Enhydro agates are round to egg-shaped nodules composed of banded microcrystalline to cryptocrystalline quartz that have a water-filled internal cavity. Enhydro agates form as agate material precipitates from silica-rich groundwater flowing through volcanic rock to form concentric layers inside vesicles. As vesicles fill with successive agate layers, portions of groundwater from which the agate precipitates can become sealed in the cavity, thus preserving a volume of ancient water inside the agate. Microorganisms present in groundwater at the time of agate formation were trapped in the agate’s internal cavity producing a “microbial time capsule.” These microbes survived by adapting to near-starvation conditions at a subsistence level.
Enhydro agates used in this study are from the agate geode deposits in volcanic rocks of the Serra Geral Magmatic Province, Rio Grande do Sul, Brazil. Each enhydro agate was aseptically drilled to obtain a sample of trapped water. Direct microscopic analysis of extracted water revealed cell-like shapes including small (~ 1μm) diplococcoid and coccobacillus forms displaying erratic movement consistent with bacterial motility.
Basalt geochemistry and SEM/EDX analyses of agate accessory minerals (manganite) were used to develop a chemoautotrophic enrichment media that simulated the natural environment of groundwater microbes in a volcanic province. A basal inorganic medium amended with supplements including olivine, basalt, and Fe- and Mn-oxyhydroxides were used for initial enrichments at various temperatures under a CO2 atmosphere. Based on growth in the enrichments, cultures were transferred to a complex medium supplemented with olivine. Microscopy yielded Gram-negative motile cells of pleomorphic nature that ranged from rod-shaped to ovoid (~ 1-5μm).
Pure cultures were subjected to DNA isolation and PCR amplification of 16S rRNA genes. (BLAST) DNA sequence analysis results of isolates using GenBank indicate the bacterial DNA are most closely related to the Alphaproteobacterium Phyllobacterium myrsinacearum. These species are most commonly found in the soil rhizosphere associated with the roots of plants native to tropical regions. Related Phyllobacterium sp. have also been discovered associated with basaltic lava flows in caves and catacombs of Europe.
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December 1994 · Molecular and Biochemical Parasitology
A monoclonal antibody, 7A9, specific for antigens in the subventral esophageal glands of adult female Meloidogyne incognita and for antigens in the longitudinal muscles of second-stage juveniles, was used to isolate a clone from a M. incognita cDNA expression library. The corresponding genomic DNA was isolated by hybridization and the gene designated sec-1. DNA sequence analysis of sec-1 revealed
... [Show full abstract] the presence of 9 introns having structural similarities to introns from the free-living nematode Caenorhabditis elegans. The sec-1 message was also trans-spliced and the leader sequence differed in one position from the sequence of the C. elegans trans-spliced leader SL1. Sequences analogous to sec-1 were specific to the genus Meloidogyne, and regulation of sec-1 expression did not involve differences in overall rates of transcript accumulation. The deduced amino acid sequence of the protein encoded by sec-1 had some similarities to the rod portions of several myosin heavy chains. Read more Article
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April 2018 · Plant Disease
Current detection methodologies for Agrobacterium vitis, causing crown gall of grapevines, are time intensive and lack the ability to quantify pathogen abundance in nursery stock and soil. Information on pathogen abundance is a key component to develop management strategies. The aim of this study was to develop a rapid and sensitive quantification assay for grapevine nursery stock and vineyard
... [Show full abstract] soil via droplet digital polymerase chain reaction targeting the virA gene. DNA isolated from roots of dormant grapevines originating from nurseries in Germany, California, and Ontario were tested for virA abundance. Bacterial numbers varied with grapevine origin; plants from California had the highest numbers. In addition, rhizosphere soil from two vineyards in the Okanagan valley in British Columbia was tested over a growing season. Sampling time during the season did not affect virA gene abundance. The older vineyard had higher soil A. vitis populations than the younger vineyard. The assay developed here has potential for use in national clean plant programs to prevent import of infected grapevine nursery stock and to test vineyard soil for abundance of the pathogen before planting. View full-text February 2018 · Plant Disease
Apple (Malus domestica) is an important fruit crop in the Netherlands, with a total production area of 7600 ha in 2016, and 1600 ha of fruit tree nurseries. For decades fruit growers and nurseries have reported a leaf blotch, characterized by irregular light brown spots, bordered by a dark brown to purple margin, and premature leaf drop, mainly on ‘Golden Delicious’. The disease causes severe
... [Show full abstract] defoliation, which may lead to almost complete defoliation. Leaf blotch typically appears in early July and is most severe in the latter part of the growing season. In a survey in August 2014 affected ‘Golden Delicious’ leaves were collected from different orchards and taken to the laboratory. Next, 20 leaves per location were rinsed with sterile water, sprayed with 70% ethanol until droplet runoff and rinsed again with sterile water, and tissue sections were excised from lesions using a sterile scalpel blade and placed onto Potato Dextrose Agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were transferred to fresh PDA plates. These isolates produced fast-growing colonies of irregular shape, tan brown to black and felty. Sporulation patterns showed long conidiophores with extensive terminal branching. Conidia were ovoid with a tapering apical beak and a size range of 10 to 30 × 5 to 10 μm, with 1 to 5 septa. The isolated fungi were morphologically identical to small spored Alternaria spp. (Simmons 2007). However, small spored Alternaria spp. cannot be identified based on morphological characteristics (Woudenberg et al. 2015). The identity of 12 representative isolates from two different orchards in their 4th growing season, and located in the central part of the Netherlands was determined by multi-locus gene sequencing. To this end, genomic DNA was extracted using the UltraCleanTM Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA). Sequences of the ITS region, the endoPG gene and the anonymous region OPA10-2 locus were amplified and sequenced as described in Woudenberg et al. (2013, 2015) and deposited under GenBank accession numbers MG744449-MG744460 (ITS), MG744473-MG744484 (endoPG), and MG744461-MG744472 (OPA10-2). MegaBLAST analysis revealed that our ITS, endoPG and OPA10-2 sequences matched with >99%-100% identity to A. arborescens species complex (SC) isolates in GenBank (AF347033 & KP124400 (ITS), AY295028 & KP124104 (endoPG) and KP124712 & KP124714 (OPA10-2)). Subsequently, Koch's postulates for three A. arborescens SC isolates were performed in the laboratory on ‘Golden Delicious’ leaves. Surface sterilized leaves were inoculated on the abaxial side with 10 μl of a suspension of 10^5 conidiospores/ml water, prepared from a 14-day-old PDA culture, after wounding with a needle, with four inoculations per leaf. Inoculated leaves were sealed in a plastic box and incubated in darkness at 20°C. The experiment was carried out in five replicates. Symptoms appeared within 7 days on 100% of the leaves, while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions cultured on PDA morphologically resembled the original isolate from the infected leaves. The identity of the re-isolations was confirmed as A. arborescens SC by sequencing. To our knowledge, this is the first report of Alternaria arborescens SC causing leaf blotch and subsequent premature leaf drop of apple cultivars in the Netherlands. Multiple Alternaria species groups are associated with leaf blotch diseases of apple in Australia and Italy (Harteveld et al. 2012; Rotondo et al. 2012). Alternaria arborescens-like isolates are most prevalent in Australia and are mostly associated with leaf blotch symptoms (Harteveld et al. 2012). Read more Article
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March 1996 · Microbiology
The bacterial mercury resistance determinant carried on the IncJ plasmid pMERPH has been characterized further by DNA sequence analysis. From the sequence of a 4097 bp Bg/II fragment which confers mercury resistance, it is predicted that the determinant consists of the genes merT, merP, merC and merA. The level of DNA sequence similarity between these genes and those of the mer determinant of
... [Show full abstract] Tn21 was between 56 center dot 4 and 62 center dot 4%. A neighbour-joining phylogenetic tree of merA gene sequences was constructed which suggested that pMERPH bears the most divergent Gram-negative mer determinant characterized to date. Although the determinant from pMERPH has been shown to be inducible, no regulatory genes have been found within the Bg/II fragment and it is suggested that a regulatory gene may be located elsewhere on the plasmid. The cloned determinant has been shown to express mercury resistance constitutively. Analysis of the pMERPH mer operator/promoter (O/P) region in vivo has shown constitutive expression from the mer PTCPA promoter, which could be partially repressed by the presence of a trans-acting MerR protein from a Tn21-like mer determinant. This incomplete repression of mer PTCPA promoter activity may be due to the presence of an extra base between the -35 and -10 sequences of the promoter and/or to variation in the MerR binding sites in the O/P region. Expression from the partially repressed mer PTCPA promoter could be restored by the addition of inducing levels of Hg2+ ions. Using the polymerase chain reaction with primers designed to amplify regions in the merP and merA genes, 1 center dot 37 kb pMERPH-like sequences have been amplified from the IncJ plasmid R391, the environmental isolate SE2 and from DNA isolated directly from non-cultivated bacteria in River Mersey sediment. This suggests that pMERPH-like sequences, although rare, are nevertheless persistent in natural environments. View full-text Article
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January 2007 · Pakistan Veterinary Journal
The myogenic factors (MYF) 5 and 6 are integral to the initiation and development of skeletal muscles and to the maintenance of their phenotypes. Thus, they are candidate genes for growth and meat quality-related traits. The MYF5 gene is expressed during proliferation of myoblasts and comprises 3 exons: 500, 76 and 191 bp long. Genomic DNA was isolated from the camel hair using NucleoSpin Tissue
... [Show full abstract] kit. Two animals of each of the six breeds namely, Marecha, Dhatti, Larri, Kohi, Sakrai and Cambelpuri were used for sequencing. For PCR amplification of the gene, a primer pair was designed from homolog regions of already published sequences of farm animals from GenBank. Results showed that exon 1 comprising of 422 bp of the dromedary MYF5 gene was more homologous (94%) to the cattle than the dog and human. However, phylogram showed that a small number of mutations had been experienced by dromedary camels at their MYF5 gene and was more near to human than other farm animals. View full-text January 2012 · World Applied Sciences Journal
The present study dealt with the identification of a bacterium having dominance in earthworm (Lennogaster pusillus) midden by 16S rDNA based genomic analysis. The bacterium identified was Aeromonas punctata strain JM10 (Genbank Accession No: GU205197.1). Isolated DNA of the bacterium showed amplicon band of 1500 bp, when resolved on agarose gel. Amplification of the 16S rDNA gene from the
... [Show full abstract] Aeromonas sp. was done with forward primer having 814 bp and reverse primer having 913 bp, which further provide consensus sequence of 1418 bp. The query sequence of 1418 bp matched the alignments scores of >= 200 in the distribution of 100 BLAST hits. About 10 types of homolog strains of Aeromonas punctata were found as its closest relatives on the basis of sequence producing significant alignments. Read more Article
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January 1995 · JOURNAL OF PLANT PATHOLOGY
Bermuda grass (Cynodon dactylon) plants showing symptoms of light brown to blackish leaf spots were observed during spring 2012 in the parks and grassy lawns of Lahore (Pakistan). Samples from infected plants were collected and a fungus consistently isolated on potato dex-trose agar (PDA) at 28±1°C. The fungus was identified as Bipolaris dactylon (telomorph Cochliobolus australiensis) based on
... [Show full abstract] its morphological and molecular features. The pathogen identity was verified by the First Fungal Culture Bank of Pakistan (FCBP) and a culture deposited (accession No. FCBP-1288). In addition, the fungal DNA was isolated and the internally transcribed spacer (ITS) region amplified (White et al., 1990), cloned, sequenced and deposited in GenBank (accession No. HE962033). Sequenc-ing of the ITS region of ribosomal DNA showed a 99.8% nucleotide sequence identity with C. australiensis isolates from Texas (HQ608034) and India (AY923860), respectively. Reproduction of symptoms in inoculated healthy plants fulfilled Koch's postulates and confirmed patho-genicity. B. australiensis had previously been isolated from infected roots of C. dactylon during a survey in Pakistan (Shahzad and Ghaffar, 1995). However, no details were given of foliar symptoms, nor any pathogenicity test was conducted. Therefore, to the best or our knowledge, this is the first confirmed report of B. dactylon causing a leaf spot on Bermudagrass in Pakistan. View full-text January 2007 · Journal of Applied Microbiology
The repetitive extragenic palindromic-PCR (rep-PCR) subtyping technique, which targets repetitive extragenic DNA sequences in a PCR, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA short variable region (SVR) DNA sequence analysis as a tool for molecular epidemiology.
Uprime Dt, Uprime B1 or Uprime RI primers were
... [Show full abstract] utilized to generate gel-based fingerprints from a set of 50 Campylobacter spp. isolates recovered from a variety of epidemiological backgrounds and sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean, of the generated fingerprints demonstrated that the Uprime Dt primers were effective in providing reproducible patterns (100% typability, 99% reproducibility) and at placing isolates into epidemiological relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under nonselective, short-term transfer conditions revealed a Pearson's correlation approaching 99%. These same 50 Campylobacter spp. isolates were analysed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships.
The Uprime Dt primer-generated rep-PCR phenogram was compared with a phenogram generated from flaA SVR DNA sequence analysis of the same isolates. Comparison of the two sets of resulting genomic relationships revealed that both methods segregated isolates into similar groups.
These results indicate that rep-PCR analysis performed using the Mo Bio Ultra Clean Microbial Genomic DNA Isolation Kit for DNA isolation and the Uprime DT primer set for amplification is a useful and effective tool for accurate differentiation of Campylobacter spp. for subtyping and epidemiological analyses. Read more Conference Paper
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November 2016
trabajado durante siete años en el desarrollo de prácticas sostenibles en el cultivo de cebolla. Una parte fundamental de esta estrategia se ha basado en el uso de microorganismos para el control de las principales enfermedades: raíz rosada (Setophoma terrestris) y pudrición blanca (Sclerotium cepivorum). Diferentes microorganismos obtenidos de varias de la zona norte de la provincia de Cartago
... [Show full abstract] han sido aislados. Los mejores resultados se han obtenido usando Trichoderma spp, y de esta especie se ha trabajado con diferentes aislamientos. El objetivo de este trabajo fue realizar una caracterización e identificación molecular de varias de las cepas de Trichoderma probadas en este programa. Los microorganismos fueron conservados en cultivo puro en la colección de microorganismos del del CIB. Se realizaron cultivos líquidos en caldo papa dextrosa, se incubaron durante 4 días a 28º C a 100 r.p.m. Posteriormente se filtró el micelio, se congeló con el uso de nitrógeno líquido y se trituró. El polvo obtenido fue usado para la extracción de ADN. Esta se realizó usando el kit PowerSoil®. Con el ADN y usando como imprimadores el GC-FUNG y NS1 para Dominio Eukarya, se realizó un PCR de punto final con el siguiente perfil térmico: 1 ciclo de desnaturalización inicial (2 minutos, 94ºC), 35 ciclos (desnaturalización, 1 minuto a 94º C; alineamiento, 1minuto a 51.5º C; extensión, 1 minuto a 72º C), un ciclo de extensión final (72º C, 5 minutos) y un ciclo de almacenamiento (4º C, 10 minutos). Los fragmentos obtenidos se visualizaron en un gel de agarosa al 1%. Los productos de PCR fueron secuenciados. Con las secuencias se realizó la identificación molecular de los aislamientos usando la base de datos GenBank por medio de un BLAST. La identificación se resume a continuación: View full-text Article
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August 2005 · World Journal of Gastroenterology
To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments.
DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD) with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious
... [Show full abstract] differences in tumor tissue DNA corresponding to that of normal tissue were separated, purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data.
A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene.
Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcinogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis. View full-text January 2008 · Journal of Arabic Literature
*Agricultural Genetic Engineering Research Institute (AGERI), ARC
**Department of Genetics, Faculty of Agriculture, Cairo University
Leaf (brown) rust caused by Puccinia triticina is a fungal disease of wheat (Triticum aestivum L.) that causes significant yield losses annually in many wheat-growing regions of the world. Host plant resistance is the most economically viable and environmentally
... [Show full abstract] responsible method for controlling Puccinia triticina, the causal agent of leaf rust in wheat. The identification and utilization of new resistance sources is critical to continue the development of improved cultivars. The objective of this work was to identify defense-related genes against rust in the Egyptian rust resistant cultivar Giza168. Specific primers were designed on the basis of converse motifs of cloned resistance genes of the resistance gene analog (RGA) and leaf rust resistance gene (Lr21) in wheat (Triticum aestivum L.). The designed PCR primers were subsequently used for RT-PCR using RNA isolated from a resistant variety to amplify fragments of 445 bp and 235 bp for RGA and Lr21 genes, respectively. The amplified products were cloned, sequenced and submitted to the GenBank. The nucleotide sequences of the amplified fragments were aligned with their corresponding genes using the BLAST. The expressions of the two genes in the infected and healthy plants were studied using RT-PCR. The RGA expression was induced and detected by RT-PCR, which is up-regulated by fungal infection. The Lr21 expression was detected on both healthy and infected plants, although the expression was higher in infected plants. Key words: RGA gene, Lr21 gene, wheat cv. Giza168. Read more Last Updated: 05 Jul 2022
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