镰孢菌属实时荧光定量PCR检测方法的建立及应用

doi:10.7668/hbnxb.20190656

摘要/Abstract

摘要： 为了建立一种快速检测镰孢菌属的方法。根据Fusarium翻译延伸因子（TEF-1α）基因序列，设计并筛选其特异性引物F8-1/F8-2，能从靶标基因组DNA中特异性扩增出大小为187 bp的目的片段，建立Fusarium荧光定量PCR （RT-PCR）检测体系的灵敏度比常规PCR高10⁴倍，且特异性良好。利用该反应体系检测不同湿度环境下病残体和土壤病残体DNA含量的动态变化。结果表明：病残体及土壤病残体DNA初始拷贝数分别为6.90×10¹¹，1.06×10¹²拷贝数/g，经过温度27℃、80%湿度下处理30 d病残体DNA含量下降至5.55×10⁸和0拷贝数/g，而在温度27℃、20%湿度下含量分别为8.04×10⁹，1.30×10⁹拷贝数/g。因此，建立的Fusarium实时荧光定量PCR检测体系具有特异性强、灵敏度高的特点，可以快速准确地定量检测病残体DNA的含量，为瓜类根腐病的早期预防和流行监测提供了有效的技术手段。

关键词: 镰孢菌, 实时荧光定量PCR, 湿度, 病残体

Abstract: To establish a method for rapid detection of Fusarium. Real-time PCR assay for quantitative detection of Fusarium in cucumber was developed and a pair of specific primers were designed based on the genome sequence of cucumber Fusarium and a target fragment of 187 bp was specifically amplified from the genomic DNA of Fusarium. The detection sensitivity of RT-PCR established in this study was 10⁴ times higher than that of conventional PCR. RT-PCR method was used to investigate the variation of crop residues DNA and residues DNA of soil in different humidity. The results showed that the initial copy numbers of DNA of crop residues and crop residues of soil was 6.90×10¹¹,1.06×10¹² copies/g,respectively. After treatment at temperature 27 ℃ and 80% humidity for 30 days, the DNA content of the crop residues decreased to 5.55×10⁸ and 0 copies/g,respectively. The content was 27 ℃ and 20% humidity was 8.04×10⁹ and 1.30×10⁹ copies/g,respectively. Therefore, these studies showed that RT-PCR assay is a highly rapid and reliable method to quantify Fusarium in cucumber crop residues. Application of the assay may potentially improve pathogen identification and disease management.

Key words: Fusarium, Real-time PCR, Humidity, Crop residues

中图分类号:

S432.4

陈利达, 袁军海, 李磊, 石延霞, 柴阿丽, 谢学文, 李宝聚. 镰孢菌属实时荧光定量PCR检测方法的建立及应用[J]. 华北农学报, 2019, 34(S1): 296-301. doi: 10.7668/hbnxb.20190656.

CHEN Lida, YUAN Junhai, LI Lei, SHI Yanxia, CHAI Ali, XIE Xuewen, LI Baoju. Development and Application of Quantitative PCR for Detection of Fusarium[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(S1): 296-301. doi: 10.7668/hbnxb.20190656.

http://www.hbnxb.net/CN/Y2019/V34/IS1/296

参考文献

[1] Nguyen D M C, Seo D J, Lee H B, Kim I S, Kim K Y, Park R D, Jung W J. Antifungal activity of gallic acid purified from Terminalia nigrovenulosa bark against Fusarium solani[J]. Microbial Pathogenesis, 2013, 56(3):8-15. doi:10.1016/j.micpath.2013.01.001.
[2] Armengol J, José C M, Moya M J, Sales R, Vicent A, García-Jiménez J. Fusarium solani f. sp. cucurbitae race 1, a potential pathogen of grafted watermelon production in Spain[J]. Eppo Bulletin, 2000, 30(2):179-183. doi:10.1111/j.1365-2338.2000.tb00875.x.
[3] Li B J, Liu Y, Shi Y X, Xie X W, Guo Y L. First report of crown rot of grafted cucumber caused by Fusarium solani in China[J]. Plant Disease,2010, 94(11):1377-1377. doi:10.1094/PDIS-03-10-0217.
[4] 刘洋. 茄病镰孢菌瓜类专化型的鉴定及其侵染途径研究[D]. 北京:中国农业科学院, 2010.
Lu Y. Study on indentification and infection routes of Fusarium solani f.sp. cucurbitae[D]. Beijing:Chinese Academy of Agricultural Sciences, 2010.
[5] 王继华,陆琳,唐开学. 香石竹尖孢镰孢菌PCR检测[J]. 西南大学学报(自然科学版),2004, 26(4):417-419. doi:10.3969/j.issn.1673-9868.2004.04.011.
Wng J H, Lu L, Tang K X. Detection of carnation wilt(Fusarium oxysporum f.sp.diathi) by PCR[J]. Journal of Southwest Agricultural University(Natural Science), 2004, 26(4):417-419.
[6] 王瑜,马建忠,张伟杰,高恺,冯海霞,张莹,车团结. 腐皮镰孢菌SYBR Green实时荧光定量PCR快速检测方法的建立[J]. 微生物学免疫学进展,2018, 46(2):34-39. doi:10.13309/j.cnki.pmi.2018.02.007.
Wng Y, Ma J Z, Zhang W J, Gao W, Feng H X, Zhang Y, Che T J. Rapid identification of Fusarium solani by SYBR Green Real time quantitative PCR[J].Progress in Microbiology and Immunology,2018, 46(2):34-39.
[7] 李文学,肖瑞刚,吕苗苗,丁宁,石华荣,顾沛雯. 葡萄霜霉病菌实时荧光定量PCR检测体系的建立和应用[J]. 中国农业科学,2019, 52(9):1529-1540.doi:10.3864/j.issn.0578-1752.2019.09.005.
L W X, Xiao R G, Lü M M, Ding N, Shi H R, Gu P W. Establishment and application of Real-time PCR for quantitatively detecting Plasmopara viticola inVitis vinifera[J]. Scientia Agricultura Sinica,2019, 52(9):1529-1540.
[8] 肖姬玲,张屹,李基光,朱菲莹,魏林,梁志怀. 实时荧光定量PCR检测土壤西瓜枯萎病菌体系的建立[J]. 植物保护学报,2018,45(4):921-922. doi:10.13802/j.cnki.zwbhxb.2018.2017032.
Xao J L, Zhang Y, Li J G, Zhu F Y, Wei L, Liang Z H. Establishment of Real-time PCR system for quantitatively detecting Fusarium oxysporum f.sp.niveum in soil[J]. Journal of Plant Protection,2018,45(4):921-922.
[9] Luo Y, Ma Z, Reyes H C, Morgan D, Michailides T J. Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using Real-time PCR[J]. European Journal of Plant Pathology, 2007, 118(2):145-154. doi:10.1007/s10658-007-9124-x.
[10] 陈璐. 黄瓜细菌性角斑病菌和多主棒孢菌PCR检测技术的建立[D]. 北京:中国农业科学院, 2014.
Cen L. PCR-based specific detection of Pseudomonas syringae pv. lachrymans and Corynespora cassiicola[D]. Beijing:Chinese Academy of Agricultural Sciences, 2014.
[11] 史建荣,王秀宇,林凡云,祭芳,董飞,曹欢. 通过荧光时实定量PCR建立转基因小麦生长期内根际土壤中镰孢菌拷贝数的方法[P]. CN102517385A. 2012-06-27.
Si J R, Wang X Y, Lin F Y, Ji F, Dong F, Cao H. Method for establishing Fusarium copy number in rhizosphere soil of transgenic wheat during growth period by real-time quantitative PCR[P]. CN102517385A. 2012-06-27.
[12] Hietala A M, Eikenes M, Kvaalen H, Solheim H, Fossdal C G. Multiplex Real-time PCR for monitoring Heterobasidion annosum Colonization in norway spruce clones that differ in disease resistance[J]. Applied and Environmental Microbiology, 2003, 69(8):4413-4420. doi:10.1128/AEM.69.8.4413-4420.2003.
[13] 贲海燕,石延霞,谢学文,柴阿丽,李宝聚. 氰氨化钙土壤消毒对黄瓜根腐病及土壤病原菌的控制效果[J]. 园艺学报,2016, 43(11):2173-2181. doi:10.16420/j.issn.0513-353x.2016-0148.
Bn H Y, Shi Y X, Xie X W, Chai A L, Li B J. Studies of soil improvement effect of calcium cyanamide and its control efficiency on soil-borne diseases of vegetable crops[J]. Acta Horticulturae Sinica,2016, 43(11):2173-2181.
[14] 程颖超,康华军,石延霞,柴阿丽,张红杰,谢学文,李宝聚. 辣椒疫霉菌RT-PCR检测技术的建立及应用[J]. 园艺学报,2018, 45(5):997-1006.
Ceng Y C, Kang H J, Shi Y X, Chai A L, Zhang H J, Xie X W, Li B J. Development and application of Real-time fluorescent quantitative PCR for detection of Phytophthora capsici[J]. Acta Horticulturae Sinica,2018, 45(5):997-1006.
[15] 陈长军,李俊,赵伟,王建新,周明国. 利用实时荧光定量PCR技术检测油菜菌核病菌[J]. 植物病理学报,2011, 41(5):516-525. doi:10.13926/j.cnki.apps.2011.05.008.
Cen C J, Li J, Zhao W, Wang J X, Zhou M G. Detection of Sclerotinia sclerotiorum by a quantitative real-time PCR[J]. Acta Phytopathologica Sinica,2011, 41(5):516-525.
[16] 谢学文,程颖超,王锟,张红杰,杨杰,石延霞,柴阿丽,李宝聚. 短密木霉菌RT-PCR检测体系的建立及其在南瓜根际的动态分析[J]. 中国生物防治学报,2018,34(4):574-581. doi:10.16409/j.cnki.2095-039x.2018.04.012.
Xe X W, Cheng Y C, Wang K, Zhang H J, Yang J, Shi Y X, Chai A L, Li B J. Establishment of Real-time fluorescent quantitative PCR forTrichoderma brevicompactum and its dynamic change in pumpkin rhizosphere[J]. Chinese Journal of Biological Control,2018,34(4):574-581.
[17] 贺字典,宋士清,高玉峰,石延霞,李宝聚. 棘孢木霉Trichoderma asperellum 在土壤中定殖量的荧光定量PCR检测[J]. 植物保护学报,2016,43(4):552-558. doi:10.13802/j.cnki.zwbhxb.2016.04.004.
H Z D, Song S Q, Gao Y F, Shi Y X, Li B J. Detection of Trichoderma asperellum colonization in soils by Real-time fluorescent quantitative PCR[J]. Acta Phytophylacica Sinica,2016,43(4):552-558.
[18] Gill J S, Sivasithamparam K, Smettem K R J.Effect of soil moisture at different temperatures on Rhizoctonia root rot of wheat seedlings[J]. Plant and Soil, 2001,231(1):91-96. doi:10.1023/a:1010394119522.
[19] 杨秀荣,刘水芳,孙淑琴,刘亦学,张学文,张惟. 天津地区设施菜地主要土传病害的综合防治技术[J]. 中国蔬菜,2008(2):58-59.
Yng X R, Liu S F, Sun S Q, Liu Y X, Zhang X W, Zhang W. Comprehensive prevention and control technology of main soil-borne diseases in facility vegetable fields in Tianjin area[J]. Chinese Vegetables,2008(2):58-59.
[20] Oritsejafor J J. Influence of moisture and pH on growth and survival of Fusarium oxysporum f. sp. elaeidis in soil[J]. Transactions of the British Mycological Society,1986, 87(4):511-517. doi:10.1016/S0007-1536(86)80091-2.
[21] Hide G A, Firmager J P. Effects of soil temperature and moisture on stem canker(Rhizoctonia solani) disease of potatoes[J]. Potato Research, 1989, 32(1):75-80. doi:10.1007/BF02365819.
[22] Lindahl T. Instability and decay of the primary structure of DNA[J]. Nature,1993, 362(6422):709-715. doi:10.1038/362709a0.
[23] Bakshi B K. Wilt disease of Shisham(Dalbergia sissoo Roxb.). IV. The effect of soil moisture on the growth and survival of Fusarium solani in the laboratory[J]. Indian Forester, 1957,83(8):505-11.
[24] Boer W D, Folman L B, Summerbell R C, Boddy L. Living in a fungal world:impact of fungi on soil bacterial niche development[J]. FEMS Microbiology Reviews, 2005, 29(4):795-811. doi:10.1016/j.femsre.2004.11.005.

选择文件类型/文献管理软件名称

选择包含的内容