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

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

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

• Research Papers • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S634.1
S 635

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.

Figures/Tables 8

References 34

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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	+	-	-	+	-	-	-	-	+	+	+	+	-	+	-	+

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	+	-	-	+	-	-	-	-	+	+	+	+	-	+	-	+

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

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

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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

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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[4]	ZHANG Hui, ZHANG Shujiang, LI Fei, ZHANG Shifan, LI Guoliang, MA Xiaochao, LIU Xitong, and SUN Rifei. Research Progress on Clubroot Disease Resistance Breeding of Brassica rapa [J]. Acta Horticulturae Sinica, 2020, 47(9): 1648-1662.
[5]	PIAO Zhong-yun;WU Di;WANG Miao;and ZHANG Teng. Marker-assisted Selection of Near Isogenic Lines for Clubroot Resistant Gene in Chinese Cabbage [J]. ACTA HORTICULTURAE SINICA, 2010, 37(8): 1264-1272.