Pseudomonas stutzeri YPL-1 Genetic Transformation and Antifungal Mechanism against Fusarium solani, an Agent of Plant Root Rot.
ABSTRACT An actively antagonistic bacterium that could be used as a biocontrol agent against Fusarium solani, which causes root rots with considerable losses in many important crops, was isolated from a ginseng rhizosphere and identified as a strain of Pseudomonas stutzeri. In several biochemical tests with culture filtrates of P. stutzeri YPL-1 and in mutational analyses of antifungal activities of reinforced or defective mutants, we found that the anti-F. solani mechanism of the bacterium may involve a lytic enzyme rather than a toxic substance or antibiotic. P. stutzeri YPL-1 produced extracellular chitinase and laminarinase when grown on different polymers such as chitin, laminarin, or F. solani mycelium. These lytic extracellular enzymes markedly inhibited mycelial growth rather than spore germination and also caused lysis of F. solani mycelia and germ tubes. Scanning electron microscopy revealed degradation of the F. solani mycelium. Abnormal hyphal swelling and retreating were caused by the lysing agents from P. stutzeri YPL-1, and a penetration hole was formed on the hyphae in the region of interaction with the bacterium; the walls of this region were rapidly lysed, causing leakage of protoplasm. Genetically bred P. stutzeri YPL-1 was obtained by transformation of the bacterium with a broad-host-range vector, pKT230. Also, the best conditions for the transformation were investigated.
- 11/2003; 6:263-294.
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ABSTRACT: THE enzyme contained in a culture fluid of Bacillus circulans has been shown to exert lytic activity towards Aspergillus oryzae and that a polymer of melibiose was liberated from the cell wall. However, the cell wall was not completely lysed by the enzyme owing to the large amount of chitin present. During the search for enzymes causing lysis of fungal cells, it was found that a mixed preparation of chitinase and lytic enzyme exerted strong lytic activity on the cell wall of A. oryzae.Nature 02/1959; 183(4655):186-7. · 38.60 Impact Factor
Vol. 57, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 510-516
Copyright © 1991, American Society for Microbiology
Pseudomonas stutzeri YPL-1 Genetic Transformation and Antifungal
Mechanism against Fusarium solani, an Agent of Plant Root Rot
HO-SEONG LIM, YONG-SU KIM, AND SANG-DAL KIM*
Department ofApplied Microbiology, Yeungnam University, Gyongsan 713-749, Korea
Received 24 July 1990/Accepted 4 December 1990
An actively antagonistic bacterium that could be used as a biocontrol agent against Fusarium solani, which
causes root rots with considerable losses in many important crops, was isolated from a ginseng rhizosphere and
identified as a strain of Pseudomonas stutzeri. In several biochemical tests with culture filtrates of P. stutzeri
YPL-1 and in mutational analyses of antifungal activities of reinforced or defective mutants, we found that the
anti-F. solani mechanism of the bacterium may involve a lytic enzyme rather than a toxic substance or
antibiotic. P. stutzeri YPL-1 produced extracellular chitinase and laminarinase when grown on different
polymers such as chitin, laminarin, or F. solani mycelium. These lytic extracellular enzymes markedly
inhibited mycelial growth rather than spore germination and also caused lysis of F. solani mycelia and germ
tubes. Scanning electron microscopy revealed degradation of the F. solani mycelium. Abnormal hyphal
swelling and retreating were caused by the lysing agents from P. stutzeri YPL-1, and a penetration hole was
formed on the hyphae in the region of interaction with the bacterium; the walls of this region were rapidly
lysed, causing leakage of protoplasm. Genetically bred P. stutzeri YPL-l was obtained by transformation of the
bacterium with a broad-host-range vector, pKT230. Also, the best conditions for the transformation were
Antagonistic microorganisms, by their interactions with
various soil-borne plant pathogens, play a major role in
microbial equilibrium and serve as powerful agents for
biological disease control (2, 5, 8, 15, 39). The interactions
between biocontrol agents and plant pathogens have been
studied extensively, and the application of biocontrol agents
in the protection of some commercially important crops is
promising (38, 47). Biocontrol of plant pathogens provides
an alternative means of reducing the incidence of plant
disease without the negative aspects of chemical controls
like pesticides (6). Chemical fungicides are costly, can cause
environmental pollution, and may induce pathogen resist-
ance (18, 24). Additionally, they can cause stunting and
chlorosis of young seedlings (18). Fusarium solani, a patho-
genic plant fungus, causes root rots, which results in con-
siderable economic losses in many important crops (7, 9).
The use of antagonists to control diseases incited by F.
solani is being intensively studied (31, 47), but the mecha-
nism involved in lysis of the fungus by bacteria is not well
The objectives of the present study were to (i) isolate,
select, and identify potentially useful bacterial antagonists
for biocontrol of F. solani;
mechanism with biochemical tests, mutational anialyses, and
microscopic observations; and (iii) attempt transformation of
the antagonist to create a model system for the further
genetic development of multifunctional biocontrol agents.
(ii) determine its antifungal
MATERIALS AND METHODS
Isolation and identification of an antagonistic bacterium.
Antagonistic bacteria were isolated from rhizospheres in
ginseng root rot-suppressive soils in Yeungpung-gun, Korea.
To isolate bacteria, the rhizosphere soils were suspended in
0.01 M phosphate buffer (pH 7.2) with a mortar, and then
serial dilutions were plated on nutrient agar. Each isolate
was tested for its inhibition of F. solani, a pathogenic plant
fungus, as described below. The fungus F. solani was
provided by the Korea Ginseng and Tobacco Research
Institute and was grown on potato dextrose agar (PDA). The
most efficient antagonistic bacterium was selected and iden-
tified according to criteria in Bergey's Manual ofSystematic
In vitro antifungal activity tests. Two different techniques
were used for testing the antagonistic effect against F.
solani. In the first, which assayed the antifungal activity of
bacterial strains on plates, samples (5 ,ul, containing approx-
imately 106 cells) from overnight cultures of bacterial strains
in nutrient broth were inoculated 1 cm from the edge ofpetri
plates and allowed to soak into the agar. A small plug (about
5 mm square) of F. solani inoculum from the leading edge of
a culture of F. solani grown at 28°C for 3 days on PDA
containing 0.2% chitin was placed in the center of the plate.
Plates were incubated at 28°C and scored after 4 or 5 days by
measuring the distance between the edges of the bacterial
colony and fungal mycelium. In the second assay, which
tested the antifungal activity in broth culture, bacterial
cultures were grown at 30°C for 84 h with aeration. Cells
were removed by centrifugation at 12,000 x g for 20 min.
The culture supernatants were then filtered aseptically
through 0.45-,um-pore-size membrane filters. The resulting
filtrates were stored at 4°C. Petri plates were filled with
molten PDA with 1% culture filtrate. After the plates were
cooled, the F. solani inoculum was placed on the agar
surface, and the plates were incubated for 4 or 5 days. The
diameters of the F. solani colonies were recorded, and the
inhibition ratios were calculated relative to that of a control
without incorporated culture filtrate. Small plugs taken from
2- to 3-day-old cultures of F. solani and added to 250-ml
Erlenmeyer flasks containing 2.64% potato dextrose broth
(PDB) were incorporated aseptically with 5% culture super-
natants and incubated on a rotary shaker at 28°C for 5 days.
ACTION OF P. STUTZERI AGAINST F. SOLANI
Fungal mycelia were collected on oven-dried preweighed
paper (Toyo filter paper no. 2) and dried at 90°C, and dry
weights were determined. The inhibition ratio was expressed
relative to a control (H20).
Enzymatic activity tests. The cell wall-degrading enzymes
such asexo-1,3-0-D-glucosidase(,B-1,3-glucanase) and ,B-N-
acethyl-D-glucosaminidase (chitinase) were assayed in cul-
ture filtrates of Pseudomonas stutzeri YPL-1. For the prep-
aration of crude chitinase, the bacterium was grown at
30°C for 84 h on a rotary shaker in chitin-peptone medium
(pH 6.8) containing 0.5% glucose, 0.2% peptone, 0.2%
chitin, (from crab shells; Sigma), 0.1% K2HPO4, 0.05%
7H2O, and 0.05% NaCl. For the preparation of
crude laminarinase, bacteria were grown at 30°C for 72 h on
a rotary shaker in peptone medium containing laminarin
(from Eisenia arborea; Tokyo Chemical Co.). The cultures
were centrifuged aseptically at 12,000 x g for 20 min at 4°C.
The lytic enzymes were prepared by salting them with
ammonium sulfate and dialyzing them with buffer.
The activities of chitinase and laminarinase were deter-
mined by measuring the release of reducing sugar by the
method of Nelson (36). One unit of chitinase (laminarinase)
activity was determined as 1 ,umol of glucose per mg of
protein per h. The reaction mixture of chitinase contained
0.3 ml of 1 M sodium acetate buffer (pH 5.3), 0.5 ml of 0.1%
colloidal chitin prepared by the method of Bemiller (4) or F.
solani mycelium prepared by the method of Morrissey et al.
(34), and 0.25 ml of enzyme solution. The reaction was
carried out at 50°C for 4 h. The reaction mixture of lamina-
rinase contained 0.3 ml of 1/15 M phosphate buffer (pH 5.5),
0.5 ml of 0.2% soluble laminarin or F. solani mycelium, and
0.25 ml of enzyme solution. The reaction was carried out at
40°C for 2 h.
Mutation and selection. The antagonist was mutated with
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment or
UV radiation. The NTG mutagenesis procedure was based
on the method ofMiller (30). For the UV treatment, bacterial
suspensions of 108 CFU/ml were prepared in 0.1 M phos-
phate buffer. Then 5 ml of each suspension was placed in a
glass petri dish and exposed 30 cm below a 10-W germicidal
UV lamp for 30 s. Chitinase-reinforced or -defective mutants
were characterized by growing the mutagen-exposed cells on
minimal agar plates containing colloidal chitin as a sole
Siderophore assay. Siderophore assay procedures were
based on the methods of Meyer and Abdallah (28, 29) and
Scher and Baker (41). Siderophore production was deter-
mined by growing bacteria in iron-deficient succinate mini-
mal medium (SMM) containing 0.6% K2HPO4, 0.3%
KH2PO4, 0.1% (NH4)2SO4, 0.02% MgSO4 7H20, and 0.4%
succinic acid. The test bacteria were introduced into 50 ml of
medium and incubated on a rotary shaker at 30°C for 40 h.
The bacterial cells were removed by centrifugation at 12,000
x g for 20 min. Then 50[lIof 2 M FeCl3 was added to sample
tubes of the culture supernatants (4 ml per tube); other tubes
to which no iron was added served as the blanks. Insoluble
iron salts were removed by centrifugation at 3,000 x g for 5
min. The A435 of the supernatants was measured with a
spectrophotometer against water as the blank.
Spore germination assay. Germination of chlamydospores
and growth of germ tubes of F. solani were assayed by
mixing a 50-,ul drop of the chlamydospore suspension pre-
pared by the method of Elad and Baker (10) with an equal
volume of P. stutzeri YPL-1 filtrate in the wells of acid-
washed depression slides. The slides were incubated for 24 h
in a sterile petri dish containing filter paper moistened with
distilled water. Fifty spores per well were counted for each
of the three replicates per treatment. Germination rates of
the chlamydospores and lysis of germ tubes were examined
under a light microscope at x400 magnification.
Scanning electron microscopy. Microscopic observations
were made in the interacting regions ofF. solani grown with
P. stutzeri YPL-1 in dual culture. The samples were fixed
with 3% glutaraldehyde in 0.2 M phosphate buffer (pH 6.5)
for 3 h, washed with the same buffer for 15 min, fixed with
2% OS04 for 2 h, and finally washed again with the buffer.
The material was dehydrated with ethanol at 4°C by using a
series of steps for 10 min each. The specimens were dried in
a Hitachi HCP-2 critical point drier with CO2 as the carrier
gas. The dried specimens were mounted on stubs with
Television Tube Koat to prevent charging. The specimens
were sputter coated with gold palladium in a Ion Coater Giko
IB-5 and observed with a scanning electron microscope (ISI
Genetic transformation. The transformation procedure
was based on the method of Bagdasarian and Timmis (1).
Bacterial cells were grown in LB broth at 30°C for 24 h on a
rotary shaker. A portion (0.5 ml) of such a culture was
reinoculated in 50 ml of fresh LB broth and grown to an
optical density at 660 nm of 0.25, which equals 7 x 105
CFU/ml. The cells were then chilled, harvested by centrifu-
gation at 12,000 x g for 3 min, and washed once with 25 ml
of 10 mM MOPS (morpholinepropanesulfonic acid) (pH
7.0)-10 mM RbCI-100 mM CaCl2. The cells were again
centrifuged and suspended in 25 ml ofMRC (100 mM MOPS
[pH 6.5], 10 mM RbCl, 100 mM CaCl2). The cells were kept
on ice for 45 min, harvested by centrifugation, and sus-
pended in 5 ml ofMRC. An aliquot (0.2 ml) ofthese cells was
then mixed with 1 pug of pKT 230 DNA isolated from
Pseudomonas putida(pKT230) by the method of Kado and
Liu (19) and incubated at 0°C for 60 min. The cell-DNA
mixture was then subjected to a heat pulse at 42.5°C for 2
min, chilled, and finally diluted in 10 volumes of fresh LB
broth. The cells were allowed to grow at 30°C on a rotary
shaker, and then aliquots were plated on LB agar plates
containing 100 ,ug of kanamycin sulfate per ml.
Isolation and identification of antagonistic bacterium. For
the selection ofthe antagonistic bacterium most inhibitoryto
F. solani, over 300 isolates of bacteria were originally
obtained from soils in which ginseng was cultivated. Among
these isolates, 45 isolates that had similar colony character-
istics were investigated for their anti-F. solani activities.
Only 10 isolates produced a zone of inhibition of 15 mm or
more with F. solani on PDA. The most active antagonistic
bacterium, YPL-1, was selected and identified as P. stutzeri
based on the following characteristics: it was gram negative,
motile by polar flagella, catalase positive, gelatin liquefac-
tion negative, denitrification positive, and fluorescent pig-
Activity of P. stutzeri YPL-1 against F. solani. To investi-
gate the antifungal substance from P. stutzeri YPL-1 culture,
the bacterial cells were grown in chitin-peptone medium at
30°C for 84 h with aeration. The antifungal activity according
to in vitro tests was compared for the following: (i) culture
filtrate dialyzed with a cellulose dialysis sack (molecular
weight cutoff, 12,000), (ii) evaporated nonprotein solution of
the supernatant after the culture filtrate was treated with
cold ethanol, and (iii) heat-treated culture filtrate. After 5
days of incubation at 28°C, the culture filtrate inhibited
VOL. 57, 1991
APPL. ENVIRON. MICROBIOL.
TABLE 1. Antifungal activity of P. stutzeri YPL-1
against F. solani
Fungal dry weight'
Fungal colony sizeb
aDry weight of F. solani with the treatment of P. stutzeri YPL-1 in PDB
after 5 days of incubation at 28°C.
bColony circle diameter of F. solani on PDA plates with treatment P.
stutzeri YPL-1 after 5 days of incubation at 28°C.
' Completed inhibition ratio (100%)-dry weight ofF. solani cultured with
solutions relative to those cultured with water.
dCompleted inhibition ratio (100%)-colony size ofF. solani cultured with
solutions relative to those cultured with water.
ep. stutzeri YPL-1 was grown in the chitin-peptone medium at 30'C for 84
fCulture filtrate was dialyzed at 4°C for 48 h through a cellulose dialysis
sack (molecular weight cutoff, 12,000).
g Culture filtrate was precipitated by the addition of cold ethanol, and the
supernatant was vacuum evaporated at 55°C.
h Culture filtrate was heated at 80°C for 1 h.
growth of F. solani by 57.7%, whereas the nonprotein
solution inhibited growth by 10.4%. Losses of antifungal
activity after treatment with nonprotein solution and heat-
treated solution were 82 and 80.7%, respectively, compared
with the activity of the culture filtrate. However, only 10.9%
ofthe antifungal activity was lost when F. solani was treated
with the dialyzed solution. According to these results, anti-
fungal substances involved in inhibition of F. solani by P.
stutzeri YPL-1 were presumed to be heat unstable, macro-
molecular substances such as hydrolytic enzymes (Table 1).
Production of extracellular hydrolytic enzymes. Chitinase
and laminarinase (,B-1,3-glucanase) activities produced by P.
stutzeri YPL-1 in the presence of different carbon sources
are presented in Table 2. Chitinase activity was observed in
media containing chitin, colloidal chitin, laminarin with
chitin, N-acetylglucosamine, or dried F. solani mycelium as
carbon sources. Laminarinase activity was observed in
media containing laminarin, laminarin with chitin, or dried
F. solani mycelium. The enzyme produced was capable of
degrading laminarin or chitin as well as F. solani mycelium.
Mutational analysis. Since the reduced suppressiveness of
chitinase-defective mutants could be due to their inability to
establish a significant mechanism of lysis against F. solani,
studies were carried out to evaluate their ability to inhibit the
growth ofF. solani. Mutants ofP. stutzeri YPL-1 that lacked
TABLE 2. Effect of various carbon sources on chitinase and
laminarinase production by P. stutzeri YPL-la
Enzymatic activity (U)
Laminarin with chitin
F. solani mycelium
aP. stutzeri was grown in a synthetic medium containing the various carbon
sources for 84 h. Units ofchitinase and laminarinase activity were determined
as micromoles of glucose per milligram of protein per hour.
TABLE 3. Comparison of antifungal abilities of P. stutzeri YPL-1
and its chitinase-reinforced or -defective mutants
against F. solani"
Enzymatic activity (U)
Antifungal activity (%)
" P. stutzeri YPL-1 and its mutants were grown in chitin-laminarin-peptone
medium at 30°C for 84 h. The culture filtrates were used for assays of
enzymatic and antifungal activities.
bDistance between the edges of the bacterial colony and fungal mycelium
after 5 days of incubation at 28°C.
chitinase and laminarinase ability (chi lam) or chitinase (chi)
were obtained by NTG mutagenesis. P. stutzeri YPL-M122
(chi lam) did not inhibit fungal growth at all, whereas P.
stutzeri YPL-M153 (chi) inhibited growth only a little. In
addition, we observed that mutants (P. stutzeri YPL-M26
and YPL-M178) produced larger inhibition zones after mu-
tagenesis with UV or NTG. These are presumed to have
These results indicate that the antifungal mechanism of P.
stutzeri YPL-1 depends more on enzymatic lysis of the cell
wall components of F. solani by chitinase than on laminar-
inase (Table 3).
Production ofsiderophore byP. stutzeri YPL-1. To evaluate
the antifungal mechanism by which the extracellular sidero-
phore efficiently chelates environmental iron, making it less
available to certain native microflora (20, 21, 25, 35, 42), the
possibility of siderophore production by P. stutzeri YPL-1
was investigated after 40 h of growth in SMM containing
different concentrations of FeCl3. In our experiments, P.
stutzeri YPL-1 was not able to produce an extracellular
siderophore in iron-deficient medium; this result is entirely
different from that reported previously (29).
Antifungal effect of lytic enzymes on mycelial growth of F.
solani. To evaluate the effects of extracellular lytic enzymes
of P. stutzeri YPL-1 and its mutant on mycelial growth ofF.
solani, each of the lytic crude enzymes (10 ,ug of chitinase
per ml, 17 ,ug of laminarinase per ml) of the bacteria was
added to PDB medium preinoculated with F. solani for 3
days, and the culture was grown at 28°C for 4 days. A
decrease of the mycelial volume of F. solani could be
observed from the first day after the enzymes were added.
Crude chitinase of P. stutzeri YPL-1 inhibited the fungal
mycelial growth by 87.1% compared with that of the un-
treated control after 24 h of incubation, whereas crude
laminarinase inhibited growth by only 50%. In addition, the
antifungal activity of a crude chitinase of mutant P. stutzeri
YPL-M26 was observed at a much higher level than that of
the original strain (Fig. 1).
Effect of lytic enzymes on the germination of F. solani
chlamydospores. To confirm whether P. stutzeri YPL-1 in-
hibits germ tube growth of the germinated chlamydospore or
spore germination of F. solani, chlamydospore suspensions
of the culture filtrate of the bacterium were added to PDB.
An increase of mycelial volume of F. solani was not ob-
served, and the mycelial mass could scarcely be seen until 4
activities (chitinase, laminarinase).
512 LIM ET AL.
ACTION OF P. STUTZERI AGAINST F. SOLANI513
FIG. 1. Antifungal effect of lytic enzymes on mycelial growth of
F. solani. The 3-day-old F. solani cultures were treated with 10 ,ug
of crude chitinase from P. stutzeri YPL-M26 per ml (0), 10 ,ug of
crude chitinase from P. stutzeri YPL-1 per ml (0), 17 ,ug of crude
laminarinase from P. stutzeri YPL-M26 per ml (A), or 17 p.g ofcrude
laminarinase from P. stutzeri YPL-1 per ml (A) or not treated (O).
days after incubation (data not shown). The germination of
F. solani chlamydospores on culture disks with the bacterial
filtrate was inhibited by only 17.2% after 24 h of incubation
compared with that of the untreated control, but the growth
of germ tubes was inhibited; about 89.5% of the germ tube
underwent lysis. The length ofthe germ tube ofF. solani was
10 ,um, whereas that of the control was 380 ,um (Table 4).
Interaction between P. stutzeri YPL-1 and F. solani in dual
culture. To investigate the antagonism involved in the inter-
action between P. stutzeri YPL-1 and F. solani in dual
culture, the bacteria and fungi were grown in PDB at 28°C.
The mass ofthe fungus was reduced and mycelial propagules
were decreased about 90% compared with those of the
control without the bacterium from day 1 to day 5. However,
the fungus had no influence on bacterial growth (Fig. 2).
Scanning electron microscopic observations revealed the
degradation of F. solani mycelium when its cell wall com-
ponents served as the sole carbon source for P. stutzeri
YPL-1. Abnormal haphal swelling and retreating were
caused by excretion of lytic enzymes from the bacterium; a
hole was formed on the hyphae with accumulation of the
TABLE 4. Effect of P. stutzeri YPL-1 on germination of
chlamydospores of F. solania
(% of control)
Germ tube growth
P. stutzeri YPL-1
380 ± 50
aChlamydospore germination and germ tube growth were observed after 24
h of incubation with filtrate from P. stutzeri YPL-1 grown in chitin-peptone
medium at 30°C for 84 h.
bLysis ofgerminated spores was determined by the reduction in percentage
of chlamydospores with germ tubes and observation of lysed germ tubes.
FIG. 2. Antifungal effect of P. stutzeri YPL-1 on growth of F.
solani by dual culture: 0, F. solani and P. stutzeri YPL-1; 0, F.
solani; 0, P. stutzeri YPL-1.
bacterial cells around the site, and the walls of this region
were rapidly lysed, causing leakage of protoplasm (Fig. 3).
Genetic transformation of P. stutzeri YPL-1. For develop-
ment of a multifunctional biocontrol agent, we obtained
genetically bred P. stutzeri YPL-1 by transformation with
the broad-host-range vector pKT230 (Fig. 4). The maximum
frequency of the transformation was achieved when the
bacterialcells were harvested
growth phase (Fig. 5). The highest transformation efficiency
was obtained when the competent cells were exposed to
chilled transformation buffer containing 20 mM RbCl and 100
mM CaCl2 before the addition of 1 ,ug of plasmid DNA (Fig.
6). The optimal pH for transformation was 6.5. When
competent bacterial cells that were incubated for 1 h were
brought in contact with plasmid DNA, the transformants
were obtained at the highest frequency. It was calculated
that the transformation frequency was 2 x 10-6 to 6 x 10-6
under these optimal conditions.
at the early-exponential
Major objectives of this study were to isolate a potentially
useful bacterial antagonist for biocontrol of F. solani, to
evaluate in detail its antifungal mechanism, and to develop a
more powerful antagonist by genetic transformation. Selec-
tion of these bacterial isolates was facilitated by the use of
the in vitro antifungal activity test. In our results, an actively
antagonistic bacterium that was strongly inhibitory to F.
solani was isolated from ginseng rhizospheres and identified
as a strain of P. stutzeri.
Selection procedures gave some indication of the mecha-
nism of interaction between P. stutzeri YPL-1 and F. solani.
In several biochemical tests with culture filtrates of P.
stutzeri YPL-1, the antifungal substances involved in the
inhibition of F. solani appeared to be heat-labile, macromo-
lecular proteins (Table 1). Hence, the mechanism could
VOL. 57, 1991
LIM ET AL.
FIG. 3. Scanning electron micrographs of F. solani hyphae interacting with P. stutzeri YPL-1 in dual culture. (A) Abnormal hyphal
swelling and outflow of protoplasm (arrow) caused by disintegration of the hypha. (B) Hyphal retreat. (C) Lysed hole on a hypha with the
bacterium (arrow). (D) Irregular wall of a "stunted" hypha and lysis of the tip.
involve a lysing agent rather than a toxic substance or
antibiotic. This was confirmed by mutational analysis of the
antifungal activities of mutants with reinforced or defective
chitinase and/or laminarinase productivity (Table 3). Also,
P. stutzeri YPL-1 released extracellular ,-1,3-glucanase and
chitinase, which are key enzymes in the lysis of fungal cell
walls, when grown on polymers such as chitin, laminarin, or
F. solani mycelium as carbon sources (Table 2). Several
studies have shown that efficient parasitic biocontrol agents
excrete extracellular lytic enzymes that are capable of
degrading chitin and laminarin (12, 13, 16, 17, 31-34, 37,
43-46). Other similar studies have suggested that sidero-
phore production rather than lytic ability could be effective
in control of F. solani. However, this P. stutzeri YPL-1
isolate was not able to produce extracellular siderophores in
iron-deficient medium, which conflicts with other reports
(29). The lysing agents of P. stutzeri YPL-1 markedly
inhibited mycelial and germ tube growth rather than spore
germination of F. solani (Table 4, Fig. 1). Furthermore, the
antifungal activity ofcrude chitinase on mycelial growth was
much higher than that ofcrude laminarinase (Fig. 1). The cell
walls ofF. solani are composed mostly of chitin (47%), with
14% glucan (44). It seems, therefore, that chitinase is more
important than laminarinase in the degradation of F. solani
cell walls. Similarly, Ordentlich et al. showed that chitinase
FIG. 4. Agarose gel electrophoresis of pKT230 plasmid DNA
obtained from the transformant of P. stutzeri YPL-1. Lanes: A,
standard pKT230; B, P. stutzeri YPL-1; C, transformant by P.
stutzeri YPL-1; D, donor P. putida(pKT230).
APPL. ENVIRON. MICROBIOL.