scholarly journals Influence of a Fungus-Feeding Nematode on Growth and Biocontrol Efficacy of Trichoderma harzianum

2001 ◽  
Vol 91 (3) ◽  
pp. 301-306 ◽  
Author(s):  
Yeoung-Seuk Bae ◽  
Guy R. Knudsen
Keyword(s):  
Trichoderma Harzianum ◽  
Fluorescent Protein ◽  
Epifluorescence Microscopy ◽  
Field Soil ◽  
Untreated Soil ◽  
Trichoderma Sp ◽  
Heat Treated ◽  
C 30 ◽  
Green Fluorescent ◽  
Pellet Formulation

A fungivorous nematode, Aphelenchoides sp., was isolated from field soil by baiting with mycelium of the biocontrol fungus Trichoderma harzianum ThzID1, and subsequently was maintained on agar cultures of the fungus. Interactions between the nematode and the green fluorescent protein-producing transformant, T. harzianum ThzID1-M3, were investigated in both heat-treated (80°C, 30 min) and untreated field soil. ThzID1-M3 was identified in soil by epifluorescence microscopy. When ThzID1-M3 was added to soil as an alginate pellet formulation, addition of the nematode (10 per gram of soil) significantly reduced radial growth and recoverable populations of the fungus, and the effect was greater in heat-treated soil than in untreated soil. Addition of ThzID1-M3 to soil pretreated with the nematode (10 per gram of soil) stimulated nematode population growth for approximately 10 to 20 days, whereas nematode populations decreased in the absence of added Trichoderma sp. When sclerotia of Sclerotinia sclerotiorum were added to soil (10 per 200 g of soil) with ThzID1-M3 (40 pellets per 200 g of soil), addition of Aphe-lenchoides sp. (2,000 per 200 g of soil) reduced the number of sclerotia colonized by ThzID1-M3. These results suggest that fungivorous nematodes may be a significant biotic constraint on activity of biocontrol fungi in the field.

Use of Green Fluorescent Protein and Image Analysis to Quantify Proliferation of Trichoderma harzianum in Nonsterile Soil

2004 ◽  
Vol 94 (12) ◽  
pp. 1383-1389 ◽  
Author(s):  
K. A. Orr ◽  
G. R. Knudsen
Keyword(s):  
Biological Control ◽  
Image Analysis ◽  
Green Fluorescent Protein ◽  
Trichoderma Harzianum ◽  
Fluorescent Protein ◽  
Biological Control Agent ◽  
Hyphal Growth ◽  
Epifluorescence Microscopy ◽  
Nonsterile Soil ◽  
Green Fluorescent

One drawback of traditional methods for fungal biomass measurement is the inability to distinguish biomass of an introduced fungus from that of the indigenous microbial community in nonsterile soil. We quantified biomass of a specific fungal biological control agent in nonsterile soil using epifluorescence microscopy and image analysis of green fluorescent protein (GFP)-expressing Trichoderma harzianum (ThzID1-M3). Numbers of colony forming units on a semiselective medium were compared with biomass estimates from image analysis, after ThzID1-M3 was incubated in soil that either remained moist (-0.05 MPa) for 14 to 21 days or remained moist for approximately 5 days and then was allowed to dry to <-3.0 MPa. Recovery of significant numbers of ThzID1-M3 propagules lagged approximately 3 days behind initiation of hyphal growth. Reductions in both colony counts and biomass were observed over time when soil was allowed to dry. However, in soil that remained moist, colony counts increased over a 14- to 21-day period even though biomass declined after approximately 3 to 5 days. Our results confirm that use of GFP, along with epifluorescence microscopy, is a useful tool to distinguish active hyphal biomass, the form of the fungus that is functional for biological control, from inactive propagules such as conidia or chlamydospores that are enumerated by plate counts.

scholarly journals Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor

2018 ◽  
Vol 96 (5) ◽  
pp. 459-470 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Robert Lodge ◽  
François Marceau
Keyword(s):  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Denaturation ◽  
Epifluorescence Microscopy ◽  
Terminal Sequence ◽  
Protein Ligands ◽  
C Terminus ◽  
Bona Fide ◽  
Green Fluorescent

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50–600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

scholarly journals Visualizing Microtubule-Dependent Vasopressin Type 2 Receptor Trafficking Using a New High-Affinity Fluorescent Vasopressin Ligand

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3893-3904 ◽  
Author(s):  
Sylvia Chen ◽  
Matthew J. Webber ◽  
Jean-Pierre Vilardaga ◽  
Ashok Khatri ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Receptor Trafficking ◽  
Epifluorescence Microscopy ◽  
Intracellular Camp ◽  
Green Fluorescent ◽  
Perinuclear Area ◽  
Major Target ◽  
High Binding Affinity ◽  
Dependent Mechanism

The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)2-Suc-TMR8]VP or (VPTMR). This ligand is fully functional as revealed by its high binding affinity toward V2R [(Kd) =157 ± 52 nm] and ability to increase intracellular cAMP 32-fold. VPTMR induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VPTMR and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VPTMR and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VPTMR-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VPTMR, and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VPTMR ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism.

An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1819-1828 ◽  
Author(s):  
James A. Carroll ◽  
Philip E. Stewart ◽  
Patricia Rosa ◽  
Abdallah F. Elias ◽  
Claude F. Garon
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Epifluorescence Microscopy ◽  
Reporter System ◽  
Environmental Signals ◽  
Gfp Reporter ◽  
Green Fluorescent

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

scholarly journals Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

2001 ◽  
Vol 75 (4) ◽  
pp. 1941-1948 ◽  
Author(s):  
Patrice Dunoyer ◽  
Etienne Herzog ◽  
Odile Hemmer ◽  
Christophe Ritzenthaler ◽  
Christiane Fritsch
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Viral Rna ◽  
Epifluorescence Microscopy ◽  
Scanning Confocal Microscopy ◽  
Virus Rna ◽  
Green Fluorescent ◽  
Rna Accumulation

ABSTRACT RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

scholarly journals Heat-Induced Oxidation of the Nuclei and Cytosol

2021 ◽  
Vol 11 ◽  
Author(s):  
Richa Babbar ◽  
Barbara Karpinska ◽  
Anil Grover ◽  
Christine H. Foyer
Keyword(s):  
Fluorescent Protein ◽  
Plant Responses ◽  
Specific Information ◽  
Redox Potentials ◽  
Heat Treated ◽  
Ros Accumulation ◽  
Stomatal Guard Cells ◽  
Transcript Profiles ◽  
Green Fluorescent ◽  
Induced Changes

The concept that heat stress (HS) causes a large accumulation of reactive oxygen species (ROS) is widely accepted. However, the intracellular compartmentation of ROS accumulation has been poorly characterized. We therefore used redox-sensitive green fluorescent protein (roGFP2) to provide compartment-specific information on heat-induced redox changes of the nuclei and cytosol of Arabidopsis leaf epidermal and stomatal guard cells. We show that HS causes a large increase in the degree of oxidation of both compartments, causing large shifts in the glutathione redox potentials of the cells. Heat-induced increases in the levels of the marker transcripts, heat shock protein (HSP)101, and ascorbate peroxidase (APX)2 were maximal after 15 min of the onset of the heat treatment. RNAseq analysis of the transcript profiles of the control and heat-treated seedlings revealed large changes in transcripts encoding HSPs, mitochondrial proteins, transcription factors, and other nuclear localized components. We conclude that HS causes extensive oxidation of the nucleus as well as the cytosol. We propose that the heat-induced changes in the nuclear redox state are central to both genetic and epigenetic control of plant responses to HS.

scholarly journals Cotransformation of Trichoderma harzianum with β-Glucuronidase and Green Fluorescent Protein Genes Provides a Useful Tool for Monitoring Fungal Growth and Activity in Natural Soils

2000 ◽  
Vol 66 (2) ◽  
pp. 810-815 ◽  
Author(s):  
Yeoung-Seuk Bae ◽  
Guy R. Knudsen
Keyword(s):  
Green Fluorescent Protein ◽  
Trichoderma Harzianum ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Marker Genes ◽  
Natural Soil ◽  
Nondestructive Monitoring ◽  
Wild Type ◽  
Glass Slides ◽  
Green Fluorescent

ABSTRACT Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), β-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introducedTrichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.

Temperature and Nutrient Effects on Campylobacter jejuni Attachment on Multispecies Biofilms on Stainless Steel

2008 ◽  
Vol 71 (2) ◽  
pp. 271-278 ◽  
Author(s):  
SHERIASE Q. SANDERS ◽  
JOSEPH F. FRANK ◽  
JUDY W. ARNOLD
Keyword(s):  
Stainless Steel ◽  
Surface Area ◽  
Campylobacter Jejuni ◽  
Intestinal Tract ◽  
Fluorescent Protein ◽  
Area Coverage ◽  
Epifluorescence Microscopy ◽  
Nutrient Effects ◽  
Green Fluorescent ◽  
Multispecies Biofilms

Campylobacter jejuni is a thermophilic microaerophilic pathogen that is commonly found in the intestinal tract of chickens. In this study, attachment of C. jejuni 1221gfp in biofilms on stainless steel was assessed at various temperatures and with reduced nutrients. Bacteria collected from a saline rinse of processed broiler chicken carcasses were used to form initial biofilms. The whole carcass rinse (WCR) biofilms were formed by incubation of the bacteria for 16 h at 13, 20, 37, and 42°C on stainless steel coupons in tryptic soy broth (TSB). The resulting biofilms were stained with Hoechst 33258 stain and visualized by epifluorescence microscopy. WCR biofilms formed at 13°C yielded the highest surface area coverage (47.6%), and the lowest coverage (2.1%) was attained at 42°C. C. jejuni transformed to produce green fluorescent protein (gfp) was allowed to attach to the preexisting biofilms (from WCR incubated for 16 h) at each of the four temperatures, and attached cells were enumerated by visualization with an epifluorescence microscope. Attachment of C. jejuni 1221gfp did not significantly differ (P &gt; 0.05) among the four temperatures. C. jejuni 1221gfp was cultured only from coupons with biofilms formed at 13 and 20°C. For nutrient limitation experiments, WCR biofilms were allowed to grow in 10- and 50-fold diluted TSB at 20 and 37°C for 48 h. The WCR biofilm surface area coverage (approximately 2%) was greater at 37°C than at 20°C for both TSB concentrations. C. jejuni 1221gfp was incubated with the WCR biofilm for 48 h at 20 and 37°C, and attached cells were enumerated. Attachment was significantly higher (P &lt; 0.05) only for the treatments with 1:10 TSB at 20°C and 1:50 TSB at 37°C. Under reduced-nutrient conditions, C. jejuni 1221gfp was cultured only from biofilms formed at 20°C. Under the conditions tested, the attachment of C. jejuni 1221gfp on stainless steel and biofilms was affected by a combination of temperature and nutrient availability, but C. jejuni culturability was affected solely by temperature.

scholarly journals Interactions Between Trichoderma harzianum Strain T22 and Maize Inbred Line Mo17 and Effects of These Interactions on Diseases Caused by Pythium ultimum and Colletotrichum graminicola

2004 ◽  
Vol 94 (2) ◽  
pp. 147-153 ◽  
Author(s):  
Gary E. Harman ◽  
Rixana Petzoldt ◽  
Alfio Comis ◽  
Jie Chen
Keyword(s):  
Inbred Line ◽  
Trichoderma Harzianum ◽  
Root Hair ◽  
Sandy Loam ◽  
Pythium Ultimum ◽  
Maize Inbred Line ◽  
Systemic Resistance ◽  
Field Soil ◽  
Colletotrichum Graminicola ◽  
Untreated Soil

Seed treatment with Trichoderma harzianum strain T22, which results in colonization of plant roots but little or no colonization of shoots or leaves, had substantial effects on growth of and disease expression in maize inbred line Mo17. Shoots and roots of 10-day-old seedlings grown in a sandy loam field soil were larger (roots were nearly twice as long) in the presence of T22 than in its absence. Both main and secondary roots were increased in size and area and the root hair area was greater with T22. However, root hair area per unit of root length was greater in control plants. Increased growth probably was due to direct stimulation of plant growth in addition to effects from biological control of deleterious microflora. Seedlings of Mo17 grown in autoclaved or mefenoxamtreated sandy loam field soil were larger than those produced in untreated soil. However, seedlings grown in the presence of T22, either in treated or untreated soil, were larger than those produced in its absence. Infestation of soil with Pythium ultimum had little effect upon growth of Mo17. The presence of T22 increased protein levels and activities of β-1,3 glucanase, exochitinase, and endochitinase in both roots and shoots, even though T22 colonized roots well but colonized shoots hardly at all. With some enzymes, the combination of T22 plus P. ultimum gave the greatest activity. Plants grown from T22-treated seed had reduced symptoms of anthracnose following inoculation of leaves with Colletotrichum graminicola, which indicates that root colonization by T22 induces systemic resistance in maize.

scholarly journals Synergistic Ability of Chitosan and Trichoderma harzianum to Control the Growth and Discolouration of Common Sapstain Fungi of Pinus radiata

Forests ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 542
Author(s):  
Tripti Singh ◽  
Colleen Chittenden
Keyword(s):  
Trichoderma Harzianum ◽  
Fluorescent Protein ◽  
Biological Control Agent ◽  
Radiata Pine ◽  
Control Agent ◽  
Depth Of Penetration ◽  
Ophiostoma Piceae ◽  
Green Fluorescent ◽  
Albino Strain ◽  
Microscopic Techniques

An environmentally compatible method for controlling sapstain fungi in wood was evaluated, using a combination of chitosan and an albino strain of Trichoderma harzianum, a biological control agent (BCA). The growth and penetration into the wood of the sapstain fungi Ophiostoma piceae, Leptographium procerum, and Sphaeropsis sapinea were assessed in radiata pine wafers treated with chitosan and BCA, both alone and in combination. Several mycological and microscopic techniques were used, including a gfp (green fluorescent protein) transformed strain of O. piceae for assessing the depth of penetration in the wood samples. The synergy between the chitosan and BCA was evident, and for two tested fungi, only the combination of chitosan and BCA afforded protection. The synnemata (recognized by erect conidiogenous cells bearing conidia) was observed on the surface of the wafers inoculated with L. procerum and O. piceae, but the hyphae were unable to penetrate and melanise. The results suggest that the limited ability of chitosan to penetrate deeply into the wood was compensated by the fast growth of T. harzianum in the inner wood.

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