scholarly journals Visualization of ‘Candidatus Liberibacter asiaticus’ Cells in Citrus Seed Coats with Fluorescence In Situ Hybridization and Transmission Electron Microscopy

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
M.E. Hilf ◽  
K.R. Sims ◽  
S.Y. Folimonova ◽  
D.S. Achor
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
In Situ Hybridization ◽  
Candidatus Liberibacter Asiaticus ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Seed ◽  
Seed Coats

scholarly journals Visualization of ‘Candidatus Liberibacter asiaticus’ Cells in the Vascular Bundle of Citrus Seed Coats with Fluorescence In Situ Hybridization and Transmission Electron Microscopy

2013 ◽  
Vol 103 (6) ◽  
pp. 545-554 ◽  
Author(s):  
Mark E. Hilf ◽  
Kenneth R. Sims ◽  
Svetlana Y. Folimonova ◽  
Diann S. Achor
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Candidatus Liberibacter Asiaticus ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Seed ◽  
Seed Coats

‘Candidatus Liberibacter asiaticus’ is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for ‘Ca. L. asiaticus’ were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the ‘Ca. L. asiaticus’ 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to ‘Ca. L. asiaticus’. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no ‘Ca. L. asiaticus’ sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as ‘Ca. L. asiaticus’, providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were ‘Ca. L. asiaticus’.

scholarly journals Colonization of Citrus Seed Coats by ‘Candidatus Liberibacter asiaticus’: Implications for Seed Transmission of the Bacterium

2011 ◽  
Vol 101 (10) ◽  
pp. 1242-1250 ◽  
Author(s):  
Mark E. Hilf
Keyword(s):  
Sweet Orange ◽  
Seed Transmission ◽  
Candidatus Liberibacter Asiaticus ◽  
Real Time Quantitative Pcr ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Varieties ◽  
Citrus Seed ◽  
Pathogen Dna ◽  
Seed Coats

Huanglongbing is an economically damaging disease of citrus associated with infection by ‘Candidatus Liberibacter asiaticus’. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of ‘Ca. Liberibacter asiaticus’ DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from ‘Sanguenelli’ sweet orange and ‘Conners’ grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from ‘Sanguenelli’ and from ‘Conners’ fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of ‘Sanguenelli’ and ‘Conners’, respectively. Small amounts of pathogen DNA were detected in 10% of ‘Sanguenelli’ seedlings grown in the greenhouse, but in none of 204 extracts from ‘Conners’ seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of ‘Sanguenelli’ and ‘Conners’ which were germinated on agar, and in 5% of ‘Sanguenelli’ but in none of 164 ‘Conners’ seedlings which grew from these seeds on agar. No pathogen DNA was detected in ‘Ridge Pineapple’ tissue at 3 months post-grafting onto ‘Sanguenelli’ seedlings, even when pathogen DNA had been detected initially in the ‘Sanguenelli’ seedling. Though the apparent colonization of ‘Conners’ seeds was more extensive and nearly uniform compared with ‘Sanguenelli’ seeds, no pathogen DNA was detected in ‘Conners’ seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.

scholarly journals Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy.

1995 ◽  
Vol 43 (7) ◽  
pp. 665-674 ◽  
Author(s):  
M V Macville ◽  
A G Van Dorp ◽  
K C Wiesmeijer ◽  
R W Dirks ◽  
J A Fransen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
In Situ Hybridization ◽  
Messenger Rna ◽  
Detection Efficiency ◽  
Rna Detection ◽  
Ultrastructural Morphology ◽  
Transmission Electron ◽  
Contrast Microscopy

We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti-digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH.

Epitaxy and texture analysis of Bi-Sr-Ca-Cu-O thin films on (100) MgO using Transmission Electron Microscopy

1990 ◽  
Vol 48 (4) ◽  
pp. 68-69
Author(s):  
J. T. Sizemore ◽  
D. G. Schlom ◽  
Z. J. Chen ◽  
J. N. Eckstein ◽  
I. Bozovic ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Transmission Electron Microscopy ◽  
Critical Currents ◽  
Unit Cell ◽  
X Ray Diffraction ◽  
Cell Axis ◽  
Transmission Electron ◽  
Synthesis Procedure

Investigators observe large critical currents for superconducting thin films deposited epitaxially on single crystal substrates. The orientation of these films is often characterized by specifying the unit cell axis that is perpendicular to the substrate. This omits specifying the orientation of the other unit cell axes and grain boundary angles between grains of the thin film. Misorientation between grains of YBa2Cu3O7−δ decreases the critical current, even in those films that are c axis oriented. We presume that these results are similar for bismuth based superconductors and report the epitaxial orientations and textures observed in such films.Thin films of nominally Bi2Sr2CaCu2Ox were deposited on MgO using molecular beam epitaxy (MBE). These films were in situ grown (during growth oxygen was incorporated and the films were not oxygen post-annealed) and shuttering was used to encourage c axis growth. Other papers report the details of the synthesis procedure. The films were characterized using x-ray diffraction (XRD) and transmission electron microscopy (TEM).

Transmission Electron Microscopy of Epitaxial silicides grown by ultra high vacuum deposition

1989 ◽  
Vol 47 ◽  
pp. 462-463
Author(s):  
D. Loretto ◽  
J. M. Gibson ◽  
S. M. Yalisove
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Diffraction ◽  
High Vacuum ◽  
High Energy ◽  
Energy Electron ◽  
Ultra High Vacuum ◽  
Ex Situ ◽  
Transmission Electron

The silicides CoSi2 and NiSi2 are both metallic with the fee flourite structure and lattice constants which are close to silicon (1.2% and 0.6% smaller at room temperature respectively) Consequently epitaxial cobalt and nickel disilicide can be grown on silicon. If these layers are formed by ultra high vacuum (UHV) deposition (also known as molecular beam epitaxy or MBE) their thickness can be controlled to within a few monolayers. Such ultrathin metal/silicon systems have many potential applications: for example electronic devices based on ballistic transport. They also provide a model system to study the properties of heterointerfaces. In this work we will discuss results obtained using in situ and ex situ transmission electron microscopy (TEM).In situ TEM is suited to the study of MBE growth for several reasons. It offers high spatial resolution and the ability to penetrate many monolayers of material. This is in contrast to the techniques which are usually employed for in situ measurements in MBE, for example low energy electron diffraction (LEED) and reflection high energy electron diffraction (RHEED), which are both sensitive to only a few monolayers at the surface.

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