全 文 ：第 50 卷 第 6 期
2 0 1 4 年 6 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol. 50，No. 6
Jun．，2 0 1 4
doi:10．11707 / j．1001-7488．20140621
Ｒecevied date: 2013 － 10 － 09; Ｒevised date: 2014 － 01 － 23．
Foundation project: Technology Infrastructure Subprojects of Fungi Ｒesources Standardization Finishing Integration in Heilongjiang Province
(2005DK20127 － 9) ．
* Corresponding author: Song Ｒuiqing． The authors wish to thank Mark Gwynn and Sara Gwynn for their editoring this paper．
中国 10 省金黄壳囊孢菌的菌落特征和遗传多样性*
杨明秀1，2 宋瑞清1
(1．东北林业大学林学院 哈尔滨 150040; 2．东北农业大学农学院 哈尔滨 150030)
关键词: 金黄囊壳孢菌; ＲAPD; 菌落特征
中图分类号: S763. 15 文献标识码: A 文章编号: 1001 － 7488(2014)06 － 0160 － 07
Colony Characteristics and Genetic Diversity of Cytospora chrysosperma
Strains from Ten Provinces in China
Yang Mingxiu1，2 Song Ｒuiqing1
(1 ． Forestry School，Northeast Forestry University Harbin 150040; 2 ． Agricultural School，Northeast Agricultural University Harbin 150030)
Abstract: Totally 30 strains of Cytospora chrysosperma causing poplar canker，derived from ten provinces in China were
classified into two clusters based on the cluster analysis of colony characteristics． The first cluster was separated into the
geographic groups of Xinjiang，Beijing-Northeast and Shandong and the second cluster was separated into the geographic
groups of Inner Mongolia and Sichuan-Gansu-Shaanxi-Qinghai． In ferm of ＲAPD makers， the 30 strains were also
separated into two clusters by the cluster analysis． The first cluster was classified into the geographic groups of Beijing，
Xinjiang，Liaoning，Gansu-Qinghai and Shandong-Heilongjian-Jilin-Shaanxi． The second cluster was separated into the
geographic groups of Inner Mongolia and Sichuan． The results showed a clear relationship between the geographical origin
and the colony characteristics or genetic diversity of the pathogenic fungus．
Key words: Cytospora chrysosperma; ＲAPD; colony characteristics
Poplar canker is common in many countries of the
world ( Adams et al．，2005)，affecting the growth of
trees and causing the death of infected trees． In China
17 kinds of pathogens of poplar canker have been
recorded (Zhang et al．，2003) ． Poplar canker disease
is mostly caused by three pathogens， Cytospora
chrysosperma，Phomopsis macrospora and Fusicoccum
aesculi (Ｒen et al．，2013; Wang et al．，2008) ． We
tested the colony characteristics of 30 C． chrysosperma
strains and analyzed their genetic diversity using
random amplification polymorphic DNA ( ＲAPD )
method． Although the cultural conditions can cause
changes of pathogenic fungus in some colony
characteristics， the differences in genetic，
physiological and pathological characteristics can be
reflected from the colony characteristics． Previous
research on colonial characters of C． chrysosperma on
various culture media has been carried out ( Jiang et
al．，2012 ) ． Zang et al． ( 2007 ) investigated the
diversity of 7 isolates with different colony
characteristics on PDA among 61 isolates of C． spp．
obtained from different apple producing areas of
Shaanxi province． The results indicated that C． spp．
isolated from apple trees had diversity and different
virulence in Shaanxi． Zhang et al． ( 2007 )
investigated the genetic change and diversity of 30
isolates of C． chrysosperma from 9 provinces in China，
the results showed that there were significant
第 6 期 杨明秀等: 中国 10 省金黄壳囊孢菌的菌落特征和遗传多样性
differences of colony characteristics among these
isolates． Xu ( 2010 ) tested the relationship between
ＲAPD genetic diversity and geographical distribution of
32 strains of C． chrysosperma from 10 provinces in
China．
The study of the colony characteristics，genetic
diversity and the relationship between geographical
distribution of C． chrysosperma has important
theoretical and practical significance in poplar disease-
resistant breeding and the control of the disease．
1 Materials and methods
1. 1 Source of strains Thirty of C． chrysosperma
strains were provided by the China Forestry Microbial
Strains Preserved Management Center ( Ｒesearch
Institute of Forest Ecological Environment and
Protection，Chinese Academy of Forestry) ． The host
plants and geographical positions of isolated strains are
shown below in Tab． 1．
Tab． 1 The information of tested strains①
Strain Host Source Strain Host Source
S01 Populus tomentosa Beijing S16 P． bolleana Kuche，Xinjiang
S02 P． tomentosa Beijing S17 P． bolleana Shache，Xinjiang
S03 P． alba Pinggu，Beijing S18 P． canescens Shawan，Xinjiang
S04 P． simonii × P． deltoides Jiagedaqi，Inner Mongolia S19 P． sp． Shuide，Shaanxi
S05 P． alba × P． berolinensis Jiagedaqi，Inner Mongolia S20 P． tomentosa Yangling，Shaanxi
S06 P． simonii × P． cathayana Daqing，Heilongjiang S21 P． liaoningensis Xinmin，Liaoning
S07 P． simonii × P． deltoides Daqing，Heilongjiang S22 P． × euramericana Longjing，Jilin
S08 P． ussuriensis Mohe，Heilongjiang S23 P． pyramidalis Jingtai，Gansu
S09 P． davidiana Hohhot，Inner Mongolia S24 P． pyramidalis Jingtai，Gansu
S10 P． sp． Inner Mongolia S25 P． pyramidalis Jingtai，Gansu
S11 P． cathayana Ulaan Chab，Inner Mongolia S26 P． gansuensis Zhangye，Gansu
S12 P． ussuriensis Mangui，Inner Mongolia S27 P． bolleana Ledu，Qinghai
S13 P． sp． Hongya，Sichuan S28 P． sp． Ningyang，Shandong
S14 P． sp． Xinwen，Sichuan S29 P． sp． Ningyang，Shandong
S15 P． sp． Kangding，Sichuan S30 P． tomentosa Zhucheng，Shandong
①Jiagedaqi is attributable to the Inner Mongolia Autonomous Ｒegion geographically but is attributed to the Heilongjiang Ｒegion for administration．
1. 2 Analysis of colony characteristics C．
chrysosperma strains were cultured on potato dextrose
agar ( PDA) in Petri dishes at 25 ℃ in the dark for
four days． Three 9 mm diameter mycelial plugs were
cut randomly from the margin of each colony and
placed in PDA medium． The colony shape，color，and
production of fructification of each strain were observed
after seven days of incubation． The fruiting bodies were
investigated after 30 days (Zhang et al．，2007; Fang，
2007) ． Observations were made from three replicates
of each strain．
Cluster analysis of the strains was carried out
using NTSYS (Version 2. 10 software) ．
1. 3 Analysis of genetic diversity 1 ) DNA
extraction The strains were cultured in PDA medium
at 25 ℃ for 7 days for mycelia collection． Before
extracting the DNA，the mycelia (100 － 150 mg) were
frozen at － 80 ℃ and then ground to a fine powder in
liquid nitrogen with a mortar and pestle． DNA was
extracted with the CTAB method ［2% (W /V) CTAB;
100 mmol·L － 1 Tris-HCl， pH8. 0; 20 mmol·L － 1
EDTA，pH8. 0; 1. 4 mol·L － 1 NaCl］( Duraisamy et
al．，2012; Jiang et al．，2008; Zhang et al．，2004; Gu，
1998) ．
2) Screening ＲAPD primers 60 random 10-base
primers (Operon Technology Inc． ) and 10 random 10-
base primers (Xu，2010) were independently used for
the polymerase chain reaction of ＲAPD analysis． The
DNA extracted from 10 strains that were isolated from
10 provinces ( Inner Mongolia，Heilongjiang，Sichuan，
Xinjiang，Shaanxi，Liaoning，Jilin，Gansu，Qinghai，
Shandong) ．
The PCＲ reaction mixtures contained 10 ng of
template DNA，10 μmol·L － 1 of primers，10 μL of
Premix Taq Version 2. 0 ( TaKaＲa) and sterile water
up to 20 μL． The amplifications were performed in a
Biometra DNA thermal cycle programmed for the
following conditions: an initial denaturation at 94 ℃
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林 业 科 学 50 卷
for 5 min，35 cycles of 94 ℃ for 1 min，44 ℃ for 1
min，and 72 ℃ for 2 min and a final extension step of
72 ℃ for 10 min． The amplification products were
separated by electrophoresis at 110 V on ethidium-
bromide-stained (0. 5 mg·mm － 1)，1. 5% agarose gels
run in 0. 5 × TBE buffer and visualized under UV light
(Duraisamy et al．，2012; Jain et al．，2007; Abreu et
al．，2012; Denoyes-Ｒothan et al．，2003) ． Each strain
was analyzed twice by PCＲ reaction．
3) ＲAPD and data analysis PCＲ amplification
of DNA was performed with each of the primers that
were screened in 1. 3. 2． The method of PCＲ
amplification of DNA is same to 1. 3. 2． Each strain
was scored for the presence or absence of each
amplicon． When a primer revealed a polymorphism，
all amplified DNA fragments were used for analysis and
treated as binary characters． The character of each
ＲAPD band was scored for each individual strain of C．
chrysosperma and the genetic similarities were
computed between all pairs of strains． Dissimilarities
were computed as a similarity coefficient and the data
was used to construct a phenogram using the
unweighted pair group method with arithmetic average
(UPGMA) (Zhang et al．，2006; Belabid et al．，2004;
Bayraktar et al．，2009) ．
2 Ｒesults
2. 1 Colony characteristics of C． chrysosperma strains
The colonial traits of C． chrysosperma strains were
shown in Fig． 1 and Tab． 2． The clustering analysis
was performed and a dendrogram was obtained using
the UPGMA． The 30 strains were grouped into two
clusters． The first cluster included all of the strains
from Beijing，Xinjiang，Liaoning， Jilin，Shandong，
Heilongjiang and one strain from each of Sichuan，
Shaanxi and Inner Mongolia，and was further separated
into the geographic groups of Xinjiang， Beijing-
Northeast ( Heilongjiang， Jilin， Liaoning ) and
Shandong at a similarity coefficient of 0. 71． The
second cluster included the five of the strains of Inner
Mongolia，four strains of Gansu，two strains of Sichuan
and one strain each of Qinghai and Shaanxi，and was
further separated into the geographic groups of Inner
Mongolia and the geographic group including Sichuan，
Gansu，Shaanxi，Qinghai at a similarity coefficient of
0. 62． Five strains (S01，S09，S11，S15，S20) were
not classified into the geographic group of sources
(Fig． 2) ．
Fig． 1 The colony characteristics of C． chrysosperma strains in PDA medium
Colonial color: A. White; B. Light gray; C. Gray; D. Dark gray; E. Beige; F. Orange． Colonial shape: A，C. Sparse and equatorial; B，
D. Dense and bulged． Colonial edge: A，C . Ｒegular; B，D. Irregular． Fruiting bodies: G. No fruiting bodies; H. Produced fruiting bodies．
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第 6 期 杨明秀等: 中国 10 省金黄壳囊孢菌的菌落特征和遗传多样性
Tab． 2 The colony characteristics of different strains of C． chrysosperma in PDA medium①
Strain
Colonial
color
Colonial
shape
Colonial
edge
Fructification Strain
Colonial
color
Colonial
shape
Colonial
edge
Fructification
S01 3 0 0 0 S16 3 0 0 1
S02 0 0 0 0 S17 3 0 0 1
S03 0 0 0 1 S18 3 0 0 1
S04 5 1 1 0 S19 1 0 0 1
S05 5 1 1 0 S20 2 0 0 1
S06 0 0 0 1 S21 0 0 0 0
S07 0 1 1 0 S22 0 0 0 1
S08 0 0 0 0 S23 4 1 1 1
S09 2 1 1 0 S24 1 1 1 0
S10 5 1 1 0 S25 1 1 1 0
S11 5 0 0 0 S26 1 1 1 0
S12 5 1 1 0 S27 0 1 1 0
S13 4 1 1 1 S28 0 1 0 0
S14 4 1 1 0 S29 0 1 0 0
S15 2 1 0 1 S30 0 1 0 0
①Colonial color: 0. White; 1. Light gray; 2. Gray; 3. Dark gray; 4. Beige; 5. Orange． Colonial shape: 0. Sparse and equatorial; 1. Dense and
bulged． Colonial edge: 0. Ｒegular; 1. Irregular． Fructification: 0. No fruiting bodies; 1. Produced fruiting bodies．
Fig． 2 Clustering analysis of the colony characteristics of 30 strains of C． chrysosperma from different geographic origins
2. 2 Screening ＲAPD primers DNA of 10 strains
that isolated from 10 provinces were amplified by using
70 random primers，9 of them were shown to yield
strong and clear bands with good reproducibility and
specificity (Tab． 3) ．
2. 3 ＲAPD analysis of C． chrysosperma strains
ＲAPD analysis of 30 strains demonstrated that the
length of the amplified DNA fragments ranged from 120
to 2 200 bp ( Tab． 3) ． By re-sequencing using the 9
primers， 58 polymorphisms were identified． The
number of polymorphic bands ranged from 5 to 9 by
using single primer． Cy-6 primer that amplified
polymorphic bands up to 9 (Fig． 3)，all of the ＲAPD
bands detected were polymorphic． These results
showed that the C． chrysosperma strains from ten
provinces in China had rich genetic diversity．
Clustering analysis was performed and a
dendrogram was constructed using the UPGMA
method． The 30 strains of C． chrysosperma were
classified into two clusters． The first cluster included
all of the strains from Beijing，Xinjiang，Liaoning，
Shaanxi， Jilin， Qinghai， Gansu， Heilongjiang and
Shandong; and one strain from Inner Mongolia． The
first cluster was further separated into the geographic
groups of Beijing，Xinjiang，Liaoning，Gansu-Qinghai
and the geographic groups include Shandong，
Heilongjiang， Jilin and Shaanxi at a similarity
coefficient of 0. 66． The second cluster included all of
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林 业 科 学 50 卷
the strains from Sichuan and five strains from Inner
Mongolia， and was further separated into the
geographic groups of Inner Mongolia and Sichuan at the
same similarity level． Four strains ( S12，S25，S23，
S30) were not separated into either of the geographic
groups (Fig． 4) ．
The results also showed that strains from the same
province had a high genetic similarity，and most of
these values were greater than 0. 7． The genetic
similarity coefficients were 0. 780 between the S01
(Beijing) and S03 (Pinggu，Beijing)，0. 720 between
the S09 (Hohhot，Inner Mongolia) and S12 (Mangui，
Inner Mongolia)，0. 840 between the S13 ( Hongya，
Sichuan) and S15 ( Kangding，Sichuan) and 0. 880
between the S6 ( Daqing， Heilongjiang ) and S8
(Mohe，Heilongjiang)，respectively ( Fig． 4) ． Thus，
C． chrysosperma strains from ten provinces in China
showed abundant genetic variation and low genetic
differentiation in geographically similar strains of same
province．
Tab． 3 PCＲ amplification ability of selected ＲAPD primers
Primer
Sequence
(5→3)
Number of
amplified
bands
Length of
amplification
Primer
Sequence
(5→3)
Number of
amplified
bands
Length of
amplification
Cy-1 AAAGTGCGGC 5 180 － 1 500 OPD-5 TGAGCGGACA 5 400 － 1 800
Cy-3 TGCTCTGCCC 6 220 － 2 000 OPD-13 GGGGTGACGA 5 300 － 2 000
Cy-5 CTGCTGGGAC 7 220 － 1 900 OPE-7 AGATGCAGCC 6 220 － 2 000
Cy-6 TGGACCGGTG 9 120 － 2 200 OPE-15 ACGCACAACC 6 300 － 2 000
OPC-2 GTGAGGCGTC 5 250 － 1 800
Fig． 3 ＲAPD patterns of C． chrysosperma strains amplified by primer Cy-6
Fig． 4 Dendrogram derived from UPGMA cluster analysis based on the similarity coefficient of ＲAPD
markers of 30 C． chrysosperma strains
2. 4 The colony characteristics and ＲAPD genetic
diversity analysis The colony characteristics and
genetic diversity of most C． chrysosperma strains were
related to the geographical origin and environments of
the strains． The geographical environment of Inner
Mongolia is predominantly grassland， but although
Beijing is close to Inner Mongolia，the strains from these
two regions were not in the same cluster in the genetic
461
第 6 期 杨明秀等: 中国 10 省金黄壳囊孢菌的菌落特征和遗传多样性
diversity analyses． Most of the strains were grouped into
the same cluster in the colony characteristics and
genetic diversity analyses except the strains from Gansu
and Qinghai Provinces． It is shown in Tab． 4．
Tab． 4 Colony characteristics and ＲAPD genetic clusters
of tested strains①
Cluster Strain
Colony
characteristics
ＲAPD cluster
The first
cluster
S01 + +
S02 + +
S03 + +
S06 + +
S07 + +
S08 + +
S11 +
S12 +
S15 +
S16 + +
S17 + +
S18 + +
S19 +
S20 + +
S21 + +
S22 + +
S23 +
S24 +
S25 +
S26 +
S27 +
S28 + +
S29 + +
S30 + +
The second
cluster
S4 + +
S5 + +
S9 + +
S10 + +
S11 +
S12 +
S13 + +
S14 + +
S15 +
S19 +
S23 +
S24 +
S25 +
S26 +
S27 +
①“ + ”indicates that the strains belonged to the groups of
morphological characteristics or ＲAPD clustering analysis
3 Discussion
In this research， the colony characteristics and
genetic diversity of C． chrysosperma strains from ten
provinces in China were tested． The results showed
that the C． chrysosperma had abundant diversity in
morphological characters，and the 30 strains could be
classified into two geographic groups． The first group
mainly included the strains from Xinjiang，Beijing-
Northeast ( Heilongjiang， Jilin， Liaoning ) and
Shandong，and the second group mainly included the
strains from Inner Mongolia， Sichuan and Gansu-
Qinghai． The results are similar to the results found by
Zhang et al． ( 2007 ) in which the 30 strains of
Cytospora chrysosperma isolated from poplar in China
were grouped into Northeast and Northwest geographic
groups based on colony characteristics．
The results of the ＲAPD clustering showed that the
C． chrysosperma strains were also classified into two
geographic groups． The first group includes Beijing，
Xinjiang，Northeast (Heilongjiang，Jilin，Liaoning)，
Qinghai，Gansu and Shaanxi． The second group mainly
included the strains from Inner Mongolia and Sichuan．
The results are similar to some views of Xu (2010) ．
The clustering analysis of the colony
characteristics and the ＲAPD genetic diversity of the
strains reflected that there was a certain relationship
between the colony characteristics and the genetic
diversity of C． chrysosperma． For example，the strains
S16，S17 and S18 from Xinjiang were clustered into
the same group in both cluster analyses． Five strains in
the study of colony characteristics and four strains in
research of ＲAPD analysis were not separated into the
geographic group of sources． Most of the 30 strains
were in the same cluster in both cluster analyses，
indicated that it is nearly reasonable to classify strains
based on colony characteristics and ＲAPD genetic
traits． The results showed that the colony
characteristics and genetic diversity of C． chrysosperma
were related to geographical origin of the pathogenic
fungus． Strains from Heilongjiang and Jilin had the
closest genetic traits and the Inner Mongolia strains had
the least genetic traits to the other province strains．
In summary， this study showed that C．
chrysosperma from China has abundant genetic
diversity，and there are more differences in population
structure and genetic variation of C． chrysosperma from
different geographic origins． Therefore， in order to
better understand the occurrence and epidemic，and to
be able to predict the development of this disease，we
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林 业 科 学 50 卷
must have a timely and accurate understanding of the
population’s genetic structure and variation laws of C．
chrysosperma． So as to provide a scientific basis for
formulating effective control measures to poplar canker．
Comparing to the results of colony characteristics
and genetic diversity，some strains which are from the
same province were not in the same geographic group
what is the reason needs to be further researched．
Because ＲAPD technology is based on genomic DNA
as a template，so the results may have been affected by
the differences of genome． At present，five molecular
marker technologies ( ＲFLP，ＲAPD，AFLP， ISSＲ，
SSＲ) ( Liao et al．， 2008; Belabid et al．， 2004;
Bayraktar et al．，2009; Duraisamy et al．，2012; Cao et
al．，2012 ) and most commonly used to analyze the
population’s genetic diversity of pathogens． ＲAPD
molecular marker technology was adopted in this study．
We aim to carry out further research on the relationship
of the genes of C． chrysosperma using other molecular
marker technologies， combined with morphological
characteristics， pathogenicity and geographical
environment．
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