A LAMP Assay for Rapid Detection of Stem Rot Fusarium oxysporum on Succulent Plant, Echeveria Perle von Nürnberg
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摘要:
目的 建立一种景天科多肉植物红心莲茎腐病菌尖孢镰刀菌（Fusarium oxysporum）快速、简便、灵敏的LAMP可视化检测方法，在茎腐病发生初期实现病原菌的快速准确检测，为病害的早期监测、诊断及防治提供依据。 方法 以EF-1a（Elongation factor-1α）基因序列为靶序列，设计LAMP（Loopmediated isothermal amplification）特异性引物；以红心莲尖孢镰刀菌DNA为模板，优化反应温度和反应时间，建立LAMP检测反应体系，开展特异性和灵敏度验证及田间病株检测。 结果 建立的LAMP反应体系可有效检测出多肉植物红心莲茎腐病尖孢镰刀菌，最佳反应温度、反应时间分别为65 ℃，60 min；特异性验证表明红心莲尖孢镰刀菌DNA呈绿色阳性反应，灵敏度验证表明LAMP检测最小质量浓度是10 fg·μL−1，比常规PCR提高了10倍；采用LAMP法对15份疑似红心莲茎腐病田间样本进行快速检测，检出率为100%。 结论 本研究建立的多肉植物红心莲茎腐病菌LAMP快速检测方法不仅特异性强、灵敏度高，而且检测结果可视、假阳性低，适合田间尖孢镰刀菌引起的红心莲茎腐病的快速检测。 -
关键词:
- 红心莲 /
- 茎腐病 /
- 尖孢镰刀菌 /
- 环介导等温核酸扩增技术（LAMP） /
- 检测
Abstract:Objective A LAMP (loop-mediated isothermal amplification) assay for rapid, visible and sensitive detection of Fusarium oxysporum on the succulent plant, Echeveria Perle von Nürnberg, was developed for early prevention and control of the disease. Method Based on the elongation factor 1α (EF-1α ) sequence of F. oxysporum , LAMP primers were designed. Template DNA from the infected stems were used to optimize the reaction temperature and time. Specificity and sensitivity of the assay were verified on the detection on infected plants from the field. Result The newly developed LAMP assay at 65 °C for 60 min effectively detected F. oxysporum on Echeveria . The assay gave the positive yellowish green result only on F. oxysporum of Echeveria . It had a detection limit of 10 fg·μL−1 and was 100% positive on detecting F. oxysporum from 15 Echeveria samples showing typical stem rot symptoms. Conclusion The established LAMP assay had high specificity and sensitivity with visual and low false positive results. It was considered suitable for rapid detection of F. oxysporum on Echeveria Perle von Nürnberg in the field. -
图 3 LAMP检测红心莲尖孢镰刀菌Fusarium oxysporum
注：a: LAMP产物凝胶电泳；b: LAMP产物颜色反应。1：Fusarium oxysporum, 2：CK阴性对照。
Figure 3. Detection of F. oxysporum on Echeveria by LAMP assay
Note: a: agarose gel electrophoresis analysis of LAMP products; b: visual result of LAMP test. Lane 1: F. oxysporum; Lane 2: CK, negative control.
图 4 LAMP检测特异性验证
注：a: LAMP产物2.0%凝胶电泳特异性检测；M：marker DL2000, 1：Fusarium oxysporum, 2：Fusarium moniliforme, 3：Colletotrichum destructivum, 4：Corynespora cassiicola, 5：Alternaria alternate, 6: Fusarium sporotrichioides, 7：Fusarium solani, 8：CK阴性对照。 b: LAMP产物特异性颜色反应。1~8试管病原菌样品顺序同（a）.
Figure 4. Specificity of LAMP assay
Note: a: specificity of LAMP for F. oxysporum and other pathogens determined by 2.0% agarose gel electrophoresis. Lane M: marker DL2000; Lane 1: F. oxysporum; Lane 2: F. moniliforme; Lane 3: Colletotrichum destructivum; Lane 4: Corynespora cassiicola; Lane 5: Alternaria alternate; Lane 6: F. sporotrichioides; Lane 7: F. solani; Lane 8: CK, negative control. b: specificity of LAMP for F. oxysporum determined by visual results. 1–8 codes for pathogens in test tubes as shown under Note a.
图 5 LAMP灵敏度检测验证
注: a: LAMP产物琼脂溏凝胶电泳，M：marker DL2000，泳道1~8：DNA质量浓度分别为10 ng·μL−1、1 ng·μL−1、100 pg·μL−1、10 pg·μL−1、1 pg·μL−1、100 fg·μL−1、10 fg·μL−1、1 fg·μL−1; b: LAMP产物灵敏度颜色反应，1~8 PCR管DNA浓度顺序同（a）；c：PCR产物琼脂糖凝胶电泳。
Figure 5. Sensitivity of LAMP and PCR
Note: a: LAMP assay determined by 2.0% agarose gel electrophoresis. Lane M: marker DL 2 000 DNA; Lanes 1–8: amplified products of LAMP reaction using DNA at concentrations of 10 ng·μL−1, 1 ng·μL−1, 100 pg·μL−1, 10 pg·μL−1, 1 pg·μL−1, 100 fg·μL−1, 10 fg·μL−1, 1 fg·μL−1. b: visual results of LAMP reactions from diluted genomic DNA. Concentrations of 1–8 pathogens correspond to Note a. c: agarose gel electrophoresis analysis on PCR products.
表 1 用于LAMP试验的菌株
Table 1. Fungal isolates tested by LAMP assay
菌株
Species寄主
Host菌株数量
Number of isolates采集地
LocalityLAMP反应
LAMP reactionFusarium oxysporum 尖孢镰刀菌 Echeveria Perle Von Nürnberg 红心莲 5 福建 Fujian + Fusarium oxysporum 尖孢镰刀菌 Echeveria Perle Von Nürnberg 红心莲 3 云南 Yunnan + Fusarium oxysporum 尖孢镰刀菌 Cymbidium ensifolium 建兰 1 福建 Fujian + Fusarium oxysporum 尖孢镰刀菌 Citrullus lanatus 西瓜 1 福建 Fujian + Colletotrichum destructivum 毁灭炭疽菌 Echeveria Perle Von Nürnberg 红心莲 1 福建 Fujian − Corynespora cassiicola 山扁豆生棒孢 Sedum morganianum 玉珠帘 1 福建 Fujian − Alternaria alternata 链格孢菌 Echeveria spp. 石莲花属 1 福建 Fujian − Fusarium sporotrichioides 拟分枝孢镰刀菌 Pisum sativum 豌豆 1 福建 Fujian − Fusarium solani 腐皮镰刀菌 Solanum lycopersicum 番茄 1 福建 Fujian − Fusarium moniliforme 串珠镰刀菌 Oryza sativa 水稻 1 江苏 Jiangsu − Fusarium nivale 雪腐镰刀菌 Triticum aestivum 小麦 1 江苏 Jiangsu − Fusarium sambucinum 接骨木镰刀菌 Solanum tuberosum 马铃薯 1 黑龙江 Heilongjiang − Fusarium acuminatum 锐顶镰刀菌 Medicago sativa 苜蓿 1 甘肃 Gansu − Fusarium avenaceum 燕麦镰刀菌 Avena sativa 燕麦 1 甘肃 Gansu − Botryosphaeria rhodina 葡萄座腔菌 Psidium guajava 番石榴 1 福建 Fujian − Verticillium dahliae 大丽轮枝菌 Solanum melongena 茄子 1 福建 Fujian − Colletotrichum gloeosporioides 胶孢炭疽菌 Cymbidium ensifolium 建兰 1 福建 Fujian − Alternaria kikuchiana 梨黑斑病菌 Pyrus pyrifolia 梨 1 福建 Fujian − Alternaria alternata 链格孢菌 Cymbidium ensifolium 建兰 1 福建 Fujian − Phomopsis asparagi 茎枯病菌 Asparagus officinalis 芦笋 1 福建 Fujian − Magnaporche oryzae 稻瘟病菌 Oryza sativa 水稻 1 福建 Fujian − 注：所有镰刀菌及其他真菌菌株均保存在福建省农业科学院； +表示LAMP反应阳性，− 表示LAMP反应阴性。
Note: All isolates of F. oxysporum and other fungi were collection of the Fujian Academy of Agricultural Science; positive (+) or negative (−) results were based on presence or absence of a LAMP product of expected size.
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表 2 红心莲尖孢镰刀菌LAMP及PCR检测引物
Table 2. LAMP and PCR primers of F. oxysporum in Echeveria
引物
Primer序列
Sequence（5′−3′）F3 CACAACCTCAATGAGTGCGT B3 GCATGAGCGACAACATACCA FIP（F1c+F2） ACCCTTACCGAGCTCAGCGG-CGTCACGTGTCAAGCAGT BIP（B1c+B2） TGACAAGCTCAAGGCCGAGC-AGGAGTCTCGAACTTCCAGA PCR F-CTCTTGGTTCTGGCATCG R-GTTCAGCGGGTATTCCTA
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