番茄枯萎病菌RT-PCR检测技术的建立与应用

doi:10.16420/j.issn.0513-353x.2021-0606

摘要/Abstract

摘要：

根据番茄枯萎病病原菌尖孢镰刀菌番茄专化型（Fusarium oxysporum f. sp. lycopersici，FOL）蛋白激酶基因序列，设计1对番茄枯萎病特异性引物209F/361R，构建番茄枯萎病菌的RT-PCR（real-time PCR）检测体系，并利用该体系定量检测盆栽番茄茎基部病原菌。RT-PCR结果表明该引物只对番茄枯萎病菌有唯一产物吸收峰，对其他供试菌株都未检到荧光信号。利用该引物建立的RT-PCR检测体系线性关系良好，灵敏度为5.76 × 10³ copies · μL^-1，是普通PCR的10倍。人工接种番茄枯萎病菌定植后7 d即可在茎基部检测到病原菌;田间采集的24个自然发病样本中有16个能够检测到番茄枯萎病菌。基于RT-PCR技术构建的番茄枯萎病菌快速检测方法检测速度快、灵敏度高、特异性强、重现性好，能够为该病的早期诊断和流行监测提供理论依据。

关键词: 番茄枯萎病, 尖孢镰刀菌番茄专化型, 实时荧光定量PCR, 早期检测技术

Abstract:

In this study，a pair of specific primers，209F/361R，were designed to establish a real-time PCR（RT-PCR）reaction system of Fusarium oxysporum f. sp. lycopersici（FOL）based on the protein kinase gene sequences of FOL. FOL was detected at the base of tomato stems using this assay. Results of RT-PCR assays showed that only one absorption peak was detected with the primers for F. oxysporum and no absorption peak was produced for other tested strains. The RT-PCR detection system established with the primers showed a good linear relationship. The detection sensitivity was 5.76 × 10³ copies · μL^-1，which was 10 times more sensitive than the conventional PCR. Moreover，FOL was detected at the base of the tomato stems at the 7th day after artificial inoculation. Twenty-four naturally infected tomato samples were collected from field condition and the RT-PCR results showed that FOL was detected in 16 samples. The established RT-PCR detection system for F. oxysporum was fast，highly specific，sensitive，and reproducible，providing scientific tools for epidemic monitoring and early diagnosis of Fusarium wilt of tomato.

Key words: Tomato Fusarium wilt, Fusarium oxysporum f. sp. lycopersici, RT-PCR, early detection technology

中图分类号:

S641.2

孟臻, 张伟萍, 王莹, 李龙, 姬小雪, 董贝, 乔康. 番茄枯萎病菌RT-PCR检测技术的建立与应用[J]. 园艺学报, 2022, 49(11): 2479-2488.

MENG Zhen, ZHANG Weiping, WANG Ying, LI Long, JI Xiaoxue, DONG Bei, QIAO Kang. Establishment and Application of Real-Time PCR for Quantitative Detection of Fusarium oxysporum f. sp. lycopersici[J]. Acta Horticulturae Sinica, 2022, 49(11): 2479-2488.

图/表 8

表1 供试菌株信息

Table 1 Tested strains used in this study

寄主 Host	菌株编号 Number	病原菌名称 Strain name
番茄 Tomato	1 ~ 3	尖孢镰刀菌番茄专化型 Fusarium oxysporum f. sp. lycopersici
	4	黄枝孢菌 Fulvia fulva
	5	灰葡萄孢菌 Botrytis cinerea
	6	尖孢镰刀菌颈腐根腐专化型 Fusarium oxysporum f. sp. radicis-lycopersici
	7	青枯假单胞菌 Pseudomonas solanacearum
	8	立枯丝核菌 Rhizoctonia solani
	9	链格孢菌 Alternaria sesamicola
烟草 Tobacco	10	寄生疫霉菌 Phytophthora parasitica var. nicotianae
小麦 Wheat	11	禾谷镰孢菌 Fusarium graminearum
生防菌 Biocontrol bacteria	12	甲基营养型芽孢杆菌TA-1 Bacillus methylotrophicus TA-1

图1 普通PCR检测引物特异性结果 M：DNA marker;1-3：尖孢镰刀菌番茄专化型;4：黄枝孢菌;5：灰葡萄孢菌;6：尖孢镰刀菌颈腐根腐专化型;7：青枯假单胞菌;8：立枯丝核菌;9：链格孢菌;10：甲基营养型芽孢杆菌;11：寄生疫霉菌;12：禾谷镰孢菌;N：阴性对照ddH2O。

Fig. 1 Results of primer-specific detection by conventional PCR M：DNA marker;1-3：Fusarium oxysporum f. sp. lycopersici;4：Fulvia fulva;5：Botrytis cinerea;6：Fusarium oxysporum f. sp. radicis-lycopersici;7：Pseudomonas solanacearum;8：Rhizoctonia solani;9：Alternaria sesamicola;10：Bacillus methylotrophicus;11：Phytophthora parasitica var. nicotianae;12：Fusarium graminearum;N：ddH2O（Negative control）.

图2 实时荧光定量PCR熔解曲线

Fig. 2 Melting curves of real-time PCR amplification

图3 普通PCR检测引物灵敏度

Fig. 3 Examination sensitivity of primer by conventional PCR

图4 实时荧光定量PCR检测引物灵敏度 1-7：质粒浓度为5.76 × 109 ~ 5.76 × 103 copies · μL-1。

Fig. 4 Sensitivity of real-time PCR 1-7：Plasmid DNA 5.76×109 ~ 5.76 × 103 copies · μL-1.

图5 番茄枯萎病菌实时荧光定量PCR标准曲线

Fig. 5 Real-time PCR standard curve of Fusarium oxysporum f. sp. lycopersici

表2 盆栽番茄枯萎病菌实时荧光定量PCR检测结果及病情指数

Table 2 Results of real-time PCR detection and disease index of Fusarium wilt of tomato in greenhouse experiment

接种后时间/ d Days after inoculation	病情指数 Disease index	Ct值 Ct value	带菌量/（copies · μL^-1） Quantity of FOL
7	0	24.76	1.10 × 10⁴
14	42.59	21.40	1.36 × 10⁵

表3 田间自然发病样本番茄枯萎病菌实时荧光定量PCR检测结果

Table 3 Results of real-time PCR detection of Fusarium wilt of tomato samples under the natural field condition

症状 Symptom	样本编号 Sample code	带菌量/（copies · μL^-1） Quantity of FOL	症状 Symptom	样本编号 Sample code	带菌量/（copies · μL^-1） Quantity of FOL
明显症状	1	1.99 × 10⁴	疑似症状	9	5.76 × 10³
Obvious symptom	2	1.26 × 10⁵	Suspect symptom	10	6.42 × 10³
	3	1.35 × 10⁵		11	7.02 × 10³
	4	1.48 × 10⁵		12	7.43 × 10³
	5	2.43 × 10⁵		13	8.15 × 10³
	6	3.54 × 10⁵		14	8.43 × 10³
	7	4.78 × 10⁵		15	2.01 × 10⁴
	8	6.94 × 10⁵		16	2.31 × 10⁴

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