全 文 ：广 西 植 物 Guihaia 28(4)：531— 533 2008年 7月
First report Emilia sonchifolia wilt caused
by Ralstonia solanacearum
LIAo Yong-MeP，NONG Yan-Xian1，ZHOU Zhi-Quan2
(1．Colege ofAgriculture，Gu~ngxi University，Nanning 530004，China；2．Institute of
Biology，Guangxi Academy of Sciences，Nanning 530003，China)
Abstract：Emilia sonchifolia bacterial wilt was severely occurred in the field in Guangxi，China．The pathogenic bac—
terium was confirmed by Koch postulate．Then Polymerase chain reaction(PCR)amplification was conducted using
1 6s rDNA universal primers to identify the pathogenic bacterium．PCR products sequencing results showed the path—
ogenic bacterium had 99 homology to Ralstonia solanacearum．This is the first report of E sonchifolia wilt caused
by R．solanacearum．
Key words：Emilia sonchifolia wilt；Ralstonia solanacearum
CLC Number：s432．4 Docum ent Code：A Article ID：1000—3142(2OO8)O4—0531—03
Emilia sonchifolia is an annual or perennial corn—
positae plant，widely distributed in China(Zhang eta1．，
2005)．E．sonchifolia is a traditional medicinal herb
used to cure pneumonia，infectious hepatitis，diarrhea，
acute conjunctivitis，etc(Li et a1．，1993)．It is reported
that E sonchifolia contained flavonoids which showed
stronger antibacterial activity against Staphylococcus
aureus and weaker antibacterial activitiy against Esche—
richia coli and Bacillus subtilis(Li甜口Z．，2007)．In re—
cent years，E sonchifolia is artificially planted widely
in Guangxi of China．Surveys on E sonchifolia disea—
ses were conducted during 2004—2006(Nong et a1．，
2006)and a bacteria1 wiit disease was discovered which
was severely occurred in the field． The disease inci—
dence were 25％ to 50％，causing significant economi—
cally loss．It is the first report of E sonchi folia bacte—
rial wilt occurred in the field and the pathogen identifi—
cation was conducted in this paper．
1 Materials and methods
1．1 Bacteria isolation and pathogenicity testing
Diseased stems of E．sonchifolia were collected
from the field and surface sterilizations were conducted
using 75％ alcohol for 1 rain．Then the sterilized stems
were cut into pieces about 5 cm in length． Put the
stem pieces into sterile distilled water for 10 min then
stirred for 1 min to obtain the bacteria suspension．
Added 1 mL bacteria suspension into 10 mL sterile dis—
tilled water，diluted it into three concentrations． Re—
moved 1 mL suspension of each concentration into cul—
ture dish，then poured into the 45 ℃ nutrient agar
(NA，beef extract 3 g，peptone 10 g，glucose 2O g，agar
16 g，add water to 1 000 mL)medium and incubated for
3 d at 30℃ ．Di fferent single colonies were selected for
streaking cultivation． After 3 times of single colony
purification，the purified bacteria were kept in sterile
distilled water under the room temperature
．
Cultivated the purified bacteria for 48 h，then add—
ed sterile distilled water to prepare 5×10。cfu／mL bac-
teria suspension． Inoculated two month old seedlings
of E sonchifolia by injecting the stem base or watering
the roots．Each seedling was injected 1 mL bacteria
suspension into stem base or watered 10 mL bacterl。a
suspension into its rhizosphere soil． Sterile distilled
Received date：2008—02—21 Accepted date：2008—04—28
Foundation item ：Supported by Natural Science Foundation of Guang)(i(O575OO9
，0447012)
Bj0鲫phy；L o Y0ng- (I963一)，femaIe，B0m in Xiangzhou County 0f Guangx／，Ph．D，Asociate profesor，mainly study on Phytopath-
ologY’(E-mar1)liaoyrn@ gxu．edu．cn。
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532 广 西 植 物 28卷
water was inoculated as the contro1． Re-isolated and
re-inoculated were conducted in accordance with Koch
postulate(Fang，1998)．Gram stain was conducted ac—
cording to the reference(Dong et a1．，2001)．
I．2 Prqp ng bae~xia DI template
Single colony directly for PCR amplification meth—
od(Dai et a1．，2000)was cited in this paper．Bacteria
cells were cultured on NA plate for overnight at 28℃．
Selected a single colony and removed the cells into a
1．5 rTlL microfuge tube containing 10 L 1 SDS(so—
dium dodecyl sulfate)solution，then swirled it thor—
oughly．Added 300 L TE(1O mM s—C1，pH7．5，1
mM E])1A)buffer to obtain DNA stock solution．Re—
moved 10 L DNA stock solution into a new 1．5 mL
microfuge tube，which contained 9 L sterile distilled
water to dilute the solution．The diluted solution was
used as PCR template directly．
1．3 Polymerase chain reaction(PCR)amplifcation
16s ribosomal DNA (rDNA)sequence analysis
methods were widely used on bacteria idenfificafion
(Macrae，2000；Jiao et a1．，2001)．In this paper，16s
rDNA universal primers were used for PCR(P1：5
AGA G1vr TGA TCc TGG CTC AG 3 ；P2：5 AAG
GAG GTG ATc cAG CC 3 )．The PCR param eters
were as follows：initial denaturation at 95℃ for 4 mA n，
35 cycles of 95℃ 30 s，56℃ 40 s，and 72℃ 2 man．
Final extension was 72 ℃ for 10 ma n．PCR product
was sequenced by TaKaRa biotechnology(Dalian)Co．，
Ltd using PCR primers． Se quence alignments were
conducted bv BLAST online software．
2 Results and analysis
2．1 Symptom of E．sonchifolia bacterial wilt
In the field，diseased plant showed green wilt
(Fig．1)，and then leaves became yelow wilt from bot—
tom to above． Finally plant died and stem became
black．In vertical section，black stem vascular tissues
were showed，and in cross section，whitish bacterial ex—
udates oozed．Artificialy injecting bacteria suspension
into stem base for 3 d，the leaves begin to appear green
wi lting，sometimes appear one-sided wilting．After in—
oculated for 5 d．the plant died and also appeared whit—
ish bacterial ooze in the cross section． Re-isolated bac—
teria from diseased plant then re-inoculated health
plant，the same symptom appeared．
Fig．1 Symptom of E sonchi
．folia bacterial wilt in the field
1．5kb
Fig．2 PCR product electrophoresis pattern
M ；lkb DNA ladder；S：Sample PCR product．
2．2 Pathogenic bacteria
The colony of bacteria was milky white on NA
plate，roundness or sub-round in shape，moist，smooth
on surface． The colony turned brown after 5-——8 d
grown on medium and the medium nearby becam e deep
brown ．Gram stain was negative．
2．3 PCR amplification and sequencing
PCR product was about 1．5 kb(Fig．2)．Two ter—
mA nals sequencing reactions were conducted using PCR
primers respectively．6 1 7bp of forward sequencing re—
action showed 99 homology to Ralstonia so—
Zanacearum strain TW56(DQ924957．1)16s ribosomal
RNA gene 5’terminal fragment(from 24nt to 641nt)．
788bp of reverse sequencing reaction showed 99％ ho—
mology to R．solanacearum strain TW56 16s ribosomal
RNA gene 3’ terrninal fragm ent(from 1451nt to
663nt)．According to the sequencing results，bacteria
colony morphological character and negative gram
stain。it can be confirmed that E sonchifolia bacterial
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4期 廖咏梅等：首次报道一点红青枯病由茄青枯茵引起 533
wilt was caused by R．solanacearum．
3 Discussion
R．solanacga~gm，one of the world’s most impor～
tant phytopathogenic bacteria，causes lethal wi lting dis～
eases of over 200 plant species belonging to over 28 bo—
tanical families(Denny，2000)．Common crops affected
by bacterial wi lt include eggplant，capsicum，tomato，
potato，tobacco，sweet potato，banana，ginger，onion，
peanut，mung bean，cashew，papaya，cassava and sesa—
me．E．sonchifolia usually grows wildly in the field
and could be the potential host in original soil． In
Guangxi of China，several important economi cal crops
suffered severely by R．solanacearum wilt，such as to—
mato，tobacco，capsicum ，ginger and so on(Huang，
2002)．Due to E sonchifolia growing wildly in the
field，R．solanacearum could keep alive longer in the
soil by parasitizing in the rhizosphere of E．sonchifolia
after harvesting the crops．In recent years，E sonchi—
folia is artificialy planted widely in the field，occur-
rence of R．solanacearurn wi lt not only causes economi —
cal loss directly but also becomes the potential danger
for the other economi cal plants growi ng nearby．
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首次报道一点红青枯病由茄青枯菌引起
廖咏梅1，农彦贤1，周志权2
(1．广西大学 农学院，南宁 530004；2．广西科学院 生物研究所，南宁 630003)
摘 要：一点红青枯病在广西种植区严重发生。通过柯赫氏法则证实病原菌为细菌。用 16s rDNA通用引物进
行PCR鉴定，测序结果表明，PCR产物与茄青枯菌有99Z的同源性。首次报道一点红青枯病由茄青枯菌引起。
关键词：一点红青枯病；茄青枯菌
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