Establishment and Application of Real-Time PCR for Quantitative Detection of Fusarium oxysporum f. sp. lycopersici

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

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (11): 2479-2488.doi: 10.16420/j.issn.0513-353x.2021-0606

• New Technology and New Methods • Previous Articles Next Articles

Establishment and Application of Real-Time PCR for Quantitative Detection of Fusarium oxysporum f. sp. lycopersici

MENG Zhen¹, ZHANG Weiping¹, WANG Ying¹, LI Long³, JI Xiaoxue¹, DONG Bei²^,^**(), QIAO Kang¹^,^**()

¹College of Plant Protection，Shandong Agricultural University，Tai’an，Shandong 271018，China
²Jinan Academy of Agricultural Sciences，Jinan 250316，China
³Tai’an Institute for Food and Drug Control，Tai’an，Shandong 271000，China

Received:2022-06-24 Revised:2022-08-16 Online:2022-11-25 Published:2022-11-25
Contact: DONG Bei,QIAO Kang E-mail:1985dongbei@163.com;qiaokang@sdau.edu.cn

Abstract

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

CLC Number:

S641.2

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.

Figures/Tables 8

References 31

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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

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

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

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

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

Establishment and Application of Real-Time PCR for Quantitative Detection of Fusarium oxysporum f. sp. lycopersici

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