柑橘黑点病菌(Diaporthe citri)快速分子检测技术

doi:10.3969/j.issn.1004-1524.2022.07.13

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (7): 1457-1465.DOI: 10.3969/j.issn.1004-1524.2022.07.13

柑橘黑点病菌(Diaporthe citri)快速分子检测技术

曾雅婷(), 熊桃, 李红叶()

浙江大学生物技术研究所,浙江杭州 310058

收稿日期:2021-01-22 出版日期:2022-07-25 发布日期:2022-07-26
通讯作者: 李红叶
作者简介:* 李红叶,E-mail: hyli@zju.edu.cn
曾雅婷(1997—),女,湖南娄底人,硕士研究生,研究方向为资源利用与植物保护。E-mail: 3157867950@qq.com
基金资助:
浙江省重点研发计划(2019C02022);现代农业(柑橘)产业技术体系(CARS-26)

Rapid molecular detection of Diaporthe citri, the pathogen of citrus melanose

ZENG Yating(), XIONG Tao, LI Hongye()

Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China

Received:2021-01-22 Online:2022-07-25 Published:2022-07-26
Contact: LI Hongye

摘要/Abstract

摘要：

柑橘黑点病是一种严重危害柑橘生产的世界性真菌病害,快速及时地检测出柑橘黑点病病菌(Diaporthe citri)对柑橘黑点病早期诊断和科学防控具有重要的实践意义。本研究基于β-微管蛋白序列,设计柑橘黑点病病菌的特异引物对,基于常规PCR和SYBR Green I实时荧光定量PCR(qRT-PCR)建立了快速分子检测体系,对柑橘黑点病病菌的菌丝和典型带病叶片进行检测验证。结果发现,在常规PCR检测中,该引物可从柑橘黑点病病菌获得244 bp的特异性扩增片段,检测灵敏度达0.78 ng·μL^-1;在实时qRT-PCR中,该引物也只有在柑橘黑点病病菌中获得唯一的产物吸收峰,检测灵敏度达0.35 ng·μL^-1。利用PCR检测体系对发病黑点叶片进行检测,结果显示,常规PCR的阳性检出率为30%,qRT-PCR的阳性检出率为60%,表明qRT-PCR比常规PCR的灵敏度更高。因此,研究设计的柑橘黑点病病菌特异性引物,以及所建立的常规和实时qRT-PCR检测方法具有速度快、特异性强等特点,可以用于柑橘黑点病病菌的分子鉴定,对病害诊断具有参考价值。

关键词: 柑橘黑点病菌, β-微管蛋白序列, SYBR Green I实时荧光定量PCR, 常规PCR检测

Abstract:

Citrus melanose is a worldwide fungal disease which seriously harms citrus production. Rapid detection of Diaporthe citri, the pathogen of citrus melanose, is of great practical significance for early diagnosis and scientific prevention and control of citrus melanose. In this study, a specific primer pair for D.citri was designed based on β-tubulin gene sequence, and a rapid molecular detection system was established based on conventional PCR and SYBR Green I Real-time PCR(qRT-PCR) to detect and verify the mycelia and typical infected leaves of D.citri. The results showed that in conventional PCR, the primers could obtain a 244 bp specific amplification fragment from D.citri, and the detection sensitivity was up to 0.78 ng·μL^-1. In qRT-PCR, the primers could only obtained a unique product absorpation peak from D.citri, and the detection sensitivity was up to 0.35 ng·μL^-1.The typical infected leaves were detected by PCR detection system. The results showed that the positive detection rate of conventional PCR was 30%, and that of qRT-PCR was 60%, indicating that qRT-PCR was more sensitive than conventional PCR. Therefore, the specific primers designed in this study and the established conventional and qRT-PCR detection method have the advantages of fast speed, strong specificity and high sensitivity, which can be used for molecular identification of D. citri and also have reference value for disease diagnosis.

Key words: Diaporthe citri, β-tubulin sequence, SYBR Green I Real-time PCR, Conventional PCR detection

中图分类号:

S436.66

曾雅婷, 熊桃, 李红叶. 柑橘黑点病菌(Diaporthe citri)快速分子检测技术[J]. 浙江农业学报, 2022, 34(7): 1457-1465.

ZENG Yating, XIONG Tao, LI Hongye. Rapid molecular detection of Diaporthe citri, the pathogen of citrus melanose[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1457-1465.

图/表 9

表1 供试菌株信息

Table 1 Information of tested strains

菌株 Strains	寄主 Host	器官和症状 Organ and symptom	来源地 Origin	基因号 Gene code
Diaporthe citri	本地早 Citrus reticulata cv. Bendizao	果实,黑点 Fruit, melanose	浙江黄岩 Huangyan, Zhejiang	KJ490395
D. citri	椪柑 C. reticulata Blanco	果实,褐色蒂腐 Fruit, brown blossom-end rot	浙江丽水 Lishui, Zhejiang	KJ490399
D. citri	椪柑 C. reticulata Blanco	果实,黑点 Fruit, melanose	湖南湘西 Xiangxi, Hunan	KJ490400
D. biguttulata	柠檬 C. limon(L.) Burm. f.	枝干,无症状 Branch, symptomless	云南瑞丽 Ruili, Yunnan	KJ490403
D. unshiuensis	金橘 Fortunella margarita(L.) Swingle	嫩枝,无症状 Twig, symptomless	广西桂林 Guilin, Guangxi	KJ490406
D. arecae	琯溪蜜柚 C. grandis(L.) Osbeck	嫩枝,无症状 Twig, symptomless	福建漳州 Zhangzhou, Fujian	KJ490412
D. biconispora	琯溪蜜柚 C. grandis(L.) Osbeck	枝干,无症状 Branch, symptomless	福建漳州 Zhangzhou, Fujian	KJ490418
D. sojae	温州蜜柑 C. unshiu	嫩枝,无症状 Twig, symptomless	浙江衢州 Zhangzhou, Fujian	KJ490424
D. endophytica	柠檬 C. limon(L.) Burm. f.	枝干,无症状 Branch, symptomless	云南瑞丽 Ruili, Yunnan	KJ490450
D. hongkongensis	南丰蜜桔 C. reticulata cv. Nanfengmiju	嫩枝,无症状 Twig, symptomless	江西南丰 Nanfeng, Jiangxi	KJ490432
D. citriasiana	沙田柚 C. maxima(Burm.) Merr. cv. Shatian Yu.	叶片,非典型病斑 Leaf, anonymous spot	浙江江山 Jiangshan, Zhejiang	KJ490438
D. citrichinensis	金橘 F. margarita(L.) Swingle	枝干,无症状 Branch, symptomless	广西桂林 Guilin, Guangxi	KJ490441
Diaporthe sp.	金橘 F. margarita(L.) Swingle	枝干,无症状 Branch, symptomless	广西桂林 Guilin, Guangxi	KJ490442
D. discoidispora	温州蜜柑 C. unshiu	嫩枝,无症状 Twig, symptomless	江西谭东 Tandong, Jiangxi	KJ490445
D. eres	金橘 F. margarita(L.) Swingle	枝干,无症状 Branch, symptomless	广西桂林 Guilin, Guangxi	KJ490440
D. subclavata	温州蜜柑 C. unshiu	叶片,类似疮痂 Leaf, scab-like	福建永春 Yongchun, Fujian	KJ490451
D. multigutullata	琯溪蜜柚 C. grandis(L.) Osbeck	枝干,无症状 Branch, symptomless	福建漳州 Zhangzhou, Fujian	KJ490454

表1 供试菌株信息

Table 1 Information of tested strains

菌株 Strains	寄主 Host	器官和症状 Organ and symptom	来源地 Origin	基因号 Gene code
Diaporthe citri	本地早 Citrus reticulata cv. Bendizao	果实,黑点 Fruit, melanose	浙江黄岩 Huangyan, Zhejiang	KJ490395
D. citri	椪柑 C. reticulata Blanco	果实,褐色蒂腐 Fruit, brown blossom-end rot	浙江丽水 Lishui, Zhejiang	KJ490399
D. citri	椪柑 C. reticulata Blanco	果实,黑点 Fruit, melanose	湖南湘西 Xiangxi, Hunan	KJ490400
D. biguttulata	柠檬 C. limon(L.) Burm. f.	枝干,无症状 Branch, symptomless	云南瑞丽 Ruili, Yunnan	KJ490403
D. unshiuensis	金橘 Fortunella margarita(L.) Swingle	嫩枝,无症状 Twig, symptomless	广西桂林 Guilin, Guangxi	KJ490406
D. arecae	琯溪蜜柚 C. grandis(L.) Osbeck	嫩枝,无症状 Twig, symptomless	福建漳州 Zhangzhou, Fujian	KJ490412
D. biconispora	琯溪蜜柚 C. grandis(L.) Osbeck	枝干,无症状 Branch, symptomless	福建漳州 Zhangzhou, Fujian	KJ490418
D. sojae	温州蜜柑 C. unshiu	嫩枝,无症状 Twig, symptomless	浙江衢州 Zhangzhou, Fujian	KJ490424
D. endophytica	柠檬 C. limon(L.) Burm. f.	枝干,无症状 Branch, symptomless	云南瑞丽 Ruili, Yunnan	KJ490450
D. hongkongensis	南丰蜜桔 C. reticulata cv. Nanfengmiju	嫩枝,无症状 Twig, symptomless	江西南丰 Nanfeng, Jiangxi	KJ490432
D. citriasiana	沙田柚 C. maxima(Burm.) Merr. cv. Shatian Yu.	叶片,非典型病斑 Leaf, anonymous spot	浙江江山 Jiangshan, Zhejiang	KJ490438
D. citrichinensis	金橘 F. margarita(L.) Swingle	枝干,无症状 Branch, symptomless	广西桂林 Guilin, Guangxi	KJ490441
Diaporthe sp.	金橘 F. margarita(L.) Swingle	枝干,无症状 Branch, symptomless	广西桂林 Guilin, Guangxi	KJ490442
D. discoidispora	温州蜜柑 C. unshiu	嫩枝,无症状 Twig, symptomless	江西谭东 Tandong, Jiangxi	KJ490445
D. eres	金橘 F. margarita(L.) Swingle	枝干,无症状 Branch, symptomless	广西桂林 Guilin, Guangxi	KJ490440
D. subclavata	温州蜜柑 C. unshiu	叶片,类似疮痂 Leaf, scab-like	福建永春 Yongchun, Fujian	KJ490451
D. multigutullata	琯溪蜜柚 C. grandis(L.) Osbeck	枝干,无症状 Branch, symptomless	福建漳州 Zhangzhou, Fujian	KJ490454

图1 Diaporthe citri与其近似种TUB基因的核苷酸序列比对

Fig.1 Nucleotide sequence alignments of the TUB gene for Diaporthe citri and its similar species

图2 DC-F/DC-R引物对D. citri的特异性扩增

Fig.2 Specific amplification of primers pair DC-F/DC-R for D. citri M,DNA 2 000 plus Marker;1,Diaporthe citri;2,D. biguttulata;3,D. unshiuensis;4,D. arecae;5,D. biconispora;6,D. sojae;7,D. citri;8,D. endophytica;9,D. hongkongensis;10,D. citriasiana;11,D. citrichinensis;12,Diaporthesp;13,D. citri;14,D. discoidispora;15,D. eres;16,D. subclavata;17,D. multigutullata;18,ddH2O。

图3 引物对D. citri的qRT-PCR扩增的熔解曲线

Fig.3 Melting curves of qRT-PCR amplification of D. citri based on primer

图4 运用常规PCR检测引物Dc-F/Dc-R的灵敏度

Fig.4 Sensitivity of conventional PCR detection using primer pair Dc-F/Dc-R M,DNA 2 000 plus marker; 1,45 ng·μL-1; 2,20 ng·μL-1; 3,8.89 ng·μL-1; 4,3.95 ng·μL-1; 5,1.76 ng·μL-1; 6,0.78 ng·μL-1; 7,0.35 ng·μL-1; 8,0.15 ng·μL-1; 9,0.07 ng·μL-1; 10,0.03 ng·μL-1; 11,0.01 ng·μL-1; 12,ddH2O。

图5 运用qRT-PCR检测引物Dc-F/Dc-R的灵敏度

Fig.5 Sensitivity of qRT-PCR detection using primer pair Dc-F/Dc-R 1,45 ng·μL-1; 2,20 ng·μL-1; 3,8.89 ng·μL-1; 4,3.95 ng·μL-1; 5,1.76 ng·μL-1; 6,0.78 ng·μL-1; 7,0.35 ng·μL-1; 8,0.15 ng·μL-1; 9,0.07 ng·μL-1; 10,0.03 ng·μL-1;11,0.01 ng·μL-1; 12,ddH2O。

图6 qRT-PCR标准曲线

Fig.6 Standard curve for qRT-PCR

图7 典型黑点病叶片中D. citri的常规PCR检测验证 M,DL2 000 plus DNA marker; 1,阳性对照(培养D. citri提取的DNA); 2~11,疑似黑点病病斑叶片; 12,ddH2O; 13,健康的柑橘叶片。

Fig.7 Conventional PCR detection of typical D. citri-infected citrus leaves M, DL2 000 plus DNA Marker, 1, Positive control (DNA extracted from lab-cultured Diaporthe citri); 2-11, Citrus leaves showing melanose-like symptom; 12, ddH2O; 13, Healthy citrus leaves.

图8 典型黑点病叶片中的D. citri的qRT-PCR检测验证 1,阳性对照(培养D. citri提取的DNA); 2~11,典型黑点病病斑的柑橘叶片; 12,ddH2O; 13,健康的柑橘叶片。

Fig.8 qRT-PCR detection of typical D.citri-infected citrus leaves 1, Positive control (DNA extracted from lab-cultured D. citri); 2-11, Citrus leaves showing melanose-like symptom; 12, ddH2O; 13, Healthy citrus leaves.

参考文献 17

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柑橘黑点病菌(Diaporthe citri)快速分子检测技术

Rapid molecular detection of Diaporthe citri, the pathogen of citrus melanose

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