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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (4): 407-417    DOI: 10.3785/j.issn.1008-9209.2018.11.262
植物保护     
茄子黄萎病病原菌致病型分化及其生物防治
刘晶晶(),庞叶洲,张敬泽()
浙江大学农业与生物技术学院生物技术研究所，杭州 310058
Pathogenic differentiation and biological control of Verticillium wilt of eggplant
Jingjing LIU(),Yezhou PANG,Jingze ZHANG()
Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
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摘要：

为了有效防治在浙江一些地区严重发生的茄子黄萎病，本文进行了病害观察、病原菌致病性试验、病原菌致病型和生理小种鉴定，测定了多粘类芽孢杆菌对病原菌菌丝和分生孢子萌发的抑制活性，开展了病害生物防治和促生长试验，并测定了多粘类芽孢杆菌产生的挥发性物质和脂肽。病害观察和致病性试验结果表明，茄子黄萎病在浙江温室大棚中于1月中旬发生，由大丽轮枝菌引起。用7个代表性菌株对病原菌致病型和生理小种进行鉴定表明，所有分离系均属于落叶型株系和2号生理小种。用原先筛选的5株多粘类芽孢杆菌对病原菌菌丝和分生孢子萌发进行体外拮抗试验表明，株系ShX301具有最高的抑制活性。体内接种和促生长试验显示，株系ShX301也具有最高的生物活性，接种株系ShX301使病害发病率和病害严重度比率分别减少30%和20.02%，对茄苗的茎长、地上生物量和地下生物量的增长率分别达59.24%、69.41%和54.11%。挥发性物质测定结果表明，5株多粘类芽孢杆菌都能产生挥发性物质，它们对病原菌菌丝生长都具有显著的抑制作用，但株系间没有显著性差异。基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）检测和分析揭示，杀镰孢菌素是拮抗病原菌的主要活性物质。这些研究结果为菌株ShX301作为生防或促生长制剂开发提供了科学理论依据。
关键词： 多粘类芽孢杆菌;  生物防治;  拮抗机制;  挥发性物质;  脂肽    
Abstract:

Verticilliumwilt of eggplant occurs seriously in some areas of Zhejiang Province. To control the disease effectively, the disease observation and pathogenicity tests of pathogen were carried out, and the pathotype and physiological races of pathogen were identified. Furthermore, inhibitory activity of Paenibacillus polymyxa strains against hypha growth and conidial germination of pathogen were determined, and inoculation assays for controlling Verticillium wilt of eggplant and plant growth promotion were conducted, and volatile metabolites and lipopeptides produced by P. polymyxa strains were analyzed. Results of disease observation and pathogenicity tests indicated that Verticillium wilt of eggplant in greenhouse occurred usually in mid-January, and it was caused by Verticillium dahliae Kleb. Identification results of pathotype and physiological races of pathogen with the representative strains demonstrated that seven isolates from Zhejiang Province all belonged to the defoliation pathotype and physiological race-2, respectively. In vitro experiments showed that the strain ShX301 had a highest inhibitory activity against hypha growth and conidial germination of pathogens isolated the diseased eggplant plants among five P. polymyxa strains. The in vivo experiments for controlling Verticillium wilt of eggplant and plant growth promotion displayed that the strain ShX301 had a highest inhibitory activity. Inoculation by the strain ShX301 reduced disease incidence and severity by 30% and 20.02%, respectively, and promoted eggplant plant growth with enhancing aboveground seedling stem length and biomass by 59.24% and 69.41%, as well as underground biomass by 54.11%, respectively. The determination of volatile metabolites showed that the five strains of P. polymyxa were able to produce volatile metabolites, which inhibited hypha growth of pathogen, but there were no significant differences among the strains. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis revealed that the fusaricidins were main activity compounds of antagonistic pathogen. Therefore, this study provides a scientific theory basis for development of the strain ShX301 as a biocontrol or plant growth promotion agent.
Key words: Paenibacillus polymyxa    biological control    antagonistic mechanism    volatile compounds    lipopeptides
收稿日期: 2018-11-26 出版日期: 2019-09-17
CLC:  S 432.4  
基金资助: 国家公益性行业（农业）科研专项资金(201503109);浙江省重点研发计划(2015C02023)
通讯作者: 张敬泽     E-mail: 457771128@qq.com;jzzhang@zju.edu.cn
作者简介: 刘晶晶（https://orcid.org/0000-0002-2274-5930），E-mail：457771128@qq.com
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引用本文:

刘晶晶,庞叶洲,张敬泽. 茄子黄萎病病原菌致病型分化及其生物防治[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(4): 407-417.

Jingjing LIU,Yezhou PANG,Jingze ZHANG. Pathogenic differentiation and biological control of Verticillium wilt of eggplant. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(4): 407-417.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.11.262    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I4/407

图1  茄子黄萎病症状
图2  落叶型和非落叶型特异性引物PCR产物的琼脂糖凝胶电泳图
图3  生理小种1号和生理小种2号特异性引物PCR产物的琼脂糖凝胶电泳图

株系

Strains

生长抑制率1）

PGI1)/%

抑菌圈直径

IZD/mm
ShX301 85.23±0.3a 33.23±0.77a
Hb6 76.52±1.9b 25.33±1.20b
Hb1 70.85±1.7c 23.45±0.61c
ShX302 69.15±1.3cd 15.66±0.67d
ShX303 68.11±0.3d 14.70±1.46d
表1  5株多粘类芽孢杆菌对大丽轮枝菌的抑制效果
图4   5株多粘类芽孢杆菌挥发性物质对大丽轮枝菌菌丝生长的影响

株系

Strains

发病率

Incidence/%

病害严重度比率1）

Disease severity index1)/%

防治效果2）

Control efficacy2)/%
ShX301 36.67±2.89d 23.17±0.44e 46.36
Hb6 41.67±2.89cd 26.16±0.24d 39.44
Hb1 43.33±2.89c 27.19±1.10d 37.05
ShX302 51.67±2.89b 34.43±0.67c 20.28
ShX303 53.33±5.77b 36.25±2.10b 16.06
CK 66.67±2.89a 43.19±0.20a
表2  接种50 d后5株多粘类芽孢杆菌对茄子黄萎病的防治效果
图5  接种50 d后5株多粘类芽孢杆菌对茄苗生长的促进效果

株系

Strains

 茎 Stem 根 Root 地上部 Aboveground 地上部 Aboveground 地下部 Underground 地下部 Underground

长度

Length/mm

促生

长率

PD/%

长度Length/

cm

促生

长率

PD/%

鲜质量

Fresh mass/

g

促生

长率

PD/%

干质量

Dry mass/

g

促生

长率

PD/%

鲜质量

Fresh mass/

g

促生

长率

PD/%

干质量

Dry mass/

g

促生

长率

PD/%
ShX301

17.98±

0.85a

 59.24

11.07±

0.71d

 24.87

10.25±

0.96a

 69.41

1.15±

0.19a

 96.83

1.06±

0.14a

 54.11

0.21±

0.05a

 73.33
Hb6

15.25±

1.56b

 35.09

12.89±

1.13a

 45.38

9.56±

0.85b

 58.07

0.86±

0.11b

 46.75

0.96±

0.11b

 38.51

0.18±

0.02b

 44.31
Hb1

14.68±

1.43bc

 30.07

11.32±

1.46c

 27.61

9.13±

1.06bc

 50.97

0.76±

0.14c

 28.91

0.79±

0.12c

 14.22

0.15±

0.03c

 41.96
ShX302

14.61±

1.71bc

 29.38

11.92±

1.70b

 34.47

8.82±

0.94c

 45.80

0.69±

0.13c

 18.10

0.81±

0.1c

 17.18

0.17±

0.03b

 21.96
ShX303

14.16±

1.40c

 25.47

11.86±

1.34b

 33.79

8.73±

1.04c

 44.32

0.71±

0.12c

 20.21

0.84±

0.09c

 21.88

0.14±

0.03c

 16.86
CK

11.29±

0.73d

8.87±

0.60e

6.05±

0.40d

0.59±

0.16d

0.69±

0.07d

0.12±

0.03d
表3  5株多粘类芽孢杆菌对茄子生长促进作用的测定结果
图6  多粘类芽孢杆菌非核糖体脂肽的MALDI-TOF-MS分析图

杀镰孢菌素

Fusaricidins

 质荷比m/z AMM 强度 Intensity [a.u.]
ShX301 ShX302 ShX303 Hb1 Hb6
Fusaricidin A 883 [M+H]+ 27 889 51 669 88 927 143 47 821
905 [M+Na]+ 5 560 5 453 4 655 — 11 633
921 [M+K]+ — — 19 337 — —
Fusaricidin B 897 [M+H]+ 29 826 64 997 93 385 196 49 006
919 [M+Na]+ 8 434 12 641 6 927 — 15 589
935 [M+K]+ 10 228 26 640 27 890 — 27 274
LI-F05b 911 [M+H]+ 6 100 33 006 56 402 2 018 16 388
933 [M+Na]+ 1 053 3 424 — — 3 445
949 [M+K]+ 1 170 6 538 — — 5 632
LI-F08b 925 [M+H]+ 1 109 22 794 35 894 2 063 5 165
947 [M+Na]+ 2 682 30 747 58 057 888 16 271
963 [M+K]+ — 5 912 — — 1 951
LI-F07a 931 [M+H]+ 3 415 5 356 17 837 616 9 656
953 [M+Na]+ 526 — 1 381 540 —
969 [M+K]+ — — — — 4 325
LI-F07b 945 [M+H]+ 3 066 6 796 16 666 574 8 406
967 [M+Na]+ 454 — 509 — —
983 [M+K]+ 966 3 895 4 801 — 4 257
Fusaricidin C 947 [M+H]+ 2 682 30 747 58 057 888 16 271
969 [M+Na]+ — — — — 4 325
985 [M+K]+ 273 3 607 7 290 — 3 645
Fusaricidin D 961 [M+H]+ 3 400 37 584 — 1 075 13 745
983 [M+Na]+ 966 3 895 4 801 — 4 257
999 [M+K]+ 547 77 113 9 480 — 4 104
表4  MALDI-TOF-MS对多粘类芽孢杆菌培养液的测定结果
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