A PCR-based method to detect species of Gondwanamyces and Ophiostoma on surfaces of insects colonizing Protea flowers

2006 ◽  
Vol 84 (6) ◽  
pp. 989-994 ◽  
Author(s):  
Francois Roets ◽  
Michael J. Wingfield ◽  
Léanne L. Dreyer ◽  
Pedro W. Crous ◽  
Dirk U. Bellstedt
Keyword(s):  
South African ◽  
Dna Sequences ◽  
Detection System ◽  
Fungal Species ◽  
Ophiostomatoid Fungi ◽  
Universal Primer ◽  
African Species ◽  
Polymerase Chain ◽  
The Relationship ◽  
28S Ribosomal Dna

Flower heads of economically important members of the genus Protea L. mature into conspicuous, often long-lived infructescences, which in South Africa are commonly colonized by species of the ophiostomatoid fungi Gondwanamyces G.J. Marais & M.J. Wingfield and Ophiostoma Syd. & P. Syd. It is suspected that these fungi are transported between infructescences by insects. To develop techniques that would enable detection of ophiostomatoid fungi on insects, primers GPR1 and OSP1 were designed based on unique 28S ribosomal DNA sequences of Gondwanamyces and Ophiostoma from Protea. Multiplex polymerase chain reaction of these primers, combined with universal primer LR6, yielded fragment lengths of 885 and 637 bp. Positive amplification was achieved from as little as 30 and 45 pg of fungal genomic DNA for Gondwanamyces and Ophiostoma, respectively, and fragments of identical lengths were amplified from insects artificially inoculated with these fungi. No other tested fungal species showed amplification with GPR1 or OSP1 and LR6. Using these primers two insect species ( Genuchus hottentottus Fabricius and Oxycarenus maculates Stal.) collected from Protea repens L. infructescences were confirmed as carriers of Gondwanamyces proteae (M.J. Wingfield et al.) G.J. Marais & M.J. Wingfield and Ophiostoma splendens G.J. Marais & M.J. Wingfield, respectively. The method developed in this study represents a rapid detection system that can be used to understand the relationship between insects and ophiostomatoid fungi found associated with flowers of South African species of Protea .

scholarly journals Filamentous Fungi and Yeasts Associated with Mites Phoretic on Ips typographus in Eastern Finland

Forests ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 743
Author(s):  
Riikka Linnakoski ◽  
Ilmeini Lasarov ◽  
Pyry Veteli ◽  
Olli-Pekka Tikkanen ◽  
Heli Viiri ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Dna Sequences ◽  
Yeast Species ◽  
Phylogenetic Analyses ◽  
Fungal Species ◽  
Ips Typographus ◽  
Ophiostomatoid Fungi ◽  
Spruce Bark ◽  
Blue Stain ◽  
Fungal Associates

The European spruce bark beetle (Ips typographus) has become a major forest pest in Finland in recent years. The beetle is a well-known vector of mainly ophiostomatoid fungi causing blue-stain of timber and pathogens that have the ability to amplify the insect damage. It also vectors other associated organisms, such as phoretic mites. The ecology of these mites remains poorly understood, including their associations with fungi. In this study, we considered filamentous fungi and yeasts associated with mites phoretic on I. typographus. Fungal identifications were based on DNA sequences and phylogenetic analyses of the ITS and/or partial β-tubulin gene regions. Fifteen fungal species were detected, including eight yeasts and seven filamentous fungi. Eleven percent of the beetles carried mites and of these 74% carried at least one fungal species. An average of two fungal species were carried per mite. The most commonly found filamentous fungi were Grosmannia penicillata (25%), Ophiostoma bicolor (19%), O. ainoae (12%) and O. brunneolum (12%). Of the yeast species, the most commonly found was Wickerhamomyces bisporus (47%). This study is the first to report yeasts associated with I. typographus and its phoretic mites in Finland. Majority of the filamentous fungal species found are those previously reported in association with I. typographus. The results also confirmed that many of the fungal species commonly found on I. typographus are also associated with its phoretic mites. However, the nature of the symbiosis between the mites, beetles and fungal associates remains to be understood.

scholarly journals First Report of Clover Yellow Edge and STRAWB2 Phytoplasmas in Strawberry in Maryland

Plant Disease ◽  
1999 ◽  
Vol 83 (11) ◽  
pp. 1072-1072 ◽  
Author(s):  
R. Jomantiene ◽  
J. L. Maas ◽  
E. L. Dally ◽  
R. E. Davis
Keyword(s):  
Dna Sequences ◽  
Infected Plant ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Universal Primer ◽  
First Report ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Phytoplasma Infection ◽  
Polymerase Chain

Commercial strawberry (Fragaria × ananassa Duchesne) plants that were either chlorotic and severely stunted or exhibiting fruit phyllody were collected in Maryland. The plants were assessed for phytoplasma infection by nested polymerase chain reactions primed by phytoplasma universal primer pairs R16mF2/R1 and F2n/R2 (2) or P1/P7 (3) and F2n/R2 for amplification of phytoplasma 16S ribosomal (r) DNA (16S rRNA gene) sequences. Phytoplasma-characteristic 1.2-kbp DNA sequences were amplified from all diseased plants. No phytoplasma-characteristic DNAs were amplified from healthy plants. Restriction fragment length polymorphism patterns of rDNA digested with AluI, KpnI, HhaI, HaeIII, HpaII, MseI, RsaI, and Sau3A1 endonucleases indicated that chlorotic and stunted plants were infected by a phytoplasma that belonged to subgroup 16SrIII-B (clover yellow edge [CYE] subgroup) and that the plant exhibiting fruit phyllody was infected by a phytoplasma that belonged to subgroup 16SrI-K (STRAWB2 subgroup). The STRAWB2 phytoplasma was first reported from strawberry plants grown in Florida and characterized as representative of a new subgroup of the aster yellows group, 16SrI (3); this is the first report of this phytoplasma occurring in strawberry outside of Florida. A STRAWB2-infected plant produced phylloid fruits in two consecutive years of observation in the greenhouse; the plant previously had been field-grown in a breeder's evaluation plots in Beltsville, MD. The CYE phytoplasma was first experimentally transmitted by leafhopper to commercial strawberry and F. virginiana Duchesne in Ontario Canada (1); this is the first report of natural CYE phytoplasma infection of strawberry in Maryland. CYE phytoplasma-infected plants, representing ≈5% of the total number of plants of one advanced sselection, were located in a breeder's evaluation plots in Beltsville. References: (1) L. N. Chiykowski. Can. J. Bot. 54:1171, 1976. (2) D. E. Gunderson and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

Co-creating the Psychological “White Picket Fence”: Three Gay Couples’ Relationship Reconfiguration

2020 ◽  
Vol 8 (2) ◽  
Author(s):  
Prevan Moodley ◽  
Francois Rabie
Keyword(s):  
Grounded Theory ◽  
South African ◽  
Gay Couples ◽  
Core Category ◽  
Picket Fence ◽  
Open Relationship ◽  
The Individual ◽  
The Relationship ◽  
Porous Boundaries

Many gay couples engage in nonmonogamous relationships. Ideas about nonmonogamy have historically been theorised as individual pathology and indicating relational distress. Unlike mixed-sex couples, boundaries for gay couples are often not determined by sexual exclusivity. These relationships are built along a continuum of open and closed, and sexual exclusivity agreements are not restricted to binaries, thus requiring innovation and re-evaluation. Three white South African gay couples were each jointly interviewed about their open relationship, specifically about how this is negotiated. In contrast to research that uses the individual to investigate this topic, this study recruited dyads. The couples recalled the initial endorsement of heteronormative romantic constructions, after which they shifted to psychological restructuring. The dyad, domesticated through the stock image of a white picket fence, moved to a renewed arrangement, protected by “rules” and imperatives. Abbreviated grounded theory strategies led to a core category, “co-creating porous boundaries”, and two themes. First, the couple jointly made heteronormative ideals porous and, second, they reconfigured the relationship through dyadic protection. The overall relationship ideology associated with the white picket fence remained intact despite the micro-innovations through which the original heteronormative patterning was reconfigured.

MiR-125a-5p Alleviates Dysfunction and Inflammation of Pentylenetetrazol- induced Epilepsy Through Targeting Calmodulin-dependent Protein Kinase IV (CAMK4)

2019 ◽  
Vol 16 (4) ◽  
pp. 365-372 ◽  
Author(s):  
Qishuai Liu ◽  
Li Wang ◽  
Guizhen Yan ◽  
Weifa Zhang ◽  
Zhigang Huan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Target Gene ◽  
Luciferase Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inflammatory Factor ◽  
Dependent Protein Kinase ◽  
Expression Levels ◽  
Dependent Protein ◽  
Polymerase Chain ◽  
The Relationship

Background: MicroRNAs (miRNA) are known to play a key role in the etiology and treatment of epilepsy through controlling the expression of gene. However, miR-125a-5p in the epilepsy is little known. Epilepsy in rat models was induced by Pentylenetetrazol (PTZ) and miR- 125a-5p profiles in the hippocampus were investigated in our experiment. Also, the relationship between miR-125a-5p and calmodulin-dependent protein kinase IV (CAMK4) was identified and the related mechanism was also illustrated. Methods: The miR-125a-5p mRNA expression levels were evaluated by quantitative real time polymerase chain reaction (qRT-PCR). Western Blot (WB) was used to analyze the CAMK4 protein expression levels. Seizure score, latency and duration were determined based on a Racine scale. The enzyme-linked immunosorbent assay (ELISA) was used to analyze the inflammatory factor expression. The relationship between miR-125a-5p and CAMK4 was detected through dual luciferase assay. Results: Downregulation of miR-125a-5p was observed in the hippocampus of PTZ-induced epilepsy rats. The overexpression of miR-125a-5p attenuated seizure and decreased inflammatory factor level in the hippocampus of PTZ-induced rats. The miR-125a-5p alleviated epileptic seizure and inflammation in PTZ-induced rats by suppressing its target gene, CAMK4. Conclusion: miR-125a-5p may represent a novel therapeutic treatment for PTZ-induced epilepsy by preventing the activation of CAMK4.

scholarly journals Knockdown of Circ_SLC39A8 protects against the progression of osteoarthritis by regulating miR-591/IRAK3 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jizhe Yu ◽  
Yushuang Qin ◽  
Naxin Zhou
Keyword(s):  
Cell Viability ◽  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Oa Chondrocytes ◽  
Polymerase Chain ◽  
Apoptosis And Inflammation ◽  
The Relationship

Abstract Background The dysregulation of circular RNAs (circRNAs) has been identified in various human diseases, including osteoarthritis (OA). The purpose of this study was to identify the role and mechanism of circ_SLC39A8 in regulating the progression of OA. Methods The expression levels of circ_SLC39A8, miR-591, and its potential target gene, interleukin-1-receptor-associated kinase 3 (IRAK3), were identified by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were determined by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The relationship between miR-591 and circ_SLC39A8 or IRAK3 was predicted by bioinformatics tools and verified by dual-luciferase reporter. Results Circ_SLC39A8 and IRAK3 were upregulated and miR-591 was downregulated in OA cartilage tissues. Knockdown of circ_SLC39A8 inhibited apoptosis and inflammation in OA chondrocytes, while these effects were reversed by downregulating miR-591. Promotion cell viability effects of miR-591 were partially reversed by IRAK3 overexpression. Conclusion Our findings indicated that knockdown of circ_SLC39A8 delayed the progression of OA via modulating the miR-591-IRAK3 axis, providing new insight into the molecular mechanisms of OA pathogenesis.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

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