全 文 :植物病理学报
ACTA PHYTOPATHOLOGICA SINICA 45(4): 356 ̄360(2015)
Received date: 2014 ̄12 ̄06ꎻ Revised date: 2015 ̄04 ̄15
Foundation item: National Natural Science Foundation of China (31270067)
Corresponding author: HUANG Qiongꎬ professorꎬ majors in plant pathology researchꎻ E ̄mail:huangqiong88hs@163.com
First author: HAN Yong ̄huaꎬ femaleꎬ postgraduate studentꎬ majors in plant pathologyꎻ E ̄mail:d20080514226@163.com.
doi:10.13926 / j.cnki.apps.2015.04.003
Occurrence of Fruit Rot Caused by Ceratocystis
fimbriata in Ziziphus jujube in China
HAN Yong ̄hua1ꎬ LI Xiao2ꎬ YANG Jun ̄yu1ꎬ LI Jie3ꎬ XU Ke ̄cheng1ꎬ
ZHANG Yu1ꎬ HUANG Qiong1∗
(1College of Plant Protectionꎬ Yunnan Agricultural Universityꎬ Kunming 650201ꎬ Chinaꎻ 2Dow AgroSciences China Ltd.ꎬ Jianshui 654300ꎬ Chinaꎻ
3Yunnan Tobacco Research Instituteꎬ Yuxi 653100ꎬ China)
Abstract:A fruit rot was found in jujube in August 2013 in Kunmingꎬ China. The isolates obtained from the
symptomatic tissues were confirmed as the pathogen responsible for the fruit rot via pathogenicity test. The
pathogenic isolates were identified as Ceratocystis fimbriata based on the morphological characteristics and mo ̄
lecular analyses. This is the first report of C. fimbriata causing jujube fruit rot in China.
Key words: jujubeꎻ fruit rotꎻ Ceratocystis fimbriata
中国发现甘薯长喙壳菌引起枣子果实腐烂 韩永花1ꎬ李 晓2ꎬ杨俊誉1ꎬ李 婕3ꎬ徐可成1ꎬ张 彧1ꎬ黄 琼1
( 1云南农业大学植物保护学院ꎬ昆明 650201ꎻ2陶氏益农中国有限公司ꎬ建水 654300ꎻ3云南省烟草研究院ꎬ玉溪 653100)
摘要:2013年 8月ꎬ在中国昆明发现一种枣子果实的腐烂病ꎮ 通过致病性测试ꎬ证明从发病组织分离出的病菌能引起枣子
果实腐烂ꎮ 经过形态学和分子鉴定ꎬ确定该病原菌为甘薯长喙壳菌ꎮ 这是在中国首次报道由甘薯长喙壳菌引起的枣子果
实腐烂病ꎮ
关键词: 枣子ꎻ果实腐烂ꎻ甘薯长喙壳
中图分类号: S432.1 文献标识码: A 文章编号: 0412 ̄0914(2015)04 ̄0356 ̄05
Introduction
The jujube (Ziziphus jujube Mill.) is a major
fruit crop cultivated in Indiaꎬ Russiaꎬ the Middle
Eastꎬ Southern Europe and China [1] . As the jujube
fruit provides people with rich nutritive materials like
ascorbic acidꎬ carotenoids[2] and has some composi ̄
tions with a role in treating insomniaꎬ hyperpiesiaꎬ
nervous debility and other diseases[3ꎬ 4]ꎬ it is quite
enjoyed by people all over the world. Among jujube
diseasesꎬ jujube anthracnoseꎬ jujube white rotꎬ ju ̄
jube fruit rust diseaseꎬ ascochyta spot of jujube and
jujube witches broom have already been documen ̄
ted[5] . Jujube fruit rot is usually caused by many
pathogensꎬ such as Rhizopus speciesꎬ Aspergillus
species and Penicillium speciesꎬ which are the major
causes of fruit postharvest diseases[6] . During August
of 2013ꎬ a new disease was observed in jujube fruit at
a farmer′ s market in Kunming of Yunnan Provin ̄
ceꎬ China ( the detailed origin place was un ̄
known) . The symptom of the disease appeared as
water ̄soaked lesions on the fruitsꎬ later became
brownish and coalescingꎬ which resulted in rotten
and sunken fruits. The inner tissue of the fruits were
also rotten and brownish (Fig. 1 ̄Aꎬ B) . The objec ̄
tive of this study was to determine the etiology of
4期 HAN Yong ̄huaꎬ et al:Occurrence of Fruit Rot Caused by Ceratocystis fimbriata in Ziziphus jujube in China
jujube fruit rot by pathogenicity testꎬ morphological
observation and molecular analysis.
1 Materials and Methods
1.1 Sample isolation
The jujube fruits with the typical rot symptoms
were from a stall in a farm market. The isolates were
obtained from the symptomatic tissues incubated be ̄
tween slices of fresh carrot root[7] . Spore masses
were removed from the apices of peritheciaꎬ trans ̄
ferred to malt extract agar medium (MEA) and in ̄
cubated at 25°C. For identificationꎬ the growth rate
was recordedꎬ and morphology characteristics of the
pathogen were observed.
1.2 Pathogenicity test
Single ̄spore cultures from two isolates were
used to inoculate in our laboratoryꎬ and fresh jujubes
were inoculated by injecting 200 μL the fungal spore
suspension (2.0×105 sporesmL ̄1) [8]ꎬ the control
jujubes were injected with sterile water. Each treat ̄
ment ( two isolates and one control) contained three
jujubes. The inoculated and control jujubes were
placed in freshness protection packages in the lab for
12 days at room temperatureꎬ degreasing cotton
dipped sterile water was used for keeping wet in the
packages.
During the inoculation periodꎬ the symptomatic
change of jujubes was observed and recorded daily.
1.3 Morphological observation
Two isolates respectively from both the original
diseased and inoculated jujubes were examined under
a microscopy. For microscopy examinationꎬ each i ̄
solate was cultured on two plates and slide samples
of black particles and mycelial masses were pre ̄
pared. Morphology and size of the peritheciaꎬ asco ̄
sporesꎬ chlamydospores and endoconidia were ob ̄
served and measured under the microscope (30 for
each) .
1.4 Molecular identification
DNA samples of the pathogen were extracted
from isolates ZY ̄4 and ZY ̄5 using DNA extract rea ̄
gent chelex ̄100ꎬ and the ITS regions of rDNA were
sequenced using the procedures reported by Bakeret
et al.[9]ꎬ the ITS and 5.8S rDNA genes were ampli ̄
fied using primers ITS1F ( 5 ’  ̄CTTGGTCATT ̄
TAGAGGAAGTAA ̄3’) and ITS4 ( 5 ’  ̄TCCTC ̄
CGCTTATT ̄GATATGC ̄3’) . A system of 30 μL
volume that consisted of 11 μL ddH2Oꎬ 15 μL 2×
Taq PCR StarMixꎬ 10 μM of each primer and 10 ng
of genomic DNA was used for PCR amplification.
The PCR amplification was performed using the fol ̄
lowing thermal profile: 94℃ for 95 sꎬ 35 cycles of
94℃ for 35 sꎬ 52℃ for 1 minꎬ 72℃ for 1 min and a
final extension cycle of 72℃ for 15 min. The se ̄
quences of ITS ̄5.8S rDNA were blasted in the Gen ̄
Bank database (http: / / www.ncbi.nlm.nih.gov / gen ̄
bank / ) . The phylogenetic analysis was performed u ̄
sing MEGA5.
2 Results and Discussion
In pathogenicity testꎬ all the inoculated jujubes
showed typical symptoms 10 ̄12 days post inocula ̄
tionꎬ while control jujubes remained symptomless.
The typical symptoms were that the fruit was rotten
and sunken partly (Fig.1 ̄C) and the interior tissue
became reddish brown (Fig.1 ̄D) . The pathogen that
caused typical jujube rotten and sunken symptoms as
in the field was re ̄isolated from the inoculated disea ̄
sed jujube tissuesꎬ and no organism was isolated from
the control. This result was in line with the theory
of Koch′s postulates.
The two isolates had the same culture characteri ̄
stics. Greyish ̄green myceliaꎬ white spores and black
perithecia could be seen on the malt extract agar me ̄
dium. The diameter of the mycelial growth reached
45. 5 ̄46. 7 mm within 10 days and at 14 days it
reached 63.4 ̄66.7 mm. Under the microscopeꎬ the
perithecium was a globose shape (161.9 ̄217.8 μm ×
143.3 ̄248.7 μm) with a long black neck (580.9 ̄
699.9 μm)ꎬ ascospores were globose or had a typi ̄
cal " hat" morphology with a dimension of 4.0 ̄6.9
μm × 5.5 ̄8.6 μmꎬ endoconidia were cylindrical and
753
植物病理学报 45卷
clavate (3.2 ̄15.9 μm × 7.5 ̄29.4 μm)ꎬ chlamydo ̄
spores growing on media were smoothꎬ black
brownꎬ ovoid or obpyriform (9.9 ̄12.4 μm × 15.7 ̄
18.7 μm) (Fig.2) . All morphological characteristics
of the isolates were consistent with the descriptions
of Ceratocystis fimbriata [10] .
Fig. 1 The symptoms of jujube fruit rot caused by Ceratocystis fimbriata
Aꎬ B: Natural infected jujube fruitsꎻ Cꎬ D: The inoculated jujubesꎬ the left jujubes in the figure are control groups.
Fig. 2 Morphological characteristics of Ceratocystis fimbriata
A: Conidiaꎻ B: Ascosporesꎻ C: Peritheciumꎻ D: Chlamydospore.
853
4期 HAN Yong ̄huaꎬ et al:Occurrence of Fruit Rot Caused by Ceratocystis fimbriata in Ziziphus jujube in China
Blast analysis indicated that the ITS rDNA se ̄
quences of isolates ZY ̄4ꎬ ZY ̄5 (GenBank acces ̄
sions KM272195ꎬ KM272196) showed 100% and
99% homologous to those of the isolates of C. fim ̄
briata from diseased Eriobotrya japonica in China
and Colocasia esculenta in Hawaiiꎬ USA (GenBank
accessions No. KF963102 and No. AY526307)ꎬ re ̄
spectively. In the phylogenetic analysisꎬ the two iso ̄
lates were grouped with other members of C. fimbri ̄
ata in a monophyletic clade with 100% bootstrap
value (Fig.3) .
Fig. 3 Phylogenetic tree of rDNA ̄ITS sequences of the pathogenic isolates and the related species
Based on the symptomsꎬ pathogenicity to the
host plantsꎬ morphological characteristics and the
molecular dataꎬ the pathogen that caused jujube fruit
rot was identified as C. fimbriata.
C. fimbriata has been previously reported cau ̄
sing diseases on Syngonium cultivars in Australia in
1988[11]ꎬ Punica granatum in China in 2003[12]ꎬ C.
esculenta in Brazil in 2005[8]ꎬ and there are other
host plants in other placeꎬ but to our knowledgeꎬ
this is the first report of C. fimbriatia causing jujube
fruit rot in China.
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植物病理学报 45卷
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