草莓根腐病病原足赤壳菌TaqMan实时荧光定量PCR检测方法的建立

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

摘要/Abstract

摘要：

足赤壳菌（Dactylonectria spp.）是引起草莓根腐病的重要土传病原真菌之一。为实现该病原菌的快速、准确检测，基于足赤壳菌保守n-ethylammeline chlorohydrolase基因序列，设计特异性引物Da6-F/Da6-R及TaqMan探针Da6-P，建立了足赤壳菌TaqMan实时荧光定量PCR（qPCR）检测方法。该检测方法特异性强、灵敏度高，能特异性区分足赤壳菌与其他常见草莓土传病原真菌及健康草莓组织或根围土壤；对质粒DNA、病原菌基因组DNA和土壤中孢子的检测灵敏度分别为10 copies · μL^-1、0.5 pg · μL^-1和10³孢子 · g^-1。人工拌土下，土壤发病阈值为9.17 × 10³ copies · g^-1（发病率30%）。通过检测29份自然发病样本，根围土壤qPCR检测和组织平板分离结果一致性达极显著水平（Cohen’s Kappa值0.592，P = 0.0002）。

关键词: 草莓, 根腐病, 足赤壳菌, 实时荧光定量PCR

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

张家齐, 董丽红, 付一帆, 王培培, 郭庆港, 马平. 草莓根腐病病原足赤壳菌TaqMan实时荧光定量PCR检测方法的建立[J]. 园艺学报, 2026, 53(3): 857-866.

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.

图/表 8

参考文献 22

[1]	Botha A, Denman S, Lamprecht S C, Mazzola M, Crous P W. 2003. Characterisation and pathogenicity of Rhizoctonia isolates associated with black root rot of strawberries in the Western Cape Province,South Africa. Australasian Plant Pathology,32:195-201.
[2]	Cao Xueren, Zhou Yilin. 2020. Research progress in quantitative detection of plant pathogens using PCR technique. Plant Protection, 46 (4):7-11. (in Chinese)
	曹学仁, 周益林. 2020. 基于PCR技术的植物病原菌分子定量检测技术研究进展. 植物保护, 46 (4):7-11.
[3]	Chen Q, Yin S L, Zhang X G, Ma X Y, Zhong S, Zhang G Z. 2021. Dactylonectria species associated with black root rot of strawberry in China. Australasian Plant Pathology,43-46:1-11.
[4]	Cheng Yingchao, Kang Huajun, Shi Yanxia, Chai Ali, Zhang Hongjie, Xie Xuewen, Li Baoju. 2018. Development and application of real-time fluorescent quantitative PCR for detection of Phytophthora capsica. Acta Horticulturae Sinica, 45 (5):997-1006. (in Chinese) doi: 10.16420/j.issn.0513-353x.2017-0862
	程颖超, 康华军, 石延霞, 柴阿丽, 张红杰, 谢学文, 李宝聚. 2018. 辣椒疫霉菌RT-PCR检测技术的建立及应用. 园艺学报, 45 (5):997-1006. doi: 10.16420/j.issn.0513-353x.2017-0862
[5]	Fang Zhongda. 1998. Research method for plant pathology. 2nd ed. Beijing: China Agriculture Press. (in Chinese)
	方中达. 1998. 植病研究方法. 2版. 北京: 中国农业出版社.
[6]	Golzar H, Phillips D, Mack S. 2007. Occurrence of strawberry root and crown rot in Western Australia. Australasian Plant Disease Notes,2:145-147.
[7]	Haegi A, Luongo L, Vitale S, Tizzani L, Belisario A. 2024. TaqMan qPCR detection and quantification of Phytophthora cinnamomi in soil and plant tissues for walnut disease management. Agriculture, 14 (7):999. doi: 10.3390/agriculture14070999 URL
[8]	Katoh K, Standley D M. 2013. MAFFT multiple sequence alignment software version 7:improvements in performance and usability. Molecular Biology and Evolution,30:772-780.
[9]	Landis J R, Koch G G. 1977. Measurement of observer agreement for categorical data. Biometrics,33:159-174.
[10]	Lastra E D L, Maria J B, Santos B D L, Miranda L, Vela-Delgado M D, Capote N. 2018. A TaqMan real-time polymerase chain reaction assay for accurate detection and quantification of Fusarium solani in strawberry plants and soil. Scientia Horticulturae,237:128-134.
[11]	Liang Ziying, Liu Fang. 2020. Research progress on real-time quantitative PCR technology and its application. Modern Agricultural Science and Technology,(6):1-3,8. (in Chinese)
	梁子英, 刘芳. 2020. 实时荧光定量PCR技术及其应用研究进展. 现代农业科技,(6):1-3,8.
[12]	Löhrer M, Vogel L, Petrescu Dita Singer S, Erwes S, Heupel M, Schaffrath U. 2022. PCR diagnostics for rapid detection of fungi associated with black root rot of strawberries. Journal of Plant Diseases and Protection, 129 (5):1053-1062. doi: 10.1007/s41348-022-00594-8
[13]	Lombard L, vender Merwa A, Groenewald J Z, Crous P W. 2014. Lineages in Nectriaceae:re-evaluating the generic status of Ilyonectria and allied genera. Phytopathologia Mediterranea, 53 (3):340-357.
[14]	Lu J Z, Ehsani R, Shi Y Y, Abdulridha J, de Castro A I, Xu Y J. 2017. Field detection of anthracnose crown rot in strawberry using spectroscopy technology. Computers and Electronics in Agriculture,135:289-299.
[15]	Singh S N, Lal A A. 2024. Efficacy of Pseudomonas Fluorescens and organic amendments against black root rot disease caused by Rhizoctonia solani in strawberry (Fragaria × ananassa Duch.). Journal of Experimental Agriculture International, 46 (10):338-350. doi: 10.9734/jeai/2024/v46i102955 URL
[16]	Sun Aiqing, Wang Lihua, Zhang Yiping, Yang Xiumei, Xu Feng, Su Yan. 2024. Simultaneous detection of cymbidium mosaic virus and odontoglossum ringspot virus by TaqMan duplex real-time fluorescence quantitative PCR. Plant Protection, 50 (2):219-227. (in Chinese)
	孙爱青, 王丽花, 张艺萍, 杨秀梅, 许凤, 苏艳. 2024. TaqMan探针双重实时荧光定量PCR法同步检测兰花中的建兰花叶病毒和齿兰环斑病毒. 植物保护, 50 (2):219-227.
[17]	Sun Qian, Zhang Wei, Yan Jiye, Li Xinghong. 2022. Identification of pathogen causing strawberry Dactylonectria root rot. Acta Phytopathologica Sinica, 52 (2):276-280. (in Chinese) doi: 10.13926/j.cnki.apps.000753
	孙倩, 张玮, 燕继晔, 李兴红. 2022. 草莓根腐病的Dactylonectria病原菌鉴定. 植物病理学报, 52 (2):276-280. doi: 10.13926/j.cnki.apps.000753
[18]	Tewoldemedhin Y T, Mazzola M, Mostert L, McLeod A. 2011. Cylindrocarpon species associated with apple tree roots in South Africa and their quantification using real-time PCR. Europe Joural Plant Pathology,129:637-651.
[19]	Weber R W, Entrop A P. 2017. Dactylonectria torresensis as the main component of the black root rot complex of strawberries and raspberries in Northern Germany. Erwerbs-Obstbau,59:157-169.
[20]	Xu Yingzhi, Zhao Limei, Zu Weixi, Gao Fen. 2024. Fusarium solani causing Astragalus membranaceus var. mongholicus root rot:establishment of RT-qPCR detection method. Chinese Agricultural Science Bulletin, 40 (15):110-116. (in Chinese)
	徐英芝, 赵丽梅, 祖未希, 高芬. 2024. 黄芪根腐病菌腐皮镰刀菌的实时荧光定量PCR检测方法建立. 中国农学通报, 40 (15):110-116. doi: 10.11924/j.issn.1000-6850.casb2023-0496
[21]	Yuen G Y, Schroth M N, Weinhold A R, Hancock J G. 1991. Effects of soil fumigation with methyl bromide and chloropicrin on root health and yield of strawberry. Plant Disease,75:416-420.
[22]	Zhou You, Xie Fangling, Xiao Rong, Zhang Zhengbing, Zhou Xinyu, Zhu Hongjian. 2023. Application of real-time fluorescence quantitative PCR technology in detection of pathogen of rapeseed clubroot in Hunan. Plant Protection, 49 (4):233-238,275. (in Chinese)
	周游, 谢芳玲, 肖蓉, 张政兵, 周鑫钰, 朱宏建. 2023. 实时荧光定量PCR技术在湖南油菜根肿病菌检测中的应用. 植物保护, 49 (4):233-238,275.

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	+	+