scholarly journals Responses of Six Eurasian Ulmus Cultivars to a North American Elm Yellows Phytoplasma

Plant Disease ◽  
2000 ◽  
Vol 84 (12) ◽  
pp. 1266-1270 ◽  
Author(s):  
W. A. Sinclair ◽  
A. M. Townsend ◽  
H. M. Griffiths ◽  
T. H. Whitlow
Keyword(s):  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Phloem Necrosis ◽  
Fluorescence Test ◽  
Polymerase Chain ◽  
Elm Yellows ◽  
Control Tree ◽  
Eurasian Species ◽  
Dapi Fluorescence

Elms (genus Ulmus) of six clonal cultivars representing Eurasian species and hybrids were grafted when 2 to 3 years old with bark patches from U. rubra infected with an elm yellows phytoplasma or were left untreated as controls. The cultivars were U. glabra × minor ‘Pioneer’, U. minor × parvifolia ‘Frontier’, U. parvifolia ‘Pathfinder’, U. wilsoniana ‘Prospector’, and the complex hybrids ‘Homestead’ and ‘Patriot’. Trees were evaluated for infection and symptoms 1 or 2 years after inoculation. Infection was detected via the 4′,6-diamidino-2-phenylindol e·2HCl (DAPI) fluorescence test in 26 of 86 grafted trees representing five cultivars. Infection of selected trees was confirmed by polymerase chain reaction (PCR) amplification of a fragment of phytoplasmal rDNA, and the phytoplasma was identified by restriction fragment length polymorphism (RFLP) analysis of the amplified DNA using restriction enzymes AluI, RsaI, and TaqI. Elm yellows phytoplasma was also identified by nested PCR and RFLP analysis in two of seven inoculated, healthy-appearing, DAPI-negative trees and one noninoculated control tree. All RFLP profiles were identical to that of reference strain EY1. Phytoplasma-associated symptoms, observed in five cultivars, included suppressed growth, progressive size reduction of apical shoots and leaves, chlorosis, foliar reddening, witches'-brooms, and dieback. Phyto-plasma was not detected in cv. Homestead. Possible resistance of this cultivar to elm yellows phytoplasma was indicated by localized phloem necrosis in stems below inoculum patches.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

scholarly journals Taxa of Leafhoppers Carrying Phytoplasmas at Sites of Ash Yellows Occurrence in New York State

Plant Disease ◽  
2000 ◽  
Vol 84 (2) ◽  
pp. 134-138 ◽  
Author(s):  
G. T. Hill ◽  
W. A. Sinclair
Keyword(s):  
New York ◽  
New York State ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Disease Group ◽  
York State ◽  
Aster Yellows ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Abundant Genus

Leafhopper (Homoptera: Cicadellidae) populations were sampled and leafhopper carriers of ash yellows (AshY) phytoplasmas were identified as first steps toward vector identification. Nearly 5,000 leafhoppers were collected in malaise traps at two sites of high AshY incidence in New York state in 1996 and 1997. These insects comprised 33 taxa, including representatives of 13 genera known to contain phytoplasma vectors. The most abundant genus was Scaphoideus, with numbers about six times greater than any other genus. A total of 1,632 insects were assayed individually for phytoplasmas by polymerase chain reaction (PCR) amplification of phytoplasmal 16S rDNA and restriction fragment length polymorphism analyses of PCR products using restriction enzymes TaqI and RsaI separately. Phytoplasmas were detected in 35 insects, all but one in the subfamily Deltocephalinae. AshY phytoplasmas were detected in 19 of 812 individuals of Scaphoideus spp. and 1 of 87 of Colladonus clitellarius. Phytoplasmas of the Prunus X-disease group were detected in 1 Scaphoideus sp., 4 individuals of C. clitellarius, and 4 of 83 Scaphytopius acutus individuals. Phytoplasmas of the aster yellows group were detected in 1 of 68 individuals of Gyponana spp. and 5 of S. acutus. AshY phytoplasma carriers merit testing for possible vector ability.

scholarly journals Discovery of a New Self-incompatibility Allele in Apple

HortScience ◽  
2000 ◽  
Vol 35 (7) ◽  
pp. 1329-1332 ◽  
Author(s):  
Shogo Matsumoto ◽  
Kentaro Kitahara
Keyword(s):  
Preliminary Investigation ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Unknown Origin ◽  
Analysis Method ◽  
Apple Cultivars ◽  
Total Dna ◽  
Polymerase Chain ◽  
Or History ◽  
Allele Identification

A polymerase chain reaction (PCR)-based method for identifying the S-alleles in the Asian pear [Pyrus pyrifolia (Burm) Nak.] was applied to apple (Malus ×domestica Borkh.) cultivars. With minor modifications in one of the primers, the fragments from S-genes (S-RNases) with introns were amplified from total DNA of apple cultivars possessing S2-, S3-, S5-, S7-(=Sd-), S9-(=Sc-), Sf- and Sg-allele genotypes. S-genes within S24-(=Sh-) and S26-alleles were also amplified. The PCR amplification step of this method appears to be useful for preliminary investigation of apple S-genotypes, especially for species or cultivars of unknown origin or history. Using the primers, which are a part of a new S-allele, the Se-allele encoding Se-RNase with an intron in the Se-allele was amplified. We cloned the cDNA of Se-RNase, and developed a PCR-restriction fragment length polymorphism (RFLP) analysis method for Se-allele identification. S-allele genotypes of seven apple cultivars were investigated.

scholarly journals Isolation and characterization of Listeria monocytogenes from environmental and clinical sources by culture and PCR-RFLP methods

2019 ◽  
Author(s):  
Hossein Meghdadi ◽  
Azar Dokht Khosravi ◽  
Ahmad Farajzadeh Sheikh ◽  
Ameneh Alami ◽  
Nerssy Nassirabady
Keyword(s):  
Listeria Monocytogenes ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Clinical Complications ◽  
Isolation And Characterization ◽  
Rflp Profile ◽  
Pcr Products

Background and Objectives: Due to the widespread distribution of Listeria monocytogenes in environmental and animal sources and serious clinical complications in human, this study was aimed to isolate L. monocytogenes from water and clin- ical specimens by culture and PCR methods and to investigate the presence of hlyA and inlA virulence genes. Materials and Methods: Water and clinical samples of vaginal and fecal were screened for the presence of L. monocyto- genes by phenotypic and standard biochemical tests. PCR amplification was performed on extracted DNA using primers based on the hlyA and inlA genes. A 733-bp fragment of inlA gene was used for investigation of polymorphism using RFLP analysis. Results: In total, 45 phenotypically and molecularly confirmed L. monocytogenes strains were isolated from different sourc- es including 30 (16.7%) from water, 9 (11.3%) from vaginal swabs and 6 (7.5%) from fecal samples. RFLP analysis of PCR products using AluI and Tsp509I restriction enzymes, generated two profiles with 8 to 10 bands ranging in size from 15 to 210 bp. The majority of water and clinical isolates were classified in profile 2. Conclusion: We demonstrated 45 L. monocytogenes isolates from tested water and clinical samples by phenotypic and mo- lecular tests. The majority of the isolates were classified in the same RFLP profile, showing the water as a potential source of clinical complications in patients in the region of study.

scholarly journals A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

2021 ◽  
Vol 9 (12) ◽  
pp. 2505
Author(s):  
Hiroki Hayashi ◽  
Tsutomu Kishi
Keyword(s):  
Dna Sequences ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Epitope Tagging ◽  
One Step ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The One ◽  
Step Polymerase Chain Reaction

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

scholarly journals Detection of Phytoplasmas in Declining Pears in Southern Australia

Plant Disease ◽  
1997 ◽  
Vol 81 (3) ◽  
pp. 254-258 ◽  
Author(s):  
B. Schneider ◽  
K. S. Gibb
Keyword(s):  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Pcr Assay ◽  
Specific Primers ◽  
Pear Tree ◽  
Pear Trees ◽  
Polymerase Chain

Forty-nine pear tree samples collected in Victoria, most of them showing decline symptoms, were tested by polymerase chain reaction (PCR) analysis to detect phytoplasmas. Two universal phytoplasma-specific primer pairs, fP1/rP7 and fU5/rU3, were tested, but only fU5/rU3 amplified the phytoplasma DNA adequately. Nested PCR with universal and group-specific primers, however, proved more effective. Thirty pear trees reacted positively in a nested PCR assay. Restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes MseI and AluI of the PCR fragment amplified with the primer pair fU5/rU3 revealed patterns identical to those from the sweet potato little leaf phytoplasma. This is the first report of a phytoplasma in pear in Australia.

scholarly journals Rhizoctonia spp. Recovered from Strawberry Roots in Central Coastal California

2000 ◽  
Vol 90 (4) ◽  
pp. 345-353 ◽  
Author(s):  
Frank N. Martin
Keyword(s):  
Polymerase Chain Reaction ◽  
Fragment Length Polymorphism ◽  
Coastal Region ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Molecular Techniques ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Collection Sites ◽  
Restriction Fragment Length

Rhizoctonia spp. were commonly recovered from the roots of strawberry plants growing in nonfumigated soil in the central coastal region of California. With the exception of one multinucleate isolate of R. solani (frequency of recovery of 0.8%), all other isolates were binucleate and were in anastomosis groups (AG) A, G, or I. AGs-A and -I were recovered from all five collection sites, whereas AG-G was recovered from only two sites. AG-A was the most commonly isolated AG, followed by AGs-I and -G. Similar levels of virulence were observed among the different AGs, but differences in virulence were observed among isolates in the same AG. Evaluating anastomosis grouping by pairing isolates recovered from strawberry with known tester isolates did not always yield a positive anastomosis reaction, even though both isolates anastomosed with other members of the same AG. Subsequent investigations with multiple isolates in the same AG from the same collection location confirmed that there was a lack of anastomosis or weak anastomosis reactions for some combinations of pairings, highlighting the need for to use multiple tester isolates or molecular techniques for AG determination. Restriction fragment length polymorphism (RFLP) analysis of a polymerase chain reaction-amplified region of the rDNA was effective for differentiating AGs. Sixteen RFLP groups were observed after cluster analysis with data for the size of the amplified products and fragment sizes after digestion with four restriction enzymes. Although each AG had isolates in multiple RFLP groups, any one individual RFLP group contained isolates of only a single AG. There was no consistent correlation between RFLP group and location of isolate collection.

The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia

Parasitology ◽  
1998 ◽  
Vol 117 (1) ◽  
pp. 1-13 ◽  
Author(s):  
K. VICTOIR ◽  
A. L. BAÑULS ◽  
J. AREVALO ◽  
A. LLANOS-CUENTAS ◽  
R. HAMERS ◽  
...  
Keyword(s):  
Gene Locus ◽  
Genetic Characterization ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Natural Isolates ◽  
Phenotype Diversity

In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63–RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR–RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni: reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63–RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63–RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63–RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR–RFLP). PCR–RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63–RFLP and PCR–RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.

scholarly journals Differences in Virulence and Genomic Features of Strains of ‘Candidatus Phytoplasma mali’, the Apple Proliferation Agent

2007 ◽  
Vol 97 (8) ◽  
pp. 964-970 ◽  
Author(s):  
Erich Seemüller ◽  
Bernd Schneider
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Apple Proliferation ◽  
Candidatus Phytoplasma Mali ◽  
Polymerase Chain

Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with ‘Candidatus Phytoplasma mali’ were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52°C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in ‘Ca. P. mali’. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.

scholarly journals The Application of Polymerase Chain Reaction – Restriction Fragment Polymorphisms (PCR-RFLP) to Determine Genetic Diversity of Madura Cattle in Sapudi Island

2015 ◽  
Vol 18 (1) ◽  
pp. 70
Author(s):  
Tety Hartatik ◽  
Slamet Diah Volkandari ◽  
S. Sumadi ◽  
W. Widodo
Keyword(s):  
Genetic Diversity ◽  
Polymerase Chain Reaction ◽  
Restriction Fragment ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Cytb Gene ◽  
Chain Reaction ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Haplotype A

The aim of this study was to determine genetic diversity of Madura cattle using Polymerase Chain Reaction – Restriction Fragment Length Polymorphisms (PCR-RFLP) analysis of the cytochrome b (cytb) gene. Samples used for the experiments were blood of 43 cattle that consist of 15 cattle obtained from Madura Island, 23 cattle from Sapudi Island, and 5 Limousin-Madura (Limura) cattle. A fragment of 464 base pair of cytb gene was amplifi ed by forward primer L14735 and reverse primer H15149. The PCR product was digested with TaqIand HinfI restriction enzymes to identify genetic patterns. Data of PCR-RFLP showed two haplotypes, that were A and B, in cattle obtained from both Madura Island and Sapudi Island. The frequencies of haplotype A and B of cattle from Sapudi Island were 69.57% and 30.47%, respectively. More diverse frequencies were observed in cattle obtained from Madura Island, where haplotype A and B were 86.67% and 13.33%, respectively. In this experiment, Limura cattle had only haplotype A. As a conclusion, PCR-RFLP of the cytb gene had been able to determine a genetic diversity of Madura cattle. Key words: Genetic diversity, Madura cattle, haplotype.

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