Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (22): 4277-4287.doi: 10.3864/j.issn.0578-1752.2018.22.006

• PLANT PROTECTION • Previous Articles     Next Articles

Resistance Detection and Mechanism of Strawberry Botrytis cinerea to Fludioxonil in Sichuan Province

GONG ChangWei(),QIN YiMan,QU JinSong,WANG XueGui()   

  1. College of Agronomy/Biorational Pesticide Research Laboratory, Sichuan Agricultural University, Chengdu 611130
  • Received:2018-06-04 Accepted:2018-07-27 Online:2018-11-16 Published:2018-11-16


【Objective】Grey mold is an important disease in strawberry production, which seriously affects the yield and quality of strawberry. The objective of this study is to clarify the resistance frequency and resistance mechanism of Botrytis cinerea in different strawberry-growing areas in Sichuan Province, and to provide theoretical basis for the fungicide control of strawberry grey mold.【Method】The disease samples were collected from Chengdu, Deyang, Meishan, Leshan and Yaan in Sichuan Province from 2016 to 2017, and 188 strains of B. cinerea were isolated and purified. The sensitivity of 188 strains of B. cinerea was classified with the distinguish measurement method. The toxicity and osmotic pressure sensitivity of fludioxonil to some representative strains were assayed using the method of mycelial growth-inhibition capacity. The glycerol content of the resistant and sensitive strains treated with fludioxonil was determined by the method of glycerol-copper colorimetric assay. The sequences of type III histidine kinase gene BOS1 (BC1G_00374) in the resistant- and sensitive-fludioxonil strains were piecewise amplified and sequenced. The effects of mutations on the structure of BOS1 were predicted and evaluated by Swissmodle and I-TASSER, respectively.【Result】Of 188 strains, 8 strains showed high resistance, 9 strains showed medium resistance, 43 strains showed low resistance and the rest were sensitive. The EC50 of representative strains ranged from 0.03 to 0.62 μg·mL -1, and the resistance multiple of the representative strains ranged from 2.2 to 45.9. The concentrations of 1.25-10 g·L -1 and 1.25-20 g·L -1 NaCl could stimulate the hypha growth of the sensitive- and resistant-fludioxonil strains, respectively, whereas the concentration of >40 g·L -1 inhibited the hypha growth, especially in the resistant strains, and the higher the resistant level, the stronger the inhibition rate. The glycerol content of representative strains ranged from 0.0025 to 0.0148 μg·mL -1 under normal conditions, and there was no significant correlation between glycerol content and fludioxonil resistance of the strain, but the glycerol content of the resistant and sensitive strains increased after the treatment of fludioxonil (0.1 μg·mL -1). The increase of glycerol content in resistant strains was significantly lower than that in sensitive strains. The low resistance strains YAHY-13, CDCZ-2 and medium resistance strain CDCZ-42 mutated in the TAR and HAMP regions, meanwhile the medium resistance strain CDCZ-20 and high resistance strains MYFC-10 and CDCZ-43 mutated in TAR and REC regions, whereas the mutation site of TAR region in CDCZ-20 was I365N, and which of MYFC-10 and CDCZ-43 was I365S. Different mutation positions showed different effects on the region structure of BOS1, in which the F127S, I365N, I365S, V1136I, A1259T were all in the irregular curl of BOS1 structure, but the I365N and I365S in TAR region made the overall deviation of the region structure irregular curl. 【Conclusion】In some areas of Sichuan Province, B. cinerea has developed resistance to fludioxonil. Compared with the sensitive strains, the tolerance ability of field resistant strains to osmotic pressure increased, but when the concentrations exceeded the tolerance range, they were highly sensitive to osmotic stress and the increase of glycerol content in the field resistant strains under the fludioxonil stress was significantly lower than that of the sensitive strains. The mutation position and mode of histidine kinase BOS1 are closely related to the resistance level of B. cinerea to fludioxonil.

Key words: Botrytis cinerea, fludioxonil, resistance, osmotic pressure, glycerol content, BOS1

Table 1

The tested strains of strawberry B. cinerea in Sichuan Province"

Sampling area
Sampling time
Latitude and longitude
Strain code
Strain number
CDCZ1(成都崇州市Chongzhou, Chengdu) 2017-02-16 103o41′, 30o34′ CDCZ (1-15) 52
CDCZ2(成都崇州市Chongzhou, Chengdu) 2017-02-16 103o37′, 30o36′ CDCZ (16-30)
CDCZ3(成都崇州市Chongzhou, Chengdu) 2017-02-16 103o44′, 30o40′ CDCZ (31-52)
CDSL(成都双流市Shuangliu, Chengdu) 2016-04-10 103o35′, 30o14′ CDSL (1-9) 9
CDPZ(成都彭州市Pengzhou, Chengdu) 2017-01-05 104o10′, 30o59′ CDPZ (1-10) 10
DYGH(德阳广汉市Guanghan, Deyang) 2017-03-02 104o20′, 31o3′ DYGH (1-11) 11
MSRS1(眉山仁寿县Renshou, Meishan) 2017-03-14 104o5′, 29o33′ MSRS (1-10) 26
MSRS2(眉山仁寿县Renshou, Meishan) 2017-03-14 104o4′, 29o31′ MSRS (11-26)
MSDP1(眉山东坡区Dongpo, Meishan) 2017-03-14 102o36′, 29o30′ MSDP (1-8) 18
MSDP2(眉山东坡区Dongpo, Meishan) 2017-03-22 103o36′, 30o0′ MSDP (9-18)
LSJY1(乐山井研县Jingyan, Leshan) 2017-03-14 104o1′, 29o24′ LSJY (1-4) 11
LSJY2(乐山井研县Jingyan, Leshan) 2017-03-14 103o34′, 29o18′ LSJY (5-11)
YAHY(雅安汉源县Hanyuan, Yaan) 2017-03-25 102o36′, 29o30′ YAHY (1-16) 16
MYFC(绵阳涪城区Fucheng, Mianyang) 2017-03-27 104o49′, 31o20′ MYFC (1-17) 17
MYJY(绵阳江油市Jiangyou, Mianyang) 2017-03-27 104o44′, 31o47′ MYJY (1-18) 18

Table 2

The primers for the segment amplification of target gene BOS1"

引物 Primer 序列 Sequence

Table 3

Resistance level of strawberry B. cinerea to fludioxonil in different sampling sites of Sichuan Province"

Strain number
敏感菌株数 Number of sensitive strains 敏感菌株比例
Proportion of sensitive strains (%)
低抗菌株数 Number of low resistance strains 低抗菌株比例
Proportion of low resistance strains (%)
中抗菌株数Number of medium resistance strains 中抗菌株比例Proportion of medium resistance strains (%) 高抗菌株数Number of high resistance strains 高抗菌株比例Proportion of high resistance strains (%)
CDCZ 52 37 70.59 8 15.69 4 7.84 3 5.88
CDSL 9 8 88.89 1 11.11 0 0 0 0
CDPZ 10 3 30.00 6 60.00 1 10.00 0 0
DYGH 11 6 54.55 4 36.36 1 9.09 0 0
MSRS 26 20 76.92 3 11.54 2 7.69 1 3.85
MSDP 18 16 88.88 2 11.11 0 0 0 0
LSJY 11 6 54.54 5 45.45 0 0 0 0
YAHY 16 9 56.25 7 43.75 0 0 0 0
MYFC 17 10 58.82 2 11.76 1 5.88 4 23.53
MYJY 18 13 72.22 5 27.78 0 0 0 0
总计Total 188 128 68.08 43 22.87 9 4.78 8 4.25

Table 4

Toxicity of fludioxime to resistant and sensitive strains of strawberry B. cinerea"

Strain code
回归方程 Regression equation (y=a+bx) EC50(95%置信区间)
95% confidence interval
Resistance multiple
a b r
CDCZ-2 5.7333 0.7442 0.9011 0.10 (0.06-0.17) 7.4
CDCZ-11 6.5566 1.1984 0.9801 0.05 (0.03-0.08) 3.7
CDCZ-20 5.8440 0.7696 0.9631 0.08 (0.05-0.12) 5.9
CDCZ-42 6.4192 1.4323 0.8460 0.10 (0.07-0.14) 7.4
CDCZ-43 5.4371 1.1802 0.8918 0.43 (0.27-0.68) 31.9
CDPZ-8 6.2100 0.8161 0.9945 0.03 (0.02-0.06) 2.2
YAHY-13 6.5496 1.6160 0.9865 0.11 (0.09-0.14) 8.1
MYFC-10 5.1480 0.7054 0.9456 0.62 (0.31-1.23) 45.9

Fig. 1

The inhibition rate of the representative strains at different concentrations of NaCl"

Table 5

Effect of fludioxonil on the glycerol content of representative strains (μg·mL-1)"

Strain code
未加咯菌腈No fludioxonil added 加入0.1 μg·mL-1咯菌腈1 mL
Add 1 mL fludioxonil (0.1 μg·mL-1)
CDCZ-2 0.0067±0.0008f 0.0143±0.0001c
CDCZ-11 0.0108±0.0001d 0.0300±0.0012e
CDCZ-20 0.0058±0.0002b 0.0099±0.0001b
CDCZ-42 0.0070±0.0005bc 0.0109±0.0001b
CDCZ-43 0.0081±0.0002c 0.0096±0.0001b
CDPZ-8 0.0077±0.0015g 0.0246±0.0001d
YAHY-13 0.0025±0.0002a 0.0057±0.0001a
MYFC-10 0.0148±0.0003e 0.0155±0.0016c

Fig. 2

Tertiary structure diagram of different mutation parts of BOS1 protein"

Fig. 3

Sequence comparison of BOS1 amino acids"

Table 6

The sequencing results of type III histidine kinase gene BOS1 of representative strains"

Strain code
Resistance level
Mutant domain
YAHA-13 低抗
Low resistance
127 F-S
287 V-G
365 I-N

CDCZ-2 低抗
Low resistance
365 I-S
287 V-G
1136 V-I (+)
1259 A-T
CDCZ-42 中抗
Medium resistance
127 F-S
287 V-G
365 I-N

CDCZ-20 中抗
Medium resistance
365 I-N
1136 V-I (+)
1259 A-T
MYFC-10 高抗
High resistance
365 I-S
1136 V-I (+)
1259 A-T
CDCZ-43 高抗
High resistance
365 I-S
1136 V-I (+)
1259 A-T
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