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: piceae@dankook.ac.kr

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.

References

1. Badham ER. 1991. Growth and competition between Lentinus

edodes and Trichoderma harzianum on sawdust media. Mycologia

83: 455-463.

2. Blanchette R. 1991. Delignification by wood-decay fungi.

Annu. Rev. Phytopathol. 29: 281-403.

3. Carbone I, Kohn LM. 1999. A method for designing primer

sets for speciation studies in filamentous ascomycetes. Mycologia

91: 553-556.

4. Chaverri P, Samuels GJ. 2003. Hypocrea/Trichoderma (Ascomycota,

Hypocreales, Hypocreaceae): species with green ascospores.

Stud. Mycol. 48: 1-116.

Fig. 5. Artificial inoculation test of the four Trichoderma species on oak bed-logs pre-colonized by the mycelia of shiitake strain

Sanzo 701HO.

Surface features of each debarked oak bed-log 54 days after inoculation with sawdust media-grown T. harzianum (B), T. atroviride (C), T.

citrinoviride (D), and T. deliquescens (E), respectively. Control inoculation was performed with sterilized sawdust media (A). No brown zone is

formed on the surrounding area of the control inoculated hole (A). The arrowheads indicate the formation of a browning/black zone around the

inoculated holes (B, C, D, E), which is a typical sign found on the debarked surface of oak bed-logs infested by Camptomyia pest. Scale bar =

15 mm.

Trichoderma Species Damaging Shiitake Mushroom Bed-Logs 917

May 2016⎪Vol. 26⎪No. 5

5. Jaklitsch WM. 2011. European species of Hypocrea Part II:

species with hyaline ascospores. Fungal Divers. 48: 1-250.

6. Kim CS, Park MS, Kim SC, Maekawa N, Yu SH. 2012.

Identification of Trichoderma, a competitor of shiitake

mushroom (Lentinula edodes), and competition between

Lentinula edodes and Trichoderma species in Korea. Plant

Pathol. J. 28: 137-148.

7. Kim JY, Kwon HW, Tang L, Kim SH. 2012. Identification

and characterization of Trichoderma citrinoviride isolated

from mushroom fly-infested oak log beds used for shiitake

cultivation. Plant Pathol. J. 28: 219.

8. Kim JY, Yun YH, Hyun MW, Kim MH, Kim SH. 2010.

Identification and characterization of Gliocladium viride

isolated from mushroom fly infested oak log beds used for

shiitake cultivation. Mycobiology 38: 7-12.

9. Kim SH, Uzunovic A, Breuil C. 1999. Rapid detection of

Ophiostoma piceae and O. quercus in stained wood by PCR.

Appl. Environ. Microbiol. 65: 287-290.

10. Kimura MA. 1980. Simple method for estimating evolutionary

rates of base substitutions through comparative studies of

nucleotide sequences. J. Mol. Evol. 16: 111-120.

11. Kredics L, Kocsubé S, Nagy L, Komo -Zelazowska M,

Manczinger L, Sajben E, et al. 2009. Molecular identification

of Trichoderma species associated with Pleurotus ostreatus and

natural substrates of the oyster mushroom. FEMS Microbiol.

Lett. 300: 58-67.

12. Lee SH. 2012. Investigation on key insect pests of shiitake

mushroom collapse and development of environment-friendly

insecticides. Korea Forest Service.

13. Mamaev M, Krivosheina NP. 1992. The Larvae of the Gall

Midges. CRC Press, USA.

14. Panelius S. 1965. A revision of the European gall midges of

the subfamily Porricondylinae (Diptera: Itonididae). Acta

Zool. Fennica 113: 1-157.

15. Reczey K, Szengyel Z, Eklund R, Zacchi G. 1996. Cellulase

production by T. reesei. Bioresour. Technol. 57: 25-30.

16. Royse DJ. 1997. Specialty mushrooms: consumption, production

and cultivation. Rev. Mex. Mic. 13: 1-11.

17. Samuels GJ, Dodd SL, Gams W, Castlebury LA, Petrini O.

2002. Trichoderma species associated with the green mold

epidemic of commercially grown Agaricus bisporus. Mycologia

94: 146-170.

18. Seaby D. 1998. Trichoderma as a weed mould or pathogen in

mushroom cultivation, pp. 267-272. In Kubicek CO, Harman

GE (eds.). Trichoderma and Gliocladium. Vol. 2. Enzymes,

Biological Control and Commercial Applications. Taylor and

Francis, London.

19. Shin S, Lee H, Lee S. 2011. Two cecidomyiid gall midge

(Diptera: Cecidomyiidae) pests of shiitake mushrooms

(Agaricales: Marasmiaceae). J. Asia Pac. Entomol. 14: 387-391.

20. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013.

MEGA6: molecular evolutionary genetics analysis version

6.0. Mol. Biol. Evol. 30: 2725-2729.

21. Tokimoto K. 1985. Physiological studies on antagonism

between Lentinula edodes and Trichoderma spp. in bed-logs of

the former (in Japanese). Rep. Tottori Mycol. Inst. 23: 1-54.

22. Tokimoto K, Fujita T, Takeda Y, Takaishi Y. 1987. Increased

or induced formation of antifungal substances in cultures of

Lentinula edodes by the attack of Trichoderma spp. Proc. Jp.

Acad. 63: 277-280.

23. Ulhoa CJ, Peberdy JF. 1992. Purification and some properties

of the extracellular chitinase produced by Trichoderma

harzianum. Enzyme Microb. Technol. 14: 236-240.

24. Yoon JH, Park JE, Suh DY, Hong SB, Ko SJ, Kim SH. 2007.

Comparison of dyes for easy detection of extracellular

cellulases in fungi. Mycobiology 35: 21-24.

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