Characterization of Species of Cladobotryum which Cause Cobweb Disease in Edible Mushrooms Grown in Korea

Abstract

Four Cladobotryum isolates were collected from four different commercially grown mushroom types infected with cobweb disease in Cheongdo-gun and Chilgok-gun of Gyeongbuk Province, Korea in 2010. The isolates were identified as C. mycophilum from Agaricus bisporus and Pleurotus eryngii, C. varium from Flammulina velutipes and Hypsizygus marmoreus. The cultural characteristics of the four isolates were investigated using potato dextrose agar (PDA) media under nine different temperatures ranging from 5~32℃. Rapid growth of the isolates to colony diameters of 47~82 mm was observed at conditions of 18~22℃. No growth was observed at 32℃. C. mycophilum produced a yellowish red pigment while C. varium produced a cream colored pigment after cultivation for 25 days on PDA. Phylogenetic analysis of the internal transcribed spacer region and partial 28S rDNA from the four isolates confirmed they were C. mycophilum and C. varium. Cross pathogenicity tests revealed that the two isolates of C. mycophilum were highly pathogenic toward three mushroom types, but not toward H. marmoreus. The two isolates of C. varium were less pathogenic than those of C. mycophilum, but were pathogenic toward all mushroom types evaluated.

Full text

189
Mycobiology 40(3) : 189-194 (2012) http://dx.doi.org/10.5941/MYCO.2012.40.3.189
© The Korean Society of Mycology pISSN 1229-8093
eISSN 2092-9323
RESEARCH ARTICLE
Characterization of Species of Cladobotryum which Cause Cobweb Disease
in Edible Mushrooms Grown in Korea
Chang-Gi Back1, Chang-Yun Lee2, Geon-Sik Seo3 and Hee-Young Jung1*
1School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Korea
2Greenpeace Mushroom Co., Cheongdo 714-853, Korea
3Department of Industrial Crops and Mushrooms, Korea National College of Agriculture and Fisheries, Hwaseong 445-760, Korea
(Received July 13, 2012. Revised August 3, 2012. Accepted August 22, 2012)
Four Cladobotryum isolates were collected from four different commercially grown mushroom types infected with cobweb
disease in Cheongdo-gun and Chilgok-gun of Gyeongbuk Province, Korea in 2010. The isolates were identified as C. myco-
philum from Agaricus bisporus and Pleurotus eryngii, C. varium from Flammulina velutipes and Hypsizygus marmoreus. The
cultural characteristics of the four isolates were investigated using potato dextrose agar (PDA) media under nine different
temperatures ranging from 5~32oC. Rapid growth of the isolates to colony diameters of 47~82 mm was observed at conditions
of 18~22oC. No growth was observed at 32oC. C. mycophilum produced a yellowish red pigment while C. varium produced
a cream colored pigment after cultivation for 25 days on PDA. Phylogenetic analysis of the internal transcribed spacer region
and partial 28S rDNA from the four isolates confirmed they were C. mycophilum and C. varium. Cross pathogenicity tests
revealed that the two isolates of C. mycophilum were highly pathogenic toward three mushroom types, but not toward H.
marmoreus. The two isolates of C. varium were less pathogenic than those of C. mycophilum, but were pathogenic toward
all mushroom types evaluated.
KEYWORDS : Cobweb disease, Cross pathogenicity, ITS region, Phylogenetic analysis, 28S rDNA
Introduction
Cobweb disease is caused by several species of Cladobotryum
and is characterized by the growth of coarse mycelium
over the affected mushrooms [1]. The disease is found in
all mushroom-growing countries worldwide and causes
economic loss in areas it impacts [2-4]. There have been
numerous reports of cobweb disease affecting Agaricus
bisporus, which is known to be infected by species of
Cladobotryum including C. dendroides, C. mycophilum,
C. varium, C. multiseptatum, and C. verticillatum [2, 5].
C. mycophilum and C. varium are known to be the
dominant pathogens for A. bisporus. In Korea, numerous
types of mushrooms including A. bisporus, Pleurotus
eryngii, Flammulina velutipes and Hypsizygus marmoreus
are commercially cultivated for domestic consumption.
In recent years, two species of cobweb fungi, C.
mycophilum on A. bisporus, and C. varium on P. eryngii
and F. velutipes, have been reported in Korea [6, 7].
Cladobotryum was identified by the morphological and
cultural characteristics of its sporocarp and spores, as well
as its internal transcribed spacer (ITS) region and partial
28S rDNA genetic characteristics. However, these two
species produce nearly identical symptoms during mushroom
cultivation. In order to manage cobweb disease effectively,
correct identification of pathogens is important as cobweb
disease can be spread through spore distribution. However,
the possible infection by Cladobotryum species of different
types of mushrooms has yet to be investigated. Therefore,
in this study we investigated Cladobotryum isolates from
four mushroom types based on their morphological and
genetic characteristics, and their cross pathogenic ability.
Materials and Methods
Fungal isolation and identification. Cladobotryum
isolates were collected from the fruiting bodies of four
different cobweb disease infected mushrooms: A. bisporus,
P. eryngii, F. velutipes and H. marmoreus, which were
obtained from Cheongdo-gun and Chilgok-gun in Gyeongbuk
Province, Korea in 2010. All cultures used in the experiments
were derived from a single spore and were grown on
potato dextrose agar (PDA) at 20oC in the dark for 3~4
days. The shape, size and color of 100 conidia and
conidiophores of the isolates were microscopically observed.
The isolates were then identified based on the morphological
*Corresponding author <E-mail : heeyoung@knu.ac.kr>
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.

190 Back et al.
characteristics of the conidia and conidiophores according
to the descriptions from Gams and Hoozemans [8].
Growth conditions. The isolates were cultured on PDA
media at 20oC for four days under dark conditions. From
these cultures, small mycelia plugs (5 mm in diameter)
were punched out from the actively growing area using a
cork borer and placed at the center of a culture plate
(90 mm in diameter) containing PDA media. The influence
of temperature on their growth was investigated by
incubating the plates at 5, 10, 15, 18, 20, 22, 25, 28, and
32oC, and measuring the resulting colony diameters after
four days. Data are presented as the means of three
replicates. To investigate the resulting pigmentation on the
PDA, the cultures were further kept at 22oC for 5~25 days.
DNA extraction and PCR amplification. Total genomic
DNA was extracted from each fungal isolate using lysis
buffer according to the procedure described by Liu et al.
[9]. Total genomic DNA was used to amplify the ITS
region and partial 28S ribosomal DNA (rDNA). The ITS
rDNA regions and the partial 28S rDNA were amplified
using the primer pairs ITS1F (5'-CTT GGT CAT TTA
GAG GAA GTA A-3')/ITS4 (5'-TCC TCC GCT TAT
TGA TAT GC-3') [10] and NL1 (5'-GCA TAT CAA TAA
GCG GAG GAA AAG-3')/NL4 (5'-GGT CCG TGT TTC
AAG ACG G-3') [11], respectively. PCR amplification was
conducted in a 20 µL reaction mixture containing 2 µL of
fungal DNA (20 ng), 0.2µL Taq polymerase (5 units/µL),
2µL 10 reaction buffer (100 mM Tris-HCl, 400 mM KCl,
15 mM MgCl2, pH 9.0), 0.4 µL dNTPs mixture (10mM)
and 2 µL of each primer (5 pmol/µL) using an Applied
Biosystems 2720 thermal cycler (Applied Biosystems, Foster
City, CA, USA). The PCR conditions included preheat at
94oC for 3 min followed by 35 cycles of 94oC for 30 sec,
52oC for 30 sec, 72oC for 1 min and final extension at
72oC for 7 min. The amplified DNA fragments were purified
using ExoSAP-IT (GE Healthcare, Buckinghamshire, UK)
and then subjected to direct sequencing (Solgent, Daejeon,
Korea) using the same primers.
Sequence and phylogenetic analysis. The obtained
sequences were aligned using the DNASTAR computer
package (DNASTAR Inc., Madison, WI, USA) and
phylogenetic trees were constructed using the neighbor-
joining method in ClustalW [12]. The phylogenetic trees
based on the ITS region and partial sequence of the 28S
rDNA were generated using TreeView (Win32 ver. 1.6.1).
Bootstrap analysis with 100 replications was performed in
order to determine the support the data will provide for
various clades.
Cross pathogenicity test. The infection ability of the
isolates was studied by inoculating each isolate onto four
mushroom types. Inoculums were prepared from 10~15-
day-old isolation cultures on PDA media and adjusted to
5×10
5conidia/mL. Inoculation was conducted by spraying
the fruiting bodies of each mushroom thoroughly with the
spore suspension (50 mL). After being kept in plastic bags
in order to maintain 100% humidity for 24 hr, the inoculated
mushrooms were incubated at 20oC. The development of
disease symptoms was observed visually one day after
inoculation and disease severity was rated based on the
following score index: +, 1~30% disease severity; ++,
31~50%; +++, > 51%; and nd, no disease development.
Results and Discussion
Morphological characteristics of the isolates and their
identification. The shape and size of the conidia of the
four isolates were observed for 100 conidia (Table 1). The
shapes of the four isolates were almost obovoid and
consisted of 2~4 cells. The conidia of the four isolates
were divided into two groups based on size, one ranging
from 11~26 × 7~12 µm and the other from 8~14 × 6~
11 µm. These morphological characteristics corresponded
to the characteristics of C. mycophilum isolated from A.
bisporus and C. varium isolated from F. velutipes [4, 7].
The characteristics of the four isolates also agreed with
the description of C. mycophilum and C. varium offered
by Gams and Hoozemans [8]. Based on the observed
morphological characteristics of the conidia, two isolates
from A. bisporus and P. eryngii were identified as C.
mycophilum while two isolates from F. velutipes and H.
marmoreus were identified as C. varium.
The color of the mycelia from the two Cladobotryum
species differed over time. The mycelia of all isolates
were initially white or grayish as grown on PDA media at
22oC. The mycelia of C. mycophilum became yellowish
after 5 days of growth and gradually turned reddish over
Table 1. Morphological characteristics of Cladobotryum mycophilum and C. varium isolated from four mushrooms on potato
dextrose agar media
Characteristics C. mycophilum
(Agaricus bisporus)C. mycophilum
(Pleurotus eryngii)C. varium
(Flammulina velutipes)C. varium
(Hypsizygus marmoreus)
Conidia shape 2~4 cell, obovoid 2~4 cell, obovoid 2~3 cell, obovoid 2~3 cell, obovoid
Conidia size (µm) 11.1~26.6 × 7.7~12.2 11.~23.7 × 10.5~12.7 10.1~17.3 × 6.6~10.1 8.0~14.8 × 6.1~11.5
Mycelial color Yellow, reddish Yellow, reddish White, cream White, cream

Cladobotryum Species Occurring to Edible Mushrooms 191
25 days. The mycelia of the cultures of C. varium remained
white or cream color.
Effect of temperature on the cultures. The optimum
growth temperature for the four isolates was investigated
at temperatures ranging from 5~32oC (Table 2, Fig. 1).
The growth of the isolates was favored at temperatures of
Table 2. Growth of Cladobotryum mycophilum and C. varium isolated from four mushrooms at different temperatures after 4 days
incubation on PDA media
Isolate Length of mycelial growth (mm/4 days)
5oC10
oC15
oC18
oC20
oC22
oC25
oC28
oC32
oC
C. mycophilum (Agaricus bisporus) 0.0 13.6 39.5 65.3 79.0 82.6 67.6 45.0 0.0
C. mycophilum (Pleurotus eryngii) 0.0 14.5 41.6 72.0 74.5 79.6 63.5 12.5 0.0
C. varium (Flammulina velutipes) 0.0 11.8 36.8 50.8 53.6 60.0 44.6 00.0 0.0
C. varium (Hypsizygus marmoreus) 0.0 14.8 36.3 47.0 52.8 54.1 44.3 09.5 0.0
Fig. 1. Effect of temperature on mycelial growth of Cladobotryum mycophilum and C. varium isolated from four mushroom
species on potato dextrose agar at 4 days after incubation.
Fig. 2. Pigments produced by Cladobotryum mycophilum (A) and C. varium (B) on PDA media kept in darkness at 22oC for 5, 8,
11, 18 and 25 days (f, front view; b, back view of plates).
18 to 22oC with the optimum for all isolates at 22oC. Of
the four isolates, only C. mycophilum from A. bisporus
grew at 28oC while the other three types could not grow
at this temperature. The growth rate of C. mycophilum
(> 79.6 mm at 22oC) was faster than that of C. varium
(> 54.1 mm at 22oC). No mycelia grew at less than 10oC,
or at a temperature as high as 28oC, except for C.

192 Back et al.
mycophilum from A. bisporus.
Pigment production. Pigments produced by two
Cladobotryum species grown at 22oC were observed on
PDA media (Fig. 2). After five days of growth, two
isolates of C. mycophilum produced yellowish pigment in
the growth media and gradually turned reddish over 25
days (Fig. 2A). The color of the two isolates of C. varium
were cream and white (Fig. 2B). Similarly, red pigment
was reportedly produced by C. mycophilum from P. eryngii
cultured on PDA [13]. C. varium did not exhibit the
pinkish red mycelium coloration such as observed with C.
mycophilum from A. bisporus [14]. The red coloration
was due to the pigment aurofusarin, a secondary metabolite
associated with the Cladobotryum species [15].
Molecular analysis. Sequences of the ITS region (596~
600 bp) and partial 28S rDNA (~563 bp) from the four
Fig. 3. Phylogenetic trees constructed by the neighbor-joining method based on comparison of the internal transcribed spacer
region (A) and partial 28S rDNA (B) sequences of Cladobotryum sp. with those of other Cladobotryum species from
GenBank. C. cubitense and C. odorum were used as the outgroup. Cladobotryum species observed in this study are shown
in bold. Numbers on branches are the confidence values obtained for 100 replicates (only values above 80% are shown).
The bar represents a phylogenetic distance of 1%.

Cladobotryum Species Occurring to Edible Mushrooms 193
isolates were compared using the GENETYX program.
Two isolates from C. mycophilum exhibited 100% homology
in their ITS regions and partial 28S rDNA sequences, as
did the two isolates from C. varium. The sequence
from the C. mycophilum ITS region and partial sequence
of 28S rDNA from A. bisporus and P. eryngii showed
high homology (100%) with those from C. mycophilum
(FN859436 and FN859434, respectively). The sequences
from the ITS region and partial 28S rDNA of C. varium
from F. vel ut ip es and H. marmoreus revealed 98.5%
and 100% homology with Hypomyces aurantius and
the anamorphs of C. varium (AF055297, AF160230,
respectively).
The phylogenetic relationship between the two
Cladobotryum species was analyzed based on comparison
of their ITS regions (Fig. 3A) and partial 28S rDNA
sequences (Fig. 3B) with those of other Cladobotryum
sequences obtained from Genbank. The ITS regions of the
two isolates of C. mycophilum (AB527074) from this study
were clustered with that of C. mycophilum (JF693809),
while C. varium (AB591044) clustered with H. aurantius
(AB298700, anamorph of C. varium). Similarly, the sequence
of the partial 28S rDNA of the two isolates of C. mycophilum
Table 3. Pathogenicity of two isolates of Cladobotryum mycophilum and C. varium inoculated on four mushrooms
Pathogenicity testaAgaricus bisporus Pleurotus eryngii Flammulina velutipes Hypsizygus marmoreus
C. mycophilum (A. bisporus) +++ +++ +++ nd
C. mycophilum (P. eryngii) +++ +++ +++ nd
C. varium (F. velutipes) ++ + ++ +++
C. varium (H. marmoreus) ++ + + +++
aDisease rate: +, 1~30% disease severity; ++, 31~50%; +++, > 50%; nd, no disease development.
Fig. 4. Cross pathogenicity of two isolates of Cladobotryum mycophilum and two isolates of C. varium on four mushrooms at
20oC. Arrows indicate black spots on cap. DAI, days after inoculation.
clustered with H. odoratus (AF160240, anamorph of C.
mycophilum) while that of C. varium clustered with H.
aurantius (AF160230, anamorph of C. varium).
Cross pathogenicity test. The cross pathogenicity of
the isolates was tested using four types of mushrooms.
The two isolates of C. mycophilum were pathogenic to
three of the mushroom types, but not H. marmoreus
(Table 3, Fig. 3). The two isolates of C. varium were
pathogenic to all mushroom types. The severity of disease
caused by C. mycophilum was more severe than that of C.
varium. In the period after inoculation, disease severity
was observed to be highest against the original host, e.g.,
the isolate obtained from H. marmoreus caused the most
severe infection in H. marmoreus (Fig. 4). Typical cobweb
symptoms including small brown spots were observed
2~3 days after inoculation (DAI). White mycelia were
observed at 3 DAI, and after 5 DAI, the fruiting bodies
were rotten and covered with massive spores. In this
study, C. mycophilum isolates from A. bisporus and P.
eryngii were unable to directly infect H. marmoreus.
Conversely, C. varium could infect all four mushroom
types even though they were less pathogenic. These findings

194 Back et al.
suggest that F. v el ut ipes was a potential host for C.
mycophilium and that A. bisporus and P. eryngii were
potential hosts for C. varium.
In this study, the four isolates of Cladobotryum were
identified as C. mycophilum or C. varium based on their
morphological and genetic characteristics. Previously, cobweb
fungi on mushrooms were reported as C. mycophilum on A.
bisporus based on morphological and genetic characteristics
[7], as C. mycophilum on P. eryngii, and C. varium on F.
velutips based on the morphological characteristics of
their spores and conidiophores [4,6]. Based on the results
of the morphological assessments and phylogenetic analyses
conducted in this study, we confirmed that the Cladobotryum
isolates from the four mushroom types belonged to C.
mycophilum and C. varium. The growth of both species
was favored at 18~22oC and C. mycophilum grew faster
than C. varium.
C. mycophilum exhibited greater pathogenicity than C.
varium against the mushroom types evaluated in this
study. The observed cross pathogenic ability of the two
Cladobotryum species should aid in designing control
measure for cobweb disease as mushrooms are grown
year round in Korea. This disease can spread rapidly
because Cladobotryum sp. produce masses of spores.
Moreover, some species of Cladobotryum from A.
bisporus were observed to be fungicide resistant [16] and
fungicide application is restricted for edible mushrooms
due to its residual toxicity. Disinfection using near-UV
irradiation has been described as an effective method for
reducing pathogenic fungi and bacteria in mushroom
growing spaces [17]. However, further studies are needed
in order to ensure prevention of outbreaks of cobweb
disease during mushroom cultivation.
Acknowledgements
This research was supported by Kyungpook National
University Research Fund, 2012.
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