Establishment of a TaqMan Real-Time Fluorescence Quantitative PCR Detection Method for Strawberry Root Rot Pathogen Dactylonectria spp.

doi:10.16420/j.issn.0513-353x.2025-0125

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (3): 857-866.doi: 10.16420/j.issn.0513-353x.2025-0125

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Establishment of a TaqMan Real-Time Fluorescence Quantitative PCR Detection Method for Strawberry Root Rot Pathogen Dactylonectria spp.

ZHANG Jiaqi, DONG Lihong, FU Yifan, WANG Peipei^*(), GUO Qinggang, MA Ping

Key Laboratory of Integrated Pest Management on Crops in Northern Region of North China，Ministry of Agriculture and Rural Affairs，IPM Innovation Center of Hebei Province，International Science and Technology Joint Research Center on IPM of Hebei Province，Plant Protection Institute，Hebei Academy of Agriculture and Forestry Sciences，Baoding，Hebei 071000，China

Received:2025-06-06 Revised:2025-12-09 Online:2026-03-25 Published:2026-03-20
Contact: WANG Peipei

Abstract

Abstract:

Dactylonectria spp. are among the most destructive soil-borne fungal pathogens causing strawberry root rot. To achieve rapid and accurate detection of this pathogen，this study developed a TaqMan-based real-time fluorescence quantitative PCR（qPCR）assay targeting the conserved n-ethylammeline chlorohydrolase gene sequence of Dactylonectria spp. Specific primers Da6-F/Da6-R and a TaqMan probe Da6-P were designed and validated. This method exhibits strong specificity and high sensitivity，could specifically distinguish Dactylonectria spp. from other common strawberry soil-borne fungal pathogens，as well as healthy strawberry tissues and root zone soils. The detection sensitivity were 10 copies · μL^-1 for plasmid DNA，0.5 pg · μL^-1 for genomic DNA，and 10³ spores · g^-1 in soil samples. Artificial soil inoculation experiments revealed disease onset（30% disease incidence）occurred at a threshold of 9.17 × 10³ copies · g^-1in root zone soil. Comparative analysis of 29 naturally infected samples showed significant agreement between qPCR and conventional tissue plating methods（Cohen’s Kappa value = 0.592，P = 0.0002）.

Key words: strawberry, root rot, Dactylonectria spp., qPCR

ZHANG Jiaqi, DONG Lihong, FU Yifan, WANG Peipei, GUO Qinggang, MA Ping. Establishment of a TaqMan Real-Time Fluorescence Quantitative PCR Detection Method for Strawberry Root Rot Pathogen Dactylonectria spp.[J]. Acta Horticulturae Sinica, 2026, 53(3): 857-866.

Figures/Tables 8

References 22

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序号 No.	编号 Isolate code	DNA种类 DNA species	来源 Geographic region	C_t
1	35-2-1-1	Dactylonectria alcacerensis	衡水武强Wuqiang，Hengshui	25.60
2	7-2-1-2	D. alcacerensis	邢台巨鹿Julu，Xingtai	24.36
3	18-2-2-2	D. novozelandica	保定满城Mancheng，Baoding	26.37
4	22-2-1-1	D. novozelandica	邯郸馆陶Guantao，Handan	25.22
5	HB3-4	D. torresensis	衡水桃城Taocheng，Hengshui	25.63
6	32-2-8-2	D. pauciseptata	邯郸永年Yongnian，Handan	25.38
7	T2-3-1	D. macodidyma	唐山滦州Luanzhou，Tangshan	30.18
8	26-2-1-2	腐皮镰刀菌 Fusarium solani	廊坊广阳Guangyang，Langfang	-
9	42-5-1	胶孢炭疽菌 Colletotrichum gloeosporioides	保定满城Mancheng，Baoding	-
10	25-2-2-1	尖孢镰刀菌草莓专化型 F. oxysporum f. sp. fragariae	邯郸魏县WeiXian，Handan	-
11	50c-1	森林腐霉 Pythium sylvaticum	沧州河间Hejian，Cangzhou	-
12	37-2-1-1	旋柄腐霉 Phytopythium helicoides	沧州肃宁Suning，Cangzhou	-
13	43A-1-1	新拟盘多毛孢玫瑰变种 Neopestalotiopsis rosea	保定满城Mancheng，Baoding	-
14	2345-1	新拟盘多毛孢玫瑰变种 N. rosea	保定顺平Shunping，Baoding	-
15	7-1-4-1	黄瓜织球壳菌 Plectosphaerella cucumerina	邢台巨鹿Julu，Xingtai	-
16	HS-1	大田中健康草莓植株根围土 Rhizosphere soil from filed-grown healthy strawberry plants	保定满城Mancheng，Baoding	-
17	HS-2	温室种植土 Green house growing medium	保定莲池区Lianchi，Baoding	-
18	DP-1	组织分离确定的带病植株根茎组织1 Crown tissue 1 from a diseased plant identified via tissue isolation	保定顺平Shunping，Baoding	32.39
19	DP-2	组织分离确定的带病植株根茎组织2 Crown tissue 2 from a diseased plant identified via tissue isolation	保定满城Mancheng，Baoding	30.72
20	DP-3	组织分离确定的带病植株根茎组织3 Crown tissue 3 from a diseased plant identified via tissue isolation	保定满城Mancheng，Baoding	30.92
21	CM-L	健康草莓植株叶片组织 Leaf tissue from healthy strawberry plants	保定顺平Shunping，Baoding	-
22	CM-N	健康草莓植株根茎组织 Crown tissue from healthy strawberry plants	保定顺平Shunping，Baoding	-

序号 No.	编号 Isolate code	DNA种类 DNA species	来源 Geographic region	C_t
1	35-2-1-1	Dactylonectria alcacerensis	衡水武强Wuqiang，Hengshui	25.60
2	7-2-1-2	D. alcacerensis	邢台巨鹿Julu，Xingtai	24.36
3	18-2-2-2	D. novozelandica	保定满城Mancheng，Baoding	26.37
4	22-2-1-1	D. novozelandica	邯郸馆陶Guantao，Handan	25.22
5	HB3-4	D. torresensis	衡水桃城Taocheng，Hengshui	25.63
6	32-2-8-2	D. pauciseptata	邯郸永年Yongnian，Handan	25.38
7	T2-3-1	D. macodidyma	唐山滦州Luanzhou，Tangshan	30.18
8	26-2-1-2	腐皮镰刀菌 Fusarium solani	廊坊广阳Guangyang，Langfang	-
9	42-5-1	胶孢炭疽菌 Colletotrichum gloeosporioides	保定满城Mancheng，Baoding	-
10	25-2-2-1	尖孢镰刀菌草莓专化型 F. oxysporum f. sp. fragariae	邯郸魏县WeiXian，Handan	-
11	50c-1	森林腐霉 Pythium sylvaticum	沧州河间Hejian，Cangzhou	-
12	37-2-1-1	旋柄腐霉 Phytopythium helicoides	沧州肃宁Suning，Cangzhou	-
13	43A-1-1	新拟盘多毛孢玫瑰变种 Neopestalotiopsis rosea	保定满城Mancheng，Baoding	-
14	2345-1	新拟盘多毛孢玫瑰变种 N. rosea	保定顺平Shunping，Baoding	-
15	7-1-4-1	黄瓜织球壳菌 Plectosphaerella cucumerina	邢台巨鹿Julu，Xingtai	-
16	HS-1	大田中健康草莓植株根围土 Rhizosphere soil from filed-grown healthy strawberry plants	保定满城Mancheng，Baoding	-
17	HS-2	温室种植土 Green house growing medium	保定莲池区Lianchi，Baoding	-
18	DP-1	组织分离确定的带病植株根茎组织1 Crown tissue 1 from a diseased plant identified via tissue isolation	保定顺平Shunping，Baoding	32.39
19	DP-2	组织分离确定的带病植株根茎组织2 Crown tissue 2 from a diseased plant identified via tissue isolation	保定满城Mancheng，Baoding	30.72
20	DP-3	组织分离确定的带病植株根茎组织3 Crown tissue 3 from a diseased plant identified via tissue isolation	保定满城Mancheng，Baoding	30.92
21	CM-L	健康草莓植株叶片组织 Leaf tissue from healthy strawberry plants	保定顺平Shunping，Baoding	-
22	CM-N	健康草莓植株根茎组织 Crown tissue from healthy strawberry plants	保定顺平Shunping，Baoding	-

孢子浓度/（spore · g^-1） Spore concentration	检测值/（copies · g^-1） Resultes of detection	发病率/% Disease incidence
1 × 10⁶	（6.98 ± 0.26）× 10⁶	53.3 ± 12.5
1 × 10⁵	（1.81 ± 0.04）× 10⁵	36.7 ± 4.7
1 × 10⁴	（1.55 ± 0.16）× 10⁴	36.7 ± 4.7
1 × 10³	（9.17 ± 0.98）× 10³	30.0 ± 8.2
1 × 10²	—	3.30 ± 4.7

孢子浓度/（spore · g^-1） Spore concentration	检测值/（copies · g^-1） Resultes of detection	发病率/% Disease incidence
1 × 10⁶	（6.98 ± 0.26）× 10⁶	53.3 ± 12.5
1 × 10⁵	（1.81 ± 0.04）× 10⁵	36.7 ± 4.7
1 × 10⁴	（1.55 ± 0.16）× 10⁴	36.7 ± 4.7
1 × 10³	（9.17 ± 0.98）× 10³	30.0 ± 8.2
1 × 10²	—	3.30 ± 4.7

采样点 Geographic region	编号 Sample number	qPCR		组织平板分离 Tissue plating method
采样点 Geographic region	编号 Sample number	检测值/（copies · g^-1） Detection value	检测结果 Detection result	组织分离结果 Tissue plating result	备注^a Note
石家庄平山	SJZ1	2.96 × 10⁵	+	+
Pingshan，Shijiazhuang	SJZ2	4.08 × 10⁵	+	+
石家庄栾城	SJZ3	6.99 × 10⁵	+	+
Luancheng，Shijiazhuang	SJZ4	1.41 × 10⁵	+	+
	SJZ5	1.44 × 10⁵	+	+
	SJZ6	1.35 × 10⁵	+	+
	SJZ7	-	-	-	尖孢镰刀菌 Fusarium oxysporum
	SJZ8	2.38 × 10⁵	+	-	尖孢镰刀菌 F. oxysporum
	SJZ9	8.21 × 10⁵	+	+
石家庄辛集 Xinji，Shijiazhuang	SJZ10	8.12 × 10⁵	+	+
保定顺平 Shunping，Baoding	BD1	1.38 × 10⁶	+	+
保定满城 Mancheng，Baoding	BD2	6.03 × 10⁵	+	+
	BD3	5.58 × 10⁵	+	+
沧州吴桥 Wuqiao，Cangzhou	CZ1	2.94 × 10⁶	+	+
	CZ2	7.61 × 10⁵	+	-	腐皮镰刀菌 F. solani
	CZ3	4.81 × 10⁵	+	+
沧州盐山 Yanshan，Cangzhou	CZ4	3.98 × 10⁵	+	+
邯郸馆陶 Guantao，Handan	HD1	6.22 × 10⁵	+	+
	HD2	8.15 × 10⁵	+	+
邯郸永年 Yongnian，Handan	HD3	4.27 × 10⁵	+	-	尖孢镰刀菌 F. oxysporum
衡水饶阳 Raoyang，Hengshui	HS1	6.76 × 10⁵	+	-	炭疽菌Colletotrichum gloeosporioides
衡水枣强 Zaoqiang，Hengshui	HS2	2.86 × 10⁵	+	+
	HS3	-	-	-	炭疽菌 C. gloeosporioides
廊坊广阳 Guangyang，Langfang	LF1	-	-	-	炭疽菌 C. gloeosporioides
	LF2	1.01 × 10⁵	+	+
	LF3	4.46 × 10⁵	+	+
	LF4	3.77 × 10⁵	+	+
唐山开平 Kaiping，Tangshan	TS1	-	-	-	尖孢镰刀菌 F. oxysporum
邢台巨鹿 Julu，Xingtai	XT1	2.49 × 10⁵	+	+

Establishment of a TaqMan Real-Time Fluorescence Quantitative PCR Detection Method for Strawberry Root Rot Pathogen Dactylonectria spp.

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