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Applied and Environmental Microbiology, February 2000, p. 810-815, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cotransformation of Trichoderma harzianum with
-Glucuronidase and Green Fluorescent Protein Genes Provides a
Useful Tool for Monitoring Fungal Growth and Activity in
Natural Soils
Yeoung-Seuk
Bae and
Guy R.
Knudsen*
Department of Plant, Soil, and Entomological
Sciences, University of Idaho, Moscow, Idaho 83844-2339
Received 4 June 1999/Accepted 13 October 1999
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ABSTRACT |
Trichoderma harzianum was cotransformed with genes
encoding green fluorescent protein (GFP), 
-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 introduced
Trichoderma 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--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.
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INTRODUCTION |
Trichoderma spp. have
received considerable attention as potential biological control agents
against a wide range of soil-borne plant-pathogenic fungi (7,
23) in the greenhouse (8, 16, 20, 24) and field
(12, 13, 15, 19). However, the efficacy of
Trichoderma spp. as biocontrol agents in natural soils may be limited by soil fungistasis (23), competition by other
soil microorganisms (17), poor plant root colonization
(2), or unfavorable environmental conditions. Identification
and quantification of ecological factors affecting the establishment
and the population dynamics of introduced Trichoderma
strains in natural habitats may provide more predictable and effective
biocontrol of plant diseases.
Although several methods have been used to study the occurrence and
distribution of Trichoderma in soils (1, 25), few methods have allowed quantitative evaluation of population dynamics and
survival. For example, Knudsen and coworkers (11, 18) quantified the influences of temperature, soil matric potential, nutrient source, and antagonistic bacteria on the hyphal growth and
biocontrol efficacy of pelletized T. harzianum in soils.
However, it was not possible to differentiate the hyphal growth of this fungal agent from that of indigenous Trichoderma strains
(3, 18). Knudsen et al. (18) pointed out that the
use of dilution plating for numerical estimation of fungal populations
does not differentiate among the different propagules (hyphal
fragments, conidia, and chlamydospores) that may generate colonies when
plated on agar, and thus it is not a true estimate of fungal biomass. The use of mutant strains resistant to specific fungicides may partially overcome problems related to nonspecific recovery (1, 2), but this method does not allow for in situ monitoring of growth dynamics and survival structures of introduced
Trichoderma strains or in situ differentiation of introduced
Trichoderma strains from indigenous strains.
Recently, genetic engineering of biocontrol agents with reporter or
marker genes has provided useful tools for detection and monitoring of
introduced biocontrol agents in natural environments (14,
21). For example, the selectable hygromycin B phosphotransferase (hygB) gene, coding for resistance to this antibiotic, has
been used to detect fungal biocontrol agents in the rhizosphere and phyllosphere (21, 22). The -glucuronidase (GUS) marker
gene also is a promising tool for ecological studies of biocontrol agents, because of the low background activity of GUS in fungi and
plants, the relative ease and sensitivity of detection (29), and the apparent lack of influence of GUS expression on biocontrol efficacy (36). However, some background GUS activity may be present in unsterile systems or natural soils. Aspergillus
niger has some indigenous acidic GUS activity, and T. harzianum strain T3 showed indigenous acidic
-galactosidase activity (36). Therefore, for study of
growth patterns and sporulation of an introduced fungus in
natural ecosystems, the GUS system may have limited usefulness.
The green fluorescent protein (GFP) of the jellyfish Aequorea
victoria also has been developed as a reporter for gene expression (5). It has been successfully cloned and expressed in
several organisms (6, 9, 31). GFP was shown to be a useful
tool for studying plant-fungus interactions in vivo (34) and
has been used to assess the colonization and dispersal of
Aureobasidium pullulans in the phyllosphere (37).
GFP requires only UV or blue light and oxygen to induce green
fluorescence. An exogenous substrate, such as GUS requires, is not
needed for the detection of GFP, thus avoiding problems related to cell
permeability and substrate uptake (10, 31).
The objectives of this study were (i) to produce a mitotically stable
cotransformant of T. harzianum isolate ThzID1 expressing the
hygB gene as well as both marker genes, GUS and GFP, and
(ii) to determine whether the cotransformant provides a useful
tool for in situ monitoring of T. harzianum hyphal growth
and activity in soil, including the ability to colonize the resting
form (sclerotia) of the plant-pathogenic fungus Sclerotinia
sclerotiorum.
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MATERIALS AND METHODS |
Transformation vectors and preparation of plasmid DNA.
Plasmids pAN7-2, containing the Escherichia coli hygB gene
(27), and pNOM102, containing the E. coli
GUS gene (29), were obtained from H. Leung (Department of
Plant Pathology, Washington State University). Both plasmids contained
the constitutive Aspergillus nidulans
glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter. Plasmid pTEFEGFP containing the E. coli engineered GFP
(EGFP) gene (37) was provided by D. Cullen (Forest Products
Laboratory, U.S. Department of Agriculture, University of
Wisconsin
Madison). It contains the Aureobasidium pullulans
translation elongation factor promoter and Aspergillus
awamori glucoamylase terminator. Each plasmid was propagated in
E. coli strain HB101, and purification of plasmid DNA was
performed by the method of Sambrook et al. (30).
Protoplast preparation.
Protoplasts were generated by
combining the protocols described by Thrane et al. (36) and
Sivan et al. (33) as detailed below, except that potato
dextrose broth (PDB) was replaced by glucose yeast extract broth (GYEB;
15 g of glucose, 3 g of yeast extract, 1 liter of water).
Conidia (2 × 106 to 5 × 106) of
T. harzianum isolate ThzID1 were added to 100 ml of GYEB for
23 h at 26 C with shaking (100 rpm). The resultant mycelium was
collected on four layers of cheesecloth and washed with 1.2 M
MgSO4. A 1-g portion of fresh mycelium was transferred to a 50-ml flask containing 20 ml of sterile-filtered (0.45-µm-pore-size filter) lysing enzyme (Sigma L-2265) solution (7.5 mg per ml in 1.2 M
MgSO4-10 mM sodium phosphate buffer [pH 5.8]). The flask was incubated for 3 to 4 h at 26°C with shaking at 100 rpm.
Protoplasts were separated from mycelial debris by filtering through
six layers of cheesecloth and washed twice in 1.2 M MgSO4
by centrifugation at 500 × g for 5 min at 4°C. The
protoplasts then were rinsed, suspended in STC medium (0.6 M sorbitol,
10 mM Tris-HCl [pH 7.5], 10 mM CaCl2), and kept on ice
until used.
Transformation of protoplasts.
The transformation procedures
were based on the methods of Penttilä et al. (26) and
Shi et al. (32). Circular plasmid DNA (1 µg each of
pAN7-2, pNOM102, and pTEFEGFP) in 25 µl of Tris-EDTA buffer
(30) was mixed into 100 µl of protoplast suspension
(1.2 × 108/ml) in a 50-ml centrifuge tube. The
mixture was incubated for 20 min on ice. In a dropwise manner, 2 ml of polyethlene glycol (PEG) solution (60% [wt/vol] PEG 3350 in 25 mM CaCl2-25 mM Tris-HCl [pH 7.5]) was added after
incubation. The suspension was gently combined with 30 ml of ice-cold
STC medium. The protoplasts were concentrated by centrifugation at
2,500 rpm for 10 min, and the pelleted protoplasts were resuspended in
4 ml of PDB containing 1 M sorbitol. After incubation at 26 C for
9 h, 0.5 ml of the suspension was plated with 10 ml of molten
potato dextrose agar (PDA). The solidified plate was overlaid with 10 ml of molten PDA (0.7%) containing 300 µg of hygromycin B per ml
(150-µg/ml final concentration).
Colonies were chosen after incubation at 25°C for 5 to 7 days and
transferred to PDA containing 150 µg of hygromycin B per ml. Colonies
were tested for GUS activity as follows. A mycelial segment from each
colony was placed in a microtiter plate containing 200 µl of 10mM
sodium phosphate buffer (pH 7.5) supplemented with 4 µl of substrate
(4 mg of 5-bromo-4-chloro-3-indolyl--D-glucuronic acid
[X-Gluc; Sigma Chemical Co. or Clonetech] in 1 ml of 50 mM sodium
phosphate buffer [pH 7.5]) and incubated in the dark for 48 h
(36).
Colonies that developed blue pigment were examined for GFP activity.
Cotransformants that showed both GUS and GFP activities
were selected
for further
tests.
Selection of stable cotransformants.
To eliminate isolates
containing heterokaryons, each transformant was incubated on a PDA
slant containing 150 µg of hygromycin B per ml, under a fluorescent
light, for 7 days at 25°C. Conidia of each transformant then were
collected through four layers of cheesecloth and plated on PDA
containing 150 µg of hygromycin B per ml. After incubation for 2 to 3 days, several distinct colonies (i.e., single-spore isolates) were
transferred to new PDA slants containing 150 µg of hygromycin B per
ml. After testing for GUS and GFP activities, one isolate was chosen to
conduct the selection process again; this was repeated five times. To
test the stability of cotransformants selected in this manner, a
mycelial plug of each cotransformant was transferred to a PDA plate
without antibiotic and the plate was incubated at 25°C. After 4 days
of incubation, cotransformants were subcultured on new PDA plates
without antibiotic for an additional 40 days. A final subculture of
each cotransformant was tested to confirm HygBr and GUS and
GFP activity.
Southern blot analysis.
Wild-type and cotransformant
isolates were grown from conidia in PDB without antibiotic. Genomic DNA
was isolated from 72-h-old thalli that were lyophilized and ground
in liquid nitrogen (28). Genomic and plasmid DNAs were
digested with restriction enzymes as follows: pAN7-2 with
HindIII-EcoRI, pNOM102 with NcoI,
and pTEFEGFP with HindIII-BamHI. DNA
digests were electrophoresed in 1% agarose and blotted on nylon
membranes (Boehringer Mannheim, Indianapolis, Ind.) using standard
techniques (30). DNA fragments of each plasmid were
separately labeled with digoxigenin-dUTP (Boehringer Mannheim) and used
to probe genomic blots of wild-type and cotransformant M3 as specified
by the manufacturer. Hybridization signals were detected
colorimetrically with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate as substrates for alkaline phosphatase (Boehringer Mannheim).
Growth on PDA and in soil.
Mycelial disks (5 mm in diameter)
of wild-type and cotransformant strains were transferred to PDA plates
with or without hygromycin B and incubated at 25°C for 3 days in the
dark. Radial growth was measured under a light microscope and recorded
daily, beginning 24 h after incubation. There were five replicates
for each strain. Hyphal extension of cotransformant ThzID1-M3 also was
measured in soil. Palouse silt loam soil was obtained from the
University of Idaho Parker Farm near Moscow. Soil analysis (University
of Idaho Analytical Services Laboratory) indicated that the soil contained 20% sand, 20% clay, and 60% silt by weight, with 82.2 µg
of plant-available iron per g. Soil pH in soil-water (2:1) solution was
approximately 5.9. The soil was sieved through a 2-mm mesh and air
dried prior to use. The soil was adjusted to a soil moisture content of
100 kPa.
Alginate pellets of the transformant strain were made using previously
described methods (
18). ThzID1-M3 was grown on PDA
for 7 days, and three 1-cm
2 pieces from the culture margins were
placed in 500 ml of PDB
in a 1-liter flask. The flasks were incubated
at approximately
22°C on a rotary shaker (120 rpm), with 12 h of
light per day,
for 1 week. The mycelial biomass was then strained,
rinsed with
sterile distilled water, and added to 100 ml of 1% aqueous
sodium
alginate solution. The mixture of fungus-alginate solution was
added as drops to 0.25 M aqueous CaCl
2. Pellets formed in
the
CaCl
2 solution were removed, rinsed with sterile
distilled water,
and allowed to air dry on waxed paper. The pellets
were stored
in glass beakers at 4°C.
A glass petri dish (15 cm in diameter) was about half filled with soil,
and a glass slide precoated with 1.8% water agar was
placed on the
soil surface. A single pellet was glued in the middle
of the glass
slide with cyanoacrylate glue, which previously was
determined not to
influence the growth of
Trichoderma (G. R. Knudsen,
unpublished results). The glass slide and pellet were covered
with soil
to fill the plate, and then the plates were placed in
a covered plastic
container lined with moist paper towels and
incubated at 25°C for 3, 5, and 10 days. At each sampling time,
glass slides were removed and
examined using epifluorescence microscopy.
Radial growth was quantified
by measuring colony diameters from
captured video images at
magnifications of ×250 or ×400. The experiment
was repeated once,
with five replicates each
time.
Colonization of sclerotia in soil.
To produce sclerotia of
S. sclerotiorum, mycelial disks grown on PDA for 7 days were
transferred to sterilized sliced carrots in 2-liter Erlenmeyer flasks.
After 6 to 8 weeks of incubation at 16°C, sclerotia were harvested,
rinsed with sterile distilled water, and air dried for 2 to 3 days. The
sclerotia were attached to plastic toothpicks with cyanoacrylate glue
and allowed to dry overnight. All sclerotia then were surface
disinfected with a sterile solution (10% ethanol-10% bleach in
water) for 1 min and stored at 4°C before use.
A quantity of a Palouse silt loam soil was prepared as described above.
Pellets of ThzID1 and ThzID1-M3 were produced as described
above. A
plastic container (25 by 25 by 10 cm) was filled with
1 kg of soil to
which five pellets of each isolate had been added.
Twenty sclerotia
were randomly placed in the soil at the depth
of 2 cm. The containers
were covered and sealed with plastic film
to maintain a relatively
constant moisture content. Treatments
were as follows: untreated
(no-
Trichoderma) control, ThzID1 alginate
pellets, and
ThzID1-M3 alginate pellets. There were four replicates.
After 7 and 14 days of incubation at 25°C, half of the sclerotia
from each container
were recovered, surface sterilized, and placed
on PDA containing 50 µg of streptomycin per ml. The plates were
incubated at 25°C for 7 days, and sclerotia were observed to determine
whether
S. sclerotiorum,
Trichoderma spp., or other fungi were
growing from them. For treatment with cotransformant ThzID1-M3,
mycelia
grown from sclerotia were examined for GUS and GFP activities.
Proportions of sclerotia colonized by
Trichoderma spp. or
ThzID1-M3
were
recorded.
GFP and GUS expression of the cotransformant after inoculation
into soil.
A pellet (as described above) of cotransformant
ThzID1-M3 and a pellet of the wild-type ThzID1 were glued onto a glass
microscope slide, 0.5 cm apart. The glass slide was incubated in a
glass petri dish (15 cm in diameter) containing soil as described
above. After 2, 3, and 5 days of incubation at 25°C, the glass slides were carefully removed and observed microscopically. GFP activity of
cotransformant ThzID1-M3 was observed as described above, using epifluorescence. To detect GUS activity, 400 µl of 10mM sodium phosphate buffer (pH 7.5) supplemented with 16 µl of X-Gluc substrate was carefully placed on the glass slide with a glass coverslip and then
incubated at 37°C for 4 h in the dark prior to observation.
Statistical analysis.
All experiments were conducted as
completely randomized designs. Analysis of variance was performed for
each experiment, and the least-significant-difference (LSD) test was
used for means separation, where appropriate, using PROC GLM (SAS
Institute Inc., Cary, N.C.).
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RESULTS AND DISCUSSION |
Fungal cotransformation and stability.
Before transformation,
the effect of the dose of hygromycin B on the regeneration frequency of
protoplasts was determined. Regeneration of untransformed protoplasts
was completely inhibited at 150 µg of hygromycin B per ml (data not
shown). Therefore, growth at this concentration was used as the
selection criterion for hygromycin B-tolerant transformants. The
average frequency of transformation to hygromycin tolerance was 15 transformants per µg of DNA. All transformants were examined for GUS
and GFP activities. A total of five expressed both GUS and GFP
activities. In PEG-mediated transformations, protoplasts generated from
young mycelia of Trichoderma typically contain 2 to 12 nuclei per protoplast (35). During transformation,
protoplasts fuse to form aggregates, which develop into thalli
containing more than 30 nuclei per cell (33). Sivan et al.
(33) reported that stabilization of transformants by
single-conidium selection was essential to obtain stable transformants, due to the heterokaryotic nature of putative transformants. To select a
monokaryon from each cotransformant, several colonies developed from a
single conidium of each cotransformant were chosen and tested for GUS
and GFP activities. Four cotransformants showed the ability to express
both GUS and GFP activities. To test the stability of expression in
cotransformants, these cotransformants were subcultured successively on
PDA without selection pressure, up to 10 times. Cotransformant
ThzID1-M3 was the only isolate that exhibited stable expression of all
three foreign genes.
Southern hybridizations were performed to confirm integration of
the foreign plasmid DNA into the genome of cotransformant
ThzID1-M3.
Total genomic DNAs from cotransformant ThzID1-M3 and
wild-type ThzID1
were isolated, digested, and probed with the
HindIII-
EcoRI pAN7-2 fragment, the
NcoI-
NcoI pNOM102 fragment,
and the
HindIII-
BamHI pTEFEGFP fragment. There
were no background
signals of the wild-type genomic DNA (Fig.
1). This isolate showed
no reversion to
the wild-type phenotype over a 6-month period.
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FIG. 1.
Southern blot analysis of genomic DNAs from T. harzianum wild-type ThzID1 and cotransformant ThzID1-M3.
(A) Probed with HindIII-EcoRI fragments from
pAN7-2 including the hygromycin B resistance gene. (B) Probed with
NcoI-NcoI fragments from pNOM102 including the
GUS gene. (C) Probed with HindIII-BamHI
fragments from pTEFEGFP including the GFP gene. Lanes: 1,  -HindIII; 2, 3, and 4: pAN7-2, wild type, and
cotransformant, respectively, digested with
HindIII-EcoRI; 5, 6, and 7, pNOM102, wild
type, and cotransformant, respectively, digested with
NcoI-NcoI; 8, 9, and 10, pTEFEGFP, wild type, and
cotransformant, respectively, digested with
HindIII-BamHI.
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Hyphal growth and colonization on sclerotia.
The morphology of
cotransformant ThzID1-M3 was similar to that of the wild type (data not
shown). However, the mycelial growth of the cotransformant on PDA was
slower than that of the wild type (Fig.
2). The wild-type ThzID1 did not grow on
100 µg of hygromycin B per ml, whereas the cotransformant grew at 300 µg of hygromycin B per ml (data not shown). GFP was a useful tool for
monitoring the growth of introduced T. harzianum in soil. It
provided a nondestructive sampling for visualization of hyphae in
situ. The mean colony diameter increased rapidly up to 5 days in soil
but subsequently decreased by day 10 (Fig.
3). Previous work in our laboratory
demonstrated several environmental factors that influence radial growth
and density of hyphae originating from alginate pellets of T. harzianum (11, 18, 19). However, previous experiments
necessarily were conducted using steamed soil, because it was
impossible to differentiate hyphae from different sources in raw soil.
When compared with those results, our present results indicate a lower
growth rate in nonsterile soil, possibly resulting from reduced growth
ability of the cotransformant and/or from the influence of the soil
microbiota in an untreated soil.
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FIG. 2.
Growth of T. harzianum wild-type ThzID1
and cotransformant ThzID1-M3 on PDA after 1, 2, and 3 days of incubation at 25°C in the dark. All values for days and
isolates were significantly different (P < 0.05)
according to an LSD analysis.
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FIG. 3.
Colony diameter of T. harzianum
ThzID1-M3 originating from alginate pellets in soil at 3, 5, and 10 days. Colony diameters were measured at a magnification of ×250
or ×400 under an epifluorescence microscope. Vertical bars represent
±1 standard error of the mean.
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Using alginate pellets, the ability of cotransformant
ThzID1-M3 to colonize sclerotia of
S. sclerotiorum, compared to the
wild-type ThzID1, in soil was not
significantly different after
7 or 14 days (
P > 0.05).
Therefore, only results after 14 days
of incubation are
presented (Fig.
4). Treatments with
alginate
pellets of either the wild-type ThzID1 or cotransformant
ThzID1-M3
resulted in higher percentages of sclerotia
colonized by
Trichoderma spp. compared to nontreated
controls (
P < 0.05). There were no
significant
differences between treatments with the introduced
wild type
compared to the cotransformant (mean, 58 and 60%, respectively;
P > 0.05). However, while approximately 60% of
sclerotia were
colonized by
Trichoderma spp. in
cotransformant treatments, the
recombinant phenotype (GFP + GUS)
accounted only for about 18%
of sclerotial colonization, with the
remaining 42% being attributable
to (presumably indigenous)
Trichoderma spp. without the recombinant
phenotype.
Bin et al. (
3) reported that incorporation of pellets
of
T. harzianum into field soil significantly increased the
colonization
of sclerotia by
Trichoderma spp. Knudsen et al.
(
19) concluded
that, based on sampled populations of
Trichoderma propagules in
field trials, the majority of
sclerotial colonization resulted
from hyphal growth of introduced
T. harzianum. However, in the
present study, we were able
for the first time to quantify the
relative contribution of an
introduced
Trichoderma strain to total
sclerotial
colonization, because of its unique phenotype. Our
results indicate a
relatively smaller contribution of the introduced
strain to total
colonization than was previously estimated, but
it should be noted that
soil environmental conditions in previous
studies (
3,
19)
probably were less conducive to activation
of dormant propagules of
indigenous
Trichoderma, since they were
hotter and drier.
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FIG. 4.
Percentage of sclerotia colonized by
Trichoderma spp. in soil at 14 days. Five alginate pellets
of T. harzianum ThzID1 or ThzID1-M3 were applied in
a 25-cm2 pot containing 1 kg of natural soil. The dashed
portion of ThzID1-M3 treatment represents the proportion of
sclerotia colonized by T. harzianum ThzID1-M3. Means
followed by the same letter are not significantly different
(P > 0.05) according to an LSD analysis.
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GFP and GUS expression of the cotransformant after inoculation into
soil.
GFP expression in soil was observed using epifluorescence
with a BPF510 illuminator slide set including 2X KP 490 and B 229 for
the excitation filter and a G 247 barrier filter. Thus viewed, hyphae
of the wild-type T. harzianum were barely visible. In
contrast, the cotransformant exhibited distinct green fluorescence
(Figs. 5A to C). Hyphal growth from
pellets was first observed after 2 days of incubation and continued for
approximately 5 days. GFP activity of hyphae revealed that green
fluorescence was more intense near septa in older hyphae (Fig.
5C). After 5 days of incubation, most structures apart from pellets of
ThzID1-M3 were terminal or intercalary chlamydospores. Older
thick-walled chlamydospores were hardly seen due to low
intensity of GFP activity. Conidia or conidiophores of
ThzID1-M3 were visible mostly around pellets only after 3 days
of incubation.
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FIG. 5.
Photomicrographs of GFP (A to C) and GUS (D to F)
activities of T. harzianum ThzID1-M3 in natural
soil. (A) Conidia; (B) conidial germination; (C and D) hyphal growth;
(E and F) chlamydospore formation. Magnifications, ×400 (A to D and F)
and ×250 (E).
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To view GUS activity, glass slides first were removed and treated with
the X-Gluc substrate. After a 4-h incubation at 37°C
in the dark, the
glass slides were examined microscopically. GUS
was a less useful tool
for in situ monitoring of growth or structures
of the introduced fungus
under these experimental conditions,
since addition of the X-Gluc
substrate to glass slides resulted
in some sample disruption. Low
activity of GUS was also observed
in most resting conidia and several
hyphal segments. In hyphae
of cotransformant ThzID1-M3 (Fig.
5D), chlamydospores were terminal
or intercalary. Older hyphae appeared
to be degraded partially
or completely after forming a
chlamydospore under the experimental
conditions (Fig.
5E and F).
These chlamydospores may play an important
role as survival structures
of introduced
Trichoderma spp. in
natural ecosystems
(
23), but little is known about their survival
and
ecological
importance.
In conclusion, cotransformation with GFP and GUS provides a potentially
useful tool for monitoring hyphal growth patterns
and population
dynamics of
Trichoderma isolates introduced into
natural
systems and will help provide insight into the important
abiotic and
biotic factors affecting biocontrol efficacy. Significant
biotic
factors may include soil bacteria (
3,
11,
15), indigenous
soil fungi, and soil microfauna (
4) that can influence the
growth and efficacy of the introduced fungus. These are areas
for
future
investigation.
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ACKNOWLEDGMENTS |
We thank D. Cullen and H. Leung for providing vectors. Matt
Morra, Maury Wiese, and three anonymous reviewers provided helpful comments and criticisms.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Plant, Soil, and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339. Phone: (208) 885-7933. Fax: (208) 885-7760. E-mail: gknudsen{at}uidaho.edu.
Published as Idaho Agricultural Experiment Station paper 99712.
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Applied and Environmental Microbiology, February 2000, p. 810-815, Vol. 66, No. 2
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
