全 文 ：Screening of Pathogenic Bacteria X46 (Micromonospora)
of Crabgrass
SUN Yi-min * ，JI Ying-guang，LI Nan
Hebei Chemical ＆ Pharmaceutical College，Shijiazhuang 050026，China
Abstract ［Objective］The screening method and the pathogenic function of pathogenic bacteria X46 (Micromonospora)of crabgrass was studied．
［Method］ Improved Gaoshi No． I medium was adopted to isolate a strain of pathogenic bacteria X46 of crabgrass from the rhizosphere soil of crab-
grass and barnyardgrass，the optimal toxin production condition was determined，and the preliminary assessment of its security was conducted．
［Result］Pathogenic bacteria X46 (Micromonospora)of crabgrass was identified to be Micromonospora． When the liquid volume of 300 ml flask was
30 ml，initial pH was 6． 0 －6． 5，inoculation amount was 10%，fermentation was conducted at 180 r /min，28 ℃ for 120 h，the production amount of
toxin in improved Gaoshi No． I medium was the largest，these were the optimum culture condition． The bacteria had significant promotion effect on
the growth of maize，tomato and soybean，which had slight inhibition effect on wheat growth and significant inhibition effect on cucumber growth．
［Conclusion］X46 is a potential strain that can be developed as the biological herbicide in North China．
Key words Biological herbicide;Micromonospora;Crabgrass;Screening
Received:August 5，2010 Accepted:September 16，2010
Supported by Funded Project in Department of Education，Hebei Prov-
ince:Research on Microorganism Herbicide of Crabgrass Pathogenic
bacteria(No． Z2007443)．
* Corresponding author． E-mail:sun3299@sohu． com
Swart weeds have wide distribution all around the world
with strong reproduction capacity and long growth season，es-
pecially the crabgrass (Digitaria sanguinalis (L．)Scop．)and
barnyardgrass (Echinochloa crusgalli (L．)Beauv．) ，which of-
ten lead to yield reduction of many crops in the drier regions in
northern China［1］． At present，the oversea scholars in the Unit-
ed States and Argentina have made certain progress in biologi-
cal control methods of crabgrass［2］，while the similar reports in
China is few． A strain of pathogen was isolated from the rhizo-
sphere soil of crabgrass and barnyardgrass，the optimal toxin
production condition was determined，and the preliminary as-
sessment of its security was conducted，in order to provide evi-
dence for further research and application of the bacteria．
1 Materials and Methods
1．1 Materials
1． 1． 1 Strains． The samples were collected from the suburb
county of Shijiazhuang City，the suburb county of Handan City
and the suburb county of Tianjin City，which were isolated to
obtain the strains．
1． 1． 2 Medium． Improved Gaoshi No． I medium:NaCl 0．5 g/L，
KH2PO4 0． 5 g /L，K2HPO4 1． 5 g /L，MgSO4 0． 5 g /L，KNO3
1. 0 g /L，FeSO4·7H2O 0．01 g /L，starch 10 g/L，cellulose
(smash and screen through 60 mesh)5 g/L，pH 6．0，agar 20
g /L． Fermentation medium:improved Gaoshi No． I medium
without addition of agar．
1． 1． 3 Model weed seeds． The seeds of crabgrass and barn-
yardgrass were collected in midsummer，and the germination
rate was above 92%．
1． 1． 4 Model plant seeds． The seeds of maize (Zea mays
L．) ，soybean (Glycine max．) ，Wheat (Triticum aestivum
L．) ，cucumber (Cucumis sativus L．)and tomato (Solanum
lycopersicum)were purchased from the seed market，and the
germination rate was greater than 99%．
1． 2 Methods
1． 2． 1 Isolation of strain． According to the strong decomposi-
tion ability of Micromonospora bacteria on the organic matters
such as starch and cellulose，improved Gaoshi No． I medium
was used to screen．
1． 2． 2 Determination of optimum culture conditions． The pa-
rameters of the isolated strains such as the initial pH，culture
temperature，aeration quantity，culture time and the inoculation
amount were determined，and its effect on secretion of herbi-
cidal toxin was monitored．
1． 2． 3 Assessment of herbicidal activity of potential strains．
1． 2． 3． 1 Preparation of toxin． The isolated strains were fully
developed under the optimal culture conditions，the culture liq-
uid was centrifuged and precipitated at 3 000 r /min for 20 min，
the supernatant was taken and placed into 100 ml flask，and
conducted water bath sterilization at (90 ± 1)℃ for 15 min．
1． 2． 3． 2 Inhibition test of spray in vivo． According to the pro-
portion of nutrient soil∶ vermiculite = 1∶ 1，the soil used for test
was prepared． Small plastic cup was loaded with the soil with
the volume 70% of the whole cup，full model seeds with the
same number were implanted into each cup，which were cul-
tured in greenhouse simulated the natural light after being moist
by spraying with watering can． When the seedlings grew to 1
leaf stage，spraying was conducted，the seedlings in control
was treated with clean water with the pH value of 6． 0． Fermen-
tation liquid was diluted according to the proportion of 1 ∶ 40，
spraying volume was 10 ml，each treatment had three repeats．
The seedlings were kept moisture for two days under the
condition of alternative light and dark，28 ℃ and 60% humidity，
Plant Diseases and Pests 2010，1(5) :50 －53
which were moved into the greenhouse in the third day． After
12 d，the aboveground parts were taken to measure their fresh
weight and the roots were taken to measure their length，and
the inhibition rate was calculated［3］．
1． 2． 3． 3 Security assessment． The process of security as-
sessment referred to the method in reference［3 －4］．
1． 2． 4 Strain identification． Based on the morphology charac-
teristics，physiological and biochemical characteristics of the
grown individual and colony of the strain，identification was con-
ducted according to Bergey Identification Manual (Ninth
Edition)［5 －6］．
2 Results and Analysis
2． 1 Strain isolation Improved Gaoshi No． I medium was a-
dopted to isolate the strain，resulting in production of relatively
clear and transparent circle in plates． The starch in transparent
circle were almost decomposed completely，and about 37% of
the cellulose were decomposed and utilized． Finally，the strain
X46 only with substrate mycelia were obtained through screening．
2． 2 Optimum culture conditions
2． 2． 1 Selection of the initial pH value． The improved culture
liquid with the initial pH value of 6． 0 was selected to culture
strain X46，the inhibitory effect of the produced toxin reached
80% (Fig． 1) ，and the raw materials were relatively cheaper，
which was suitable for large-scale development．
Fig． 1 Effect of initial pH value on toxin production of X46
2． 2． 2 Determination of culture temperature and ventilation
quantity． Toxin production effect of X46 strain was better under
the temperature condition of 28 ℃ (Fig． 2) ，the inhibitory effect
reached 75% with significant difference (P ＜0． 05)． As shown
in Fig． 3，when the liquid volume of 250 ml flask was 20 and 30
ml，and the revolution speed of shaking table was 180 r /min，
the inhibitory effect against crabgrass was the strongest，being
78% and 79%，respectively，and the difference was significant
(P ＞0． 05)． Therefore，the liquid volume was also one of the
major factors affecting the production of toxin．
2． 2． 3 Determination of culture time and inoculation amount．
As shown in Fig． 4，the inhibitory effect basically reached the
peak when the culture time was 5 d，and the inhibition rate was
81． 5%;when the culture exceeded 5 d，the inhibitory effect
was not obviously increased，and the difference was not signifi-
cant (P ＞0． 05)． As shown in Fig． 5，when the inoculation a-
mount was 10%，the inhibition rate of toxin produced by X46
strain was 70%，and the difference was significant (P ＜0．05)．
Fig． 2 Effect of culture temperature on toxin production of
X46
Fig． 3 Effect of ventilation quantity on toxin production of
X46
Therefore，10% inoculation amount and fermentation for 5 d
was the best choice．
Fig． 4 Effect of culture time on toxin production of X46
2． 2． 4 Growth curve chart． When the strain was cultured in
liquid medium for long time，the mycelium of strain assumed
micro bulbus． Therefore，two methods including counting and
absorbance determination were adopted to determine the
growth curve，and two curves all entered the plateau phase
near 120 h (Fig． 6) ，the fermentation results were basically
consistent．
2． 3 Activity assessment
2． 3． 1 Inhibitory results of spraying． After spraying，the inhibi-
tion rate of X46 strain against the fresh weight of crabgrass was
determined to be more than 70%，the length of root shortened
for 65%，and the difference was significant through difference
analysis (P ＜0． 05)． No variation appeared after spraying for
48 h，and the leaves would be chlorosis in the third day，but if
15SUN Yi-min et al． Screening of Pathogenic Bacteria X46 (Micromonospora)of Crabgrass
Fig． 5 Effect of inoculation amount on toxin production of
X46
Fig． 6 Determination of growth curve of X46 strain
sterile water was sprayed again，the plants could recover to
grow after two days． Although the chlorosis leaves continued to
turn yellow，the newly grown leaves would had normal growth，
and the growth of crabgrass would not be affected． This indica-
ted that the toxin only had contact toxicity on crabgrass，so it
was not the ideal bioassay method． However，when spraying
volume was increased，the inhibition effect was more apparent．
It could be conjectured that when the spraying volume was in-
creased，the toxin had a surplus to penetrate into rhizome part，
the decomposition effect of the toxin on the fiber would be final-
ly worked，so the normal physiological functions of the roots
would be limited，thus the fresh weight above ground was inhib-
ited． The plants would recover to grow after spraying with ster-
ile water，if toxin liquid was spraying again，the newly grown
leaves would suffer certain inhibition，and the inhibition rate was
about 12%．
2． 3． 2 Safety assessment． The main crops in north upland
field were selected，monocots such as wheat， tomato and
corn，and dicotyledonous plants such as soybean and cucum-
ber were selected to conduct safety determination． The results
showed that the toxin had certain inhibition effect on wheat and
cucumber，the inhibition rate on bud was 7． 5% and 14． 0%，
and the inhibition rate on root was 12% and 31%，the effect on
the germination rate was not obvious;the inhibition effect on
corn，tomato and soybean was very weak，which could be neg-
lected，some test samples even had promotion effect on their
growth，the inhibition rates on root were 9． 4%，－13% and －
34%，respectively，and the inhibition rates on bud length were
－43．75%，－32% and －47． 5%;the promotion effect of the
toxin on the model crops were all greater than 32%．
Therefore，it could be concluded that X46 strain had cer-
tain herbicidal activity，which was basically harmless to the
crops in north upland field in China，while it has slight inhibition
effect on winter wheat［7］．
2．4 Strain identification X46 strain only had substrate my-
celia in agar medium with good growth，the sporophore disinte-
grated by substrate mycelia all had growth of single spores，
which could decompose and utilize starch and cellulose． It was
Gram-positive，cell wall was typeⅡwithout resistance to acid，
the colony assumed walnut-shape，which was suitable to grow
under the temperature of 28 ℃． It belonged to medium temper-
ature bacteria，which liked acidic environment and was aerobic．
According to Bergey Identification Manual (Eighth Edition)［5］，
it belonged to Micromonospora，Actinomycetales．
3 Discussion
When the liquid volume of 300 ml flask was 30 ml，initial
pH was 6． 0 －6． 5，inoculation amount was 10%，fermentation
was at 180 r /min，28 ℃ for 120 h，the production amount of
toxin in improved Gaoshi No． I medium was the largest;when
the revolution speed of shaking table was up to 250 r /min，the
liquid volume was also raised to 60 ml，and the other conditions
were the same，the results were similar with that under the opti-
mum condition． This indicated that the production of toxin was
accompanied with the growth of strain，to some extent，it had
the accumulation of quantity．
There havent been any formal research reports about pro-
duction of pathogenic toxin when the actinomycetes were used
in microorganism herbicide［8］． This is because that the resear-
ches of microorganism herbicide are mainly focused on the de-
velopment of spore preparation in fungal field［9］ so as to form
products as soon as possible in market development;the re-
searches on pathogenic mechanism of actinomycete have not
been fully carried out，which can only consult the researches on
some bacteria and toxin of fungal pathogens，and the research
on toxin have the inherent difficulty［10］． At present，although
scientists have been striving to research and develop，the a-
chievements of this research are very slow．
Inhibition effect of toxin is often only one aspect of the
pathogenic process，the substance is might be the heterocyclic
imide compound［11］，the extraction and verification of active in-
gredients still needs further study． Herbicidal mode of actino-
mycetes is not stick to one pattern，the bacteria can decom-
pose and utilize cellulose from the studied action effect，which
also can be adopted as biocontrol potential bacteria from the
contact effect with weeds root and seedling cotyledons;on the
other hand，they also can produce strong inhibition effect on
root，causing the deformation，atrophy and division halt of cells
in growing points of root and bud，this indicates that the action
of toxin is in full range［12］，which will provide foundation for fur-
ther study．
References
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selection of Nimbya alternantherae SF-193［J］． Chinese Journal of
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screening［J］． Chinese Journal of Pesticide Science，2002，4(4):3
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natural products for control of weeds［J］． Pesticide Science and Ad-
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小单孢菌属 X46 马唐致病菌的筛选
孙祎敏，冀营光，李 楠 (河北化工医药职业技术学院，河北石家庄 050026)
摘要 ［目的］研究小单孢菌属 X46马唐致病菌的筛选方法及致病作用。［方法］采用改良的高氏 I号培养基从马唐、稗草根际土壤中分离出 1
株马唐致病菌 X46，对其最优化产毒素条件进行测定并对其安全性进行评估。［结果］马唐致病菌 X46经鉴定为小单孢菌属，其最优化培养条件
为，在高氏 I号培养液中，300 ml三角瓶装液量 30 ml，初始 pH值 6． 0 ～ 6． 5，采用 10%接种量，180 r /min 28 ℃发酵 120 h即可使毒素产生量最
大。该菌对玉米、番茄和黄豆具有明显的促生长作用，对小麦有轻微的抑制作用，对黄瓜抑制生长作用明显。［结论］X46是一株具有开发为中
国北方用生物除草剂的潜力菌株。
关键词 生物除草剂;小单孢菌属;马唐;筛选
基金项目 河北省教育厅资助项目“马唐致病菌微生物除草剂的研究”(Z2007443)。
作者简介 孙祎敏(1969 － )，女，河北沧州人，硕士，副教授，从事菌种选育研究。
收稿日期 2010-08-05 修回日期
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2010-09-16
(Continued from page 39)
χ2 =(442 －425． 45)
2
425． 45 +
(460 －408． 27)2
408． 27 +
(478 －419． 73)2
419． 73 +
(376 －551． 45)2
551． 45 +
(494 －445． 45)2
445． 09 +
(78 －94． 55)2
94． 55 +
(39 －90． 73)2
90． 73 +
(35 －93． 27)2
93． 27 +
(298 －122． 55)2
122． 55 +
(50 －98． 91)2
98． 91
=0. 644 +6. 554 +8. 089 +55. 821 +5. 375 +2. 897 +
29. 494 +36. 404 +251. 185 +24. 186 =420. 7
or
χ2 = T
2
R1R2
［(
O 21j
Cj
)－
R 21
T］
= 2 750
2
2 250 ×500 ×(
4422
520 +
4602
499 +
4782
513 +
3762
674 +
4942
544 －
2 2502
2 750)
=420. 7
ⅳ)χ2 table is checked，and χ20. 975(4)=11. 143，if actu-
ally χ2 = 420. 7 ＞ χ20. 975(4) ，so H0 is rejected，and HA is ac-
cepted，indicating that the condition of five wheat cultivars in-
fected by head blight is inconsistent．
References
［1］LI CX，WANG ZH，WANG WL． Bio-statistics［M］． Beijing:Scientific
Press，2004． (in Chinese)．
［2］MAO SS，CHENG YM，PU XL． Stochastik and mathematical statis-
tics［M］． Beijing:Advanced Education Press，2008． (in Chinese)．
［3］ZHAO RR． Farm test method［M］． Beijing:China Agricultural Press，
1979． (in Chinese)．
小麦品种感染赤霉病的独立性检验
夏海峰 (淮阴师范学院数学科学学院，江苏淮安 223300)
摘要 讨论了不同的小麦品种是否与赤霉病的发生有关，并利用列联表方法进行了独立性检验。
关键词 假设检验;样本;独立性
作者简介 夏海峰(1961 －)，男，江苏淮安人，副教授，从事概率论与数理统计研究。
收稿日期 2010-04-21 修回日期 2010-09-19
35SUN Yi-min et al． Screening of Pathogenic Bacteria X46 (Micromonospora)of Crabgrass