Development of High-Throughput SNP Genotyping Assays for Rapid Detection of Strawberry Colletotrichum Species and the G143A Mutation

Colletotrichum spp. cause major diseases of strawberry and disease management depends on the species present. However, species identification based on symptoms and spore morphology is difficult. Therefore, development of molecular techniques for trustworthy and high-throughput identification of Colletotrichum spp. is vital for the accurate diagnosis. A high-resolution melting (HRM) assay was developed for simultaneous identification and differentiation of Colletotrichum spp. from fungal colonies or from symptomatic strawberry tissue. HRM markers were designed based on the internal transcribed spacer region of Colletotrichum acutatum and C. gloeosporioides from strawberry, and accurately identified and differentiated the two species. In addition, for the rapid detection of a single-nucleotide polymorphism (SNP) in the cytochrome b (cytb) gene of C. acutatum and C. gloeosporioides associated with resistance to quinone-outside inhibitor fungicides, an endpoint SNP genotyping analysis was developed. The HRM and endpoint SNP genotyping assays are useful methods that can be implemented in plant diagnostic clinics for the rapid and accurate identification of Colletotrichum spp. and detection of the G143A mutation in the cytb gene of C. acutatum and C. gloeosporioides.

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Sensitivity of Colletotrichum Species, Including C. fioriniae and C. nymphaeae, from Peach to Demethylation Inhibitor Fungicides

Plant Disease ◽

10.1094/pdis-04-16-0574-re ◽

2016 ◽

Vol 100 (12) ◽

pp. 2434-2441 ◽

Cited By ~ 15

Author(s):

S. N. Chen ◽

C. X. Luo ◽

M. J. Hu ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Colletotrichum Acutatum ◽

In Vitro Activity ◽

Internal Transcribed Spacer Region ◽

Control Efficacy ◽

Dmi Fungicides ◽

Colletotrichum Spp ◽

Colletotrichum Acutatum Species Complex ◽

Demethylation Inhibitor

Few fungicides are effective against anthracnose, caused by Colletotrichum spp., and emerging resistance makes the search for chemical alternatives more relevant. Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards and phylogenetic analysis of the combined internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and β-tubulin gene sequences separated the isolates into C. nymphaeae and C. fioriniae. The sensitivity of these and three other previously reported Colletotrichum spp. from peach, including C. fructicola, C. siamense, and C. truncatum, to demethylation inhibitor (DMI) fungicides difenoconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole was determined based upon mycelial growth inhibition. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole and C. nymphaeae was resistant to flutriafol and fenbuconazole based on 50% effective concentration (EC50) values >100 μg/ml. C. fructicola and C. siamense were sensitive to all DMI fungicides (EC50 values of 0.2 to 13.1 μg/ml). C. fioriniae subgroup 2 isolates were less sensitive to DMI fungicides (EC50 values of 0.5 to 16.2 μg/ml) compared with C. fioriniae subgroup 1 (EC50 values of 0.03 to 2.1 μg/ml). Difenoconazole and propiconazole provided the best control efficacy in vitro to all five species, with EC50 values of 0.2 to 2.7 μg/ml. Tebuconazole and metconazole were effective against all Colletotrichum spp., except for C. truncatum. The strong in vitro activity of some DMI fungicides against Colletotrichum spp. may be exploited for improved anthracnose disease management of peach.

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Characterization of Colletotrichum Species Causing Bitter Rot of Apple in Kentucky Orchards

Plant Disease ◽

10.1094/pdis-10-15-1144-re ◽

2016 ◽

Vol 100 (11) ◽

pp. 2194-2203 ◽

Cited By ~ 34

Author(s):

M. Munir ◽

B. Amsden ◽

E. Dixon ◽

L. Vaillancourt ◽

N. A. Ward Gauthier

Keyword(s):

Species Complex ◽

Common Species ◽

Apple Fruit ◽

Colletotrichum Acutatum ◽

Individual Species ◽

Accurate Identification ◽

Fungicide Sensitivity ◽

Molecular Approaches ◽

Bitter Rot ◽

Colletotrichum Spp

Multiple species of Colletotrichum can cause bitter rot disease of apple, but the identities and relative representation of the species causing the disease in Kentucky are unknown. In total, 475 Colletotrichum isolates were collected from diseased apple fruit in 25 counties and characterized both morphologically and by using various molecular approaches. Multigene sequence analyses revealed that sample isolates belonged to several newly erected species within the Colletotrichum acutatum and C. gloeosporioides species complexes. The isolates were identified as C. fioriniae and C. nymphaeae, which reside within the C. acutatum species complex, and C. siamense, C. theobromicola, and C. fructicola, which are placed within the C. gloeosporioides species complex. C. fioriniae was the most common species causing bitter rot in Kentucky, comprising more than 70% of the isolates. Infectivity tests on detached fruit showed that C. gloeosporioides species-complex isolates were more aggressive than isolates in the C. acutatum species complex. However, isolates within the C. acutatum species complex produced more spores on lesions compared with isolates within the C. gloeosporioides species complex. Aggressiveness varied among individual species within a species complex. C. siamense was the most aggressive species identified in this study. Within the C. acutatum species complex, C. fioriniae was more aggressive than C. nymphaeae, causing larger, deeper lesions. Apple cultivar did not have a significant effect on lesion development. However, Colletotrichum spp. produced more spores on ‘Red Stayman Winesap’ than on ‘Golden Delicious’. Fungicide sensitivity tests revealed that the C. acutatum species complex was more tolerant to thiophanate-methyl, myclobutanil, trifloxystrobin, and captan compared with the C. gloeosporioides species complex. The study also revealed that mycelial growth of C. siamense was more sensitive to tested fungicides compared with C. fructicola and C. theobromicola. These research findings emphasize the importance of accurate identification of Colletotrichum spp. within each species complex, because they exhibit differences in pathogenicity and fungicide sensitivity.

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The Use of Essential Oils from Thyme, Sage and Peppermint against Colletotrichum acutatum

Plants ◽

10.3390/plants10010114 ◽

2021 ◽

Vol 10 (1) ◽

pp. 114

Author(s):

Armina Morkeliūnė ◽

Neringa Rasiukevičiūtė ◽

Lina Šernaitė ◽

Alma Valiuškaitė

Keyword(s):

Essential Oils ◽

Fungal Pathogens ◽

Control Plant ◽

Antimicrobial Activities ◽

Plant Diseases ◽

Colletotrichum Acutatum ◽

Low Toxicity ◽

Colletotrichum Spp ◽

Biological Protection

The Colletotrichum spp. is a significant strawberry pathogen causing yield losses of up to 50%. The most common method to control plant diseases is through the use of chemical fungicides. The findings of plants antimicrobial activities, low toxicity, and biodegradability of essential oils (EO), make them suitable for biological protection against fungal pathogens. The aim is to evaluate the inhibition of Colletotrichum acutatum by thyme, sage, and peppermint EO in vitro on detached strawberry leaves and determine EO chemical composition. Our results revealed that the dominant compound of thyme was thymol 41.35%, peppermint: menthone 44.56%, sage: α,β-thujone 34.45%, and camphor: 20.46%. Thyme EO inhibited C. acutatum completely above 200 μL L−1 concentration in vitro. Peppermint and sage EO reduced mycelial growth of C. acutatum. In addition, in vitro, results are promising for biological control. The detached strawberry leaves experiments showed that disease reduction 4 days after inoculation was 15.8% at 1000 μL L−1 of peppermint EO and 5.3% at 800 μL L−1 of thyme compared with control. Our findings could potentially help to manage C. acutatum; however, the detached strawberry leaves assay showed that EO efficacy was relatively low on tested concentrations and should be increased.

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ChemInform Abstract: High-Throughput SNP Genotyping

ChemInform ◽

10.1002/chin.200238287 ◽

2010 ◽

Vol 33 (38) ◽

pp. no-no

Author(s):

Suzanne Jenkins ◽

Neil Gibson

Keyword(s):

High Throughput ◽

Snp Genotyping

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Yeast Species Associated with Orange Juice: Evaluation of Different Identification Methods

Applied and Environmental Microbiology ◽

10.1128/aem.68.4.1955-1961.2002 ◽

2002 ◽

Vol 68 (4) ◽

pp. 1955-1961 ◽

Cited By ~ 87

Author(s):

Covadonga R. Arias ◽

Jacqueline K. Burns ◽

Lorrie M. Friedrich ◽

Renee M. Goodrich ◽

Mickey E. Parish

Keyword(s):

Orange Juice ◽

Restriction Pattern ◽

Partial Sequence ◽

Rrna Gene ◽

Correct Identification ◽

Internal Transcribed Spacer Region ◽

Accurate Identification ◽

Hanseniaspora Uvarum ◽

Classical Methodology ◽

26S Rrna

ABSTRACT Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.

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Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

Journal of Food Research ◽

10.5539/jfr.v7n1p32 ◽

2017 ◽

Vol 7 (1) ◽

pp. 32 ◽

Cited By ~ 2

Author(s):

Dimitra Houhoula ◽

Stamatios Koussissis ◽

Vladimiros Lougovois ◽

John Tsaknis ◽

Dimitra Kassavita ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Food Products ◽

Molecular Techniques ◽

Food Allergens ◽

Pcr Assay ◽

Peanut Allergen ◽

Pcr Method ◽

Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

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Duplex Real-Time PCR Assay for Rapid Detection of Ampicillin-Resistant Enterococcus faecium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.2.556-560.2004 ◽

2004 ◽

Vol 48 (2) ◽

pp. 556-560 ◽

Cited By ~ 14

Author(s):

Stein Christian Mohn ◽

Arve Ulvik ◽

Roland Jureen ◽

Rob J. L. Willems ◽

Janetta Top ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Enterococcus Faecium ◽

Rapid Detection ◽

Resistant Bacteria ◽

Pcr Assay ◽

Culture Methods ◽

Accurate Identification ◽

Antibiotic Resistant ◽

Highly Correlated

ABSTRACT Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the d-Ala-d-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

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Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications

PLoS ONE ◽

10.1371/journal.pone.0206085 ◽

2018 ◽

Vol 13 (10) ◽

pp. e0206085 ◽

Cited By ~ 25

Author(s):

Peter W. Inglis ◽

Marilia de Castro R. Pappas ◽

Lucileide V. Resende ◽

Dario Grattapaglia

Keyword(s):

High Throughput ◽

Snp Genotyping ◽

High Quality ◽

Dna And Rna

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Assessing and Maximizing Cultivated Diversity with Plate-Wash PCR and High Throughput Sequencing

10.1101/2020.11.19.390864 ◽

2020 ◽

Author(s):

Emily N. Junkins ◽

Bradley S. Stevenson

Keyword(s):

High Throughput ◽

Drug Targets ◽

High Throughput Sequencing ◽

Multidrug Resistant ◽

Molecular Techniques ◽

Bioactive Metabolites ◽

Plate Count ◽

Molecular Tools ◽

Vast Number ◽

Sequencing Studies

AbstractMolecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed “the great plate count anomaly” by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Molecular methods indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were not detected in the original inoculum. In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete, albeit more complex, understanding of which organisms were present compared to what was eventually cultivated. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against a multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.ImportanceCultivation is the definitive tool to understand a microorganism’s physiology, metabolism, and ecological role(s). Despite continuous efforts to hone this skill, researchers are still observing yet-to-be cultivated organisms through high-throughput sequencing studies. Here, we use the very same tool that highlights biodiversity to assess cultivation efficiency. When applied to drug discovery, where screening a vast number of isolates for bioactive metabolites is common, cultivating redundant organisms is a hindrance. However, we observed that cultivating in combination with molecular tools can expand the observed diversity of an environment and its community, potentially increasing the number of microorganisms to be screened for natural products.

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