中国农业科学 ›› 2018, Vol. 51 ›› Issue (22): 4277-4287.doi: 10.3864/j.issn.0578-1752.2018.22.006
收稿日期:
2018-06-04
接受日期:
2018-07-27
出版日期:
2018-11-16
发布日期:
2018-11-16
基金资助:
GONG ChangWei(),QIN YiMan,QU JinSong,WANG XueGui(
)
Received:
2018-06-04
Accepted:
2018-07-27
Online:
2018-11-16
Published:
2018-11-16
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。
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.
表1
四川省草莓灰霉病菌供试菌株"
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) | |
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 |
表2
靶标基因BOS1分段扩增所需引物"
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 |
表3
四川省不同采集地点的草莓灰霉病菌对咯菌腈的抗性水平"
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 |
表4
咯菌腈对抗性及敏感草莓灰霉病菌菌株毒力"
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 | ||
---|---|---|---|---|---|
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 |
表5
咯菌腈对代表性菌株甘油含量影响"
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 |
图2
BOS1蛋白不同突变部分三级结构图绿色Green:没有突变位点的结构Structure without mutation site;红色Red:突变位点结构Structure of mutation site;1、3、5、8、10:野生型BOS1蛋白在125—233、267—347、289—628、1111—1228、1229—1310 aa区域的三级结构Tertiary structure of wild type BOS1 protein in 125-233, 267-347, 289-628, 1111-1228, 1229-1310 aa regions;2:BOS1蛋白F127S在125—233 aa的三级结构Tertiary structure of BOS1 proteinF127S in 125-233 aa;4:BOS1蛋白V287G在267—347 aa的三级结构Tertiary structure of BOS1 proteinV287G in 267-347 aa;6、7:BOS1蛋白I365N及BOS1蛋白I365S在289—628 aa的三级结构Tertiary structure of BOS1 proteinI365N and BOS1 proteinI365S in 289-628 aa;9:BOS1蛋白V1136I在1111—1228 aa的三级结构Tertiary structure of BOS1 proteinV1136I in 1111-1228 aa;11:BOS1蛋白A1259T在1229—1310 aa的三级结构Tertiary structure of BOS1 proteinA1259T in 1229-1310 aa"
表6
代表性菌株Ⅲ型组氨酸激酶基因BOS1测序结果"
菌株编号 Strain code | 抗性水平 Resistance level | 突变点 Discontinuity | 突变结构域 Mutant domain |
---|---|---|---|
YAHA-13 | 低抗 Low resistance | 127 F-S 287 V-G 365 I-N | — HAMP TAR |
CDCZ-2 | 低抗 Low resistance | 365 I-S 287 V-G 1136 V-I (+) 1259 A-T | TAR HAMP REC — |
CDCZ-42 | 中抗 Medium resistance | 127 F-S 287 V-G 365 I-N | — HAMP TAR |
CDCZ-20 | 中抗 Medium resistance | 365 I-N 1136 V-I (+) 1259 A-T | TAR REC — |
MYFC-10 | 高抗 High resistance | 365 I-S 1136 V-I (+) 1259 A-T | TAR REC — |
CDCZ-43 | 高抗 High resistance | 365 I-S 1136 V-I (+) 1259 A-T | TAR REC — |
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