Article

J. Korean Soc. Appl. Biol. Chem. 54(1), 99-104 (2011)

Bacteriophages of Pseudomonas tolaasii for the Biological Control ofBrown Blotch Disease
Min-Hee Kim, Seong-Wan Park, and Young-Kee Kim*
Department of Environmental and Biological Chemistry, Chungbuk National University, Cheongju 361-763, Korea
Received October 8, 2010; Accepted October 19, 2010
Pseudomonas tolaasii causes brown blotch disease in cultivated mushrooms by producing tolaasin,
a peptide toxin, which forms pores on the membrane and disrupts the cellular and fruiting bodystructures of mushrooms. For the biological control of this mushroom disease, virulent
bacteriophages of P. tolaasii were isolated from the sewage of Cheongju, Korea. Twenty-one phages
were isolated from four different locations, and their toxicities to host bacteria were measured byinspecting the turbidity and size of their plaques. They were divided into three categories on thebasis of their toxicities to host bacteria. In order to test if these phages can be used for the biologicalcontrol of mushroom diseases, a pitting test was performed. The surfaces of mushroom caps were
inoculated with both pathogenic bacteria and their phages. Phage toxicity was analyzed bymeasuring the size of the blotches that formed on the surface of mushrooms, because these sizes are
representative of the amount of tolaasin peptide produced by pathogenic bacteria in the presenceof bacteriophages. The formation of blotches was completely blocked by co-incubated phages.
These results show that phages can sterilize pathogenic bacteria in mushroom tissues as well as be
useful for the biological control of brown blotch disease. The optimum conditions for thebactericidal activity of the phages were also determined.
Key words: bacteriophage, brown blotch disease, oyster mushroom, phage therapy, Pseudomonas
tolaasii

Oyster mushroom (Pleurotus ostreatus) is a commercially
important crop in Korea. It ranks first in terms of
cultivation area and third in terms of production in themushroom industry. Cultivation of oyster mushroom isoften interrupted by major outbreaks of infection with
Pseudomonas tolaasii, Trichoderma, and mushroom
flies. P. tolaasii causes brown blotch disease of oyster
mushroom [Tolaas, 1915]. This pathogenic bacterium
produces tolaasin [Peng, 1986], a peptide toxin, whichforms pores on the cellular membranes, resulting in the
disruption of mushroom tissue and blotches on themushroom cap.
Brown blotch disease often makes mushroom cultivation
very difficult, because the pathogenic bacteria spread easily
through the cultivation shelf and then the cultivation
room, especially during the growth phase of the fungus
and the early budding period. The tolaasin moleculecomprises 18 amino acids and an octanoic acid at the N*Corresponding author
Phone: +82-43-261-2560; Fax: +82-43-271-5921
E-mail: ykkim10@chungbuk.ac.kr
doi:10.3839/jksabc.2011.014

terminus of the peptide. Since tolaasin molecules behave
as a detergent and have pore-forming properties, they canspread easily on the surface of fungi and mushroom caps
[Hutchison and Johnstone, 1993]. When the caps ofgrowing mushroom are infected, tissues are rapidly turned
into brown-colored blotches and eventually melted down,
leaving mucus and liquids with unpleasant smell. The
disease spreads rapidly, making mushroom cultivation
impossible in farms and eventually, the whole village[Kim et al., 2007].
In order to prevent the disease, underground water used
for cultivation should be sterilized and the cultivation
room should be clean. Furthermore, during the preparationof cultivation shelves, the room should be fumigated with
hot air [Wong and Preece, 1985; Geels et al., 1991]. For
the biological control, antagonistic bacteria [Soler-Rivas
et al., 1999] and specific bacteriophages [Guillaumes etal., 1998] were tested, and crop losses were reduced[Munsch et al., 1991]. Nevertheless, problems of resistantbacteria made large-scale experiments invalid, and nofurther experiments were reported. Antibiotics cancontrol diseases [Geels, 1995]; however, they cannot be
used to cultivate edible mushrooms. Therefore, no useful

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tools are presently available for disease control.
There are many advantages of using bacteriophages,
such as preventing contamination by pesticides andsuppressing the development of bacterial drug-resistance[Chanishvili et al., 2001]. Recently, the usefulness ofphage therapy for preventing the bacterial resistance toantibiotics was reevaluated. Since phages are highly
specific to host bacteria and have a rapid sterilization
effect, they were found to be more effective thanchemical therapy [Greer, 2005; Matsuzaki et al., 2005].
Since phages are specific to their host bacteria, various
phages of pathogenic bacteria are needed. In the present
study, various phages of P. tolaasii 6264 were isolated
and characterized for use to control brown blotch disease.

Materials and Methods
Isolation and culture conditions of P. tolaasii. P.
tolaasii 6264 was originally isolated in Korea from thefruiting bodies of oyster mushrooms severely damagedby brown blotch disease [Lee et al., 1997]. Mushroom
tissues were homogenized, and the homogenate was
extracted with sterilized distilled water. The extract was
evenly spread onto a Pseudomonas agar F (PAF; Difco,
Lawrence, KS, USA; Bacto-peptone, 10 g; Bacto-tryptone,
10 g; K2HPO4, 1.5 g; MgSO4, 1.5 g; glycerol, 10 mL;
agar, 15 g/L) plate, and P. tolaasii 6264 was isolated andidentified by the white line test and pitting test on
mushroom tissue [Kim et al., 1994] as well as bybacteriological characteristics using the method of Tsuneda
et al. [1995]. The isolated bacteria were stored at 80oC in
PAF broth contaning 20% (v/v) glycerol until use.
Bacteriophage isolation. Phages were isolated fromvarious sewage samples obtained from Cheongju, Korea
by using the double agar layer technique [Adams, 1959].
The hard agar layer was made with 1.5% agar, and thesoft agar layer contained 0.7% agar. The supernatant fluidof each sewage sample was added to the culture of hostbacteria. This solution was mixed with soft agar medium
at a ratio of 1:2 and poured on to the hard agar medium.
The double layer medium was incubated for 15 h at 25oC.
One phage plaque was chosen for phage amplification,
picked with a sterilized toothpick, and inoculated in 5 mL
of PAF broth together with 1% of overnight culture of the
P. tolaasii 6264 strain. In keeping with the method of
Chibani-Chennoufi et al. [2004], NaCl was added to thelysate to a final concentration of 10% and incubated for
1 h at 0oC. After centrifugation at 8,000 rpm for 10 min,polyethylene glycol 6000 was added to the supernatant toa final concentration of 10%, and the lysate wasincubated for 1 h at 0oC. Polyethylene glycol-precipitatedphages were collected by centrifugation at 8,000 rpm for

10 min. Phage precipitates were resuspended in phage
buffer (50 mM Tris-HCl, pH 7.3, 150 mM NaCl, 20 mM
NH4Cl, 10 mM MgCl2, 1 mM CaCl2, 0.2% gelatin).
Phage count and lysis activity. Phage titers were
determined using the double agar layer technique[Sambrook and Russell, 2001]. Crude lysates wereobtained by removing bacterial debris. The phage lysates
were diluted 104-108 times, mixed with host bacteria in 3
mL of soft agar, and poured over solidified PAF hard agar
medium. These plates were incubated for 15 h at 25oC
under stationary conditions, and phage plaques werecounted. In order to measure the lysis activity of phages,
PAF broth was inoculated with 5% culture supernatant of
P. tolaasii 6264 and 1% of diluted phage lysate. The broth
was incubated at 25oC under aerobic condition (150 rpm)
in a shaking incubator for 15 h. The absorbance wasmeasured using a spectrophotometer at 600 nm.
Pitting test. The fresh fruiting bodies of Agaricus
bisporus mushrooms were collected and inoculated with
P. tolaasii 6264 following the method of Gandy [1968].
Mushroom caps were cut off horizontally, and 10 μL ofbacterial culture was dropped on the surface. The
mushrooms were incubated at 25oC in a plastic box
saturated with water vapor. Brown discoloration aroundthe inoculation spot was considered as a positive reaction.
In order to evaluate the phage-induced inhibitory effects
on brown blotch disease, the properly diluted phagelysates were mixed with P. tolaasii 6264 at a 1:1 ratio,
and 10 μL of the mixture was dropped on the surface ofthe mushrooms. After 15 h incubation, the degree of
discoloration was evaluated by measuring the area of
blotch. A control experiment was performed using mixturesof P. tolaasii 6264 and sterile water.

Results
Bactericidal activity of bacteriophages. Twenty-one
bacteriophages were isolated from sewage samples collectedfrom four different locations in Cheongju, Korea. On thebasis of their toxicities to the host bacterium, P. tolaasii
6264, phages were divided into three groups, according to
the shapes of their plaques: big and clear, small and clear,and turbid, and bactericidal activities were evaluated asvery toxic, toxic, and mildly toxic, respectively (Fig. 1).
In order to characterize host toxicities, three phages,
namely, hb1a, hb2d, and bp5e, were chosen from each
toxicity group. Bacterial lysis was measured on the basisof absorbance decrease at 600 nm. When 5% of hostbacteria and 1% of diluted phages at a 100:1 ratio were
cultured in PAF medium, complete bacterial lyses wereobtained with hb1a and hb2d after 12 h (Fig. 2). The
addition of phage bp5e slowly decreased the absorbanceBacteriophages for the control of brown blotch disease

101

Fig. 1. Plaques formed by the isolated phages. (A) Clear and large plaques formed by highly toxic phages. (B) Clear andsmall plaques formed by toxic phages. (C) Large but turbid plaques formed by mildly toxic phages.

Fig. 2. Bacterial lyses caused by phages having various
bacterial lysis activities. Host bacteria and phages wereadded simultaneously at a 100:1 ratio.

after 15 h, but no complete lysis was observed.
When complete bacterial lyses were obtained withphages hb1a, hb2d, and bp5e, their titers were determined
to be 1.12×109, 1.35×109 and 7.93×109 pfu·mL−1, respectively,
implying that the densities of the phages were similar,
regardless of their toxicities. These results show that thesize and clearance of plaques represent the host toxicityof the phages. The phage hb1a decreased absorbanceafter a 5-h incubation, and complete bacterial lysis wasachieved after 11 h. Similarly, the phage hb2d decreased
absorbance at 7 h, and complete bacterial lysis wasachieved after 13 h. The mildly virulent phage bp5e
decreased absorbance at 9-10 h, and the decrease inabsorbance was very slow.
Suppression of blotch formation by phages. The
direct effect of phages on brown blotch disease was
evaluated using the pitting test. This method measures theamount of collapsing on the surface of the mushroom
after the surface is inoculated with the pathogen. P.
tolaasii made big brown blotches on the cut surface ofmushrooms after 12 h (Fig. 3). Blotch formation was
suppressed when phage hb1a was simultaneously added,
implying that hb1a can prevent brown blotch formation.
Similar effects of phages were obtained with the phagesof both very toxic and toxic groups. The effect of theFig. 3. Brown blotch formation by P. tolaasii. The
diluted lysate of phage hb1a by 1,000 times was diluted
again as indicated and added to the surface of a mushroom
along with P. tolaasii. The mushroom was incubated for
15 h. P. tol: only pathogenic bacteria were added. The 1-1/
64 hb1a: equal volumes of diluted phage lysate andpathogenic bacteria were added.

phages decreased when the phage solution was diluted.
More dilutions of phage solution increased the size of the
blotches (Fig. 4). As the number of phages decreased
with dilution, blotches were formed at dilutions of morethan 1/8 times, and the size of the blotches increased as
the dilution of the phage solution increased. Blotches
formed by pathogen in the presence of phages at all
dilutions were not dense and deep enough compared tothose formed by only pathogen. These results show thatsimultaneous treatments of a pathogen and its phage
suppress or decrease the formation of blotches, suggestingthat the phage can be a good candidate for disease control.
Size of phage inoculation and bacterial lysis. The
relationship between time taken for complete bacterial
lysis and the size of phage inoculation was examined.

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Fig. 4. Effects of bacteriophages on the formation ofbrown blotches. The phage hb1a was diluted as indicatedand added to the surface of a mushroom. The sizes of the
blotches were compared with those formed after inoculation
with only host bacteria.

When 10, 100, and 500 μL of the diluted phage lysate of
hb1a (1×105 pfu·mL−1) were added, at bacteria:phageratios corresponding to 5,000:1, 1,000:1, and 100:1, theabsorbance decreased after 8, 6, and 5 h of incubation,
respectively (Fig. 5A). After 13 h of incubation, complete
bacterial lysis was observed. The diluted toxic phagehb2d (1×105 pfu·mL−1) could not lyse bacteria completely
at the bacteria:phage ratio of 5,000:1; however, at 1,000:1
and 100:1, complete lyses were observed (Fig. 5B). In thecase of the diluted mildly toxic phage bp5e (7×105
pfu·mL−1), the bacteria:phage ratios corresponding to
5,000:7, 1,000:7, and 100:7 showed decreases in absorbanceafter 7-9 h, and no complete lysis was observed after 15h, regardless of the size of the inoculation (Fig. 5C).
Growth phase-dependent bacterial lysis. In order tomeasure host toxicity of bacteriophages at various growthphases, hb1a (105 pfu·mL1) was added 0, 1.5, 3, 4.5, and 6
h after the beginning of incubation (Fig. 6). When phages
were inoculated simultaneously with bacteria at 0 h,
almost no bacterial growth was observed. Inoculations ofphages at 1.5 and 3 h also resulted in complete bacterial
lyses after 8 and 12 h of incubation, respectively. Afteradding phages at 4.5 h, the early stationary phase wasobserved at 12 h, but lysis was measured at 16 h.
However, phage inoculation at 6 h resulted in incomplete
bacterial lysis and maximal bacterial growth was obtained.
These results showed that the bactericidal effect of thephage decreases as the ratio between a host bacterium andits phage increases. Therefore, as bacterial density increases,
the host toxicity of phages appears to weaken, and achieving
complete bacterial lysis becomes increasingly difficult.
Temperature effect on phage toxicity. After 4 h ofincubation of P. tolaasii, phage solution (105 pfu·mL−1)
was added at various incubation temperatures: 20, 25, 30,

Fig. 5. Inoculation size-dependent bacterial lyses. Phages
diluted by 104 times were added to the 50 mL of bacterial
culture. The indicated volumes of diluted phages, (A)hb1a, (B) hb2d, and (C) bp5e, were added to the P. tolaasii
culture.

and 37oC (Fig. 7). In the absence of phage treatment, P.
tolaasii showed normal growth curves at 20, 25, and
30oC. However, bacterial growth was suppressed at 37oC,
because the conditions were not optimal. The absorbance
did not increase after 8 h of cultivation and was below 1.5
at 600 nm. In the presence of phages, complete bacterial
lyses were observed at 20 and 25oC after 16 h ofcultivation. At 30oC, the bactericidal effect of phages was
slow. The absorbance decreased after 16 h of incubation
and was 1.2 at 20 h. No bacterial lysis was observed at
37oC. The toxicity of the phages was maximal at the
optimum temperature of the host bacteria.

Bacteriophages for the control of brown blotch disease

Fig. 6. Effect of phage inoculations at different growthphases. The phage hb1a was added to the culture at theindicated times.

Fig. 7. Temperature-dependent bacterial lyses. Bacteria
were cultured at the indicated temperature, and the phagehb1a was added after incubation for 4 h.

Discussion
Twenty-one phages of P. tolaasii 6264 were isolated,
and their bactericidal activities were analyzed. Other
characteristics of these phages, such as morphology,
restriction endonuclease digestion patterns, and immunological criteria, are currently under investigation. They
showed various toxicity levels toward the host bacterium.
A positive relationship was observed between thevirulence of phages and the size and clearness of theirplaques. Phages that formed clear and big plaques werevery toxic to their host bacteria and achieved complete
bacterial lyses. However, phages that formed small andclear plaques or turbid plaques had relatively weakbactericidal activities (Figs. 1 and 2). They needed alonger incubation time to achieve bacterial lysis and often
showed incomplete lysis. These results suggest that thehost toxicities of phages can be evaluated qualitatively byanalyzing their plaques.

103

Host toxicity of bacteriophages also depends on thegrowth phase of bacteria. Bactericidal activity of phages
was maximal in the early logarithmic phase of hostbacteria. In the late logarithmic phase and stationary
phase, incomplete bacterial lyses were observed after
inoculations with the toxic phage hb1a (Fig. 6). Phage
hb1a-induced bacterial lyses were also dependent on the
nutritional growth state of the host bacteria. These results
were consistent with those of previous reports, showingthat the growth of the phage depends on the physiologicalconditions of the host bacterium [Hadas et al., 1997;
Cooper and Heinemann, 2000].
The incubation temperature was also one of the major
factors determining phage toxicity (Fig. 7). The bactericidal
activity of hb1a was maximal at or near the optimum
temperatures of host bacteria. Since the optimum
temperature of P. tolaasii is 25oC, complete bacterial lysis
was also achieved at this temperature. The influence ofbacterial growth phase on phage infection was similar tothat of the bacteriophage of P. fluorescens [Sillankorva etal., 2004]. Although there was a report that the amount ofphage inoculated is inversely related to the amount ofphage production [Moebus, 1996], hb1a-induced bacterial
lysis appears to depend on the amount of phage inoculated(Fig. 2). Therefore, when disease symptoms are detected,
phage treatment should be administered as early as
possible for the best disease control.
During the characterization of various bacteriophagesof P. tolaasii 6264, the phages were found to control thedensity of pathogenic bacteria in cultivation farm area,
and thus probably decrease the rate of disease outbreaks.
Phage therapy appears to be more beneficial for controllingmushroom diseases due to the toxicity of antibiotics andagricultural chemicals on mushrooms. Nevertheless,difficulty in phage application still remains due to its highspecificity to host bacteria. At present, we have more than
80 strains of P. tolaasii-like bacteria collected from farms
damaged by brown blotch disease. These strains will beclassified as pathogenic strains in terms of phage sensitivity[Chakrabarti et al., 2000] and specific bacteriophages will
be isolated for each type of bacterial strain. Since various
phages of P. tolaasii 6264 from sewage were isolated, we
foresee no difficulty in isolating more phages from otherlocations. Once the phage sensitivities of these strains arecompletely determined, mixtures of phages will be available
for disease control.
Acknowledgments. This study was supported by theTechnology Development Program for Agriculture andForestry, Ministry for Agriculture, Forestry and Fisheries,
Republic of Korea.

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