多主棒孢SdhB-H278R突变位点AS-real-time PCR 定量检测体系的建立

doi:10.3864/j.issn.0578-1752.2018.24.006

Abstract

Abstract:

【Objective】 The objective of this study is to establish a rapid and efficient allele specific real-time PCR (AS-real-time PCR) method for quantitative detection of the H278R mutation in the SdhB associated with Corynespora cassiicola in cucumber, and to verify the effect. 【Method】 A total of 24 single conidial strains of C. cassiicola were isolated from Daxing, Beijing. The method of mycelial growth inhibition was used to determine the EC₅₀ value to boscalid. Mycelium growth rate, spore outputs and pathogenicity of 4 susceptible (S) and 8 resistant (R) strains were measured in vitro. Primer pair Cc-SdhB-F/R was used to detect the sequence of SdhB and the base change of SdhB. Based on the sequencing results in SdhB by Cc-SdhB-F/R primers, specific primer pair B-H278R-2F/2R14 and internal primers B-H278R-TY-F/R were designed. An AS-real-time PCR reaction system was established and optimized. The specificity, sensitivity and melt curve of the system were also evaluated. DNA and spore suspension containing different proportions of H278R mutation were detected with the optimized reaction system, respectively. 【Result】 The sensitive frequency of C. cassiicola to boscalid was 66.67% in 24 strains and the EC₅₀ values ranged from 0.057 to 0.563 μg·mL ^-1. The resistance frequency of C. cassiicola to boscalid was 33.33% and the EC₅₀ values ranged from 5.395 to 11.710 μg·mL ^-1. Resistant and susceptible strains only significantly differed in terms of mycelium growth rate, and there was a significant negative correlation between the rate of mycelial growth and EC₅₀ values, but no significant difference in spore outputs and pathogenicity. Sequencing analysis showed that all the resistant strains carried SdhB-H278R mutation. The primers B-H278R-2F/2R14 were of great specificity, the specific fragment was amplified from the DNA of H278R strains, but not from the DNA of other fungal strains. The sensitivity of ordinary AS-PCR was 91 pg·μL ^-1, while that of AS-real-time PCR was 9.1 pg·μL ^-1, which was 10 times higher than that of ordinary AS-PCR. The standard curve established by AS-real-time PCR showed a fine linear relationship between ΔCT value and lg of H278R frequency, the correlation coefficient of the standard curve was 0.9857 and with high amplification efficiency (92.59%). The absorption peak of melting curve was single. The internal reference primers B-H278R-TY-F/R and the specific primer B-H278R-2F/2R14 had a single specific peak at 87.81℃and 91.62℃, respectively. The result of mixtures DNA and mixtures spore suspension to verify the standard curve showed a fine linear relationship between expected percentage and detected percentage (R ²=0.9998 and R ²=0.9922). As the proportions of H278R mutation in DNA and spore suspension decreased, the accuracy of the system gradually increased, and the detection limit was 5%. 【Conclusion】 An efficient, quantitative, and rapid AS-real-time PCR detection system was established for the detection of SdhB-H278R mutation site, which can provide a theoretical basis for the SDHIs resistance management.

Key words: cucumber, Corynespora cassiicola, AS-real-time PCR, resistance, SdhB, boscalid

SUN BingXue,SHI YanXia,ZHU FaDI,XIE XueWen,CHAI ALi,LI BaoJu. Establishment of AS-real-time PCR for Quantitatively Detecting the H278R Allele in the SdhB Associated with Corynespora cassiicola in Cucumber[J].Scientia Agricultura Sinica, 2018, 51(24): 4647-4658.

Figures/Tables 13

Fig. 1

Table 1

Fig. 2

Table 2

Sensitivity of tested strains"

序号 Number	菌株编号 Strain code	采集时间 Collection time	采集地 Collection area	EC₅₀ (μg·mL^-1)	毒力回归方程Toxicity regression equation (y=)	相关系数 Coefficient (R²)	突变类型Mutation type	敏感性 Sensitivity
1	HG17031006-1	2017	北京Beijing	9.261	1.2509x+3.7941	0.9995	+	R
2	HG17031006-2	2017	北京Beijing	5.925	1.1082x+4.1659	0.9788	+	R
3	HG17031006-3	2017	北京Beijing	0.482	0.4848x+5.1535	0.9899	-	S
4	HG17031006-4	2017	北京Beijing	0.227	0.4565x+5.2941	0.9910	-	S
5	HG17031006-5	2017	北京Beijing	5.395	0.6544x+4.4397	0.9276	+	R
6	HG17031007-1	2017	北京Beijing	0.337	0.4846x+5.2290	0.9905	-	S
7	HG17031007-2	2017	北京Beijing	0.057	1.6824x+7.0839	0.9777	-	S
8	HG17031007-3	2017	北京Beijing	0.261	2.1962x+4.9202	0.9905	-	S
9	HG17031007-4	2017	北京Beijing	0.383	0.4912x+5.2047	0.9331	-	S
10	HG17031007-5	2017	北京Beijing	0.221	0.5069x+5.9325	0.9746	-	S
11	HG17031008-1	2017	北京Beijing	7.585	0.9496x+4.1644	0.9998	+	R
12	HG17031008-2	2017	北京Beijing	0.535	1.5481x+5.8387	0.9219	-	S
13	HG17031008-3	2017	北京Beijing	6.089	1.0695x+4.1608	0.9998	+	R
14	HG17031008-4	2017	北京Beijing	7.408	1.0990x+4.0752	0.9961	+	R
15	HG17031008-5	2017	北京Beijing	7.309	1.3476x+3.8337	0.9967	+	R
16	HG17031009-1	2017	北京Beijing	0.495	1.1440x+5.6638	0.9038	-	S
17	HG17031009-2	2017	北京Beijing	0.469	1.4358x+6.3270	0.9929	-	S
18	HG17031009-3	2017	北京Beijing	0.550	1.1084x+5.9661	0.9944	-	S
19	HG17031009-4	2017	北京Beijing	0.484	0.5298x+5.1666	0.9791	-	S
20	HG17031009-5	2017	北京Beijing	11.710	0.6667x+4.2859	0.9154	+	R
21	HG17031010-1	2017	北京Beijing	0.216	0.5158x+5.3436	0.9991	-	S
22	HG17031010-2	2017	北京Beijing	0.563	1.3191x+5.6942	0.9225	-	S
23	HG17031010-4	2017	北京Beijing	0.296	0.6165x+5.3264	0.9897	-	S
24	HG14102524-4	2014	河北Hebei	0.316	2.9056x+3.5592	0.9945	-	S
25	HG17031010-5	2017	北京Beijing	0.274	1.0630x+5.7384	0.9129	-	S
26	HG14102430-1	2014	河北Hebei	22.371	0.9039x+3.7312	0.9652	SdhB-H278Y	R
27	HG15050729-5	2015	辽宁Liaoning	3.954	0.9434x+4.4106	0.9960	SdhB-I280V	R
28	HG14102415-1	2014	河北Hebei	1.111	0.8091x+4.4362	0.9967	SdhB-P199S	R
29	HG15050701-1	2015	辽宁Liaoning	19.526	0.7921x+4.0567	0.9967	SdhC-P73S	R

Table 2

Fig. 3

Table 4

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Table 5

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引物名称 Primer name	序列 Sequence	引物长度 Length of primer (bp)	产物长度 Length of product (bp)
Cc-SdhB-F	CACTCTTCTTCGCCATCC	18	1422
Cc-SdhB-R	CATCACACTCACGGTCAC	18	1422
B-H278R-2F	GTGAATACCGCCAGTCCAA	19	244
B-H278R-2R14	CAGTTGAGAATCGCGC	16	244
B-H278R-TY-F	GACCTTTAGGCGAAGTTGC	19	246
B-H278R-TY-R	CTGCTTGTAGAAGAGCGTCAT	21	246

菌株编号 Strain code	敏感性 Sensitivity	菌丝生长速率（11d） Mycelium growth rate (cm·d^-1)	产孢量 Spore outputs (×10⁵/mL)	病斑直径 Lesion diameter (cm)
HG17031007-2	S	0.60±0.09f	0.92±1.38a	0.78±0.09efg
HG17031010-1	S	0.61±0.09f	7.00±4.61e	0.65±0.26de
HG14102524-4	S	0.61±0.01f	3.85±1.99bcd	0.84±0.16fg
HG17031008-2	S	0.49±0.06de	0.33±0.52a	0.38±0.10ab
HG17031006-2	R	0.49±0.04de	5.15±2.41de	0.70±0.26def
HG17031008-4	R	0.45±0.05cd	4.25±2.67bcd	0.75±0.17efg
HG17031006-5	R	0.30±0.03a	2.31±2.06abc	0.56±0.19cd
HG17031008-1	R	0.37±0.02ab	1.33±1.87a	0.45±0.22abc
HG17031008-3	R	0.56±0.08ef	7.46±2.70e	0.91±0.12g
HG17031008-5	R	0.52±0.01de	4.82±2.93cd	0.54±0.05bcd
HG17031006-1	R	0.37±0.02ab	1.85±1.28ab	0.80±0.09efg
HG17031009-5	R	0.40±0.01bc	4.00±2.80bcd	0.33±0.07a

	EC₅₀	生长速率 Growth rate	产孢量 Spore outputs	致病性 Pathogenicity
EC₅₀	1	-0.700*	0.141	-0.21
生长速率Growth rate			0.42	0.457
产孢量Spore outputs				0.51
致病性Pathogenicity				1

预期值Expected percentage (%)		80	40	20	10	5
检测值Detected percentage (%)	DNA	85.95±1.45	44.64±0.69	22.24±0.85	11.86±0.60	6.40±0.24
检测值Detected percentage (%)	孢子Spore	116.45±4.83	48.39±6.15	26.38±4.59	11.86±0.98	6.83±1.43

Establishment of AS-real-time PCR for Quantitatively Detecting the H278R Allele in the SdhB Associated with Corynespora cassiicola in Cucumber

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