scholarly journals Using PCR to Distinguish Diaporthe phaseolorum and Phomopsis longicolla from Other Soybean Fungal Pathogens and to Detect Them in Soybean Tissues

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1143-1149 ◽  
Author(s):  
A. W. Zhang ◽  
G. L. Hartman ◽  
L. Riccioni ◽  
W. D. Chen ◽  
R. Z. Ma ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Nuclear Ribosomal Dna ◽  
Specific Primers ◽  
Phomopsis Longicolla ◽  
Tissue Samples ◽  
Diaporthe Phaseolorum ◽  
Pcr Products ◽  
Fungal Dna ◽  
Dna Fragment ◽  
Polymerase Chain

Restriction fragment length polymorphism analyses of polymerase chain reaction (PCR) amplified DNA were used to distinguish Diaporthe phaseolorum and Phomopsis longicolla isolates from other soybean fungal pathogens. Primers made to the conserved sequences of nuclear ribosomal DNA amplified the internal transcribed spacer (ITS) regions of D. phaseolorum var. meridionalis and P. longicolla. The PCR products were cloned and then sequenced. Specific-primers, Phom.I and Phom.II, were designed from the polymorphic regions of D. phaseolorum and P. longicolla isolates from soybean to distinguish them from other soybean fungal pathogens. These ITS-derived primers amplified a 337-bp-specific DNA fragment from P. longicolla, D. phaseolorum var. meridionalis, D. phaseolorum var. caulivora, D. phaseolorum var. sojae, and Phomopsis spp. from 20 different hosts. No amplified product was observed using DNA of seven other soybean fungal pathogens or soybean DNA. The detection limit of PCR using primers Phom.I and Phom.II was 2.5 × 10-7 dilution of fungal DNA extracted from samples of 10 pooled seeds and as low as a 1:15 (Phomopsis:soybean) ratio when using 10 ng of DNA per μl from each P. longicolla and soybean. PCR did not produce products using primers Phom.I and Phom.II with DNA extracted from noninfected seeds, but specific bands were observed from samples of 10 pooled seeds and from individually infected seeds. A specific band was observed as well from DNA extracts of tissue samples from symptomless plants inoculated with P. longicolla and D. phaseolorum var. sojae.

Variation in bacterial endosymbionts associated with the date palm hopper,Ommatissus lybicuspopulations

2017 ◽  
Vol 108 (2) ◽  
pp. 271-281 ◽  
Author(s):  
S. Karimi ◽  
H. Izadi ◽  
M. Askari Seyahooei ◽  
A. Bagheri ◽  
P. Khodaygan
Keyword(s):  
Date Palm ◽  
Multiple Infections ◽  
Infection Frequency ◽  
Pcr Assay ◽  
Specific Primers ◽  
Consensus Sequences ◽  
Pcr Products ◽  
Bacterial Endosymbionts ◽  
Different Populations ◽  
Polymerase Chain

AbstractThe date palm hopper,Ommatissus lybicus, is a key pest of the date palm, which is expected to be comprised of many allopatric populations. The current study was carried out to determine bacterial endosymbiont diversity in the different populations of this pest. Ten date palm hopper populations were collected from the main date palm growing regions in Iran and an additional four samples from Pakistan, Oman, Egypt and Tunisia for detection of primary and secondary endosymbionts using polymerase chain reaction (PCR) assay with their specific primers. The PCR products were directly sequenced and edited using SeqMan software. The consensus sequences were subjected to a BLAST similarity search. The results revealed the presence of ‘CandidatusSulcia muelleri’ (primary endosymbiont) andWolbachia,ArsenophonusandEnterobacter(secondary endosymbionts) in all populations. This assay failed to detect ‘CandidatusNasuia deltocephalinicola’ andSerratiain these populations. ‘Ca. S. muelleri’ exhibited a 100% infection frequency in populations andWolbachia,ArsenophonusandEnterobacterdemonstrated 100, 93.04 and 97.39% infection frequencies, respectively. The infection rate ofArsenophonusandEnterobacterranged from 75 to 100% and 62.5 to 100%, respectively, in different populations of the insect. The results demonstrated multiple infections by ‘Ca. Sulcia muelleri’,Wolbachia,ArsenophonusandEnterobacterin the populations and may suggest significant roles for these endosymbionts on date palm hopper population fitness. This study provides an insight to endosymbiont variation in the date palm hopper populations; however, further investigation is needed to examine how these endosymbionts may affect host fitness.

scholarly journals Analysis of Genetic and Molecular Identity Among Field Isolates of the Rice Blast Fungus with an International Differential System, Rep-PCR, and DNA Sequencing

Plant Disease ◽  
2013 ◽  
Vol 97 (4) ◽  
pp. 491-495 ◽  
Author(s):  
Junjie Xing ◽  
Yulin Jia ◽  
James C. Correll ◽  
Fleet N. Lee ◽  
Richard Cartwright ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Dna Sequencing ◽  
Differential System ◽  
Repetitive Element ◽  
Chain Reaction ◽  
Specific Primers ◽  
Molecular Identity ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Avr Gene

The Pi-ta gene deployed in southern U.S. rice germplasm is effective in preventing the infection by strains of Magnaporthe oryzae isolates that carry the avirulence (AVR) gene AVR-Pita1. In the present study, 169 isolates from rice (Oryza sativa) cultivars, with and without Pi-ta, were analyzed for their genetic identity using an international differential system, repetitive element-based polymerase chain reaction (Rep-PCR), and sequence analysis of PCR products of AVR-Pita1. These isolates belong to the races IA1, IB1, IB17, IC1, and IC17 of M. oryzae. These isolates were further classified into 15 distinct groups by Rep-PCR. There was a predominant group within each race. Pathogenicity assays on ‘Katy’ (Pi-ta) and ‘M202’ (pi-ta) rice determined that IC1 was virulent to Katy and M202; IB17, IC17, and most of IA1 and IB1 were avirulent to Katy and virulent to M202, suggesting that the Pi-ta gene in Katy is responsible for preventing infection by these isolates. Consistently, AVR-Pita1 was not amplified from 28 virulent isolates. One AVR-Pita1 allele was amplified by AVR-Pita1-specific primers in 78 avirulent isolates. Interestingly, different AVR-Pita1 alleles were found in each of the 12 avirulent isolates, as determined by DNA sequencing. Sequence analysis of 90 PCR products revealed 10 AVR-Pita1 haplotypes, 4 of which were new. In total, 12 amino acid changes were identified in the new variants when compared with the first described AVR-Pita sequence (AF207841). The finding of isolates with altered AVR-Pita1 from rice cultivars with and without Pi-ta suggests that these virulent isolates were adapted to the field environments in the southern United States. Further research will be needed to verify this prediction.

scholarly journals Development of Sequence-specific Primers that Amplify a 1.5-kb DNA Marker for Race 1 Fusarium Wilt Resistance in Cucumis melo L.

HortScience ◽  
1998 ◽  
Vol 33 (2) ◽  
pp. 291-292 ◽  
Author(s):  
W. Patrick Wechter ◽  
Ralph A. Dean ◽  
Claude E. Thomas
Keyword(s):  
Fusarium Wilt ◽  
Cucumis Melo ◽  
Specific Primers ◽  
Breeding Programs ◽  
Breeding Lines ◽  
Race 1 ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Cucumis Melo L

Two 24-mer primers, MUSKFOM I and MUSKFOM II, were developed that amplify a 1.5-kb DNA fragment in race 1 Fusarium wilt resistant muskmelon (Cucumis melo L.), but not in race 1 susceptible germplasm tested. Three race 1 resistant cultivars and two race 1 resistant breeding lines as well as eight race 1 susceptible lines were analyzed using the two sequence-specific primers in the polymerase chain reaction. These primers should prove valuable for nondestructive determination of Fom 2 gene introgression in breeding programs.

The occurrence of the filarial nematode Dirofilaria repens in canine hosts from Maio Island, Cape Verde

2016 ◽  
Vol 91 (1) ◽  
pp. 87-90 ◽  
Author(s):  
R. Marcos ◽  
C. Pereira ◽  
J.P. Maia ◽  
M. Santos ◽  
C. Luzzago ◽  
...  
Keyword(s):  
Dirofilaria Immitis ◽  
Parasite Species ◽  
Cape Verde ◽  
Control Measures ◽  
Chain Reaction ◽  
Species Identity ◽  
Specific Primers ◽  
Pcr Products ◽  
Males And Females ◽  
Polymerase Chain

AbstractThe prevalence of canine Dirofilaria infection in Maio Island (Cape Verde) was analysed by serology, morphological and molecular identification of the parasite species. Blood and sera were collected from 150 dogs and 80 cats aged over 6 months from various localities of the island. DNA was extracted from blood and samples were screened by polymerase chain reaction (PCR) using microfilaria-specific primers. No Dirofilaria immitis was found in dogs while D. repens microfilariae were found in 5.3% of dogs and 6% were positive by PCR. The species identity was confirmed by sequencing of PCR products, which showed almost 100% homology with D. repens European sequences published in GenBank. No difference in Dirofilaria infection was observed between males and females or in dogs with different weights. However, older dogs and those from the western part of Maio Island were more frequently infected. No Dirofilaria was found in cats. This study represents the first evidence of D. repens in Cape Verde (West Africa) and highlights the need for implementing control measures and for a better surveillance of dirofilariosis in Africa.

scholarly journals Karakterisasi Molekuler Papaya ringspot virus tipe P pada Tanaman Mentimun di Jawa

2018 ◽  
Vol 14 (3) ◽  
pp. 75
Author(s):  
Listihani Listihani ◽  
Tri Asmira Damayanti ◽  
Sri Hendrastuti Hidayat ◽  
Suryo Wiyono
Keyword(s):  
Ringspot Virus ◽  
Mosaic Symptom ◽  
Papaya Ringspot Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Specific Primers ◽  
Central Java ◽  
Dna Fragment ◽  
Polymerase Chain

Moleculer Characterization of Papaya ringspot virus type P on Cucumber in JavaInfection of Papaya ringspot virus (PRSV) on cucumber plants showing mosaic symptom was detected using specific antibody.  Further investigation was conducted to determine molecular characters and status of PRSV infecting cucumber in Java.  Infection of PRSV was detected from leaf samples collected from the field using dot immunobinding assay (DIBA).  Disease frequency caused by PRSV infection reached 81.11%, 95.86%, 91.66%, and 92.3% in East Java, Central Java, Yogyakarta, and West Java, respectively.  Characterization of PRSV isolates was conducted by reverse transcription polymerase chain reaction (RT-PCR) using specific primers for PRSV-P and PRSV-W, followed by cloning, and DNA sequencing.  DNA fragment of 470 bp was successfully amplified using specific primers for PRSV-P from several samples from Nganjuk, Brebes, Kulon Progo, and Subang; but no amplification was achieved using specific primers for PRSV-W.  Nucleotide and amino acid analysis showed high homology among PRSV-P isolates from Nganjuk, Brebes, Kulon Progo, and Subang, i.e. 98.6%-99.7% and 99.3%-100%, respectively.  This is an indication of a low genetic variation among PRSV-P from Java. Further phylogenetic analysis indicated that PRSV-P isolate cucumber is in the same cluster with PRSV-P isolate papaya from Bali, Indonesia.  This is the first report of PRSV-P infecting cucumber in Indonesia.

scholarly journals Development of Nested Polymerase Chain Reaction Detection of Mycosphaerella spp. and Its Application to the Study of Leaf Disease in Eucalyptus Plantations

2007 ◽  
Vol 97 (2) ◽  
pp. 132-144 ◽  
Author(s):  
M. Glen ◽  
A. H. Smith ◽  
S. R. H. Langrell ◽  
C. L. Mohammed
Keyword(s):  
Polymerase Chain Reaction ◽  
Sequence Similarity ◽  
Nuclear Ribosomal Dna ◽  
Chain Reaction ◽  
Specific Primers ◽  
Nested Polymerase Chain Reaction ◽  
Double Blind ◽  
Leaf Disease ◽  
Eucalyptus Plantations ◽  
Polymerase Chain

Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylo-genetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambi phylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm2 or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.

Detection ofLawsonia intracellularisin faeces of swine from the main producing regions in Brazil

1999 ◽  
Vol 45 (3) ◽  
pp. 230-234 ◽  
Author(s):  
Adriana E.C. Nascimento Chiriboga ◽  
Walter V Guimarães ◽  
Maria Cristina D. Vanetti ◽  
Elza F Araújo
Keyword(s):  
Polymerase Chain Reaction Method ◽  
Lawsonia Intracellularis ◽  
Specific Primers ◽  
Poor Growth ◽  
Mato Grosso ◽  
Proliferative Enteropathy ◽  
Total Incidence ◽  
Faecal Samples ◽  
Dna Fragment ◽  
Polymerase Chain

Swine proliferative enteropathy is an enteric disease caused by Lawsonia intracellularis which affects animals between 6 and 20 weeks of age, causing diarrhea, anorexia, and poor growth. The presence of L. intracellularis was evaluated in the faecal samples of 636 swine from 75 randomly chosen herds in the main swine-producing regions of Brazil. The pathogen was detected by the polymerase chain reaction method (PCR) using L. intracellularis specific primers. A 319-bp DNA fragment specific for L. intracellularis was produced on amplification of DNA from the faeces of pigs with proliferative enteropathy. Equal amounts of DNA extracted from the faeces of animals from the same herd were pooled together and, once L. intracellularis was detected, the faecal material of each animal was analyzed separately. The incidence of L. intracellularis was 33.4% in the state of Santa Catarina, 29.4% in Paraná, 26.3% in Minas Gerais, 16.7% in Mato Grosso, and 7.1% in São Paulo. The presence of the pathogenic agent was detected in samples from 15 farms, representing a total incidence of 20%. Although 46 animals (7.2%) were shown to be infected, 11% did not present any symptoms of swine proliferative enteropathy. The use of PCR allowed the detection of L. intracellularis in swine farms and the evaluation of the incidence of proliferative enteropathy in different regions of Brazil.Key words: proliferative enteropathy, diagnosis, Lawsonia intracellularis, PCR, incidence.

scholarly journals Detection of Gaeumannomyces graminis Varieties Using Polymerase Chain Reaction with Variety-Specific Primers

Plant Disease ◽  
2000 ◽  
Vol 84 (9) ◽  
pp. 947-951 ◽  
Author(s):  
H. M. Fouly ◽  
H. T. Wilkinson
Keyword(s):  
Polymerase Chain Reaction ◽  
Fungal Pathogens ◽  
Gaeumannomyces Graminis ◽  
Universal Primers ◽  
Middle Region ◽  
Chain Reaction ◽  
Specific Primers ◽  
Grass Roots ◽  
Polymerase Chain ◽  
Take All

The polymerase chain reaction (PCR) was used for detection of Gaeumannomyces graminis, the causal agent of take-all disease in wheat, oats, and turfgrass. NS5 and NS6 universal primers amplified the middle region of 18S ribosomal DNA of Gaeumannomyces species and varieties. Primers GGT-RP (5′ TGCAATGGCTTCGTGAA 3′) and GGA-RP (5′ TTTGTGTGTGAC CATAC 3′) were developed by sequence analysis of cloned NS5-NS6 fragments. The primer pair NS5:GGT-RP amplified a single 410-bp fragment from isolates of G. graminis var. tritici, a single 300-bp fragment from isolates of G. graminis var. avenae, and no amplification products from isolates of G. graminis var. graminis or other species of Gaeumannomyces. The primer pair NS5:GGA-RP amplified a single 400-bp fragment from isolates of varieties tritici and avenae. Two sets of primer pairs (NS5:GGT-RP and NS5:GGA-RP) were used in PCR reactions to detect and identify the varieties tritici and avenae either colonizing wheat, oats, or grass roots, or in culture. No amplification products were observed using DNA extracted from plants infected with eight other soilborne fungal pathogens or from uninoculated plants.

Sequencing Herbarium Specimens of a Common Detrimental Plant Disease (Powdery Mildew)

2020 ◽  
Vol 110 (7) ◽  
pp. 1248-1254 ◽  
Author(s):  
Michael Bradshaw ◽  
Patrick C. Tobin
Keyword(s):  
Powdery Mildew ◽  
Fungal Pathogens ◽  
Plant Disease ◽  
Large Subunit ◽  
Genetic Identification ◽  
Herbarium Specimens ◽  
Powdery Mildews ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Genomic Regions

Powdery mildew (Erysiphaceae) is a detrimental plant disease that occurs on a variety of economically important crops. Powdery mildew consists of over 873 species of fungal pathogens that affect over 10,000 plant species. Genetic identification of powdery mildew is accomplished using the internal transcribed spacer (ITS) and large subunit (LSU) regions of the nuclear ribosomal RNA gene cluster. The ITS and LSU regions of powdery mildews can be useful in ecological, epidemiological, phylogenetic, and taxonomic investigations. However, sequencing these regions is not without its challenges. For example, powdery mildew sequences are often contaminated with plant and/or fungal DNA. Also, there tends to be a limited amount and older specimens’ DNA can fragment over time. The success of sequencing powdery mildew often depends on the primers used for running polymerase chain reaction (PCR). The primers need to be broad enough that they match the majority of powdery mildew DNA yet specific enough that they do not align with other organisms. A review of the taxonomy and phylogeny of the powdery mildews is presented with an emphasis on sequencing the ITS + LSU genomic regions. Additionally, we introduce a new nested primer protocol for sequencing powdery mildew herbarium samples that includes six new powdery mildew-specific primers. The new sequencing protocol presented allows specimens up to 130 years old to be sequenced consistently. Sequencing herbarium specimens can be extremely useful for addressing many ecological, epidemiological, phylogenetic, and taxonomic problems in multiple plant pathogenic systems including the powdery mildews.

scholarly journals Survey of mutations in prolificacy genes in Santa Ines and Morada Nova sheep

2017 ◽  
Vol 69 (4) ◽  
pp. 1047-1053
Author(s):  
G.M.L. Holanda ◽  
J.C. Oliveira ◽  
D.M.F. Silva ◽  
S.S.N. Rocha ◽  
V. Pandolfi ◽  
...  
Keyword(s):  
Restriction Site ◽  
Fragment Length Polymorphism ◽  
Nucleotide Sequencing ◽  
Chain Reaction ◽  
Specific Primers ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Santa Inês ◽  
Restriction Fragment Length

ABSTRACT Polymorphisms in the BMP-15 gene related to Galway (FecXG) and Inverdale (FecXI) and in the BMPR-1B gene known as Booroola (FecB) mutations were investigated using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, on sheep from the breeds Santa Inês (n= 574) and Morada Nova (n=282). DNA was extracted and amplified through PCR with specific primers that introduced a restriction site in association with the mutation. The PCR products were submitted to endonucleases. The experiment found no FecXG and FecXI mutations. Six samples of animals with multiple offspring/birth history presented polymorphism for FecB similar to control samples, but this pattern was not confirmed by nucleotide sequencing. Although the absence of these mutations in the studied breeds, other factors related to prolificacy should be investigated to explain the inherent prolificity mechanisms.

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