Establishment and Application of Real-time Quantitative PCR for Detecting of Streptomyces acidiscabies
CHEN Li-Da1, XIE Xue-Wen1, SHI Yan-Xia1, CHAI A-Li1, PAN Hao-Qin2, LI Lei1,*, LI Bao-Ju1,*
1 Insititute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 Weifang University of Science and Technology, Shouguang 262700, China
Abstract Potato common scab, a seed tuber-born disease, is caused by Streptomyces in temperate regions, which seriously threatens the potato (Solanum tuberosum) production and tuber quality. In this study, the specific primer pairs Aci-3/4 were designed according to the toxin gene sequence of Streptomyces acidiscabies, which is one of the pathogens causing potato common scab. A target fragment of 192 bp was specifically amplified using S. acidscabies genomic DNA as a template. The sensitivity of real-time quantitative PCR (qPCR) method obtained in this work showed 1 000 times higher than that of conventional PCR. The results of genomic DNA amplification of the tested samples showed that the cycle threshold (Ct) values in qPCR detection of diseased potato and diseased soil were in the range of 25~32, while conventional PCR did not detect DNA bands in diseased soil. These results suggested that the PCR detection method established in this work had the advantage of strong specificity and high sensitivity, which could quickly detect S. acidiscabies from diseased potato tissues and soil samples. This detection method for potato scab pathogens could provide an effective technical means for early monitoring and effective control of potato scab.
CHEN Li-Da,XIE Xue-Wen,SHI Yan-Xia, et al. Establishment and Application of Real-time Quantitative PCR for Detecting of Streptomyces acidiscabies[J]. 农业生物技术学报, 2020, 28(7): 1314-1321.
[1] 白晓东, 杜珍, 范向斌, 等. 2002. 基质对马铃薯疮痂病抑制效果研究初报[J]. 中国马铃薯, 16(6): 332-334. (Bai X D, Du Z, Fan X B, et al.2002. A study on control over Streptomyces scabies of virus-free microtuber with substratum[J]. Chinese Potato Journal, 16(6): 332-334.) [2] 陈长军, 李俊, 赵伟, 等. 2011. 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, 41(5): 516-525. (Chen C J, Li J, Zhao W, et al.2011. Detection of Sclerotinia sclerotiorum by a quantitative real-time PCR[J]. Acta Phytopathologica Sinica, 41(5): 516-525.) [3] 陈璐, 谢学文, 石延霞, 等. 2013. 实时荧光定量PCR检测蔬菜病原细菌技术[J]. 中国蔬菜, (4): 1-5. (Chen L, Xie X W, Shi Y X, et al. 2013. Real-time quantitative PCR technology for detecting vegetable bacterial disease[J]. China Vegetables, (4): 1-5.) [4] 程颖超, 康华军, 石延霞, 等. 2018. 辣椒疫霉菌RT-PCR检测技术的建立及应用[J]. 园艺学报, 45(5): 997-1006. (Cheng Y C, Kang H J, Shi Y X, et al.2018. Development and application of real-time fluorescent quantitative PCR for detection of Phytophthora capsici[J]. Acta Horticulturae Sinica, 45(5): 997-1006.) [5] 邓宽平, 丁海兵, 雷尊国. 2012. 马铃薯疮痂病的实时定量PCR检测方法[J]. 浙江农业科学, (11): 66-69. (Deng K P, Ding H B, Lei Z G. 2012. Real-time quantitative PCR detection method for potato scab[J]. Journal of Zhejiang Agricultural Sciences, (11): 66-69.) [6] 郭凤柳, 张海颖, 于秀梅, 等. 2013. 中国马铃薯疮痂病菌快速PCR检测技术[J]. 中国农业科学, 46(23): 4926-4932. (Guo F L, Zhang H Y, Yu X M.2013. Rapid PCR detection technology of common scab pathogens in China[J]. Scientia Agricultura Sinica, 46(23): 4926-4932.) [7] 李文学, 肖瑞刚, 吕苗苗, 等. 2019. 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, 52(09): 1529-1540. (Li W X, Xiao R G, Lv M M, et al.2019. Establishment and application of real-time PCR for quantitatively detecting Plasmopara viticola in Vitis vinifera[J]. Scientia Agricultura Sinica, 52(9): 1529-1540.) [8] 史建荣, 王秀宇, 林凡云, 等. 2012. 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]. 中国: 102517385A. (Shi J R, Wang X Y, Lin F Y, et al.2012. Method for establishing Fusarium copy number in rhizosphere soil of transgenic wheat during growth period by real-time quantitative PCR[P]. China: 102517385A.) [9] 王瑜, 马建忠, 张伟杰, 等. 2018. 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, 46(2): 34-39. (Wang Y, Ma J Z, Zhang W J, et al.2018. Rapid identification of Fusarium solani by SYBR Green real time quantitative PCR[J]. Progress in Microbiology and Immunology, 46(2): 34-39.) [10] 肖姬玲, 张屹, 李基光, 等. 2018. 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, 45(4): 921-922. (Xiao J L, Zhang W, Li J G, et al.2018. Establishment of real-time PCR system for quantitatively detecting Fusarium oxysporum f. sp. niveum in soil[J]. Journal of Plant Protection, 45(4): 921-922.) [11] 赵伟全, 刘大群, 杨文香, 等. 2005. 马铃薯疮痂病菌毒素及其致病性的研究[J]. 植物病理学报, 35(4): 317-321. (Zhao W Q, Liu D Q, Yang W X, et al.2005. The relationship between the toxin produced by scab related Streptomyces and the pathogenicity[J]. Acta Phytopathologica Sinica, 35(4): 317-321.) [12] 赵伟全, 杨文香, 李亚宁, 等. 2006. 中国马铃薯疮痂病菌的鉴定[J]. 中国农业科学, 39(2): 313-318. (Zhao W Q, Yang W X, Li Y N, et al.2006. Characterization and identification on the pathogen of potato scab in China[J]. Scientia Agricultura Sinica, 39(2): 313-318.) [13] 张海颖, 郭凤柳, 许华民, 等. 2014. 河北省张北地区马铃薯疮痂病的病菌鉴定[J]. 江苏农业科学, 42(10): 131-134. (Zhang H Y, Guo F L, Xu H M, et al.2014. Identification of pathogens of potato scab in Zhangbei area of Hebei province[J]. Journal of Jiangsu Agricultural Sciences, 42(10): 131-134.) [14] Bouchek-Mechiche K, Gardan L, Normand P, et al.2000. DNA relatedness among strains of Streptomyces pathogenic to potato in France: Description of three new species, S. europaeiscabiei sp. nov. and S. stelliscabiei sp. nov. associated with common scab, and S. reticuliscabiei sp. nov. associated with netted scab[J]. International Journal of Systematic and Evolutionary Microbiology, 50(1): 91-99. [15] Duvivier M, Dedeurwaerder G, Bataille C, et al.2016. Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Puccinia triticinain Belgium[J]. European Journal of Plant Pathology, 145(2): 405-420. [16] Kieser T, Bibb M J, Buttner M J, et al.2000. Practical Streptomyces Genetices[M]. The John Innes Foundation, UK, pp. 613. [17] Kreuze J F, Suomalainen S, Paulin L, et al.1999. Phylogenetic analysis of 16S rRNA genes and PCR analysis of the nec1 gene from Streptomyces spp. causing common scab, pitted scab and netted scab in Finland[J]. Phytopathology, 89(6): 462-469. [18] Lambert D H, Loria R.1989a. Streptomyces acidiscabies sp. nov[J]. International Journal of Systematic Bacteriology, 39(4): 393-396. [19] Lambert D H, Loria R.1989b. Streptomyces scabies sp. nov., nom. rev[J]. International Journal of Systematic Bacteriology, 39(4): 387-392. [20] Lee J H, Park M H, Lee S.2016. Identification of Pseudoperonospora cubensisusing real-time PCR and high resolution melting (HRM) analysis[J]. Journal of General Plant Pathology, 82(2): 110-115. [21] Lehtonen M J, Rantala H, Kreuze J F, et al.2004. Occurrence and survival of potato scab pathogens (Streptomyces species) on tuber lesions: Quick diagnosis based on a PCR-based assay[J]. Plant Pathology, 53: 280-287. [22] Liu D, Anderson N A, Kinkel L L.1995. Biological control of potato scab in the field with antagonistic Streptomyces scabies[J]. Phytopathology, 85(7): 827-831. [23] Loria R, Bukhalid R A, Creath R A.1995. Differential production of thaxto mins by pathogenic Streptomyces species in vitro[J]. Phytopathology, 85(5): 537-541. [24] Luo Y, Ma Z, Reyes H C, et al.2007. Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using real-time PCR[J]. European Journal of Plant Pathology, 118(2): 145-154. [25] Miyajima K, Tanaka F, Takeuchi T, et al.1998. Streptomyces turgidiscabies sp. nov[J]. International Journal of Systematic and Evolutionary Microbiology, 48(2): 495-502. [26] Sharp P J, Kreis M, Shewry P R, Gale M D.1988. Location of β-amylase sequence in wheat and its relatives[J]. Theoretical and Applied Genetics, 75(2): 286-290. [27] Wanner L A .2006. A survey of genetic variation in streptomyces isolates causing potato common scab in the United States[J]. Phytopathology, 96(12): 1363-1371. [28] Wanner L A, Haynes K G.2009. Aggressiveness of Streptomyces on four potato cultivars and implications for common scab resistance breeding[J]. American Journal of Potato Research, 86(5): 335-346.
