全 文 ：12期
Received date：2014-10-25
Foundation item ：Guangxi Natural Science Foundation（2013GXNSFBA019068）；China Agriculture Research System（CARS-20-
2-3）；Agricultural Public Welfare Research Item（200903004）；Key Project for Guangxi Academy of Agricultural Sciences
（2013JZ12）
Biography：*forcorrespondingauthor，HUANGCheng-hua（1974-），associateresearcher，interestinginintegratedcontrolofsugarcane
insect and pests， E-mail： chenghuahuang@sina.com. LIN Shan-hai（1979-）， Ph.D.， interesting in integrated control of
sugarcane diseases， E-mail： sh nhailin79@163.com
Identification and sequences analysis of Ceratocystis
paradoxa causing sugarcane pineapple disease based
on rDNA-ITS and large subunit
LINShan-hai1，ZHOUZhu-gui2，PANXue-hong1，SHANGXian-kun1，
WEIJi-li1，HUANGCheng-hua1*
（1Sugarcane Research Institute，Guangxi Academy of Agricultural Sciences/Sugarcane Research Center，Chinese Academy of
Agricultural Sciences/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement（Guangxi），Ministry of
Agriculture，Nanning 530007，China；2Flowers Research Institute，Guangxi Academy of Agricultural Sciences，
Nanning 530007，China）
Abstract： 【Objective】The nucleotide sequences of rDNA -ITS and large subunit of Ceratocystis paradoxa （de
Seynes） Moreau causing sugarcane pineapple disease， were analyzed in order to provide technological supports for rapid
and effective molecular detection of the pathogen in sugarcane production. 【Method】A representative strain FL-1 was
isolated from ROC22 underground seedcane with pineapple disease. The pathogenicity of strain FL -1 to sugarcane
plants was determined by artificial inoculation based on Koch’s postulates. The morphological characteristics of FL-1
colony grown on PDA medium were observed， as well as its conidiophores， aleurioconidia and conidia under light mi-
croscope. The nucleotide sequence regions of rDNA-ITS and large subunit were amplified by using primers ITS1/4 and
NL1/4. The phylogenetic tree based on ITS and large subunit sequences of strain FL-1， respectively， were constructed
by using Neighbor -joining method， and their phylogenetic distance were analyzed using the p -distance program in
software MEGA. 【Result】The isolate FL-1 was determined as the pathogen causing sugarcane pineapple disease ac -
cording to the pathogenicity test， and it was charactered by typical arthrospores and aleurioconidia， which was consisted
with that of C. paradoxa. The nucleotide sequences of rDNA-ITS and large subunit amplified from genome DNA of
isolate FL-1 were 499 and 563 bp in length， respectively. And they shared 100.0% of homology with the fungus of C.
paradoxa. The rDNA-ITS sequence was clustered with C. paradoxa in an absolute evolution group in the phylogenetic
tree， as well as the large subunit sequence. The genetic distance matrix results showed that rDNA-ITS and large sub-
unit sequences of isolate FL-1 were genetically clustered with C. paradoxa， and the intraspecific genetic distance were
0-0.017 and 0-0.008， respectively. While， the interspecific genetic distance of Ceratocystis were 0.015-0.106 and
0.002-0.059， respectively. Therefore， FL-1 strain was identified as C. paradoxa based on morphological characteris-
tics and molecular detection. 【Conclusion】The isolate FL-1 from sugarcane was considered as member of the C. para-
doxa complex and primarily classified into the Clade 1 of complex. rDNA-ITS and large subunit were fit for PCR de-
tection and molecular marker of sugarcane pineapple disease.
Key words： Ceratocystis paradoxa complex； morphology characteristic； rDNA-ITS； large subunit； phylogenetic
analysis
CLC number： S566.1 Document code：A Article：2095-1191（2014）12-2103-07
基于rDNA-ITS和大亚基的甘蔗凤梨病菌
分子鉴定及序列分析
林善海1，周主贵2，潘雪红1，商显坤1，魏吉利1，黄诚华1*
（1广西农业科学院 甘蔗研究所/中国农业科学院 甘蔗研究中心/农业部广西甘蔗生物技术与遗传改良重点实验室，南宁 530007；
2广西农业科学院 花卉研究所，南宁 530007）
摘要：【目的】分析甘蔗凤梨病菌［Ceratocystis paradoxa（de Seynes）Moreau］核酸rDNA-ITS和大亚基区域序列，
DOI：10.3969/j：issn.2095-1191.2014.12.2103
南方农业学报 Journal of Southern Agriculture 2014，45（12）：2103-2109
ISSN 2095-1191；CODEN NNXAAB http：//www.nfnyxb.cn
南 方 农 业 学 报 45卷
为甘蔗凤梨病菌的快速、有效分子检测提供技术支持。【方法】从发生凤梨病的新台糖22号（ROC22）地下种茎分离获
得1株菌株FL-1，根据柯赫氏法则测定其致病性；将该病菌在PDA培养基进行培养，观察其菌落形态特征，并在光学
显微镜下观察分生孢子梗、厚垣孢子、分生孢子的形态特征。用引物ITS1/4和NL1/4分别对该菌株的核酸rDNA-ITS和
大亚基序列进行扩增，以MEGA软件的Neighor-joining方法分别构建系统发育树，并用p-distance进行遗传距离分析。
【结果】分离菌株FL-1为甘蔗凤梨病致病菌，具有典型的节孢子和厚垣孢子，形态特征与奇异长喙壳菌（C. paradoxa）
一致。从菌株FL-1基因组DNA中分别扩增获得499 bp rDNA-ITS和563 bp大亚基片段，其序列均与C. paradoxa其他菌
株的同源性为100.0%。系统聚类分析结果表明，两条序列均与C. paradoxa聚为一个独立簇。遗传矩阵分析结果表明，
rDNA-ITS和大亚基均与C. paradoxa聚合在一起，种内遗传距离分别为0～0.017和0~0.008，而Ceratocystis属内种间遗
传距离分别为0.015~0.106和0.002~0.059。结合分子检测结果和形态学特征，将FL-1鉴定为C. paradoxa。【结论】来自
甘蔗的FL-1菌株是C. paradoxa复合群的成员，并被划分在进化复合群的分枝1上。rDNA-ITS和大亚基适合用于甘蔗
凤梨病的PCR检测及分子标记。
关键词：奇异长喙壳菌复合群；形态特征；rDNA-ITS；大亚基；遗传分析
0 Introduction
【Research signi ficance】Sugarcane is one of the
important commercial crops in tropical and subtropical
countries and contributes nearly 70% of global sugar
production. Although sugar is the most important
product of sugarcane， various byproducts in sugar
production can be served as industrial raw material.
China is the third largest producer of sugarcane next
to Brazil and India. A large number of pathogens
damage sugarcane generation after generation， which
reduces the yield and quality and results in genetic
characteristics degeneration of sugarcane. Among the
major diseases， sett borne diseases play an important
role in restraining the production of sugarcane
（Byther and Moore， 1974；Wu et al.， 2007；Begum
et al.，2008） . Therefore， analyzing rDNA -ITS and
large subunit genes of C. paradoxa is important to
rapid detection of sugarcane pineapple disease at
molecular level. 【Research progress】Pineapple disease
and black rot of sugarcane， caused by ascomycetous
fungi C. paradoxa and C. adiposum， respectively， are
the primarily diseases of underground seedcane. Of
the two sett rot diseases， pineapple disease is a
destructive rot on sugarcane sett worldwide. Pineapple
disease occurred frequently in all sugarcane growing
areas of China （Feng， 1996； Wu et al . ， 2007 ） .
Furthermore， the infected canes decay before buds
germination and seedlings die back shortly after
emergence. Sugarcane tissue infected by pineapple
disease may contain ethyl acetate that was sufficient
to inhibit buds germination （Kuo et al.， 1969； Yadahalli
et al.， 2007） . The fungus of C. paradoxa was well
known formerly as the pathogen causing sugarcane
pineapple disease， and the pathogen on sugarcane
usually was identified based on morphological characters.
Really， the fungus has been redefined as a complex
population which comprises a group of fungal pathogens.
At present， at least six species in the C. paradoxa
complex were recognized with multigene DNA phylogeny，
morphology and mating tests， which included C.
musarum， C. paradoxa， C. radicicola， Theielaviopsis
euricoi， C. ethacetica and C. cerberus （Harrington，
2009； Wingfield et al.， 2013； Mbenoun et al.， 2014） .
The genealogical structure of C. paradoxa complex is
divided into three clades based on ITS， β -tubulin
and TEF1-α gene phylogeny. 【Research breakthrough
point】Generally， at least two pathogens were known
to cause sugarcane underground seedcane rot disease，
and their symptoms were confused. At present， few
researches on molecular detection of sugarcane
pineapple disease were found to be reported. 【Solving
problems 】 In this study ， a representative strain of
C. paradoxa causing sugarcane pineapple disease
was isolated and identified based on morphological
characteristic and molecular detection. The sequences of
rDNA-ITS and large subunit of the isolated strain were
analyzed in order to provide technological reference for
molecular detection of sugarcane pineapple disease.
1 Materials and methods
1. 1 Experimental materials
The used sugarcane variety ROC22 in the
experiment was cultivated in Sugarcane Research
Institute， Guangxi Academy of Agricultural Sciences，
the pathogen was isolated from its underground seed-
cane with pineapple disease.
1. 2 Experimental methods
1. 2. 1 Pathogen isolation and its DNA extraction
The seedcanes with classical symptom of sugarcane
pineapple disease were washed to remove surface
soil and dried in the air. The diseased tissue from
infected canes was surface -sterilized for 1 min with
5% sodium hypochlorite solution and 30 s in 75%
ethanol， followed by rinsing for three times with
sterile distilled water. After drying out， the diseased
tissue was placed into potato dextrose agar （PDA）
plate and incubated for 2-3 days at 28 ℃ in the dark.
Journal of Southern Agriculture2104· ·
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LIN et al.： Identification and sequences analysis of Ceratocystis paradoxa causing sugarcane pineapple disease based on
rDNA-ITS and large subunit
The fungal single -spore derived from culture
was obtained and grown on PDA medium at 28 ℃
for 5 days for DNA extraction. Approximately 30 mg
of fungal tissue were scraped from colony surface
and placed into a sterile 2.0 mL centrifuge tube
freezed with liquid nitrogen and grinded into a fine
powder using tissue grinding apparatus. The DNA
extraction protocol was performed using method as
described by Lin et al. （2011a） .
1. 2. 2 Morphological characteristic identification of
pathogen A little fresh fungal tissue was inoculated
into a new PDA plate and incubated at 28 ℃ for 3
days. The morphological characteristics of colony ， and
more than 100 conidiophores， conidia along with the
spore -germinating patterns of pathogen were ob-
served under light microscope.
1. 2. 3 Amplification of rDNA-ITS and large sub-
unit sequences The conservative target regions of
the Internal Transcribed Spacer regions 1 （ITS1）
and 2 （ITS2） including 5.8S rRNA， 28S rRNA and
large subunit of ribosome were amplified using the
primers listing in Tab .1 ， which were synthesized
by TaKaRa Biotechnology （Dalian） Co.，Ltd.， China.
Polymerase chain reaction （PCR） was done using the
following ingredients for each 20.0 μL reaction sys-
tem viz.， 0.2 μL of Taq DNA polymerase （5 U/μL），
0.5 μL of 10 μmol/L of each forward and reverse
primer， 1.0 μL of dNTP （10 mmol/L）， 2.0 μL of
10×PCR Buffer （25 mmol/L MgCl2）， 2.0 μL of ge-
nomic DNA （25 ng/μL）， and the nuclease-free dou-
ble distilled water was added to 20.0 μL of total
volume. The PCR amplification protocol of ITS and
large subunit genes were as follows： initial denatura-
tion at 94 ℃ for 5 min； 34 cycles of denaturation
for 30 s at 94 ℃ ， annealing for 30 s at 56 ℃ ，
with a final extension for 1 min at 72 ℃ ； final ex-
tension for 10 min at 72 ℃ . The amplification size
and purity of PCR products were analyzed by 1%
agarose electrophoresis gels stained with GelRedTM
（Biotium Inc.） in a 1 ×TAE Buffer and visualized
under UV light. Then the PCR products were puri-
fied using the PCR Product Purification Kit ［pro-
duced by TaKaRa Biotechnology （Dalian） Co.， Ltd.，
China］， and sequenced （both strands） by Invitrogen
（Shanghai） Co.， Ltd.， China. The nucleotide sequence
was edited with software MEGA Ver.5.10. All the
sequences were checked manually and nucleotides with
ambiguous positions were clarified using both primer
direction sequences. The sequences obtained from
isolate FL -1 were deposited in GenBank and com-
pared with sequences of ITS and large subunit in other
species.
Tab.1 Primers used for amplifying ribosomal RNA of isolated pathogen
Amplification region
rDNA-ITS
Large subunit rDNA
Primer name
ITS1
ITS4
NL1
NL4
Gene primer
5-TCCGTAGGTGAACCTGCGG-3
5-TCCTCCG CTTATTGATATGC-3
5-GCATATCAATAAGCGGAGGAAAAG-3
5-GGTCCGTGTTTCAAGACGG-3
Reference
Lin et al.（2011a）
Kurtzman and Robnett
（1998）
1. 2. 4 Sequences and phylogenetic analysis The
nucleotide sequences of rDNA-ITS and large subunit
were aligned and analyzed， respectively， by using
the CLUSTAL multiple alignment program in the
software MEGA Ver.5.10. The neighbor -joining
phylogenetic trees based on rDNA -ITS and large
subunit aligned sequences were constructed using
NEIGHBOR in MEGA Ver.5.10. The phylogenetic
distance matrix of the two nucleotide sequences were
calculated with p-distance program， and their topologies
were tested with 1000 replications on bootstrap level
（Lin et al.， 2011b） .
2 Results and analysis
2. 1 Morphological characteristics identification of
isolate FL-1
The fungus isolated from diseased sugarcane
seed -cane showed white colonies on PDA medium
at first and turned into deep black in the center
with gray - white edge after incubating 2 days .
Conidiophores produced laterally from the hyphae was
characterized by slender， septate， phialidic， hyaline to
very pale brown， 86.0 -228.0 μm × 6.5 -10.5 μm
in size， tapering tip， and continuously producing
conidia through the open end （Fig.1）. Conidia showed
cylindrical at first， followed by oval at mature stage ，
and were with hyaline to dark brown color ， smooth-
walled， and 6.5 -15.5 μm × 2.5 -5.0 μm in size.
Aleurioconidia presented terminal ， in chains， obovate
to oval， thick -walled， brown， and 8.5 -26.0 μm ×
5.0-11.0 μm in size（Fig.1）. The conidia characteristics
was consistent with that of C. paradoxa described by
Huang et al.（1990） .
2. 2 Analysis of rDNA-ITS and large subunit se-
quences
Using primers of rDNA-ITS and large subunit ，
approximate 500 and 560 bp DNA segments of
nucleotide ITS and large subunit were amplified from
genomic DNA of strain FL-1 isolated from sugarcane
seedcane（Fig.2）. Through sequence comparison of ITS
and large subunit sequences （Accession No. KP017525
and KP017524）， it was found that the rDNA -ITS
2105· ·
南 方 农 业 学 报 45卷
Fig.1 Conidia morphological characteristics of isolate causing sugarcane pineapple disease （bar=20 μm）
A：arthrospores; B：aleurioconidia
nucleotide sequence of isolate FL -1 was 499 bp in
length， which existed simple base substitution
compared with those of C. paradoxa in GenBank
database. The large subunit sequence was 563 bp in
length， which also existed simple base substitution
and absence of one base located at 102 locus of
Internal Transcribed Spacer regions 1 （data not showed） .
Both rDNA -ITS and large subunit sequences of
isolate FL-1 shared 99.0%-100.0% of homology with
those of C. paradoxa isolates in GenBank databse.
2. 3 Phylogenetic analysis of rDNA-ITS and large
subunit
The phylogenetic tree was constructed based on
comparison of strain FL-1 rDNA-ITS sequence and
the other fungi from GenBank database （Fig . 3 ） .
The results showed that isolate FL -1 was clustered
with 5 isolates of C. paradoxa from GenBank， which
clearly distinguished from other species of Ceratocystis
in ITS phylogenetic tree. The phylogenetic distance
of isolate FL-1 with C. paradoxa isolates from GenBank
Fig.2 Amplification results of rDNA-ITS and large sub-
unit from strain FL-1
M：DL3000 DNAMarker；1：rDNA-ITS region；2：large subunit
Fig.3 Phylogenetic tree based on comparing rDNA-ITS sequences of C. paradoxa isolates and other Ceratocystis species
from GenBank
The value in each branch indicated the percentage supported at bootstrap level; the scale bar denoted 0.005 substitutions per nucleotide
position. The same was applied in the subsequent figure
Journal of Southern Agriculture
M 1 2
3000 bp
2000 bp
1500 bp
1000 bp
800 bp
700 bp
600 bp
500 bp
400 bp
300 bp
200 bp
100 bp
2106· ·
12期
Fig.4 Phylogenetic tree based on comparing large subunit sequences of C. paradoxa isolates and the other Ceratocystis
species from GenBank
Tab.2 Phylogenetic distance matrix of C. paradoxa based on rDNA-ITS region
Fungal isolate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 FL-1
2 DQ318203_C. paradoxa 0.006
3 KC305159_C. paradoxa 0.006 0.000
4 HM131605_C. paradoxa 0.017 0.023 0.023
5 KF939052_C. paradoxa 0.000 0.006 0.006 0.017
6 FN668726_C. paradoxa 0.013 0.015 0.015 0.030 0.013
7 KC305129_C. rufipenni 0.089 0.087 0.087 0.106 0.089 0.083
8 KC305128_C. rufipenni 0.089 0.087 0.087 0.106 0.089 0.083 0.000
9 DQ318205_C. resinifera 0.074 0.072 0.072 0.091 0.074 0.068 0.019 0.019
10 DQ318201_C. polonica 0.083 0.081 0.081 0.100 0.083 0.078 0.034 0.034 0.034
11 DQ318200_C. polonica 0.083 0.081 0.081 0.100 0.083 0.078 0.034 0.034 0.034 0.000
12 DQ318199_C. polonica 0.083 0.081 0.081 0.100 0.083 0.078 0.034 0.034 0.034 0.000 0.000
13 DQ318198_C. pinicola 0.081 0.078 0.078 0.097 0.081 0.074 0.030 0.030 0.019 0.023 0.023 0.023
14 KC305119_C. coerulescens 0.081 0.078 0.078 0.097 0.081 0.074 0.034 0.034 0.025 0.032 0.032 0.032 0.015
15 KC305118_C. coerulescens 0.081 0.078 0.078 0.097 0.081 0.074 0.034 0.034 0.025 0.032 0.032 0.032 0.015 0.000
16 KC305117_C. coerulescens 0.081 0.078 0.078 0.097 0.081 0.074 0.034 0.034 0.025 0.032 0.032 0.032 0.015 0.000 0.000
The block with grey shading denoted intraspecific matrix of C. paradoxa; the block with dashed line denoted the lowest value of interspecific genetic dis-
tance and those with solid line were the highest value. The same was applied in the subsequent table
LIN et al.： Identification and sequences analysis of Ceratocystis paradoxa causing sugarcane pineapple disease based on
rDNA-ITS and large subunit
ranged from 0 to 0.017 in the phylogenetic distance
matrix of rDNA - ITS sequence （Tab . 2 ） . The C .
paradoxa isolates HM131605 and FN668726 were
found with largest intraspecific distance （ 0 . 030 ）
（Tab.2） . The results shown in Tab.2 also indicated
that， the evolutionary distance among sequences of
interspecies isolates in Ceratocystis genus ranged
from 0.015 to 0.106. The smallest distance （of
interspecies was found between isolates C. pinicola
（DQ318198） and C. coerulescens （KC305117， KC305
118_and KC305119）， and the largest distance was
found between C. paradoxa （HM131605） and C.
rufipenni （KC305128 and KC305129） （Tab.2） .
In the phylogenetic tree constructed with large
subunit sequences （Fig . 4 ） ， the isolate FL-1 was
clustered with two isolates C. paradoxa （AF275498 and
AF043607） from GenBank， and clearly distinguished
from other Ceratocystis species. Based on phylogenetic
distance matrix， the evolutionary distance among isolate
FL-1 with two C. paradoxa isolates （AF275498 and
AF043607 ） from GenBank was 0 -0 . 008 （Tab . 3 ） .
However， the evolutionary distance between sequences
of interspecies isolates in Ceratocystis genus was
0.002 -0.059. The smallest distance of interspecies
was found between isolates C. pinicola （AF275511 ）
and C. coerulescens （AF275529 and AF275510）， isolates
C. eucalypti （AF222482） and Thielaviopsis australis
（AF222450）， and the largest distance between isolates
C. pinicola （AF27 5511） and C. moniliformis （AF222
487_and AF275499） （Tab.3） .
2107· ·
南 方 农 业 学 报 45卷
Tab.3 Phylogenetic distance matrix of C. paradoxa based on large subunit
Fungal isolate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
1 FL-1
2 AF275498_C. paradoxa 0.000
3 AF043607_C. paradoxa 0.008 0.008
4 AY283562_C. adiposa 0.043 0.043 0.037
5 AF222481_C. adiposa 0.043 0.043 0.037 0.000
6 AF275529_C. coerulescens 0.039 0.039 0.035 0.057 0.057
7 AF275510_C. coerulescens 0.039 0.039 0.035 0.057 0.057 0.000
8 AF222482_C. eucalypti 0.035 0.035 0.039 0.055 0.055 0.012 0.012
9 AF222483_C. fagacearum 0.028 0.028 0.028 0.020 0.020 0.047 0.047 0.047
10 AF275499_C. moniliformis 0.041 0.041 0.041 0.024 0.024 0.059 0.059 0.051 0.020
11 AF222487_C. moniliformis 0.043 0.043 0.039 0.026 0.026 0.057 0.057 0.053 0.018 0.006
12 AF275511_C. pinicola 0.041 0.041 0.037 0.057 0.057 0.002 0.002 0.014 0.049 0.059 0.059
13 AF222489_C. virescens 0.035 0.035 0.039 0.059 0.059 0.014 0.014 0.010 0.047 0.055 0.057 0.016
14 CVU47824_C. virescens 0.035 0.035 0.039 0.059 0.059 0.014 0.014 0.010 0.047 0.055 0.057 0.016 0.000
15 AF222450_Thielaviopsis australis 0.033 0.033 0.037 0.057 0.057 0.010 0.010 0.002 0.045 0.053 0.055 0.012 0.008 0.008
16 AF275509_T. basicola 0.024 0.024 0.024 0.043 0.043 0.039 0.039 0.035 0.035 0.039 0.041 0.041 0.035 0.035 0.033
17 AF222459_T. basicola 0.024 0.024 0.024 0.043 0.043 0.039 0.039 0.035 0.035 0.039 0.041 0.041 0.035 0.035 0.033 0.000
18 AF275502_T. ovoidea 0.024 0.024 0.020 0.039 0.039 0.035 0.035 0.035 0.030 0.039 0.037 0.037 0.030 0.030 0.033 0.012 0.012
19 AF222471_T. neocaledoniae 0.039 0.039 0.043 0.057 0.057 0.016 0.016 0.004 0.051 0.053 0.057 0.014 0.014 0.014 0.006 0.039 0.039 0.039
20 AF222475_T. populi 0.028 0.028 0.024 0.039 0.039 0.039 0.039 0.039 0.030 0.039 0.037 0.041 0.039 0.039 0.037 0.012 0.012 0.008 0.043
21 AF222480_T. thielavioides 0.024 0.024 0.020 0.039 0.039 0.035 0.035 0.035 0.030 0.039 0.037 0.037 0.030 0.030 0.033 0.012 0.012 0.000 0.039 0.008
3 Discussion
Pineapple sett rot is a major soil -born disease
in sugarcane， and the causing organism of C.
paradoxa is a weak parasite that is commonly found
in soil . When sugarcane seedcanes are invaded by
the fungus， buds are often destroyed before growing
to seedlings （Aberdeen and Patil -Kulkarni， 1969；
Mahalingam et al.， 2012） . Besides sugarcane， C.
paradoxa also could cause post-harvest rot disease in
coconut， banana， carambola， guava and pineapple
（Huang et al.， 1990； Reyes et al.， 1998； Junqueira
et al.， 2001； Ploetz， 2003； Tzeng and Sun， 2009；
Warwick and Passos， 2009； Pinho et al.， 2013）. The
fungus was found causing post-harvest rots on coconut
matched well with the morphological description and
multilocus genes of C. paradoxa， including ITS， β-
tubulin and TEF1-α genes （Pinho et al.， 2013） . In
the present study， although ITS sequence of isolate
FL-1 closely hitted the sequence of C. paradoxa in
GenBank database with BLASTn program， ITS
sequence （No. JQ963886） from coconut was diverged
significantly with that of isolate FL -1， resulting in
the two sequences could not be clustered into a branch
in phylogenetic tree （data not showed ） . Therefore ，
the ITS sequence （No. JQ963886） was not selected
for phylogenetic analysis in this study. It was indicated
that ITS region of isolate FL-1 was different obviously
with those of C. paradoxa from other natural host
plants. Since ITS sequences had limited phylogenetic
value for Ceratocystis species （Harrington et al.，2011），
large subunit analysis was used additionally to compare
with the isolates from other natural host plants.
Recently， at least six species were recognized in
the C. paradoxa complex， including C. musarum，
C. paradoxa， C. radicicola， Theielaviopsis euricoi，
C. ethacetica and C. cerberus with multigene DNA
phylogenies， morphology and mating tests （Harrington，
2009； Wingfield et al.， 2013； Mbenoun et al.， 2014） .
Besides other five species， C. cerberus is the only
known homothallic species in C. paradoxa complex.
The genealogical structure of C. paradoxa complex is
divided into three clades based on ITS， β -tubulin
and TEF1-α phylogenies. Clade 1 included C. paradoxa，
C. euricoi， Clade 2 included C. ethacetica and C.
musarum， and Clade 3 was composed of C. radicicola
and C. cerberus （Mbenoun et al.， 2014） . No DNA
sequences of this species in sugarcane were available
in GenBank to check whether the taxon belongs to
C. paradoxa or not. Thus， it was decided to identify
this isolate based on rDNA-ITS and large subunit in
this study. The results showed that the sequences of
rDNA -ITS and large subunit were high similarity
with those of C. paradoxa from other natural host in
GenBank database. The isolate FL -1 should be
classified in the Clade 1 according to the description
of Mbenoun et al.（2014）. Since C. paradoxa complex
comprises greater species variation， few morphological
differences can be used as a diagnostic method to
discriminate species in the C. paradoxa complex，
and sexual structure is not known to all species and
not generally observed in nature or culture conditions.
Thus， an effective and rapid molecular marker technology
is expected to be developed to discriminate the species
within C. paradoxa complex.
Journal of Southern Agriculture2108· ·
12期
4 Conclusion
It is suggested that isolate FL-1 could be consi-
dered as one member of the C. paradoxa complex
and classified primarily into Clade 1. rDNA-ITS and
large subunit of ribosome were fit for PCR detection
and molecular marker of sugarcane pineapple disease.
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LIN et al.： Identification and sequences analysis of Ceratocystis paradoxa causing sugarcane pineapple disease based on
rDNA-ITS and large subunit 2109· ·