identification and characterization of trichoderma species ...910 kim et al.j. microbiol....
TRANSCRIPT
May 2016⎪Vol. 26⎪No. 5
J. Microbiol. Biotechnol. (2016), 26(5), 909–917http://dx.doi.org/10.4014/jmb.1602.02012 Research Article jmbReview
Identification and Characterization of Trichoderma Species DamagingShiitake Mushroom Bed-Logs Infested by Camptomyia PestJun Young Kim, Hyuk Woo Kwon, Yeo Hong Yun, and Seong Hwan Kim*
Department of Microbiology and Institute of Biodiversity, Dankook University, Cheonan 31116, Republic of Korea
Introduction
Shiitake (Lentinula edodes, Berk. Pegler) is an economically
important edible and medicinal mushroom that is widely
cultivated in Asian countries, including Korea, Japan, and
China. Its cultivation has been increased around the world
owing to its unique flavor, tastes, and nutritional values
[16]. In general, shiitake is cultivated either by sawdust
media-based methods or by bed-log-based methods. Bed-
logs using oak timber have been used for the cultivation of
high-quality shiitake. Recently, huge economic loss has
occurred nationwide in Korea in shiitake farms where
mushroom production was performed using bed-logs [19].
Farmers in the shiitake farms have blamed mushroom flies
that newly emerged in the cultivation houses as the cause
of bed-log damage. The flies’ influence has been investigated
on several shiitake farms where disturbance of shiitake’s
mycelial growth and fruit body formation occurred in
mushroom fly-infested bed-logs. Large colonies of unknown
dipteran insects were found under the bark of the damaged
bed-logs and were identified as gall midges that
taxonomically belong to the genus Camptomyia Kieffer (Diptera:
Cecidomyiidae). Camptomyia corticalis and C. heterobia have
been identified in damaged shiitake bed-logs [19]. The
larvae of some Camptomyia species in this genus are known
to feed on the fungi colonized in dead trees [14], and the
larvae of the two Camptomyia flies were found to live on
shiitake mushroom that colonized the oak bed-logs. Thus,
these Camptomyia pests were associated with the shiitake
damage in bed-logs. Because these mushroom fly species
had not previously been recorded as mushroom pests, they
were reported as such [19].
Green molds are the fungi that cause green mold diseases
on cultivated mushrooms such as button mushroom (Agaricus
Received: February 11, 2016
Revised: February 20, 2016
Accepted: February 25, 2016
First published online
February 29, 2016
*Corresponding author
Phone: +82-41-550-3454;
Fax: +82-41-523-3454;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2016 by
The Korean Society for Microbiology
and Biotechnology
The shiitake mushroom industry has suffered from Camptomyia (gall midges) pest, which
feeds on the mycelium of shiitake mushroom during its cultivation. It has been postulated that
fungal damage of shiitake bed-logs is associated with infestation by the insect pest, but this is
not well understood. To understand the fungal damage associated with Camptomyia pest,
various Trichoderma species were isolated, identified, and characterized. In addition to two
previously known Trichoderma species, T. citrinoviride and T. deliquescens, two other
Trichoderma species, T. harzianum and T. atroviride, were newly identified from the pest-
infested bed-log samples obtained at three mushroom farms in Cheonan, Korea. Among these
four species, T. harzianum was the most evident. The results of a chromogenic media-based
assay for extracellular enzymes showed that these four species have the ability to produce
amylase, carboxyl-methyl cellulase, avicelase, pectinase, and ß-glucosidase, thus indicating
that they can degrade wood components. A dual culture assay on PDA indicated that T.
harzianum, T. atroviride, and T. citrinoviride were antagonistic against the mycelial growth of a
shiitake strain (Lentinula edodes). Inoculation tests on shiitake bed-logs revealed that all four
species were able to damage the wood of bed-logs. Our results provide evidence that the four
green mold species are the causal agents involved in fungal damage of shiitake bed-logs
infested by Camptomyia pest.
Keywords: Camptomyia, shiitake mushroom bed-logs, Trichoderma damage
910 Kim et al.
J. Microbiol. Biotechnol.
bisporus), shiitake mushroom, and oyster mushroom
(Pleurotus ostreatus) [1, 11, 17]. Hypocrea, Trichoderma, and
Gliocladium are well-known taxonomic names for green
molds. Among these, ascomycete Hypocrea is the teleomorph
name of some of the anamorphic species of Trichoderma and
Gliocladium. These anamorphic species produce huge
numbers of green-colored spores that can easily disperse in
mushroom cultivation houses. Since Camptomyia pests are
mycophagous and signs of green mold are frequently
observed in shiitake bed-logs, it has been postulated that
the damage of shiitake mushroom bed-logs might be caused
not only by the mushroom flies but also by green mold. In a
previous work on green mold, Trichoderma citrinoviride and
Gliocladium viride (an anamorph of Hypocrea lutea) were
identified in shiitake bed-logs infested by mushroom flies
[7, 8]. G. viride was later renamed to Trichoderma deliquescens
[5]. Recently, T. harzianum and T. citrinoviride were isolated
from Camptomyia larvae and adults that were caught from
damaged shiitake bed-logs [12]. This isolation data
displayed that there is a relationship between Trichoderma
species and Camptomyia. Because Trichoderma spp. are known
for their ability to degrade wood and their antagonistic
properties on mushroom fungi [17, 20], they have been
presumed possible agents of fungal damage on shiitake
bed-logs. However, the damage to Camptomyia-infested
shiitake bed-logs associated with Trichoderma species has
not been fully elucidated yet.
The purpose of this study was to understand the fungal
damage of shiitake bed-logs associated with the Camptomyia
pest that occurred in Korea. Four Trichoderma species,
which were possibly responsible for wood damage of
Camptomyia-infested shiitake bed-logs, were isolated, identified,
and evaluated for shiitake damage. We analyzed the
competitiveness of the isolated Trichoderma species over
shiitake mycelia growing on culture media plates and bed-
logs, as well as their ability to produce extracellular enzymes
that are responsible for wood degradation. Thus, we obtained
evidence that they are the causal agents of fungal damage.
Materials and Methods
Sampling of Shiitake Bed-Logs Infested by Camptomyia Pests
and Fungal Isolation
Three shiitake bed-logs infested by C. corticalis and C. heterobia
were sampled in the summer season of 2009 from each cultivation
house of three shiitake mushroom farms located in Cheonan,
Korea. The damaged parts of the oak bed-log were easily
distinguished from the normal parts by debarking the oak logs.
The insect pest-infested parts of the oak bed-logs were cut, placed
in plastic bags, and transferred to the laboratory. The transferred
samples were chopped up into small pieces (1 cm × 1 cm × 0.5 cm)
using a sterile chisel and hammer. To isolate the green mold
species, the chopped small wood pieces were surface sterilized by
soaking in 1% sodium hypochlorite solution for 2 min. After
washing with sterile water three times, the wood pieces were
placed on potato dextrose agar (PDA) plates supplemented with
streptomycin (100 µg/ml) and the PDA plates were incubated at
25°C for 3-5 days. Mycelia that grew out from the small wood
pieces and started to grow on PDA were separated with a sharp
sterile needle and transferred to new PDA plates. After growing
for 3-5 days, single spore isolates were obtained from the PDA-
grown fungi. Single colony isolation was undertaken at least three
times from the single spore-grown fungi. The fungal colonies thus
obtained were initially grouped based on colony morphology and
then randomly selected colonies from each group were subjected
to species identification. The selected fungal colonies were kept on
PDA plates for the experiment and stored at -70oC for preservation.
Morphology Observation
For the observation of fungal colony morphology, each single
spore isolate was pre-cultured on a PDA plate at 25°C for 3 days.
Agar plugs (5 mm diameter) were taken from the cultured fungal
isolate and inoculated at an edge position on PDA plates and
Czapek yeast extract agar plates, respectively, and incubated at
25°C for 3 days. Five replicates were prepared for each single spore
isolate. Colony color and shape were examined by the naked eye.
The fungal microstructures such as conidiophores and conidia
were observed using the phase contrast microscope Axioskop 40
(Karl Ziess, Germany) and scanning electron microscope (SEM)
S4300 (Hitachi, Japan). For SEM observation, the specimens were
prepared using 1% osmium tetroxide as described by Kim et al. [8].
For all isolates, morphological characteristics were compared with
those of reference isolates [4]. For the observation of damaged
wood pieces, a SZ61 stereo microscope (Olympus Opitical Co.,
Japan) was used.
Molecular Identification of Fungal Isolates
For fungal DNA preparation, the subject isolates were grown
on PDA at 25°C for 3-5 days and their genomic DNAs were
extracted by the drilling method described by Kim et al. [9]. The
translation elongation factor 1-alpha gene (tef1) was PCR
amplified using the TEF1 and TEF728 primer pair [3, 17], in a 50 µl
reaction mixture using EmeraldAmp PCR Master Mix (Takara,
Japan). The PCR was performed with the following directions: one
cycle of pre-denaturation at 95°C for 5 min, followed by 30 cycles
of denaturation at 95°C for 1 min, annealing at 56°C for 30 sec,
and extension at 72°C for 1 min, and one cycle of final extension at
72°C for 5 min. The PCR products were purified using a PCR
purification kit (NaviBiotech, Korea), cloned into pGEM T-easy
vector (Promega Corp., USA), and sequenced by Macrogen Inc.
(Korea). The determined nucleotide sequences were compared
with known sequences of Trichoderma species through BLASTN at
the GenBank database of the National Center for Biotechnology
Trichoderma Species Damaging Shiitake Mushroom Bed-Logs 911
May 2016⎪Vol. 26⎪No. 5
Information (http://www.ncbi.nlm.nih.gov/genbank) and Tricho-
BLAST at the website of the International Subcommission on
Trichoderma and Hypocrea Taxonomy (http://www.isth.info/). The
determined nucleotide sequences of tef1 were manually edited and
aligned using the Biological Sequence Alignment Editor ver. 7.0.5.
Molecular phylogenetic analysis was performed using the MEGA
program [20], and a neighbor-joining tree was constructed using
Kimura’s two-parameter model [10]. Bootstrap values were
generated with 1,000 replicates. The reference tef1 sequences of
related taxa were obtained from the GenBank database.
Extracellular Enzyme Test
To test the ability to degrade wood cell components, the fungi
were grown on media containing 0.5% avicel (Sigma-Aldrich, USA),
CM-cellulose (Sigma-Aldrich), D-cellobiose (Sigma-Aldrich), starch
(Sigma-Aldrich), skim milk (Sigma-Aldrich), polygalacturonic acid
(MP Biomedicals, USA), xylan (Sigma-Aldrich) as a carbon source,
0.1% yeast nitrogen base as their fundamental nitrogen source,
and 1.5% agar powder. Congo red dye (0.5%) was used for
chromogenic reaction due to its better performance in extracellular
enzyme activity detection [24]. After 7 days of culturing at 25°C,
we could observe the clear zone formed by reaction between the
secreted extracellular enzymes and chromogenic substrates. The
diameter of clear zone was measured and used for the basis of
relative activity comparison of the tested extracellular enzymes.
Antagonistic Ability and Inoculation Test on Oak Bed-Logs
Lentinula edodes (shiitake strain Sanzo 701Ho) was grown at
25°C on one side of PDA for 7 days to investigate the antagonistic
behavior of the isolated Trichoderma species in vitro. Then, each
test species was inoculated on the other side of the L. edodes-
grown PDA and incubated for 14 days at 25°C. The dual cultured
samples were examined for the existence or absence of antagonistic
growth inhibition between L. edodes and each Trichoderma species.
The experiment was repeated with three replicates.
To investigate the invasive behavior of the isolated green mold
species on shiitake bed-logs in vivo, each Trichoderma species was
grown on sawdust media containing 10% rice bran and used as
inoculum for the bed-log inoculation. After making inoculation
holes using a sterile drill on Mongolian oak bed-log (50 cm in
length, 15 cm in diameter), which was pre-colonized by the
shiitake strain, 1 g of the sawdust media-grown inoculum of each
Trichoderma species was inoculated into each prepared hole.
Control inoculation was performed with sterilized sawdust
media. Three bed-logs and three holes per bed-log were prepared
for each Trichoderma species inoculation. The inoculated shiitake
bed-logs were incubated in a mushroom cultivation house during
the summer season (average temperature 26oC, relative humidity
85%) [7]. After 54 days incubation, we removed the bark around
the inoculation holes on the bed-logs to check for the presence of
brown and/or black colored zones formed on the surface of the
log wood by the growth and colonization of the inoculated
Trichoderma species. To check Koch’s postulate, we tried to re-
isolate the inoculated Trichoderma fungal species from the brown
and/or black colored zones on the bed-logs.
Results and Discussion
The sampled shiitake bed-logs infested by Camptomyia
pest were easily debarked and discolored to black (Fig. 1A)
and/or brown (Fig. 1B). Severe infestation resulted in a
dried and yellowish texture of the wood in the log and
some areas of the wood were blackened (Fig. 1C). These
features demonstrated that the shiitake bed-logs were not
in good condition for the spreading of shiitake mycelia
through the wood cells in the log that is required before the
shiitake mycelia start to produce a fruiting body. In
addition, the inside part of the log was not dense but
loosened and easily breakable, indicating the presence of
wood degradation (Fig. 1E). The SEM image of the wood
tissue in Fig. 1E showed that fungal mycelia had colonized
the tissues (Fig. 1F). In contrast, the microscopic image of a
part of wood tissues in the undamaged bed-log showed
white healthy shiitake mycelia, well colonized and evenly
spread on the wood (Fig. 1D). Considering that the
Camptomyia larvae move to eat shiitake mycelia and
multiply in the wood tissues underneath of the bark, their
activity is likely to hinder the colonization and spreading
out of shiitake mycelia through the wood cells.
Camptomyia larvae can degrade wood and develop only
under the bark [13]. Thus, the insect larvae would not
penetrate into the inside of wood cells. However, fungi are
well known as wood decomposers that biochemically
break down wood cells [2]. Thus, the fungi in the wood
tissue are assumed to be the main source of organisms that
are involved in wood decomposition. From the observations
in Fig. 1, it was inferred that Camptomyia pest infestation
accompanied wood tissue decomposition by fungi.
Wood degradation is the main reason for wood damage
and damaged wood is not suitable for shiitake cultivation.
Therefore, the presence of wood-degrading fungi inside of
shiitake bed-logs could aggravate the damage of shiitake
bed-logs infested by Camptomyia pest. To verify that fungi
do play a role in the damage of shiitake bed-logs, we first
tried to isolate them from the bed-logs samples. According
to the information in Fig. 1F, fungal isolation was performed
from the inside part of the oak bed-logs samples infested
by Camptomyia pest. From the isolation work, 42 fungal
isolates were obtained (Table 1). Based on microscopic
observation of microstructures such as conidia and
conidiophores, all 42 fungal isolates were identified as
Trichoderma, imperfect fungi with teleomorphs belonging
912 Kim et al.
J. Microbiol. Biotechnol.
to the ascomycete order Hypocreales. The 42 Trichoderma
isolates were divided into four groups through further
identification with colony morphology and morphological
characters. Since Trichoderma is known to be a complex
fungal group that is difficult to identify solely based on
morphological characters, molecular analysis was additionally
performed with the partial translation elongation factor 1α
gene (tef1), which is known to be a suitable target gene for
identification of Trichoderma spp. [17]. For the molecular
analysis, each representative isolate was chosen from the
four Trichoderma groups and coded as DUCC001, DUCC003,
DUCC005, and DUCC007, respectively. By PCR with TEF728
and TEF1 primers, 651, 628, 635, and 608 bp amplicons were
obtained from the four representative isolates. These PCR
amplicons were sequenced and searched for homologous
DNA sequences through BLAST programs. The determined
nucleotide sequences of the four representative isolates
DUCC001, DUCC003, DUCC005, and DUCC007 shared
99% sequence identity with tef1 sequences of T. citrinoviride
YNKM5010 (Accession No. JQ040469), T. harzianum
TAMA04131 (Accession No. AB856677), T. atroviride 8234
(Accession No. KJ624780), and T. deliquescens GJS89-129
(Accession No. AY737731) registered on the GenBank DNA
database. The BLAST search results agreed with the
results of the four nucleotide sequences in Tricho-BLAST.
Consequently, based on these results of tef1 sequence
homology, Trichoderma isolates DUCC001, DUCC003,
DUCC005, and DUCC007 were identified as T. citrinoviride,
T. harzianum, T. atroviride, and T. deliquescens (synonym
G. viride), respectively. We deposited the determined tef1
sequences of the four Trichoderma isolates to the GenBank
DNA database. The tef1 sequence of T. harzianum DUCC003
was deposited with its teleomorph name Hypocrea lixii
(Accession No. HQ602998). The tef1 sequence of T. atroviride
Fig. 1. Microscopy of shiitake bed-logs infested by Camptomyia pest.
Photos of examples of the shiitake bed-logs infested by Camptomyia pest (A, B, C) and stereoscopic microscopic (E) and SEM (F) images of a part of
wood tissues in the damaged bed-logs. A stereoscopic microscopic image of a part of wood tissues in the undamaged bed-logs (D) is showing
well-colonized and evenly spread white healthy shiitake mycelia.
Table 1. Trichoderma species and number of isolates identified in this study from the shiitake bed-logs infested by Camptomyia pest.
Source T. harzianum T. atroviride T. citrinoviride T. deliquescens
Mushroom farm 1 6 4 8 -
Mushroom farm 2 4 - - -
Mushroom farm 3 10 - - 10
Total 20 4 8 10
-: No isolation.
Trichoderma Species Damaging Shiitake Mushroom Bed-Logs 913
May 2016⎪Vol. 26⎪No. 5
DUCC005 was deposited as Accession No. HQ603000 and
that of T. citrinoviride DUCC001 as Accession No. JF700485.
The tef1 sequence of T. deliquescens DUCC007 was deposited
as synonym G. viride (Accession No. GU903312). Combining
the results of molecular identification and morphological
characters, the 42 Trichoderma isolates were identified as
T. citrinoviride, T. harzianum, T. atroviride, and T. deliquescens,
respectively (Table 1). Among the four Trichoderma species,
T. harzianum was dominantly isolated and found on three
mushroom farms. Three Trichoderma species were found in
one of the three mushroom farms. These isolation data
suggest that the occurrence of Trichoderma species on the
oak bed-logs infested by Camptomyia pest is not confined to
a single Trichoderma species and varies depending on
mushroom farms.
Reportedly, T. citrinoviride and T. deliquescens were identified
from shiitake bed-logs infested by mushroom flies by Kim
et al. [7, 8]. Our results confirmed the previous reports.
However, T. harzianum and T. atroviride have not been
reported before as species associated with the mushroom
fly Camptomyia. Therefore, we further studied these species
morphologically using a SEM and genetically using a
phylogenetic tree. The colony morphology grown on PDA
and CYA at 25oC for 7 days is displayed in Fig. 2. On PDA,
T. harzianum revealed a grayish green color and T. atroviride
showed a white color (Table 2, Fig. 2). On CYA, both species
revealed green colors. The SEM images of microstructures
of the two Trichoderma species are given in Fig. 2 and their
mycological characters are presented in Table 2 with
comparison with known references [3, 4]. Overall, these two
species morphologically matched well with the reported
references. A phylogram based on the tef1 sequences clearly
Fig. 2. Colony characteristics of Trichoderma harzianum DUCC003 and T. atroviride DUCC005 grown on PDA and CYA at 25oC for
7 days and SEM images of conidiophores and conidia of the two fungi.
Table 2. Morphological characters of Trichoderma harzianum DUCC003 and T. atroviride DUCC005 isolated in this study from the
shiitake bed-logs infested by Camptomyia pest.
Species T. harzianum
(anamorph of H. lixii) [2]
T. harzianum
(DUCC003 this study)
T. atroviride (anamorph of
H. atroviridis) [3]
T. atroviride
(DUCC005 this study)
Colony color Dark green Grayish green White White
Conidiophores
Color Hyaline Hyaline Hyaline Hyaline
Shape Branched in a pyramidal fashion Branched in a pyramidal fashion Lageniform often curved Lageniform often curved
Size 4.0~7.0 µm × 2.5~3.5 µm 5.0~7.0 µm × 1.7~2.6 µm 7.7~8.0 µm × 2.0~2.1 µm 8.0~10 µm × 1.4~2.0 µm
Conidia
Color Pale green Pale green Pale green Pale green
Shape (sub)Spherical shape (sub)Spherical shape Globose Globose
Size 2.5~3.0 µm × 2.0~2.5 µm 1.5~2.0 µm × 1.5~2.0 µm 2.8~3.5 µm × 3.0~3.8 µm 1.0~1.5 µm × 1.0~1.5 µm
914 Kim et al.
J. Microbiol. Biotechnol.
displayed the position of T. harzianum DUCC003 and
T. atroviride DUCC005 among the related Trichoderma taxa
(Fig. 3). As T. harzianum belongs to an awkward fungal
group that is not easy to identify, our results will be very
useful for the identification of T. harzianum and T. atroviride
from shiitake bed-logs infested by Camptomyia pest.
Some of the Trichoderma taxa possess mycoparasitic
behavior towards mushrooms [18]. T. harzianum and
T. polysporum are known to attack shiitake mycelia in bed-
logs used for mushroom cultivation by producing antifungal
substances and mycolytic enzymes [21-23]. Their attack
leads to yield loss of shiitake production. To obtain evidence
that the four Trichoderma species are antagonists that inhibit
the mycelium growth of shiitake mushroom, each of the
four Trichoderma species was dual cultured with a shiitake
strain on PDA plate. The mycelia of T. harzianum DUCC003,
T. atroviride DUCC005, and T. citrinoviride DUCC001 invaded
the mycelial growth territory of the shiitake strain (Fig. 4).
However, the mycelial growth territory of T. deliquescens
DUCC007 was aerially invaded by shiitake mycelia.
These results showed that T. harzianum, T. atroviride, and
T. citrinoviride are antagonists of shiitake mushroom. The
antagonistic effect of T. harzianum, T. atroviride, and T. citrinoviride
on shiitake strains was also reported previously [6]. This
means that the presence of these antagonistic Trichoderma
species in bed-logs for shiitake cultivation would be also
harmful to the shiitake mycelium as the Camptomyia larvae
do harm. It is expected that the coexistence of Camptomyia
larvae and each of the antagonistic Trichoderma species in
shiitake bed-logs will be more detrimental to the development
of shiitake mushroom.
Kim et al. [6] morphologically and phylogenetically defined
several Trichoderma spp. from the shiitake cultivation
environment in Korea. In their work, the Trichoderma spp
Fig. 3. Phylogenetic position of the two green molds Trichoderma harzianum DUCC006 and T. atroviride DUCC005 isolated from the
shiitake bed-logs infested by Camptomyia pest.
The phylogram was constructed based on tef1 gene sequences by the neighbor-joining method in the MEGA program. Numbers above branches
represent bootstrap values with 1,000 replications.
Trichoderma Species Damaging Shiitake Mushroom Bed-Logs 915
May 2016⎪Vol. 26⎪No. 5
inhibited the mycelial growth of shiitake strains on both
culture plates and sawdust media. These data informed
that Trichoderma spp. are important in shiitake cultivation.
However, there was no analysis about Trichoderma spp.
isolated from the problematic Camptomyia-infested bed-
logs. Thus, the report did not answer what caused the
damage of shiitake bed-logs infested by Camptomyia.
Additionally, their biochemical properties and colonizing
ability on log have not been tested. Because the two
Camptomyia species that infested shiitake bed-log are newly
described species in Korea [19], work on the associated
Trichoderma fungi and their enzymatic properties and
colonization ability on living wood will be new and
valuable information.
As some Trichoderma species are very good cellulase
producers [15], we also evaluated their ability to produce
extracellular enzymes in T. harzianum DUCC003, T. atroviride
DUCC005, T. citrinoviride DUCC001, and T. deliquescens
DUCC007. Enzymes that could degrade wood components
were evaluated on chromogenic media: amylase, avicelase,
β-glucosidase, carboxylmethyl cellulase, pectinase, xylanase,
and protease. Their ability to degrade cellulose, pectin, and
xylan was found in all the tested species (Table 3). All the
four species showed very strong activity in extracellular
protease, and production of extracellular amylase was also
seen in them. There were some differences in the degree of
enzyme activities among the four Trichoderma species.
Overall, these results suggested that the four Trichoderma
species have the ability of wood degradation by producing
extracellular enzymes. Regarding the four Trichoderma
species, an inoculation test was performed on Mongolian
oak log that was pre-colonized by the mycelia of the
shiitake strain. This test resulted in a brown-colored line
that clearly formed around the inoculation hole (Fig. 5).
The brown-colored line is a barrier formed by incompatible
interactions between Trichoderma species and the shiitake
mycelium. The line formation means the two different fungi
Fig. 4. Antagonistic behavior against shiitake strain Sanzo
701HO (Lentinula edodes) shown on PDA by the four green
mold Trichoderma species isolated from the shiitake bed-logs
infested by Camptomyia pest.
① : Inoculated position of L. edodes. ② : Inoculated position of green
mold Trichoderma species. T. harzianum (A), T. citrinoviride (B), and
T. atroviride (C) invaded into the mycelial growth territory of L. edodes.
The mycelial growth territory of T. deliquescens (D) was aerially
invaded by L. edodes. The arrow indicates the barrier (brown-colored
line) formed by incompatible interactions between L. edodes and the
four green mold Trichoderma species.
Table 3. Extracellular enzyme activity shown on chromogenic media by the four Trichoderma species obtained from the shiitake
bed-logs infested by Camptomyia pest.
EnzymeT. harzianum
DUCC003
T. atroviride
DUCC005
T. citrinoviride
DUCC001
T. deliquescens
DUCC007
Amylase Sb Mc VSa M
Avicelase M M M Wd
β-Glucosidase VS M S VS
Carboxylmethyl cellulase S M S W
Pectinase M W M W
Xylanase W M W W
Protease VS VS VS VS
aVS: very strong activity (clear zone diameter > 9 cm); bS: strong activity (clear zone diameter 5-8 cm); cM: moderate activity (clear zone diameter 2-5 cm); dW: weak
activity (clear zone diameter 0-2 cm).
916 Kim et al.
J. Microbiol. Biotechnol.
are competing. Brown-colored lines formed between
Trichoderma species and shiitake mycelium were also
observable on culture media (Fig. 4B) [6], but no brown line
was found in the control inoculation. We re-isolated the
four Trichoderma species from each of the brown-colored
lines. These results satisfied Koch's postulate, indicating
that the four Trichoderma species colonized and generated
brown line symptom in the shiitake bed-log. This wood
pathological data underlined the significance of the
Trichoderma in shiitake cultivation. Together with the
results of Table 3 and Figs. 4 and 5, we concluded that T.
harzianum, T. atroviride, T. citrinoviride, and T. deliquescens
are also fungal pests that damage oak bed-logs for shiitake
mushroom cultivation. Among the four species, T. citrinoviride
was also reported in a previous work [5]: its ability for
colonizing on oak log has been confirmed again in this
study.
In conclusion, this study confirmed that Trichoderma
species exist in shiitake bed-logs infested by Camptomyia
and demonstrated that they are able to invade shiitake bed-
logs. This is no table information that the four Trichoderma
spp. are truly damaging agents of shiitake bed-logs infested
by the mushroom fly. Since T. harzianum and T. citrinoviride
were isolated from the body of Camptomyia adults [12], it is
assumed that Camptomyia introduced Trichoderma into the
shiitake bed-logs during its infestation process. Consequently,
the coexistence of Camptomyia and Trichoderma species is
expected to aggravate the damage of shiitake bed-logs.
Acknowledgments
The present research was supported by the research fund
of Dankook University in 2013.
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