreement

ABSTRACT In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.

scholarly journals Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil

2002 ◽  
Vol 68 (8) ◽  
pp. 3818-3829 ◽  
Author(s):  
Christopher Rösch ◽  
Alexander Mergel ◽  
Hermann Bothe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nitrite Reductase ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Cloning And Sequencing ◽  
Genetic Traits ◽  
Uncultured Bacteria ◽  
Pcr Products ◽  
The 16S Rrna Gene

ABSTRACT Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd 1-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.

scholarly journals Characterization of cytomegalovirus isolates from patients with AIDS by DNA restriction analysis

1988 ◽  
Vol 101 (3) ◽  
pp. 483-494 ◽  
Author(s):  
D. L. Taylor ◽  
D. Taylor-Robinson ◽  
D. J. Jeffries ◽  
A. S. Tyms
Keyword(s):  
Restriction Analysis ◽  
Genome Structure ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Migration Patterns ◽  
Disease Pattern ◽  
Dna Restriction ◽  
Acquired Immunodeficiency ◽  
Different Strains ◽  
Immunodeficiency Syndrome

SUMMARYThirty-seven isolates of cytomegalovirus (CMV) were obtained from a group of 20 promiscuous homosexual men, either suffering from the acquired immunodeficiency syndrome (AIDS) at the time of CMV isolation, or who developed AIDS subsequently. The isolates of CMV were characterized by the method of DNA restriction analysis. All epidemiologically unrelated strains of CMV exhibited different fragment migration patterns and no one strain appeared to be associated with AIDS or any particular disease pattern in these patients.Sequential isolates of CMV were obtained from nine patients in the study group either from different sites at the same time or from the same site on different dates. In the case of seven of the men, viruses with minor differences in restriction profile were obtained, possiblyrepresenting sub-populations of an endogenous strain of CMV. In two of the patients, reinfection with different strains was apparent. We conclude that reinfections with CMV in AIDS patients can occur, but the isolation of strains exhibiting major differences in genome structure seen by restriction enzyme analysis was uncommon.

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