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 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.

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.

scholarly journals Stable Expression of Modified Green Fluorescent Protein in Group B Streptococci To Enable Visualization in Experimental Systems

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Matthew J. Sullivan ◽  
Glen C. Ulett
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Group B Streptococcus ◽  
Experimental Studies ◽  
Bacterial Attachment ◽  
Stable Expression ◽  
Epifluorescence Microscopy ◽  
Group B ◽  
Green Fluorescent

ABSTRACTGroup B streptococcus (GBS) is a Gram-positive bacterium associated with various diseases in humans and animals. Many studies have examined GBS physiology, virulence, and microbe-host interactions using diverse imaging approaches, including fluorescence microscopy. Strategies to label and visualize GBS using fluorescence biomarkers have been limited to antibody-based methods or nonspecific stains that bind DNA or protein; an effective plasmid-based system to label GBS with a fluorescence biomarker would represent a useful visualization tool. In this study, we developed and validated a green fluorescent protein (GFP)-variant-expressing plasmid, pGU2664, which can be applied as a marker to visualize GBS in experimental studies. The synthetic constitutively active CP25 promoter drives strong and stable expression of the GFPmut3 biomarker in GBS strains carrying pGU2664. GBS maintains GFPmut3 activity at different phases of growth. The application of fluorescence polarization enables easy discrimination of GBS GFPmut3 activity from the autofluorescence of culture media commonly used to grow GBS. Differential interference contrast microscopy, in combination with epifluorescence microscopy to detect GFPmut3 in GBS, enabled visualization of bacterial attachment to live human epithelial cells in real time. Plasmid pGU2664 was also used to visualize phenotypic differences in the adherence of wild-type GBS and an isogenic gene-deficient mutant strain lacking CovR (thecontrolofvirulenceregulator) in adhesion assays. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications. We discuss the advantages and consider the limitations of this fluorescent biomarker system developed for GBS.IMPORTANCEGroup B streptococcus (GBS) is a bacterium associated with various diseases in humans and animals. This study describes the development of a strategy to label and visualize GBS using a fluorescence biomarker, termed GFPmut3. We show that this biomarker can be successfully applied to track the growth of bacteria in liquid medium, and it enables the detailed visualization of GBS in the context of live human cells in real-time microscopic analysis. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications.

Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria

1998 ◽  
Vol 64 (7) ◽  
pp. 2554-2559 ◽  
Author(s):  
Serina Stretton ◽  
Somkiet Techkarnjanaruk ◽  
Alan M. McLennan ◽  
Amanda E. Goodman
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Marine Bacteria ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Bacterial Species ◽  
Epifluorescence Microscopy ◽  
Broad Host Range ◽  
Green Fluorescent

ABSTRACT Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87–94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or thenpt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when thegfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed toN′-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.

Use of Green Fluorescent Protein Expressing Salmonella Stanley To Investigate Survival, Spatial Location, and Control on Alfalfa Sprouts

2001 ◽  
Vol 64 (12) ◽  
pp. 1891-1898 ◽  
Author(s):  
MEGHA GANDHI ◽  
SHERENE GOLDING ◽  
SIMA YARON ◽  
KARL R. MATTHEWS
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Plasmid Stability ◽  
Constitutive Expression ◽  
Epifluorescence Microscopy ◽  
Initial Population ◽  
Alfalfa Sprouts ◽  
Green Fluorescent ◽  
Alfalfa Seeds

Laser scanning confocal microscopy (LSCM) was used to observe the interaction of Salmonella Stanley with alfalfa sprouts. The green fluorescent protein (gfp) gene was integrated into the chromosome of Salmonella Stanley for constitutive expression, thereby eliminating problems of plasmid stability and loss of signal. Alfalfa seeds were inoculated by immersion in a suspension of Salmonella Stanley (ca. 107 CFU/ml) for 5 min at 22°C. Epifluorescence microscopy demonstrated the presence of target bacteria on the surface of sprouts. LSCM demonstrated bacteria present at a depth of 12μm within intact sprout tissue. An initial population of ca. 104 CFU/g seed increased to 7.0 log CFU/g during a 24-h germination period and then decreased to 4.9 log CFU/g during a 144-h sprouting period. Populations of Salmonella Stanley on alfalfa seeds decreased from 5.2 to 4.1 log CFU/g and from 5.2 to 2.8 log CFU/g for seeds stored 60 days at 5 and 22°C, respectively. The efficacy of 100, 200, 500, or 2,000 ppm chlorine in killing Salmonella Stanley associated with sprouts was determined. Treatment of sprouts in 2,000 ppm chlorine for 2 or 5 min caused a significant reduction in populations of Salmonella Stanley. Influence of storage on Salmonella Stanley populations was investigated by storing sprouts 4 days at 4°C. The initial population (7.76 log CFU/g) of Salmonella Stanley on mature sprouts decreased (7.67 log CFU/g) only slightly. Cross-contamination during harvest was investigated by harvesting contaminated sprouts, then directly harvesting noncontaminated sprouts. This process resulted in the transfer of ca. 105 CFU/g Salmonella Stanley to the noncontaminated sprouts.

scholarly journals Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

2004 ◽  
Vol 39 (4) ◽  
pp. 403-405 ◽  
Author(s):  
Paulo Roberto Queiroz ◽  
Maria Cléria Valadares-Inglis ◽  
Peter Ward Inglis
Keyword(s):  
Green Fluorescent Protein ◽  
Trichoderma Harzianum ◽  
Nested Pcr ◽  
Fluorescent Protein ◽  
Culture Media ◽  
Soil Samples ◽  
Egfp Gene ◽  
Soil Extracts ◽  
Green Fluorescent ◽  
Enrichment Step

The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.

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.

scholarly journals Rapid Screening Method for Mycobactericidal Activity of Chemical Germicides That Uses Mycobacterium terrae Expressing a Green Fluorescent Protein Gene

2001 ◽  
Vol 67 (3) ◽  
pp. 1239-1245 ◽  
Author(s):  
Ahmed A. Zafer ◽  
Yvonne E. Taylor ◽  
Syed A. Sattar
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Pearson Correlation ◽  
Screening Method ◽  
Rapid Screening ◽  
Epifluorescence Microscopy ◽  
Green Fluorescent Protein Gene ◽  
Bacterial Inactivation ◽  
Culture Methods ◽  
Green Fluorescent

ABSTRACT The slow growth of mycobacteria in conventional culture methods impedes the testing of chemicals for mycobactericidal activity. An assay based on expression of the green fluorescent protein (GFP) by mycobacteria was developed as a rapid alternative. Plasmid pBEN, containing the gene encoding a red-shifted, high-intensity GFP mutant, was incorporated into Mycobacterium terrae (ATCC 15755), and GFP expression was observed by epifluorescence microscopy. Mycobactericidal activity was assessed by separately exposing a suspension of M. terrae(pBEN) to several dilutions of test germicides based on 7.5% hydrogen peroxide, 2.4% alkaline glutaraldehyde, 10% acid glutaraldehyde, and 15.5% of a phenolic agent for contact times ranging from 10 to 20 min (22°C), followed by culture of the exposed cells in broth (Middlebrook 7H9) and measurement of fluorescence every 24 h. When the fluorescence was to be compared with CFU, the samples were plated on Middlebrook 7H11 agar and incubated for 4 weeks. No increase in fluorescence or CFU occurred in cultures in which the cells had been inactivated by the germicide concentrations tested. Where the test bacterium was exposed to ineffective levels of the germicides, fluorescence increased after a lag period of 1 to 7 days, corresponding to the level of bacterial inactivation. In untreated controls, fluorescence increased rapidly to reach a peak in 2 to 4 days. A good Pearson correlation coefficient (r ≥0.85) was observed between the intensity of fluorescence and the number of CFU. The GFP-based fluorescence assay reduced the turnaround time in the screening of chemical germicides for mycobactericidal activity to ≤7 days.

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