全 文 ：Isolation and Preliminary Identification of Antibacterial
Active Substances of Antagonistic Actinomycetes
against Peach Crown Gall(Agrobacterium tumefaciens)
JI Jing-lin，MA Huan-pu* ，LIU Zhi-min
Department of Plant Science and Technology，Beijing University of Agriculture，Beijing 102206，China
Abstract ［Objective］The paper was to isolate and preliminarily identify the antibacterial active substances of antagonistic actinomycete strain G19 obtained from
the soil highly affected by peach crown gall (Agrobacterium tumefaciens)． ［Method］The antibacterial substances of antagonistic actinomycete strain G19 were ex-
tracted using protein precipitation method，then isolated and purified using high performance liquid chromatography and medium-pressure preparative chromatogra-
phy． Its molecular weight was determined by MALDI-TOFMS method，and the related functional groups were verified through chemical color reaction． ［Result］
Seven peptide portions were produced from the antibacterial substances of antagonistic actinomycete strain G19 with the molecular weights of 900 － 1 300 Da after
isolation and purification． It could be also inferred that it contained Cys，and carried with H2O and Na
+ ． Color reaction of functional groups verified that the sub-
stance was polypeptide containing glycosyl． ［Conclusion］The result provided basis for the final definition of the structure of antibacterial substances in antagonistic
actinomycete strain G19．
Key words Peach crown gall;Actinomycetes;Antibacterial peptides;China
Received:March 24，2011 Accepted:June 3，2011
Supported by Beijing Natural Science Foundation(5112010)＆ Beijing Munic-
ipal Education Commission Grant (KM200910020001)．
* Corresponding author． E-mail:liuzhimin600@ sina． com
Peach crown gall is a genetic disease caused by Agrobacteri-
um tumefaciens with special function in the way of natural genetic
transformation，which is a soil-borne bacterial disease difficult to
control［1 －2］． Fruit crown gall has common occurrence in China，
resulting in great economic losses． About 13 provinces and cities
in northern region have different levels of incidence，the disease in
grape in the places of Liaoning，Inner Mongolia，Hebei，Shandong
and Beijing is more serious，and the incidence rate is 30%－100%
with yield reduction of 30%，or even the whole garden is de-
stroyed without harvest［3］．
Inducement of peach crown gall is diverse，in addition to soil
texture，temperature and root wounds，continuous cropping is also
an important inducement． If peach trees are planted in the soil
with continuous cropping，most plants will suffer crown gall． Com-
bined with years of practice，LI Ren-fang et al． considered that if
seedlings were cultivated in the seedling cultivation field which
had been used for 1 －2 years，the incidence rate of crown gall ob-
viously increased，especially the incidence rate of crown gall in
peach tree was up to 90%［4］． Through years of production prac-
tice，LIU Jia-bin et al． found that the root system of pervious
peach secreted large amounts of metabolic substance after years of
development such as amygdalin，which produced toxic substances
after the decomposition by soil microorganisms，and caused toxic
effect on the root system of newly planted trees，thus resulting in
the occurrence of continuous cropping disease［5］． It was later
proved that this toxic substance was hydrocyanic acid (HCN) ，
which had toxicity on the growth of peach trees［6 －7］．
At present，the most effective control measure against crown
gall is biological control． In 1980，Kerr et al． found that K84
strain had good control efficacy on the crown gall of stone fruit
trees such as peach，as well as the flowers with relative re-
sources［8］． In 1998，WANG Hui-min et al． found that K84 could
effectively control crown gall of sweet cherry［9］． In 2004，WANG
Guan-lin et al． found that WJK84-1 strain and P-2001 bacteriocins
produced by the strain showed significant inhibition effect on C58
pathogen of crown gall tumor［10］． LI Jin-yun et al． found that ag-
rocin produced by Agrobacterium vitis E26 had significant inhibi-
tion effect against crown gall of grape，which also showed certain
inhibition effect against Ralstonia solanacearum，Xanthomonas
Campestris，Xanthomonas oryzae，Xanthomonas campestris and Er-
winia carotovora［11］． Hammami et al． extracted antibacterial pep-
tide from Bacillus subtilis 14B using (NH4)2SO4 precipitation
method，and found the peptide could inhibit crown gall［12］． How-
ever，such biocontrol agents could survive in the soil with high
toxic substances (HCN)was unknown． The author screened the
biocontrol strain of actinomycetes G19 with cyanide-resistant effect
from the replanted soil of peach with high incidence of crown gall;
the fermentation broth of the strain showed good inhibition effect
against Agrobacterium tumefaciens，which had stable quality and
identified to have 97% similarity with Streptomyces by 16S
rRNA［13］． Thus，the isolation and identification of antibacterial
substances of the actinomycetes strain had great significance．
1 Materials and Methods
1． 1 Materials The pathogen used in the test was Agrobacteri-
um tumefaciens，which was purchased from China Agricultural
University． Biocontrol strain used in the test was cyanide-resistant
actinomycetes G19 isolated from the soil with high incidence of
crown gall．
Plant Diseases and Pests 2011，2(3) :1 －5
1． 2 Crude extraction of antibacterial active substances of
biocontrol actinomycetes Ammonium sulfate precipitation
method was adopted． The actinomycetes strain was placed in
Gaoshi liquid medium，shook at 28 ℃，180 r /min for 8 d，and
the fermentation broth was centrifuged at 10 000 r /min for 15 min．
The supernatant was filtered with sterile membrane， added
(NH4)2SO4 to 60% saturation，placed stilly for 2 h，and centri-
fuged at 10 000 r /min for 10 min． The precipitation was put into
the dialysis bag with cutoff molecular weight of 8 000 －14 000 Da
for overnight dialysis in 0． 1 mol /L phosphate buffer (pH 7． 0) ，
and centrifuged at 10 000 r /min for 10 min under low tempera-
ture． The insoluble substances were abandoned，and the superna-
tant after freezing and drying was the crude extract of antibacterial
peptides．
1． 3 Determination of antibacterial activity The plate coated
with A． tumefaciens was punched around using the sterilized
puncher，and injected with 100 μl original fermentation broth of
actinomycetes，100 μl supernatant of actinomycetes，100 μl su-
pernatant after precipitation of (NH4)2SO4，100 μl crude extracts
and 100 μl sterile water． Those injected with phosphate buffer was
set as control． After incubation at constant temperature of 28 ℃
for 12 h，the diameter of inhibition zone was observed．
1． 4 Purification of antibacterial active substances of biocon-
trol actinomycetes The crude extracts of antibacterial active
substances were isolated and purified by high performance liquid
chromatography and preparative chromatography． The samples
were filtered through 0． 22 μm microporous membrane，and detec-
ted by ultraviolet rays at 268 nm with column temperature of 25
℃，the mobile phase was acetonitrile∶ water = 3∶ 7 (V ∶ V)． The
column of high performance liquid chromatography was zorboxX-
DB-C18 (4． 6 mm × 150 mm，5 μm) ，the flow rate was 1． 0
ml /min，and the injection volume was 10 μl． The column of pre-
parative chromatography was zorboxXDB-C18 (4． 6 mm × 250
mm，20 μm) ，the flow rate was 10 ml /min，and the injection vol-
ume was 200 μl． The freeze-dried powder was then collected for
later use．
1． 5 Identification of antibacterial active substances of bio-
control actinomycetes
1． 5． 1 Determination of molecular weight of antibacterial active
substances of biocontrol actinomycetes． A small amount of sample
was taken and dissolved with small amount of matrix(acetonitrile∶
water = 3∶ 7 (V ∶ V) )． 0． 5 μl water solution was absorbed and
transferred into MALDI (matrix-assisted laser desorption ionisati-
on)target board，and carried out MS /MS detection after air dry．
When the molecular weight of the sample was obtained，blast com-
parison was carried out in CNBI database．
1． 5． 2 Determination of relevant functional groups of antibacteri-
al active substances of biocontrol actinomycetes．
1． 5． 2． 1 Determination of characteristic of amino acids and pro-
tein． 0． 1 g sample powder was weighed，dissolved with water，
and prepared into the test samples with mass fraction of 1% ． 1 ml
test sample was taken in the test tube，added with 2 － 3 drops of
1% ninhydrin，and placed in boiling water for 10 － 15 min． The
color changes were observed． 0． 1% bovine serum albumin and
sterile water was set as control．
1． 5． 2． 2 Determination of sugar characteristic． The test samples
were prepared according to the method described in 1． 5． 2． 1 ． 1
ml test sample was added with 2 drops of Molish agent (2． 5 g
a-naphthol in 50 ml ethanol，diluted to 100 ml with ethanol)． Af-
ter mixing，the tube was tilted，slowly added with 1 ml H2SO4
along the tube wall． After stratification，the tube was placed
stilly，and the color changes were observed． 0． 1% glucose and
sterile water was as control．
1． 5． 2． 3 Determination of phenol characteristic． The test sam-
ples were prepared according to the method described in
1．5．2．1 ． 0． 5 ml test sample was added with 2 －3 drops of 1%
FeCl3 solution，and the color changes were observed． 0． 1% sali-
cylic acid and sterile water was set as control．
Note:A represents high performance liquid chromatogram at 268 nm with flow speed of 1． 0 ml /min;B represents medium pressure preparative chromato-
gram when flow speed is 10 ml /min and the other conditions are the same．
Fig． 1 Chromatogram of antibacterial active substances of actinomycetes strain G19
2 Results and Analysis
2． 1 Extraction，purification and activity determination of
antibacterial active substances When the crude extracts of an-
tibacterial active substances were carried out high performance liq-
uid chromatography analysis，a peak appeared as the retention
time was 1． 244 min，and two small peaks appeared as the reten-
tion times were 1． 625 and 2． 069 min (Fig． 1-A)． In preparative
2 Plant Diseases and Pests 2011
chromatography，the peak appeared at 1． 244 min was a single
peak(Fig． 1-B)． The activity determination results of antibacterial
active substances showed that original fermentation broth of G19
(Fig． 2-A) ，the supernatant of actinomycetes (Fig． 2-B) ，crude
extract (Fig． 2-C) and the supernatant after precipitation of
(NH4)2SO4(Fig． 2-E)showed certain inhibition effect against
A． tumefaciens，while PBS buffer (Fig． 2-D)and sterile water
(Fig． 2-F)had no inhibitory effect．
Note:A is original fermentation broth of G19;B is supernatant;C is
crude extract;D is PBS buffer;E is supernatant after precipita-
tion of 60% ammonium sulfate;F is sterile water．
Fig． 2 Antibacterial activity determination of actinomycetes
strain G19
2． 2 Identification of antibacterial active substances of bio-
control actinomycetes
2． 2． 1 Determination of molecular weight of antibacterial active
substances of biocontrol actinomycetes． The samples purified by
preparative chromatography were attacked by MALDI-TOFMS la-
ser． As shown in Fig． 3，a total of seven peptides appeared with
molecular weight range of 900 － 1 300 Da，of which molecular
weights of 1 078． 5，1 096. 5 and 1 118． 5 Da were the main part．
Fig． 3 MALDI-TOFMS chromatogram of actinomycetes strain
G19
The difference between 1 078． 5 Da and 1 096. 5 Da was 18
Da，and the interval should be H2O;the interval between 1 096． 5
Da and 1 118． 5 Da should be Na +，because MS /MS had the
difference of + 1． Similarly，the difference between 1 037 Da and
Note:A． Bovine serum albumin;B． Purified antimicrobial peptide;
C． Sterile water．
Fig． 4 Reaction between antibacterial peptide and ninhydrin
Note:A． Sterile water;B． Purified antimicrobial peptide;C． Glucose．
Fig． 5 Reaction between antibacterial peptide and a-naphhthol
Note:A． Sterile water;B． Purified antimicrobial peptide;C． Sale-
cylic acid．
Fig． 6 Reaction between antimicrobial peptide and FeCl3
1 199 Da was 162 Da，it could be inferred that a glucose residue
was between them;the interval between 1 096 Da and 1 199 Da
was 103 Da，indicating that it might be combined with a Cys．
Blast comparison results showed that its score was low，and no
matching amino acid sequence was found in database．
2． 2． 2 Determine of relevant functional groups of antibacterial
active substances of biocontrol actinomycetes． As shown in Fig． 4，
the reaction between antibacterial active substances and ninhydrin
3Plant Diseases and Pests 2011，2(3) :1 －5
was positive，so it contained proteins，peptides or amino acid struc-
ture． It reaction with a-naphthol was also positive，indicating that
the molecular structure contained a carbohydrate group (Fig． 5)．
But its reaction with 1% FeCl3 was negative，indicating that the mo-
lecular structure did not contain phenolic groups (Fig． 6)．
3 Discussion
The researches on biocontrol agent of Streptomyces and its re-
lated groups are more in biological control of plant diseases．
ZHANG Yong-chun isolated actinomycetes strains A4 and A24
from soil，and found that they had good control effect against Phy-
tophthora parasitica［14］． HU Jun et al． isolated 8 actinomycetes
strains with antagonistic effect against Sclerotinia sclerotiorum from
sunflower soil carrying S． sclerotiorum［15］． CHEN Jie isolated K5
actinomycete from soil with strong antagonistic effect against Fu-
sarium oxysporum，and the strain also had certain inhibition effect
against Magnaporthe grisea［16］． ZHANG Lu et al． found that the
control effect of actinomycetes SG-126 against cucumber wilt dis-
ease was 42． 86%［17］． TU Xuan et al． found that antagonistic acti-
nomycetes of 0108 and 0110 could control Phytophthora capsici via
affecting the root microorganisms of pepper，which also had signif-
icant promotion effect on root growth of pepper［18］． YUAN Ben-
ben et al． screened 8 actinomycetes with strong antagonistic effect
against Valsa sordida［19］． By confrontation test，YAO Min et al．
screened 26 strains of endophytic actinomycetes with antagonistic
effect against Cladosporium fulvum，Alternaria solani and Botrytis
cinerea［20］． Actinomycete strains of Z-6，Z-8，Z-23，Z-42 and 5-1
isolated from soil by ZHAN Li-ran et al． showed certain antagonis-
tic effect against Botryospuaeria berengeriana［21］． LIU Hong-liang
et al． isolated 6 strains of biocontrol actinomycetes with antagonis-
tic effect against Sphacelotheca reiliana［22］． LI Zeng-bo et al． iso-
lated actinomycetes AL-04 from the soil samples of Qinghai-Tibet
Plateau，and the antibacterial substances produced by the strain
had strong inhibition effect against many species of pathogenic fun-
gi and bacteria［23］． WANG Zhuo et al． isolated 3 actinomycetes
strains with obvious antagonistic effect against B． cinerea from 20
soil samples collected from 10 kinds of crops in Jinan City［24］．
The extraction of antibacterial peptide commonly uses ammo-
nium sulfate precipitation method． HAN Dong-mei et al． isolated
and purified Apn5 protein from fermentation supernatant of
B． subtilis BSn5 using 30% saturation of ammonium sulfate pre-
cipitation method and ultrafiltration centrifugation method［25］．
ZHANG Yu-fen et al． extracted antibacterial protein from antago-
nistic bacteria of B． cinerea using 50% saturation of ammonium
sulfate precipitation method［26］． LI Yong-gang et al． extracted an-
tibacterial protein from fermentation broth of Bacillus L1 using dif-
ferent gradients of ammonium sulfate;when the saturation of am-
monium sulfate was 50%－60%，the antibacterial protein showed
inhibition effect against M． grisea;when the saturation was
60%－70%，it showed inhibition effect against Thanatephorus
cucumeris［27］．
However，for the purification of antibacterial protein，there
are many different methods． ZHANG Yu-fen et al． used DEAE-
cellulose ion exchange chromatography［26］;HAN Dong mei et al．
purified using dialysis bag with different cutoff molecular
weights［25］;LV Shu-xia et al． used Sephadex G-50 gel column
chromatography to purify nisin and detected at 280 nm［28］． The
column used in the tests is also reported． QIU Hong-duan et al．
used CM-Sephadex C-50 column to purify the antagonistic sub-
stances against salt-resistant Rhodospirillum，eluted by acetic acid
buffer with flow rate of 0． 3 ml /min，and detected at UV 280
nm［29］． CHEN Bei et al． used DEAE-650 column to purify anti-
fungal protein MAP13，eluted by 2 mmol /L phosphate buffer with
flow rate of 2． 0 ml /min，and detected at UV 280 nm［30］． In the
paper，the author extracted antibacterial protein using ammonium
sulfate precipitation method，the column was zorboxXDB-C18，the
detection was at UV 268 nm，and medium-pressure preparative
chromatography was used for the preparation of purified
substances．
4 Conclusion
Using ammonium sulfate protein precipitation method，the
crude extract of antibacterial substances of antagonistic actinomy-
cetes G19 is obtained． It is isolated and purified using high per-
formance liquid chromatography and medium-pressure preparative
chromatography，and its structure and characteristic have been
confirmed by MALDI-TOFMS and a series of chemical reactions．
The antibacterial substance of antagonistic actinomycetes G19 can
be inferred to be antibacterial peptide containing glycosyl with mo-
lecular weight range of 900 －1 300，it can also be inferred that it
contains Cys and carries H2O and Na
+;the color reaction of its
functional groups verifies that the substance is polypeptides contai-
ning glycosyl．
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桃根癌病菌拮抗放线菌抑菌活性物质的分离及其初步鉴定
季敬霖，马焕普* ，刘志民 ( 北京农学院植物科学技术学院，北京102206)
摘要 ［目的］分离并初步鉴定从桃树高发根癌病土壤中获得的拮抗线菌 G19 菌株的抑菌活性物质。［方法］采用蛋白沉淀法对拮抗放线菌
G19菌株的抑菌活性物质进行粗提，利用高效液相色谱仪、中压制备色谱仪对其进行分离、纯化，应用 MALDI-TOFMS法进行分子量的测定，最后
通过化学显色反应进行相关官能团的验证。［结果］经分离纯化后拮抗放线菌 G19的抑菌物质被抨击出 7个肽段，是分子量范围为 900 ～ 1 300
Da，同时推断其含有一个 Cys并携带 H2O，Na
+;官能团显色反应验证该物质为多肽且含有糖基。［结论］研究结果为拮抗放线菌 G19菌株的抑
菌物质结构的最终确定奠定了基础。
关键词 桃树根癌病;放线菌;抑菌肽
基金项目 北京市自然科学基金(5112010);北京市教委资助项目(KM200910020001)。
作者简介 季敬霖(1986 －)，女，安徽巢湖人，硕士研究生，研究方向:果品品质生理与生态安全。* 通讯作者，教授，硕士生导师，从事果品品质生理与
生态安全研究工作。
收稿日期 2011-03-24 修回日期 2011-06-03
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