芸薹根肿菌单孢亚甲基蓝琼脂糖分离法及其对生理小种的鉴定

doi:10.16420/j.issn.0513-353x.2021-0421

园艺学报 ›› 2022, Vol. 49 ›› Issue (6): 1290-1300.doi: 10.16420/j.issn.0513-353x.2021-0421

芸薹根肿菌单孢亚甲基蓝琼脂糖分离法及其对生理小种的鉴定

芮婷婷¹^,², 高青云¹^,³, 李晓菁¹, 石延霞¹, 谢学文¹, 李磊¹, 张红杰³, 徐文娟², 柴阿丽¹^,^**(), 李宝聚¹^,^**()

¹中国农业科学院蔬菜花卉研究所,北京 100081
²安徽农业大学,合肥 230031
³河北北方学院,河北张家口 075061

收稿日期:2021-12-20 修回日期:2022-02-21 出版日期:2022-06-25 发布日期:2022-07-05
通讯作者: 柴阿丽,李宝聚 E-mail:chaiali@caas.cn;libaojuivf@163.com
基金资助:
“十三五”国家重点研发计划项目(2017YFD0101802);中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS);农业农村部园艺作物生物学与种质创制重点实验室开放课题项目(IVF2017);国家现代农业产业技术体系建设专项资金项目(CARS-25)

Methylene Blue Combined Agarose Assay for Single-spore Isolation of Plasmodiophora brassicae and Pathotype Differentiation

RUI Tingting¹^,², GAO Qingyun¹^,³, LI Xiaojing¹, SHI Yanxia¹, XIE Xuewen¹, LI Lei¹, ZHANG Hongjie³, XU Wenjuan², CHAI Ali¹^,^**(), LI Baoju¹^,^**()

¹The Institute of Vegetable and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China
²Anhui Agricultural University,Hefei 230031,China
³Hebei North University,Zhangjiakou,Hebei 075061,China

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

摘要/Abstract

摘要：

对传统芸薹根肿菌（Plasmodiophora brassicae）单孢分离的水琼脂法进行改进,确定了亚甲基蓝和琼脂糖的最佳浓度,将0.01%亚甲基蓝染色液与1.5%琼脂糖溶液按体积比1︰300混匀,制成亚甲基蓝琼脂糖凝层,在显微镜下可成功分离芸薹根肿菌单孢。大白菜单孢接种最佳苗龄为催芽24 h,接种后暗培养24 h,再移栽至育苗钵,发病率最高。利用该方法分离获得中国11个省十字花科（大白菜、甘蓝、油菜）芸薹根肿菌单孢系菌株79株,采用Williams鉴别系统鉴定出P2、P3、P4、P5、P6、P7、P8、P9、P10、P12、P13和P14等12个不同的生理小种类型,其中P4为优势生理小种,出现频率为49.37%。明确了中国11个省份根肿菌的生理小种类型及其分布,为十字花科蔬菜抗病育种提供重要资源。

关键词: 十字花科蔬菜, 根肿病, 芸薹根肿菌, 单孢分离, 生理小种鉴定

Abstract:

The traditional water agar method for single-spore isolation of Plasmodiophora brassicae was improved,and the optimum concentrations of methylene blue and agarose were determined. Methylene blue agarose gel layers were prepared by mixing 0.01% methylene blue and 1.5% agarose solution with a volume ratio of 1︰300,on which single-spore of P. brassicae could be successfully isolated under the microscope. The best seedling age for single-spore inoculation was germinating for 24 h. After inoculation,the seedlings were then cultured in darkness for 24 h before transplanted to bowls. A total of 79 single-spore strains of P. brassicae were isolated from 11 provinces in China by using methylene blue combined agarose assay developed in this study. And 12 pathotypes such as P2,P3,P4,P5,P6,P7,P8,P9,P10,P12,P13 were identified using the Williams identification system. Among which,P4 was the dominant pathotype,with a frequency of 49.37%. The pathotype differentiates and distributions of P. brassicae from 11 provinces of China were clarified,and the results may provide important resources for breeding disease-resistant varieties of crucifer vegetables.

Key words: Crucifer vegetable, clubroot disease, Plasmodiophora brassicae, single-spore isolation, pathotype differentiation

中图分类号:

S634.1
S 635

芮婷婷, 高青云, 李晓菁, 石延霞, 谢学文, 李磊, 张红杰, 徐文娟, 柴阿丽, 李宝聚. 芸薹根肿菌单孢亚甲基蓝琼脂糖分离法及其对生理小种的鉴定[J]. 园艺学报, 2022, 49(6): 1290-1300.

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.

图/表 8

参考文献 34

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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

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

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

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

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
催芽时间/h Germination time	光培养Light	暗培养Darkness	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
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
36	0	48	84	300	177	3	1.69 b
48	0	24	72	300	204	1	0.49 d
48	0	48	96	300	155	1	0.65 c

芸薹根肿菌单孢亚甲基蓝琼脂糖分离法及其对生理小种的鉴定

Methylene Blue Combined Agarose Assay for Single-spore Isolation of Plasmodiophora brassicae and Pathotype Differentiation

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鉴别寄主 Differential host		生理小种 Pathotype
鉴别寄主 Differential host		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	+	-	-	+	-	-	-	-	+	+	+	+	-	+	-	+

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
菌株来源 Origin			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
	湖北宜昌市 Yichang,Hubei	HB110728	-	-	+	-	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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芸薹根肿菌单孢亚甲基蓝琼脂糖分离法及其对生理小种的鉴定

Methylene Blue Combined Agarose Assay for Single-spore Isolation of Plasmodiophora brassicae and Pathotype Differentiation

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