芸薹根肿菌活细胞PMAxx-qPCR快速定量检测方法的建立与应用

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doi:10.3864/j.issn.0578-1752.2022.10.005

摘要：

【目的】由芸薹根肿菌（Plasmodiophora brassicae）侵染引起的十字花科根肿病是一种世界性土传病害,病原菌长期存在于土壤中,对十字花科作物造成严重威胁。改良叠氮溴化丙锭（propidium monoazide xx,PMAxx）可选择性地穿透受损的死细胞膜,并抑制死细胞DNA的实时荧光定量PCR（qPCR）扩增。本文将PMAxx与qPCR技术相结合,建立一种快速检测芸薹根肿菌活菌的方法,为根肿病的早期诊断及制定科学的防控措施提供依据。【方法】配置浓度分别为0、5、10、20、40、60 µmol·L^-1的叠氮溴化丙锭PMA和改良叠氮溴化丙锭PMAxx,比较两种核酸染料对芸薹根肿菌死细胞DNA扩增的抑制效果,确定最佳核酸染料及工作浓度;设置光照时间分别为0、2、5、10、15和20 min,进行最佳光照时间的优化,建立芸薹根肿菌活细胞PMAxx-qPCR快速检测体系。设置芸薹根肿菌活孢子百分比为0、0.01%、0.1%、1%、10%、25%、50%、75%和100%的混合体系,验证PMAxx-qPCR体系的准确性,并应用于田间土壤样本中芸薹根肿菌活孢子的定量检测。【结果】PMAxx对芸薹根肿菌死细胞DNA的扩增抑制效果更好,当芸薹根肿菌浓度为1×10⁸个孢子/mL,PMAxx预处理的最适终浓度为4 µmol·L^-1,最佳光照时间为10 min时,可有效地抑制死孢子DNA的扩增,仅以有活力孢子DNA为靶标选择性地扩增。利用PMAxx-qPCR技术检测已知不同活孢子比例的菌悬液样品,各样品实测孢子存活率和理论存活率之间呈正相关（R²=0.992）。对田间采集的25份土壤样本,采用PMAxx-qPCR方法检测到11份样本中携带芸薹根肿菌,活细胞DNA浓度为32.35—6.97×10³ fg·g^-1。【结论】建立了基于PMAxx-qPCR的芸薹根肿菌活细胞定量检测技术,该技术具有快速、准确、灵敏的特点,解决了qPCR不能仅对活体病原菌进行准确鉴别和定量分析的问题,为制定有效的根肿病防控策略提供了依据。

关键词: 芸薹根肿菌, 根肿病, 改良叠氮溴化丙锭, 实时荧光定量PCR, 活性孢子

Abstract:

【Objective】Plasmodiophora brassicae is an obligate endoparasite that causes clubroot disease, which is the most devastating soil-borne disease in brassica crops. The propidium monoazide xx (PMAxx) could selectively bind to the chromosomal DNA of dead spores and therefore block DNA amplification by real-time fluorescent quantitative PCR (qPCR). In the present study, a strategy involving a PMAxx pre-treatment followed by the qPCR (PMAxx-qPCR) assay was developed for quantifying viable spores of P. brassicae, so as to provide a basis for early detection and prevention measurement of cloobroot disease. 【Method】 PMA and PMAxx with concentrations of 0, 5, 10, 20, 40 and 60 µmol·L^-1 were prepared, respectively, and were used to pre-treat P. brassicae prior to DNA extraction, followed by qPCR. The inhibitory effects of PMA and PMAxx on DNA amplification of P. brassicae dead spores were compared, and the optimal nucleic acid dye and concentration to distinguish between live and dead spores were determined. The illumination time was set as 0, 2, 5, 10, 15 and 20 min, respectively, and the optimal exposure time was optimized to establish a PMAxx-qPCR assay for selectively detection of viable spores of P. brassicae. The mixed suspensions with different ratios of dead and viable spores (0, 0.01%, 0.1%, 1%, 10%, 25%, 50%, 75% and 100% viable spores) were prepared to determine the suitability of PMAxx-qPCR assay for distinguishing viable and dead spores. The assay was also applied to quantitative detection of viable spores of P. brassicae in 25 field soil samples. 【Result】 PMAxx showed a better discrimination effect than PMA on the viable and dead spores of P. brassicae. When the concentration of P. brassicae was 1×10⁸spores/mL, the optimal PMAxx concentration and light exposure time were 4 μmol·L^-1 and 10 min, respectively. The amplification of dead spores could be inhibited effectively, and only the DNA of living spores was targeted for selective amplification. For pre-defined ratio of viable spores, there was a good linear relationship between the lg of the P. brassicae DNA concentration assessed by PMAxx-qPCR and the theoretical viability (R²=0.992). For soil samples, viable P. brassicae was quantified in 11 of 25 samples, with infestation levels of approximately 32.35-6.97×10³ fg·g^-1. 【Conclusion】 The established method could quantitatively detect the viable spores of P. brassicae, with advantages of rapid, efficiency and sensitivity, which could be useful for avoiding the inability of qPCR method to distinguish between viable and nonviable spores. Application of the assay may potentially improve P. brassicae control and disease management.

Key words: Plasmodiophora brassicae, clubroot disease, propidium monoazide xx (PMAxx), real-time fluorescent quantitative PCR (qPCR), viable spore

李晓菁,张思雨,刘迪,袁晓伟,李兴盛,石延霞,谢学文,李磊,范腾飞,李宝聚,柴阿丽. 芸薹根肿菌活细胞PMAxx-qPCR快速定量检测方法的建立与应用[J]. 中国农业科学, 2022, 55(10): 1938-1948.

LI XiaoJing,ZHANG SiYu,LIU Di,YUAN XiaoWei,LI XingSheng,SHI YanXia,XIE XueWen,LI Lei,FAN TengFei,LI BaoJu,CHAI ALi. Establishment and Application of Rapid Quantitative Detection of Viable Plasmodiophora brassicae by PMAxx-qPCR Method[J]. Scientia Agricultura Sinica, 2022, 55(10): 1938-1948.

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序号No.	病原菌Pathogen	菌株编号Strain code	寄主Host	采集地Geographic origin
1	芸薹根肿菌Plasmodiophora brassicae	BC18052103	大白菜Chinese cabbage	云南省昆明市Kunming, Yunnan
2	尖镰孢Fusarium oxysporum	GL17061203	甘蓝 Cabbage	陕西省太白县Taibai, Shaanxi
3	茄镰孢Fusarium solani	BC10120305	大白菜Chinese cabbage	辽宁省丹东市Dandong, Liaoning
4	半裸镰孢Fusarium incarnatum	HYC15171302	花椰菜Broccoli	河北省承德市Chengde, Hebei
5	立枯丝核菌Rhizoctonia solani	BC16072809	普通白菜Pakchoi	河北省张家口市Zhangjiakou, Hebei
6	核盘菌Sclerotinia sclerotiorum	YC19081601	油菜Rape	山东省临沂市Linyi, Shandong
7	芸薹链格孢Alternaria brassicae	BC15110628	大白菜Chinese cabbage	河北省廊坊市Langfang, Hebei
8	长孢轮枝菌Verticillium longisporum	BC13091524	大白菜Chinese cabbage	河北省张家口市Zhangjiakou, Hebei
9	瓜果腐霉Pythium aphanidermatum	BC12073101	大白菜Chinese cabbage	江苏常州市Changzhou, Jiangsu
10	灰葡萄孢Botrytis cinerea	HYC20051608	花椰菜Broccoli	北京市顺义区Shunyi, Beijing
11	胡萝卜软腐果胶杆菌胡萝卜亚种 Pectobacterium carotovorun subsp. carotovorum	BC14120625	大白菜Chinese cabbage	内蒙古自治区乌兰察布市 Ulanqab, Inner Mongolia
12	野油菜黄单胞菌野油菜致病变种 Xanthomonas campestris pv. campestris	GL18080621	甘蓝 Cabbage	湖南省长沙市Changsha, Hunan

光敏染料 Photoactivatable dye	浓度 Concentration (µmol∙L^-1)	Ct （活孢子Viable spores）	Ct （死孢子Dead spores）	dCt （活孢子Viable spores）	dCt （死孢子Dead spores）	ddCt
PMA/PMAxx	0	14.53a	14.86a	/	/	/
PMA	5	15.53b	25.82b	1.00	10.96	9.96
	10	15.90b	27.43c	1.37	12.57	11.20
	20	16.84c	28.92d	2.31	14.06	11.75
	40	17.90d	29.71e	3.37	14.85	11.48
	60	18.52e	29.84e	3.99	14.98	10.99
PMAxx	5	15.56b	27.91c	1.03	13.05	12.02
	10	16.21bc	29.57d	1.68	14.71	13.03
	20	17.18c	30.59e	2.65	15.73	13.08
	40	18.21d	32.10f	3.68	17.24	13.56
	60	18.58e	32.61f	4.05	17.75	13.70

活孢子百分比 Ratio of viable spores (%)	活孢子浓度 Concentration of viable spores (spores/mL)	qPCR		PMAxx-qPCR
活孢子百分比 Ratio of viable spores (%)	活孢子浓度 Concentration of viable spores (spores/mL)	Ct值 Ct value	DNA浓度对数值 Lg DNA concentration	Ct值 Ct value	DNA浓度对数值 Lg DNA concentration
100	1.00×10⁸	10.04±0.28	6.74±0.08a	10.34±0.21	6.66±0.06a
75	7.50×10⁷	10.76±0.56	6.58±0a	11.10±0.24	6.45±0.07b
50	5.00×10⁷	10.26±0.23	6.72±0.12a	12.13±0.47	6.16±0.13c
25	2.50×10⁷	10.48±0.65	6.66±0.10a	13.31±0.37	5.83±0.10d
10	1.00×10⁷	10.19±0.53	6.74±0.10a	14.66±0.25	5.46±0.07e
1	1.00×10⁶	10.40±0.39	6.68±0.14a	18.01±0.39	4.53±0.11f
0.1	1.00×10⁵	10.81±0.62	6.57±0.13a	20.63±0.49	3.80±0.13g
0.01	1.00×10⁴	10.57±0.52	6.63±0.10a	23.59±0.34	2.98±0.09h

样本 Sample No.	采集地 Geographic origin	种植作物 Crop	采样时间Sampling time	qPCR		PMAxx-qPCR		病情指数Disease index
样本 Sample No.	采集地 Geographic origin	种植作物 Crop	采样时间Sampling time	Ct值 Ct value	浓度<BOLD>C</BOLD>oncentration (fg DNA·g^-1 soil)	Ct值 Ct value	浓度<BOLD>C</BOLD>oncentration (fg DNA·g^-1 soil)	病情指数Disease index
1	四川省绵阳市 Mianyang, Sichuan	甘蓝Cabbage	2020-07	18.59±0.16	2.32×10⁴	20.86±0.29	5.43×10³	47.61±0.58
2	四川省绵阳市 Mianyang, Sichuan	甘蓝Cabbage	2020-07	20.65±0.26	6.23×10³	22.62±0.34	1.76×10³	38.20±0.30
3	四川省广元市 Guangyuan, Sichuan	甘蓝Cabbage	2020-08	21.12±1.36	4.61×10³	23.50±0.25	1.01×10³	32.10±0.44
4	四川省广元市 Guangyuan, Sichuan	甘蓝Cabbage	2020-08	23.59±0.31	9.52×10²	25.48±0.28	2.84×10²	26.11±0.52
5	湖北省恩施州 Enshi, Hubei	大白菜Chinese cabbage	2020-07	17.00±0.50	6.44×10⁴	20.47±0.70	6.97×10³	50.48±0.63
6	湖北省恩施州 Enshi, Hubei	大白菜Chinese cabbage	2020-07	19.95±0.89	9.74×10³	20.95±0.53	5.14×10³	44.85±0.63
7	山东省青岛市 Qingdao, Shandong	大白菜Chinese cabbage	2020-07	23.08±0.42	1.31×10³	25.03±0.33	3.78×10²	26.36±0.72
8	山东省青岛市 Qingdao, Shandong	小白菜Pakchoi	2020-07	25.21±0.89	3.37×10²	28.88±0.30	32.35	0
9	河南省南阳市 Nanyang, Henan	甘蓝Cabbage	2020-08	20.68±0.77	6.12×10³	24.55±0.41	7.86×10²	27.94±0.58
10	江苏省无锡市 Wuxi, Jiangsu	甘蓝Cabbage	2020-09	24.80±0.67	4.39×10²	26.71±0.23	1.29×10²	20.07±0.32
11	辽宁省沈阳市 Shenyang, Liaoning	大白菜Chinese cabbage	2020-10	24.14±0.79	6.70×10²	25.95±0.55	2.10×10²	21.62±0.60
其余14份土壤样本 The other 14 samples				>35.00	0	>35.00	0	0