四川省草莓灰霉病菌对咯菌腈的抗性测定及其机制

doi:10.3864/j.issn.0578-1752.2018.22.006

摘要/Abstract

摘要：

【目的】灰霉病是草莓生产过程中的一种重要病害,严重影响了其产量和品质。论文旨在明确四川省草莓灰霉病菌对咯菌腈的抗性频率以及抗性机制,为草莓灰霉病的药剂防治提供理论依据。【方法】2016—2017年从四川成都、德阳、眉山、乐山及雅安等地采集草莓灰霉病样本,并分离纯化得到188株草莓灰霉病菌菌株。采用区分计量法测定188株灰霉病菌对咯菌腈的敏感性水平,采用菌丝生长速率抑制法测定咯菌腈对代表性菌株的毒力和渗透压敏感性,采用甘油铜比色法测定经咯菌腈处理的抗性菌株和敏感菌株甘油含量,采用分段测序对抗性菌株和敏感菌株Ⅲ型组氨酸激酶基因BOS1（BC1G_00374）扩增测序,采用Swissmodle和I-TASSER预测和评价突变对BOS1的结构影响。【结果】188株灰霉病菌菌株中有8株表现为高抗,9株为中抗,43株为低抗,其余表现为敏感;咯菌腈对代表性菌株的EC₅₀介于0.03—0.62 μg·mL ^-1,代表性菌株的抗性倍数范围为2.2—45.9。NaCl浓度在1.25—10 g·L ^-1可刺激敏感菌株生长,浓度在1.25—20 g·L ^-1范围可刺激抗性菌株生长,但>40 g·L ^-1时则抑制菌丝生长,尤其对抗性菌株,抗性越高抑制作用越强;在正常条件下各代表性菌株甘油含量介于0.0025—0.0148 μg·mL ^-1,且菌株的甘油含量与咯菌腈抗性没有明显的关联性,但在使用咯菌腈处理(0.1 μg·mL ^-1)抗性菌株和敏感菌株后,甘油含量均上升,且抗性菌株甘油含量上升幅度明显低于敏感菌株。低抗菌株YAHY-13、CDCZ-2以及中抗菌株CDCZ-42在TAR和HAMP区域均发生突变,中抗菌株CDCZ-20和高抗菌株MYFC-10、CDCZ-43在TAR和REC区域均有突变,但CDCZ-20菌株在TAR区域位点是I365N,而两株高抗菌株在TAR区域位点是I365S。不同突变位置对BOS1区域结构有不同程度的影响,其中F127S、I365N、I365S、V1136I、A1259T均处于BOS1区域结构的无规则卷曲,但TAR区域I365N和I365S使区域结构无规则卷曲发生整体偏移。【结论】四川省已有部分地区草莓灰霉病菌对咯菌腈产生了抗性;相比敏感菌株,田间抗性菌株对渗透压的耐受能力增加,但当浓度超过耐受范围后对渗透胁迫高度敏感,药剂胁迫下田间抗性菌株甘油含量增加量显著小于敏感菌株;组氨酸激酶BOS1突变的位置和方式与灰霉病菌菌株对咯菌腈的抗性水平存在必然联系。

关键词: 灰霉病菌, 咯菌腈, 抗药性, 渗透压, 甘油含量, BOS1

Abstract:

【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

贡常委,秦旖曼,屈劲松,王学贵. 四川省草莓灰霉病菌对咯菌腈的抗性测定及其机制[J]. 中国农业科学, 2018, 51(22): 4277-4287.

GONG ChangWei,QIN YiMan,QU JinSong,WANG XueGui. Resistance Detection and Mechanism of Strawberry Botrytis cinerea to Fludioxonil in Sichuan Province[J]. Scientia Agricultura Sinica, 2018, 51(22): 4277-4287.

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采样地区 Sampling area	采样时间 Sampling time	经纬度 Latitude and longitude	菌株编号 Strain code	菌株数 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)	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)	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)	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

引物 Primer	序列 Sequence
BFl	TACCGATCGAAAAACCCAAC
BRl	TGGGCTGGTCTCTCAATCTT
BF2	CAACGTTATGGCACAAAATCTCA
BR2	AAGTTTCTGGCCATGGTGTTCA
BF3	GGTCGGAACTGATGGAACTC
BR3	CGCGGTAAGTGAGGTCTAGG
BF4	GCAAACCGTATGATCATGGA
BR4	AGCTCGATTCTCCAAAGCAG
BF5	TCCCGTTATTCATGTCAGCTT
BR5	AAGTACTCGCAGTCGGTGGT

地区 Area	菌株数 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

菌株编号 Strain code	回归方程 Regression equation (y=a+bx)			EC₅₀（95%置信区间） 95% confidence interval	抗性倍数 Resistance multiple
菌株编号 Strain code	a	b	r	EC₅₀（95%置信区间） 95% confidence interval	抗性倍数 Resistance multiple
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

菌株编号 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