Bacterial endophyte communities of two wheatgrass varieties following propagation in different growing media

2012 ◽  
Vol 58 (1) ◽  
pp. 67-80 ◽  
Author(s):  
D. Ringelberg ◽  
K. Foley ◽  
C.M. Reynolds
Keyword(s):  
Military Training ◽  
Pcr Amplification ◽  
Mature Plant ◽  
Plant Tissues ◽  
Growing Media ◽  
Bacterial Endophyte ◽  
16S Rdna Pcr ◽  
Culture Independent ◽  
Slender Wheatgrass ◽  
Root Tissues

Bacterial endophyte communities of two wheatgrass varieties currently being used in the revegetation of military training ranges were studied. Culturable and direct 16S rDNA PCR amplification techniques were used to describe bacterial communities present in Siberian and slender wheatgrass seeds, leaf tissues, and root tissues following propagation in either sand or a peat-based growing mix. Our hypothesis was that the resulting plant endophytic communities would be distinct, showing not only the presence of endophytes originating from the seed but also the characteristics of growth in the two different growing media. Both culture and culture-independent assays showed the likely translocation of Actinobacteria, Firmicutes, and Gammaproteobacteria from seed to mature plant tissues as well as subsequent colonization by exogenous organisms. Statistical analysis of 16S terminal restriction fragment profiles identified growing media as having a greater significant effect on the formation of the endpoint endophytic communities than either plant tissue or wheatgrass variety. In silico digests of the ribosomal database produced putative identifications indicating an increase in overall species diversity and increased relative abundances of Firmicutes and Cyanobacteria following propagation in sand and Betaproteobacteria following propagation in the peat-based growing mix. Results indicated a substantial translocation of endophytes from seed to mature plant tissues for both growing media and that growing medium was a dominant determinant of the final taxonomy of the endpoint plant endophytic communities.

scholarly journals Effect of Selenium-Arabinogalactan Nanocomposite on the Colonization of Potato Plants in vitro by the Ring Rot Pathogen

2020 ◽  
Vol 32 ◽  
pp. 3-17
Author(s):  
A. I. Perfileva ◽  
◽  
O. A. Nozhkina ◽  
I. A. Graskova ◽  
N. S. Zabanova ◽  
...  
Keyword(s):  
Shoot Apex ◽  
Plant Tissues ◽  
Similar Data ◽  
Potato Root ◽  
Potato Plants ◽  
Ring Rot ◽  
Negative Effect ◽  
Pathogen Colonization ◽  
Root Tissues

It has been previously shown that the chemically synthesized nanocomposite of selenium with arabinogalactan (NC Se/AG) is characterized by antibacterial effect upon the agent of ring rot – gram-positive bacterium Clavibacter sepedonicus (Cms), with the NC Se/AG having no negative effect on potato plants. In the present paper, it has been found that, 1 hour after the treatment of the NC Se/AG, a substantial elevation of lipid peroxidation products was observed in potato root tissues. This supports earlier results on the increase in reactive oxygen species (ROS) production in potato root tissues under the influence of NC Se/AG. It is proposed that the increased ROS content in potato may inhibit pathogen colonization of plants. This has been tested by seeding homogenised plant tissues of various potato zones (roots, stems, shoot apex zone) onto the nutrient medium. In plants infected with Cms and untreated with the NC, the number of colony forming units (CFUs) of Cms has been shown to be numerous both in potato culture medium and in root and stem tissues. In shoot apex zone of such plants, it has been revealed, bacteria also present, but in smaller quantities. Similar data have been obtained by seeding homogenised tissues from roots and stems of potato plants treated with the NC followed by infection with Cms. However, seeding from shoot apex zones of the plants has been given 4 times less CFUs than from potato plants not treated with the NC. The effect of the NC Se/AG upon the pathogen colonization of plants appears to depend on the titre of the microorganism. In shoot apex zone of plants, characterized with small number of CFUs of Cms, the pathogen growth has been decreased. For the first time, Cms bacteria in potato plant tissues in vitro have been visualized with the aid of scanning microscopy.

scholarly journals LAMBDR: Long-range amplification and Nanopore sequencing of the Mycobacterium bovis direct-repeat region. A novel method for in-silico spoligotyping of M. bovis directly from badger faeces

2019 ◽  
Author(s):  
R.S. James ◽  
E.R. Travis ◽  
A. D. Millard ◽  
P.C. Hewlett ◽  
L. Kravar-Garde ◽  
...  
Keyword(s):  
Long Range ◽  
Mycobacterium Bovis ◽  
Pcr Amplification ◽  
Direct Repeat ◽  
Repeat Region ◽  
Culture Independent ◽  
Long Read ◽  
Novel Method ◽  
Long Range Pcr ◽  
Template Dna

AbstractThe environment is an overlooked source of Mycobacterium bovis, the causative agent of bovine TB. Long read, end to end sequencing of variable repeat regions across the M. bovis genome was evaluated as a method of acquiring rapid strain level resolution directly from environmental samples. Eight samples of M. bovis, two BCG strains (Danish and Pasteur), and a single M. tuberculosis type culture (NCTC 13144) were used to generate data for this method. Long range PCR amplification of the direct repeat region was used to synthesize ∼5kb template DNA for onward sequence analysis. This has permitted culture independent identification of M. bovis spoligotypes present in the environment. Sequence level analysis of the direct repeat region showed that spoligotyping may underestimate strain diversity due to the inability to identify both SNPs and primer binding mutations using a biotinylated hybridisation approach.

scholarly journals Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis

2003 ◽  
Vol 69 (1) ◽  
pp. 220-226 ◽  
Author(s):  
R. Temmerman ◽  
I. Scheirlinck ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Human Consumption ◽  
Probiotic Potential ◽  
Conventional Culture ◽  
Freeze Dried ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis ◽  
Culture Dependent

ABSTRACT In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.

scholarly journals Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

2000 ◽  
Vol 66 (10) ◽  
pp. 4372-4377 ◽  
Author(s):  
Bo Normander ◽  
Jim I. Prosser
Keyword(s):  
Community Composition ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Populations ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

ABSTRACT An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.

Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis

2005 ◽  
Vol 54 (11) ◽  
pp. 1031-1035 ◽  
Author(s):  
Niclas Grahn ◽  
Mounira Hmani-Aifa ◽  
Karin Fransén ◽  
Peter Söderkvist ◽  
Hans-Jürg Monstein
Keyword(s):  
16S Rdna ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Limited Information ◽  
Rdna Sequences ◽  
Biopsy Specimens ◽  
16S Rdna Pcr ◽  
H Pylori ◽  
Species Specific ◽  
Pyrosequencing Analysis

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27 %). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes’ classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens.

scholarly journals Beringian Paleoecology Inferred from Permafrost-Preserved Fungal DNA

2005 ◽  
Vol 71 (2) ◽  
pp. 1012-1017 ◽  
Author(s):  
Magnus C. Lydolph ◽  
Jonas Jacobsen ◽  
Peter Arctander ◽  
M. Thomas P. Gilbert ◽  
David A. Gilichinsky ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Frozen Soil ◽  
Ice Age ◽  
Rrna Genes ◽  
Parasitic Fungi ◽  
Northeastern Siberia ◽  
Culture Independent ◽  
Severe Change ◽  
18S Rrna Genes

ABSTRACT The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.

scholarly journals Interactive effect of sulphur and lead on the growth of muskmelon (Cucumis melo L.) seedling

2000 ◽  
Vol 6 (1) ◽  
Author(s):  
A. El. B. Ragab
Keyword(s):  
Growth Medium ◽  
Cucumis Melo ◽  
Lead Concentration ◽  
Interactive Effect ◽  
Plant Tissues ◽  
Pb Toxicity ◽  
Root Absorption ◽  
Root Tissues ◽  
Cucumis Melo L ◽  
Pb Concentration

The effect of sulphur and lead on growth of muskmelon (Cucmis melo L.) sweet ananas was measured under plastic tunnel. Lead (Pb) was applied at the rate of 0, 75, 300, 600 and 1200 pg/g of soil in factorial combination with treatments of 0, 48, 80, 112 and 224 mg S/kg soils. Muskmelon growth was reduced by increasing Pb concentration and/or by removing S from the growth medium. Lead concentration in plant tissues increased linearly with increasing Pb concentration in the growth medium. Increasing S concentration in solution reduced the effects of the 75-1200 pg Pb /g soil treatments. Increasing S concentration in soil increased shoot and root growth at high levels of Pb. Increased levels of S in the growth medium decreased Pb concentration in shoot and root tissues and increased growth of muskmelon. The reduction in Pb toxicity at high S supply may be due to reduced root absorption of Pb.  

A universal method for direct PCR amplification of plant tissues

2017 ◽  
Vol 9 (11) ◽  
pp. 1800-1805 ◽  
Author(s):  
Yuping Li ◽  
Huanhuan Zhao ◽  
Xuefen Yan ◽  
Meng Li ◽  
Peng Chen ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Dna Purification ◽  
Plant Tissues ◽  
Universal Method ◽  
Direct Pcr ◽  
Modern Biology

PCR is a vital tool in modern biology; however, it can be costly owing to the price of commercial DNA purification kits.

Interactomics: Development of an efficient and improved Agrobacterium tumefaciens-mediated transformation method for transient expression of heterologous protein in recalcitrant plant tissues in planta

2020 ◽  
Author(s):  
Nurhafizah Dahniar Affandi ◽  
Nur Hikmah Mostaffa ◽  
Aisyafaznim Al-Idrus
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Western Blot ◽  
Plant Pathogen ◽  
Heterologous Protein ◽  
Affinity Column ◽  
Plant Tissues ◽  
Plant Proteins ◽  
Sds Page ◽  
Plant Pathogen Interaction ◽  
Root Tissues

Abstract Background: Since more researches leaned towards a holistic approach of “omics” to understand PPI, interactomics studies pivoting on AP-MS approach are getting popular amongst plant scientists. However, to date, AP-MS approach has yet to be adopted to study plant-pathogen interaction even in model plants, hence, the needs to design and develop an optimised transformation procedure for successful isolation of pathogen-targeted plant proteins.Methods: Banana plants were grown in soil or hydroponic media for 2-3 months before subjected to transformation procedure. As a bait, m16D10 , a mature parasitism gene isolated from Meloidogyne incognita was transiently expressed in banana root tissues cv. Grand naine (ITC1256) to prey for interacting plant proteins. Subsequently, the plant-pathogen interacting protein complex was “pulled” at the purification step using histidine affinity column, electrophoresed in SDS-PAGE gel and subjected to western blot assay.Results: We first compared Agrobacterium tumefaciens -mediated transformation quality on root tissues grown in soil and hydroponic media and found that the latter yielded higher area of transformed tissues (99% - 100%). We found hydroponic-grown root tissues were amenable to syringe-based agroinfiltration method and required lesser acclimatisation period compared to the soil-grown root tissues. Our result showed that western blot minimum detection limit of heterologous protein amount in the purified root protein product was at 1:1 ratio, with minimum concentration of 9 mg/mL of purified protein. Using these parameters, western blot assay confirmed the expression of m16D10 gene in transformed plant root tissues and SDS-PAGE gel revealed that we had isolated putative plant proteins interacting with the bait protein.Conclusion: An improved, efficient, and cost-effective Agrobacterium tumefaciens -mediated transformation protocol was developed for transient expression of heterologous protein in recalcitrant plant tissues, suitable for AP-MS-based plant-pathogen interaction studies. Confirming our hypothesis, we demonstrated that the improved method had successfully “pulled-out” banana root proteins interacting with m16D10 protein using histidine affinity column.

Examining a synchrotron-based approach for in situ analyses of Al speciation in plant roots

2020 ◽  
Vol 27 (1) ◽  
pp. 100-109
Author(s):  
Zhigen Li ◽  
Peng Wang ◽  
Neal W. Menzies ◽  
Brigid A. McKenna ◽  
Chithra Karunakaran ◽  
...  
Keyword(s):  
Plant Tissues ◽  
Tetrahedral Coordination ◽  
Fagopyrum Tataricum ◽  
Edge Structure ◽  
X Ray ◽  
Al Speciation ◽  
The Common ◽  
Root Tissues ◽  
X Ray Absorption

Aluminium (Al) K- and L-edge X-ray absorption near-edge structure (XANES) has been used to examine Al speciation in minerals but it remains unclear whether it is suitable for in situ analyses of Al speciation within plants. The XANES analyses for nine standard compounds and root tissues from soybean (Glycine max), buckwheat (Fagopyrum tataricum), and Arabidopsis (Arabidopsis thaliana) were conducted in situ. It was found that K-edge XANES is suitable for differentiating between tetrahedral coordination (peak of 1566 eV) and octahedral coordination (peak of 1568 to 1571 eV) Al, but not suitable for separating Al binding to some of the common physiologically relevant compounds in plant tissues. The Al L-edge XANES, which is more sensitive to changes in the chemical environment, was then examined. However, the poorer detection limit for analyses prevented differentiation of the Al forms in the plant tissues because of their comparatively low Al concentration. Where forms of Al differ markedly, K-edge analyses are likely to be of value for the examination of Al speciation in plant tissues. However, the apparent inability of Al K-edge XANES to differentiate between some of the physiologically relevant forms of Al may potentially limit its application within plant tissues, as does the poorer sensitivity at the L-edge.

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