scholarly journals Development and Evaluation of PCR-Based Diagnostic Assays for the Bacterial Speck and Bacterial Spot Pathogens of Tomato

Plant Disease ◽  
2006 ◽  
Vol 90 (4) ◽  
pp. 451-458 ◽  
Author(s):  
Diane A. Cuppels ◽  
Frank J. Louws ◽  
Teresa Ainsworth
Keyword(s):  
Pseudomonas Syringae ◽  
Disease Diagnosis ◽  
Bacterial Spot ◽  
Pcr Assay ◽  
Diagnostic Assays ◽  
Bacterial Speck ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Group D

Bacterial speck and bacterial spot lesions can easily be confused with each other and with those formed by other tomato pathogens. To facilitate disease diagnosis, we developed and evaluated polymerase chain reaction (PCR)-based lesion assays using crude DNA extracts and primer sets COR1/2 (bacterial speck) and BSX1/2 (bacterial spot). All 29 pathogenic Pseudomonas syringae pv. tomato strains tested produced a 689-bp amplicon with COR1/2; 28 of the 37 geographically diverse bacterial spot-causing xanthomonad (BSX) strains that were tested generated the 579-bp BSX1/2 amplicon. The detection limit with plant samples was 30 to 50 CFU/reaction. In a 6-year study, the COR1/2 PCR assay diverged from the culture-based classical assay for only 3 of 70 bacterial speck lesion samples collected from Ontario greenhouses and tomato fields; the BSX1/2 assay was positive for 112 of the 124 confirmed bacterial spot lesions sampled. The majority (66%) of the BSX strains isolated from these lesions belonged to group D; the 12 strains that were BSX1/2-negative belonged to group C. Group D strains produced a 425-bp PCR product with crude DNA extracts but a 579-bp product with purified DNA; the former was identical to the latter except that it was missing 150 bp from the middle of the 579-bp sequence.

scholarly journals Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

1997 ◽  
Vol 87 (12) ◽  
pp. 1192-1196 ◽  
Author(s):  
M. Sato ◽  
K. Watanabe ◽  
M. Yazawa ◽  
Y. Takikawa ◽  
K. Nishiyama
Keyword(s):  
Pseudomonas Syringae ◽  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Enzyme Gene ◽  
Indigenous Plasmid ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Primer Sets

Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the “kudzu strain”) and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.

scholarly journals Field Control of Bacterial Spot and Bacterial Speck of Tomato Using a Plant Activator

Plant Disease ◽  
2001 ◽  
Vol 85 (5) ◽  
pp. 481-488 ◽  
Author(s):  
F. J. Louws ◽  
M. Wilson ◽  
H. L. Campbell ◽  
D. A. Cuppels ◽  
J. B. Jones ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Field Experiments ◽  
Production Systems ◽  
Acquired Resistance ◽  
Dry Weight ◽  
Bacterial Spot ◽  
Population Densities ◽  
Bacterial Speck ◽  
Plant Activator

Acibenzolar-S-methyl (CGA 245704 or Actigard 50WG) is a plant activator that induces systemic acquired resistance (SAR) in many different crops to a number of pathogens. Acibenzolar-S-methyl was evaluated for management of bacterial spot (Xanthomonas axonopodis pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato) of tomato in 15 and 7 field experiments, respectively. Experiments were conducted over a 4-year period in Florida, Alabama, North Carolina, Ohio, and Ontario using local production systems. Applied at 35 g a.i. ha-1, acibenzolar-S-methyl reduced foliar disease severity in 14 of the 15 bacterial spot and all 7 bacterial speck experiments. Disease control was similar or superior to that obtained using a standard copper bactericide program. Acibenzolar-S-methyl also reduced bacterial fruit spot and speck incidence. Tomato yield was not affected by using the plant activator in the field when complemented with fungicides to manage foliar fungal diseases, but tomato transplant dry weight was negatively impacted. X. axonopodis pv. vesicatoria population densities on greenhouse-grown tomato transplants were reduced by acibenzolar-S-methyl treatment. Bacterial speck and spot population densities on leaves of field-grown plants were not dramatically affected. Acibenzolar-S-methyl can be integrated as a viable alternative to copper-based bactericides for field management of bacterial spot and speck, particularly where copper-resistant populations predominate.

Polymerase Chain Reaction Assay for Detection of Escherichia coli O157:H7 and Escherichia coli O157:H−

2002 ◽  
Vol 65 (1) ◽  
pp. 5-11 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
NATSUKO ICHIOKA ◽  
CHIE SASAKI ◽  
HIROSHI KOBAYASHI ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Pcr Assay ◽  
E Coli ◽  
Pcr Product ◽  
Genomic Dnas ◽  
Polymerase Chain

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non–E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H− and E. coli O157:H7. Since the DNA sequence from base 15 to base 1008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H− and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 × 100 to 3.5 × 102 CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42°C for 18 h and for the conventional cultural method.

scholarly journals Specific and Sensitive Detection of Enterobacter mori Using Reliable RT-PCR

Plant Disease ◽  
2011 ◽  
Vol 95 (9) ◽  
pp. 1070-1074 ◽  
Author(s):  
M. M. Lou ◽  
G. L. Jin ◽  
W. X. Tian ◽  
G. Q. Zhang ◽  
X. Y. Fan ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Infected Plant ◽  
Rpob Gene ◽  
Effective Control ◽  
Bacterial Cells ◽  
Pcr Assay ◽  
Strong Positive Reaction ◽  
Polymerase Chain ◽  
Serious Disease ◽  
Type Strains

Enterobacter mori, the causal agent of bacterial wilt in mulberry, is becoming a serious disease in mulberry orchards in China. Because no effective control strategy has been devised for this disease, the reliable screening of mulberry material for latent infection became necessary. Hence, a fast polymerase chain reaction (PCR) assay for the detection of E. mori was developed in this study. The primers were designed within regions of the RNA polymerase β-subunit (rpoB) gene. The method is fast and simple and showed 100% sensitivity (no false negatives) and 100% specificity (no false positives), which was tested with 4 representative E. mori strains, 9 Enterobacter type strains, 2 strains of the other major mulberry bacterial pathogens (Ralstonia solanacearum and Pseudomonas syringae pv. mori) in China, 7 strains of other plant-associated pathogens, and 50 unidentified epiphytic bacterial isolates from mulberry plants. The real-time PCR assays reliably detected the DNA at at least 10 fg/μl and the bacterial cells at 102 CFU/ml from mulberry shoots and roots suspension. The strong positive reaction in testing of all symptomatic plants (with 100% positive) and parts of asymptomatic latent infected plant samples (with 36.4% positive) provided proof that this method is reliable and sensitive and suitable for screening plant material with latent infections of E. mori.

scholarly journals Application of RT-PCR for Indexing Avocado Sunblotch Viroid

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1023-1026 ◽  
Author(s):  
R. J. Schnell ◽  
D. N. Kuhn ◽  
C. M. Ronning ◽  
D. Harkins
Keyword(s):  
Rt Pcr ◽  
Amplification Product ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Dideoxynucleotide Chain Termination Method ◽  
Slab Gels ◽  
Avocado Sunblotch Viroid

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

scholarly journals A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato

Plant Disease ◽  
2016 ◽  
Vol 100 (8) ◽  
pp. 1660-1668 ◽  
Author(s):  
A. L. Strayer ◽  
A. Jeyaprakash ◽  
G. V. Minsavage ◽  
S. Timilsina ◽  
G. E. Vallad ◽  
...  
Keyword(s):  
Real Time ◽  
Infected Plant ◽  
Multiplex Assay ◽  
Bacterial Spot ◽  
Pcr Assay ◽  
Tomato Production ◽  
Xanthomonas Species ◽  
Pcr Products ◽  
Pure Cultures ◽  
Primer Sets

Bacterial spot of tomato, a major problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hrpB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB7 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB7 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB7 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.

Polymerase Chain Reaction Detection of Listeria monocytogenes on Frankfurters Using Oligonucleotide Primers Targeting the Genes Encoding Internalin AB

2003 ◽  
Vol 66 (2) ◽  
pp. 237-241 ◽  
Author(s):  
YONG SOO JUNG ◽  
JOSEPH F. FRANK ◽  
ROBERT E. BRACKETT ◽  
JINRU CHEN
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Meat Products ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Oligonucleotide Primers ◽  
Pcr Product ◽  
Genes Encoding ◽  
Pure Cell ◽  
Polymerase Chain

A polymerase chain reaction (PCR) assay targeting the genes encoding internalin AB (inlAB) was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated frankfurters. Four sets of oligonucleotide primers were evaluated. The set targeting a 902-bp region of the inlAB gene was the most specific. This PCR product was detected in 51 L. monocytogenes strains belonging to four different serogroups (1/2a, 1/2b, 1/2c, and 4b). In contrast, the PCR product was not detected in other Listeria spp. (Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, or Listeria grayi) or in gram-positive, non-Listeria bacteria, indicating that the primer set was highly specific for L. monocytogenes. The detection limit of the PCR assay was 105 CFU per ml of pure cell culture. However, the assay could detect as few as 101 CFU of L. monocytogenes in 25 g of frankfurter with 16 h of enrichment in modified Listeria enrichment broth at 30°C. The total assay time including enrichment was approximately 24 h. These results suggest that the PCR assay can be used to rapidly detect L. monocytogenes on frankfurters and possibly other types of ready-to-eat meat products.

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