Ultrastructural localization of β-(1 → 4)-D-glucans in two pathogenic fungi and in their host tissues by means of an exoglucanase–gold complex

1987 ◽  
Vol 33 (5) ◽  
pp. 405-417 ◽  
Author(s):  
Nicole Benhamou ◽  
Hélène Chamberland ◽  
G. B. Ouellette ◽  
F. J. Pauze
Keyword(s):  
Close Contact ◽  
Middle Lamella ◽  
Pathogenic Fungi ◽  
Ultrastructural Localization ◽  
Dutch Elm Disease ◽  
Gold Complex ◽  
Tomato Root ◽  
Gold Particles ◽  
Infected Tomato ◽  
Crown And Root Rot

An exoglucanase, purified from a cellulase produce by the fungus Trichoderma harzianum Rifai., was successfully bound to colloidal gold and used for ultrastructural detection of intracellular cellulosic β-(1 → 4) glucans. These saccharides were found to be present in great amount in the walls of Ophiostoma ulmi (Buism.) Nannf., the Dutch elm disease agent, whereas they were randomly distributed in the walls of Fusarium oxysporum Schlecht f. sp. radicis-lycopersici Jarvis and Shoemaker (FORL), the agent of tomato crown and root rot. In O. ulmi cell walls, the β-(1 → 4) glucans were predominantly concentrated over the central portion of the inner walls. In both colonized elm wood and infected tomato root tissues, the compound middle lamella and secondary walls of parenchyma cells, fibers (absent in tomato roots), and vessel members were always intensely labeled, but gold particles appeared somewhat irregularly distributed. In fibers having an S3 gelatinous layer, the latter was always preferentially labeled. Penetration of O. ulmi in elm wood tissues resulted in the digestion of host wall areas free of labeling. Such areas were not observed in infected tomato tissues; instead, an accumulation of gold particles was noted along the fungal portion that was in close contact with the host wall. Results of this study confirm the potential value of gold-labeled exoglucanase and provide some new information about wall topochemistry during host-pathogen interactions.

scholarly journals Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes.

1981 ◽  
Vol 29 (4) ◽  
pp. 531-541 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Secretory Granules ◽  
Ultrastructural Localization ◽  
Rnase A ◽  
Gold Complex ◽  
Gold Complexes ◽  
Thin Sections ◽  
Gold Particles ◽  
Enzyme Substrate ◽  
Cellular Compartments ◽  
Cytochemical Technique

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.

Ultrastructural localization in host tissues of a toxic glycopeptide produced by Ophiostoma ulmi, using monoclonal antibodies

1985 ◽  
Vol 63 (7) ◽  
pp. 1185-1195 ◽  
Author(s):  
Nicole Benhamou ◽  
J. G. Lafontaine ◽  
J. R. Joly ◽  
G. B. Ouellette
Keyword(s):  
Monoclonal Antibodies ◽  
Protein A ◽  
Middle Lamella ◽  
Protective Layer ◽  
Ultrastructural Localization ◽  
Wall Layer ◽  
Dutch Elm Disease ◽  
Ophiostoma Ulmi

Monoclonal antibodies against a phytotoxic glycopeptide from Ophiostoma ulmi (Buism.) Nannf., the Dutch elm disease pathogen, were used in a postembedding protein A – gold labeling technique to localize this toxin in experimentally infected tissues of young elm seedlings. Significant labeling was observed over pit membranes, over the innermost wall layer (protective layer) of paratracheal parenchyma cells, and over the intercellular spaces and, adjacently, the middle lamella. Host secondary walls, cytoplasm, and various organelles, except regions of amyloplastids, were free of labeling. From day 1 to day 4 after inoculation an intensification of the labeling reaction was noted that corresponded to an increase in the disease symptoms. This specific and sensitive technique has thus proved to be highly suitable for the in situ identification of antigenic macromolecules in diseased elm tissues. The classification of the toxic glycopeptide among the group of vivotoxins is discussed.

Fungi on leaves, branches and stems of trees and shrubs of Saint Petersburg suburban parks

2017 ◽  
Author(s):  
М.В. Сидельникова ◽  
А.В. Тобиас ◽  
Д.Ю. Власов
Keyword(s):  
Pathogenic Fungi ◽  
Dutch Elm Disease ◽  
City Center ◽  
Saint Petersburg ◽  
Xylotrophic Fungi ◽  
Fungi Infection ◽  
Mycological Examination ◽  
The Moment ◽  
Available Information ◽  
Trees And Shrubs

Проведены микологические обследования древесной и кустарниковой растительности на территории парковой зоны Санкт-Петербурга и пригородов. Сбор материала проводился в парках южных пригородов Санкт-Петербурга (Павловский парк, Екатерининский парк, Нижний сад и Верхний парк Ораниенбаума, Верхний сад и Нижний парк ГМЗ «Петергоф»). В сравнительных целях был обследован парк при Обуховской больнице в центре Санкт-Петербурга. На древесно-кустарниковых породах парковой зоны нами выявлено 230 видов грибов (микро- и макромицетов). На листьях выявлено 28 видов микромицетов, в числе которых возбудители мучнистой росы, ржавчины и пятнистостей. На ветвях и стволах древесных пород выявлено 150 видов микромицетов, среди которых есть как часто встречающиеся, так и редкие виды грибов. Большинство из них обнаруживается в анаморфной стадии. Наибольшее разнообразие и развитие микромицетов отмечено на сухих ветвях. Высокой вредоносностью характеризуются тиростромоз липы и голландская болезнь вязов. Выявлены устойчивые патогенные комплексы грибов, развитие которых приводит к заметному ухудшению состояния растений. На стволах живых и усыхающих деревьев, а также растительных остатках отмечено 52 вида макромицетов. Среди них выявлены доминирующие и редкие виды. Среди источников заражения древесных растений ксилотрофными грибами выделяются отмершие вязы, усохшие стволы которых можно наблюдать как в пригородных парках, так и в центральной части Санкт-Петербурга. Полученные данные существенно расширяют имеющиеся сведения по микобиоте парков Санкт-Петербурга. Mycological examination of tree and shrub vegetation on the territory of Saint Petersburg park zone and its suburbs was conducted. Material was collected in the parks of southern suburbs of Saint Petersburg (Pavlovsk Park, Catherine Park, Lower Garden and Upper Park in Oranienbaum, Upper Garden and Lower Park in Peterhof). For comparative purposes Park of Obukhov Hospital in Saint Petersburg city center was also examined. At the moment, 230 fungi species (micro- and macrofungi) were identified on trees and shrubs of the park zone. Among them, 28 species of microfungi, including powdery mildew, rust and blights pathogens were found on leaves. Also, 150 species of microfungi, including both common and rare fungi species, were found on branches and trunks. Most of them were found in the anamorphic stage. The greatest diversity and microfungi development were noted on dry branches. Thyrostromose of linden and Dutch elm disease are the most harmful. Stable complexes of pathogenic fungi, which development leads to clear decline of plants' condition, were identified. In addition, 52 species of macrofungi, including dominant and rare species, were observed on trunks of living and drying trees and vegetation residues. Among the sources of xylotrophic fungi infection of woody plants, dead elms are the most distinguished. Their dead trunks can be found in both suburban parks and the central part of Saint Petersburg. The presented data significantly expand available information on mycobiota Saint Petersburg parks.

scholarly journals Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface.

1993 ◽  
Vol 121 (1) ◽  
pp. 201-212 ◽  
Author(s):  
G M Bressan ◽  
D Daga-Gordini ◽  
A Colombatti ◽  
I Castellani ◽  
V Marigo ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Chick Embryo ◽  
Culture Medium ◽  
Close Contact ◽  
Corneal Stroma ◽  
Ultrastructural Level ◽  
Elastic Fibers ◽  
Specific Antibodies ◽  
Gold Particles ◽  
Immunofluorescence Studies

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).

scholarly journals Cytological Aspects of Compost-Mediated Induced Resistance Against Fusarium Crown and Root Rot in Tomato

2002 ◽  
Vol 92 (4) ◽  
pp. 424-438 ◽  
Author(s):  
Benoît Pharand ◽  
Odile Carisse ◽  
Nicole Benhamou
Keyword(s):  
Induced Resistance ◽  
Root Rot ◽  
Disease Incidence ◽  
Disease Suppression ◽  
Gold Complex ◽  
Peat Moss ◽  
Fungal Colonization ◽  
Pythium Oligandrum ◽  
Vascular Elements ◽  
Crown And Root Rot

The potential of a pulp and paper mill residues compost for the control of crown and root rot of greenhouse-grown tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici was ultrastructurally investigated. Peat moss amended with compost substantially reduced disease-associated symptoms. Addition of Pythium oligandrum to either peat moss alone or peat moss amended with compost resulted in a considerable reduction in disease incidence compared with controls grown in peat moss alone. Histological and cytological observations of root samples from Fusarium-inoculated plants revealed that the beneficial effect of compost in reducing disease symptoms is associated with increased plant resistance to fungal colonization. One of the most prominent facets of compost-mediated induced resistance concerned the formation of physical barriers at sites of attempted fungal penetration. These structures, likely laid down to prevent pathogen ingress toward the vascular elements, included callose-enriched wall appositions and osmiophilic deposits around the sites of potential pathogen ingress. Invading hyphae, coated by the osmiophilic material, showed marked cellular disorganization. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin was altered in severely damaged hyphae. A substantial increase in the extent and magnitude of the cellular changes induced by compost was observed when P. oligandrum was supplied to the potting substrate. This finding corroborates the current concept that amendment of composts with specific antagonists may be a valuable option for amplifying their beneficial properties in terms of plant disease suppression.

scholarly journals A new lectin-gold complex for ultrastructural localization of galacturonic acids.

1988 ◽  
Vol 36 (11) ◽  
pp. 1403-1411 ◽  
Author(s):  
N Benhamou ◽  
N Gilboa-Garber ◽  
J Trudel ◽  
A Asselin
Keyword(s):  
Binding Sites ◽  
Ricinus Communis ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Gold Complex ◽  
Carbohydrate Binding ◽  
Wheat Coleoptile ◽  
Ultrastructural Studies ◽  
Pectic Substances ◽  
Considerable Loss

We report the development of a cytochemical affinity technique for detection of galacturonic acids at the ultrastructural level. The highly purified gonad lectin from Aplysia depilans (AGL) was tagged with colloidal gold particles and used for labeling carbohydrates in resin-embedded sections of various plant and fungal tissues. Patterns of AGL binding sites were compared to those obtained with a D-galactose-specific lectin, Ricinus communis agglutinin I. Differences in labeling patterns were noted, indicating that the lectins exhibited differential carbohydrate binding. In addition, the considerable loss of labeling over isolated wheat coleoptile walls treated for removal of pectin, after incubation with the AGL-gold complex, strongly suggested an affinity of AGL for pectic substances. A series of cytochemical controls, including sugar inhibition tests, has proven the specificity of the technique and the high affinity of AGL towards galacturonic acids. The potential value of this new lectin for ultrastructural studies on cell wall pectic substances in plant biology and pathology is demonstrated.

scholarly journals Phalloidin-gold complexes: a new tool for ultrastructural localization of F-actin.

1988 ◽  
Vol 36 (9) ◽  
pp. 1197-1202 ◽  
Author(s):  
M Lachapelle ◽  
H C Aldrich
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Colloidal Gold ◽  
Bovine Serum ◽  
Physarum Polycephalum ◽  
Ultrastructural Localization ◽  
Slime Mold ◽  
Gold Complex ◽  
Cell Compartment ◽  
Nuclear Regions

We used a phalloidin-gold complex to study the distribution of F-actin in the myxamoebae and macroplasmodia of the slime mold Physarum polycephalum. After incubation of Lowicryl- or Quetol-embedded specimens with this complex, significantly different labeling intensities were found over the various cytoplasmic and nuclear regions of the cells. The nucleoplasm was the most heavily labeled cell compartment, followed in decreasing order of labeling intensity by the cytoplasm, the nucleolus, and the chromocenters. The labeling observed over the latter area did not appear significantly different from that of the background. Sections incubated in the phalloidin-gold complex to which an excess of F-actin was added showed no significant labeling over any of the above-mentioned cell regions. Other control experiments included incubation of the sections with a phalloidin solution followed by the phalloidin-gold complex, PEG-stabilized colloidal gold, and a bovine serum albumin-gold complex. There was no or very little labeling of the preparations.

scholarly journals Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body.

1988 ◽  
Vol 36 (6) ◽  
pp. 693-696 ◽  
Author(s):  
T Uchida ◽  
T Endo
Keyword(s):  
Basal Body ◽  
Colloidal Gold ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Cell Organelles ◽  
Gold Particles ◽  
Microscopic Demonstration ◽  
S 100 ◽  
And Function

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.

Acid phosphatase activity in Pinus pinea – Tuber albidum ectomycorrhizal association

1992 ◽  
Vol 70 (7) ◽  
pp. 1377-1383 ◽  
Author(s):  
S. Pasqualini ◽  
F. Panara ◽  
M. Antonielli
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Middle Lamella ◽  
Ultrastructural Localization ◽  
Ectomycorrhizal Fungus ◽  
Pinus Pinea ◽  
Inorganic Pyrophosphate ◽  
Mycorrhizal Roots ◽  
Substrate Concentrations

Acid phosphatase activity of pine (Pinus pinea L.) roots was investigated in the presence or absence of the ectomycorrhizal fungus Tuber albidum Pico. Acid phosphatase activity was higher in mycorrhizal roots than in roots of uncolonized control plants. The optimum pH values for acid phosphatase were 3.5 and 5.0 for mycorrhizal roots and 5.0 for control roots. The acid phosphatase activity was inhibited by tartrate, fluoride, and molybdate ions, but a lower inhibition was exerted by orthophosphate. Mycorrhizal roots of pine possessed active acid phosphatases that hydrolyzed a wide variety of natural and synthetic phosphate esters. In particular, the enzyme was active against phytate and inorganic pyrophosphate. Two different Km values were estimated: about 0.22 mM and 2.78 mM at low and high substrate concentrations, respectively. The ultrastructural localization of acid phosphatase in mycorrhizal roots showed that the activity in the Hartig net was mainly localized in the plasmalemma of hyphae. Some lead phosphate precipitates were also observed in the middle lamella of the host cell. Key words: Pinus pinea, Tuber albidum, acid phosphatase, ectomycorrhiza, histochemical localization.

scholarly journals Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings.

1984 ◽  
Vol 32 (11) ◽  
pp. 1185-1191 ◽  
Author(s):  
M Bendayan ◽  
E Puvion
Keyword(s):  
Nucleic Acids ◽  
Osmium Tetroxide ◽  
Phosphotungstic Acid ◽  
Sodium Tungstate ◽  
Ethylenediaminetetraacetic Acid ◽  
High Specificity ◽  
Ultrastructural Localization ◽  
Gold Complex ◽  
Sodium Metaperiodate ◽  
Tissue Sections

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.

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