Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (6): 1290-1300.doi: 10.16420/j.issn.0513-353x.2021-0421
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RUI Tingting1,2, GAO Qingyun1,3, LI Xiaojing1, SHI Yanxia1, XIE Xuewen1, LI Lei1, ZHANG Hongjie3, XU Wenjuan2, CHAI Ali1,**(), LI Baoju1,**(
)
Received:
2021-12-20
Revised:
2022-02-21
Online:
2022-06-25
Published:
2022-07-05
Contact:
CHAI Ali,LI Baoju
E-mail:chaiali@caas.cn;libaojuivf@163.com
CLC Number:
RUI Tingting, GAO Qingyun, LI Xiaojing, SHI Yanxia, XIE Xuewen, LI Lei, ZHANG Hongjie, XU Wenjuan, CHAI Ali, LI Baoju. Methylene Blue Combined Agarose Assay for Single-spore Isolation of Plasmodiophora brassicae and Pathotype Differentiation[J]. Acta Horticulturae Sinica, 2022, 49(6): 1290-1300.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2021-0421
鉴别寄主 Differential host | 生理小种 Pathotype | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | P11 | P12 | P13 | P14 | P15 | P16 | ||
结球甘蓝Cabbage | Jersey Queen | + | + | + | + | - | + | + | - | - | + | - | + | - | - | - | - |
Badger Shipper | - | + | - | + | - | - | + | - | - | + | + | - | + | + | + | - | |
芜菁甘蓝Rutabaga | Laurentian | + | + | + | + | - | - | - | + | + | - | + | - | + | - | - | - |
Wilhelmsburger | + | - | - | + | - | - | - | - | + | + | + | + | - | + | - | + |
Table 1 Host reactions to Plasmodiphora brassicae pathotypes using differential sets of Williams
鉴别寄主 Differential host | 生理小种 Pathotype | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | P11 | P12 | P13 | P14 | P15 | P16 | ||
结球甘蓝Cabbage | Jersey Queen | + | + | + | + | - | + | + | - | - | + | - | + | - | - | - | - |
Badger Shipper | - | + | - | + | - | - | + | - | - | + | + | - | + | + | + | - | |
芜菁甘蓝Rutabaga | Laurentian | + | + | + | + | - | - | - | + | + | - | + | - | + | - | - | - |
Wilhelmsburger | + | - | - | + | - | - | - | - | + | + | + | + | - | + | - | + |
浓度/% Concentration | 透明度 Transparency | 质地 Texture | 其他 Other |
---|---|---|---|
1.0 | 透明Transparent | 很软Very soft | 不易分离Difficult separation |
1.2 | 透明Transparent | 较软Softer | 较易分离Separable |
1.3 | 透明Transparent | 软硬适中 Moderate hardness and softness | 容易分离,最省材料 Easy separation,the most economical materials |
1.5 | 透明Transparent | 软硬适中 Moderate hardness and softness | 容易分离Easy separation |
1.8 | 亚透明Sub transparent | 较硬Harder | 较易分离Separation |
2.0 | 半透明Translucent | 较硬Harder | 凝胶易干The gel is easy to dry |
2.5 | 半透明Translucent | 较硬Harder | 凝胶易干The gel is easy to dry |
3.0 | 微透明Microtransparent | 很硬Very hard | 凝胶易干The gel is easy to dry |
Table 2 Physical properties of agarose gel with different concentrations
浓度/% Concentration | 透明度 Transparency | 质地 Texture | 其他 Other |
---|---|---|---|
1.0 | 透明Transparent | 很软Very soft | 不易分离Difficult separation |
1.2 | 透明Transparent | 较软Softer | 较易分离Separable |
1.3 | 透明Transparent | 软硬适中 Moderate hardness and softness | 容易分离,最省材料 Easy separation,the most economical materials |
1.5 | 透明Transparent | 软硬适中 Moderate hardness and softness | 容易分离Easy separation |
1.8 | 亚透明Sub transparent | 较硬Harder | 较易分离Separation |
2.0 | 半透明Translucent | 较硬Harder | 凝胶易干The gel is easy to dry |
2.5 | 半透明Translucent | 较硬Harder | 凝胶易干The gel is easy to dry |
3.0 | 微透明Microtransparent | 很硬Very hard | 凝胶易干The gel is easy to dry |
Fig. 1 Viability determine of Plasmodiphora brassicae resting spores using Hochest 33342-PI dual fluorescence assay The active spores showed blue fluorescence and the inactive spores showed red fluorescence.
染色剂 Stain | 活孢子率/% Live spores rate | 染色剂 Stain | 活孢子率/% Live spores rate |
---|---|---|---|
0.05%酸性品红 Acid fuschin | 87.82 b | 0.01%番红 Safranine O | 60.95 e |
0.01%刚果红 Congo red | 81.03 c | 0.01%亚甲基蓝 Methylene blue | 97.31 a |
0.01%伊红Y纳 Eosin Y | 87.76 b | 0.02%溴酚蓝 Bromophenol blue | 87.57 b |
0.05%中性红 Neutral red | 71.20 d | 0.01%甲基绿 Methyl green | 77.98 c |
对照 Control | 97.92 a |
Table 3 Effect of different dyes to Plasmodiphora brassicae resting spores vitality
染色剂 Stain | 活孢子率/% Live spores rate | 染色剂 Stain | 活孢子率/% Live spores rate |
---|---|---|---|
0.05%酸性品红 Acid fuschin | 87.82 b | 0.01%番红 Safranine O | 60.95 e |
0.01%刚果红 Congo red | 81.03 c | 0.01%亚甲基蓝 Methylene blue | 97.31 a |
0.01%伊红Y纳 Eosin Y | 87.76 b | 0.02%溴酚蓝 Bromophenol blue | 87.57 b |
0.05%中性红 Neutral red | 71.20 d | 0.01%甲基绿 Methyl green | 77.98 c |
对照 Control | 97.92 a |
Fig. 2 Optical micrograph of zoospores of Plasmodiaphora brassicae A is the picture of spores on 1.3% agarose and 0.01% methylene blue mixed agar;B is the picture of spores on water agar.
催芽时间/h Germination time | 接种后培养时间/h Culture time after inoculation | 总苗龄/h Total seedling age | 接种株数 Inoculated plants | 成活株数 Surviving plants | 发病株数 Diseased plants | 发病率/% Disease incidence | |
---|---|---|---|---|---|---|---|
光培养Light | 暗培养Darkness | ||||||
24 | 0 | 24 | 48 | 300 | 286 | 21 | 10.19 a |
12 | 12 | 48 | 300 | 287 | 2 | 0.70 c | |
16 | 8 | 48 | 300 | 287 | 1 | 0.35 e | |
0 | 48 | 72 | 300 | 199 | 1 | 0.50 d | |
36 | 0 | 24 | 60 | 300 | 160 | 1 | 0.63 c |
0 | 48 | 84 | 300 | 177 | 3 | 1.69 b | |
48 | 0 | 24 | 72 | 300 | 204 | 1 | 0.49 d |
0 | 48 | 96 | 300 | 155 | 1 | 0.65 c |
Table 4 Plant disease incidence at different seedling ages and under different culture conditions
催芽时间/h Germination time | 接种后培养时间/h Culture time after inoculation | 总苗龄/h Total seedling age | 接种株数 Inoculated plants | 成活株数 Surviving plants | 发病株数 Diseased plants | 发病率/% Disease incidence | |
---|---|---|---|---|---|---|---|
光培养Light | 暗培养Darkness | ||||||
24 | 0 | 24 | 48 | 300 | 286 | 21 | 10.19 a |
12 | 12 | 48 | 300 | 287 | 2 | 0.70 c | |
16 | 8 | 48 | 300 | 287 | 1 | 0.35 e | |
0 | 48 | 72 | 300 | 199 | 1 | 0.50 d | |
36 | 0 | 24 | 60 | 300 | 160 | 1 | 0.63 c |
0 | 48 | 84 | 300 | 177 | 3 | 1.69 b | |
48 | 0 | 24 | 72 | 300 | 204 | 1 | 0.49 d |
0 | 48 | 96 | 300 | 155 | 1 | 0.65 c |
分离方法 Separation Method | 接菌株数 Inoculated plants | 成活株数 Surviving plants | 发病株数 Diseased plants | 发病率/% Disease incidence |
---|---|---|---|---|
亚甲基蓝琼脂糖法 Methylene blue combined agarose assay | 600 | 477 | 85 | 17.45 a |
水琼脂法 Water agar assay | 600 | 403 | 3 | 0.74 b |
液滴法 Liquid drop assay | 600 | 380 | 3 | 0.79 b |
Table 5 Comparison of single-spore isolation methods of Plasmodiophora brassicae
分离方法 Separation Method | 接菌株数 Inoculated plants | 成活株数 Surviving plants | 发病株数 Diseased plants | 发病率/% Disease incidence |
---|---|---|---|---|
亚甲基蓝琼脂糖法 Methylene blue combined agarose assay | 600 | 477 | 85 | 17.45 a |
水琼脂法 Water agar assay | 600 | 403 | 3 | 0.74 b |
液滴法 Liquid drop assay | 600 | 380 | 3 | 0.79 b |
菌株来源 Origin | 鉴别寄主 Differential Hos | 生理小种 Pathotype | 单孢菌株数 Single-spore isolates Number | 本地占比/% Proportion of region | |||||
---|---|---|---|---|---|---|---|---|---|
结球甘蓝Cabbage | 芜菁甘蓝Rutabaga | ||||||||
寄主Host | 地区Region | 编号Code | JQ | BS | LT | WB | |||
大白菜 Brassica rapa ssp. Pekinensis | 云南昆明 Kunming,Yunnan | YN111202 | + | + | + | - | P2 | 1 | 6.25 |
+ | + | + | + | P4 | 7 | 43.75 | |||
- | - | - | - | P5 | 2 | 12.50 | |||
+ | - | - | - | P6 | 1 | 6.25 | |||
+ | + | - | - | P7 | 1 | 6.25 | |||
- | - | + | + | P9 | 1 | 6.25 | |||
+ | + | - | + | P10 | 1 | 6.25 | |||
+ | - | - | + | P12 | 1 | 6.25 | |||
- | + | - | + | P14 | 1 | 6.25 | |||
河南新野 Xinye,Henan | HN170807 | + | + | + | - | P2 | 1 | 11.11 | |
+ | + | + | + | P4 | 4 | 44.44 | |||
- | - | - | - | P5 | 3 | 33.33 | |||
+ | + | - | - | P7 | 1 | 11.11 | |||
四川广元 Guangyuan,Sichuan | SC161203 | - | - | - | - | P5 | 2 | 100.00 | |
辽宁丹东 Dandong,Liaoning | LNDD131026 | + | + | + | - | P2 | 2 | 50.00 | |
+ | + | + | + | P4 | 1 | 25.00 | |||
- | - | - | - | P5 | 1 | 25.00 | |||
山东青岛 Qingdao,Shandong | SD180531 | + | + | + | - | P2 | 1 | 10.00 | |
+ | + | + | + | P4 | 6 | 60.00 | |||
- | - | - | - | P5 | 1 | 10.00 | |||
+ | + | – | - | P7 | 1 | 10.00 | |||
- | - | + | - | P8 | 1 | 10.00 | |||
陕西太白 Taibai,Shaanxi | SX120803 | + | + | + | + | P4 | 1 | 100.00 | |
湖北宜昌市 Yichang,Hubei | HB110728 | + | + | + | + | P4 | 5 | 71.43 | |
- | - | + | - | P8 | 2 | 28.57 | |||
重庆武隆 Wulong,Chongqing | CQ120809 | + | - | + | - | P3 | 1 | 25.00 | |
甘蓝 Brassica oleracea | 重庆武隆 Wulong,Chongqing | CQ180616 | + | + | + | - | P4 | 3 | 75.00 |
北京顺义 Shunyi,Beijing | BJ180503 | + | + | + | - | P2 | 1 | 12.50 | |
+ | + | + | + | P4 | 4 | 50.00 | |||
- | - | - | - | P5 | 2 | 25.00 | |||
+ | + | - | + | P10 | 1 | 12.50 | |||
黑龙江哈尔滨 Harbin,Heilongjiang | HLJ180929 | + | + | + | - | P2 | 2 | 25.00 | |
+ | + | + | + | P4 | 3 | 37.50 | |||
- | - | - | - | P5 | 2 | 25.00 | |||
- | + | + | - | P13 | 1 | 12.50 | |||
油菜 Brassica napus | 福建漳州 Zhangzhou,Fujian | FJ19073101 | + | + | + | - | P2 | 2 | 20.00 |
+ | + | + | + | P4 | 5 | 50.00 | |||
- | - | - | - | P5 | 2 | 20.00 | |||
- | - | + | - | P8 | 1 | 10.00 | |||
合计 Total | / | / | / | / | / | / | / | 79 | / |
Table 6 Pathotypes of 79 single-spore strains of Plasmodiphora brassicae from different regions of China
菌株来源 Origin | 鉴别寄主 Differential Hos | 生理小种 Pathotype | 单孢菌株数 Single-spore isolates Number | 本地占比/% Proportion of region | |||||
---|---|---|---|---|---|---|---|---|---|
结球甘蓝Cabbage | 芜菁甘蓝Rutabaga | ||||||||
寄主Host | 地区Region | 编号Code | JQ | BS | LT | WB | |||
大白菜 Brassica rapa ssp. Pekinensis | 云南昆明 Kunming,Yunnan | YN111202 | + | + | + | - | P2 | 1 | 6.25 |
+ | + | + | + | P4 | 7 | 43.75 | |||
- | - | - | - | P5 | 2 | 12.50 | |||
+ | - | - | - | P6 | 1 | 6.25 | |||
+ | + | - | - | P7 | 1 | 6.25 | |||
- | - | + | + | P9 | 1 | 6.25 | |||
+ | + | - | + | P10 | 1 | 6.25 | |||
+ | - | - | + | P12 | 1 | 6.25 | |||
- | + | - | + | P14 | 1 | 6.25 | |||
河南新野 Xinye,Henan | HN170807 | + | + | + | - | P2 | 1 | 11.11 | |
+ | + | + | + | P4 | 4 | 44.44 | |||
- | - | - | - | P5 | 3 | 33.33 | |||
+ | + | - | - | P7 | 1 | 11.11 | |||
四川广元 Guangyuan,Sichuan | SC161203 | - | - | - | - | P5 | 2 | 100.00 | |
辽宁丹东 Dandong,Liaoning | LNDD131026 | + | + | + | - | P2 | 2 | 50.00 | |
+ | + | + | + | P4 | 1 | 25.00 | |||
- | - | - | - | P5 | 1 | 25.00 | |||
山东青岛 Qingdao,Shandong | SD180531 | + | + | + | - | P2 | 1 | 10.00 | |
+ | + | + | + | P4 | 6 | 60.00 | |||
- | - | - | - | P5 | 1 | 10.00 | |||
+ | + | – | - | P7 | 1 | 10.00 | |||
- | - | + | - | P8 | 1 | 10.00 | |||
陕西太白 Taibai,Shaanxi | SX120803 | + | + | + | + | P4 | 1 | 100.00 | |
湖北宜昌市 Yichang,Hubei | HB110728 | + | + | + | + | P4 | 5 | 71.43 | |
- | - | + | - | P8 | 2 | 28.57 | |||
重庆武隆 Wulong,Chongqing | CQ120809 | + | - | + | - | P3 | 1 | 25.00 | |
甘蓝 Brassica oleracea | 重庆武隆 Wulong,Chongqing | CQ180616 | + | + | + | - | P4 | 3 | 75.00 |
北京顺义 Shunyi,Beijing | BJ180503 | + | + | + | - | P2 | 1 | 12.50 | |
+ | + | + | + | P4 | 4 | 50.00 | |||
- | - | - | - | P5 | 2 | 25.00 | |||
+ | + | - | + | P10 | 1 | 12.50 | |||
黑龙江哈尔滨 Harbin,Heilongjiang | HLJ180929 | + | + | + | - | P2 | 2 | 25.00 | |
+ | + | + | + | P4 | 3 | 37.50 | |||
- | - | - | - | P5 | 2 | 25.00 | |||
- | + | + | - | P13 | 1 | 12.50 | |||
油菜 Brassica napus | 福建漳州 Zhangzhou,Fujian | FJ19073101 | + | + | + | - | P2 | 2 | 20.00 |
+ | + | + | + | P4 | 5 | 50.00 | |||
- | - | - | - | P5 | 2 | 20.00 | |||
- | - | + | - | P8 | 1 | 10.00 | |||
合计 Total | / | / | / | / | / | / | / | 79 | / |
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