scholarly journals Identification of Pantoea stewartii subsp. stewartii by PCR and Strain Differentiation by PFGE

Plant Disease ◽  
2002 ◽  
Vol 86 (3) ◽  
pp. 304-311 ◽  
Author(s):  
David L. Coplin ◽  
Doris R. Majerczak ◽  
Yongxiang Zhang ◽  
Won-Sik Kim ◽  
Susanne Jock ◽  
...  
Keyword(s):  
Sweet Corn ◽  
Low Frequency ◽  
Its Region ◽  
Enteric Bacteria ◽  
Pantoea Stewartii ◽  
Seed Health ◽  
Field Samples ◽  
Pure Cultures ◽  
Primer Sets ◽  
Pigmented Bacteria

Stewart's bacterial wilt and leaf blight of sweet corn and maize is caused by Pantoea stewartii subsp. stewartii. This bacterium can be seed transmitted at a low frequency, so it is subject to quarantine restrictions by many countries. To develop a polymerase chain reaction assay for the identification of this pathogen from field samples and for use in seed health tests, four primer pairs were tested. These were selected from the sequences of hrpS, cpsDE, and the 16S rRNA intergenic transcribed spacer (ITS) region. Under optimal reaction conditions, about 20 and 200 cells of P. stewartii could be detected in pure cultures and leaf lesions, respectively. Other plant-associated enteric bacteria (e.g., P. agglomerans pv. herbicola, P. ananas, Erwinia amylovora, and E. carotovora) either did not produce amplicons or they were not the correct size for P. stewartii. To test further for possible false positives, 29 yellow-pigmented bacteria, mainly other Pantoea spp., were isolated from lesions on old corn leaves and assayed with the ITS primer sets. Except for weak, variable reactions with three P. ananas strains, the bacteria did not test positive. Pulsed field gel electrophoresis (PFGE) was evaluated as an additional test to confirm the identity of P. stewartii. After digestion with SpeI and XbaI, P. stewartii strains could be easily distinguished from related Erwinia and Pantoea spp. and each other.

scholarly journals Development of Molecular Markers for Detection of Acidovorax citrulli Strains Causing Bacterial Fruit Blotch Disease in Melon

2019 ◽  
Vol 20 (11) ◽  
pp. 2715 ◽  
Author(s):  
Md. Rafiqul Islam ◽  
Mohammad Rashed Hossain ◽  
Hoy-Taek Kim ◽  
Denison Michael Immanuel Jesse ◽  
Md. Abuyusuf ◽  
...  
Keyword(s):  
Infected Leaf ◽  
Bacterial Fruit Blotch ◽  
Early Disease Detection ◽  
Acidovorax Citrulli ◽  
Seed Health ◽  
Field Samples ◽  
Whole Genomes ◽  
Pathogenesis Related ◽  
Primer Sets ◽  
Inoculation Assay

Acidovorax citrulli (A. citrulli) strains cause bacterial fruit blotch (BFB) in cucurbit crops and affect melon significantly. Numerous strains of the bacterium have been isolated from melon hosts globally. Strains that are aggressively virulent towards melon and diagnostic markers for detecting such strains are yet to be identified. Using a cross-inoculation assay, we demonstrated that two Korean strains of A. citrulli, NIHHS15-280 and KACC18782, are highly virulent towards melon but avirulent/mildly virulent to the other cucurbit crops. The whole genomes of three A. citrulli strains isolated from melon and three from watermelon were aligned, allowing the design of three primer sets (AcM13, AcM380, and AcM797) that are specific to melon host strains, from three pathogenesis-related genes. These primers successfully detected the target strain NIHHS15-280 in polymerase chain reaction (PCR) assays from a very low concentration of bacterial gDNA. They were also effective in detecting the target strains from artificially infected leaf, fruit, and seed washing suspensions, without requiring the extraction of bacterial DNA. This is the first report of PCR-based markers that offer reliable, sensitive, and rapid detection of strains of A. citrulli causing BFB in melon. These markers may also be useful in early disease detection in the field samples, in seed health tests, and for international quarantine purposes.

scholarly journals Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

2000 ◽  
Vol 66 (9) ◽  
pp. 4131-4135 ◽  
Author(s):  
A. Vishnubhatla ◽  
D. Y. C. Fung ◽  
R. D. Oberst ◽  
M. P. Hays ◽  
T. G. Nagaraja ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Pcr Amplification ◽  
Enteric Bacteria ◽  
Taqman Assay ◽  
Virulent Strains ◽  
Field Samples ◽  
Ground Pork ◽  
Standard Culture ◽  
Pure Cultures ◽  
Nuclease Assay

ABSTRACT We have developed a rapid procedure for the detection of virulentYersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species ofYersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri,Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia,Salmonella, Citrobacter, andFlavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥102 CFU/ml in pure cultures and ≥103 CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked withY. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocoliticawith both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.

Development and Application of a New PCR Method for Detection of Blumeria graminis f. sp. tritici

2018 ◽  
Vol 28 (3) ◽  
pp. 137-146 ◽  
Author(s):  
Adam Kuzdraliński ◽  
Hubert Szczerba ◽  
Anna Kot ◽  
Agnieszka Ostrowska ◽  
Michał Nowak ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Puccinia Triticina ◽  
Blumeria Graminis ◽  
Pyrenophora Tritici Repentis ◽  
Field Samples ◽  
Expected Length ◽  
Pcr Assays ◽  
Primer Sets ◽  
Wheat Leaves ◽  
Species Specific

We developed new PCR assays that target beta-tubulin (<i>TUB2</i>) and 14 alpha-demethylase (<i>CYP51</i>) genes and used them for the species-specific detection of <i>Blumeria graminis</i> f. sp. <i>tritici</i> (<i>Bgt</i>). Based on fungi DNA sequences available in the NCBI (National Center for Biotechnology Information) GenBank database we developed simplex and duplex PCR assays. The specificities of the primer sets were evaluated using environmental samples of wheat leaves collected during the 2015/2016 growing season across Poland. Primer sets<i></i> LidBg17/18 and LidBg21/22 strongly amplified fragments of the expected length for all 67 tested samples. Primer specificity was confirmed using field samples of <i>Zymoseptoria tri­tici</i>, <i>Puccinia triticina</i> (syn.<i> P. recondita</i> f. sp.<i> tritici</i>), <i>P. striiformis</i> f. sp.<i> tritici</i>, and <i>Pyrenophora tritici-repentis</i>.

scholarly journals Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus

Plant Disease ◽  
2007 ◽  
Vol 91 (7) ◽  
pp. 865-870 ◽  
Author(s):  
J. W. Hyun ◽  
N. A. Peres ◽  
S.-Y. Yi ◽  
L. W. Timmer ◽  
K. S. Kim ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Sweet Orange ◽  
Random Amplified Polymorphic Dna ◽  
Its Region ◽  
The United States ◽  
Specific Primer ◽  
Identification Of Species ◽  
Orange Fruit ◽  
Pcr Assays ◽  
Primer Sets

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

scholarly journals First report of Colletotrichum siamense Causing Blossom Blight on Thai basil (Ocimum basilicum L.) in Malaysia

Plant Disease ◽  
2020 ◽  
Author(s):  
Siti Izera Ismail ◽  
Nur Adlina Rahim ◽  
Dzarifah Zulperi
Keyword(s):  
Disease Incidence ◽  
Its Region ◽  
Ocimum Basilicum ◽  
Distilled Water ◽  
Single Spore ◽  
First Report ◽  
Plastic Bags ◽  
Colletotrichum Siamense ◽  
Primer Sets ◽  
Acca Sellowiana

Thai basil (Ocimum basilicum L.) is widely cultivated in Malaysia and commonly used for culinary purposes. In March 2019, necrotic lesions were observed on the inflorescences of Thai basil plants with a disease incidence of 60% in Organic Edible Garden Unit, Faculty of Agriculture in the Serdang district (2°59'05.5"N 101°43'59.5"E) of Selangor province, Malaysia. Symptoms appeared as sudden, extensive brown spotting on the inflorescences of Thai basil that coalesced and rapidly expanded to cover the entire inflorescences. Diseased tissues (4×4 mm) were cut from the infected lesions, surface disinfected with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, placed onto potato dextrose agar (PDA) plates and incubated at 25°C under 12-h photoperiod for 5 days. A total of 8 single-spore isolates were obtained from all sampled inflorescence tissues. The fungal colonies appeared white, turned grayish black with age and pale yellow on the reverse side. Conidia were one-celled, hyaline, subcylindrical with rounded end and 3 to 4 μm (width) and 13 to 15 μm (length) in size. For fungal identification to species level, genomic DNA of representative isolate (isolate C) was extracted using DNeasy Plant Mini Kit (Qiagen, USA). Internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), and chitin synthase-1 (CHS-1) were amplified using ITS5/ITS4 (White et al. 1990), CL1C/CL2C (Weir et al. 2012), ACT-512F/783R, and CHS-79F/CHS-345R primer sets (Carbone and Kohn 1999), respectively. A BLAST nucleotide search of ITS, CHS-1, CAL and ACT sequences showed 100% similarity to Colletotrichum siamense ex-type cultures strain C1315.2 (GenBank accession nos. ITS: JX010171 and CHS-1: JX009865) and isolate BPDI2 (CAL: FJ917505, ACT: FJ907423). The ITS, CHS-1, CAL and ACT sequences were deposited in GenBank as accession numbers MT571330, MW192791, MW192792 and MW140016. Pathogenicity was confirmed by spraying a spore suspension (1×106 spores/ml) of 7-day-old culture of isolate C onto 10 healthy inflorescences on five healthy Thai basil plants. Ten infloresences from an additional five control plants were only sprayed with sterile distilled water and the inoculated plants were covered with plastic bags for 2 days and maintained in a greenhouse at 28 ± 1°C, 98% relative humidity with a photoperiod of 12-h. Blossom blight symptoms resembling those observed in the field developed after 7 days on all inoculated inflorescences, while inflorescences on control plants remained asymptomatic. The experiment was repeated twice. C. siamense was successfully re-isolated from the infected inflorescences fulfilling Koch’s postulates. C. siamense has been reported causing blossom blight of Uraria in India (Srivastava et al. 2017), anthracnose on dragon fruit in India and fruits of Acca sellowiana in Brazil (Abirami et al. 2019; Fantinel et al. 2017). This pathogen can cause a serious threat to cultivation of Thai basil and there is currently no effective disease management strategy to control this disease. To our knowledge, this is the first report of blossom blight caused by C. siamense on Thai basil in Malaysia.

scholarly journals First Report of Root and Stem Rot Caused by Phytophthora nicotianae on Peperomia tetraphylla in China

Plant Disease ◽  
2010 ◽  
Vol 94 (9) ◽  
pp. 1171-1171 ◽  
Author(s):  
D. X. Zeng ◽  
X. L. Wu ◽  
Y. H. Li
Keyword(s):  
Southern China ◽  
Aerial Mycelium ◽  
Its Region ◽  
Soil Extract ◽  
Phytophthora Nicotianae ◽  
Stem Rot ◽  
Molecular Characteristics ◽  
Its Sequence ◽  
First Report ◽  
Pure Cultures

Peperomia tetraphylla, an evergreen herb, is becoming increasingly popular as a potted ornamental plant in southern China. In the summer of 2008, in some commercial flower nurseries in Shenzhen, Guangdong Province, P. tetraphylla showed extensive black stem and root rot, with leaves dropping from the rotten stem. Small pieces (approximately 3 mm2) of stems and leaves were excised from the margins of the black lesions, surface disinfected for 30 s to 1 min in 0.1% HgCl2, plated onto potato dextrose agar (PDA), and incubated at 25°C in the dark. All the plated samples yielded Phytophthora, and microscopic examination of pure cultures grown on PDA plates showed arachnoid colonies with abundant aerial mycelium, chlamydospores, and a few sporangia. Numerous sporangia were formed in sterile soil extract. Sporangia were ovoid or obpyriform, noncaducous, with prominent solitary papillae, and measured 31 to 52 μm (average 38 μm) × 21 to 34 μm (average 27 μm). Chlamydospores were spherical and 21 to 34 μm in diameter (average 28 μm). The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using primers ITS4/ITS5 and sequenced (2). The ITS sequence, when submitted for a BLAST search in the NCBI database, showed 100% homology with the sequences of two reference isolates of Phytophthora nicotianae (Accession Nos. AY833526 and EU433396) and the consensus ITS sequence was deposited in the NCBI as Accession No. GQ499373. The isolate was identified as Phytophthora nicotianae on the basis of morphological and molecular characteristics (1). Pathogenicity of the isolate was confirmed by inoculating 1-year-old plants of P. tetraphylla growing in pots. The isolate was grown for 7 days on PDA plates and mycelial plugs, 5 mm in diameter and taken from the advancing margins of the colonies, were buried approximately 1 cm deep near the base of the stem in such a way that the mycelium on the plugs was in contact with the surface of the stem, which had been wiped earlier with 70% ethanol and gently wounded with a needle. Plants treated the same way but inoculated with sterile PDA plugs served as control plants. Three plants in each pot were inoculated and there were five replications each for the treatment and the control. All plants were kept in a greenhouse at 22 to 32°C. After 6 to 7 days, the inoculated plants showed black lesions around the mycelial plugs; symptoms of root and stem rot developed rapidly thereafter and the plants collapsed within 2 weeks. All symptoms on the inoculated plants were identical to those observed in naturally diseased plants, whereas the control plants remained healthy. The same fungus was consistently reisolated from the inoculated plants. To our knowledge, this is the first report of Phytophthora nicotianae on P. tetraphylla in China. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) J. B. Ristaino et al. Appl. Environ. Microbiol. 64:948, 1998.

Identification of Pathogenic Fusarium spp. Responsible for Root Rot of Angelica sinensis and Characterization of their Biological Enemies in Dingxi, China

Plant Disease ◽  
2022 ◽  
Author(s):  
Liu Yang ◽  
Tian Yuan ◽  
Xia Zhao ◽  
Yue Liang ◽  
UWAREMWE CONSTANTINE ◽  
...  
Keyword(s):  
Root Rot ◽  
Elongation Factor ◽  
Its Region ◽  
Fungal Species ◽  
Cortical Cells ◽  
Translation Elongation ◽  
Field Samples ◽  
Fusarium Pathogens ◽  
Rot Disease ◽  
Serious Disease

Root rot is a serious disease in plantations of A. sinensis, severely affecting yield and quality and threatening sustainable production. Fusarium isolates (n=32) were obtained from field samples of root rot tissue, leaves and infected soil. Isolates were identified by comparing the sequences of their internal transcribed spacer (ITS) region and translation elongation factor 1-ɑ (TEF-1ɑ) to sequences of known species in the NCBI-database. These Fusarium isolates include F. tricinctum (43.75%), F. equiseti (31.25%), F. solani (9.37%), F. oxysporum (6.25%), F. acuminatum (6.25%), and F. incarnatum (3.12%). For pathogenicity testing under greenhouse conditions, seven isolates were selected based on a phylogenetic analysis, including four strains of F. tricinctum and one strain each of F. solani, F. oxysporum, and F. acuminatum. The seven isolates were all pathogenic but differed in their ability to infect: the four F. tricinctum strains were capable pathogens causing root rot in A. sinensis at 100% incidence and the highly aggressive. Furthermore, the symptoms of root rot induced by those seven isolates were consistent with typical root rot cases in the field, but their disease severity varied. Observed histopathological preparations of F. tricinctum-infected seedlings and tissue-slides results showed this fungal species can penetrate epidermal cells and colonize the cortical cells where it induces necrosis and severe plasmolysis. Plate confrontation experiments showed that isolated rhizosphere bacteria inhibited the Fusarium pathogens that cause root rot in A. sinensis. Our results provide timely information for informing the use of biocontrol agents for suppression of root rot disease.

scholarly journals Molecular Verification of Bloom-forming Aphanizomenon flos-aquae and Their Secondary Metabolites in the Nakdong River

2018 ◽  
Vol 15 (8) ◽  
pp. 1739 ◽  
Author(s):  
Hae-Kyung Park ◽  
Mi-Ae Kwon ◽  
Hae-Jin Lee ◽  
Jonghee Oh ◽  
Su-Heon Lee ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Water Samples ◽  
Its Region ◽  
Cyanobacterial Blooms ◽  
Specific Primer ◽  
Nakdong River ◽  
Off Flavors ◽  
Primer Sets ◽  
The Nakdong River ◽  
Harmful Cyanobacterial Blooms

Aphanizomenon spp. have formed harmful cyanobacterial blooms in the Nakdong River during spring, autumn, and now in winter, and the expansion of blooming period and area, associated with the global warming is predicted. The genus Aphanizomenon has been described to produce harmful secondary metabolites such as off-flavors and cyanotoxins. Therefore, the production of harmful secondary metabolites from the Aphanizomenon blooms in the Nakdong River needs to be monitored to minimize the risk to both water quality and public health. Here, we sampled the cyanobacterial blooms in the Nakdong River and isolated ten Aphanizomenon strains, morphologically classified as Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888. Phylogenetic analysis using 16S rRNA and internal transcribed spacer (ITS) region nucleotide sequences confirmed this classification. We further verified the harmful secondary metabolites-producing potential of A. flos-aquae isolates and water samples containing cyanobacterial blooms using PCR with specific primer sets for genes involved in biosynthesis of off-flavor metabolites (geosmin) and toxins (microcystins, saxitoxins and cylindrospermopsins). It was confirmed that these metabolite biosynthesis genes were not identified in all isolates and water samples containing only Aphanizomenon spp. Thus, it is likely that there is a low potential for the production of off-flavor metabolites and cyanotoxins in Aphanizomenon blooms in the Nakdong River.

scholarly journals Development of the Automated Primer Design Workflow Uniqprimer and Diagnostic Primers for the Broad-Host-Range Plant Pathogen Dickeya dianthicola

Plant Disease ◽  
2019 ◽  
Vol 103 (11) ◽  
pp. 2893-2902 ◽  
Author(s):  
Shaista Karim ◽  
R. Ryan McNally ◽  
Afnan S. Nasaruddin ◽  
Alexis DeReeper ◽  
Ramil P. Mauleon ◽  
...  
Keyword(s):  
Host Range ◽  
Bacterial Pathogen ◽  
The United States ◽  
Broad Host Range ◽  
Software Pipeline ◽  
Widespread Occurrence ◽  
Field Samples ◽  
Primer Sets ◽  
Genome Analyses ◽  
User Friendly

Uniqprimer, a software pipeline developed in Python, was deployed as a user-friendly internet tool in Rice Galaxy for comparative genome analyses to design primer sets for PCRassays capable of detecting target bacterial taxa. The pipeline was trialed with Dickeya dianthicola, a destructive broad-host-range bacterial pathogen found in most potato-growing regions. Dickeya is a highly variable genus, and some primers available to detect this genus and species exhibit common diagnostic failures. Upon uploading a selection of target and nontarget genomes, six primer sets were rapidly identified with Uniqprimer, of which two were specific and sensitive when tested with D. dianthicola. The remaining four amplified a minority of the nontarget strains tested. The two promising candidate primer sets were trialed with DNA isolated from 116 field samples from across the United States that were previously submitted for testing. D. dianthicola was detected in 41 samples, demonstrating the applicability of our detection primers and suggesting widespread occurrence of D. dianthicola in North America.

scholarly journals WtsE, an AvrE-Family Effector Protein from Pantoea stewartii subsp. stewartii, Causes Disease-Associated Cell Death in Corn and Requires a Chaperone Protein for Stability

2006 ◽  
Vol 19 (10) ◽  
pp. 1092-1102 ◽  
Author(s):  
Jong Hyun Ham ◽  
Doris R. Majerczak ◽  
Angel S. Arroyo-Rodriguez ◽  
David M. Mackey ◽  
David L. Coplin
Keyword(s):  
Cell Death ◽  
Sweet Corn ◽  
Coli Strain ◽  
Effector Proteins ◽  
Erwinia Chrysanthemi ◽  
Endoplasmic Reticulum Membrane ◽  
E Coli ◽  
Pantoea Stewartii ◽  
C Terminus ◽  
Efficient Delivery

The pathogenicity of Pantoea stewartii subsp. stewartii to sweet corn and maize requires a Hrp type III secretion system. In this study, we genetically and functionally characterized a disease-specific (Dsp) effector locus, composed of wtsE and wtsF, that is adjacent to the hrp gene cluster. WtsE, a member of the AvrE family of effector proteins, was essential for pathogenesis on corn and was complemented by DspA/E from Erwinia amylovora. An intact C-terminus of WtsE, which contained a putative endoplasmic reticulum membrane retention signal, was important for function of WtsE. Delivery of WtsE into sweet corn leaves by an Escherichia coli strain carrying the hrp cluster of Erwinia chrysanthemi caused water-soaking and necrosis. WtsE-induced cell death was not inhibited by cycloheximide treatment, unlike the hypersensitive response caused by a known Avr protein, AvrRxo1. WtsF, the putative chaperone of WtsE, was not required for secretion of WtsE from P. stewartii, and the virulence of wtsF mutants was reduced only at low inoculum concentrations. However, WtsF was required for full accumulation of WtsE within the bacteria at low temperatures. In contrast, WtsF was needed for efficient delivery of WtsE from E. coli via the Erwinia chrysanthemi Hrp system.

Sign in / Sign up

Export Citation Format

Share Document