全 文 ：植物病理学报
ACTA PHYTOPATHOLOGICA SINICA　 42(2): 206-213(2012)
Received date: 2011-01-23; Revised date: 2011-12-10
Foundation item: National Natural Science Foundation of China(31100479)
Corresponding author: ZHOU Guo-ying, Professor, focus on the biological control of plant diseases; E-mai:csuftlihe@163. com.
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Resistance of Colletotrichum gloeosporioides to
Benzimidazole Fungicide Carbendazim in
Camellia oleifera Nurseries
LI He1,2, ZHOU Guo-ying1,2*, ZHANG Huai-yun1,2,
LIU Jun-ang1,2, PENG Kuan1,2
( 1Central South University of Forestry and Technology, Hunan Forestry Biotechnology Key Laboratories, Changsha 410004, China;
2Central South University of Forestry and Technology, Biotechnology Core Facilities, College of Forestry, Changsha 410004, China)
Abstract: The fungicide resistance of anthracnose in Camellia oleifera nurseries was investigated in Liuyang,
Changning, and other regions in Hunan Province. The Colletotrichum gloeosporioides strains from the former
two regions were highly resistant to carbendazim. After subcultured for 10 generations on fungicide-free medi-
um, the resistant strains grew well on the medium containing carbendazim 450 μg / mL and suggesting that its
resistance was stable. The β-tubulin genes from the resistant and susceptible strains were cloned and se-
quenced. The coding region was 1 344 bp nucleotides and predicted to encode a protein with 447 amino acids.
Comparison of the β-tubulin amino acid sequences between resistant and susceptible strains of Colletotrichum
gloeosporioides revealed that a mutation leading to an amino acid substitution at the position 198 from glutamic
acid in the susceptible strain to alanine in the resistant strain. Finally, the main morphological characteristics of
C. gloeosporioides were descripted,but it could not be used to determine the resistance to carbendazim.
Key Words: Camellia oleifera Abel. ; Colletotrichum gloeosporioides Penz; β-tubulin gene; Carbendazim-
resistance; Single base-pair point mutation
油茶苗圃炭疽病菌抗药性研究　 李 河1,2, 周国英1,2, 章怀云1,2, 刘君昂1,2, 彭 宽1,2 　 ( 1中南林业科
技大学,林业生物技术湖南省重点实验室, 长沙 410004; 2中南林业科技大学,生物技术中心实验室,林学院, 长沙
410004)
文章编号: 0412-0914(2012)02-206-08
　 　 Camellia oleifera ( tea oil camellia) is an im-
portant edible oil woody plant, native to China. Ca-
mellia oil is rich in vitamin E, fatty acids, and ca-
mellin. It is also known as the East Olive because its
unsaturated fatty acid content can exceed 85% ,
ranking first among various vegetable oils. Colleto-
trichum gloeosporioides may cause serious fruit,
flower bud and leaf drops, as well as shoot blight
and even the death of whole plants [1] . The disease
harms the fruits, leaves, shoots, flower buds, and
leaf buds of Camellia oleifera (Fig. 1) . In China,
the control of Colletotrichum gloeosporioides has
　
　 2 期 LI He,et al.:Resistance of Colletotrichum gloeosporioides to Carbendazim in Camellia oleifera Nurseries
mainly relied on benzimidazole fungicides such as
carbendazim and achieved very good effect. However,
these systemic fungicides only have a single action
site; hence the fungus may easily develop fungicide
resistance. For the prolonged and frequent usage of a
large number of fungicides, a significant reduction in
sensitivity to these fungicides has been caused in
Camellia oleifera nurseries in some of the main pro-
ducing provinces. Resistance to benzimidazole could
develop from point mutations in the drug target,
β-tubulin gene, altering the three-dimensional struc-
ture of the protein and reducing the affinity for the
agent[2] . Zhang, et al. compared β-tubulin amino
acid sequences from the resistant and susceptible
strains of mango anthracnose, and found that the
181 st, 237 th, and 363 rd amino acids mutated, where-
as the other sites ( such as 198 th or 200 th) remained
unchanged[3] . At present, there is no report on the
resistance of Colletotrichum gloeosporioides of oil-
tea camellia anthracnose to carbendazim. Therefore,
the resistant strains tested in the Camellia oleifera
nurseries in Liuyang, Changning, and other regions
in Hunan were used as experimental materials. The
β-tubulin genes from resistant and susceptible strains
were cloned and their complete sequences were de-
termined in the study to examine whether the muta-
tions occurred in β-tubulin genes and to explore the
molecular mechanism of fungicide resistance of
Colletotrichum gloeosporioides to carbendazim.
1　 Material and Methods
1. 1　 Plasmids, transformation recipient strains
and agents
The pMD18-T vector,Escherichia coli JM109,
T4DNA ligase, X-Gal, IPTG, λDNA / Hind Ⅲ,
Markers, BamHⅠ, Premix Taq, and dNTPs were
all purchased from TaKaRa [TaKaRa Biotechnology
(Dalian)Co. , Ltd. ] .
1. 2　 Tested strains
Seedling leaves of Camellia oleifera with symp-
toms of anthracnose from the nurseries were obtained
in Liuyang, Changning, and other regions of Hunan
where the carbendazim was used as fungicide for
long time. The fungi were isolated and purified in
potato dextrose agar (PDA) medium, and the Col-
letotrichum gloeosporioides was identified according
to Koch ’ s Postulates. Based on the report by
Zhang, et al. [3], the present study used susceptible
strains, which could not grow under 0. 5 μg / mL
carbendazim, and resistant strains, which could
grow under 20. 0 μg / mL carbendazim. Ten Colleto-
trichum gloeosporioides strains including six resistant
and four susceptible strains used for sequencing were
separated and preserved from Liuyang Camellia ole-
ifera seedling base and Changning Camellia oleifera
Fig. 1　 The symptoms of anthracnose of Camellia oleifera
A: Fruit infected with Colletotrichum gloeosporioides; B:Leaf infected with C. gloeosporioides.
702
　
植物病理学报 42 卷
nursery base in Hunan. The six resistant strains grew
well on plates containing 450 μg / mL carbendazim,
whereas the minimum inhibitory concentration for
four susceptible strains were only 0. 25 μg / mL.
1. 3 　 Sensitivity determination of the fungus
to carbendazim in PDA medium
The strains of Colletotrichum gloeosporioides
were inoculated onto PDA plates. After culture of
strains for 7 d (25 ℃), a punch was used on the
colony edge to obtain 5 mm-diameter sample discs,
which were transplanted into the PDA plates contai-
ning 0. 25, 0. 5, 10. 0, and 20. 0 μg / mL carbenda-
zim respectively, with three replicates for each con-
centration and each strain. After they were cultured
for 4 d, the cross method was used to measure colo-
ny diameter, and the diameters of the discs were de-
ducted from the corresponding colony.
1. 4 　 Determination of the stability of resis-
tant strains
Six resistant strains were randomly selected and
cultured in fungicide-free PDA for 5 d. The conidia
from the cultures were chosen and transplanted into
fungicide-free PDA medium for reculturing. After
the resistant strain had been subcultured for 10 gene-
rations, according to the transplanting method in
Section 1. 3, the discs were transplanted into PDA
media containing carbendazim 0 (CK), 5, 20, 50,
100, 400, and 450 μg / mL respectively, with 3 rep-
lications for each concentration of each strain. After
culturing for 4 d, the average inhibition ratios of the
2nd and the 10 th generation were compared.
1. 5　 DNA extraction
DNA from the pathogens was extracted using
the method by Li[1] . The mycelia at the colony edge
of the tested strain cultured for 5 d were selected
with a sterile inoculating needle, placed in PD liquid
medium, and subsequently cultured with agitation
for 6 d. Then, the thalli were collected after centri-
fugation at 4 000 r / min for 10 min, and placed in a
vacuum freeze dryer. Then, the dried thalli were
further used for DNA extraction.
1. 6　 Primer design
The primers, originally designed by Zhang,
were as follows: tub2F 5′-CCTATCCTCGGT-
CAAGCCCA-3′ and tub2R 5′-GAAGCCCATGT-
TCTGGCAAA-3′ for β-tubulin gene tub2 from Col-
letotrichum gloeosporioides. The complete se-
quences of the β-tubulin gene were amplified from
the resistant and susceptible strains. Theoretically,
the amplified fragment length for this pair of primers
was 1 995 bp. The primers were synthesized by
Shanghai Sunny Biotechnology Co. , Ltd on a com-
mission basis.
1. 7 　 Cloning and identification of β-tubulin
genes
The PCR reaction system of the β-tubulin genes
(50 μL) was 25 μL Premix Taq (1. 25 U / 25 μL),
1 μL (100 ng) DNA templates ,4 μL tub2F primer
(5 mmol / L), 4 μL tub2R primer(5 mmol / L) and
16 μL ddH2O. The reaction conditions were as fol-
lows: 94℃ for 5 min; 94℃ for 1 min, 55℃ for 30
s, 72℃ for 2 min, for a total of 30 cycles; 72℃ for
10 min, and the reaction products were preserved at
4℃. Then, 10 μL of the reaction products were ob-
tained for electrophoretic analysis. DNA Gel Extrac-
tion Mini Kits from TaKaRa were used to recycle
and purify the PCR target fragments of each tested
strain, which were cloned into the pMD18-T vector.
The DNA were transformed into E. coli JM109, and
coated onto the LB culture plates containing ampicil-
lin, X-Gal, and IPTG. After an overnight culture,
the white colonies were selected for culture, and
plasmid DNA was extracted. Finally, the recombi-
nant plasmids were identified by restriction enzyme
digests. Determination of the β-tubulin gene se-
quences from the target fragments of different strains
was performed by Shanghai Sunny Biotechnology
Co. , Ltd.
1. 8　 Comparison of β-tubulin gene se-
quences from resistant and susceptible
strains
The DNAMAN software was used to translate
the obtained β-tubulin gene sequences into amino
802
　
　 2 期 LI He,et al.:Resistance of Colletotrichum gloeosporioides to Carbendazim in Camellia oleifera Nurseries
acid sequences. Using BLAST, the amino acid se-
quences of the β-tubulin genes from six resistant
strains and four susceptible strains were compared,
and their mutational sites were determined.
1. 9　 Comparison of the three-dimensional
structures of β-tubulin from resistant
and susceptible strains
Based on amino acid sequences, Sweet Molly
Grace 1. 3 and SWISS-MODEL were used to con-
struct the three-dimensional structures of β-tubulin
from resistant and susceptible strains. The two struc-
tures were compared, and their differences were
identified.
1. 10　 Measuring the spore germination rate
of resistant and sensitive strains
100 μL conidial suspension of resistant and sen-
sitive strains with the same concentration (1 × 105
spores·mL-1)were coated on 2% agar plate and in-
cubated at 25 ℃. After 24 h dark cultured, the num-
bers of colonies were detected. The rate of spore
germination was determined using the following
formula:
Rate of spore germination (% ) = Number of
spore germination / Total of spore ×100%
2　 Results
2. 1　 The sensitivity of Colletotrichum gloeos-
porioides to carbendazim
According to the growth of each strain in the
fungicide-containing media, the strains that could
not grow under 0. 5 μg / mL carbendazim were listed
as susceptible, and those could grow at 20. 0 μg / mL
carbendazim were listed as resistant strains. Through
determination of the sensitivity of Colletotrichum
gloeosporioides strains to carbendazim, the results
showed that the longer carbendazim was used in Ca-
mellia oleifera nurseries, the higher probability of
producing resistant strains. The growth ratio of the
strains from Changning and Liuyang in the medium
containing 30 μg / mL carbendazim was more than
62% , indicating that Colletotrichum gloeosporioides
in the two regions were highly resistant to carbenda-
zim(Table 1) .
2. 2　 Genetic stability of the resistant strains
After being subcultured for 10 generations in
fungicide-free medium, resistant strains grew well
on the plate containing carbendazim 450 μg / mL,
and its inhibition ratio did not increase with fewer
changes compared with the 2nd generation, it indica-
ting that the resistance of this strain was relatively
stable. Such stability will cause great difficulty in
these fields to use benzimidazole fungicides to con-
trol anthracnos(Table 2) .
2. 3　 Cloning and identification of β-tubulin
genes from pathogens
For the six resistant and four susceptible strains,
25 -30 blue and white colonies grew in each 100 μL
ligation and conversion liquor, of which were 4 - 6
blue colonies. Through electrophoresis and restric-
tion enzyme digests, specific fragments of about 2
kb were obtained from the extracted plasmid DNA,
similar to the theoretical amplified fragment size
(1 995 bp), thus the target fragments were properly
inserted into the vector.
Table 1　 Percentage of Colletotrichum gloeosporioides isolation with
growing capacity under different carbendazim stress
Origin
Number of
isolate
Percentage of isolation with growing capacity under carbendazim stress / %
0. 5 μg / mL 20 μg / mL 30 μg / mL
Chang ning, Hunan 83 69. 8 63. 7 62. 7
Liu yang, Hunan 86 76. 9 65. 3 62. 8
902
Table 2　 Resistant genetic stability of Colletotrichum gloeosporioides tolerate to carbendazim
Isolate
Second generation
Average colony diameter under dosage of carbendazim 4 d / mm
0 5 20 50 100 450
Average rate
of inhibition
/ %
Tenth generation
Average colony diameter under dosage of carbendazim 4 d / mm
0 5 20 50 100 450
Average rate
of inhibition
/ %
COR21 55 52 51 51 50 49 8. 0 53 52 52 52 50 50 3. 4
COR37 57 56 54 53 53 50 6. 7 56 55 54 55 55 53 2. 9
COR56 53 52 52 51 49 45 6. 0 53 53 51 51 52 51 2. 6
COR57 58 58 56 57 52 53 4. 8 56 55 53 54 53 53 4. 3
COR61 50 49 49 50 47 46 3. 6 49 49 48 47 49 47 2. 0
COR69 59 58 57 58 56 54 4. 1 57 56 56 56 55 54 2. 8
COS82 43 0 0 0 0 0 100 42 0 0 0 0 0 100
　
　 2 期 LI He,et al.:Resistance of Colletotrichum gloeosporioides to Carbendazim in Camellia oleifera Nurseries
2. 4 　 Comparison of the amino acid se-
quences of the β-tubulin genes from
the resistant and susceptible strains
After sequencing of the target fragments of the
ten strains, the DNA length obtained was about 2 kb
and contained 6 introns. The coding nucleotide se-
quence length was 1 344 bp, which encoded 447 a-
mino acids that was 99. 6% homologous to the ami-
no acids of tub2 gene from existing Colletotrichum
gloeosporioides sequence in GenBank. Thus, all the
target fragments contained tub2 gene β-tubulin. The
results showed that the six resistant strains had exact-
ly the same amino acid sequence and the four sus-
ceptible strains had a different amino acid sequence.
Comparison of the tub2 amino acid sequences
between the resistant and susceptible strains showed
that most of the amino acid sequences were the
same, except the 198 th amino acid. The 198 th amino
acid of susceptible strains was E (Glu), and that of
resistant strains was A (Ala), which were trans-
formed from Glutamic acid into alanine (Fig. 2) .
2. 5 　 Morphological characters and rate of
spore germination
Table 3 indicated the germination rates of resis-
tant strains were slightly lower than those from sus-
ceptible strains. However, Duncan’ s significance
test showed that there was no significant difference
between these two groups at significance levels of
5% and 1% (Table 3) .
Fig. 2　 Comparison of amino acid sequences of the tub2 gene
from the resistant and susceptible strains
In the amino acid sequence,glutamic acid (E) is transformed into alanine (A) .
112
　
植物病理学报 42 卷
Table 3 　 Comparison of the spore germination rate between resistant and sensitive isolates
Isolate Germination / %
Duncan’s significance test
0. 05 0. 01
COS12 23. 2 a A
COS13 23. 5 a A
COS73 23. 3 a A
COS82 23. 0 a A
COR21 22. 5 a A
COR37 21. 9 a A
COR56 22. 3 a A
COR57 22. 1 a A
COR61 21. 8 a A
COR69 22. 2 a A
a: A indicate P >0. 05, P >0. 01 is the 5 % significance level; A is the 1 % very significant level.
　 　 The morphological characters showed that the
colonies of sensitive and resistant strains were circu-
lar, with regular edge, concentric rings, and greyish
white aerial hypha. Most strains produced pink spore
caboodle. Conidia were all cylindrical (10 - 25 )
μm × (3 - 4. 5) μm. The results showed that the
morphological characteristics of C. gloeosporioides
could not be used to determine the resistance to car-
bendazim.
3　 Discussions
Benzimidazole fungicides are systemic and
broad-spectrum fungicides with good control effects.
However, at present, many pathogens have deve-
loped resistance to this type of fungicide because it
has a single action site. If a single gene locus mu-
tates, the pathogen can easily develop resistance.
The resistance of most pathogens to this fungicide
mainly focuses on the mutations in the 198 th and
200 th amino acids of the β-tubulin genes, but the re-
sistance mechanism of pathogens to this fungicide is
very complex. Katan [4] indicated that mutation of
the 198 th amino acid from glutamic acid to alanine or
lysine produced highly resistant strains, and mutation
of the 200 th amino acid from phenylalanine to tyro-
sine produced moderate resistance. However, the
molecular mechanism of low resistance remained un-
clear. The common phenomenon was that the coding
base of the 198 th amino acid from the β-tubulin gene
changes, e. g. , in the resistance mutation of Neuros-
pora crassa, the original coding glycine was mutated
to glutamic acid [5] . In addition to the changes in the
198 th amino acid gene sequence, different plant
pathogens could also mutate in other locations to de-
velop fungicide resistance. For Aspergillus nidulans,
the gene mutation causing its fungicide resistance oc-
cured in the benA gene of its tubulin. In the present
study, comparison of the amino acid sequences of
the tub2 gene between resistant and susceptible
strains at the Camellia oleifera nurseries showed mu-
tation of the 198 th amino acid. This study obtained
the complete gene sequence of β-tubulin from the re-
sistant and susceptible strains of Camellia oleifera,
and preliminarily elucidated the mechanism of the
carbendazim resistance. The results will further veri-
fy the findings through directed mutation and other
methods, and further use the technology in molecu-
lar biology such as PCR to monitor the occurrence
and development of fungicide resistance rapidly in
large colonies of Colletotrichum gloeosporioides.
Such ability will be very important in guiding the use
of fungicides in Camellia oleifera nurseries and
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　 2 期 LI He,et al.:Resistance of Colletotrichum gloeosporioides to Carbendazim in Camellia oleifera Nurseries
improving their effects.
References
[1] 　 Li H, Zhou G Y, Lu L L, et al. Isolation and identifi-
cation of endophytic bacteria antagonistic to Camellia
oleifera anthracnose[ J] . Afr. Journal of Microbiology
Research. 2009, 3(6): 315 -318.
[2] 　 Zhou M G, Ye Z Y. The resistance of plant pathogens
to benzimidazole and related fungicides ( in Chinese)
[J] . Plant Protection(植物保护), 1987, 13(2):31
-33.
[3] 　 Zhan R L, Zhang F C. Cloning of β-tubulin gene and
their correlation with conferred carbendazim resistance
of Colletotrichum gloeosporioides Penz in mango ( in
Chinese) [J] . Acta Microbiologica Sinica(微生物学
报), 2004, 4(6): 827 -829.
[4] 　 Katan T, Yunis H. Resistance to diethofencarb
(NPC) in benomyl-resistant field isolates of Botrytis
cinerea[J] . Plant Pathogy, 1989, 38: 86 -92.
[5] 　 Yanden O, KatanT. Mutations leading to substitutions
at amino acids 198 and 200 of beta-tubulin that corre-
late with benomyl-resistance phenotypes of field strains
of Botrytis cinerea [ J ] . Phytopathology, 1993, 83
(12): 1478 -1483.
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