Molecular Characterization of Fusarium oxysporum Causing Chickpea Wilt by Internal Transcribed Spacer (ITS) Marker

2019 ◽  
Author(s):  
Bharati Jadhav ◽  
Sarika R Bhalerao ◽  
Priya George
Keyword(s):  
Genetic Distance ◽  
Fusarium Oxysporum ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Chickpea Wilt ◽  
Chickpea Plant ◽  
Minimum Genetic Distance ◽  
Serious Disease

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris is one of the serious disease causes tremendous loss to crop throughout the world and has assumed serious proportions in the recent years. Wilt affected chickpea plant roots samples were collected from forty eight different regions of Maharashtra. The collected samples were characterized by designed ITS primers. The Fusarium oxysporum f. sp. ciceris (Foc) showed about 302 bp amplicon when subjected to PCR with these primers. Further the amplified product was digested with five restriction enzymes viz. AluI, EcoRI, HaeIII, MboI and MspI and restriction fragment length polymorphism (RFLP) pattern were analysed. A dendrogram derived from ITS-RFLP analysis of the rDNA region divided the Foc isolates into four clusters. The maximum genetic distance 0.75 exhibited by five Foc isolates viz. Foc-30, Foc-36, Foc-24, Foc-25 and Foc-48 belonging to Shahartakli, Shelur, Nashik, Satara, Kolhapur regions respectively and found to be more diverse among the other Foc isolates. Whereas isolate no. Foc-20, Foc-21 and Foc-8 from Ambajogai, Karjat and Rahata shown minimum genetic distance (0.13). This showed the genetic variability among the Foc isolates collected from different regions of Maharashtra.

scholarly journals Characterization of Ascaris from Ecuador and Zanzibar

2014 ◽  
Vol 89 (4) ◽  
pp. 512-515 ◽  
Author(s):  
A.M. Sparks ◽  
M. Betson ◽  
G. Oviedo ◽  
C. Sandoval ◽  
P.J. Cooper ◽  
...  
Keyword(s):  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Its Region ◽  
Restriction Enzymes ◽  
Zoonotic Transmission ◽  
Pcr Rflp ◽  
Double Digestion ◽  
Polymerase Chain ◽  
Shed Light

AbstractTo shed light on the epidemiology of ascariasis in Ecuador and Zanzibar, 177 adult worms retrieved by chemo-expulsion from either people or pigs were collected, measured and subjected to polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of the ribosomal internal transcribed spacer (ITS) region. Upon double digestion with RsaI and HaeIII, PCR-RFLP analysis revealed the presence of A. lumbricoides in people and A. suum in pigs in Ecuador. In contrast, while there are no pigs on Zanzibar, of the 56 worms obtained from people, one was genotyped as A. suum. No additional genetic variation was detected upon further PCR-RFLP analysis with several other restriction enzymes. Upon measurement, worm mass and length differed by location and by species, A. suum being lighter and longer. While there is no evidence to suggest zoonotic transmission in Ecuador, an enduring historical signature of previous zoonotic transmission remains on Zanzibar.

scholarly journals Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 196 ◽  
Author(s):  
García-Suárez ◽  
González-Rodríguez ◽  
Cima-Cabal ◽  
Yuste ◽  
Vazquez ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Dna Sequences ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

scholarly journals Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

2003 ◽  
Vol 69 (5) ◽  
pp. 2555-2562 ◽  
Author(s):  
Markus Egert ◽  
Michael W. Friedrich
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragments ◽  
Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

scholarly journals Detection and characterization of Leptospira spp. isolated from aborted bovine clinical samples

2011 ◽  
Vol 81 (1) ◽  
pp. 21-25 ◽  
Author(s):  
Hassan Momtaz ◽  
Saadat Moshkelani
Keyword(s):  
Public Health Problem ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Clinical Samples ◽  
Dairy Herds ◽  
Important Public Health Problem ◽  
Pcr Rflp ◽  
Beef Herds ◽  
Bovine Abortion

Leptospira is recognized as an important public health problem worldwide, especially in tropical countries, and is a common cause of abortion in dairy and beef herds. The aim of the present study was to detect and characterize Leptospira as the causative agent of abortion in cattle using a PCR-RFLP in Chaharmahal va Bakhtiari and Isfahan provinces, Iran. A total of 220 bovine aborted foetuses and 120 vaginal discharges from an aborted calf were collected from 64 commercial dairy herds. After isolation of 60 Leptospira spp. from samples, RFLP analysis was carried out with HindIII and HaeIII restriction enzymes in reference strains and isolated for characterization. In a total of 340 specimens, 46 (20.9%) and 14 (11.66%) were identified positive for Leptospira spp. from aborted bovine foetuses and vaginal discharges, respectively. The present results also suggest that L. interrogans serovar hardjo has the highest prevalence in the region under study and L. hardjo is a major pathogen causing bovine abortion in Chaharmahal va Bakhtiari and Isfahan provinces of Iran.

scholarly journals Molecular characterization of papaya genotypes using AFLP markers

2011 ◽  
Vol 33 (3) ◽  
pp. 849-858 ◽  
Author(s):  
Eder Jorge de Oliveira ◽  
Juliana Leles Costa ◽  
Lucas Ferraz dos Santos ◽  
Fabiana Moraes de Carvalho ◽  
Aline dos Santos Silva ◽  
...  
Keyword(s):  
Genetic Variability ◽  
Genetic Distance ◽  
Carica Papaya ◽  
Fragment Length Polymorphism ◽  
Distance Matrix ◽  
Inbred Lines ◽  
Genetic Distance Matrix ◽  
Pair Method ◽  
Polymorphic Bands

Due to the low genetic variability reported in the commercial plantations of papaya (Carica papaya L.), the objective of this study was analyze the genetic diversity of 32 genotypes including cultivars, landraces, inbred lines, and improved germplasm using the AFLP technique (Amplified Fragment Length Polymorphism). The genetic distance matrix was obtained using the Nei and Li genetic distance and clustering was performed using the unweighted pair-method with arithmetic mean (UPGMA). Using 11 combinations of EcoRI/MseI primers, 383 polymorphic bands were obtained. On average, 34.8 polymorphic bands were obtained per primer combination. Five clusters were formed. The traditional cultivar 'Sunrise' and the inbred line CMF-L30-08 were the closest genotypes, and the improved germplasm (CMF041) and landrace (CMF233) the most distant. The main papaya cultivars commercially grown in Brazil, as well as four inbred lines and three improved germplasm, were clustered together, however, were not grouped in the same branch. The genetic distance between the Sunrise and Golden cultivars was 0.329, and even arising from mutation and selection within the Sunrise variety, the Golden stores considerable genetic variability. Additional variability was observed in the inbred lines derived from papaya breeding program at Embrapa Cassava and Fruits.

scholarly journals Characterization of the Archaeal Community in a Minerotrophic Fen and Terminal Restriction Fragment Length Polymorphism-Directed Isolation of a Novel Hydrogenotrophic Methanogen

2008 ◽  
Vol 74 (7) ◽  
pp. 2059-2068 ◽  
Author(s):  
Hinsby Cadillo-Quiroz ◽  
Erica Yashiro ◽  
Joseph B. Yavitt ◽  
Stephen H. Zinder
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
New York State ◽  
Rflp Analysis ◽  
Archaeal Community ◽  
Restriction Fragment Length

ABSTRACT Minerotrophic fen peatlands are widely distributed in northern latitudes and, because of their rapid turnover of organic matter, are potentially larger sources of atmospheric methane than bog peatlands per unit area. However, studies of the archaeal community composition in fens are scarce particularly in minerotrophic sites. Several 16S rRNA-based primer sets were used to obtain a broad characterization of the archaeal community in a minerotrophic fen in central New York State. A wide archaeal diversity was observed in the site: 11 euryarchaeal and 2 crenarchaeal groups, most of which were uncultured. The E1 group, a novel cluster in the order Methanomicrobiales, and Methanosaetaceae were the codominant groups in all libraries and results of terminal restriction fragment length polymorphism (T-RFLP) analysis. Given its abundance and potential hydrogenotrophic methane contribution, the E1 group was targeted for culture attempts with a low-ionic-strength medium (PM1). Initial attempts yielded Methanospirillum-dominated cultures. However, by incorporating a T-RFLP analysis as a quick selection tool for treatments and replicates, we were able to select an enrichment dominated by E1. Further dilutions to 10−9 and tracking with T-RFLP yielded a strain named E1-9c. E1-9c is a novel coccoid hydrogenotrophic, mesophilic, slightly acidophilic methanogen and is highly sensitive to Na2S concentrations (requires <0.12 mM for growth). We propose E1-9c as the first representative of a novel genus in the Methanomicrobiales order.

scholarly journals Characterization of Phytoplasmas Related to Aster Yellows Group Infecting Annual Plants in Iran, Based on the Studies of 16S rRNA and RP Genes

2014 ◽  
Vol 54 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Fereshteh Vali Sichani ◽  
Masoud Bahar ◽  
Leila Zirak
Keyword(s):  
16S Rrna ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Annual Plants ◽  
Universal Primer ◽  
Aster Yellows ◽  
Central Regions ◽  
Group Specific Primer

Abstract Several annual field crops, vegetables, ornamentals, oilseed crops, and weeds showing phytoplasma diseases symptoms were collected to detect phytoplasmas related to ‘Candidatus Phytoplasma asteris’. The collecting was done in the central regions of Iran. For general detection of phytoplasmas, 16S rRNA gene fragments were amplified using phytoplasma universal primer pair P1/P7 in polymerase chain reaction (PCR) followed by primer pair R16F2n/R16R2 in nested PCR. Then, for finer detection of phytoplasmas related to ‘Ca. P. asteris’, DNA samples were used to extend the rp and tuf gene fragments by PCR using aster yellows group specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy, respectively. Restriction fragment lenght polymorphism (RFLP) analysis of rp gene fragments using digestion with AluI, MseI, and Tsp509I restriction enzymes indicated that aster yellows group related phytoplasmas in these Iranian regions, belong to rpI-B subgroups. Sequence analysis of partial 16S rRNA and rp genes from representative phytoplasma isolates confirmed the RFLP results. This research is the first report of annual plants infected with phytoplasmas related to subgroup rpI-B in Iran.

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.

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