scholarly journals Colonization of Tomato Root by Pathogenic and Nonpathogenic Fusarium oxysporum Strains Inoculated Together and Separately into the Soil

2006 ◽  
Vol 72 (2) ◽  
pp. 1523-1531 ◽  
Author(s):  
Chantal Olivain ◽  
Claude Humbert ◽  
Jarmila Nahalkova ◽  
Jamshid Fatehi ◽  
Floriane L'Haridon ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Laser Scanning ◽  
Root Surface ◽  
Laser Scanning Microscopy ◽  
Pathogenic Strain ◽  
Confocal Laser ◽  
Fungal Colonization ◽  
Tomato Root ◽  
Tomato Roots ◽  
Nonpathogenic Strain

ABSTRACT In soil, fungal colonization of plant roots has been traditionally studied by indirect methods such as microbial isolation that do not enable direct observation of infection sites or of interactions between fungal pathogens and their antagonists. Confocal laser scanning microscopy was used to visualize the colonization of tomato roots in heat-treated soil and to observe the interactions between a nonpathogenic strain, Fo47, and a pathogenic strain, Fol8, inoculated onto tomato roots in soil. When inoculated separately, both fungi colonized the entire root surface, with the exception of the apical zone. When both strains were introduced together, they both colonized the root surface and were observed at the same locations. When Fo47 was introduced at a higher concentration than Fol8, it colonized much of the root surface, but hyphae of Fol8 could still be observed at the same location on the root. There was no exclusion of the pathogenic strain by the presence of the nonpathogenic strain. These results are not consistent with the hypothesis that specific infection sites exist on the root for Fusarium oxysporum and instead support the hypothesis that competition occurs for nutrients rather than for infection sites.

scholarly journals Visualization of Interactions Between a Pathogenic and a Beneficial Fusarium Strain During Biocontrol of Tomato Foot and Root Rot

2005 ◽  
Vol 18 (7) ◽  
pp. 710-721 ◽  
Author(s):  
Annouschka Bolwerk ◽  
Anastasia L. Lagopodi ◽  
Ben J. J. Lugtenberg ◽  
Guido V. Bloemberg
Keyword(s):  
Root Rot ◽  
Laser Scanning ◽  
Root Colonization ◽  
Pathogenic Fungus ◽  
Laser Scanning Microscopy ◽  
Systemic Resistance ◽  
Confocal Laser ◽  
Tomato Root ◽  
Tomato Roots

The soilborne fungus Fusarium oxysporum f. sp. radicislycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50- fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.

scholarly journals Novel Aspects of Tomato Root Colonization and Infection by Fusarium oxysporum f. sp. radicis-lycopersici Revealed by Confocal Laser Scanning Microscopic Analysis Using the Green Fluorescent Protein as a Marker

2002 ◽  
Vol 15 (2) ◽  
pp. 172-179 ◽  
Author(s):  
Anastasia L. Lagopodi ◽  
Arthur F. J. Ram ◽  
Gerda E. M. Lamers ◽  
Peter J. Punt ◽  
Cees A. M. J. J. Van den Hondel ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusarium Oxysporum ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Microscopic Analysis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Tomato Root ◽  
Green Fluorescent

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F. oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

scholarly journals In planta Activity of Novel Copper(II)-Based Formulations to Inhibit the Esca-Associated Fungus Phaeoacremonium minimum in Grapevine Propagation Material

2021 ◽  
Vol 12 ◽  
Author(s):  
Enrico Battiston ◽  
Stéphane Compant ◽  
Livio Antonielli ◽  
Vincenzo Mondello ◽  
Christophe Clément ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Defense Responses ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Fungal Colonization ◽  
Post Inoculation ◽  
In Planta ◽  
Experimental Treatments ◽  
Propagation Material

Grapevine trunk diseases (GTDs) are a serious and growing threat to vineyards worldwide. The need for innovative control tools persists since pesticides used against some GTDs have been banned and only methods to prevent infections or to reduce foliar symptoms have been developed so far. In this context, the application of imaging methods, already applied to study plant–microbe interactions, represents an interesting approach to understand the effect of experimental treatments applied to reduce fungal colonization, on GTD-related pathogens activity. To this aim, trials were carried out to evaluate the efficacy of copper-based treatments, formulated with hydroxyapatite (HA) as co-adjuvant with innovative delivery properties, loaded with two different copper(II) compounds (tribasic sulfate and sulfate pentahydrate), and applied to grapevine propagation material to inhibit fungal wood colonization. The treated rootstock (Vitis berlandieri × Vitis riparia cv. K5BB) and scion cuttings (Vitis vinifera L., cv. Chardonnay) had been inoculated with a strain of Phaeoacremonium minimum (Pmi) compared to uninoculated rootstocks. Experimental treatments were applied during the water-soaking process, comparing the copper(II) compounds pure or formulated with HA, to hydrate the cuttings. After callusing, grafted vines were grown under greenhouse conditions in a nursery and inoculated with Pmi::gfp7 or with Pmi wild-type. Fifteen weeks post-inoculation, woody tissues close to the inoculation site were sampled to evaluate the efficiency of the treatments by studying the plant–microbe interaction by confocal laser scanning microscopy (CLSM). Copper and further elements were also quantified in the same tissues immediately after the treatments and on the CLSM samples. Finally, the grapevine defense responses were studied in the leaves of cuttings treated with the same formulations. The present investigation confirmed the relevant interaction of Pmi and the related transformed strain on the vascular tissues of grafted vines. Furthermore, in vitro assay revealed (i) the fungistatic effect of HA and the reduced effect of Cu fungicide when combined with HA. In planta assays showed (ii) the reduction of Pmi infection in propagation material treated with HA-Cu formulations, (iii) the movement of HA-Cu formulations inside the plant tissues and their persistence over time, and (iv) the plant defense reaction following the treatment with pure HA or Cu, or combined.

scholarly journals Endophytic Bacillus cereus Effectively Controls Meloidogyne incognita on Tomato Plants Through Rapid Rhizosphere Occupation and Repellent Action

Plant Disease ◽  
2017 ◽  
Vol 101 (3) ◽  
pp. 448-455 ◽  
Author(s):  
Hai-Jing Hu ◽  
Ya-Li Chen ◽  
Yu-Fang Wang ◽  
Yun-Yun Tang ◽  
Shuang-Lin Chen ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Meloidogyne Incognita ◽  
Endophytic Bacteria ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Egg Mass ◽  
Root Knot Nematodes ◽  
Tomato Roots ◽  
Repellent Action

Root-knot nematodes (Meloidogyne spp.), which cause severe global agricultural losses, can establish a special niche in the root vascular cylinder of crops, making them difficult to control. Endophytic bacteria have great potential as biocontrol organisms against Meloidogyne incognita. Three endophytic bacteria were isolated from plant tissues and showed high nematicidal activity against M. incognita second-stage juveniles (J2) in vitro. The gyrB gene sequence amplification results indicated that the three isolates were Bacillus cereus BCM2, B. cereus SZ5, and B. altitudinis CCM7. The isolates colonized tomato roots rapidly and stably during the colonization dynamic experiment. Three pot experiments were designed to determine the potential of three endophytic bacterial isolates on control of root-knot nematodes. The results showed that the preinoculated B. cereus BCM2 experiment significantly reduced gall and egg mass indexes. The inhibition ratio of gall and egg mass was up to 81.2 and 75.6% on tomato roots and significantly enhanced shoot length and fresh weight. The other two experiments with inoculated endophytic bacteria and M. incognita at the same time or after morbidity had lower inhibition ratios compared with the preinoculated endophytic bacteria experiment. The confocal laser-scanning microscopy method was used to further study the possible mechanism of endophytic bacteria in the biocontrol process. The results showed the localization pattern of the endophytic bacteria B. cereus BCM2-(str′)-pBCgfp-1 in tomato root tissues. Root tissue colonized by endophytic bacteria repelled M. incognita J2 infection compared with the untreated control in a repellence experiment. We isolated an endophytic B. cereus strain that stably colonized tomato and controlled M. incognita effectively. This strain has potential for plant growth promotion, successful ecological niche occupation, and M. incognita J2 repellent action induction. It plays an important role in endophytic bacteria against root-knot nematodes.

scholarly journals Interactions in the Tomato Rhizosphere of Two Pseudomonas Biocontrol Strains with the Phytopathogenic Fungus Fusarium oxysporum f. sp. radicis-lycopersici

2003 ◽  
Vol 16 (11) ◽  
pp. 983-993 ◽  
Author(s):  
Annouschka Bolwerk ◽  
Anastasia L. Lagopodi ◽  
André H. M. Wijfjes ◽  
Gerda E. M. Lamers ◽  
Thomas F. C. Chin-A-Woeng ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Laser Scanning ◽  
Hyphal Growth ◽  
Confocal Laser Scanning Microscope ◽  
Systemic Resistance ◽  
Phytopathogenic Fungus ◽  
Confocal Laser ◽  
Bacterial Strains ◽  
Tomato Root ◽  
Biocontrol Bacteria

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carbox-amide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Use of confocal laser scanning microscopy and a computer model to understand ink cavitation and filamentation

TAPPI Journal ◽  
2010 ◽  
Vol 9 (10) ◽  
pp. 7-15
Author(s):  
HANNA KOIVULA ◽  
DOUGLAS BOUSFIELD ◽  
MARTTI TOIVAKKA
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Print Quality ◽  
Length Scale ◽  
Offset Printing ◽  
Significant Difference ◽  
Printing Speed

In the offset printing process, ink film splitting has an important impact on formation of ink filaments. The filament size and its distribution influence the leveling of ink and hence affect ink setting and the print quality. However, ink filaments are difficult to image due to their short lifetime and fine length scale. Due to this difficulty, limited work has been reported on the parameters that influence filament size and methods to characterize it. We imaged ink filament remains and quantified some of their characteristics by changing printing speed, ink amount, and fountain solution type. Printed samples were prepared using a laboratory printability tester with varying ink levels and operating settings. Rhodamine B dye was incorporated into fountain solutions to aid in the detection of the filaments. The prints were then imaged with a confocal laser scanning microscope (CLSM) and images were further analyzed for their surface topography. Modeling of the pressure pulses in the printing nip was included to better understand the mechanism of filament formation and the origin of filament length scale. Printing speed and ink amount changed the size distribution of the observed filament remains. There was no significant difference between fountain solutions with or without isopropyl alcohol on the observed patterns of the filament remains.

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