全 文 ：收稿日期:2008-07-10
基金项目:This work was funded by Inner Mongolia Natural Scientific Foundation(200508010314)
作者简介:CAO Li-xia(1978-), Female , Inner Mongolia , phD , Senior research fellow ,Majored in plant pathology.
内蒙古中部地区苜蓿根腐病病原研究
曹丽霞1 ,赵存虎1 ,白全江1 ,邵志壮2
(1.内蒙古农牧业科学院 植物保护研究所 ,内蒙古 呼和浩特　010031;2.内蒙古农牧业科学院 玉米中心 ,内蒙古呼和浩特　010031)
　　摘要:为了查明内蒙古中部地区苜蓿根腐病的主要致病菌 , 按照柯赫氏法则对呼和浩特地区感染苜蓿根腐病的
317 个样品进行病原物分离鉴定。结果表明:呼和浩特地区的苜蓿根腐病是由多种病原菌混合侵染所致 ,从发病部位
分离到的菌株以镰孢霉(Fusarium spp.)为优势病原 , 经致病性测定和接种试验证明 , 茄病镰刀菌(F.solani)致病性最
强 ,接种后发病率达 90.0%, 其次是尖孢镰刀菌(F .oxysporum)和黄色镰刀菌(F .culmorum), 发病率分别为 61.7%和
81.7%。茄病镰刀菌是苜蓿根腐病的主要病原菌 , 与黄色镰刀菌或尖孢镰刀菌复合侵染后 , 发病率高于黄色镰刀菌或
尖孢镰刀菌单独接种的发病率。
关键词:苜蓿;根腐病;茄病镰刀菌;黄色镰刀菌;尖孢镰刀菌
中图分类号:S435.4　　文献标识码:A　　文章编号:1000-7091(2008)06-0105-03
Identification of the Pathogens Causing Root Rot of Alfalfa in Inner Mongolia
CAO Li-xia1 , ZHAO Cun-hu1 , BAI Quan-jiang1 ,SHAO Zhi-zhuang2
(1.Plant Protection Institute , Inner Mongolia Academy of Agriculture and Animal Husbandry
Sciences ,Huhhot　010031 ,China;2.Corn Research Center , Inner Mongolia Academy of
Agriculture and Animal Husbandry Sciences ,Huhhot　010031 ,China)
Abstract:To identify the major pathogen , 317 alfalfa root rot isolates sampled from Huhhot , Inner Mongolia of Chi-
na ,were determined.The results showed that:root rot of alfalfa was caused by several pathogens , where predominant
pathogen was Fusarium spp.Pathogenicity analysis indicated that F .solani had the strongest virulence with infected rate
of 90.0%,whereas infected rate of F .culmorum and F.oxysporum was 81.7% and 61.7%, respectively.When F .
solani was co-inoculated with other isolated pathogens , the infected rate of the host were higher than individual infection
of F.culmorum and F.oxysporum.F.solani was the major pathogen of root rot of alfalfa , F .culmorum and F.oxyspo-
rum played an accessory role in alfalfa.
Key words:Medicago sativa;Root rot;F.solani;F .culmorum;F .oxysporum
　　Root rot is an economic important disease on alfalfa
worldwide.In China , root rot of alfalfa mainly in Gansu ,
Beijing , Xinjiang , Jilin , Inner Mongolia etc.Root rot re-
duces alfalfa nitrogen-fixing capacity , shortens its life cy-
cle , and affects yield and quality in every period of alfalfa
growing.For the difference of local climates , soil and cul-
tivars ,many pathogens have been associated with this dis-
ease , including 13 Fusarium spp.,12 other fungi ,2 bac-
terial and 1 nematode[ 1-5] .Root rot in Inner Mongolia
was chronic disease , and more than 200 000 ha herbage
plants were destroyed by the disease each year
[ 6 ,7] .The
objectives of this study were a):to identify the predomi-
nant pathogens of root rot in alfalfa;b):to assess the
pathogenicity of the major pathogens.
1　Materials and methods
1.1　Fungal isolates and identification
100 isolates f rom root rot plants of alfalfa sampled
from HeLin County ,TuoKeTuo County and Xibazha Town.
The identification of isolates was carried out by using
monoconidial cultures , grown on potato-dextrose-agar
(PDA)at 26℃ for 12 h photoperiod[ 8] .
1.2　Plant material and growth conditions
Seeds of alfalfa(Medicago sativa L.Aljmgand)were
华北农学报·2008 , 23(5):105-107
sterilized with 5% sodium hypochlorite solution for 5
min ,washed three times with sterile distilled water , and
germinated in darkness at 20℃ for 2 d and similar sized
seedlings were transferred into vermiculite.
1.3　Pathogenicity test
The first test:After the first leaf was fully expanded ,
seedlings(5 plants)were transferred into test tube filled
with modified half-strength Hoagland′s solution:0.02
mmol/L KH2PO4 , 2.5 mmol/L KNO3 , 2.5 mmol/L
Ca(NO3)2 , 1 mmol/L MgSO4 , 0.5 mmol/L KCl , 0.5
mmol/L NaCl , 0.125 mmol/L Fe-EDTA , 23 mmol/L
H3BO3 ,4.5 mmol/L MnSO4 , 0.38 mmol/L ZnSO4 ,0.16
mmol/L CuSO4 , and 0.05 mmol/L Na2MoO4.Twenty
plugs(6 mm diameter)of mycelium were cut with a cork
borer from isolates cultures grown on PDA after 7 d , and
45 mL sterile distilled water were added into each Petri
dish , chopped to suspension.When the 2nd leaves emer-
gence ,15 mL suspension were added into each test tube.
The development of disease symptoms was observed daily
for at least 2 weeks.Seedlings growing on dH2O without
the fungus were used as a control.Three replicates for
each treatment and the experiments were repeated twice.
The second test:Healthy seedling growing in vermi-
culite in pot inoculated single isolates cultures after the
2nd leaves emergence.The inoculation method was same
as in the first test.Three replicates for each treatment and
the experiments were repeated twice.
The third test:Healthy seedlings growing in solution
inoculated by several isolates cultures after the 2nd leaves
emergence.The inoculation method was same as in the
first test.Three replicates for each treatment and the ex-
periments were repeated twice.
1.4　Pathogens identification
Root rot of alfalfa was caused by several pathogens ,
where predominant pathogen was Fusarium spp.The iden-
tification methods of Fusarium spp.was followed by Wang
Gongchen[ 9] .The culture substrates were PSA(potato-su-
crose-agar),Bilais ,WA(water-agar)and Rice.
2　Results and analysis
2.1　Symptoms characters
Although there were some differences in symptoms
caused by different pathogens , the symptoms were not al-
ways specific to any one of the pathogens.In Huhhot , In-
ner Mongolia , infected plants delay 19 d germinating , and
growing slowly.The disease is characterized by the pres-
ence of dark brown to black ,wedge shaped necrotic le-
sions that spread from the affected crowns downward into
root tips.
2.2　Fungal isolates and identification
317 alfalfa root rot isolates sampled from Huhhot
were conducted ,84 fungi strains were obtained , and clas-
sified into 6 groups , e.g.3 Fusarium spp., 1 Alternaria
spp ,1 Penicillium spp.and 1 Aspergillus spp.(Tab.1).
Tab.1　Isolation frequency of alfalfa root rot
Isolates　　　 Number of
colonies
Isolation
frequency/ %
Fusarium.spp F1 36 42.9
Fusarium.spp F2 12 14.3
Fusarium.spp F3 23 27.4
Alternaria spp. 4 4.8
Penicillium spp. 6 7.1
Aspergillus spp. 3 3.5
2.3　Pathogenicity test
2.3.1　The first test 　3 d after hydroponics healthy
seedling inoculated isolates , root and stem base were ini-
tially light brown , then became dark brown necrotic
strands in 5 d.Seedlings grown slowly and the number of
lateral root deceased.The seedlings wilt and die in 8 d
(Tab.2).From the Table 2 , the predominant pathogens
were F1 ,F2 and F3.
Tab.2　Pathogenicity test of the isolated pathogens
Pathogens Number of inoculated seedlings Number of infected seedlings Rate of infected/ %
Fusarium.spp F1 60 37 61.7
Fusarium.spp F2 60 54 90.0
Fusarium.spp F3 60 49 81.7
Alternaria spp. 30 12 40.0
Penicillium spp. 30 14 46.7
Aspergillus spp. 30 9 30.0
F1+F2 60 48 80.0
F1+F3 60 41 68.3
F1+F2+F3 60 56 93.3
F2+F3 60 53 88.3
CK 60 2 3.3
106　 华　北　农　学　报 23卷
2.3.2　The second test　The results of seedlings in pot
were the same as its in nutrient solution;its infected peri-
od was longer.Root and stem base were dark brown
necrotic strands in 18 d.
2.3.3　The third test　Healthy seedlings inoculated by
several pathogens were infected severity.Infected rate of
seedlings in combined inoculation was up to 80%, and the
highest rate was 93.3%, caused by F1+F2+F3.When
inoculated by single pathogen ,F2′s infected rate was the
highest one(Tab.2).
2.4　Pathogens identification
Predominant pathogen of root rot of alfalfa in Huhhot
was Fusarium spp , i.e.F .oxysporum , F .solani and F .
culmorum.The characters were as follows:F1(F .oxyspo-
rum), average growth speed of cultures on PSA in 4 d was
4.4 cm.Mycelium was abundant with white color at be-
ginning and became violet later.Mycelium was thin on Bi-
lai′s and light green to red color on rice.Microconidia
were generally abundant , cylindrical.Macroconidia sickle ,
tall and slender , 3 -5 separation.Chlamydospores were
generally abundant and terminal or intercalary , formed in
pairs or chains.
F2(F.solani), average growth speed of cultures on
PSA in 4 d was 4.1 cm.Mycelium was thin with white to
grey color.Mycelium was thin on Bilai′s and white to caf
color on rice.Microconidia were generally abundant , o-
vate.Macroconidia sickle , thick and strong , 2-8 separa-
tion.Chlamydospores were generally abundant , circular.
F3(F.culmorum), average growth speed of cultures
on PSA in 4 d was 4.4 cm.culture pigmentation was
claret.Mycelium was thin on Bilai′s and yellow on rice.
Only macroconidia were produced.They were 3-5 sepa-
rate , shorten and strong.Chlamydospores globose.
All of the three species were the major pathogen to
the disease in huhhot , Inner Mongolia.
3　Discussion
Root rot of alfalfa is a chronic disease worldwide.
The pathogens were different in different areas because of
local climate and environment.In recent years ,13 Fusari-
um spp.causing root rot of alfalfa were clarified:F .oxys-
porum , F.solani , F .culmorum , F .acuminatum , F.ave-
naceum , F.semitectum , F.tricinctum , F .sambucinum ,
F .fusarioides , F .equiseti , F.moniliforme , F.falcatum
and F.poae[ 10] .But in a given areas , only a few major
pathogens exist.In Huhhot , Inner Mongolia , the predomi-
nant pathogens of root rot in alfalfa were F .solani , F .
culmorum and F .oxysporum .When F.solani was co-in-
oculated with other two , the infected rate of the host were
higher than the infection of individual F.culmorum or
F .oxysporum .F .solani was the major pathogen of root
rot of alfalfa , F.culmorum and F.oxysporum played an
accessory role in alfalfa.These results coincide with the
results of Wang Duocheng
[ 11] .
F .solani was a soil fungus with different specialized
species and physiological forms.It could cause root rot in
many crops , such as pea , soybean etc.As for physiological
forms , infected mechanism and parasitical capacity of F .
solani were still unknown now , and need further study.
4　Acknowledgments
We thank the research funding from IMNSF
(200508010314), Li Ziqin for critical review of the
manuscript and Yun Xiaopeng for experimental assistance
in the laboratory.
参考文献:
[ 1] 　李敏权.苜蓿根和根颈腐烂病的病原及种质抗病性研
究[ D] .兰州:甘肃农业大学 , 2002:26-31.
[ 2] 　曹丽霞 ,赵存虎 ,孔庆全 , 等.紫花苜蓿根腐病病原及防
治研究进展[ J] .内蒙古农业科技 , 2006(3):36-37.
[ 3] 　李敏权 ,柴兆祥 ,李金花 , 等.定西地区苜蓿根和根颈腐
烂病病原研究[ J] .草地学报 , 2003 , 11(1):83-86.
[ 4] 　陈雅君 ,崔国文.黑龙江省紫花苜蓿根腐病调查及病原
分离[ J] .中国草地 , 2001 , 23(3):78-79.
[ 5] 　UddinW , Knous T R.Fusarium species associated with crown
rot of alfalfa in Nevada[ J] .Plant disease , 1991 , 75(1):51-
55.
[ 6] 　陈申宽 ,闫任沛 , 刘玉良.呼伦贝尔市人工草地主要有
害生物种类调查[ J] .内蒙古民族大学学报:自然科学
版 , 2005 , 20(1):62-65.
[ 7] 　张玉民 ,王德霞 ,赵永富 , 等.内蒙古莫旗人工草地主要
病虫害发生原因及防治对策[ J] .草业科学 , 2004 , 21
(7):52-54.
[ 8] 　方中达.植病研究方法[ M] .第 3 版.北京:中国农业出
版社 , 1998:122-125.
[ 9] 　王拱辰 , 郑　重 , 叶琪明 , 等.常见镰刀菌鉴定指南
[ M] .第 1 版.北京:中国农业科技出版社 , 1996:22-
25.
[ 10] 　孟　嫣 , 李敏权.苜蓿根和根颈腐烂病病原及防治研
究进展[ J] .草业科学 , 2005 , 22(5):89-92.
[ 11] 　王多成 ,孟有儒 , 李文明 , 等.苜蓿根腐病病原菌的分
离及鉴定[ J] .草业科学 , 2005 , 22(10):78-81.
[ 12] 　周洪友 , 杨合同 ,唐文华.沙打旺根腐病发生及病原菌
鉴定[ J] .草地学报 , 2004 , 12(4):285-288.
[ 13] 　Gray L E , Achenbach L A , Duff R J , et al.Pathogenicity of
Fusarium solani f.sp.glycines Isolates on Soybean and
Green Bean Plants[ J] .Journal of Phytopathology , 1999 , 147
(5):281-284.
6期 曹丽霞等:内蒙古中部地区苜蓿根腐病病原研究 107