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qPCR Assays for the Detection of Cylindrocladium buxicola in Plant, Water and Air Samples
Gehesquière, B. ; Haeyer, S. D'; Pham, K.T.K. ; Kuik, A.J. van; Maes, M. ; Höfte, M. ; Heungens, K. - \ 2013
Plant Disease 97 (2013)8. - ISSN 0191-2917 - p. 1082 - 1090.
real-time pcr - polymerase-chain-reaction - 1st report - buxus spp. - box blight - specificity - fusarium - belgium - primers - disease
Cylindrocladium buxicola (syn. C. pseudonaviculatum; teleomorph Calonectria pseudonaviculata) is an important fungal pathogen of Buxus spp. Although widespread in Western Europe, this pathogen has only recently been introduced into North America, where it represents a significant threat to the U.S. and Canadian boxwood industries. Trade of latently infected nursery stock is an important mode of long-distance dissemination and introduction of this pathogen but no methods for detection of latently infected material are available. Also, the pathways for short-distance dispersal of C. buxicola have not been adequately studied. Improved detection methods of this pathogen in air and water samples would benefit future research in this area. We have developed real-time polymerase chain reaction assays for the detection of C. buxicola based on the ribosomal DNA internal transcribed spacer 1 (ITS) and the ß-tubulin 2 gene (TUB). Using a TaqMan probe conjugated with a 3' minor groove binding group (TaqMan MGB probe), the ITS-based assay could reliably detect as little as 10 fg of genomic DNA or 20 copies of cloned target DNA and was approximately 70 times more sensitive than the SYBR Green TUB-based assay. The ITS-based assay provided good but not complete specificity, and is well suited for epidemiological studies. The TUB-based assay, however, proved to be fully specific and can be used for diagnostics. We developed and optimized sample processing and DNA extraction methods for detection of latently present C. buxicola in boxwood plants and quantification of conidia in water and air samples. C. buxicola could be detected in 20 g of plant material, of which only 1 ppm of the tissue was infected, in 10-ml water samples containing as low as 1 conidium/ml, and on Melinex tape pieces representing 12 h of air sampling containing 10 or more conidia. The applicability of the techniques to plant, water, and air samples of practical size was demonstrated.
Genetic diversity and population structure of Iranian wild Pleurotus eryngii species-complex strains revealed by URP-PCR markers
Behnamian, Mahdi ; Mohammadi, Seyed A. ; Sonnenberg, A.S.M. ; Goltapeh, Ebrahim M. ; Hendrickx, P.M. - \ 2010
Journal of Food, Agriculture & Environment 8 (2010)3&4. - ISSN 1459-0255 - p. 1203 - 1207.
rapd analysis - mushroom - dna - polymorphisms - amplification - primers - fungi
In the present study, a set of 68 P. eryngii wild strains collected from nine locations in northwest and west of Iran along with six commercial strains were studied using universal rice primers (URP). The wild strains were isolated from Ferula ovina, F. haussknechtii, Cachrys ferulacea, Kellusia odoratissima and Smyrniopsis aucheri plant species. Eleven URP primers amplified 188 polymorphic fragments. A total of 3, 2 and 7 bands were specific to the strains collected from F. ovina and C. ferulacea plant species and commercial strains, respectively. The highest and lowest polymorphisms were identified in populations B (66.49%) and F (24.47%), respectively. Genetic distances among populations ranged from 0.027 (between populations A and B) to 0.393 (between populations C and F) with an average of 0.210. The closest and furthest wild populations to commercial strains were populations B (0.102) and F (0.234), respectively. Analysis of molecular variance (AMOVA) revealed significant among regions, among populations within regions and within population diversity, whereas within population variation (61.6%) accounted for most of the total molecular variance.
A Bifidobacterium mixed-species microarray for high resolution discrimination between intestinal bifidobacteria
Boesten, R.J. ; Schuren, F.H. ; Vos, W.M. de - \ 2009
Journal of Microbiological Methods 76 (2009)3. - ISSN 0167-7012 - p. 269 - 277.
16s ribosomal-rna - human-milk oligosaccharides - genus bifidobacterium - rapid identification - comb-nov - sequence - longum - bacteria - primers - genome
A genomic DNA-based microarray was constructed containing over 6000 randomly cloned genomic fragments of approximately 1-2 kb from six mammalian intestinal Bifidobacterium spp. including B. adolescentis, B. animalis, B. bifidum, B. catenulatum, B. longum and B. pseudolongum. This Bifidobacterium Mixed-Species (BMS) microarray was used to differentiate between type strains and isolates belonging to a set of nine Bifidobacterium spp. Hierarchical clustering of genomic hybridization data confirmed the grouping of the Bifidobacterium spp. according to the 16S rRNA-based phylogenetic clusters. In addition, these genomic hybridization experiments revealed high homology between the type-strain B. animalis subsp. lactis LMG18314 and B. animalis subsp. animalis LMG10508 (79%) as well as between the type strains B. longum biotype longum LMG13197 and B. longum biotype infantis LMG8811 (72%) - nevertheless, discrimination between these species was possible due to the high resolution output of the BMS-array. In addition, it was shown that the BMS-array could be used for assigning unknown Bifidobacterium isolates to a species group. Finally, a set of 54 diagnostic clones for Bifidobacterium identification was selected and sequenced to advance the understanding of the species-related differences. Remarkably, a large fraction (31%) of these was predicted to encode proteins that belong to the bifidobacterial glycobiome and another 11% had functional homology with genes involved in the protection against foreign DNA. Overall, the BMS-microarray is a high-resolution diagnostic tool that is able to facilitate the detection of strain- and species-specific characteristics of bifidobacteria
Accidental importation of the mosquito Aedes albopictus into the Netherlands: a survey of mosquito distribution and the presence of dengue virus
Scholte, E.J. ; Dijkstra, E. ; Blok, H. ; Vries, A. de; Takken, W. ; Hofhuis, A. ; Koopmans, M.P.G. ; Boer, A. de; Reusken, C.B.E.M. - \ 2008
Medical and Veterinary Entomology 22 (2008)4. - ISSN 0269-283X - p. 352 - 358.
stegomyia albopictus - primers - skuse
In the summer of 2005, the Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae) was found for the first time in the Netherlands. It was collected on the premises of several horticultural companies that import the ornamental plant Dracaena sanderiana (Sparagalus: Dracaenaceae [Agavaceae]), known as Lucky bamboo, from southern China, an area endemic for this mosquito species and for arboviruses transmitted by this vector. Here we report the results of a 1-year survey of the distribution and vector status of Ae. albopictus in Lucky bamboo nurseries in the Netherlands (July 2006-June 2007). As it had been established previously that the presence of this species was linked to the import of Lucky bamboo, the survey was conducted only on sites owned by relevant import companies. In total, 569 adult Ae. albopictus were collected with mosquito traps from 15 of the 17 (88%) glasshouses used by Lucky bamboo importers, none of which were found to be infected with dengue virus. On two occasions there was evidence that Ae. albopictus had escaped from the glasshouses, but, overall, there was no evidence that a population had become established in the greenhouses or elsewhere.
Accumulation of trans C18:1 fatty acids in the rumen after dietary algal supplementation is associated with canges in the Butyrivibrio communitytrans
Boeckaert, C. ; Vlaeminck, B. ; Fievez, V. ; Maignien, L. ; Dijkstra, J. ; Boon, N. - \ 2008
Applied and Environmental Microbiology 74 (2008)22. - ISSN 0099-2240 - p. 6923 - 6930.
conjugated linoleic-acid - fish-oil - clostridium-proteoclasticum - in-vitro - ruminal bacterium - biohydrogenation - primers - chain - pcr - lipolysis
Optimization of the fatty acid composition of ruminant milk and meat is desirable. Dietary supplementation of algae was previously shown to inhibit rumen biohydrogenation, resulting in an altered milk fatty acid profile. Bacteria involved in biohydrogenation belong to the Butyrivibrio group. This study was aimed at relating accumulation of biohydrogenation intermediates with shifts in Butyrivibrio spp. in the rumen of dairy cows. Therefore, an experiment was performed with three rumen-fistulated dairy cows receiving a concentrate containing algae (9.35 g/kg total dry matter [DM] intake) for 20 days. Supplementation of the diet with algae inhibited biohydrogenation of C18:2 omega 6 (n-6) and C18:3 n-3, resulting in increased concentrations of biohydrogenation intermediates, whereas C18:0 decreased. Addition of algae increased ruminal C18:1 trans fatty acid concentrations, mainly due to 6- and 20-fold increases in C18:1 trans 11 (t11) and C18:1 t10. The number of ciliates (5.37 log copies/g rumen digesta) and the composition of the ciliate community were unaffected by dietary algae. In contrast, supplementation of the diet with algae changed the composition of the bacterial community. Primers for the Butyrivibrio group, including the genera Butyrivibrio and Pseudobutyrivibrio, were specifically designed. Denaturing gradient gel electrophoresis showed community changes upon addition of algae without affecting the total amount of Butyrivibrio bacteria (7.06 log copies/g rumen DM). Clone libraries showed that algae affected noncultivated species, which cluster taxonomically between the genera Butyrivibrio and Pseudobutyrivibrio and might play a role in biohydrogenation. In addition, 20% of the clones from a randomly selected rumen sample were related to the C18:0-producing branch, although the associated C18:0 concentration decreased through supplementation of the diet with algae
Novel Paraconiothyrium species on stone fruit trees and other woody hosts
Damm, U. ; Verkley, G.J.M. ; Crous, P.W. ; Fourie, P.H. ; Haegi, A. ; Riccioni, L. - \ 2008
Persoonia 20 (2008). - ISSN 0031-5850 - p. 9 - 17.
peach bark - fungi - microsphaeropsis - paraphaeosphaeria - primers
Coniothyrium-like fungi are common wood and soil inhabitants and hyperparasites on other fungi. They belong to different fungal genera within the Pleosporales. Several isolates were obtained on wood of different Prunus species (plum, peach and nectarine) from South Africa, on Actinidia species from Italy and on Laurus nobilis from Turkey. Morphological and cultural characteristics as well as DNA sequence data (5.8S nrDNA, ITS1, ITS2, partial SSU nrDNA) were used to characterise them. The isolates belonged to three species of the recently established genus Paraconiothyrium. This is the first report of Paraconiothyrium brasiliense on Prunus spp. from South Africa. Two new species are described, namely Paraconiothyrium variabile sp. nov. on Prunus persica and Prunus salicina from South Africa, on Actinidia spp. from Italy and on Laurus nobilis from Turkey, and Paraconiothyrium africanum sp. nov. on Prunus persica from South Africa. Although other known species of Paraconiothyrium commonly produce aseptate conidia, those of P. africanum and P. hawaiiense comb. nov. are predominantly two-celled
Detection of hybridization and species identification in domesticated and wild quails using genetic markers
Amaral, A.J. ; Silva, A.B. ; Grosso, A.R. ; Chikhi, L. ; Bastos-Silveira, C. ; Dias, D. - \ 2007
Folia Zoologica 56 (2007)3. - ISSN 0139-7893 - p. 285 - 300.
coturnix-c.-japonica - multilocus genotype data - population-structure - mitochondrial genome - dna - primers - number - individuals - vertebrates - simulation
Hybridization is particularly widespread in birds and can affect species status and recovery. The common quail Coturnix coturnix is a protected game species that has undergone significant population decrease due to habitat changes. The release of Japanese quail C. japonica and or hybrids for restocking has been occurring since the 1970¿s. Both species have not developed reproductive isolating mechanisms and hybridization is occurring. Species distinction based on morphology and male callings is difficult. In this work cytochrome b gene and five microsatellite loci were used with the aim of establishing an identification test for quails sampled in Portugal. Cytochrome b gene revealed to be of promising use to identify the quail maternal lineage. Success in species assignment with the studied microsatellite loci was moderate to identify samples with suspicion of being hybrids with common quail maternal lineage.
Susceptibility of human and probiotic Bifidobacterium spp. to selected antibiotics as determined by the Etest method
Matto, J. ; Hoek, A.H.A.M. van; Domig, K.J. ; Saarela, M. ; Flórez, A.B. ; Brockmann, E. ; Amtmann, E. ; Mayo, B. ; Aarts, H.J.M. ; Danielsen, M. - \ 2007
International Dairy Journal 17 (2007)9. - ISSN 0958-6946 - p. 1123 - 1131.
human gastrointestinal-tract - antimicrobial susceptibility - resistance genes - intestinal bifidobacteria - tetracycline resistance - rapid identification - bacteria - differentiation - products - primers
This study reports the antibiotic susceptibility of 203 strains representing human or probiotic associated Bifidobacterium species as determined by the Etest method. Strains showing minimum inhibitory concentration (MIC) for tetracycline >= 16 mu g mL(-1) were detected in all studied Bifidobacterium species/groups (prevalence 4-18%). Mostly the high MICs could be associated with a tet gene, although the correspondence between the phenotypic susceptibility and detection of let determinants was not complete. In addition to tet(W), tet(O), which has not been previously described in bifidobacteria, was detected. Occasional erythromycin (2%) and/or clindamycin (5%) resistant strains were found, while the strains were uniformly susceptible to ampicillin and vancomycin. Bifidobacterium strains displayed,generally high MICs for streptomycin and gentamicin suggesting intrinsic resistance. The Etest on lactic acid bacteria susceptibility test medium supplemented with cysteine proved to be an applicable technique for antimicrobial susceptibility testing of bifidobacteria, and is especially useful when susceptibility data of individual strains is needed. (c) 2007 Elsevier Ltd. All rights reserved.
Development of PCR-based detection methods for the quarantine phytopathogen Synchytrium endobioticum, causal agent of wart disease
Boogert, P.H.J.F. van den; Gent-Pelzer, M.P.E. van; Bonants, P.J.M. ; Boer, S.H. de; Wander, J.G.N. ; Lévesque, C.A. ; Leeuwen, G.C.M. van; Baayen, R.P. - \ 2005
European Journal of Plant Pathology 113 (2005)1. - ISSN 0929-1873 - p. 47 - 57.
real-time pcr - spongospora-subterranea - resting sporangia - soil - tubers - quantification - primers - solani - dna
Abstract PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation (HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a 472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps. A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according to EPPO guidelines
PCR detection of oxytetracycline resistance genes from diverse habitats in total community DNA and in streptomycete isolates.
Nikolakopoulou, T.L. ; Egan, S. ; Overbeek, L.S. van; Guillaume, G. ; Heuer, H. ; Wellington, E.M.H. ; Elsas, J.D. van; Collard, J.M. ; Smalla, K. ; Karagouni, A.D. - \ 2005
Current Microbiology 51 (2005)4. - ISSN 0343-8651 - p. 211 - 216.
environmental bacteria - efflux protein - mycobacterium - prevalence - validation - plasmids - clusters - rimosus - primers - soil
A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (TcR) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of TcR streptomycetes originated from bulk and rhizosphere soil. Fewer TcR streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene¿carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which TcR streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common
Molecular identification of ectomycorrhizal mycelium in soil horizons
Landeweert, R. ; Leeflang, P. ; Kuyper, T.W. ; Hoffland, E. ; Rosling, A. ; Wernars, K. ; Smit, E. - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 327 - 333.
gradient gel-electrophoresis - environmental-samples - fungal communities - ribosomal-rna - dna extraction - forest soil - diversity - primers - growth - amplification
Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (greater than or equal to99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had greater than or equal to98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.
Resolving relationships over a wide taxonomic range in Delphacidae (Homoptera) using the COI gene
Dijkstra, E.G.M. ; Rubio, J.M. ; Post, R.J. - \ 2003
Systematic Entomology 28 (2003). - ISSN 0307-6970 - p. 89 - 100.
mitochondrial-dna - spectral-analysis - cytochrome-b - sequences - organization - hemiptera - oxidase - primers - trees - code
Using a combination of different methods to investigate the suitability of a fragment of the cytochrome c oxidase I gene (COI), we succeeded in partially resolving phylogenetic relationships in Delphacidae from the level of species to subfamily. Spectral analysis applied to the relatively noisy COI data proved to be especially useful. It clearly showed when phylogenetic signals were not completely randomized and it was very helpful for identifying problem areas in the dataset. Relationships among the four sampled subfamilies were completely resolved. In contrast to the tree based on morphological characters, we found evidence that Asiraca and Ugyops are sister groups (supporting monophyly of Asiracinae) and that Stenocraninae are the sister group of Kelisiinae. Contradictory signals were observed within Delphacini, but there are characters that support a close relationship between Conomelus and Megamelus. Other than this, the COI data gave support for the monophyly of Kelisiinae, Delphacinae, Chloriona and Javesella. Although third codon positions may appear to be saturated within the 'modern' Delphacidae (Delphacini), they still contain important phylogenetic signals at the deepest taxonomic level. The easiest explanation for this is the difference in amino acid usage between Asiracinae and non-Asiracinae. Overall, this fragment of the COI gene seems to be useful for a rather wide taxonomic range in Delphacidae, except maybe for resolving generic relationships in the large tribe Delphacini.