全 文 ：浙江大学学报(农业与生命科学版)　35(3):237～ 242 , 2009
Journal of Zhejiang University(Agric.& Life Sci.)
Article ID:1008-9209(2009)03-0237-06 DOI:10.3785/j.issn.1008-9209.2009.03.001
　　Received date:2008-04-22
Foundation i tem:Suppo rted by the National Natural S cience Foundation of China(No.30571208).
Biography:SUN H u , m ale , born in 1980 in Zhumadian City , Henan Province , Ph.D., engaged in Plant Pathology.
Corresponding author:ZHANG Jing-ze , male , ass ociated profess or , engaged in Plan t Pathology.T el:0571-86971710;E-mai l:
jzzhang@zju.edu.cn.
Identification of causal agent of blossom blight of loquat in Zhejiang
Province , China
S UN Hu , ZHANG Li-li , ZHANG Jing-ze , HU Dong-wei(I nstitute o f Biotechnology , College o f Agriculture &
Biotechnology , Zhe jiang University , Hangz hou 310029 , China)
Abstract:The blo ssom blight of loquat occurr ed severely in 2007 in loquat o rchards in Deqing County ,
Jiaxing District , Zhejiang P rovince of China , causing the flow er spike wilt and death o f loquat.To
identify the causal agent , six Botry tis sp.isolates we re obtained from the diseased f low er spikes o f
loqua t.Based on mo rpho lo gical and cultura l char acte rs , the iso lates w ere identified as Botrytis cinerea
Per s , and its teleomorph is Botryotinia f uckeliana Whetze l.By analy sis of nucleo tide sequences o f the
ITS regions of ribo somal DNA(ITS1 and ITS2)and the 5.8S RNA gene , the sequences o f iso la te s we re
100% identical to tha t of B.f uckeliana , further confirming tha t the pathogen was B .cinerea.
Pathogenicity test show ed that B.cinerea iso la tes wer e able to infect the flower spikes of loquat , causing
the symptoms to be simila r to natural hosts.
Key words:blossom blight of loquat;Botry tis cinerea;identification
CLC number:S432.44;S667.3;Q93-331　　　Document code:A
孙 虎 ,张丽丽 , 张敬泽 ,胡东维(浙江大学 农业与生物技术学院 生物技术研究所 , 浙江 杭州 310029)
枇杷花疫病病原菌鉴定(英文).浙江大学学报(农业与生命科学版), 2009 , 35(3):237-242
摘　要:枇杷花疫病于 2007 年在浙江省德清县严重发生 ,造成枇杷花穗枯萎和死亡.为了鉴定其病原
菌 ,从受害枇杷花穗上分离到 6 个病原菌菌株.依据病原菌的形态学和培养特征 , 该病原菌被鉴定为灰
葡萄孢菌(Botry tis cinerea), 其有性态为富氏葡萄孢盘菌(Botryotinia f uckeliana Whetzel.).通过核糖
体 DNA ITS 序列分析 , 分离菌株序列与富氏葡萄孢盘菌序列 100%同源 , 进一步证明侵染枇杷花穗的
病原菌是灰葡萄孢菌(Botry tis cinerea).致病性试验结果表明 , B .cinerea 能侵染枇杷花穗 , 可引起与自
然寄主上相似的病害症状.
关　键　词:枇杷花疫病;灰葡萄孢菌;鉴定
　　The loquat (Eriobotrya japonica)is a f ruit
tree in the subfamily M aloideae of the family
Rosaceae , indigenous to southeastern China.In
that region , Deqing County of Zhejiang Province ,
is the most important area of Chinese cultivation.
It is a kind of fresh frui t , with a high sugar , acid
and pectin content.Similarly , its f ruit , skin ,
seeds , and leaves of the tree all have medicinal
浙江大学学报(农业与生命科学版)
uses in t radi tional Chinese medicinal herbs ,
especially being used fo r treating cough , thirst ,
constipation , asthma , nosebleeds and
pharyngolaryngitis.Due to the economic profit
and the huge amount of demands with rapidly
economic development , many loquat orchards
were established in 1997 and both the cv.
Dahongpao and cv.Shiromogi were regarded as
the majority of cultivated cultivars.However , the
blossom blight of loquat w as hardly observed
until the early January , 2007.The disease
occurrence may be close to the warm winter and
excessive rainfall during exceptional w eather
condition.
　　Due to the economic importance of blossom
blight of loquat , the causal o rganism w as
investigated.In this study , we described this
disease and , documented its morphological
characteristics , and compared it to o ther species
of Botrytis.At the same time , the analysis of
rDNA-ITS(internal t ranscribed spacer)sequences
of the isolates , biological characters and
pathogenesis test w ere undertaken as well.
1　Materials and methods
1.1 　Observation of symptoms and collection of
Botrytis isolates
　　During the early January to M arch 2007 , the
symptoms of blossom blight disease w ere
observed in loquat orchards in Deqing County ,
Zhejiang Province , China.The diseased flower
spikes with typical symptoms were collected.
Isolates w ere obtained from fresh diseased flower
spikes.Conidia were removed directly from
sporulating diseased areas on petals , or small
pieces of diseased tissue taken from the margin of
a lesion on petals , were placed on PDA (potato-
dex trose agar) containing rifampicin.Isolates
were t ransferred to PDA , and conidia f rom these
PDA culture were stored in 20%glycerol , in 1.5
mL cryotube , at -70 ℃.
1.2　Morphological and cultural studies
　　Fo r observing fungal morphology on media ,
small pieces of f rozen conidial suspensions were
removed from stock-culture cryotubes without
thawing , and transferred to Pet ri dishes
containing PDA.Single conidial i solates were
prepared and incubated on PDA.Colonies and
spo res were observed under different temperatures
with 12 h of alternate darkness and fluorescent.
Measurement of spores size was taken from slide
mounts in water.
1.3　DNA extraction
　　Fungal isolates we re cul tured on PDA at
25 ℃ under coo l f luo rescent illuminat ion.
Afte r four day s , five mycelial disks w ere excised
f rom the margins of colonies and inoculated into
100 mL of liquid growth media(PDB)in 250 mL
flasks , shaken at 200 r min-1 at 25 ℃ for 5 d.
Mycelia were harvested by filt ration , freeze-
dryed , g round to a final pow der in liquid
nit rogen , then sto red at -70 ℃.About 50 mg of
mycelial pow der was removed into a sterile 1.5
mL tube , was rehydrated in 600μL of 2×CTAB
buffer(100 mmol L-1 Tris , pH 8.0 , 1.4 mol
L-1 NaCl , 30 mmol L-1 EDTA , 2%
hexadecyltri-methy lammonium bromide)and was
incubated in a water bath at 65 ℃ for 30-60 min.
Follow ing a phenol/chloroform extraction , the
genomic DNA was precipitated by isopropanol in
the presence of sodium acetate.Genomic DNA
was visualized in 1% agro se gels af ter ethidium
bromide staining.
1.4　Amplification of ITS regions
　　The ITS regions of rDNA were amplified
using the universal primers ITS6 (5-
GAAGGTGAAGTCGTAACAAGG-3) and
ITS4 (5-TCCTCCGCTTATTGATATGC -
3)[ 1] .PCR amplifications were performed in a
total volume of 50 mL containing 40 mmol L-1
Tris-HCl (pH 8.4), 100 mol L-1 KCl , 3 mmol
L
-1
MgCl2 , 400 mol L-1 of each dN TP , 1μmol
L
-1
of each primer , and 0.5 U Taq.PCR
238 第 35卷　
孙虎 ,等:枇杷花疫病病原菌鉴定(英文)
amplification w as carried out on a DNA thermal
cycler (PTC-150 MiniCycler , MJ RESEARCH
Corp.).Following an initial denaturation at 95 ℃
for 4 min , the DNA templates were amplified for
35 cycles.Each cycle consisted of a denaturation
step at 95 ℃ for 1 min , an annealing step at 55
℃for 1 min , and an ex tension step at 72 ℃ for
1.5 min.At the end , a final ex tension step(72
℃for 10 min)was included.Aliquo ts of 4 L of
the amplification products were electrophoresed
on 1% agarose gels , and the PCR products w ere
subsequent ly stained with ethidium bromide.
Successful PCR amplification products resulted in
a sing le DNA band (corresponding to about 530
bp).The PCR products were purified using a
Biolight PCR Purification Kit (Shanghai Biolight
Technology Co., Ltd) according to the
manufactures inst ructions.
1.5　Sequencing and analysis of rDNA-ITS regions
　　The purified PCR products w ere
submit ted to Hangzhou Genomics Insti tute for
sequencing in both directions.Sequence files
were assembled and edited , and consensus
sequences w ere const ructed using DNAMAN
4.0 (Lynnon bioSof t).The ITS sequences
were submit ted to the GenBank database.A
Blast search o f the GenBank database w as
carried out for comparing thei r identity w ith
the sequences of other isolates.
1.6　Pathogenicity test
　 　 Due to the difficulty of performing
pathogenicity tests in the field , healthy flow er
spikes w ere pick up and inoculated with conidial
suspension(1 ×106 conidia mL-1)from 8 to 10-
day-old cultures in the lab.The inoculated flow er
spikes were kept in glass containers covered with
plastic membrane for tw o days and then in natural
light under outdoor temperature condition (7-15
℃, the middle January , 2007).The treatments
were replicated three times.Similarly diseased
f lower spikes were used for the fungal reisolation.
2　Results
2.1　Symptoms
　　In the loquat orchards , the E.japonica cv.
Shiromogi origining f rom Japan is mostly
susceptible to the blossom blight pathogen , while
the E.japonica cv.Dahongpao is resistant.On
the susceptible cv.Shiromogi , the symptoms
firstly appeared on petals and w ere characterized
by brow n water-soaked lesions. After an
extended period of high relative humidity , the
entire f lower spike wilted and perished after the
sepals and pedicels were infected (Fig.1).
Diseased parts w ere covered with g rey patches of
Fig.1　Botrytis sp.infected(left)and healthy(right)loquat blossom
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浙江大学学报(农业与生命科学版)
mycelium and abundant conidia of fungus.The
disease incidence of f lower spikes w as up to
80.2%.Whereas , in contrast , on the resistant
cv.Dahongpao , the fungus hardly progressed
into the sepals and pedicels and only a few lesions
were observed on petals at that time.
　　Six isolates (pipa-dq01 , pipa-dq02 , pipa-
dq03 , pipa-dq04 , pipa-dq05 , pipa-dq06) from
diseased tissues were obtained and subsequent ly
used for the species identification.
2.2　Cultural characters
　　Colonies or aerial mycelium were produced
under dif ferent temperature.They were compact ,
cottony , white , dirty w hite , grayish w hite , or
hyaline at first but soon becoming light g ray ,
dark gray to dark brow n.The growth speed test
of colonies showed that mycelium grow th ranged
from 5 to 30 ℃ and thei r optimal temperature
was at about 20 ℃ (Fig.2).The mycelium
grow th speed increased from 5 to 20 ℃ with
going-up temperature , reduced from 25 to 30 ℃
and ceased at 35 ℃.But there w as not significant
difference f rom 5 to 15 ℃ and 25 to 30 ℃.
Fig.2　Effect of temperature on growth speed of
colonies
　　Conidia usually produced over the surface of
the medium and sporulation began from the old
part of the colony.Conidia and conidiophores
produced largely f rom 5 to 20 ℃, and optimum
temperature for spore fo rmation w as found to be
about 15 ℃and ceased over 20 ℃.Conidiopho re
length w as f requent ly 1429-3207 μm , mostly
12.5-24.5 μm wide.The ultimate conidiophore
branches inflated into a swollen conidiogenous
cell , the ampulla , that bearing simultaneous
conidia on edicles (Fig.3A).The ampulla was
clavate , spherical , subspherical , o r somehow
lobate.Conidia w ere solitary and attached to the
ampulla.They were hyaline or pale brown , but
in mass they seemed ashen-gray becoming darker
with age.Conidia were ovate , globose to
subglobose.They were smooth , often wi th a
slight ly protuberant hilum and usually unicellular
(Fig.3C).Conidial dimension fell in the range of
(6.5-13.75)μm ×(6.5-10.0)μm.Sclerotia
produced largely under higher temperature (over
20 ℃).They were black , o r w hite at first ,
becoming black with the age and w ere variable in
shape and size (Fig.3B). They were
planoconvex , hemispherical , rounded , roughly
circular in shape , with the surface smooth.Their
size w as(1.0-4.5)mm×(1.8-3.0)mm.
　　According to the characters of morphology ,
the isolates w ere identified as Botry tis cinerea
Pers. and its teleomorph is Botryot inia
fuckeliana Whetzel[ 2-3] .
2.3　Sequencing and analysis of rDNA-ITS regions
　　Nucleotide sequences of the ITS regions of
ribosomal(r)DNA (ITS1 and ITS2)and the
5.8S RNA gene w ere amplified with universal
primers , generateing approximately 600 bp PCR
fragment.Sequences were determined in both
directions , and the sequence alignment of
different isolates using Clustal-w show ed that
they were identical. The sequences were
submit ted to the GenBank database (accession
No.:EU128648 , EU128649 and EU128650).A
Blast search of the GenBank nucleotide database
show ed that the ITS1 , ITS2 and 5.8S rDNA of
isolates were 100% identical to B.f uckeliana
Whetzel ( accession No.: Z73765 and
AJ716294)[ 4-5] , further confirming that the
pathogen w as B.cinerea.
240 第 35卷　
孙虎 ,等:枇杷花疫病病原菌鉴定(英文)
A.A conidiophore.B.S clerot ia formation on the surface of PDA.C.Conidia.
Fig.3　Morphological and cultural characters of Botrytis cinerea
2.4　Pathogenicity test
　　Inoculation tests we re conducted w ith six
isolates.The results of pathogenici ty tests
show ed that all six iso lates were able to infect
loquat blossom af ter inoculation 3-4 day s ,
caused the similar symptoms identical to that
in f ields , and no disease w as found in the
control f low er spikes o f loquat.By 7 d ,
sporulation w as observed on al l inoculated
flower spikes.The fungus w as re-iso lated
f rom the infected parts and its colonies and
mo rpholog y w as consistent w ith previous
isolates.The results of pathogenici ty tests
show ed that the fungus w as re-i solated f rom
the infected parts and its co lonies and
mo rpholog y w as consistent w ith previous
isolates.The results of pathogenici ty tests
confi rmed that the pathogens w as B.cinerea.
3　Discussion
　　Botry tis cinerea Pers.is a ubiquitous fungal
pathogen that causes g ray rot on a large number
of economically important agricultural and
horticultural crops.It opportunistically infects
w ounds or senescing tissue and also invades
young tissues , causing necrosis[ 6] . The B.
cinerea fungus is present in loquat o rchards as
part of the envi ronmental microflora and infection
of loquat often occurs at bloom time
[ 7] .Early
f lower spikes may be the first to become infected
because they come into bloom first.Once the
fungus becomes established on early flow er
spikes , the infected tissue can serve as a source of
spo res to infect later flower spikes.During the
f lorescence of loquat , local temperature condition
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浙江大学学报(农业与生命科学版)
is usually suitable for spore fo rmation of pathogen
and wet weather promote spore production and
germination.In January 2007 , continuous rainfall
was more than 40% that of normal years , leading
to severe occurrence of the blossom blight of
loquat.
　　This is the first repo rt of B.cinerea causing
the blossom blight on the loquat in Zhejiang
Province , China.This disease w as reco rd in
Japan as w ell[ 7] .In China , although the rot ten
disease of loquat flowers w as reported in
Chongqing City (south-west China), i t w as
caused by Pestalot ia eriobof olia and sporodochia ,
being different from our report[ 8] .
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